Current Microbiology

, Volume 55, Issue 4, pp 356–361

Porphyrobacter meromictius sp. nov., an Appendaged Bacterium, That Produces Bacteriochlorophyll a

Authors

  • Christopher Rathgeber
    • Department of MicrobiologyThe University of Manitoba
  • Natalia Yurkova
    • Department of MicrobiologyThe University of Manitoba
  • Erko Stackebrandt
    • DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH
  • Peter Schumann
    • DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH
  • Elaine Humphrey
    • Bio-Imaging FacilityThe University of British Columbia
  • J. Thomas Beatty
    • Department of Microbiology and ImmunologyThe University of British Columbia
    • Department of MicrobiologyThe University of Manitoba
Article

DOI: 10.1007/s00284-007-0275-y

Cite this article as:
Rathgeber, C., Yurkova, N., Stackebrandt, E. et al. Curr Microbiol (2007) 55: 356. doi:10.1007/s00284-007-0275-y

Abstract

Four Gram-negative strains (ML4T, ML19, ML31, ML32) of nonmotile, appendaged, budding bacteria were isolated from the meromictic Mahoney Lake in British Columbia, Canada. The strains were red to brown-red in color and produced bacteriochlorophyll a incorporated into photosynthetic pigment-protein complexes. Phylogenetic analysis has placed these strains within the class Alphaproteobacteria, with the closest relatives being members of the genera Erythrobacter, Porphyrobacter, and Erythromicrobium. Morphological features warrant their inclusion within the genus Porphyrobacter and these strains can be readily distinguished from other species of this genus on the basis of a mesophilic temperature range, a broad pH range, and tolerance to extremely high NaCl and Na2SO4 concentrations, in keeping with the environment from which they were isolated, a Na2SO4-dominated meromictic lake. These isolates utilize a variety of organic substrates for aerobic chemoheterotrophic growth and do not grow under anaerobic conditions, in either the presence or the absence of light. All strains require vitamin B12, and strains ML4T and ML19 require biotin. The DNA G + C contents ranged from 62.2 to 64.9 mol%. Phenotypic and phyletic data support the classification of strains ML4T, ML19, ML31, and ML32 as a novel Porphyrobacter species for which the name Porphyrobacter meromictius sp. nov. is proposed.

Aerobic anoxygenic phototrophic bacteria (APB) are distinguished from other bacteria by the presence of bacteriochlorophyll (BChl) a incorporated into photosynthetic units, and the inability to use these photosynthetic units for phototrophic growth under anaerobic conditions [21, 15]. APB fall primarily into the class Alphaproteobacteria, and are intermixed with both phototrophic and nonphototrophic genera. This has led to some confusion with regard to the taxonomy of this heterogeneous class [21, 22], in which nonphototrophic representatives have sometimes been classified as species within the existing phototrophic genera based primarily on phylogenetic distance, despite a lack of phenotypic similarities, for example, Erythrobacter citreus [2]. Conversely, in other cases classical taxonomic markers have been given precedence and closely related strains have been classified into novel nonphototrophic genera, as, for example, the not yet validated taxon “Lutibacterium anuloederans” [1].

The first described genus of APB, Erythrobacter, was originally defined for long, slender, rod-shaped, aerobic chemoorganotrophs that produce BChl a and carotenoids, and reproduce by binary fission [17]. The genus Porphyrobacter was designated in 1993 to include BChl containing aerobes, branching with Erythrobacter longus within the Alphaproteobacteria. Porphyrobacter differed from Erythrobacter in habitat, originating from a freshwater environment, and by an ultrastructural resemblance to the Planctomycetales, a tendency toward pleomorphism, replication by polar growth or budding, and the production of multifibrillar stalk-like structures, as well as the absence of cytochrome c oxidase and a lack of vitamin requirements [5]. This genus now includes five species, the type species Porphyrobacter neustonensis, the moderate thermophiles Porphyrobacter tepidarius [7] and Porphyrobacter cryptus [14], the budding Porphyrobacter sanguineus [8], and the marine micro-organism Porphyrobacter donghaensis [20]. In this study we report the characterization of four halotolerant reddish-brown, BChl-containing strains that form appendages and reproduce by budding as a novel species within the genus Porphyrobacter.

