Reference Work Entry

The Prokaryotes

pp 3-37

Introduction to the Proteobacteria

  • Karel Kersters
  • , Paul De Vos
  • , Monique Gillis
  • , Jean Swings
  • , Peter Vandamme
  • , Erko Stackebrandt

Introduction

Within the domain Bacteria, the phylum Proteobacteria constitutes at present the largest and phenotypically most diverse phylogenetic lineage. In 1988, Stackebrandt et al. named the Proteobacteria after the Greek god Proteus, who could assume many different shapes, to reflect the enormous diversity of morphologies and physiologies observed within this bacterial phylum. In 2002, the Proteobacteria consist of more than 460 genera and more than 1600 species, scattered over 5 major phylogenetic lines of descent known as the classes “Alphaproteobacteria,” “Betaproteobacteria,” “Gammaproteobacteria,” “Deltaproteobacteria” and “Epsilonproteobacteria.” The Proteobacteria account for more than 40% of all validly published prokaryotic genera and encompass a major proportion of the traditional Gram-negative bacteria, show extreme metabolic diversity, and are of great biological importance, as they include the majority of the known Gram-negatives of medical, veterinary, industrial and agricultural interest. Moreover, not only can the origin of mitochondria be traced back to the “Alphaproteobacteria,” but several representatives are ecologically important because they play key roles in the carbon, sulfur and nitrogen cycles on our planet. In this context, the purple nonsulfur bacteria and the rhizobia (both members of the “Alphaproteobacteria”) are two of the most apparent and well-known examples, because the former are purple-colored photosynthetic prokaryotes using light as energy source, and the latter are able to reduce atmospheric nitrogen gas when living in symbiosis with leguminous plants. Agriculture and life on earth would be very different in the absence of these nitrogen-fixing rhizobia!

The Proteobacteria, formerly known as “purple bacteria and relatives,” are characterized by a bewildering diversity of morphological and physiological types: besides rods and cocci, curved, spiral, ring-shaped, appendaged, filamentous and sheathed bacteria occur among this phylum. Most Proteobacteria are mesophilic, but some thermophilic (e.g., Thiomonas thermosulfata and Tepidomonas) and psychrophilic (e.g., Polaromonas) representatives have been described. A great number of Proteobacteria are motile by means of polar or peritrichous flagella, whereas the myxobacteria (belonging to the “Deltaproteobacteria”) display a gliding type of motility and show highly complex developmental lifestyles, whereby often remarkable multicellular and macroscopic structures (so-called “fruiting bodies”) are formed. Most of the known Proteobacteria are free-living; some (such as the rhizobia) enter in symbiotic associations with specific leguminous plants, where they fix nitrogen in root or stem nodules. Others live as intracellular endosymbionts of protozoa and invertebrates (mussels, insects and nematodes), whereas the rickettsiae are obligate intracellular parasites of humans or mammals. The extreme diversity of energy-generating mechanisms is a unique biochemical characteristic of the Proteobacteria: some are chemoorganotrophs (e.g., Escherichia coli), others are chemolithotrophs (e.g., the sulfur-oxidizing bacteria such as the thiobacilli and the ammonia-oxidizing bacteria such as Nitrosomonas) or phototrophs (e.g., the purple colored Chromatium, Rhodospirillum and many others). Concerning their relationship towards oxygen, the Proteobacteria include strictly aerobic and anaerobic species as well as facultative aerobes and microaerophiles. Denitrifiers are reported among the “Alphaproteobacteria,” “Betaproteobacteria,” “Gammaproteobacteria,” and the “Epsilonproteobacteria.” To reveal the extreme phenotypic biodiversity among the Proteobacteria, Table 1 gives an overview of the distribution of the five phylogenetic proteobacterial groups among 13 major phenotypic groups as defined in the 9th edition of Bergey’s Manual of Determinative Bacteriology (Holt et al., 1994). Table 1 clearly shows that only three of these 13 phenotypic groups are unique to any one of the current proteobacterial classes. Representatives of some of these 13 phenotypic groups occur also among several other prokaryotic phyla.

Table 1.

The occurrence of major phenotypic groups among the five classes of the Proteobacteria.

Phenotypic groupa

Group nr.a

Occurring in proteobacterial class:

Aerobic/microaerophilic, motile, helical/vibrioid Gram-negative bacteria

2

α, β, γ, δ and ɛ

Gram-negative aerobic/microaerophilic rods and cocci

4

α, β, γ, δ and ɛ

Facultatively anaerobic Gram-negative rods

5

α, β, γ, δ and ɛ

Anaerobic straight, curved, and helical Gram-negative rods

6

β, γ and δ

Symbiotic and parasitic bacteria of vertebrate and invertebrate species

9

α and γ

Anoxygenic phototrophic bacteria

10

α, β and γ

Aerobic chemolithotrophic bacteria and associated genera

12

β, γ and ɛ

Budding and/or appendaged nonphototrophic bacteria

13

α and γ

Nonphotosynthetic, nonfruiting gliding bacteria

15

β and γ

Nonmotile or rarely motile, curved Gram-negative bacteria

3

α and ɛ

Dissimilatory sulfate- or sulfur-reducing bacteria

7

δ

Sheathed bacteria

14

β

Fruiting gliding bacteria

16

δ

aPhenotypic groups and group numbers are according to Bergey’s Manual of Determinative Bacteriology (Holt et al., 1994). See also Garrity and Holt (2001).

This chapter sketches how and why the Proteobacteria were recognized as a coherent but diverse phylogenetic group and summarizes its phylogenetic structure as well as its phenotypic and ecological diversity.

Towards a Phylogenetic Definition of the “Purple Bacteria and Their Nonphototrophic Relatives”

In the context of the above mentioned extreme biodiversity of the Proteobacteria, it is not surprising that some 50 years ago—when only phenotypic techniques were available for characterization and comparison of prokaryotes—it was impossible to foresee that morphologically and physiologically highly different bacterial groups such as the Enterobacteriaceae and the photosynthetic purple sulfur bacteria (e.g., Chromatium) would be phylogenetically more closely related to each other, whereas morphologically similar bacteria such as the Enterobacteriaceae and the flavobacteria would harbor widely different genomes. Microbiologists possessed in those years only tools to look at the tips of the evolutionary branches of the tree of life. About 15 years prior to the introduction of ribosomal RNA analysis as a suitable method for determining prokaryote evolution, two different approaches were followed aiming at the same goal. The physiologists used the deductive strategy, trying to establish the phylogeny of prokaryotes from selected metabolic properties (Broda, 1970; Broda, 1975), while geneticists and biochemists followed the inductive strategy, originating from the pioneering publication by Zuckerkandl and Pauling (1965).

The question about the origin of bacterial respiration placed the photosynthetic bacteria, especially the anoxygenic “purple bacteria,” in the center of discussion. To be more precise, the problem was how respiration could evolve in parallel in so many groups of organisms and independently from that evolved in cyanobacteria. The similarity of the electron-flow chains in photosynthesis and respiration as an ordered assembly of flavoproteins, cytochromes, quinones and non-heme Fe-S proteins in connection with membranes has been noted (Olson, 1970), and it was proposed that the photosynthetic chain could have been modified and adapted to respiration. It was Broda (1970) who concluded that respiration evolved independently from different kinds of photosynthetic apparatus, and if photosynthetic bacteria themselves evolved in different lines of descent, so would the aerobic respiring bacteria. This “conversion” hypothesis placed significant emphasis on the presence of phototrophic bacteria in various lines of descent and focused on the evolution of electron chains from photosynthetic anaerobes, anaerobic respirers, and aerobes ranging from phototrophic aerobes to strictly aerobic respirers. Broda’s hypothesis, later supported (though refined) by the general phylogenetic frame of ribosomal RNA/DNA sequences, is illustrated by two examples. Firstly, he postulated the existence of a Gram-positive phototroph, later proven by the isolation of the phylogenetically Gram-positive Heliobacterium chlorum (Gest and Favinger, 1983; Woese et al., 1985a), branching deeply in the Clostridium lineage. Secondly, early 16S rRNA cataloguing data indicated the high phylogenetic similarity between the nonsulfur purple bacterium Rhodopseudomonas palustris and the aerobic Nitrobacter winogradskyi (Seewaldt et al., 1982). However, Broda (1975) was not in a position to predict the specific phylogenetic clustering of the purple sulfur and the purple nonsulfur bacteria as opposed to the lineages of green sulfur bacteria, the green nonsulfur bacteria, and the cyanobacteria. Also, as they had not been described in the 1970s, the aerobic bacteriochlorophyll a-containing bacteria (α-3 and α-4 groups and a few in the “Betaproteobacteria”) are not mentioned in the conversion hypothesis. Modifications to the outline of biochemical pathways given by Broda have been made by Schwemmler (1989), combining rRNA-based phylogenetic relatedness between species and the biochemistry of components of electron chains involved in aerobic respiration and anaerobic and aerobic photosynthesis.

Zuckerkandl and Pauling (1965) highlighted the importance of using semantides, such as genomic DNA, its primary transcript RNA, and the translation product, the proteins, as evolutionary markers. They postulated that the path of evolution is laid down in the primary structure of nucleic acids and determination of the blueprint of evolutionary conservative genes would necessarily unravel the evolutionary history of organisms. Owing to methodological restrictions, proteins rather than DNA or RNA were accessible to sequence analyses, restricting early molecular studies to those proteins easily isolated and sequenced. Besides histones and fibrinopeptides, used in the determination of the rate of gene evolution in eukaryotes, primarily proteins such as ferredoxin and cytochrome c allowed the first glimpse of the evolution of prokaryotic genes. While the ferredoxin data were obtained for mainly Gram-positive anaerobic bacteria and cyanobacteria, cytochrome c sequences were generated for respiring organisms as well as for anaerobic and aerobic photosynthetic bacteria (Schwartz and Dayhoff, 1978; Ambler et al., 1979) as well as for mitochondria, the origin of which could be traced to members of the “Alphaproteobacteria.” Surprising to taxonomists, the clustering of species according to the primary and tertiary structure of proteins (Dickerson, 1980) did not correlate with the traditional systematic groupings such as those laid down in Bergey’s Manual of Determinative Bacteriology (Buchanan and Gibbons, 1974). It was especially the phylogenetic closeness between Gram-negative, nonphototrophic organisms and phototrophs that shed doubts on the correctness of the sequence-based findings. However, shortly afterwards, results from the 16S rRNA cataloguing approach, fully supporting the protein-based studies, demonstrated the obvious failure of the phenotype to mirror phylogenetic relatedness. Some 30 years ago, the pioneering studies on 16S rRNA oligonucleotide cataloguing by Woese and coworkers (Fox et al., 1977; Woese and Fox, 1977) revolutionized the insights in microbial evolution, in particular, and evolution of life on earth, in general. The purple bacteria (nowadays named Proteobacteria) were first recognized and circumscribed as a distinct division (phylum) of the eubacteria by Woese and coworkers on the basis of signature and sequence analyses of the 16S rRNA/rDNA (Gibson et al., 1979; Fox et al., 1980; Woese et al., 1985b; Woese, 1987). Because the purple photosynthetic phenotype was distributed throughout the group the trivial name “purple bacteria” seemed to be justified for this phylogenetic group of Gram-negatives (Woese, 1987), although many nonphotosynthetic species (such as the enterics, the pseudomonads, rhizobia, rickettsiae, etc.) grouped among the photosynthetic representatives. The oligonucleotide signature analysis of 16S rRNAs revealed already in the 1980s that the purple bacteria comprised at least three major subdivisions, arbitrarily designated as α, β and γ. The purple nonsulfur (PNS) bacteria (e.g., Rhodospirillum, Rhodobacter and Rhodocyclus) were found in two subdivisions (α and β; Woese et al., 1984a; Woese et al., 1984b), whereas the purple sulfur (PS) bacteria (e.g., Chromatium and Ectothiorhodospira) belonged to the γ-subdivision (Woese et al., 1985c). When more Gram-negatives were investigated by rRNA-cataloguing, rDNA-sequencing and also by the DNA/rRNA hybridization technique, it became clear that the majority of the so-called purple bacteria were in fact nonphotosynthetic prokaryotes. Without being able to trace the deeper phylogenetic relationships, De Ley and coworkers (reviewed by De Ley, 1992) studied hundreds of Gram-negatives by the DNA/rRNA hybridization technique, which allowed their relative phylogenetic position within the various rRNA-branches to be determined. The α, β and γ groups corresponded to the rRNA superfamilies IV, III and I+II, respectively, as defined by De Ley and coworkers (De Vos and De Ley, 1983; De Vos et al., 1985; De Vos et al., 1989; De Ley et al., 1986; Jarvis et al., 1986; Rossau et al., 1986; Willems et al., 1991a; De Ley, 1992). The DNA/rRNA hybridization approach together with the application of various genomic and phenotypic techniques (i.e., the polyphasic approach; Colwell, 1970; Vandamme et al., 1996) allowed improvement of the classification of various Gram-negative taxa, such as the pseudomonads (De Vos and De Ley, 1983; De Vos et al., 1985; De Vos et al., 1989; Willems et al., 1991a; De Ley, 1992; Kersters et al., 1996), Alcaligenes and Bordetella (Kersters and De Ley, 1984; De Ley et al., 1986), the Pasteurellaceae (De Ley et al., 1990), the Neisseriaceae and Moraxellaceae (Rossau et al., 1986; Rossau et al., 1991), the Acetobacteraceae (Gillis and De Ley, 1980; De Ley and Gillis, 1984; Swings, 1992), the Campylobacteraceae (Vandamme and De Ley, 1991) and many others.

During the last 15 years, the knowledge concerning the phylogeny of the Gram-negatives gradually became more comprehensive by the availability of an increasing number of nearly complete 16S rRNA sequences, indicating that the Gram-negative sulfur- and sulfate-reducing bacteria form a fourth group or δ-subdivision among the purple bacteria together with the myxobacteria and the bdellovibrios (Oyaizu and Woese, 1985; Woese, 1987). Because the majority of the genera belonging to the purple bacteria are not purple and not photosynthetic, Stackebrandt et al. proposed in 1988 the name “Proteobacteria” for a new higher taxon (at the level of class), including these purple bacteria and their relatives; the α to δ groups were temporarily considered as subclasses, pending further studies and nomenclatural proposals. Later some microaerophilic and helical-shaped bacteria such as the campylobacters were placed in the fifth or ε-subclass, corresponding to rRNA-superfamily VI of De Ley’s research group (Vandamme and De Ley, 1991; Stackebrandt, 1992).

