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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. SapB: function before form
  5. The ram gene cluster
  6. SapB is a lanthionine (Lan)-containing molecule
  7. SapB maturation
  8. Regulation of SapB production
  9. Sequence conservation and the role of the SapB-like morphogenetic surfactants
  10. References

Withstanding environmental adversity and seeking optimal conditions for reproduction are basic requirements for the survival of all organisms. Filamentous bacteria of the genus Streptomyces produce a remarkable cell type called the aerial hyphae that is central to its ability to meet both of these challenges. Recent advances have brought about a major shift in our understanding of the cell surface proteins that play important roles in the generation of these cells. Here we review our current understanding of one of these groups of proteins, the morphogenetic surfactants, with emphasis on the SapB protein of Streptomyces coelicolor.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. SapB: function before form
  5. The ram gene cluster
  6. SapB is a lanthionine (Lan)-containing molecule
  7. SapB maturation
  8. Regulation of SapB production
  9. Sequence conservation and the role of the SapB-like morphogenetic surfactants
  10. References

Growth of soil bacteria belonging to the genus Streptomyces takes place in the context of a defined life cycle that includes the generation of differentiated cell types, the production of antibiotics, sporulation and a mechanism of programmed cell death (Miguélez et al., 2000; Chater, 2001; Challis and Hopwood, 2003). This life cycle begins with spore germination and the propagation of vegetative cells called substrate hyphae. Unlike the rod-shaped, planktonic cells of most bacterial species, the substrate hyphae are filamentous and grow by branching and extension of hyphal tips into the substrate where they form a dense mycelium (Flardh, 2003) (Fig. 1A). These cells are non-motile and therefore at risk of starvation once local sources of nutrients are depleted. Streptomyces colonies are able to withstand this challenge by producing a second filamentous cell type, the aerial hyphae that grow up from the colony surface, at the expense of substrate hyphae lysis. The aerial hyphae differentiate into spores (Fig. 1B); it is generally assumed that, in nature, aerial growth assures maximal dispersal of spores to new sources of nutrients.

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Figure 1. Structure of Streptomyces cell types. A. Substrate mycelium. B. Aerial mycelium.

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Genetic dissection of morphogenesis in the model species Streptomyces coelicolor has resulted in the identification of genes whose products are important for normal morphological differentiation. Wild-type colonies acquire a white, fuzzy appearance due to the upwardly growing aerial mycelium and, with time, this layer of cells turns grey due to a pigment deposited in the cell wall of mature spores (Chater, 2001). Mutations in the bld (bald) genes block or greatly impair the formation of the aerial mycelium and therefore confer a smooth, ‘hairless’ phenotype to colonies (Kelemen and Buttner, 1998; Chater and Horinouchi, 2003). Many of the bld genes have been cloned, and in most cases their products serve regulatory functions. The whi (white) genes are required for the completion of spore maturation but not for the formation of the aerial mycelium; whi mutants therefore form a white fuzzy aerial mycelium that fails to turn grey (Chater, 2001).

The cues that trigger the formation of the aerial mycelium are not well understood. However, as is the case for morphogenetic adaptation in other bacteria (Pottathil and Lazzazzera, 2003; Kaiser, 2004), this process is believed to involve the sensing of nutrient depletion and intercellular signalling (Kelemen and Buttner, 1998). For instance, the γ-butyrolactone known as A-factor triggers a well-characterized pathway leading to morphogenesis and streptomycin biosynthesis in Streptomyces griseus (Hara et al., 1983; Ohnishi et al., 1999; Yamazaki et al., 2003) and is the subject of several recent reviews (Horinouchi et al., 2001; Ohnishi et al., 2002; Horinouchi, 2002; Chater and Horinouchi, 2003). In S. coelicolor, multiple signals have been implicated in morphogenesis (Willey et al., 1993) including at least one peptide-signalling mechanism (Nodwell et al., 1996; Nodwell and Losick, 1998). cAMP and a number of metabolic genes are also involved in morphogenesis in S. coelicolor (Viollier et al., 2001a,b; Sprusansky et al., 2003; Gehring et al., 2004).

Major insights into the aerial hyphae have come about with the recent discoveries that cell surface proteins called chaplins and rodlins are integral to the formation and structure of this cell type (Claessen et al., 2002; 2003; 2004; Elliot et al., 2003). Together these proteins appear to form a hydrophobic sheath on the cell surface; and mutants defective in multiple chaplins are unable to produce aerial hyphae under many growth conditions. Two recent reviews (Elliot and Talbot, 2004; Gebbink et al., 2005) describe these proteins and illuminate interesting insights by comparing the streptomycetes with the fungi.

