A novel firmicute protein family related to the actinobacterial resuscitation-promoting factors by non-orthologous domain displacement
© Ravagnani et al; licensee BioMed Central Ltd. 2005
Received: 21 September 2004
Accepted: 17 March 2005
Published: 17 March 2005
In Micrococcus luteus growth and resuscitation from starvation-induced dormancy is controlled by the production of a secreted growth factor. This autocrine r esuscitation-p romoting f actor (Rpf) is the founder member of a family of proteins found throughout and confined to the actinobacteria (high G + C Gram-positive bacteria). The aim of this work was to search for and characterise a cognate gene family in the firmicutes (low G + C Gram-positive bacteria) and obtain information about how they may control bacterial growth and resuscitation.
In silico analysis of the accessory domains of the Rpf proteins permitted their classification into several subfamilies. The RpfB subfamily is related to a group of firmicute proteins of unknown function, represented by YabE of Bacillus subtilis. The actinobacterial RpfB and firmicute YabE proteins have very similar domain structures and genomic contexts, except that in YabE, the actinobacterial Rpf domain is replaced by another domain, which we have called Sps. Although totally unrelated in both sequence and secondary structure, the Rpf and Sps domains fulfil the same function. We propose that these proteins have undergone "non-orthologous domain displacement", a phenomenon akin to "non-orthologous gene displacement" that has been described previously. Proteins containing the Sps domain are widely distributed throughout the firmicutes and they too fall into a number of distinct subfamilies. Comparative analysis of the accessory domains in the Rpf and Sps proteins, together with their weak similarity to lytic transglycosylases, provide clear evidence that they are muralytic enzymes.
The results indicate that the firmicute Sps proteins and the actinobacterial Rpf proteins are cognate and that they control bacterial culturability via enzymatic modification of the bacterial cell envelope.
The growth and culturability of the actinobacteria is controlled by a family of secreted or membrane-associated proteins . The Rpf protein of Micrococcus luteus was the founder member of this family, which now comprises more than forty representatives [2–4]. Rpf is required for the resuscitation of dormant cells of M. luteus and for the growth of sparsely inoculated cultures of this organism in nutrient-poor media. M. luteus seems to contain only one rpf gene, whose product appears to be essential for bacterial growth . In contrast, most organisms contain several rpf-like genes, whose products are functionally redundant [3, 6–8]. All the proteins so far tested show cross-species activity in bioassays using laboratory cultures of several different organisms, including M. luteus, Rhodococcus rhodochrous, Mycobacterium tuberculosis, Mycobacterium bovis (BCG) and Mycobacterium smegmatis [4, 7, 9, 10]. Since they are active at minute concentrations, it was suggested that they might be involved in inter-cellular signalling [1, 3, 4].
Rpf-like proteins are not found in firmicutes (low G+C Gram-positive bacteria), although some distantly related proteins are found in Staphylococcus and Oenococcus (see below). In this article we report the results of comparative genomic and domain analyses indicating that the firmicutes contain a cognate protein family related to the actinobacterial Rpf proteins by a process of "non-orthologous domain displacement". The available evidence strongly suggests that both the firmicute and actinobacterial proteins have a catalytic function, which may be responsible for their observed activity in improving the culturability of the organisms that produce them.