Each new issue of the International Journal of Systematic and Evolutionary Microbiology adds new genera and species to the Proteobacteria, entailing a more bush-like rRNA/rDNA-tree topology. Yet, the distinctness of the subclasses seems to be maintained, although the differentiation between the β- and the γ-group is becoming less clear, and some rDNA-based trees indicate that Desulfurella and allied bacteria of the δ-group may constitute a sixth subdivision (Rainey et al., 1993). In the second edition of Bergey’s Manual of Systematic Bacteriology (Garrity, 2001a), the Proteobacteria have been elevated to the rank of phylum and the subclasses α to ε have been elevated to the rank of classes, corresponding to the names “Alphaproteobacteria,” “Betaproteobacteria,” “Gammaproteobacteria,” “Deltaproteobacteria,” and “Epsilonproteobacteria,” respectively (see Table 2). In a recently revised megaclassification of the prokaryotes, Cavalier-Smith (2002) proposes a new classification and nomenclature for the five major subgroups of the Proteobacteria (see Table 2).

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Table 2.

Three systems of Proteobacteria classification: correspondence between the names of taxa above the rank of order.

In the present chapter, we follow the classification of the major reference work, Bergey’s Manual of Systematic Bacteriology (Garrity, 2001), and will use ranks and names as listed in Table 2 (2nd column).

Is the 16S-rRNA-based Phylogeny of the Proteobacteria Confirmed by Other Experimental Approaches?

The first indications that the traditional groupings of the Gram-negative bacteria according to the previous editions of Bergey’s Manual of Systematic Bacteriology (Buchanan and Gibbons, 1974; Krieg and Holt, 1984) and Bergey’s Manual of Determinative Bacteriology (Holt et al., 1994) were not phylogenetic came from comparative sequence analysis of some redox proteins, such as cytochrome c [Introduction]. Also studies by Byng et al. (1983) on the regulation of multibranched pathways for the biosynthesis of aromatic amino acids indicated that some members of the genus Pseudomonas, such as P. testosteroni and P. solanacearum, were more similar to other genera than to the group of species around the type species Pseudomonas aeruginosa (belonging to the “Gammaproteobacteria”), which is in perfect agreement with rRNA/rDNA data. Later, Pseudomonas testosteroni and P. solanacearum (both belonging to the “Betaproteobacteria”) were indeed transferred to the genera Comamonas and Ralstonia, respectively (Tamaoka et al., 1987; Yabuuchi et al., 1995).

The definition of the Proteobacteria as a separate evolutionary lineage and the overall picture of the grouping in five classes are confirmed by comparative analysis of 23S rRNA-genes and alternative phylogenetic markers such as the genes coding for elongation factor Tu and ATPase (Ludwig et al., 1995; Ludwig and Schleifer, 1999; Ludwig and Klenk, 2001). Moreover, the comparative analysis of conserved insertions and deletions (so-called “signature sequences”) found in different bacterial proteins yielded molecular means to define the phylum Proteobacteria and its various classes and improved the understanding of their evolutionary relationships to other groups of Bacteria and Eukarya (Gupta, 2000; Gupta, 2002). Proteins studied were, e.g., alanyl-tRNA synthetase, succinyl-CoA synthetase, Hsp 60 (GroEL), Hsp70 heat shock protein, and recA protein. The listing of extensive literature on this subject would be unjustifiable in the context of this chapter (see Gupta, 2000). The proteobacterial classes emerged in the following order during evolution (Gupta, 2000): epsilon and delta → alpha → beta → gamma, which is similar to the order proposed on the basis of RNA polymerase β and β′ subunits (Klenk et al., 1999). Indeed, the ribosomal DNA tree does not clearly resolve the order in which the majority of main lineages emerged during evolution. Proteobacteria, Gram-positive bacteria, Cyanobacteria, Cytophaga/Flavobacteria and some other minor lineages appear to evolve at the same node, giving the rDNA-tree a fork-like appearance. The branching order of the various main lines of descent as depicted by Gupta (2000) is different. Gupta places the low G+C Gram-positive bacteria (clostridia) at the base of bacterial evolution, while those organisms constituting the base of the bacterial 16S rDNA tree (Aquifex, Deinococcus-Thermus, and green sulfur bacteria) are described as having evolved later. Proteobacteria appear as the last evolutionary group. The importance of the proteobacterial cell is highlighted not only as the donor of the mitochondria (Margulis, 1993; Falah and Gupta, 1994), but also as one of the fusion partners (the other being a archaeal cell) giving rise to the ancestral eukaryotic cell (see Gupta [2000] for an extensive list of references). More genes and complete genomes will have to be sequenced to determine the extent and relative time of lateral gene transfer and to highlight those genes that represent the core of genes transmitted vertically, hence representing the evolutionarily stable component of the cell.

So far, no reliable phenotypic features were found that would be characteristic for the whole phylum of the Proteobacteria and differentiate it from other phyla of the domain Bacteria. It seems also very difficult to find stable phenotypic features for the differentiation of the five classes, which is not surprising in view of the enormous variety of morphological and physiological types existing in each of these five lineages. Phototrophy cannot be used as phylogenetic marker, except that PNS- and PS-bacteria are only reported so far among the Proteobacteria, which could indicate that this type of photosynthesis was common in the proteobacterial ancestors and was lost during evolution in a great number of sublineages. Support for the validity of some of the results of nucleic acid and protein sequencing came from chemotaxonomy. Within the “Alphaproteobacteria,” Rhodopseudomonas palustris, Nitrobacter winogradskyi, Blastochloris ( Rhodopseudomonas) viridis, Phenylobacterium immobile, certain thiobacilli, Brucella melitensis and Brevundimonas ( Pseudomonas) diminuta exhibited an unusual lipid A, which lacked glucosamine but contained instead a 2,3-diamino-2,3-dideoxy-D-glucose (Stackebrandt et al., 1988a). This structure was not uniformly distributed among all members of the “Alphaproteobacteria” but was shared among closely related species. Lipid A composition also confirmed the phylogenetic distinctness of species hitherto affiliated to the same genus on the basis of their phenotype. For example, Rhodocyclus tenue and Rhodocyclus gelatinosa (members of the “Betaproteobacteria”; Imhoff et al., 1984) differed significantly in the chemistry of their lipid A, a finding that supported the reclassification of the latter species as Rubrivivax gelatinosus (Willems et al., 1991b). As far as we could determine from the literature, quinones and polyamines seem to be good marker molecules to differentiate the major proteobacterial lineages (Collins and Jones, 1981; Collins and Widdel, 1986; Busse and Auling, 1988; Auling, 1992; Hamana and Matsuzaki, 1993; Busse et al., 1996; Table 3; Directory, Databases and Dictionaries Compiled by WDCM website [{http://www.wdcm.nig.ac.jp/cgi-bin/search.cgi}]). Ubiquinones are typical for the α, β and γ lineages, whereas menaquinones are characteristic for the “Deltaproteobacteria” and “Epsilonproteobacteria,” although some photosynthetic “Alphaproteobacteria” and “Betaproteobacteria” contain also MK-8, MK-9 or MK-10 (Hiraishi et al., 1984). Some members of the Pasteurellaceae (“Gammaproteobacteria”) are characterized by demethylmenaquinones. Gas chromatographic analysis of the methylesters of cellular fatty acids (FAME) does not allow differentiation of the five major lineages. Of course, phenotypic and chemotaxonomic features (e.g., FAME) are very useful for the description and differentiation of species, genera and families, and the polyphasic taxonomic approach integrating all available data (Colwell, 1970; Vandamme et al., 1996) has been applied during the last decade to improve the classification of previously polyphyletic proteobacterial genera, such as Erwinia (Hauben et al., 1998), Pseudomonas (Kersters et al., 1996; Anzai et al., 2000), Rhizobium and allies (de Lajudie et al., 1998), Thiobacillus (Kelly and Wood, 2000), Leptothrix (Spring et al., 1996) and Oceanospirillum (Satomi et al., 2002).

Table 3.

Some selected key genera, general characteristics, and differentiating features of the five classes of the Proteobacteria.

 

Proteobacterial class

Alpha

Beta

Gamma

Delta

Epsilon

Important genera

Acetobacter

Alcaligenes

Actinobacillus b

Bdellovibrio

Campylobacter a

Agrobacterium a

Bordetella a,b

Azotobacter

Chondromyces

Helicobacter a

Bartonella a

Burkholderia b

Buchnera a

Desulfobacter

Sulfurospirillum

Bradyrhizobium

Comamonas

Chromatium

Desulfovibrio b

Wolinella

Brucella a

Neisseria a,b

Coxiella b

Geobacter b

 

Caulobacter a

Nitrosomonas b

Erwinia b

Myxococcus b

 

Ehrlichia

Ralstonia b

Escherichia a,b

Polyangium

 

Gluconobacter

Rhodocyclus

Francisella b

Syntrophus

 

Hyphomicrobium

Sphaerotilus

Haemophilus a,b

  

Mesorhizobium a

Spirillum

Legionella b

  

Methylobacterium b

Thiobacillus

Methylococcus b

  

Nitrobacter

 

Pasteurella a

  

Rhizobium

 

Pectobacterium

  

Rhodobacter b

 

Pseudomonas a,b

  

Rhodospirillum

 

Salmonella a,b

  

Sinorhizobium a

 

Shewanella b

  

Sphingomonas b

 

Shigella a,b

  

Rickettsia a,b

 

Stenotrophomonas

  

Wolbachia b

 

Vibrio a,b

  
  

Xanthomonas a,b

  
  

Xylella a,b

  
  

Yersinia a,b

  

Number of genera/number of speciesc

140/425

76/225

181/755

57/165

6/49

Major ubiquinone typed

Q-10

Q-8

Q-8, Q-9, or Q-10 to Q-14

Major mena-quinone typed

Some contain also MK-9 or MK-10

Some contain also MK-8

Some contain also MK-8 or MK-7

MK-6, MK-6(H2), MK-7, MK-7(H2) or MK-8e

MK-6, methyl-substituted MK-6f

Characteristic polyaminesg

Most contain a triamine (sym-homosper-midine or spermidine)

2-Hydroxy-putrescine

Spermidine and/or putrescine or cadaverine; or 1,3-diamino-propane

Most contain a triamine (sym-homosper-midine or spermidine)

Spermidine

Symbols and abbreviations: —, absent; DMK, demethylmenaquinone; and MK-6(H2), hydrogenated menaquinone-6.

aThe genome of at least one representative strain has been sequenced (as of mid 2002).

bSequencing of the genome of at least one representative strain is in progress (as of mid 2002; see, e.g., http://www.tigr.org/ or http://www.ncbi.nlm.nih.gov).

cOnly validly published names (situation as of mid 2002).

dCollins and Jones (1981), Hiraishi et al. (1984), http://www.wdcm.nig.ac.jp/cgi-bin/search.cgi, and H.J. Busse, personal communication.

eCollins and Widdel (1986).

fMoss et al. (1990).

gAuling (1992), Busse and Auling (1988), and Hamana and Matsuzaki (1993).

No single nucleotide signature is found in the 16S rRNA that could serve unambiguously to define the proteobacterial phylum (sensu Bergey’s Manual). However, specific 16S rRNA sequence signatures for the various classes of the Proteobacteria have been described and used for the construction of DNA probes (Woese, 1987; Stackebrandt et al., 1988b; Manz et al., 1992; Ludwig et al., 1998). Such probes were extensively applied for the detection and visualization of Proteobacteria and other prokaryotes in activated sludge (Wagner et al., 1993; Snaidr et al., 1997; Juretschko et al., 2002). Table 4 lists the sequence of some representative rRNA-targeted oligonucleotide probes.

Table 4.

Some rRNA-targeted oligonucleotide probes for fluorescent in-situ hybridization.

Probe

Position

Probe sequence (5′ → 3′)

Specificity

Reference

ALF1b

16S rRNA 19–35

CGTTCG(C/T)TCTGAGCCAG

“Alphaproteobacteria,” but not exclusive

Manz et al., 1992

BET42a

23S rRNA 1027–1043

GCCTTCCCACTTCGTTT

“Betaproteobacteria”

Manz et al., 1992

GAM42a

23S rRNA 1027–1043

GCCTTCCCACATCGTTT

“Gammaproteobacteria,” but not the deeply branching taxa

Manz et al., 1992

Delta 385

16S rDNA 385–402

CGGCGT(C/T)GCTGCGTCAGG

“Deltaproteobacteria” sulfate-reducers, but not exclusive

Rabus et al., 1996

The Proteobacterial Classes: Morphological, Physiological, Ecological and Phylogenetic Diversity

Figure 1 is a simplified phylogenetic tree of the Proteobacteria based on the nearly complete 16S rDNA sequences of the type strains of the type species of the majority of proteobacterial genera. Only the names of the families and major groups are indicated. The “Deltaproteobacteria” and “Epsilonproteobacteria” form the deeper branches of the phylum; the “Alphaproteobacteria” are also clearly separated, whereas the closer relationship between the β and γ lineages may indicate the common origin of the latter groups (Ludwig and Klenk, 2001).

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Fig. 1.

Simplified neigbor-joining phylogenetic tree of the Proteobacteria based on the 16S rDNA sequences of the type strains of the proteobacterial genera. Distances were calculated using the substitution rate calibration method in TREECON 3.1 (Van de Peer and De Wachter, 1997). The bar indicates 10% estimated sequence divergence. Bacillus subtilis was used as outgroup (not shown). The width of the triangles is proportional to the number of genera within each cluster. The purple color indicates clusters where phototrophic bacteria occur: purple sulfur photosynthetics (Chromatiaceae and Ectothiorhodospiraceae) in the “Gammaproteobacteria,” purple nonsulfur photosynthetics and aerobic phototrophs in the “Alphaproteobacteria” and “Betaproteobacteria.” Shaded purple triangles indicate that most representatives of phototrophic “Alphaproteobacteria” and “Betaproteobacteria” are phylogenetically related to nonphototrophic, strictly chemotrophic bacteria belonging to the respective families or groups. Aerobic phototrophic Proteobacteria were reported among the Acetobacteraceae (sensu lato), the Sphingomonadaceae, the Rhodobacter, Methylobacterium and Bradyrhizobium groups (all “Alphaproteobacteria”), and in the genus Roseateles (“Betaproteobacteria,” family Comamonadaceae).

Phototrophic bacteria occur only in the “Alphaproteobacteria,” “Betaproteobacteria” and “Gammaproteobacteria” (purple colored triangles in Fig. 1); these anoxygenic phototrophic purple bacteria can be subdivided into the purple sulfur (PS; e.g., Chromatium and Ectothiorhodospira) and purple nonsulfur (PNS) bacteria (e.g., Rhodospirillum). All known PS bacteria belong to the “Gammaproteobacteria” and use H2S or S° as sole electron donor. The PNS bacteria occur among the “Alphaproteobacteria” and “Betaproteobacteria.” A great number of phototrophic Proteobacteria are versatile and can easily switch from a phototrophic to a heterotrophic lifestyle in the absence of light. The results of comparative 16S rDNA sequence analysis have led to extensive taxonomic rearrangements within previously defined taxa of proteobacterial phototrophs (Imhoff, 2001a).

In this chapter, we briefly touch on the morphological, physiological, ecological and phylogenetic diversity of the major proteobacterial groups. The reader will find more detailed information in the chapters dealing with the individual families and genera of the Proteobacteria.