Strains ML19, ML31, and ML32 were isolated from the surface, and strain ML4T from a 3-m depth, of the Na2SO4-dominated meromictic Mahoney Lake in the Okanagan Valley of British Columbia, Canada [25]. Medium N1 (ML4T, ML19) and medium N4 (ML31, ML32) or modifications thereof were used for all experiments unless otherwise noted [25]. Strains could be stored on agar media at 4°C for several months. For long-term storage a thick cell suspension was supplemented with 30% glycerol and frozen at –70°C. Morphology and cytology were examined by phase contrast microscopy (Zeiss Axioskop 2 microscope) and transmission electron microscopy as described [25]. The ability to grow at different salinities was tested with concentrations of NaCl or Na2SO4 from 0% to 14% [16]. Temperature and pH range, utilization of soluble organic substrates, ability to ferment sugars and reduce nitrate, catalase and oxidase production, and antibiotic sensitivity were determined [23, 24]. Phototrophic growth under anaerobic conditions was tested in the above media, in basal media designed for purple sulfur bacteria containing H2S or Na2S2O3 and CO2 with or without acetate, and in a basal medium for purple nonsulfur bacteria containing acetate, malate, or succinate as the sole source of organic carbon [9, 16]. Spectral absorption measurements of whole cells grown under aerobic conditions in liquid culture were recorded using a Hitachi U-2010 spectrophotometer [16].

DNA G + C content was determined using HPLC (Shimadzu), as described [18], of nucleotides obtained according to Mesbah and Whitman [11]. Extraction of genomic DNA, PCR-mediated amplification of 16S rRNA gene sequences and direct sequencing of PCR products were carried out as by Rainey et al. [13]. Sequence reaction mixtures were electrophoresed using a model 373A automatic DNA sequencer (Applied Biosystems). The 16S sequences were aligned with published sequences obtained from the EMBL nucleotide sequence database and the Ribosomal Database Project, using the ae2 editor [10], and similarity values were determined. Ribotyping was carried out with the RiboPrinter microbial characterization system (Qualicon, DuPont), according to the instructions of the manufacturer.

Strains ML4T, ML19, ML31, and ML32 formed red to reddish-brown colonies on agar plates, dependent on age, and appear red-brown in liquid culture. Absorption spectra of whole cells, grown aerobically in the dark, revealed that this pigmentation was due to the presence of BChl a incorporated into reaction centers and light harvesting (LH) I complexes, evidenced by peaks at 806–808 and 866–867 nm, respectively, and carotenoid pigments giving rise to peaks at 466–468 and 489–491 nm (Fig. 1). This type of photosynthetic apparatus is common to most Porphyrobacter species [5, 7, 14, 20]. However, P. sanguineus, the sole exception, displays an absorption maximum at 814 nm that may represent an additional LH II component [8], although the cause of this absorption maximum remains unclear, as such a blue-shifted LH II has not previously been observed in APB. Cells grown aerobically in the presence of continuous illumination lack or have greatly reduced absorption peaks in both the 806- and the 867-nm regions indicating that production of the photosynthetic apparatus is strongly inhibited by light. All of the isolates were incapable of anaerobic photosynthetic growth in all media tested, confirming that these isolates are members of the APB.
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Fig. 1

Absorption spectra of strain ML4T whole cells grown aerobically in the dark show a peak at 806 nm, indicative of a photosynthetic reaction center, and at 867 nm, indicative of a light harvesting I complex.

Morphology was examined in exponential phase cells grown in liquid medium under aerobic conditions. Each of the strains formed short rods (0.5 × 1.5 μm) to ovoid-shaped cells (0.5 × 1.0 μm) and reproduced by budding or constrictions. Motility was not observed in either liquid cultures or cultures grown on agar plates, and flagella were not observed in electron microscopy. There was a tendency to form chains of about six cells, although longer chains were observed in strain ML19.