The Rpf domain
Organisms containing rpf-like genes
Part A: genes encoding proteins containing a Rpf domain
Genome size (Mb)
No. of genes
Genome Accession Number
Mukamolova et al, 1998
Mycobacterium tuberculosis H37Rv
Part B: genes encoding proteins containing a domain distantly related to the Rpf domain
Bifidobacterium longum NCC2705
Tropheryma whipplei strain Twist
Staphylococcus aureus N315
HMMs were used to create profiles of the Rpf domain alignment and these were employed to perform local and global searches of the SWISS-PROT and TrEMBL databases (downloaded from the European Bioinformatics Institute website ). In addition to the previously known Rpf domains in the various actinobacterial Rpf-like proteins, which were detected with highly significant E-values (5.7·10-56 – 4.8·10-39), these searches also identified two Staphylococcus carnosus protein precursors, SceD and SceA (054493 and 054494), with much higher, but nevertheless statistically significant E-values (7.1·10-4 and 3.9·10-2). These proteins contain a domain more distantly related to the Rpf domain. Additional hits above the level of statistical significance (E-values more than 0.1) included many c-type lysozyme precursors, which shared similarity with a 24-residue segment towards the C-terminus of the Rpf domain, as has been reported previously [2, 14, 16]. A PSI-BLAST search was also performed (Blosum62 matrix and a 0.005 E-value threshold) using the Rpf domain of M. luteus Rpf for the first iteration http://www.ncbi.nlm.nih.gov/BLAST/. No new hits were found after 3 iterations. In addition to the known Rpf-like gene products and the more distantly related SceA & SceD proteins of S. carnosus, this search revealed SceD orthologues in two strains of Staphylococcus aureus (NP_646837.1 & NP_372619.1; E-values 2·10-3 & 3·10-3) and Staphylococcus epidermidis (NP_765249.1; E-value 9·10-4) in addition to a previously undetected gene product from Oenococcus oeni (ZP_00069230.1; E-value 3·10-13). These proteins containing a domain distantly related to the Rpf domain are found in the firmicutes, whereas proteins containing the Rpf domain appear to be restricted to the actinobacteria.
Rpf protein subfamilies
Proteins more distantly related to Rpf have been grouped together in two additional families. One of these includes the O. oeni protein mentioned above; it has an inverse domain organisation compared with that of M. luteus Rpf and Rpf-like proteins from Streptomyces. The other family of proteins distantly related to Rpf contains two proteins identified following a PSI_BLAST search (3 iterations), using the large N-terminal region of M. tuberculosis RpfB (Rv1009) for the first iteration. This protein segment contains three repeats of PFAM-B DUF348 (d omain of u nknown f unction) and a G5 domain (also of unknown function, which is found in various proteins involved in cell wall metabolism). The search detected all the previously known RpfB homologues, as well as the two additional gene products from Bifidobacterium longum (BL0658 and BL1227; E-values 2·10-59 and 9·10-32). Several firmicute proteins were also detected (see below). The C-terminal region of the two previously undetected B. longum proteins was similar to part (the N-terminal portion) of the Rpf domain (Fig. 1). It was used to search the genpept database downloaded from the National Centre for Biotechnology Information website  and this revealed multiple hits in B. longum, Streptomyces avermitilis, S. coelicolor and Tropheryma whipplei. The search also detected the S. carnosus SceA protein, although this hit was not statistically significant. The actinobacterial gene products detected in these searches are grouped together as a subfamily of proteins distantly related to Rpf in Fig. 1. They were not detected in the original searches using HMMs of the profile of the Rpf domain alignment because similarity with the Rpf domain is restricted to its N-terminal portion (see additional data file 1).
Proteins similar to RpfB are found in firmicutes
YabE is a member of an extended firmicute protein family
Organisms containing sps-like genes
Part A: genes encoding proteins containing a Sps domain
Genome size (Mb)
No. of genes
Genome Accession Number
Bacillus anthracis strain A2012
Bacillus anthracis strain Ames
Bacillus cereus ATCC 10987
Bacillus cereus ATCC 14579
Listeria monocytogenes EGD-e
Enterococcus faecalis V583
Lactococcus lactis subsp lactis
Clostridium botulinum A
Clostridium perfringens str 13
Clostridium tetani E88
Part B: genes encoding proteins containing a domain distantly related to the Sps domain
Sps protein subfamilies
Two more subfamilies not represented in B. subtilis are of particular interest as they provided evidence for a link between the Sps proteins and muralytic enzymes. Bacillus anthracis and Bacillus cereus are the only organisms containing multiple sps genes that do not contain members of the spsB subfamily. Instead, they have gene products containing two copies of the SH3b domain (SpsE). In bacteria this domain is found in a number of muralytic enzymes, including endopeptidases and amidases. Several Sps proteins from a variety of firmicutes were clustered in another subfamily (SpsD) because they all contain a copy of the putative COG3883 domain. This uncharacterised conserved domain is also shared by a number of muralytic enzymes.