An incomplete survey of disease-causing Proteobacteria is given in Table 5 for the bacteria of clinical and veterinary interest and in Table 6 for the phytopathogens. Some examples of industrial and biotechnological applications are listed in Table 7.

Table 5.

Some Proteobacteria involved in human and animal disease.

Proteobacterial class and species

Familya

Disease (site of action)

Risk groupb

“Alphaproteobacteria”

Bartonella henselae

Bartonellaceae

Cat-scratch disease, and bacillary angiomatosis

2

Bartonella quintata

Bartonellaceae

Trench fever

2

Brucella melitensis

Brucellaceae

Abortion (genital tract of animals), and brucellosis in man

3

Ehrlichia chaffeensis

Ehrlichiaceae

Human ehrlichiosis

2

Orientia tsutsugamushi

Rickettsiaceae

Scrub typhus

3

Rickettsia rickettsii

Rickettsiaceae

Rocky Mountain spotted fever

3

Rickettsia prowazekii

Rickettsiaceae

Typhus fever

3

“Betaproteobacteria”

Bordetella pertussis

Alcaligenaceae

Whooping cough (respiratory tract)

2

Burkholderia mallei

“Burkholderiaceae”

Glander disease in equines

3

Burkholderia pseudomallei

“Burkholderiaceae”

Melioidosis

3

Neisseria gonorrhoeae

Neisseriaceae

Gonorrhoea (genital tract)

2

Neisseria meningitidis

Neisseriaceae

Meningitis (central nervous system)

2

Taylorella equigenitalis

Alcaligenaceae

Endometritis in mares

2

“Gammaproteobacteria”

Coxiella burnetii

“Coxiellaceae”

Q-fever

3

Escherichia coli

Enterobacteriaceae

  

intestinal variants

 

Diarrhea

2

uropathogenic variants

 

Urinary tract infections

2

verocytotoxigenic strains

 

e.g., O157:H7, causing hemorrhagic diarrhea and kidney failure

3

Francisella tularensis

“Francisellaceae”

Tularemia (skin and lymph nodes)

3

Haemophilus influenzae

Pasteurellaceae

Meningitis, pericarditis, and pneumonia

2

Legionella pneumophila

Legionellaceae

Legionnaires’ disease (respiratory tract)

2

Pseudomonas aeruginosa

Pseudomonadaceae

Nosocomial infections (skin and respiratory tract, urinary tract)

2

Salmonella typhi

Enterobacteriaceae

Typhoid fever (gastrointestinal tract)

3

Shigella dysenteriae

Enterobacteriaceae

Dysenteria (gastrointestinal tract)

3

Vibrio cholerae

Vibrionaceae

Cholera (gastrointestinal tract)

2

Yersinia pestis

Enterobacteriaceae

Plague (blood)

3

“Epsilonproteobacteria”

Campylobacter coli, C. jejuni

Campylobacteraceae

Diarrhea

2

2

Helicobacter pylori

“Helicobacteraceae”

Duodenal and gastric ulcers, and gastric carcinoma

2

aAccording to Bergey’s Manual of Systematic Bacteriology (Garrity and Holt, 2001). See also Fig. 1. Quotation marks are used for names which have not yet been validated (as of mid 2002).

bAccording to EU-directive 2000/54/EG; see also http://www.dsmz.de/.

Table 6.

Some selected plant diseases caused by Proteobacteria.

Proteobacterial class and species

Familya

Disease (symptoms)

“Alphaproteobacteria”

Agrobacterium rhizogenes

Rhizobiaceae

Hairy root

Agrobacterium tumefaciens

Rhizobiaceae

Crown gall

Candidatus Liberibacter asiaticus”

in cluster of Rhizobiaceae, Bartonellaceae, etc.

Greening disease on citrus (a phloem-restricted disease)

“Betaproteobacteria”

Acidovorax anthurii

Comamonadaceae

Leaf-spot on Anthurium

Burkholderia cepacia

“Burkholderiaceae”

Soft rot (sour skin on onion)

Burkholderia glumae

“Burkholderiaceae”

Sheath necrosis on rice

Ralstonia solanacearum

“Ralstoniaceae”

Moko disease on banana (vascular wilt)

Xylophilus ampelinus

Comamonadaceae

Necrosis and canker on grapevine

“Gammaproteobacteria”

Brenneria (Erwinia) salicis

Enterobacteriaceae

Watermark disease on willow

Brenneria nigrifluens

Enterobacteriaceae

Bark canker on Persian walnut (Juglans regia)

Erwinia amylovora

Enterobacteriaceae

Fire blight on pome fruit (vascular wilt)

Erwinia stewartii

Enterobacteriaceae

Stewart’s wilt on corn (vascular wilt)

Pectobacterium (Erwinia) carotovorum

Enterobacteriaceae

Soft rot

Pseudomonas agarici

Pseudomonadaceae

Spots on mushrooms

Pseudomonas marginalis

Pseudomonadaceae

Soft rot (pink eye) on potato

Pseudomonas savastanoi

Pseudomonadaceae

Galls on olive trees

Pseudomonas syringae

Pseudomonadaceae

Wildfire on tobacco, haloblight on beans, spots on tomato and pepper (blights and spots)

Pseudomonas syringae

Pseudomonadaceae

Canker on stone fruit

Xanthomonas campestris

“Xanthomonadaceae”

Black rot on crucifers (vascular wilt)

Xanthomonas citri

“Xanthomonadaceae”

Canker on citrus

Xanthomonas oryzae

“Xanthomonadaceae”

Blight on rice

Xanthomonas populi

“Xanthomonadaceae”

Canker on poplar trees

Xanthomonas translucens

“Xanthomonadaceae”

Blight on cereals

Xanthomonas vesicatoria

“Xanthomonadaceae”

Spots on tomato and pepper

Xylella fastidiosa

“Xanthomonadaceae”

Pierce’s disease (e.g., on grapevine)

aAccording to Bergey’s Manual of Systematic Bacteriology (Garrity and Holt, 2001). See also Fig. 1. Quotation marks are used for names which have not yet been validated (as of mid 2002).

Table 7.

Some Proteobacteria involved in industrial and biotechnological processes.

Proteobacterial class, genus or species

Familya

Industrial product or process

“Alphaproteobacteria”

Acetobacter aceti

Acetobacteraceae

Vinegar

Acetobacter xylinus

Acetobacteraceae

Cellulose membranes

Agrobacterium

Rhizobiaceae

Plant engineering (Ti-plasmid)

Gluconobacter oxydans

Acetobacteraceae

Oxidation of sorbitol (for vitamin C production)

Rhizobium

Rhizobiaceae

Inoculants for nodule formation on leguminous plants (N2-fixation)

Rhodobacter capsulatus

“Rhodobacteraceae”

Production of hydrogen gas

Zymomonas mobilis

Sphingomonadaceae

Ethanol

“Betaproteobacteria”

Ralstonia eutropha

“Ralstoniaceae”

Poly-β-hydroxybutyrate (bioplastics) and single-cell protein

“Gammaproteobacteria”

Azotobacter

Pseudomonadaceae

Alginates (polysaccharide) and poly-β-hydroxybutyrate (bioplastics)

Chromatium

Chromatiaceae

Production of hydrogen gas

Erwinia herbicola

Enterobacteriaceae

Biological control of frost damage

Escherichia coli

Enterobacteriaceae

Production of heterologous proteins (e.g., insulin, interferon, and antiviral vaccines)

Photobacterium

Vibrionaceae

Luciferase (lux-genes)

Pseudomonas

Pseudomonadaceae

Oxidation of aliphatic and aromatic compounds

Acidithiobacillus ferrooxidans

Acidithiobacillus group

Active metal mining (bioleaching)

Xanthomonas campestris

“Xanthomonadaceae”

Xanthan (polysaccharide)

aAccording to Bergey’s Manual of Systematic Bacteriology (Garrity and Holt, 2001). See also Fig. 1. Quotation marks are used for names which have not yet been validated (as of mid 2002).

The “Alphaproteobacteria”

The 16S rDNA-tree separates the α-class clearly from the other proteobacterial classes (Fig. 1). The bacterial taxa belonging to the “Alphaproteobacteria” (some140 genera and 425 species at present) are morphologically and metabolically extremely diverse. More detailed information can be found in the chapters dealing with the individual families and genera of this class. Table 8 provides an overview of the major phylogenetic groups (see also Fig. 1) and the names of the orders and families of the “Alphaproteobacteria” as they are listed in the 2nd edition of Bergey’s Manual of Systematic Bacteriology (Garrity, 2001a).

Table 8.

The “Alphaproteobacteria”: orders, families and number of genera.a

Order

Family

Number of genera

Rhodospirillales

Rhodospirillaceae

10

Acetobacteraceae

12

Rickettsiales

Rickettsiaceae

3

Ehrlichiaceae

5

“Holosporaceae”

7

“Rhodobacterales”

“Rhodobacteraceae”

20

“Sphingomonadales”

Sphingomonadaceae

9

Caulobacterales

Caulobacteraceae

4

“Rhizobiales”

Rhizobiaceae

7

Bartonellaceae

1

Brucellaceae

3

“Phyllobacteriaceae”

6

“Methylocystaceae”

3

“Beijerinckiaceae”

3

“Bradyrhizobiaceae”

8

Hyphomicrobiaceae

19

“Methylobacteriaceae”

3

“Rhodobiaceae”

1

aAccording to the second edition of Bergey’s Manual of Systematic Bacteriology (Garrity and Holt, 2001). Quotation marks are used for names which have not yet been validated (as of mid 2002).

The majority of the “Alphaproteobacteria” are rod-shaped, but cocci and curved, spiral, stalked, budding and prosthecate forms do also occur. Some are phototrophic purple nonsulfur (PNS) bacteria (such as Rhodospirillum and Rhodobacter), whereas others are chemolithotrophs (e.g., the nitrite-oxidizing Nitrobacter) or chemoorganotrophs (e.g., Sphingomonas and Brucella). Marine and halophilic phototrophic PNS bacteria seem to be restricted to the “Alphaproteobacteria” (Imhoff, 2001b), whereas a physiologically remarkable group of bacteria, containing bacteriochlorophyll (BChl) but unable to grow phototrophically under anaerobic conditions, belongs to various lineages of the “Alphaproteobacteria” (Yurkov and Beatty, 1998; Yurkov, 2001). Except for Roseateles, all aerobic BChl-containing bacteria investigated so far are exclusively found within the “Alphaproteobacteria” and appear to be related phylogenetically to aerobic purely chemoorganotrophic bacteria. This could indicate that the presence of BChl in some members of the “Alphaproteobacteria” (e.g., Erythrobacter and Roseobacter) is an atavistic trait that remained functional after the aerobic bacteria evolved from their anaerobic phototrophic ancestors (Stackebrandt et al., 1996; Yurkov, 2001). Thus, aerobic phototrophic bacteria could represent an intermediate phase of evolution from anaerobic purple phototrophs to nonphotosynthetic aerobic chemotrophs. During the last decade, the classification of a number of classical phototrophic genera, such as Rhodospirillum and Rhodopseudomonas, underwent considerable changes, which are in agreement with 16S rDNA sequence data, morphological parameters, internal membrane structures as well as important chemotaxonomic parameters, such as the composition of cellular fatty acids, ubiquinones and cytochrome c-type structures. Most of the photosynthetic PNS bacteria are also capable of nitrogen fixation.

Classical chemoorganotrophs (such as Sphingomonas), as well as typical acidophiles (e.g., Acetobacter) and methylotrophs (e.g., Methylobacterium) belong to the “Alphaproteobacteria.” A great number of α-class members live in association with eukaryotes: some are indeed pathogenic for humans and animals (e.g., Brucella) or plants (e.g., Agrobacterium), and others display an obligate parasitic lifestyle, cause diseases in humans and mammals, are transmitted by insect or tick bites (e.g., the Rickettsiaceae), or live symbiotically in the roots of leguminous plants (e.g., Rhizobium and Bradyrhizobium) and play a key role in atmospheric nitrogen fixation. Hence, these “Alphaproteobacteria” thrive in widely divergent habitats and exert a significant impact in the biosphere of our planet. The evolutionary roots of the mitochondrion are within the α-class (see Symbiotic, Parasitic and Not-Yet Cultured Proteobacteria, and the Alphaproteobacterial Origin of Mitochondria), and the understanding of the molecular aspects of plant tumor induction by Agrobacterium tumefaciens led to revolutionary applications in plant agriculture, where plant genetic engineers have used the natural transformation system of Agrobacterium as a vector for the introduction of foreign DNA into plants (Birch, 1997).

The Acetobacteraceae and the Rhodospirillaceae appear to be the deeper branching lineages among the “Alphaproteobacteria” (Ludwig and Klenk, 2001). In our analysis (Fig. 1), the Ehrlichiaceae and the Rickettsiaceae form also one of the deeper branches.

Acetobacteraceae and Related Groups

The Acetobacteraceae form a clearly separate lineage of acidophilic bacteria encompassing the classical vinegar-producing Acetobacter, together with Gluconobacter, Gluconacetobacter, Acidimonas, Acidiphilium, Asaia and some other genera, as well as the phototrophic PNS bacterium Rhodopila. Some of the Gluconacetobacter species are able to fix nitrogen and live in association with sugar cane (G. diazotrophicus) or coffee plants (G. azotocaptans; Fuentes-Ramirez et al., 2001).

Rickettsiaceae, Ehrlichiaceae and the Holospora Group

The Rickettsiaceae, the Anaplasmataceae and the Ehrlichiaceae form a distinct lineage among the “Alphaproteobacteria” and consist of small intracellular parasitic bacteria, such as Rickettsia (causing typhus and Rocky Mountain spotted fever), Ehrlichia (causing human granulocytic ehrlichiosis and Potomac fever in horses), Anaplasma (infecting erythrocytes of ruminants), and the remarkable Wolbachia, involved in parthenogenesis in various arthropods. Although these bacteria can infect various vertebrate hosts, their vectors and reservoirs are predominantly ticks and trematodes, except for the wolbachiae, which are highly promiscuous for diverse invertebrate hosts and are also found in a variety of helminths. On the basis of 16S rDNA gene and groESL operon sequence results, Dumler et al. (2001) proposed a reorganization of the genera into the families Rickettsiaceae and Anaplasmataceae: members of the Rickettsiaceae grow in the cytoplasm or nucleus of their eukaryotic host cells and the family is restricted to the genera Rickettsia and Orientia, whereas members of the Anaplasmataceae replicate while enclosed in a eukaryotic host cell membrane-derived vacuole, and the family was broadened to include all the alphaproteobacterial species of the genera Ehrlichia, Anaplasma, Cowdria, Wolbachia and Neorickettsia (Dumler et al., 2001). Various endosymbiotic bacteria of protozoa, such as Paramecium, were allocated to the genera Holospora, Caedibacter and Lyticum forming the Holospora rDNA lineage, which is distantly related to the group formed by the rickettsiae (Fig. 1). The evolutionary roots of the eukaryotic mitochondrion are among the rickettsiae (Symbiotic, Parasitic and Not-Yet Cultured Proteobacteria, and the Alphaproteobacterial Origin of Mitochondria).