In contrast to the recently discovered chaplins and rodlins, a third class of cell surface protein, exemplified by the S. coelicolor protein SapB was first described in 1988 (Guijarro et al., 1988) as one of five so-called spore-associated proteins. This molecule is produced and secreted during growth on rich media (although not on poor carbon sources such as mannitol) and appears to be important for morphogenesis under these conditions. The most compelling evidence for this is the observation that application of purified SapB peptide to bld mutants restores their ability to form aerial filaments (Willey et al., 1991). The recent demonstration that SapB is derived from the product of the developmental gene ramS and the resolution of its structure represent the culmination of 15 years of research in several laboratories. Along with a more complete understanding of the hydrophobic sheath, these advances illuminate the mechanical process involved in streptomycete sporulation and raise important questions. In this review we focus on the current understanding of SapB and its relatives and frame those questions that strike us as most important for further investigation.

SapB: function before form

  1. Top of page
  2. Summary
  3. Introduction
  4. SapB: function before form
  5. The ram gene cluster
  6. SapB is a lanthionine (Lan)-containing molecule
  7. SapB maturation
  8. Regulation of SapB production
  9. Sequence conservation and the role of the SapB-like morphogenetic surfactants
  10. References

Not long after its discovery, SapB was shown to have the intriguing property of restoring the ability to form aerial hyphae to bld mutants, albeit without subsequent spore formation. Consistent with this, SapB production was found to depend on all bld genes (Willey et al., 1991) except bldM and bldN (J. M. Willey, unpublished) and to be spatially confined to aerial hyphae and spores (Willey et al., 1991). The production of SapB by bldM and bldN mutants is consistent with the fact that both eventually form aerial hyphae after a considerable delay, and morphogenesis is correlated temporally with SapB production. Indeed, the first mutant alleles of both these genes were identified as whi mutants (Ryding et al., 1999; Molle and Buttner, 2000; Gehring et al., 2004).

Initially SapB was reported to consist of 18 amino acids and an additional unknown moiety with a total molecular mass of 2027 Da. Edman degradation suggested the N-terminal sequence T-X-G-X-R followed by a block that could not be overcome by conventional sequencing methods. Because SapB is resolutely insoluble and protease resistant, it is not amenable to structural analysis by NMR or mass spectrometry (Willey et al., 1991; Kodani et al., 2004).

Tillotson and Willey hypothesized that the role of SapB might be similar to that of the fungal hydrophobins (Tillotson et al., 1998), which similarly bring about aerial growth of spore-forming filamentous cells and fruiting bodies (Wessels, 1997). Hydrophobins self-assemble at the interface between the air and the aqueous surface of fungal colonies. Their surfactant activity reduces the surface tension at this interface, which enables the upward growth of aerial structures (Wösten and Wessels, 1997). Biophysical analysis demonstrates that SapB also has strong surfactant activity. The functional similarity between SapB and a fungal hydrophobin was further demonstrated by the capacity of the SC3 hydrophobin from Schizophyllum commune to restore aerial mycelium formation to S. coelicolor bld mutants (Tillotson et al., 1998). Strikingly, a developmental mutant of the fungus is rescued by streptofactin, a SapB-like peptide produced by Streptomyces tendae (Wösten et al., 1999). Given that SC3 and the streptomycete surfactants have no common structural features, we regard this as an extraordinary example of convergent evolution. Note, however, that there is some specificity to surfactant action: application of the Bacillus subtilis surfactants surfactin and fengicin and the Pseudomonas aeruginosa product viscosin have no effect on bld mutants. (Richter et al., 1998).

While exogenously applied SapB and SC3 stimulate S. coelicolor bld mutants to produce an aerial mycelium, it is significant that these strains do not go on to sporulate. Instead, short branching vegetative hyphae are simply released from the colony surface to stand erect. This is consistent with a role for SapB as a biosurfactant: it can release the hyphae of bld mutants from the confines of the aqueous colony surface, but cannot trigger their differentiation into true aerial hyphae with the capacity to sporulate (Tillotson et al., 1998). While this supports the hypothesis that SapB serves a primarily mechanical role, the possibility that it also serves as a signal could not be eliminated.

The ram gene cluster

  1. Top of page
  2. Summary
  3. Introduction
  4. SapB: function before form
  5. The ram gene cluster
  6. SapB is a lanthionine (Lan)-containing molecule
  7. SapB maturation
  8. Regulation of SapB production
  9. Sequence conservation and the role of the SapB-like morphogenetic surfactants
  10. References

SapB was the first, and for many years the only, developmental structural protein known to be involved in the formation of the aerial mycelium (Willey et al., 1991); most of the bld gene products are regulatory proteins. One early hypothesis that SapB might be produced non-ribosomally (Willey et al., 1993), was ruled out because the S. coelicolor genome (Bentley et al., 2002) does not encode a peptide synthetase gene cluster that could be implicated in SapB biosynthesis. Paradoxically, careful scrutiny of the entire genome failed to reveal an obvious SapB precursor-encoding gene for the peptide (G. Chandra and J. M. Willey, unpublished).