Transmission electron microscopy of strains ML4T and ML19 (both thin sections and negative stains) revealed many as yet unexplained structures [25]. Negatively stained cells of ML19 showed an unusual type of connective material between cells that appeared to consist of a bubble-like formation with a tubular structure located at the center (Fig. 2A). This type of connective material has not been observed before in the domain Bacteria. Strain ML19 also produced an appendage (Fig. 2B) similar to that seen in P. neustonensis [5]. The presence of these structures was confirmed in thin sections (Fig. 2C).
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Fig. 2

Negatively stained micrographs of strain ML19 reveal (A) unusual connective material and (B) a stalk-like structure. (C) The presence of these structures was confirmed in thin sections. Strain ML4T-negative stains show (D) an unusual extracellular protrusion and (E) an appendage detached from the cell. Thin sections (F) show a tendency toward pleomorphism and prostheca formation. Bars: (a, d) 1.0 μm; (b, f) 0.5 μm; (c, e) 0.25 μm.

Strain ML4T excreted large amounts of capsular material, causing aggregation among cells, and also produced an extracellular protrusion, although this structure resembled a chain of beads (Figs. 2D and E) [25] that was clearly different from the stalk of P. neustonensis. Thin sections of ML4T revealed cells with a tendency toward pleomorphism and possible production of a prostheca-like structure (Fig. 2F).

The Gram-negative cell wall structure was confirmed in thin sections, and as expected" for APB, intracytoplasmic membrane formations, of the type usually found in anoxygenic phototrophs, were not observed (Figs. 2C and F).

Strains ML4T, ML19, ML3, and ML32 were similar in terms of physiological properties. They tolerated a wide range of pH, with growth occurring between pH 5.5 and pH 10.0, but not at pH 5.0 or 11.0. Saline conditions were not required, and all strains grew well in the absence of added NaCl or Na2SO4. Strains ML4T, ML19, and ML32 tolerated up to 8% NaCl, whereas ML31 grew at up to 10% NaCl. Each of the strains exhibited slightly higher tolerance to Na2SO4, the dominant salt at the site of isolation [12], with ML19 tolerating 10%, ML4T and ML32 tolerating 13%, and ML31 withstanding 14% Na2SO4, the highest concentration tested. In all cases, however, optimal growth was achieved at concentrations between 0% and 2% of the respective salt. The temperature range was mesophilic, with growth occurring between 10 and 37°C but not at 5 or 45°C.

Catalase and oxidase were produced by all of the strains, as well as extracellular lipases capable of hydrolyzing Tween 60. Additionally ML4T, ML19, and ML31 produced extracellular proteinases capable of hydrolyzing gelatin, and ML19 and ML32 produced extracellular amylases.

These strains appear to be nutritionally versatile, able to use a variety of simple organic compounds as the sole source of carbon and energy for aerobic chemo-organotrophic growth. All strains use acetate, pyruvate, glutamate, butyrate, malate, succinate, and lactate. Additionally, strain ML4T can use glucose, and strains ML31 and ML32 can use glucose, fructose, citrate, and formate, and even appear capable of very weak growth on ethanol and methanol. The strains did not ferment glucose or fructose and were not capable of dissimilative nitrate reduction under anaerobic conditions. All of the strains require the addition of vitamin B12 to the growth medium, and strains ML4T and ML19 have a requirement for biotin. The addition of biotin stimulates the growth of ML31 and ML32, although they are capable of growth in its absence.

The strains were all susceptible to chloramphenicol (30 μg), tetracycline (30 μg), and kanamycin (30 μg), and ML31 and ML32 were susceptible to penicillin G (10 units). Strain ML31 alone was susceptible to ampicillin (2 μg). All were able to grow in the presence of streptomycin (10 μg), polymixin B (50 μg), and nalidixic acid (30 μg).