Rhodospirillaceae

The Rhodospirillaceae lineage corresponds to the α-1 subgroup of Woese et al. (1984a) and encompasses at least seven genera of spiral-shaped phototrophic PNS bacteria (e.g., Rhodospirillum, Phaeospirillum, Rhodospira and Rhodovibrio). Previously, these genera (except Rhodospira) were species assigned to the broad and heterogeneous genus Rhodospirillum (Imhoff et al., 1998). The phototrophic rhodospirillae practice a photoorganotrophic type of metabolism, where a simple fatty acid or amino acid is the carbon source and light the energy source. Most of the phototrophic rhodospirillae can also fix nitrogen gas, a property that is also characteristic for members of the genus Azospirillum, belonging to the Rhodospirillaceae rDNA-lineage. The azospirillae are nonphototrophic N2-fixing bacteria, widely distributed in the rhizosphere of tropical and subtropical grasses where they seem to enhance the growth of these plants. Magnetotactic bacteria belonging to the genus Magnetospirillum are also members of this phylogenetic lineage, together with some former Aquaspirillum species.

Sphingomonadaceae

Proteobacteria containing glycosphingolipids in their cell envelopes belong to the phylogenetic lineage of the sphingomonads, forming a versatile group of aerobic bacteria occurring in various environments such as soil, water and clinical specimens. This lineage corresponds to the α-4 subgroup. The genus Sphingomonas contained at least 20 species and was recently split on the basis of phylogenetic and chemotaxonomic analyses into four different genera (Sphingomonas sensu stricto, Sphingobium, Novosphingobium and Sphingopyxis; Takeuchi et al., 2001). Some of the species belonging to these genera can metabolize various aromatic compounds and possess promising biotechnological properties. The Sphingomonadaceae comprise also the genera Erythrobacter, Erythromicrobium and Porphyrobacter, which are aerobic chemoorganotrophs, although their cells contain bacteriochlorophyll a and carotenoids. The genus Zymomonas is related to the sphingomonads. Zymomonas strains occur in palm sap and pulque, where they ferment sugars to high concentrations of ethanol by using the Entner-Doudoroff pathway, leading to the alcoholic beverages palm wine and tequila, respectively (Swings and De Ley, 1977; Sahm et al., 2001).

Rhodobacter Group

The Rhodobacter group is a heterogeneous phylogenetic lineage among the “Alphaproteobacteria,” including a number of photosynthetic rod-shaped PNS bacteria as well as numerous chemoorganotrophs. It corresponds to the α-3 group (Woese et al., 1984a). Typical photosynthetic PNS bacteria of this lineage belong to the genera Rhodobacter and Rhodovulum, containing freshwater and marine species, respectively. Rhodobacter sphaeroides and R. capsulatus were extensively used for genetic studies of bacterial photosynthesis. The facultative chemolithotroph Paracoccus denitrificans belongs also here; it is a remarkable and versatile bacterium, being able to grow chemolithoautotrophically at the expense of hydrogen gas or reduced inorganic sulfur compounds as electron donors, carbon dioxide and oxygen, but it can also grow chemoorganotrophically with various organic compounds as sole carbon source. Some hyphal, budding and prosthecate aerobic bacteria such as Hyphomonas, Hirschia and Gemmobacter belong to this lineage, as well as the budding Staleya and Antarctobacter, which were isolated from a hypersaline, heliothermal antarctic meromictic lake (Labrenz et al., 1998; Labrenz et al., 2000). Gram-negative cocci typically occurring in regular packages of tetrads and isolated from activated sludge biomass were assigned to the genus Amaricoccus, belonging to the Rhodobacter lineage (Maszenan et al., 1997). The Rhodobacter group is peripherally related to some of the most abundant cultured marine species and strains from some terrestrial halophilic lake environments. These organisms, frequently referred to as the Roseobacter group, include in addition to the aerobic bacteriochlorophyll-containing genera (such as Roseobacter, Roseovarius and Rubrimonas) the genera Antarctobacter, Ketogulonicigenium, Methylarcula, Octadecabacter, Sagitulla, Silicibacter, and Sulfitobacter, as well as the phylogenetically heterogeneous genus Ruegeria.

Caulobacteraceae

The Caulobacteraceae are aquatic chemoorganotrophic and aerobic bacteria often attaching to surfaces with a stalk at one end and forming polarly flagellated swarming cells at the other end. Stalks of several cells may form rosettes. Caulobacters occur typically in freshwater and marine habitats with low nutrient levels. The rod-shaped Brevundimonas strains, which were previously allocated to the genus Pseudomonas, belong also to the Caulobacteraceae.

Rhizobiaceae, Bartonellaceae, Brucellaceae and Phyllobacterium Group

This large and complex rDNA-cluster contains at present at least 15 genera and 70 species and corresponds to the α-2 subgroup of Woese et al. (1984a), together with the adjacent lineage formed by the Hyphomicrobiaceae and the Bradyrhizobium, Methylobacterium and Methylocystis rDNA groups (Fig. 1). One common trait of bacteria belonging to this cluster is that most of them interact with eukaryotes: agrobacteria are plant pathogens causing crown-gall or hairy-root disease (tumors) on various dicotyledonous plants; the rhizobia induce nodules on roots or stems of leguminous plants and live symbiotically in these nodules where they reduce atmospheric nitrogen; and the bartonellae and brucellae are pathogenic for humans or animals. The fast growing rhizobia classified in the genera Rhizobium, Allorhizobium, Mesorhizobium and Sinorhizobium form a major rDNA cluster, together with the plant pathogenic genus Agrobacterium and the genus Phyllobacterium, whose members were isolated from leaf nodules of various plants belonging to the Myrsinaceae and Rubiaceae. The genus Mesorhizobium (more related to Phyllobacterium) and members of the genus Sinorhizobium form also a distinct clade among the rhizobia. According to 16S rDNA analysis, various Rhizobium species as well as Allorhizobium are highly related to the plant pathogenic agrobacteria. Recently, Young et al. (2001) proposed inclusion of all the species of Agrobacterium and Allorhizobium in the genus Rhizobium. The rRNA-based classification of the rhizobia is generally supported by sequence analysis of atpD and recA genes (Gaunt et al., 2001). The genes for nodule formation (nod genes), nitrogen fixation (nif genes) and tumor induction (in the case of agrobacteria) are localized on large plasmids. Species of the genus Bartonella occur in the blood of man and mammals; they are often vector borne, but can also be transmitted by animal scratches or bites. Bartonellae are considered to be emerging human pathogens. The type species Bartonella bacilliformis is a fastidious hemophilic organism that invades and destroys human red blood cells and is transmitted by a sandfly. Bartonella quintana (Brenner et al., 1993) was previously classified in the genus Rochalimaea and causes trench fever, a disease transmitted by lice and afflicting, e.g., soldiers during the First World War (1914–1918). Bartonella henselae (formerly Rochalimaea henselae) is nowadays recognized as the causative agent of cat scratch disease (Table 5). Brucellae develop intracellularly and cause worldwide infections (brucellosis) in humans and a great number of animals such as cattle, pigs, dogs, and even marine mammals.

Hyphomicrobiaceae, the Bradyrhizobium, Methylobacterium, Methylocystis and Related Groups

The family Hyphomicrobiaceae contains hyphal, prosthecate and budding bacteria; most of them (e.g., Hyphomicrobium and Pedomicrobium) are chemoorganotrophs and prefer to grow on one-carbon compounds such as methanol, whereas others are phototrophic (e.g., Rhodomicrobium). Hyphomicrobium is well adapted to grow in oligotrophic freshwater habitats. Other members of the family are Xanthobacter, a versatile soil bacterium capable of nitrogen fixation and autotrophic growth in an atmosphere of hydrogen, oxygen and carbon dioxide, and Azorhizobium caulinodans, a nitrogen-fixing bacterium living symbiotically in the stem nodules of some leguminous plants such as Sesbania.

A large subcluster within this complex lineage is formed by the Bradyrhizobium group, including the slow-growing rhizobia nodulating soy bean and other leguminous plants and the stem-nodulating phototrophic bradyrhizobia (Molouba et al., 1999; Giraud et al., 2002), the photosynthetic PNS Rhodopseudomonas, the ecologically important chemolithotrophic nitrobacters oxidizing nitrite to nitrate, as well as the opportunistic Afipia species. The Methylobacterium and Methylocystis groups contain various methanotrophic and methylotrophic bacteria, mostly utilizing the serine pathway for the assimilation of one-carbon intermediates. Some of these methanotrophs may have biotechnological applications because they can utilize chloromethanes from polluted environments, whereas others such as Methylocapsa are acidophilic and can fix atmospheric nitrogen (Dedysh et al., 2002). A subgroup of the methylotrophic methylobacteria has been shown to nodulate Crotolaria legumes and fix nitrogen (Sy et al., 2001). Other members of this rDNA lineage are the genera Beijerinckia, consisting of aerobic, acid-tolerant free-living nitrogen-fixing rods occurring mainly in tropical acidic soils, and Rhodobium, which contains marine budding phototrophic PNS bacteria.

The “Betaproteobacteria”

The “Betaproteobacteria” (at least 75 genera and 220 species) clearly represent a monophyletic group within the larger phylogenetic lineage composed of the β-γ proteobacterial complex, named Chromatibacteria by Cavalier-Smith (Cavalier-Smith, 2002; Table 2). From the metabolic, morphological and ecological viewpoint, the “Betaproteobacteria” are very heterogeneous. They contain some purple nonsulfur (PNS) phototrophs (Fig. 1, purple triangles), various chemolithotrophs, some methylotrophs, a great number of chemoorganotrophs, some nitrogen-fixing bacteria, and some important plant-, human- and animal pathogens (see Tables 5 and 6). Their morphologies can vary from rods or cocci to spiral and sheathed cells. Some members of the “Betaproteobacteria” are of biotechnological interest owing to their biodegradation properties. Recently nitrogen-fixing “Betaproteobacteria” were described that nodulate the roots of legumes (Chen et al., 2001; Moulin et al., 2001). More detailed information concerning the group can be found in the chapters dealing with the individual families and genera of the “Betaproteobacteria.” Table 9 gives an overview of the major phylogenetic groups (see also Fig. 1) and the names of the six orders and 12 families of the “Betaproteobacteria” as they are listed in the 2nd edition of Bergey’s Manual of Systematic Bacteriology (Garrity, 2001a).

Table 9.

The “Betaproteobacteria”: orders, families and number of genera.a

Order

Family

Number of genera

“Burkholderiales”

“Burkholderiaceae”

4

“Ralstoniaceae”

1

“Oxalobacteraceae”

5

Alcaligenaceae

6

Comamonadaceae

15

“Hydrogenophilales”

“Hydrogenophilaceae”

2

“Methylophilales”

“Methylophilaceae”

3

“Neisseriales”

Neisseriaceae

14

“Nitrosomonadales”

“Nitrosomonadaceae”

2

Spirillaceae

1

Gallionellaceae

1

“Rhodocyclales”

“Rhodocyclaceae”

6

aAccording to the second edition of Bergey’s Manual of Systematic Bacteriology (Garrity and Holt, 2001). Quotation marks are used for names which have not yet been validated (as of mid 2002).

Although the majority of the phototrophic PNS bacteria belong to the “Alphaproteobacteria,” some PNS species are members of the Rhodocyclus group or the family Comamonadaceae of the “Betaproteobacteria.” Similarly to the previously mentioned PNS “Alphaproteobacteria,” the PNS betaproteobacterial phototrophs are phylogenetically intermingled with nonphototrophs (Fig. 1, visualized by purple shaded areas) and can be clearly differentiated from the PNS “Alphaproteobacteria” (e.g., Rhodobacter, Rhodobium, etc.) on the basis of fatty acid and quinone composition as well as cytochrome c sequences (Imhoff, 2001a).

Alcaligenaceae, Comamonadaceae, Burkholderia, Oxalobacter and Related Groups

The Alcaligenaceae, Comamonadaceae, and the Burkholderia-, Ralstonia- and Oxalobacter-groups are phylogenetically more closely related to each other than to other 16S rDNA branches of the “Betaproteobacteria” (Fig. 1). A number of clinically important taxa belong to this lineage: members of the genus Alcaligenes are mostly saprophytic, but behave often as nosocomial bacteria, whereas the closely related Bordetella pertussis (Table 5) causes whooping cough in humans (other Bordetella species are pathogenic for animals). Taylorella is responsible for endometritis in mares, Pelistega is associated with a respiratory disease in pigeons (Vandamme et al., 1998), and Brackiella oedipodis (Willems et al., 2002) was recently reported as the causal agent of endocarditis in a small neotropical primate.

The former “acidovorans” and “solanacearum” rRNA groups of the genus Pseudomonas sensu lato (i.e., respectively, rRNA groups III and II of Palleroni [1984]) belong to the “Betaproteobacteria.” The “acidovorans” group has at present the status of the family Comamonadaceae, a phylogenetically coherent but physiologically heterogeneous group of prokaryotes encompassing genera such as Acidovorax, Comamonas, Delftia, Hydrogenophaga, Variovorax, Xylophilus, Rhodoferax, Roseateles, Rubrivivax, Leptothrix and Sphaerotilus (Willems et al., 1991a; Willems et al., 1991b). The latter two genera are heterotrophic sheathed bacteria, which may appear yellow or dark brown owing to the deposition of iron and manganese oxides, whereas Rhodoferax and Rubrivivax are typical PNS photosynthetic bacteria. Some Comamonadaceae, such as Acidovorax facilis, Hydrogenophaga flava and Variovorax paradoxus, are able to grow chemolithotrophically at the expense of hydrogen gas oxidation. Xylophilus ampelinus causes necrosis and canker on grapevines. Other representatives, e.g., belonging to Comamonas or Delftia, can degrade aromatic compounds (via the meta-cleavage of the aromatic ring) and are important in the biodegradation of toxic wastes. Roseateles was described as the first obligate aerobic betaproteobacterium containing bacteriochlorophyll a (Suyama et al., 1999). However, light does not support growth of Roseateles strains under anaerobic conditions and in this sense Roseateles resembles physiologically the aerobic bacteriochlorophyll a-containing “Alphaproteobacteria,” such as Erythrobacter and Erythromicrobium (Sphingomonadaceae).