O. ieheyensis OB0947, D. radiodurans DR0488 and T. maritima TM0568 are grouped together because they contain a domain that is only distantly related to the Sps domain (see above). DR0488 is the only known example of an Sps-like protein in an organism with high mole % GC DNA – note however, that D. radiodurans is not closely related to the Rpf-containing actinobacteria. The domain structure of TM0568, which has LysM and M23 peptidase domains, in addition to the Sps module, is reminiscent of the Rpf5 proteins from S. coelicolor and S. avermitilis that contain LysM and M23 peptidase domains in addition to the Rpf module (Fig. 1), and provides another link between these proteins and cell-wall metabolism.
The MltA-like proteins
These observations acquire even greater significance in the light of the weak similarity that has been noted between the Rpf domain and the goose-type lysozymes [2, 14, 16]. Blackburn and Clarke  identified four motifs in the consensus sequence of this type of lytic transglycosylase, and divided the family into five subclasses according to two more variable motifs 3 and 4. The C-terminus of the Rpf domain encompasses motifs 1 and 2 of the EmtA-type family 1e, which includes the absolutely conserved catalytic glutamyl residue (Fig. 4B).
Most rpfB and spsB genes lie within a very similar genomic context flanked by tatD and ksgA(with rnmV inserted between spsB and ksgA in firmicutes). The only exceptions are the duplicate spsB genes found in C. perfringens and C. tetani, one of which is located elsewhere in both organisms. Statistical analysis of the enormous amount of genome sequence information that has become available in recent years has shown that conservation of genome context may often be employed to infer functional relationships between neighbouring genes . In our case, a functional association is indeed predicted by the SNAP algorithm (Similarity Neighbourhood APproach [37, 38]), though it is not obvious what the relationship might be. TatD is a Mg2+-dependent deoxyribonuclease of unknown function , RnmV is a ribonuclease M5/primase-related protein involved in maturation of the 5S rRNA [40, 41] and KsgA is a 16S rRNA methyltransferase that may play a role in translation initiation . In B. subtilis the tatD (yabD) gene does not appear to be expressed during either vegetative growth or sporulation, whereas the rnmV (yabF) and ksgA genes appear to be co-transcribed during vegetative growth. They are highly expressed at the beginning of exponential phase and their expression declines sharply shortly afterwards, an almost identical pattern to that of yabE (data from the B. subtilis Genome Database . These observations may reflect a connection between protein synthesis (RnmV, KsgA) and cell wall expansion (RpfB or SpsB – see below) as would be required when a cell restarts growth after dormancy (in the case of Rpf) or prolonged stationary phase (in the case of Sps). The SNAP algorithm also predicts a functional association between RpfB/SpsB and the 4-diphosphocytidyl-2C-methyl-D-erythritol kinase. The gene encoding this protein (ispE) is located immediately downstream of ksgA in actinobacteria and two to four genes downstream of ksgA in Listeria and Bacillus spp., respectively (however, it appears to have a scattered distribution in clostridia). The 4-diphosphocytidyl-2C-methyl-D-erythritol kinase participates in the non-mevalonate pathway for isoprenoid synthesis, which is involved in cell wall biosynthesis in E. coli and B. subtilis .
A functional relationship between neighbouring genes is normally inferred when they also show the same phylogenetic profile. This is not universally true in the present case, since some firmicutes, e.g. S. aureus, Streptococcus agalactiae, Streptococcus pyogenes, B. anthracis and B. cereus, contain neither rpfB nor spsB although the other genes normally associated with them, tatD, ksgA and rnmV (in firmicutes) are present in the same relative order. Presumably, rpfB or yabE have been lost from these organisms (the alternative, necessitating several independent gene acquisition events, seems less likely). This is particularly evident in the mollicutes, where the occurrence of the genes in question is patchy. None of the strains sequenced contain rpfB/spsB (these organisms lack a cell wall), but some contain rnmV-ksgA (Mycoplasma capricolum and Mycoplasma mycoydes – D14983 and NC_005364, respectively), some contain tatD-ksgA (Mycoplasma pulmonis, NC_002771) and some contain only ksgA (Mycoplasma genitalium, Mycoplasma gallisepticum, Mycoplasma penetrans and Mycoplasma pneumoniae – NC_000908, NC_004829, NC_004432 and NC_000912, respectively). As mollicutes are believed to derive from bacilli by reductive evolution , it seems that this group has lost rpfB/spsB and is in the process of loosing the remaining genes in the string. Note that rpfB, yabE and ksgA are non-essential genes [6, 8, 46] (Ravagnani et al., in preparation), as are tatD and rnmV in B. subtilis [41, 43]).