In a previous study partial 16S rRNA gene sequences (>400 nucleotides) identified these and other APB isolates from Mahoney Lake as members of the class Alphaproteobacteria and revealed that the partial 16S rRNA genes of ML4T, ML19, ML31, and ML32 were nearly identical to each other and most closely related (99.7–98.8%) to an undescribed strain of Porphyrobacter [25]. Based on these data and on biochemical properties, ML4T, ML19, and ML31 were chosen for nearly complete 16S rRNA gene sequencing (>1430 nucleotides), which revealed that the 16S rRNA genes of ML4T and ML19 are identical and differ only slightly from that of ML31 (99.8%) (Fig. 3). Phylogenetic analysis based on 16S rRNA gene sequences placed these strains within the phyletic cluster of Erythrobacter-Porphyrobacter-Erythromicrobium. The nearest phylogenetic neighbors are the aerobic phototrophic species Erythrobacter longus and Erythrobacter litoralis (97.4–97.7%) and the nonphototrophic species recently ascribed to the genus Erythrobacter (92.6–98.1%), as well as phototrophs of the genera Porphyrobacter (94.5–96.7%) and Erythromicrobium (95.8–96.1%). The G + C contents of the DNA of strains ML4T, ML19, and ML31 were found to be 64.9, 62.2, and 63.6 mol%, respectively.
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Fig. 3

Neighbor-joining dendrogram of 16S rRNA gene sequence relatedness shows that strains ML4T, ML19, and ML31 form a monophyletic group within the Alphaproteobacteria supported by 100% bootstrap values (500 resamplings). The position of these sequences relative to the existing genera is unclear due to the relatively low confidence for branching points within the α-4 cluster. Zymomonas mobilis ssp. mobilis was used as an outgroup. Bar: 5 substitutions per 100 sequence positions.

Assessing the taxonomic status of new strains related to the Erythrobacter-Porphyrobacter-Erythromicrobium cluster within the Alphaproteobacteria is complicated by relatively unclear phylogenetic tree topology. Regardless of whether the algorithm of De Soete [3] or the neighbor joining or maximum parsimony algorithms [4] were applied to the sequence alignment, branches separating these genera were generally not well supported by the bootstrap values [14; this study]. Our strains could be assigned to any of the three genera Erythrobacter, Porphyrobacter, or Erythromicrobium based solely on phylogenetic distance. However, examination of classical taxonomic markers makes it apparent that these strains tend to fall within the taxonomic criteria originally described for the genus Porphyrobacter (Table 1). The production of BChl incorporated into a RC and LH I, but not LH II (as is the case for Erythromicrobium, which contains an unusual blue-shifted LH II), a tendency toward pleomorphism, production of appendages, and reproduction by budding. Whereas neither members of Erythrobacter nor members of Erythromicrobium form appendages or reproduce by budding, and neither are pleomorphic, forming relatively long rod-shaped cells and very long thread-like cells, respectively [21]. These significant morphological features strongly differentiate the genus Pophyrobacter from either Erythrobacter or Erythromicrobium.
Table 1

Comparative phenotypic properties between Porphyrobacter meromictius and closely related species of the Alphaproteobacteria

Property

Species

1

2

3

4

5

6

7

8

9

Habitat

Meromictic lake

Freshwater lake

Brackish-marine

Hotspring

Brackish hotspring

Marine

Marine

Marine

Alkaline spring

Color

Red-brown

Orange-red

Orange-red

Red-orange

Orange

Red-orange

Orange

Orange

Orange

  BChl a

+

+

+

+

+

+

+

+

  LH II

+

+

Stalk

+

+

Budding

+

+

+

na

na

na

Motility

+

+

+

+

+

+

Growth

  At 10°C

+

+

+

+

na

na

  At 45°C

+

+

+

+

na

na

  In 8% NaCl

+

na

na

+

Hydrolysis of

Gelatin

V(+)

+

+

Starch

V(–)

+

+

+

V(+)

Required

Biotin

Va(+)

na

na

+

na

na

+

na

Vitamin B12

+

na

na

na

na

na

Mol% G + C

62.2–64.9

65.7–66.4

63.8–64.0

66.2

65.0

65.9–66.8

62.2–62.9

60–64

63.6–64.2

Note. +, growth occurs, substrate is hydrolyzed, or vitamin is required; –, growth does not occur, substrate is not hydrolyzed, or vitamin is not required; V, results vary between strains; na, data not available; LH II, light harvesting II complex. Data in parentheses are for the type strain. Taxa: 1, P. meromictius; 2, P. neustonensis [5]; 3, P. sanguineus [8]; 4, P. cryptus [14]; 5, P. tepidarius [7]; 6, P. donghaensis [20]; 7, E. aquimaris [21]; 8, E. longus [17]; 9, E. ramosum [24].

aBiotin stimulates growth for strains ML31 and ML32, whereas strains ML4T and ML19 require biotin.

