The SsgA-like proteins in actinomycetes: small proteins up to a big task
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- Traag, B.A. & van Wezel, G.P. Antonie van Leeuwenhoek (2008) 94: 85. doi:10.1007/s10482-008-9225-3
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Several unique protein families have been identified that play a role in the control of developmental cell division in streptomycetes. The SsgA-like proteins or SALPs, of which streptomycetes typically have at least five paralogues, control specific steps of sporulation-specific cell division in streptomycetes, affecting cell wall-related events such as septum localization and synthesis, thickening of the spore wall and autolytic spore separation. The expression level of SsgA, the best studied SALP, has a rather dramatic effect on septation and on hyphal morphology, which is not only of relevance for our understanding of (developmental) cell division but has also been succesfully applied in industrial fermentation, to improve growth and production of filamentous actinomycetes. Recent observations suggest that SsgB most likely is the archetypal SALP, with only SsgB orthologues occurring in all morphologically complex actinomycetes. Here we review 10 years of research on the SsgA-like proteins in actinomycetes and discuss the most interesting regulatory, functional, phylogenetic and applied aspects of this relatively unknown protein family.
KeywordsStreptomyces SALP Phylogeny Peptidoglycan Cell division Development
Actinomycetes have an unusually complex life cycle, many aspects of which are globally similar to those observed in some lower eukaryotes, which makes them particularly interesting for the study of bacterial development and evolution (Chater and Losick 1997). One of the best characterized genera among the actinomycetes is Streptomyces. As producers of over half of the known antibiotics, the Gram-positive soil-dwelling filamentous streptomycetes are a paradigm of secondary metabolite-producing microorganisms (Chater and Losick 1997; Hopwood 1999), with Streptomyces coelicolor A3(2) as the most-studied streptomycete (Hopwood 1999). Development of streptomycetes is initiated by the germination of a spore, from which typically two hyphae are produced, which continue to grow and branch to form a vegetative mycelium. Exponential growth is achieved by apical (tip) growth and branching (Flärdh 2003), with a complex mycelial network as the result. The vegetative hyphae consist of syncytial cells separated by occasional cross-walls, laid down at 5–10 μm intervals (Wildermuth and Hopwood 1970). When development is initiated an aerial mycelium is produced, with hydrophobic hyphae breaking through the moist surface, erected into the air. This is the start of the reproductive phase, initiated in response to nutrient depletion and the resulting requirement of mobilization. Eventually, sporulation-programmed hyphae are formed in a process requiring a complex, spatial and temporal genetic programming scheme that is switched on upon nutrient limitation (Chater 1998). During sporulation long chains of unigenomic spores are formed from multigenomic aerial hyphal compartments.
Multiple cell division during sporulation of streptomycetes requires an unparalleled complex coordination of septum-site localization, cell division and DNA segregation and coordination (Flärdh et al.2000; McCormick et al. 1994; Schwedock et al. 1997; Wildermuth and Hopwood 1970). Penicillin-binding proteins (PBPs) are key enzymes for the synthesis of the bacterial peptidoglycan, both during growth and during cytokinesis (Errington et al. 2003; Holtje 1998; Stewart 2005). The best-studied PBPs are PBP2, which is required specifically for lateral cell-wall synthesis in E. coli (Den Blaauwen et al. 2003), and FtsI (PBP3), which is part of the divisome and essential for synthesis of the septal peptidoglycan (Botta and Park 1981). Streptomycetes contain many PBPs and a number of these are developmentally controlled, suggesting a role specifically during sporulation (Hao and Kendrick 1998; Noens et al. 2005). During maturation, spores are separated in a process that most likely resembles the separation of mother and daughter cells during cell division of unicellular bacteria, involving several autolytic enzymes such as amidases, lytic transglycosylases and endopeptidases (Heidrich et al. 2002). While the cell division machinery of streptomycetes strongly resembles that of other bacteria (Flärdh and van Wezel 2003), the control of septum formation is very different in these organisms. Streptomycetes lack the MinC, MinE and SulA proteins that control septum-site localization (Autret and Errington 2001; Marston et al. 1998), the nucleoid occlusion system (NOC) that coordinates septum synthesis and DNA segregation (Wu and Errington 2004), as well as some crucial Z-ring anchoring proteins such as FtsA and ZipA (Errington et al. 2003; Lowe et al. 2004). How cell division is controlled in streptomycetes is unclear. In sporulation-committed aerial hyphae FtsZ organizes into spiral-shaped intermediates along the length of the aerial hyphal cell, eventually forming up to a hundred Z-rings per aerial hyphae (Grantcharova et al. 2005). At this stage MreB localizes to the septa, suggesting this actin-like cytoskeletal protein may assist in cell division (Mazza et al. 2006).