Ironically, not long after the first reports of the role of SapB in morphogenesis, part of the gene cluster required for production of SapB, the ram cluster, was unwittingly identified (Ma and Kendall, 1994) and the boundaries of the entire ram gene cluster were known by 2002 (Keijser et al., 2000; 2002; Nguyen et al., 2002; O’Connor et al., 2002). Several investigators (including the authors of this review) considered the possibility that the ramS gene, which is embedded in this cluster, might encode a SapB precursor but the biochemical data available at that time did not support this hypothesis (Chater and Horinouchi, 2003).

The ram gene cluster in S. coelicolor (Ma and Kendall, 1994) is orthologous to, and organized in the same manner as the S. griseus amf gene cluster that had been identified by Ueda et al. in 1993. Overexpression of the amf genes (for aerial mycelium formation) in S. griseus restores aerial hyphae formation to a mutant deficient in A-factor (Ueda et al., 1993). Similarly, in S. coelicolor, the ram genes (for rapid aerial mycelium) were discovered in a screen for genes that stimulated precocious formation of aerial hyphae when present at higher than normal copy number (Ma and Kendall, 1994). The ram gene cluster consists of the genes, ramC, ramS, ramA and ramB, and on the opposite strand, the monocistronic gene ramR(Fig. 2); the S. griseus orthologues are, respectively, amfT, amfS, amfB, amfA and amfR (Ueda et al., 1993; 2002). RamR is a response regulator in the NarL/FixJ subgroup and ramAB encodes the components of a heterodimeric ABC transporter (Ma and Kendall, 1994). RamC was first described as a protein of unknown function having sequence similarity to serine/threonine kinases (Hudson et al., 2002; Keijser et al., 2002; Nguyen et al., 2002); its orthologue in S. griseus is similarly important for morphogenesis (Ueda et al., 2005). RamC is a membrane-associated protein, 904 amino acid residues in length, and has several distinguishable domains. Sequence motifs within the N-terminal 440 amino acid residues are related to those found in ser/thr kinases, including those that typically constitute the catalytic centre. Mutational analysis of predicted catalytic residues in this region supports the prediction that these kinase-like motifs are functionally relevant (Hudson et al., 2002). A domain C-terminal to this kinase-like domain is required for homodimerization (Hudson and Nodwell, 2004). This ensemble of features along with its membrane association gave rise to the misconception that RamC might be a receptor involved in morphogenetic signalling (Hudson et al., 2002).

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Figure 2. The ram gene cluster.

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The small open reading frame ramS encodes a 42 amino acid peptide, originally of unknown function (O’Connor et al., 2002; Keijser et al., 2002; Nguyen et al., 2002), which we now know is the SapB precursor (Kodani et al., 2004). The demonstration that an octapeptide derived from the C-terminal sequence of AmfS could restore formation of an aerial mycelium in S. griseus was an important clue that the action of this gene product is extracellular (Ueda et al., 2002). Recently the ability of purified SapB to restore morphogenesis to an S. coelicolor ramS null mutant was also demonstrated (Kodani et al., 2005). Interestingly, the extracellular complementation observed in both cases (Ueda et al., 2002; Kodani et al., 2005) results in the formation of a sporulating aerial mycelium, rather than the release of undifferentiated vegetative hyphae from the colony surface, as is the case in peptide rescue of bld mutants (Tillotson et al., 1998).

SapB is a lanthionine (Lan)-containing molecule

  1. Top of page
  2. Summary
  3. Introduction
  4. SapB: function before form
  5. The ram gene cluster
  6. SapB is a lanthionine (Lan)-containing molecule
  7. SapB maturation
  8. Regulation of SapB production
  9. Sequence conservation and the role of the SapB-like morphogenetic surfactants
  10. References

The most important clue to understanding the role of RamC was also a key observation in resolving the SapB structure and showing that ramS encodes its precursor peptide. Specifically, it was discovered that the C-terminal domain of RamC is similar in sequence to enzymes that catalyse the formation of the Lan bridges (Kodani et al., 2004), the defining structural features of antimicrobial peptides known as lantibiotics (Sahl and Bierbaum, 1998). This suggested that the role of RamC might be to modify the 42 amino acid RamS peptide and this in turn led to a crucial opportunity to reconsider the possibility that RamS might be converted post-translationally into SapB. Most importantly, the hypothesis that SapB might contain post-translational modifications similar to those found in lantibiotic peptides suggested new biochemical approaches for determining its structure. This led to the rapid discovery that the peptide is indeed derived from RamS and that it bears the structural features that characterize the Lan-containing antibiotics, lantibiotics (Kodani et al., 2004).