Information from gene fusions may also be used to predict gene function. The "Rosetta stone"  and "guilt by association"  approaches propose that if a combination of domains A and B is detected in one protein and a combination of domains B and C in another, then it may be predicted that domains A, B and C are functionally related. The "Rosetta stone" hypothesis suggests that the function of one protein domain may be predicted on the basis of its fusion to another domain of known function. Since we do not know the function of the domains connecting RpfB and SpsB (DUF348 & G5), it might be more correct to invoke "guilt by association" in the present case.
More recently, a new method based on consideration of genomic context has been employed to predict orthologous relationships between genes on the basis of anti-correlating occurrences of genes across species . Given three genes A, B and C, if A is always present in a particular group of organisms in association with either B or C, but B and C are never found in the same organism, it can be predicted that B and C fulfil the same function. Extending this approach to protein domains, we may predict that the Rpf domain of RpfB and the Sps domain of SpsB have the same function, as they are both fused to the same DUF348- and G5-containing region, but never occur in the same organism (or, at least, in those so far sequenced).
In bacteria, the DUF348 domain appears to be restricted to proteins containing either Rpf or Sps domains (but it is also found in the yeast Myb-like protein Snt1). B. anthracis and B. cereus are the only organisms containing multiple sps genes that do not have an spsB gene, despite conservation of the genes with which it is normally associated (tatD, rnmV and ksgA). These bacteria have instead four and three copies, respectively, of spsE genes encoding proteins containing two SH3b domains. SH3b is the equivalent of the eukaryotic SH3 (Src homology 3) domain, which is found in a variety of membrane-associated and cytoskeletal proteins and mediates protein-protein interactions by typically binding proline-rich polypeptides . In bacteria, SH3b domains are found in various cell wall amidases and peptidases. Although their function is unknown, the SH3b-containing region of Staphylococcus simulans lysostaphin, which cleaves peptidoglycan, mediates binding to the S. aureus cell wall . Such a function would be consistent with the occurrence of this domain in muralytic enzymes. It is tempting to suggest that the DUF348 domain has a role similar to that of the SH3b domain. Whatever their functions might be, invoking again the principle of "guilt by association" , the association of the Sps domains with other domains present in muralytic enzymes (SH3b, COG3883, LysM) points very strongly to a role for the Sps proteins in cell wall metabolism. This hypothesis is also supported by the occurrence of an M23 peptidase domain in S. coelicolor and S. avermitilis Rpf5, Thermotoga maritima TM0568 and some lytic transglycosylases, such as B. subtilis YomI.
The sequence similarity between the C-terminal region of the Sps domain and that of the Gram-negative membrane-bound lytic transglycosylase, MltA, serves to reinforce this connection. Figure 4A shows that the similarity between Sps and MltA encompasses all three aspartate residues that have been highlighted as potential catalytic residues for the lytic transglycosylase family 2 – classification according to Blackburn and Clarke . In parallel with this, there is also sequence similarity between the Rpf domain and the N-terminal region of the Gram-negative endo membrane-bound lytic transglycosylase, EmtA [2, 14]. Although quite limited, the similarity in this case encompasses the absolutely conserved catalytic glutamate residue of the lytic transglycosylase family 1 (Fig. 4B).
Lytic transglycosylases are enzymes that catalyse cleavage of the β-1,4-glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in the peptidoglycan backbone. Unlike lysozyme, they also catalyse an intramolecular glycosyltransferase reaction to form terminal 1,6-anhydromuramic acid-containing products. The exact function of these enzymes is unknown, but they are thought to be involved in cleavage of the peptidoglycan to permit the insertion of newly synthesised material during cell elongation and division. Remodelling of the cell envelope requires the concerted action of both hydrolases and synthetases, which may form large multienzyme complexes [52, 53]. Consistent with this, physical interactions between some E. coli lytic transglycosylases and penicillin-binding proteins (enzymes involved in the synthesis of peptidoglycan) have been demonstrated experimentally [54, 55].