The former “Pseudomonas solanacearum group” is composed of the genera Burkholderia, Ralstonia and a few others. Some of these bacteria are typical plant pathogens (e.g., Ralstonia solanacearum, causing wilt on many cultivated plants; Table 6), whereas others are important animal and human pathogens. Burkholderia mallei and B. pseudomallei have been classified as risk group 3 organisms (Table 5) because they cause respectively glanders disease in horses and melioidosis, a disease endemic in animals and humans in Southeast Asia. Burkholderia cepacia seems ubiquitous and is both friend and foe to humans (Govan et al., 2000): it is associated with plants (the original isolate was reported by Burkholder [1950] as the causative agent of bacterial rot of onion bulbs), occurs in soil and water, protects crops from bacterial and fungal infections, and is also frequently reported in the environment of patients, particularly cystic fibrosis patients, where B. cepacia infections have a considerable impact on their morbidity and mortality, as well as on their social life. The remarkable genomic heterogeneity among the B. cepacia strains isolated from various ecological niches makes their correct identification problematic (Coenye et al., 2001). It was demonstrated that presumed B. cepacia strains isolated from cystic fibrosis patients and other sources belong to at least nine distinct genomic species or genomovars (Vandamme et al., 1997; Coenye et al., 2001). At least two of these genomovars are often implicated in the transmission of B. cepacia between cystic fibrosis patients (Mahenthiralingam et al., 2000; LiPuma et al., 2001). The extreme metabolic versatilty of B. cepacia could be applied in the bioremediation of recalcitrant xenobiotics (Parke and Gurian-Sherman, 2001). However, more caution is undoubtedly needed in this area, because many strains used or under development for biocontrol or bioremediation purposes are taxonomically poorly characterized, causing a potential hazard to the cystic fibrosis community worldwide (Parke and Gurian-Sherman, 2001). The related genus Ralstonia is also unusual as it harbors important plant pathogens, opportunistic human pathogens, as well as organisms of considerable biotechnological interest because of their potential for biodegradation of xenobiotics and recalcitrant compounds. Moreover, Chen et al. (2001) described Ralstonia taiwanensis as a betaproteobacterium capable of root nodule formation and nitrogen fixation in a leguminous plant (Mimosa). Together with the Burkholderia-like strains described by Moulin et al. (2001), these are the first betaproteobacteria known to be capable of root nodule formation and nitrogen fixation. Ralstonia eutropha (previously classified as Alcaligenes eutrophus) is a well-known and intensely studied facultative chemolithotroph (a typical “Knallgas” bacterium) that can grow at the expense of hydrogen gas, CO2 and O2 (Aragno and Schlegel, 1992). When grown under appropriate conditions, R. eutropha is also an excellent producer of the bioplastic poly-β-hydroxybutyrate and similar polyhydroxyalkanoates (Table 7). Some toxic-metal-resistant bacteria isolated from industrial biotopes were recently allocated to the genus Ralstonia (Goris et al., 2001). The latter organisms can be exploited to recycle soils polluted by toxic metals, such as mercury, cadmium or nickel.

The Oxalobacter group is composed of Herbaspirillum (a diazotroph colonizing endophytically roots, stems or leaves of graminaceous plants), Janthinobacterium (one of the known violacein-producing organisms), Oxalobacter and a few other genera. Oxalobacter formigenes occurs in the rumen or the large bowel of humans and animals; it performs a unique fermentation of oxalate to formate and CO2, whereby ATP is generated via a membrane-linked proton-translocating ATPase.

Hydrogenophilus Group

The genus Hydrogenophilus harbors thermophilic (optimum growth temperature 50–60°C), facultatively chemolithoautotrophic hydrogen-oxidizing rods that form a distinct lineage among the “Betaproteobacteria” (Hayashi et al., 1999). The classification of the colorless chemolithotrophic S-oxidizing Thiobacillus species has been thoroughly revised by Kelly and Wood (2000). The type species T. thioparus together with a few other Thiobacillus species belongs to the “Betaproteobacteria” and is distantly related to the Hydrogenophilus group. The majority of the other Thiobacillus species belong to the “Gammaproteobacteria” and were reassigned to three other genera (see The “Gammaproteobacteria”). Because they are able to leach metals from ore, Thiobacilli (e.g., Acidithiobacillus species) are used in processing low-grade metal ores.

Methylophilus, Nitrosomonas and Related Groups

The majority of the one-carbon utilizers (methanotrophs) belong either to the “Alphaproteobacteria” or to the “Gammaproteobacteria.” However, three methylotrophic genera (Methylobacillus, Methylophilus and Methylovorus) group in the Methylophilus rRNA lineage of the “Betaproteobacteria.” These are phylogenetically distantly related to the Nitrosomonas lineage. The aerobic chemolithoautotrophic ammonia-oxidizing bacteria such as the rod-shaped Nitrosomonas and the spiral-shaped Nitrosospira constitute a monophyletic lineage among the “Betaproteobacteria.” Spirillum volutans is distantly related to this group, as well as the Fe2+-oxidizing chemolithotrophic Gallionella ferruginea, already known since 1836 for its peculiar bean-shaped cells forming very long spiral stalks composed of ferric hydroxide (Ehrenberg, 1836).

Rhodocyclus Group

The Rhodocyclus lineage harbors besides PNS bacteria (e.g., Rhodocyclus purpureus) also diazotrophic rhizosphere bacteria such as Azoarcus and Azospira. Several strains of Azoarcus and the related Thauera can degrade aromatic compounds including chlorobenzoates. Thauera selenatis is capable of nitrate and selenate respiration and another member of this group, Zoogloea ramigera (Rossellomora et al., 1993), is considered to be a typical floc-forming prokaryote during the activated sludge process in sewage treatment plants (Juretschko et al., 2002). Also Quadricoccus (a large coccus occurring in tetrads; Maszenan et al., 2002) is found in the activated sludge biomass where it synthesizes polyphosphate and polyhydroxyalkanoates.

Dechloromonas and Dechlorosoma are newly described genera of perchlorate-reducing bacteria, associated, e.g., with the manufacture and dismantling of ammunition (Achenbach et al., 2001).

Neisseriaceae

The most prominent human pathogens of the “Betaproteobacteria” belong to the genus Neisseria, containing the two well-known species N. gonorrhoeae and N. meningitidis. Strains of these organisms have been extensively studied by multi-locus-sequencing-typing (MLST; Achtman, 1998; Maiden et al., 1998).

Other clinical bacteria, such as Kingella and Eikenella, belong also to this phylogenetic group together with Chromobacterium, which produces the purple pigment violacein. The mammalian oral commensals Alysiella and Simonsiella belong also to Neisseriaceae and are unique because their filament-forming cells show a dorsal-ventral asymmetry and display a gliding motility on the epithelial cells in the oral cavity and upper respiratory tract of their host. These bacteria might have coevolved with their mammalian hosts during the past 100 million years (Hedlund and Staley, 2002).

The “Gammaproteobacteria”

Most 16S rDNA trees show that the members of the “Gammaproteobacteria” represent a monophyletic group which includes in fact also the “Betaproteobacteria” as a major line of descent (see Fig. 1). The “Gammaproteobacteria” form the largest proteobacterial group (at least 180 genera and 750 species), including the phytopathogenic Xanthomonas group as a border-line member. Depending upon the rDNA-treeing method used, the Xanthomonas rDNA-group appears peripherally linked to either the β- or the γ-class and can be considered as a sister group of the “Betaproteobacteria” (Ludwig and Klenk, 2001). The major phylogenetic groups (Fig. 1) and the names of the 13 orders and 20 families of the “Gammaproteobacteria” as listed in the 2nd edition of Bergey’s Manual of Systematic Bacteriology (Garrity, 2001a) are given in Table 10. The individual chapters on the families and genera of this class can be consulted for more detailed information.

Table 10.

The “Gammaproteobacteria”: orders, families and number of genera.a

Order

Family

Number of genera

“Chromatiales”

Chromatiaceae

22

Ectothiorhodospiraceae

5

“Xanthomonadales”

“Xanthomonadaceae”

8

“Cardiobacteriales”

Cardiobacteriaceae

3

“Thiotrichales”

“Thiotrichaceae”

9

“Piscirickettsiaceae”

5

“Francisellaceae”

1

“Legionellales”

Legionellaceae

1

“Coxiellaceae”

2

“Methylococcales”

Methylococcaceae

6

“Oceanospirillales”

“Oceanospirillaceae”

6

Halomonadaceae

6

Pseudomonadales

Pseudomonadaceae

15

Moraxellaceae

3

“Alteromonadales”

Alteromonadaceae

11

“Vibrionales”

Vibrionaceae

6

“Aeromonadales”

Aeromonadaceae

2

Succinivibrionaceae

4

“Enterobacteriales”

Enterobacteriaceae

41

“Pasteurellales”

Pasteurellaceae

6

aAccording to the second edition of Bergey’s Manual of Systematic Bacteriology (Garrity and Holt, 2001). Quotation marks are used for names which have not yet been validated (as of mid 2002).

The “Gammaproteobacteria” contain the photosynthetic purple sulfur (PS) bacteria (Chromatiaceae and Ectothiorhodospiraceae; Table 10; Fig. 1) together with a great number of familiar chemoorganotrophic bacterial groups, such as the Enterobacteriaceae, Legionellaceae, Pasteurellaceae, Pseudomonadaceae, Vibrionaceae, and also some chemolithotrophic mostly sulfur- or iron-oxidizing prokaryotes. The class harbors some important human and animal pathogens (Table 5). It is noteworthy that the family Enterobacteriaceae is known since 1937 as a classical phenotypic group (Rahn, 1937), which remains fully supported by modern molecular taxonomy. On the other hand, the tradional group of the pseudomonads turned out to be phylogenetically extremely heterogeneous, because its members are scattered over the “Alphaproteobacteria”, “Betaproteobacteria” and “Gammaproteobacteria.” The genus Pseudomonas is at present restricted to all species phylogenetically related to its type species Pseudomonas aeruginosa, a member of the “Gammaproteobacteria,” whereas all the other pseudomonads belonging to the α- and β-classes have been allocated to new genera such as Brevundimonas, Sphingomonas, Comamonas, Burkholderia, Ralstonia, etc. (see The “Alphaproteobacteria”, The “Betaproteobacteria”). An analogous taxonomic history concerns the genus Thiobacillus, whose members are chemolithotrophs oxidizing various inorganic sulfur-compounds. Its classification was recently clarified by Kelly and Wood (2000): thiobacilli related to the type species T. thioparus belong to the “Betaproteobacteria,” whereas the Thiobacillus species belonging to the “Gammaproteobacteria” were reclassified in the genera Acidithiobacillus, Halothiobacillus and Thermithiobacillus.

Chromatiaceae and Ectothiorhodospiraceae

The distribution of the anoxygenic photosynthetic purple sulfur (PS) bacteria is at present restricted to two clearly distinct phylogenetic lineages of the “Gammaproteobacteria” (Fig. 1): the Chromatiaceae (25 genera), which accumulate elemental sulfur globules inside their cells, and the Ectothiorhodospiraceae (7 genera), which deposit sulfur outside their cells. These prokaryotes are mainly found in illuminated anoxic zones of lakes (particularly meromictic lakes) where H2S accumulates and also in “sulfur springs,” where biologically or geochemically produced H2S can trigger the formation of massive blooms of purple sulfur bacteria (Imhoff, 2001a). All PS bacteria can utilize H2S as an electron donor for CO2 reduction, but many are also able to use other reduced sulfur compounds (e.g., thiosulfate) as photosynthetic electron donors. Ectothiorhodospiraceae are haloalkaliphilic sulfur bacteria possessing lamellar intracellular photosynthetic membrane structures, whereas most Chromatiaceae possess a vesicular type of intracellular membrane. Some nonphototrophic and alkaliphilic taxa belong also to the phylogenetic lineage of the Ectothiorhodospiraceae: for example, Nitrococcus is a halophilic nitrite-oxidizing chemolithotroph, Thioalkalivibrio (Sorokin et al., 2001) contains obligately chemolithotrophic sulfur-oxidizing bacteria, and Alcalilimnicola (Yakimov et al., 2001) is a halotolerant aerobic heterotroph.

Enterobacteriaceae, Aeromonadaceae, Alteromonadaceae, Pasteurellaceae, Succinivibrionaceae and Vibrionaceae

The core of the γ-class is composed of the well-known enterics (See Introduction to the Family Enterobacteriaceae in Volume 6), Vibrionaceae (See The Family Vibrionaceae in Volume 6), Aeromonadaceae (See The Genera Aeromonas and Plesiomonas in Volume 6), Pasteurellaceae (See The Genus Pasteurella in Volume 6) and the Alteromonadaceae (See The Genus Alteromonas and Related Proteobacteria in Volume 6), each including a large number of genera and species. Some representatives of these families are known as pathogens of humans and animals (Table 5). The Enterobacteriaceae include the well-known and thoroughly studied Escherichia coli, together with numerous inhabitants of the intestinal tract of warm-blooded animals (e.g., Salmonella and Shigella, the etiological agents of salmonellosis and shigellosis, respectively; see Table 5). Escherichia coli is an excellent indicator organism for water quality (fecal contamination). The Enterobacteriaceae comprise a relatively homogeneous phylogenetic group within this large cluster; they are mostly facultative anaerobic carbohydrate-degrading microorganisms; some perform a mixed acid fermentation, whereas others carry out the butanediol fermentation. The enterics comprise also plant pathogenic bacteria (e.g., Erwinia, Pectobacterium and Brenneria; see Table 6), the plague-causing Yersinia pestis, as well as Xenorhabdus and Photorhabdus, which are symbionts of entomopathogenic nematodes, where they live in the intestinal lumen. An interesting and somewhat distant member of the enterics is Buchnera aphidicola, a not-yet-cultured endosymbiont of aphids (Baumann et al., 1995). The symbiotic association between aphids and Buchnera seems to be obligate and mutualistic: Buchnera synthesizes tryptophan, cysteine and methionine and supplies these essential amino acids to the aphid host. A parallel evolution of Buchnera and aphids seems to have occurred, and Baumann et al. (1998) estimated the origin of this symbiotic association as 200–250 million years ago. The correlation of sequence diversity of 16S rDNA of symbionts with the age of their hosts has led to the calibration of the molecular clock of 16S rDNA in recent organisms (Moran et al., 1993), assumed to generate 1% sequence divergence within 25–50 million years (see also Stackebrandt, 1995). Also the endosymbionts of carpenter ants (Camponotus spp.) constitute a distinct taxonomic group within the “Gammaproteobacteria” and are phylogenetically closely related to Buchnera and symbionts of tsetse flies. Comparison of the phylogenetic trees of the bacterial endosymbionts and their host species suggests a highly synchronous cospeciation process of both partners (Sauer et al., 2000).

Vibrionaceae are facultative anaerobic inhabitants of brackish, estuarine and pelagic waters and sediments and form the dominant culturable microflora in the gut of molluscs, shrimps and fish. The family harbors several pathogens (e.g., Vibrio cholerae, the causal agent of cholera) as well as luminous bacteria (e.g., Photobacterium and several Vibrio species), occurring free-living in seawater, as well as being symbionts in the light organs of many fish and invertebrates (see Dunlap and Kita-Tsukamoto, 2001). Bioluminescent bacteria occur also among the genera Photorhabdus (Enterobacteriaceae) and Shewanella. The latter genus belongs to the Alteromonadaceae, which are strictly aerobic chemoorganotrophs requiring seawater for growth. Quorum-sensing plays an important role in the phenomenon of bioluminescence. Several representatives of the Pasteurellaceae are the etiological agents of various infectious diseases in humans and other vertebrates (cattle, sheep, goats and fowl). They encompass the following major genera: Pasteurella, Haemophilus, Actinobacillus and Mannheimia. Haemophilus influenzae was the first free-living organism whose entire genome (ca. 1.8 × 109 bp) was sequenced (Fleischmann et al., 1995). The Aeromonadaceae include the fish pathogen Aeromonas salmonicida. The Succinivibrionaceae (Hippe et al., 1999) are strict anaerobes fermenting glucose and other carbohydrates to succinate and acetate. They occur in the rumen of sheep and cattle (Ruminobacter, Succinomonas and Succinivibrio) or in the feces or colon of dogs and humans (Anaerobiospirillum).