Lantibiotics, sometimes called class I bacteriocins, are ribosomally translated peptides produced by Gram-positive bacteria that have antimicrobial activity against closely related bacteria. Typically, they are produced as 25–50 amino acid precursors peptides that are post-translationally modified in three steps (illustrated in terms of SapB in Fig. 3A). The first step is the dehydration of select serine and threonine residue side chains, giving rise to didehydroalanine (Dha) and didehydrobutyrine (Dhb) residues, respectively (Fig. 3B): RamS is subject to serine but not threonine dehydration (although the protein does possess four threonine residues). These dehydrated residues bear double bonds between Cα and Cβ. Dehydration is critical for the next step, in which the sulphydral groups in cysteines react with the dehydrated residues at the Cβ to form thioether cross-links (Fig. 3B). These acid-stable bonds are referred to as Lan bridges when made between Dha and Cys residues, and 3-methyl lanthionine (MeLan) bridges when spanning Dhb and Cys residues. Such intramolecular thioether bond formation confers structural rigidity and protease resistance to the resulting molecules. Because the number of Cys residues is often less than the number of dehydrated residues, many lantibiotics contain unreacted Dha and Dhb residues. Finally, the peptide is exported and a designated peptidase cleaves the leader peptide (Fig. 3A) to release the mature peptide (Sahl and Bierbaum, 1998). The leader in preSapB corresponds to the first 21 amino acid residues in RamS.

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Figure 3. Conversion of RamS into SapB. A. SapB maturation is believed to occur in three stages. First, serine residues (pink balls) are dehydrated giving rise to 2,3 didehydroalanine (Dha) residues (red balls). Next, nucleophillic attacks by the sulphydrals of cysteine residue (green balls) at positions 31 and 41 on the β carbon of Dha at positions 24 and 34 gives rise to the intramolecular lanthionine cross-links, each of which consists of two alanine residues (blue balls) connected by a thioether linkage. We refer to this molecule, which bears the putative RamC-mediated modifications but still has the leader, as preSapB. B. Serine dehydration is an ATP hydrolysis-dependent process that liberates water, ADP and Pi and reduces the mass of the RamS precursor by 18 Da. Lanthionine cyclization gives rise to meso-lanthionine residues and has no effect on the mass of the molecule.

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The complexity of these post-translational modifications complicates lantibiotic structural analysis. Dha and Dhb residues block Edman degradation and, while these residues can be modified to remove the sequencing block, the resulting amino acid sequence information is rarely comprehensive (Meyer et al., 1994; Martin et al., 2004). Furthermore, amino acid analysis of mature or chemically modified lantibiotics can be misleading because modified residues are lost or unrecognized resulting in an underestimate in the number and variety of constituent amino acids. These properties account for the inaccuracy of the originally published SapB amino acid composition (Willey et al., 1991) and subsequent challenges in determining the structure of the molecule (Kodani et al., 2004).

The proposed structure of SapB (Kodani et al., 2004) is shown in Fig. 4A. The deduced Lan bridging pattern yields two hydrophobic cyclic structures of eight residues each that are flanked and connected by short, relatively hydrophilic linear stretches (TG, GD and N). The loops are symmetrical in the placement of two Dha and other hydrophobic residues. As shown in Fig. 4B, the modelled SapB structure agrees with previously obtained experimental data suggesting that SapB is amphiphilic and capable of self-assembly (Tillotson et al., 1998), properties required for biosurfactant activity. As inferred by molecular modelling, the hydrophilic edge of SapB would be easily solvated, whereas the hydrophobic face would reach its lowest energy state by escaping the aqueous environment, orienting away from the cell surface. Such an alignment might also favour self-assembly of SapB through hydrophobic interaction between surface-exposed loops in adjacent SapB monomers (Kodani et al., 2004). Indeed, a similar model has been proposed for the multimerization of the fungal surfactant HFBII (Hakanpääet al., 2004).

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Figure 4. Proposed SapB structure. A. Primary amino acid sequence of SapB. Note that following dehydration of serine to Dha and lanthionine bridge formation by a nearby cysteine residue, both the Dha residue and Cys residues involved in thioether formation are, by convention, denoted as alanine residues. B. Spacefilling model of SapB. The basic N-terminal group and Arg4 side chain are coloured blue and the acidic C-terminal group and Asp12 side chain are coloured red.