Porphyrobacter was originally proposed to include APB of freshwater origin, however, the majority of species since added to the genus originate from marine or brackish environments. Because we feel that the location of origin should remain an important taxonomic marker, the strains from Mahoney Lake present an interesting quandary. Although the Mahoney Lake monomolimnion may have salinity as high as 40‰ [6], the salinity of surface waters on the day of sampling was only 5‰ [25], which is typical of freshwater systems. As all of our strains are capable of robust growth in a medium devoid of added NaCl or Na2SO4, it is appropriate to include these strains within the genus Porphyrobacter.

Although our isolates fit well within the specifics of the genus Porphyrobacter, several important properties clearly differentiate them from other known species (Table 1). The ability to grow at very high salinities and over a broad range of pH values is likely an essential property for survival in their natural environment. Riboprint analysis separates strain ML4T from Erythrobacter longus (DSM 6887T), P. neustonensis (DSM 9434T), and Erythromicrobium ramosum (DSM 8510T) (Fig. 4), clearly demonstrating species-level differences from the most closely related type strains. In addition, phylogenetic analysis indicates that strains ML4T, ML19, and ML31 form a distinct cluster clearly separate from other species within the Alphaproteobacteria.
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Fig. 4

Normalized riboprint patterns found in selected type strains of the Alphaproteobacteria, related to strain ML4T. Cluster analysis was performed by the unweighted pair group method with arithmetric averages (UPGMA) based on the Pearson correlation coefficient (BioNumerics, Kortrijk, Belgium).

Description of Porphyrobacter meromictius sp. nov.

Porphyrobacter meromictius (me.ro.mic’ti.us. Gr., neut., n. meros part, L. adj. part mictus mixed, L. adj. suffix –ius belonging to, N.L. masc. adj. meromictius belonging to meromictic environments, lakes).

Cultures are pigmented red to red-brown due to production of BChl a giving rise to absorption peaks at 806–808 and 866–867 nm, and carotenoids with peaks at 466–468 and 489–491 nm. Cells are short rods (0.5 × 1.0 to 0.5 × 1.5 μm) or pleomorphic, may occur in chains of six cells or more, and reproduce by budding or constrictions. They may form appendages or other extracellular protrusions. It is an obligate aerobic chemo-organoheterotroph and facultative photoheterotroph. Growth occurs on acetate, pyruvate, glutamate, butyrate, malate, succinate, and lactate; some strains also use citrate, formate, fructose, and glucose. Strains differ in their ability to hydrolyze starch and gelatin; all strains hydrolyze Tween 60. It exhibits a mesophilic temperature range between 10 and 37°C. It tolerates a wide range of salinity, from 0 to 8–10% NaCl or 0 to 10–14% Na2SO4; the optimum salinity is 0–2% NaCl or Na2SO4. Growth occurs between pH 5.5 and pH 10.0. It requires vitamin B12; biotin either is required or stimulates growth, depending on the strain. It is catalase and oxidase positive; sensitive to chloramphenicol, tetracycline, and kanamycin; and resistant to streptomycin, polymixin B, and nalidixic acid. Some strains may be resistant to penicillin G and ampicillin. The DNA G + C content is 62.2–64.9 mol%.

The habitat is a Na2SO4-dominated meromictic lake, in south-central British Columbia, Canada. The type strain is ML4T ( = DSM 18336T = VKM B-2405T).

Acknowledgments

This research was funded by grants from the NSERC (Canada) to V.Y. and J.T.B. We thank K. J. Hall and T. G. Northcote for collection of samples from Mahoney Lake and H. G. Trüper for assistance with the nomenclature.

Copyright information

© Springer Science+Business Media, LLC 2007