Instead of the canonical cell division control proteins, several unique protein families have been identified that play a role in the control of cell division in streptomycetes, and notably the CrgA-like proteins and the SsgA-like proteins (Chater and Chandra 2006; Flärdh and van Wezel 2003). CrgA-like proteins comprise a family of small integral membrane proteins, thought to play a role in the inhibition of FtsZ-ring formation during Streptomyces development (Del Sol et al. 2006). In this review, we will have a closer look at the SsgA-like proteins or SALPs, which play an important role in the control of sporulation-specific cell division in sporulating actinomycetes.
The cell division activator SsgA
In the mid-90s Kawamoto and Ensign identified a genomic DNA fragment of Streptomyces griseus that inhibited submerged sporulation of a hyper-sporulating S. griseus strain at multiple copies (Kawamoto and Ensign 1995). The same genomic fragment induced fragmented growth of the otherwise branching mycelial filaments and the responsible gene was designated ssgA (for sporulation of Streptomyces griseus). A direct correlation between SsgA accumulation and the onset of sporulation in wild-type cells, and the failure of some developmental mutants of S. griseus to accumulate SsgA, further demonstrated the sporulation-related function of SsgA (Kawamoto et al. 1997). SsgA is member of a family of proteins now known as the SsgA-like proteins (SALPs), which are small acidic 14–17 kDa proteins that do not carry any known protein motif (see next section). Remarkably, even to date there is not a single protein in the databases that has significant sequence similarity to hint at the possible mode of action for the SALPs.
ssgA null mutants of both S. coelicolor and S. griseus produce an aerial mycelium but fail to sporulate except on mannitol-containing media, where some spores are produced after prolonged incubation; this makes ssgA a rather unique example of a conditional white (whi) mutant (Jiang and Kendrick 2000; van Wezel et al. 2000a). Later studies showed that SsgA directly activates sporulation-specific cell division, and over-production results in a dramatic morphological change of the vegetative hyphae of S. coelicolor; the vegetative hyphae become approximately twice as wide (average of 800 nm instead of 400–500 nm) and form spore-like compartments separated by massive and aberrant septa, resulting in hyper-fragmenting hyphae in submerged culture that occasionally produce submerged spores (van Wezel et al. 2000a; van Wezel et al. 2000b). The large impact of SsgA on morphogenesis was underlined by microarray analysis, which showed that deletion of ssgA affects expression of an unprecedented large number of genes, with many hundreds of genes up- or downregulated by at least two-fold, including most developmental genes (e.g.bld, whi and ssg genes), as well as many genes involved in DNA segregation and topology (Noens et al. 2007). The remarkable upregulation of many of the known bld and whi genes, which are essential for aerial mycelium and spore formation, respectively, is best explained as a stress response to try and compensate for the absence of an important morphogen (i.c. ssgA). The same is probably true for the strong upregulation of ftsI (septum synthesis) and of divIVA (apical growth), whose functions relate to and may be assisted by SsgA. The genes that are by far the most strongly upregulated in ssgA mutants are the chaplin and rodlin genes. These genes encode hydrophobic proteins that form the water-repellent sheath of aerial hyphae that allows them to break through the soil surface (Claessen et al. 2003; Claessen et al. 2004; Elliot et al. 2003). Further research is required to explain the correlation between these developmental coat proteins and SsgA.