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Two classes of lantibiotics, type A (including types AI and AII) and type B have been recognized to date, however, our new understanding of SapB suggests that the structural and functional diversity of these molecules has been underestimated. Type A lantibiotics such as nisin, subtilin, and epidermin are basic peptides that are elongated and relatively flexible with well separated Lan and MeLan bridges. Interestingly, type A lantibiotics are also amphiphilic; in this case the separation of positively and negatively charged side chains on opposite faces of the peptide facilitates the formation of pores in the plasma membrane of target microbes (Brötz and Sahl, 2000). Type AI lantibiotics typically have leader sequences that exhibit a conserved FNLD motif that is thought to be important in peptide processing (Chatterjee et al., 2005a). Peptide maturation of type AII lantibiotics is catalysed by two distinct enzymes including LanB and LanC, a dehydratase and cyclase, respectively, which form the thioether cross-links (Lan is the generic locus symbol for homologous lantibiotic genes). In the case of nisin (Siegers et al., 1996) and subtilin (Kiesau et al., 1997), the LanB and LanC proteins are localized to the membrane in association with a LanT ABC transporter. A lantibiotic-specific peptidase, LanP, removes the leader peptide (Table 1). In contrast, type B lantibiotics such as mersacidin and cinnemycin are more globular peptides because their Lan and MeLan bridges overlap such that one might be within the other. These peptides thus have little conformational freedom, especially those featuring head to tail intramolecular bridging (Kaiser et al., 1998). They are either neutral or bear a single positive charge. Type B peptides block peptidoglycan biosynthesis by binding to the precursor molecule lipid II to prevent transglycosylation. The leader sequences of both type AII and type B are usually characterized by two glycine residues (or more rarely GS, GA, or AA) at the −2 and −1 positions and a conserved sequence of hydrophobic residues between −15 and −11 that target the prepeptide to the modification enzyme (Altena et al., 2000; Pag and Sahl, 2002). Unlike type AI peptide maturation, type AII and type B lantibiotics are processed by multifunctional enzymes. LanM-type enzymes exhibit both dehydratase and cyclase activities, and LanT enzymes export the peptide and cleave the leader (Chatterjee et al., 2005a).

Table 1.  Summary of enzymes involved in the maturation of lantibiotics.
Lantibiotic enzymeFunctionExampleReference
LanAPrepropeptideNisA, CinA, MrsASiegers et al. (1996) Widdick et al. (2003) Altena et al. (2000)
LanR,KTwo-component signal transduction regulatory proteinsSpaR,K; MrsR2,K2; CinR,K; SalK,RKiesau et al. (1997) Altena et al. (2000) Widdick et al. (2003) Upton et al. (2001)
Type A lantiobiotics
 LanBDehydrate Ser and Thr residues to Dha and Dhb respectivelyNisB, SpaBSiegers et al. (1996) Kiesau et al. (1997)
 LanCForm thioether link between Cys and Dha, Dhb residuesNisC, SpaC 
 LanTExport modified peptide  
 LanPCleave leader sequence  
Type B lantibiotics
 LanMDehydrate Ser and Thr residues and form thioether linkagesCinM, MrsMWiddick et al. (2003) Altena et al. (2000)
 LanTExport modified peptide and cleave leader sequenceMrsM, SalTAltena et al. (2000) Upton et al. (2001)
SapB-associated proteins
 RamSPrepropeptide Kodani et al. (2004)
 RamCC-terminal sequence similarity to cylase domain of CinM, MrsM; Kodani et al. (2004)
N-terminal sequence similarity to ser/thr kinases Hudson et al. (2002)
 RamABComponents of an ABC transporter (no similarity to peptidases) Ma and Kendall (1994)
 RamRResponse regulator Ma and Kendall (1994) Nguyen et al. (2002)

SapB does not fit easily into either of these groups. Like the type A peptides, it is clearly amphiphilic, but is uncharged at neutral pH, reminiscent of the type B group. Its lack of extensive intramolecular cross-linking may render it relatively flexible, like the type A peptides (the glycine residue between the two loops may act as a flexible hinge, but this is by no means certain). Examination of the leader sequence fails to clarify this distinction. Alignment of the RamS/AmfS leader sequences from S. coelicolor, S. griseus, Streptomyces avermitilis and S. scabies (which possesses two ram gene clusters) reveals three conserved sequence elements L(L/F)DLQ (M/L)(E/D) and (E/D)(E/D) between −19 and −7 (Fig. 5). While RamS (S. coelicolor), AmfS (S. griseus) and RamS1 (S. scabies) lack double glycine or alanine residues at positions −1 and −2, RamS2 (S. scabies) and AmfS (S. avermitilis;Omura et al., 2001) clearly possess this processing signal. The possibility that the conserved elements in the leader might be required for targeting the peptide to the site of modification is currently being tested. A final, significant difference between SapB and the lantibiotics is that SapB appears to lack antimicrobial activity (Kodani et al., 2004). For these reasons it should be regarded as a lantibiotic-like or Lan-containing peptide, rather than a true lantibiotic.