In E. coli there are at least six lytic transglycosylases, one soluble and five membrane-bound [56–60], with different substrate specificities. Due to the high degree of redundancy, no obvious effect on growth is observed after deletion of their genes . This is in agreement with the results obtained after disruption of three of the five rpf-like genes in S. coelicolor  and the five rpf-like genes of M. tuberculosis [6, 8]. In contrast, there is evidence for essentiality of the apparently unique rpf gene of M. luteus, whose chromosomal copy could be disrupted only in the presence of an extra plasmid-encoded copy of the gene . However, definitive proof of essentiality would require the construction of a conditional mutant and this technology is not currently available for M. luteus.
In B. subtilis the sps genes are not essential, but a clear phenotype is associated with disruption of yocH and this is much accentuated by the disruption of all four sps genes: these mutants show reduced survival after prolonged stationary phase (Ravagnani et al., ms. in preparation). This phenotype has been observed previously, associated with disruption of genes involved in cell wall metabolism, such as the E. coli nlpD, encoding an M23 endopeptidase , and surA, encoding a peptidyl-prolyl isomerase . The latter is required for the correct folding of extracytoplasmic proteins and it has been proposed to be necessary for the assembly of the murein-synthesizing complex, of which lytic transglycosylases are a component . In the Gram-positive bacteria, rpfB or spsB occupy a highly conserved genomic context, within a group of genes including ksgA (see above). Interestingly, in E. coli and related enteric bacteria, ksgA lies within the same transcription unit as surA (surA-pdxA-ksgA-apaG-apaH), suggesting again a possible association between protein synthesis and cell wall expansion.
The assignment of a muralytic function to the Sps and Rpf domains is entirely consistent with the presence of an Sps protein, YorM, in the B. subtilis prophage SPβ, and the recent discovery of the Rpf domain in a large mycobacteriophage "tape measure protein" . Muralytic transglycosylase activity is often associated with bacteriophage virions and confers upon them the highly localised muralytic activity that is required for the process of phage infection, without provoking premature lysis of the host .
The bioinformatic evidence in favour a role for the Rpf and Sps proteins in peptidoglycan metabolism is now compelling. This prediction has recently been confirmed; both M. luteus Rpf and B. subtilis YocH have murein hydrolase activity in zymograms (Mukamolova et al., ms. in preparation; Ravagnani et al., ms. in preparation).
As a result of the observed catalytic activity of the Sps and Rpf proteins, our views on the nature of bacterial non-culturability are changing. The various models of non-culturability we have developed over the years [1, 64, 65] might be explained by the disappearance of nascent peptidoglycan and its gradual replacement by inert peptidoglycan in the bacterial cell wall. This has recently been proposed as a key feature of the mechanism that determines the position of growth zones in the bacterial cell wall [66–68]. We suggest that the walls of non-culturable organisms may contain such a preponderance of inert peptidoglycan that their envelope has effectively become a "cocoon", requiring the action of specialised muralytic enzymes to make a restricted number of scissions, before growth and wall expansion can resume. The Sps and Rpf proteins may have been recruited to serve this function. Resumption of cell wall synthesis might therefore be regarded as one of the "core processes" (see above), along with re-initiation of protein synthesis, that would need to be activated by cells emerging from dormancy (in the case of Rpf) or prolonged stationary phase (in the case of Sps). Signalling could be part of such a resuscitation mechanism, mediated perhaps by a small molecule released from murein as a result of the action of Rpf / Sps proteins. This hypothesis is currently being tested.
Database searching was carried out using either the position-specific iterative BLAST (PSI-BLAST) method  or the Hidden Markov model (HMM) database searching algorithm of HMMER 2.2 g http://hmmer.wustl.edu/. Both local and global profiles of aligned sequences were generated, and searches were carried out using the default parameters. For one application, FASTA  was employed.
Phylogenetic trees were generated using MEGA v2.1 . T-coffee-aligned sequences were analysed using the neighbour-joining method (options: p-distance model, compete removal of gaps, 10,000 bootstrap replications).
This work was funded by the UK BBSRC. C.L.F. was the grateful recipient of a BBSRC studentship. We are grateful to Tim Langdon and Joe Ironside for many helpful discussions and to Eugene Koonin for drawing other examples of non-orthologous domain displacement to our attention.
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