The SsgA-like proteins exclusively occur in morpologically complex actinomycetes
Five of the SALPs, namely SsgA, SsgB, SsgD, SsgE and SsgG, have orthologues in all streptomycetes analysed (Noens et al. (2005) and our unpublished hybridization data), with the exception of SsgG, absent from the S. avermitilis genome. Additionally, a few species-specific SALPs are typically found in all streptomycetes. SALPs can be divided into phylogenetic subfamilies, namely the SsgA branch, the SsgBG branch, the SsgDE branch and the species-specific SALPs, which include SsgC and SsgF (Fig. 1). Unexpectedly, the genome of S. griseus contains an additional three much larger proteins with an approximately 120 aa C-terminal SsgA-like domain, which contains the typical sequence identity to other SALPs of approximately 30–50%. Two of these (SGR7098t and SGR41t) are identical proteins of 654 aa, while the third (SGR128) is a 651 aa protein with an end-to end sequence identity of 67% to the other two (approximately 84% in the 120 aa SsgA-like domain). Apart from the SsgA-like domain the remainder of these proteins have no significant similarity to other proteins.
Distribution of SALPs across actinomycetes
Genome size (Mbp)
Number of SALPs
Cell morphology; development
Filamentous growth, spore chains on aerial hyphae
(project ID: 20085)
Filamentous growth, multilocular sporangia either terminally or intercalary
Frankia sp. CcI3
Frankia sp. EAN1pec
Filamentous growth, short spore chains on fragmented aerial hyphae
Slender floculles, non-spore forming
Cocci with polar flagella or symmetrical multi-cell clusters, non-spore forming. Ageing colonies form an extracellular polymer shell around individual colonies
Nocardioides sp. JS614
Single rods/cocci, non-spore forming
Filamentous growth, single spores borne on substrate mycelium
Filamentous growth, single spores borne on dichotomously branched sporophores
SsgB is most likely the SALP archetype
Analysis of the genetic locus of the single SALP-encoding genes of Thermobifida, Kineococcus, Nocardioides, Acidothermus and Salinispora showed that all resembled the gene organization around ssgB in Streptomyces. In fact, all SALP-containing actinomycetes have one ssg gene with a similar genetic locus to Streptomyces ssgB (BAT and GPvW, unpublished data), which is preceded by a homologue of SCO1540 (the gene directly upstream of S. coelicolorssgB) in most actinomycetes and in all genera a gene for tRNAval somewhat further upstream. A number of other genes are conserved in several genera, for example at least three additional tRNA genes are found in all except Salinispora, and a threonine-tRNA synthetase (SCO1531) is present in Streptomyces, Acidothermus, Kineococcus, Nocardioides and Thermobifida. Hence, gene syntheny evidence strongly suggests that all SALP-encoding genes have been derived from spread and/or gene duplication of ssgB in actinomycetes and that perhaps this gene has a universally conserved function in actinomycete morphogenesis. In a phylogenetic tree nearly all of the putative SsgB orthologues group together in the above mentioned SsgBG branch (Fig. 1), with the exception of the orthologues from the non-sporulating Acidothermus and Nocardioides. Whether these two SALP orthologues are still functional in these actinomycetes remains to be elucidated. More phylogenetic evidence for the importance of SsgB is provided by the fact that SsgB orthologues found in different species within a specific genus are almost completely conserved. This is true for the SsgBs from Streptomyces, from Frankia and from the salt-water actinomycete Salinispora. The orthologues from Streptomyces are identical except for aa position 150 (Gln or Thr), those from Salinispora differ only at aa position 137 (Asn or Ser), and in Frankia two orthologues are identical while the third has three aa changes (two conserved Ile/Val changes, and more importantly Ser or Ala at aa position 105). Furthermore, many nucleotide changes occur that do not lead to changes in the predicted proteins. This extraordinary conservation within genera perhaps reflects an inflexible co-evolution with an interaction partner.