Francisella and Piscirickettsia Groups

The Francisella and Piscirickettsia groups form a deeply branching lineage among the “Gammaproteobacteria.” Francisella tularensis is the causal agent of tularemia, a plague-like zoonosis, spread to humans from rodents via direct contact or biting arthropods, whereas Piscirickettsia salmonis causes an epizootic disease in salmonid fishes. Members of the Piscirickettsia group display a great morphological and metabolic diversity: Thiomicrospira contains spiral-shaped chemolithotrophic sulfur-oxidizing bacteria, which are closely related to the alkaliphilic chemolithotrophic Thioalkalimicrobium (Sorokin et al., 2001). Hydrogenovibrio is a marine obligately chemolithoautotrophic hydrogen-oxidizing bacterium and Cycloclasticus harbors rod-shaped bacteria degrading aromatic hydrocarbons and occurring in marine sediments.

Cardiobacteriaceae

The Cardiobacteriaceae are an example of a family created (Dewhirst et al., 1990) on the basis of 16S rDNA comparisons of three taxa, whose phylogenetic relationship was at first quite unexpected. Cardiobacterium hominis is an occasional resident of the human respiratory tract and has been recovered from blood samples of patients suffering from endocarditis. Suttonella indologenes (previously classified as Kingella indologenes) has been reported in human eye infections and blood of patients with endocarditis, whereas Dichelobacter nodosus (formerly Bacteroides nodosus) is the causative agent of footrot in ruminants. Authentic kingellae belong to the “Betaproteobacteria” and authentic bacteroideae belong to the Cytophaga-Flavobacterium-Bacteroides phylum (the so-called “Bacteroidetes” according to Garrity, 2001).

Moraxellaceae

The Moraxellaceae encompass the genera Moraxella, Acinetobacter and Psychrobacter forming a separate lineage among the “Gammaproteobacteria” (Rossau et al., 1991). Moraxellae are commonly isolated from animals or humans and some of them are pathogenic, whereas the psychrophilic Psychrobacter species occur in the marine and antarctic environment (e.g., in antarctic ornithogenic soils; Bowman et al., 1996) and also in clinical material. The oxidase-negative acinetobacters are saprophytes occurring in soil, water and sewage, but also typically on the skin of healthy humans. Moreover, some acinetobacters can also cause nosocomial infections.

Methylococcaceae

The Methylococcaceae form a distinct lineage (Bowman et al., 1995) encompassing methylotrophic bacteria such as the genera Methylobacter, Methylococcus and Methylomonas, which share the ribulose monophosphate cycle for the assimilation of reduced C1-compounds. Some of these methylotrophs form cysts as resting stages and possess internal membrane systems arranged as bundles of disc-shaped vesicles distributed throughout the cell, in contrast to some methylotrophs of the “Alphaproteobacteria,” whose membrane systems run along the periphery of the cells. The moderately halophilic Methylophaga belong phylogenetically to the Piscirickettsia group.

Thiothrix Group

The Thiothrix group contains a number of filamentous and gliding sulfur-oxidizing chemolithotrophs, such as Thiothrix, Beggiatoa and Thioploca. Thiothrix forms sheathed filaments, whereas Beggiatoa forms long filaments lacking a sheath. Sulfur granules generally accumulate in the cells. The not yet axenic Thiomargarita (“sulfur pearl”) is related to Thioploca and is a real giant among the prokaryotic life forms (its spherical cells are about 500 µm wide!). Thiomargarita cells form thick mats near the coast of Namibia, where they perform an anoxic oxidation of H2S coupled to the reduction of nitrate (Schulz et al., 1999; Schulz, 2002). Leucothrix can be considered as a chemoorganotrophic counterpart of Thiothrix. Some not yet cultured sulfur-oxidizing symbiotic bacteria of bivalves are closely related to the Thiothrix group and play an important role in the nutrition of such animals, living near hydrothermal vents.

Halomonadaceae

The family Halomonadaceae is a large and complex group of moderately halophilic and marine bacteria encompassing the genera Halomonas, Chromohalobacter, Zymobacter and Carnimonas. Recent comparative sequence analysis of 16S and 23S rDNA of representative strains indicated (Arahal et al., 2002) that the genus Chromohalobacter forms a monophyletic group, whereas the genus Halomonas is clearly polyphyletic and needs a taxonomic re-evaluation based on a polyphasic taxonomic approach (Romanenko et al., 2002 [doi10.1099/ijs.0.02240-0]).

Pseudomonadaceae

In the past (Palleroni, 1984), the family Pseudomonadaceae contained an extremely heterogeneous group of aerobic, rod-shaped and mostly polarly flagellated bacteria, phylogenetically spread over the “Alphaproteobacteria,” “Betaproteobacteria” and “Gammaproteobacteria.” Nowadays the family is restricted to some 90 Pseudomonas species around the type species Pseudomonas aeruginosa and some related genera which form a separate lineage within the “Gammaproteobacteria.” The pseudomonads of the “Alphaproteobacteria” and “Betaproteobacteria” have been transferred to other genera (Willems et al., 1991a; Kersters et al., 1996; Anzai et al., 2000; Caulobacteraceae, Alcaligenaceae, Comamonadaceae, Burkholderia, Oxalobacter and Related Groups). Some authentic Pseudomonas species (e.g., P. fluorescens) produce fluorescent pigments. Most pseudomonads are versatile and can grow on a great variety of organic compounds, including aromatic hydrocarbons, because some of them possess efficient oxygenases. Such bacteria play key roles in the purification of wastewater and clean-up of oil spills. Some of the Pseudomonas species (e.g., P. syringae) are plant pathogens (Table 6), whereas P. aeruginosa and some other fluorescent pseudomonads can be involved in serious nosocomial infections (Table 5). The free-living nitrogen fixers of the genera Azotobacter and Azomonas belong also to this phylogenetic lineage, together with the cellulose-degrading Cellvibrio and a few other related genera.

Oceanospirillum Group

The Oceanospirillum group encompasses six genera whose members are aerobic, are moderately halophilic and generally possess curved cells. Some representatives (e.g., Neptunomonas) can degrade polycyclic aromatic hydrocarbons or long chain alkanes. The taxonomy of the genus Oceanospirillum has recently been revised (Satomi et al., 2002). Balneatrix alpica was reported as a bacterium associated with a case of pneumonia and meningitis in a spa therapy center in France (Dauga et al., 1993).

Legionellaceae and Coxiella Group

The clinically important legionellae occur in surface water, mud, thermally polluted lakes and streams and some (e.g., Legionella pneumophila) may enter the human respiratory tract when water is aerosolized in showers and air-conditioning systems. Legionella pneumophila is a pathogen for humans causing pneumonia (Legionnaires’ disease). An obligate intracellular bacterial parasite of small free-living amoebae (previously classified as Sarcobium lyticum; Drozanski, 1991) belongs also to the genus Legionella (Hookey et al., 1996). The genera Coxiella and Rickettsiella belong to the same phylogenetic lineage as the legionellae. Although phylogenetically distinct, Coxiella and Rickettsia (the latter belonging to the “Alphaproteobacteria”) share similarities in their parasitic lifestyle. Coxiella burnetii, an obligate parasitic bacterium growing preferentially in the vacuoles of the host cells, causes Q-fever, a pneumonia-like infection, transmitted among animals by insect bites (e.g., ticks) and occasionally causes disease in humans (originally called “abattoir fever”). Rickettsiellae are widely distributed intracellular pathogens of invertebrates, including insects, crustaceans and arachnids. They have not yet been cultivated in cell-free media and grow and multiply in cell vacuoles of the fat body and hepatopancreas of their invertebrate hosts.

Xanthomonas Group

The plant pathogenic Xanthomonas species, Frateuria (phenotypically resembling the acetic acid bacteria of the α-class), Xylella (a phytopathogen living in the xylem of various plants) and Stenotrophomonas, together with some yellow-pigmented N2O-producing bacteria isolated from ammonia-supplied biofilters (Finkmann et al., 2000), constitute a clearly separated phylogenetic lineage among the Chromatibacteria sensu Cavalier-Smith (2002), i.e., the large complex formed by the “Betaproteobacteria” and the “Gammaproteobacteria”; see Table 2). The ubiquitous Stenotrophomonas maltophilia is also involved in important nosocomial infections (Van Couwenberghe et al., 1997; Khan and Mehta, 2002). Depending on the treeing algorithms used and the number of rDNA-sequences included, the xanthomonads cluster peripherally linked either to the “Betaproteobacteria” or to the “Gammaproteobacteria” (Fig. 1). The first complete genome sequence published of a plant pathogenic bacterium (Simpson et al., 2000) was that of Xylella fastidiosa, a pathogen causing important diseases in citrus trees, grapevines and other plants.

The “Deltaproteobacteria”

From the viewpoint of lifestyle and morphology, the δ-class is most peculiar because it contains bacteria that are typical predators on other prokaryotes (bdellovibrios), whereas other “Deltaproteobacteria” belonging to the myxobacteria display complex developmental life cycles, forming multicellular structures called “fruiting bodies.” Up to now, photosynthetic bacteria have not been reported among the δ-class. Menaquinones are the major electron carriers in the respiratory chain (Table 3). The major subgroups of the “Deltaproteobacteria” are: 1) the myxobacteria displaying gliding motility and forming multicellular fruiting bodies, which are often large enough to be observed with the aid of a hand lens or even by the naked eye (e.g., Chondromyces); 2) the bdellovibrios living as predators on other Gram-negative bacteria; 3) the dissimilatory sulfate- and sulfur-reducing bacteria (their genus names are mostly prefixed by Desulfo-); and 4) some synthrophic bacteria which ferment propionate (e.g., Syntrophobacter) or benzoate (e.g., Syntrophus) to acetate, CO2 and hydrogen syntrophically in coculture with hydrogen-consuming methanogens.

Bdellovibrios and myxobacteria are strict aerobes, whereas the sulfate- and sulfur-reducers and the syntrophic bacteria are strictly anaerobic. One may speculate whether the bdellovibrios and the myxobacteria represent aerobic descendants of the sulfate- and sulfur-reducing bacteria. We consider here briefly the major phylogenetic groups of the “Deltaproteobacteria,” containing at present some 60 different genera and 160 species. Table 11 summarizes the names of taxa above the rank of genus within the “Deltaproteobacteria” according to the new edition of Bergey’s Manual of Systematic Bacteriology (Garrity, 2001).

Table 11.

The “Deltaproteobacteria”: orders, families and number of genera.a

Order

Family

Number of genera

“Desulfurellales”

“Desulfurellaceae”

2

“Desulfovibrionales”

“Desulfovibrionaceae”

3

“Desulfomicrobiaceae”

1

“Desulfohalobiaceae”

3

“Desulfobacterales”

“Desulfobacteraceae”

12

“Desulfobulbaceae”

4

“Desulfoarculaceae”

4

“Desulfuromonadales”

“Desulfuromonadaceae”

2

“Geobacteraceae”

1

“Pelobacteraceae”

2

“Synthrophobacterales”

“Synthrophobacteraceae”

4

“Synthrophaceae”

2

“Bdellovibrionales”

“Bdellovibrionaceae”

3

Myxococcales

Myxococcaceae

2

Archangiaceae

1

Cystobacteraceae

3

Polyangiaceae

3

aAccording to the second edition of Bergey’s Manual of Systematic Bacteriology (Garrity and Holt, 2001). Quotation marks are used for names which have not yet been validated (as of mid 2002).

Myxobacteria

The myxobacteria constitute a fascinating group of strictly aerobic, chemoorganotrophic, gliding, generally pigmented rod-shaped cells, which under starvation conditions aggregate, forming multicellular and macroscopic fruiting bodies (see Dworkin and Kaiser [1993] and Dawid [2000]). Some of the cells within these fruiting bodies are often converted to resting stages called myxospores. The peculiar gliding movement of the vegetative cells is not restricted to myxobacteria as it occurs also amongst members of the genera Cytophaga and Flexibacter, both belonging to the Cytophaga-Bacteroides-Flavobacterium phylum (now called “Bacteroidetes”; Garrity, 2001). New molecular and genetic insights concerning gliding motility have been gathered from studies on Myxococcus xanthus (Spormann, 1999). The myxobacteria are grouped in the order Myxococcales, which is composed of the following four families: Myxococcaceae, Archangiaceae, Cystobacteraceae and the Polyangiaceae. Those fruiting myxobacteria whose rDNA sequence could be examined form a phylogenetically coherent group (Spröer et al., 1999; Fig. 1), consisting of two major phylogenetic lineages. One fairly homogeneous group is composed of the genera Myxococcus, Angiococcus, Archangium, Cystobacter, Melittangium and Stigmatella, whereas the second lineage is much more heterogeneous and is composed of three genera belonging to the family Polyangiaceae. Within the latter group, the genus Nannocystis seems to occupy the most separate position. The classification of the myxobacteria relies heavily on morphological features whose phylogenetic significance has been confirmed (Spröer et al., 1999). The myxobacteria occur in various habitats, such as soils and particularly on decaying organic material, including dung of herbivorous animals, rotting wood, and bark of living and dead trees. They obtain their nutrients primarily by causing lysis of other bacteria. The myxobacteria are mostly mesophilic, but some psychrophilic, acidophilic and alkaliphilic species have been described (Dawid, 2000). The sporangia of Chondromyces crocatus have been found to harbor a sphingobacterium-like organism which was described as “Candidatus comitans” by Jacobi et al. (1997).

Several myxobacteria produce potentially useful secondary compounds, such as antibiotics and cytostatic peptides (Reichenbach and Höfle, 1993; Reichenbach, 2001). The fruiting myxobacteria possess the most complex behavioral patterns and life cycles of all prokaryotes known so far and are therefore considered as a very interesting study object. They typically display social behavior expressed by collective food uptake, cooperative motility (swarming), and social development (Crespi, 2001). Intercellular communication plays an important role in these phenomena. The analysis of the genome sequence of Myxococcus xanthus (9.5 Mb; twice as large as the E. coli genome) should enhance the understanding of the molecular and evolutionary basis of the peculiar life style of these myxobacteria.