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Figure 5. Alignment of RamS (S. coelicolor), AmfS (S. griseus), AmfS (S. avermitilis), RamS1 and 2 (S. scabies).

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SapB maturation

  1. Top of page
  2. Summary
  3. Introduction
  4. SapB: function before form
  5. The ram gene cluster
  6. SapB is a lanthionine (Lan)-containing molecule
  7. SapB maturation
  8. Regulation of SapB production
  9. Sequence conservation and the role of the SapB-like morphogenetic surfactants
  10. References

Re-examination of the ram gene cluster in light of the identity of SapB yields some important insights. The genes encoding lantibiotic processing enzymes are generally clustered in one or two operons encoding all of the proteins required for production, maturation, export, and leader processing of the peptide. In addition, most lantibiotic gene clusters also feature genes that encode polypeptides required for self-immunity, a sensor kinase and its associated response regulator (Altena et al., 2000; McAuliffe et al., 2000; Stein, 2005). Recall that the ram gene cluster (Fig. 2) encodes the SapB precursor (ramS), a single putative LanM-like modifying enzyme likely having multiple activities (ramC), a putative SapB exporter (ramA and ramB) and an orphan response regulator protein (ramR).

There are conflicting reports regarding the importance of RamA and RamB. A disruption of the ramB gene was shown to impair formation of aerial hyphae in Streptomyces lividans (Ma and Kendall, 1994) but subsequent insertion mutations in both ramA and ramB had little or no effect on morphogenesis in S. coelicolor (Nguyen et al., 2002), as was the case for an amfAB deletion in S. griseus (Ueda et al., 2002). In contrast, a mutant bearing a partial deletion of both amfA and amfB in S. griseus was unable to extracellularly complement the amfS null strain. In the wild type, this extracellular complementation is thought to result from the diffusion of AmfS, the S. griseus SapB orthologue (Ueda et al., 2002). Also consistent with a role for these genes in SapB function, is the report that the bldJ and citA (which has a Bld phenotype) mutants could not be genetically complemented by ramCSA overexpression; rather the entire ramCSAB cluster was required (Nguyen et al., 2002). Determination of the importance of ramA and ramB therefore awaits definitive analysis but we suggest that there may be an additional ABC transporter that can export SapB, although perhaps at reduced efficiency, in the absence of either the Ram or the Amf transporter. Consistent with this, the S. coelicolor genome encodes at least 27 ABC transporters, many of which are predicted to be exporters (Bentley et al., 2002). In any event, it is clear that neither RamA nor RamB bears sequence similarity to known lantibiotic leader peptidases of either the LanP or LanT family. At present there are no strong candidates for the RamS leader peptidase among the numerous proteolytic enzymes encoded in the S. coelicolor genome.

So far, the strongest evidence that RamC is the likely RamS modifying enzyme is the sequence similarity between the C-terminal domains of RamC and the LanM type enzymes CinM and MrsM (Kodani et al., 2004). We therefore suggest that the role of the C-terminus of RamC is to generate the Lan bridges between residues 3 and 10, and 13 and 20 of mature SapB. Like some other lantibiotic processing enzymes (Siegers et al., 1996; Kiesau et al., 1997), RamC is predicted to form a dimer (Hudson and Nodwell, 2004).

Less obvious is the role of the N-terminal domain of RamC. The sequence between residues 250 and 422 exhibits similarity to the catalytic domains of ser/thr kinases. Within this region are residues and motifs predicted to coordinate Mg++ and catalyse phosphorylation of target hydroxyl groups. While in vitro phosphorylation of a specific or model substrate has not been demonstrated for RamC, site directed mutagenesis of candidate catalytic residues generates mutants phenotypically similar to S. coelicolor strains lacking the entire ramC gene (Hudson et al., 2002). Between the kinase-like domain and the N-terminus of the protein is an additional domain of unknown function. Our favoured view is that this domain and the kinase-like domain bring about serine dehydration through a two-step mechanism. Because the hydroxyl group of serine is very poor leaving group, a reasonable hypothesis is that it is targeted for phosphorylation by the kinase-like domain and a subsequent reaction, catalysed by the N-terminal domain brings about the formation of the double bond between the α and β carbons. Confoundingly, the 422 residue N-terminal domain of RamC does not exhibit obvious similarity to either the N-terminal domains of the LanM-like enzymes or to LanB-like enzymes both of which catalyse ser/thr dehydration: it is possible, however, that the three-dimensional folds and catalytic mechanisms of RamC and these enzymes are similar. This would be consistent with the observation that in vitro serine and threonine dehydration during the maturation of lacticin 481 is dependent on ATP hydrolysis (Xie et al., 2004). In support of our two-step reaction hypothesis, it has recently been demonstrated that LctM, an enzyme involved in lacticin 481 maturation, can dehydrate target residues in the precursor peptide if they are phosphorylated. Under these circumstances, ADP can be substituted for ATP (Chatterjee et al., 2005b).