ssgB (and to a lesser extent ssgA, which is not essential for sporulation on most mannitol-containing media) is one of the very few known whi genes that are essential for sporulation but do not encode a DNA binding protein. ssgB mutants produce long aseptate aerial hyphae in seemingly ‘immortal’ white colonies (Keijser et al. 2003), indicating a possible role for SsgB in the cessation of aerial growth prior to the onset of sporulation-specific cell division. Interestingly, many electron-dense granules were seen in hyphae of both ssgB mutants and PBP2 mutants, which perhaps reflect the accumulation of peptidoglycan subunits in both mutants (Noens et al. 2005). Importantly, the ssgB genes from Salinispora tropica and Saccharopolyspora erythraea restored sporulation to the otherwise non-sporulating ssgB mutant of S. coelicolor, even though end-to-end sequence homology to S. coelicolorssgB for both is only around 50% (BAT and GPvW, unpublished data). This strongly suggests that the SsgB proteins of Salinispora and Saccharopolyspora are indeed functional orthologues of SsgB. However, defects in septum placement, DNA segregation and spore size indicate that control of septum-site localization depends on (the amino acid sequence of) SsgB. Indeed, localization of SsgB to both tips of growing aerial hyphae and to immature sporulation septa suggests the protein functions in both tip growth and cell division (E.E. Noens, J. Willemse and GPvW, unpublished data).
Phylogenetically the closest relative of SsgB is SsgG (see Fig. 1), and this SALP plays a role specifically in the control of septum-site localization. ssgG mutants have a light grey phenotype, resulting from the production of significantly fewer spores than wild-type S. coelicolor. Closer inspection revealed that sporulation septa are regularly ‘skipped’, resulting in many spores of exactly two, three or even four times the normal size. This showed that SsgG is required to ensure that the divisome is localised to all division sites (Noens et al. 2005). Interestingly, despite the lack of cell division in the multiple-sized spores of the ssgG mutant the chromosomes were segregated normally, which clearly demonstrates that septum synthesis is not a prerequisite for DNA segregation in streptomycetes.
Streptomyces coelicolorssgC-F mutants produced an abundance of grey-pigmented spores after a few days of incubation, and ssgC mutants even hypersporulate. Microscopic analysis showed that aerial hyphae of the ssgC mutant produce very long ladders of septa, resulting in seemingly endless spore chains, while at the same time chromosome segregation in aerial hyphae was disturbed. In fact, ssgC mutants in many ways resemble strains over-producing SsgA and vice versa, and it was therefore proposed that SsgC may function as an antagonist of SsgA (Noens et al. 2005). Orthologues of ssgC are so far only found in streptomycetes that have a low expression of ssgA under conditions of normal growth and as a result produce large clumps in liquid-grown cultures, namely in S. coelicolor, in S. ambofaciens and, as determined by hybridization studies, in S. lividans (GPvW, unpublished). Mutation of ssgD pleiotropically affected integrity of the cell wall in aerial hyphae and spores, with many spores lacking the typical thick peptidoglycan layer, which rather resembles the wall of aerial hyphae. Finally, correct autolytic spore separation depends on SsgE and SsgF. ssgE mutants produce predominantly single spores indicating an enhanced or accelerated autolytic activity, while mutation of ssgF leads to incomplete detachment of spores, which remain attached by a thin peptidoglycan linkage, suggesting reduced or incorrect function of the secreted lytic transglycosylase SLT (Noens et al. 2005).