Bdellovibrio Group

The bdellovibrios are small vibrioid prokaryotes living as predators on other Gram-negative bacteria. They are widespread in nature and have been isolated from soil, sewage, freshwater and marine environments. Wildtype bdellovibrios show a biphasic life cycle, alternating between an extracellular “attack-phase” flagellated form and an intracellular nonflagellated reproductive phase. After a bdellovibrio cell penetrates the wall of a Gram-negative prey cell (such as Pseudomonas or Erwinia), it performs a number of modifications on the outer membrane and the peptidoglycan of the invaded bacterium. The resulting spherical structure, consisting of the killed invaded cell and the developing bdellovibrio, is called a “bdelloplast.” The bdellovibrio cell grows and multiplies in the periplasmic space of the prey cell and digests the periplasmic and cytoplasmic contents of the invaded cell (for more details, see Jurkevitch, 2001). The parasitic life style of Bdellovibrio is reflected in the size of its genome, which is only half of that of E. coli. Genomic heterogeneity has been observed among the bdellovibrios (Seidler et al., 1972; Baer et al., 2000): the genera Bdellovibrio and Bacteriovorax constitute separate lineages among the “Deltaproteobacteria.” The 16S rDNA sequences of two other genera (Micavibrio and Vampirovibrio) have not been analyzed yet. The latter genus attacks cells of the green alga Chlorella.

Sulfate- and Sulfur-reducing Bacteria

Sulfate- and sulfur-reducing bacteria occur in various major phylogenetic lineages, such as the Archaea (e.g., Archaeglobus), the phylum of the thermophilic Thermodesulfobacteria, the order Clostridiales (the spore-forming Desulfotomaculum and Desulfosporosinus), and the “Deltaproteobacteria” (such as the genera Desulfovibrio, Desulfobacter and Desulfuromonas). The proteobacterial sulfate- and sulfur-reducing bacteria are mostly mesophilic and constitute a physiological group of an otherwise phylogenetically heterogeneous assemblage of bacteria (Fig. 1). The number of genera and species has significantly increased during the last decade: in 2002, the group encompassed some 40 validly published genera and some 120 species, as well as a number of not yet culturable bacteria which emerge from molecular environmental studies (Akkermans et al., 1994). The proteobacterial sulfate- and sulfur-reducers are widespread in aquatic and humid terrestrial environments that become anoxic as a result of microbial decomposition. They are for the most part strictly anaerobic, performing the complete or partial oxidation of small organic molecules (e.g., lactate, pyruvate, acetate, and in a few cases, also alkanes and aromatic compounds) with oxidized S-compounds (such as sulfate or elemental sulfur) as terminal electron acceptors generating hydrogen sulfide. They play a crucial role in the sulfur cycle. Some sulfate reducers (e.g., Desulfonema and Desulfosarcina) can also grow autotrophically with hydrogen gas as electron donor and CO2 as sole carbon source. Multiheme cytochromes (of the c3 type) play a key role in electron-transport reactions in the periplasm of sulfate- and sulfur-reducing bacteria. Strictly sulfur-reducing bacteria occur among the genera Desulfuromonas, Desulfuromusa, Desulfurella and Hippea (Liesack and Finster, 1994; Miroshnichenko et al., 1999).

Phylogenetically the sulfate- and sulfur-reducing proteobacterial taxa display a significant diversity; at present at least five major phylogenetic lineages can be discerned within the “Deltaproteobacteria” (Fig. 1). To make the picture perhaps even more complex, not all the taxa belonging to these phylogenetic clusters metabolize with sulfate or sulfur as terminal electron acceptor. These five major rRNA clusters are the Desulfovibrio, Desulfuromonas, Desulfobacter, Synthrophobacter and Desulfurella groups.

The Desulfovibrio rRNA group (encompassing the genera Desulfovibrio, Bilophila and Lawsonia) is phylogenetically related to the Desulfohalobium group, Desulfomicrobium and Desulfonatronum. Desulfovibrios occur in aquatic biotopes but are also common inhabitants of the intestines of mammals, including humans. Interestingly, Lawsonia intracellularis is an obligately intracellular bacterium occurring in the intestinal epithelial cells of pigs with proliferative enteropathy, a major disease affecting the economics of pig industries worldwide. Lawsonia does not reduce sulfate, but is clearly related to the Desulfovibrio group (McOrist et al., 1995). Similarly Bilophila wadsworthia is a common inhabitant of the human colon and has been associated with human appendicitis and was also reported as a common infective agent in neonatal pigs (McOrist et al., 2001). One species of Desulfomicrobium (D. orale) has been described as being involved in peridontal disease.

The Desulfuromonas group is more related to the Pelobacter group, which is mainly composed of fermenting bacteria, such as Malonomonas rubra, growing by the decarboxylation of malonate to acetate. Some of the sulfate-reducing bacteria can also utilize Fe3+ as electron acceptor, which is typical for the various Geobacter species, also belonging to this rRNA cluster.

The Desulfobacter group is quite diverse as it contains 14 genera and is phylogenetically related to the Desulfobulbus and the Desulfomonile (or “Desulfoarculus”) groups. The Desulfobacter group harbors also some psychrophilic sulfate-reducing bacteria such as Desulfofrigus and Desulfofaba, which were isolated from cold Arctic marine sediments (Knoblauch et al., 1999).

Sulfate reducers occur also among the Syntrophobacter group (Fig. 1), which is composed of the syntrophs classified in the genus Syntrophobacter and the sulfate-reducers belonging to Desulforhabdus and Desulfovirga (Tanaka et al., 2000) together with the thermophilic marine genera Desulfacinum and Thermodesulforhabdus. The latter two genera form part of a subsurface microbial community; the former was isolated from hydrothermal vent systems and the latter from hot North Sea oil field water. Other syntrophs of the “Deltaproteobacteria” such as the benzoate-degrading Syntrophus species and the propionate-degrading Smithella belong to the Syntrophus group (Fig. 1).

The genera Desulfurella and Hippea seem to occupy a separate position within the Proteobacteria; they are anaerobic, moderately thermophilic, sulfur-reducing bacteria, isolated from respectively terrestrial hot springs or submarine hot vents. Pending further studies, it is possible that in the future the Desulfurella group might be considered as a sixth (zeta) class among the Proteobacteria (Rainey et al., 1993).

The “Epsilonproteobacteria”

This is the smallest and a more recently recognized line of descent within the Proteobacteria (6 genera and some 50 species). Compared to the multiple changes in the classification of the four other proteobacterial classes, the classification of the ε-class remained quite stable, because it is a fairly new group whose taxonomy was right from its recognition based on comparisons of 16S rDNAs. Up to now, photosynthetic bacteria have not been reported and key representatives are the genera Campylobacter and Helicobacter, encompassing enteropathogens for man and/or animals. Most species of the “Epsilonproteobacteria” are microaerophilic, chemoorganotrophic nonsaccharolytic spiral-shaped or curved bacteria, typically motile with a corkscrew-like motion by means of polar flagella. They obtain their energy mainly from amino acids or tricarboxylic acid cycle intermediates. Menaquinones are the characteristic respiratory quinones (Table 3). In the 1980s, interest in campylobacters and related bacteria increased owing to the availability of adequate isolation and cultivation procedures. Some species require indeed fumarate, or formate plus fumarate, or hydrogen plus fumarate for growth in microaerobic conditions. Numerous new taxa were described, and the gastritis-causing Campylobacter pylori was transferred together with C. mustelae (isolated from the gastric mucosa of ferret) to the new genus Helicobacter by Goodwin et al. (1989). Some not yet culturable “Epsilonproteobacteria” have been reported as symbionts of shrimps and polychaetes. The complete genome sequences of Campylobacter jejuni (Parkhill et al., 2000) and Helicobacter pylori (Alm et al., 1999) have been determined. For recent reviews emphasizing taxonomic aspects of the major genera of the “Epsilonproteobacteria,” see Vandamme (2000), On (2001), and Solnick and Vandamme (2001), and also Wassenaar and Newell (2001). Table 12 summarizes the names of taxa above the rank of genus within the “Epsilonproteobacteria” according to the new edition of Bergey’s Manual of Systematic Bacteriology (Garrity, 2001).

Table 12.

The “Epsilonproteobacteria”: orders, families and number of genera.a

Order

Family

Number of genera

“Campylobacterales”

Campylobacteraceae

4

“Helicobacteraceae”

2

aAccording to the second edition of Bergey’s Manual of Systematic Bacteriology (Garrity and Holt, 2001). Quotation marks are used for names which have not yet been validated (as of mid 2002).

The Campylobacteraceae

Campylobacter coli and C. jejuni are the most frequently identified causative agents of acute infective diarrhea in humans. Campylobacters can enter the food chain via raw milk and raw meat (particularly poultry). Other Campylobacter and Arcobacter species are involved in abortion and infectious infertility in various animals (e.g., sheep and cows), whereas some Campylobacter species are also found in large numbers in the periodontal pockets of diseased gums of man. Although primarily known as human and animal associated bacteria which are of relevance in food microbiology, arcobacters were shown to be abundantly present in certain environmental niches including water reservoirs, sewage, oil field communities, and certain saline environments. Their role in the environment is not well documented, but some of these organisms were shown to be sulfide oxiders (with the production of sulfur) and it has been suggested that they play a role in the sulfur cycle by reoxidizing sulfide formed by microbial sulfate or sulfur reduction (Teske et al., 1996; Voordouw et al., 1996; Snaidr et al., 1997; Wirsen et al., 2002). The type species A. nitrofigilis occurs in the roots and the rhizosphere of salt marsh plants, where these bacteria fix nitrogen. The genus Sulfurospirillum harbors microaerophilic sulfur-reducing curved bacteria occurring in freshwater or marine surface sediments. Two recently described Sulfurospirillum species (S. barnesii and S. arsenophilum) can even use arsenate or selenate as electron acceptor (Stolz et al., 1999). The strictly anaerobic “Dehalospirillum multivorans” was isolated from activated sludge and can dechlorinate tetrachloroethene, a volatile man-made pollutant (Scholz-Muramatsu et al., 1995). This organism seems to be phylogenetically related to Sulfurospirillum.

The Helicobacter Group

The helicobacters constitute together with Wolinella a distinct phylogenetic lineage within the “Epsilonproteobacteria” (Fig. 1). Helicobacter pylori has received considerable attention for its role in gastric and duodenal ulcers and in gastric cancer in humans. Helicobacter encompasses at present some 20 species and more will likely be discovered when the stomach of other animals will be screened for bacteria similar to H. pylori. The phylogenetic and taxonomic position of various so-called “gastrospirilla” occurring in the stomachs of humans and animals will only be resolved when significant advances in the culture methods for these difficult bacteria are made. The pig and cattle strains now constitute two provisional candidate species: respectively Candidatus Helicobacter suis and Candidatus Helicobacter bovis (De Groote et al., 1999a; De Groote et al., 1999b; see also Table 13). Similarly, polyphasic investigations are needed in the group of “Flexispira rappini,” which is a provisional name for the spindle-shaped bacteria with periplasmic fibers and bipolar tufts of sheathed flagella, isolated from a variety of clinical and veterinary sources. The name “Flexispira rappini” refers to a characteristic morphotype shared by several distinct taxa and not to a distinct well-defined species (Dewhirst et al., 2001; Vandamme and On, 2001).

Table 13.

Published Candidatus taxa among the Proteobacteria (as of mid 2002).

Name of Candidatus Reference

Comments/host

References

“Alphaproteobacteria”

Candidatus Liberibacter africanus”

Phloem-restricted bacterium associated with greening disease in citrus

Jagoueix et al., 1994

Murray and Stackebrandt, 1995

Candidatus Liberibacter africanus subsp. capensis”

Phloem-restricted bacterium associated with leaf mottle symptoms in Calodendrum capense (Cape chestnut)

Garnier et al., 2000

Candidatus Liberibacter asiaticus”

Phloem-restricted bacterium associated with greening disease in citrus

Jagoueix et al., 1994

Murray and Stackebrandt, 1995

Candidatus Odyssella thessalonicensis”

Endosymbiont of Acanthamoeba spp. Phylogenetically related to Caedibacter and Holospora

Birtles et al., 2000

Candidatus Xenohaliotis californiensis”

Endosymbiont causing withering syndrome of Haliotis spp. (abalone). Belongs to the Rickettsiaceae

Friedman et al., 2000

“Betaproteobacteria”

Candidatus Procabacter acanthamoebae”

Endosymbiont of Acanthamoeba spp.

Horn et al., 2002

“Gammaproteobacteria”

Candidatus Arsenophonus triatominarum”

Endosymbiont of Triatoma infestans. Closely related to Arsenophonus nasoniae (the triatomine bug)

Hypsa and Dale, 1997

Candidatus Blochmannia floridanus”

Endosymbiont of Camponotus floridanus (carpenter ants)

Sauer et al., 2000

Candidatus Blochmannia herculeanus”

Endosymbiont of Camponotus herculeanus (carpenter ants)

Sauer et al., 2000

Candidatus Blochmannia rufipes”

Endosymbiont of Camponotus rufipes (carpenter ants)

Sauer et al., 2000

Candidatus Phlomobacter fragariae”

Phloem-restricted bacterium associated with marginal chlorosis of strawberry

Zreik et al., 1998

“Epsilonproteobacteria”

Candidatus Helicobacter bovis”

Occurs in pyloric part of the abomasum of cattle

De Groote et al., 1999a

Candidatus Helicobacter suis”

Occurs in stomach of pigs

De Groote et al., 1999b

Wolinella succinogenes (formerly Vibrio succinogenes), a commensal of the bovine rumen, belongs also to the Helicobacter group. This organism can grow on hydrogen as electron donor and fumarate as electron acceptor.

Likely the full extent of the ecological diversity of the “Epsilonproteobacteria” still remains to be discovered: 16S rDNA gene sequence data of symbionts of shrimps (Rimicaris exoculata) and polychaete annelids (Alvinella pompejana) suggest that some “Epsilonproteobacteria” may occupy important niches in the habitats of deep-sea hydrothermal vents, where they contribute to overall carbon and sulfur cycling at moderate thermophilic temperatures (Campbell et al., 2001; Corre et al., 2001). Recently, such a thermophilic sulfur-reducing bacterium isolated from the deep-sea hydrothermal vent polychaete, Alvinella pompejana, was allocated to a new genus, Nautilia (Miroshnichenko et al., 2002). Another remarkable member of the “Epsilonproteobacteria” is Thiovulum, whose phylogenetic relationship was shown by partial rDNA analysis (Romaniuk et al., 1987). The sulfur-oxidizing Thiovulum possesses large ovoid cells with a diameter often reaching 25 µm. The cytoplasm contains orthorhombic sulfur inclusions, and the bacterium is considered as a chemolithotroph occurring in marine and freshwater environments, where sulfide-containing water and mud layers are in contact with overlaying oxygen-containing water. The environmental Thiomicrospira denitrificans belongs also to the “Epsilonproteobacteria,” whereas the other six Thiomicrospira species, including the type species T. pelophila, are affiliated to the Piscirickettsia group of the “Gammaproteobacteria” (Brinkhoff et al., 1999).