RamC is associated with the S. coelicolor membrane (Hudson et al., 2002), as is the case for some lantibiotic modification enzymes such as those involved in the maturation of nisin (Siegers et al., 1996) and subtilin (Kiesau et al., 1997). We have since recognized that RamC domains originally thought to be transmembranous (Hudson et al., 2002) fall within the predicted Lan cyclase and dimerization domains. Thus, our current hypothesis is that RamC is held at the membrane indirectly, perhaps via a protein-protein interaction with the SapB exporter as suggested for the nisin (Siegers et al., 1996) and subtilin (Kiesau et al., 1997) synthetase complexes (Fig. 6). By analogy with membrane-associated lantibiotic synthetase complexes, we are currently testing the prediction that RamC and RamAB form a SapB maturation complex localized to the plasma membrane.

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Figure 6. Model for SapB maturation and export complex. The unmodified ramS gene product (RamS) is modified by dehydration and thioether formation, presumably catalysed by RamC, which is hypothesized to have LanM-like bifunctional enzyme activity. RamC is known to function as a dimer (Hudson and Nodwell, 2004) and is associated with the membrane (Hudson et al., 2002). The modified product, PreSapB, is exported and the leader sequence is cleaved to yield mature SapB. Currently there are no good candidates for the leader peptidase, so it is unclear if processing occurs before, during or following export.

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Regulation of SapB production

  1. Top of page
  2. Summary
  3. Introduction
  4. SapB: function before form
  5. The ram gene cluster
  6. SapB is a lanthionine (Lan)-containing molecule
  7. SapB maturation
  8. Regulation of SapB production
  9. Sequence conservation and the role of the SapB-like morphogenetic surfactants
  10. References

The regulation of ram gene expression is of considerable interest because it links the erection of aerial hyphae to earlier regulatory events, presumably including some mediated by bld gene products. ramCSAB transcript (Keijser et al., 2002; Nguyen et al., 2002) and the RamC protein (O’Connor et al., 2002) can be detected within 24–36 h of spore germination, consistent with the production of SapB at the onset of the aerial hyphae formation (Willey et al., 1991). There is a single promoter upstream of ramC and additional promoters may be upstream of ramS and/or ramA (Keijser et al., 2002). The promoter upstream of ramC has been shown to interact with RamR and this interaction is essential for the initiation of transcription of the ramCSAB operon (Keijser et al., 2002; Nguyen et al., 2002; O’Connor et al., 2002). Expression of the ram genes and production of SapB depends on all of the known bld genes except bldM and bldN (O’Connor et al., 2002), yet little is known about the regulatory cascade mediated by the products of these genes.

The promoter upstream of ramR is activated within 24–36 h of spore germination (depending on growth conditions) and soon reaches a stable, steady state that persists for several days. In contrast, the ramC promoter exhibits a sharp peak of activity during the first 2 days of colony growth but then falls silent. Because RamR is a response regulator, one might propose that this discrepancy is due to modulation of RamR activity by phosphorylation (Nguyen et al., 2002) however, several recent observations suggest that RamR may not have a cognate sensor kinase. The unphosphorylated protein is a dimer that interacts tightly and cooperatively with the ramC promoter. Moreover, RamR cannot be phosphorylated with the small molecule phosphodonors acetyl phosphate, carbamoyl phosphate or phosphoramadate, suggesting that its receiver domain might lack phosphotransferase activity (O’Connor and Nodwell, 2005). Consistent with this view, of the five known ramR-containing ram gene clusters (S. coelicolor, S. griseus, S. lividans, S. avermitilis and S. scabies), none encodes a cognate RamR sensor kinase. Nonetheless, mutations that alter the predicted site of phosphorylation in RamR impair its function (Nguyen et al., 2002), but this might be explained by the fact that the aspartate side chain is important for both dimer formation and DNA-binding (O’Connor and Nodwell, 2005). Interestingly, the response regulator that governs production of the lantibiotic epidermin by Staphylococcus epidermidis lacks the conserved N-terminal region and the essential aspartate residue that is phosphorylated by a cognate sensor kinase in other two-component signal transduction systems (Peschel et al., 1993). Four other streptomycetes ‘orphan’ response regulators, DnrN, RedZ, WhiI and BldM may also function without phosphorylation (Furuya and Hutchinson, 1996; Guthrie et al., 1998; Ainsa et al., 1999; Molle and Buttner, 2000).