Transcriptional regulation of the ssg genes in streptomycetes
Microarray analysis of S. coelicolor M145 and MT1110 and promoter-probe experiments in S. coelicolor indicated that all ssg genes except ssgD are developmentally regulated (Noens et al. 2005; Noens et al. 2007). In contrast to the other ssg genes, ssgD is transcribed at a much higher level and its transcription is life cycle-independent (Traag et al. 2004), suggesting a role for SsgD at different stages of the life cycle. Furthermore, all ssg genes but ssgD are catabolically repressed by glucose. The transcription of ssgA has been studied extensively (see below). The only other ssg gene whose transcription has been studied in more detail is ssgB. Transcription of ssgB is directed from a single promoter 52 nt upstream of the most likely ATG start codon, resulting in a protein of 137 aa. Expression occurs in a life-cycle dependent manner and is strongly activated towards sporulation (Kormanec and Sevcikova 2002). In an E. coli two-plasmid system the ssgB promoter was found to be active in the presence of several members of the SigB-like sigma factor family (i.e. σB, σF and σH). SigB-like σ factors, of which the S. coelicolor genome encodes nine paralogues, are implicated in morphological differentiation and/or responses to different stresses (Kelemen et al. 2001; Lee et al. 2004). Expression of ssgB was unaffected in an sigF mutant of S. coelicolor, while in a sigH mutant no ssgB transcripts were detected. Furthermore, His-tagged σH initiated transcription from the ssgB promoter in in vitro run-off transcription assays (Kormanec and Sevcikova 2002). However, since a sigH mutant of S. coelicolor still produced some spores (Sevcikova et al. 2001) and a ssgB mutant does not, sigH cannot be solely responsible for transcription of ssgB.
Transcriptional and translational control of ssgA
There has long been a controversy about the position of the ssgA translational start site. While initially S. griseusssgA was considered to encode a 145 aa protein (Kawamoto and Ensign 1995), the presence of a more likely ribosome binding site (RBS) further downstream led to reassignment of the translational start to the third of three in-frame ATG codons, that lies 30 nucleotides (10 codons) downstream of the originally predicted start (Kawamoto et al. 1997). Alignment of ten ssgA ORFs and flanking regions obtained from the sequenced genomes of S. avermitilis, S. coelicolor, S. griseus, and S. scabies and from sequenced ssgA clones in our laboratory (namely from S. albus, S. clavuligerus, S. diastatochromogenes, S. fradiae, S. roseosporus, and S. venezuelae), showed that two alternative ATG start codons and the putative RBS sequences preceding them are fully conserved (Fig. 2), with precious few differences in the sequences between these possible start sites among the different ssgA orthologues. This suggests that perhaps both ATG codons could function as start codons. Indeed, we recently obtained evidence from Western blot analysis that both start codons may be used in vivo. Interestingly, only expression of the longer version resulted in soluble protein in E. coli (our unpublished data). This surprising new regulatory aspect of ssgA requires further analysis.
How do SALPs function?
As for how the SALPs themselves are recruited, we have strong evidence that SsgA and SsgB are recruited prior to Z-ring formation, and hence their localization is not dependent on the divisome (Joost Willemse, BT and GPvW, unpublished data). We recently identified mutants in hypothetical ORFs that have an almost identical phenotype as that of SsgG, producing large spores with multiple well-segregated chromosomes, One of the genes encodes a coiled coil protein, and hence may have a cytoskeletal function, tentatively pointing at a functional relationship between SsgG and the Streptomyces cytoskeleton. Live-cell imaging and biochemical methods such as FRET-FLIM microscopy and two-hybrid screening should elucidate the interaction partners for the SALPs.