Symbiotic, Parasitic and Not-Yet Cultured Proteobacteria, and the Alphaproteobacterial Origin of Mitochondria

The application of molecular biological methods to study the diversity of genes from environmental DNA without the need to isolate prokaryotic strains prior to molecular analyses has broadened immensely our insight into community structure and must be considered a quantum leap in microbial ecology (Hugenholz et al., 1998). Though problems are still associated with its ability to qualify and quantify individual partners of the community, the molecular approach has circumvented problems recognized with pure culture studies—the “great plate count anomaly,” as phrased by Staley and Konopka (1985). Hundreds of habitats and selected environmental sites have been subjected to the direct analysis of community structure by ribosomal rDNA sequencing and, more recently, of community function by the analysis of genes involved in metabolism (sulfur, nitrogen, methanogenesis, etc.) as well as degradation, bioremediation or heavy metal resistance.

Symbiosis between prokaryotic and eukaryotic organisms seems to play a major role in evolution (Gray and Doolittle, 1982; Margulis, 1993; Margulis, 1996). A classical example is the symbiotic association between some diazotrophic “Alphaproteobacteria” (e.g., Rhizobium, Bradyrhizobium and Azorhizobium) and leguminous plants, where the bacteria reduce atmospheric nitrogen in root or stem nodules. Some Proteobacteria live as obligate intracellular symbionts or parasites in animals. To study the long-term history of symbiotic associations, the phylogenetic trees of hosts and their symbionts have been compared. Few data only are available, the most convincing study being the one on the endosymbionts of aphids. The topology of the symbiont (Buchnera aphidicola) tree is completely concordant with host phylogeny, based upon morphology (Moran et al., 1993). The fossil and biogeographic time points for the host phylogeny have been used to calibrate the 16S rDNA of the closely related endosymbionts (>8% similarity). The value of 1% fixed substitutions per 25 to 50 milllion years (Moran et al., 1993) is similar to the value of 1% per 50 million years determined by Ochman and Wilson (1987) on the basis of a broader range of nonobligate symbiotic relationships (e.g., Rhizobium/legumes; Photobacterium/fish; and enterobacteria/mammals) and 1% per 60 million years suggested by Stackebrandt (1995) for the past 500 years.

The advantage to the host of the association has been unraveled in only a few cases (e.g., the removal of hydrogen produced from hydrogenosomes of ciliates, provision of nutritional carbon to the host bivalves by sulfur-oxidizing gill symbionts [Dando and Southward, 1986] or of essential amino acids to aphids by their endosymbionts [Baumann et al., 1995; Baumann et al., 1998]). Molecular tools have also been used to elucidate the transmission route of symbionts in tropical lunicid bivalves (Gros et al., 1998) and deep-sea bivalves (Krueger et al., 1996) by application of the polymerase chain reaction (PCR) techniques in ovaries, testis and gill tissue.

We can estimate that a considerable number of new proteobacterial taxa has not yet been detected or described, because suitable in vitro cultivation techniques do not yet exist for such endosymbiotic bacteria. However, DNA-based techniques allow the detection and visualization of such endosymbionts, and PCR amplification of their 16S rDNA by oligonucleotide probes (see Table 4), applied singly or in a combination of several probes, each representing a different taxonomic level, enables the determination of their phylogenetic position (Manz et al., 1992; Amann et al., 1995; Amann and Ludwig, 2000).

An intracellular lifestyle has obvious advantages for a suitably adapted prokaryote, and the spread of intracellular bacteria seems to be guaranteed in hosts such as blood sucking insects. Hence, it is not surprising that intracellular bacteria have evolved in different phylogenetic groups. Classical examples among the Proteobacteria are Coxiella, Rickettsiella and the endosymbionts of aphids belonging to the genus Buchnera (all “Gammaproteobacteria”) and various members of the Rickettsiaceae and allied groups (e.g., Holospora and Caedibacter), belonging to the “Alphaproteobacteria.” Related to the Rickettsiaceae are the wolbachiae, which are symbionts of invertebrate hosts, affecting the reproduction of the host by induction of thelytokous parthenogenesis (i.e., male killing or feminization). The wolbachiae infect the reproductive cells of various insects and have thus the potential to be vertically transmitted by cytoplasmic inheritance. More details concerning intracellular prokaryotes can be found in the introductory chapter by Fredricks (Introduction to the Rickettsiales and other Intracellular Prokaryotes in this Volume) and some examples of symbiotic and not yet cultured Proteobacteria are shown in Table 14, together with their phylogenetic affiliation.

Table 14.

Examples of symbiotic Proteobacteria and their phylogenetic affiliation.

Host species

Host trivial name

Phylogenetic affiliation of symbiont

Reference/EMBL accession number

Muscidifurax uiniraptor

House fly

α-class

Stouthamer et al., 1993

Bangasternus orientalis

Coleoptera, weevil

α-class

M85266

Rhinocyllus conicus

Coleoptera, weevil

α-class

M85267

Trichogramma deion

Parasite wasp of Lepidoptera

α-class

Stouthamer et al., 1993

Nasonia vitripennis

Parasitic wasp

α-class

Gherna et al., 1991

Crassostrea virginica

Eastern Oyster

α-class

Boettcher et al., 2000

Dysmicoccus neobrevipes

Homoptera, mealy bug

β-class

Munson et al., 1992

Acromyrmex octospinosus

Leave cutting ant

β-class

AF459796

Antonina crawii

Bamboo pseudococcoid, Homoptera

β, γ-class

Fukatsu et al., 2000

Solemya velum

Bivalve

γ-class

M90415

Heliothis virescens

Moth, tobacco budworm

γ-class

L22481

Bathymodiolus thermophilus

Bivalve

γ-class

Distel et al., 1988

Calyptogena elongata

Bivalve

γ-class

L25719

Calyptogena magnifica

Bivalve

γ-class

Distel et al., 1988

Vesicomya cordata

Bivalve

γ-class

L25713

Anodontia phillipiana

Bivalve

γ-class

L25711

Codakia orbicularis

Bivalve

γ-class

Distel et al., 1988

Thyasira flexuosa

Bivalve

γ-class

Distel and Wood, 1992

Lucina floridana

Bivalve

γ-class

L25707

Riftia pachyptila

Tube worm

γ-class

Distel et al., 1988

Anomalops katoptron

Deep sea anglerfish

γ-class

Z19081

Cryptosaras couesi

Deep sea anglerfish

γ-class

Haygood et al., 1992

Sitophilus zeamais

Coleoptera, maize weevil

γ-class

M85270

Acyrthosiphon sp.

Pea aphid

γ-class

Unterman et al., 1989

Camponotus floridanus

Giant ants

γ-class

Schröder et al., 1996

Glossina pallidipes

Tsetse fly

γ-class

Beard et al., 1993

Most of these endosymbiotic bacteria cannot yet be cultivated and illustrate that only a very limited proportion of the bacterial species present in various habitats is actually known. Although a great part of the rDNA sequences of uncultured bacteria is available in public databases, no formal phenotypic description nor species names can be given for such organisms, and type strains cannot be made publicly available. Some of these uncultured bacteria have been partially described and provisionally allocated to the category of Candidatus (Murray and Schleifer, 1994; Murray and Stackebrandt, 1995). The proteobacterial Candidatus taxa together with some additional information are listed in Table 13 (see also the [{http://www.bacterio.cict.fr}List of Bacterial Names with Standing in Nomenclature website]).

The mitochondrion of eukaryotic cells can be considered as the ultimate prokaryotic endosymbiont. Like chloroplasts, mitochondria possess unique genomes that are distinct from the nuclear genomes of their associated eukaryotic cells, and mitochondrial ribosomes are clearly of the prokaryotic type and related to the “Alphaproteobacteria” (Lang et al., 1999). Among the many bacterial genomes that have now been sequenced, that of Rickettsia prowazekii (1.1 Mb) is most closely related to the mitochondrial genome (Andersson et al., 1998). Rickettsia prowazekii is the agent of epidemic louse-borne typhus in humans and multiplies in eukaryotic cells only. Most likely the genome types of mitochondria and rickettsiae have shared a common free-living alphaproteobacterial ancestor, which through genome reduction evolved via two descendent lineages (Gray, 1998; Gray et al., 1999). Studies on mitochondria of yeast reveal that the number of alphaproteobacterial descendents in the mitochondrial proteome is surprisingly small, and that a large number of novel mitochondrial genes likely originated from the eukaryotic nuclear genome complementing the remaining genes from the bacterial ancestor (Karlberg et al., 2000; Kurland and Andersson, 2000). Further genomic and proteomic analyses of other members of the “Alphaproteobacteria,” such as Bradyrhizobium and Rhodobacter, may yield more information concerning the metabolic versatility of the proto-mitochondrion. Whether the origin of hydrogenosomes—the ATP-producing organelles of many anaerobic protists—is related to that of mitochondria remains to be investigated in more detail (Andersson and Kurland, 1999; Dyall et al., 2000).

Genome Analysis

The G+C content of genomic DNA of the Proteobacteria varies from 30 mol% (e.g., in Campylobacter and Rickettsia) to 70% (e.g., in Alcaligenes), indicating that almost the entire span of mol% G+C variation among living microorganisms is covered. Even within each of the proteobacterial subclasses, variation in G+C content of the genome is very large. Similarly the genome sizes of various Proteobacteria differ considerably. Commensals and free-living prokaryotic strains have a larger genome, ranging from 9.4 Mb (Myxococcus xanthus) to about 2.5 Mb (Pasteurella multocida), than that of obligate symbiotic and parasitic strains, e.g., Buchnera aphidicola (0.64 Mb) and Rickettsia prowazekii (1.1 Mb). Unexpectedly, the genome size of phylogenetically closely related species and even strains of the same species can differ significantly, as shown for three species of Yersinia (3.8–4.9 Mb) and various strains of E. coli (4.6–5.5 Mb) and Burkholderia cepacia (4.7–8.1 Mb; Lessie et al., 1996). Most Proteobacteria studied contain a single circular chromosome, but the presence of multiple chromosomes has been reported, particularly among the “Alphaproteobacteria”: two different circular chromosomes in Rhodobacter sphaeroides (Suwanto and Kaplan, 1989) and Brucella melitensis (Michaux-Charachon et al., 1997); three circular chromosomes in Rhizobium meliloti (Sobral et al., 1991) and Burkholderia cepacia (Cheng and Lessie, 1994). Another unconventional organization has been detected in several strains of Agrobacterium tumefaciens, where the larger chromosome is circular (3.0 Mb), whereas the smaller (2.1 Mb) is linear (Jumas-Bilak et al., 1998). It should be noted that not all strains of Brucella melitensis or all species of the genus Rhodobacter contain multiple chromosomes, and the evolutionary significance of these peculiar genomic organizations remains to be unraveled (Jumas-Bilak et al., 1998).

A multitude of genes of various Proteobacteria have been sequenced; genetic and genomic aspects of E. coli, various Pseudomonas species, Rhizobium and many animal and human pathogenic Proteobacteria have been or are being studied in detail, and more importantly in recent years, considerable progress has been made in the sequencing of whole genomes of an increasing number of Proteobacteria. The first whole-genome sequence of a free-living microorganism was published in 1995 by Fleishmann et al. for the gammaproteobacterium Haemophilus influenzae (1.83 Mb). At present (mid 2002), the sequences have been completed for at least 9 α-, 3 β- (Bordetella and Neisseria), 17 γ- and 3 ε- (Campylobacter and Helicobacter) class members (sequenced genera are indicated by footnote “a” in Table 3; see also [{http://www.tigr.org}The Institute for Genomic Research website]). Sequencing of a great number of proteobacterial genomes is in progress (see footnote “b” in Table 3 and [{http://www.tigr.org}The Institute for Genomic Research website], [{http://www.ncbi.nlm.nih.gov}National Center for Biotechnological Information website], ([DOE Joint Genome Institute website] [http://www.jgi.doe.gov/JGI_microbial/html] and [GOLD Genome OnLine Database website] wit.integratedgenomics. com/GOLD/) and most of it pertains to bacteria of clinical or biotechnological importance. The sequencing projects will elucidate regulatory and structural functions of newly discovered genes and will also yield significant insights into the mechanisms of pathogenicity, bacterial photosynthesis, phylogeny and evolution.

Final Considerations

With the publication of the Approved Lists of Bacterial Names in 1980, the number of genera and species presently belonging in the Proteobacteria was approximately 130 and 500, respectively. Until 2002, the number of validly described genera and species increased to respectively about 460 and 1600, making the phylum Proteobacteria a heavily populated section of the phylogenetic bacterial tree. Indeed, in terms of number of genera, the Proteobacteria encompass more than 40% of all prokaryotic genera. Recent insights into the community structure of as-yet uncultivated prokaryotes reveal that the vast majority of taxa have not yet been described. This is true not only for open environments, such as the open ocean (Giovannoni et al., 1990), soil (Liesack and Stackebrandt, 1992; Felske et al., 1999), deep subsurface (Chandler et al., 1997), waste treatment plants and bioreactors (Bond et al., 1995; Juretschko et al., 2002), and sediments of oceans and lakes (Bowman et al., 2000; Urakawa et al., 2000; Brambilla et al., 2001), but also for the microflora of eukaryotic hosts. Particularly the not-yet culturable Proteobacteria, the endosymbionts and those which are difficult to grow in axenic culture such as the helicobacters and allied taxa, are a goldmine for further studies on bacterial diversity. The most promising tools to explain the role of Proteobacteria in global cycling of gases and chemicals are: 1) for elucidating the phylogenetic structure of the community, DNA fragment electrophoresis, sequence analysis of universal and taxon-specific genes, and their identification in situ by FISH hybridization, and 2) for determining the physiological capacities of its members, identification of housekeeping genes by FISH techniques and by microautoradiography (Ouverney and Fuhrman, 1999; Lee et al., 1999). Horizontal transfer of genes (Klein et al., 2001) will shed light on the possible evolutionary history of the various large groups within Proteobacteria by tracing the origin of genes to the ancestors of recent species that are phylogenetically related to other prokaryotic phyla. The number of full genome sequences will continue to rise, providing microbiologists with an unparalleled wealth of information for scientific exploitation to the benefit of clinical, environmental, evolutionary and general microbiology. It is likely that the increasing number of genera and species as well as comparative studies on other semantic macromolecules will challenge the present five major subdivisions of the Proteobacteria: as the phylogenetic radiation of the proteobacterial lineages will become more complex, boundaries between the five major subgroups will become vague and the subgroups more ambiguous. On the other hand, information on orthologous genes other than ribosomal rRNA genes will provide systematicists with sets of molecules to be included in future studies on multi-locus sequence analysis. As recommended, this information will be used in the description of the taxon species (Stackebrandt et al., 2002) and possibly of higher taxa. Molecular studies and culture attempts will continue to go hand in hand. The scientific challenge in the near future will also include molecular determination of metagenomes from proteobacteria of selected sites as well as the cultivation of hitherto uncultured proteobacterial symbiotic and pathogenic organisms.

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