The ram genes exhibit spatial regulation in addition to temporal regulation. The ramC promoter is dormant in spores, activated in the substrate hyphae and pre-septational aerial hyphae but is then shut off in the post-septational aerial hyphae (O’Connor et al., 2002). As is the case for the sharp peak in the activity of this promoter, this pattern raises questions that cannot be answered at present. Most important is the mechanism by which operon transcription shuts off following sporulation septation. While it appears that RamR is not phosphorylated as part of the mechanism that activates the promoter upstream of ramC (O’Connor and Nodwell, 2005), it cannot be ruled out that a phosphorylation dependent mechanism is involved in the de-activation of the ram genes in the post-septational aerial hyphae. Interestingly, while transcription of the amfTSBA operon is similarly activated by amfR, it is negatively regulated by the bldD gene product (Ueda et al., 2005); however, the region upstream of ramCSAB lacks an obvious BldD binding site. Thus, other possibilities include proteolytic degradation of RamR or the modulation of other, as yet unidentified regulators involved in ram gene expression. For example, at present it is not known which of the 67 S. coelicolor sigma factors is required for expression of the ramC promoter. Furthermore, the links, direct and indirect, between expression of ramR and the bld genes remain unknown.

Sequence conservation and the role of the SapB-like morphogenetic surfactants

  1. Top of page
  2. Summary
  3. Introduction
  4. SapB: function before form
  5. The ram gene cluster
  6. SapB is a lanthionine (Lan)-containing molecule
  7. SapB maturation
  8. Regulation of SapB production
  9. Sequence conservation and the role of the SapB-like morphogenetic surfactants
  10. References

Alignment of RamS to its orthologues in other streptomycetes (Fig. 5) shows strong conservation of the serine and cysteine residues involved in Lan bridging and serine residues that are present as unreacted Dha in mature SapB. This suggests that the dicyclic structure and overall hydrophobic character that characterizes SapB are also found in these four SapB-like peptides. There are, however, at least two known peptides with the capacity to promote aerial growth that differ markedly in structure from SapB. A most extraordinary departure from the SapB paradigm is the peptide ‘goadsporin’ produced by Streptomyces sp. TP-A0584 (Onaka et al., 2001). This peptide stimulates morphogenesis in S. lividans TK23 and a number of other streptomycetes but, unlike SapB, also stimulates production of pigmented antibiotics. In addition, it appears to have weak antimicrobial activity. Strikingly, the peptide is reported to be a linear oligopeptide that lacks lantibiotic modifications but possesses four oxazole and two thiazole residues (Igarashi et al., 2001; Onaka et al., 2001). In this regard, goadsporin is more similar to the microcin group of antimicrobial peptides produced by Gram-negative bacteria (Kaiser et al., 1998). It is not clear whether goadsporin acts as a surfactant like SapB and the genes for goadsporin biosynthesis have yet to be identified; these are obviously very high priorities for future research.

The Lan-containing peptide SapT was recently isolated from S. tendae. SapT has the same biological properties as SapB; i.e. it can drive aerial hyphae formation of S. coelicolor bld mutants. While SapT is functionally similar to SapB and is a Lan-containing peptide, its structure differs markedly from SapB. The 21 residues of SapT form four loops, three of which result from MeLan bridges and one by a Lan bridge (Kodani et al., 2005). Despite these structural dissimilarities, molecular modelling of both SapB and SapT predicts the macrocyclic rings limit conformational freedom and, importantly, prevent the hydrophobic side chains from forming a hydrophobic core. This confers to the peptides distinctly amphiphilic structures where the polar backbone atoms might form extensive hydrogen bonding contacts with the surrounding water molecules, while the hydrophobic side chains project out of the water layer. Thus, these molecules, which exhibit cross-species function (Kodani et al., 2005) have conserved the key amphiphilicity that allows them to function as biosurfactants.

Despite the differences in structure, the application of either SapB or SapT to a S. coelicolor ramS null mutant restores the formation of a sporulating aerial mycelium (Kodani et al., 2005). Indeed the fungal hydrophobin SC3, which bears no structural similarity to any of the streptomycete surfactants, also induces both the efficient formation of an aerial mycelium and sporulation in a ramS mutant (Kodani et al., 2005). In our view, these observations are the strongest evidence that SapB and its relatives play a strictly mechanical role in morphogenesis and that they have no signalling function. It seems unlikely, for example, that there is a SapB receptor, at least in the traditional sense of the word. We suggest therefore that the role of the surfactants is simply to release nascent aerial hyphae from the confines of the substrate mycelium. This exposes them to a myriad of new stimuli that may then trigger the expression of the sporulation genes. Identifying these cues seems like an especially important goal.

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  1. Top of page
  2. Summary
  3. Introduction
  4. SapB: function before form
  5. The ram gene cluster
  6. SapB is a lanthionine (Lan)-containing molecule
  7. SapB maturation
  8. Regulation of SapB production
  9. Sequence conservation and the role of the SapB-like morphogenetic surfactants
  10. References
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