Application of SsgA for improved industrial fermentations
Biotechnological relevance of actinomycetes in general, and streptomycetes in particular, is underlined by the fact that approximately 60% of all known antibiotics are produced by these organisms, as well as a large number of other biotechnologically interesting compounds and enzymes (Bennett 1998; Demain 1991; Hopwood et al. 1995). Productivity and hence the fermentation costs are strongly affected by the morphology of filamentous microorganisms. Models for mycelial growth have been worked out for filamentous fungi, and particularly for Penicillium chrysogenum (Krabben 1997; Nielsen et al. 1995; Trinchi 1971). Morphology is determined by the efficiency of germination, the tip extension rate, the degree of branching, and especially by fragmentation of the hyphae. When one considers that filamentous microorganisms can produce clumps of millimeters in diameter, the necessity to improve growth by reducing pellet formation becomes obvious. In mycelial pellets the bulk of the biomass is hidden at the inside of the clump, resulting in strongly reduced growth rates and inefficient transfer of nutrients and oxygen. Mycelial mats (large open structures) result in highly viscous broths, which is again undesirable from the production perspective. For streptomycetes the degree of hyphal fragmentation (the major determinant of mycelial clump size) is directly proportional to the frequency of septation (cross-wall formation) of the vegetative hyphae. In turn this directly depends on the ssgA expression level, and as a consequence the morphology of liquid-grown mycelia is dictated by SsgA (van Wezel et al. 2004; van Wezel et al. 2000a). The enhanced expression of SsgA was employed successfully to obtain fragmented and fast growth of pellet-forming species such as S. coelicolor, S. lividans and S. roseosporus in shake flasks as well as in small-scale fermentations (5–50 liter scale) (van Wezel et al. 2006). Using the secreted enzyme tyrosinase as a test enzyme, an increase in the yield of around 3-fold was achieved in significantly shorter fermentation time, underlining the promise of this technology (van Wezel et al. 2006; van Wezel and Vijgenboom 2003).
Conclusions and future perspectives
SALPs comprise a family of unique proteins, each playing an important role in the control of morphogenesis in streptomycetes. The presence of SsgB orthologues in morphologically very distinct actinomycetes which can, at least in part, complement each others functions raises a number of important points. First, since SsgB is essential for sporulation in streptomycetes, an important question that needs to be answered is what paralogous function SsgB has in actinomycetes that do not produce aerial hyphae (Salinispora and Thermobifida fusca) or even fail to produce any spores (Acidothermus cellulolyticus, Kineococcus radiotolerans, and Nocardioides sp. JS614). Second, if SALPs, as previously suggested (Noens et al. 2005; Noens et al. 2007) fulfill a recruiting role, the putative SsgB interacting partner is also likely to be conserved in the genomes of all SALP-containing actinomycetes. The genome sequences of Acidothermus cellulolyticus and Thermobifida fusca can prove very useful on the search for or elimination of such candidate partners, especially because of their considerably smaller sizes (around 2.4 and 3.6 Mbp, respectively), which allows searching for the proverbial needle in a considerably smaller haystack.
Multiple SALP-encoding genes have so far only been found in actinomycetes that produce aerial hyphae and complex multisporous structures, namely in streptomycetes, in sporangia-forming Frankia species and in Saccharopolyspora erythraea that produces short spore chains. From this one could conclude that SALPs are crucial for septum formation in actinomycetes, that at least two SALPs are required to produce more than one septum simultaneously, and that multiple (three or more) SALPs are required to coordinate the production of long spore chains or sporangia. It would be interesting to see if multiple SALPs can trigger the production of multisporous structures in actinomycetes that normally produce one spore or no spores at all. Conversely, combinations of SALP mutants in for example S. coelicolor may result in streptomycetes producing single spores or short spore chains.
Studies on the three-dimensional structure of SsgA or any other member of the SALP family will hopefully finally identify proteins with significant similarity to the SALPs, and it is to be expected that functional insights may be gleaned from such structural homologies. The overall sequence similarity is highly suggestive of an essential common aspect in their specialized functions. Further functional and localization studies on the SALPs should provide more detailed insight into the exact mode of action of the members of this still rather mysterious protein family.
We are grateful to Elke Noens and Joost Willemse for sharing unpublished data and for discussions, and to Keith Chater, Barend Kraal and Erik Vijgenboom for discussions. We would like to thank the participants at ISBA14 in August 2007 in Newcastle (UK) for stimulating discussions. This work was supported by grants from the Netherlands Society for Scientific Research (NWO) and from the Royal Netherlands Academy for Arts and Sciences (KNAW) to GPvW.
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