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Two-component regulators and genetic competence in Bacillus subtilis
BACTERIAL SENSOR KINA SE/RESPONSE REGULATOR S YSTE?vtS
403
Two-component regulators and genetic competence in Bacillus subtilis D. D u b n a u 0), j . H a h n
(1)
M. Roggiani (2), F. Piazza 0) and Y. Weinrauch O)
I1) Department o f Microbiology, Public Health Research Institute, 455 First Ave., New York, N Y 10016 (USA), ~2~Department o f Microbiology, Medical School, University o f Minnesota, 420 Delaware Street, SE-Box 196, Minneapolis, M N 55455-039 (USA), and (3~Department o f Microbiology, N Y U School o f Medicine, 550 First Ave., New York, N Y 10016 (USA)
Introduction Microorganisms adapt to external and intracellular conditions by the induction of appropriate transcriptional responses. These responses often include changes in the transcription of many genes which may or may not be genetically linked to one another, comprising so-called global regulatory responses. Bacillus subtilis is a Gram-positive soil organism which can undergo varying developmental responses during the transition from exponential growth. These responses include sporulation, the elaboration of degradative enzymes, increased motility, antibiotic production, induction of the SOS response and the expression of genetic competence. Some of these responses are mutually exclusive" sporuiation, for instance, clearly precludes the other responses. In general however, the relationship among the various possible post-exponential outcomes has not been adequately described as either sequential, mutually exclusive or simultaneous. The regulatory systems governing degradative enzyme production, motility and competence, are discussed by papers appearing in this Forum (Ordal et al., 1994; Kunst et al., 1994). One important generalization to emerge from these and other studies is that postexponential global regulons are controlled by overlapping signal transduction pathways, constituting a network of protein-protein and proteinDNA interactions which culminate in a finely tuned set of transcriptional responses. Another important generalization, which is a corollary of the first, is that few, if any, of the gene products involved in the sensing and transduction of environmental information, are specific for a single form of global response. Thus, genes originally defined as required for the initiation of sporulation may also be required for competence, and alleles of genes identified as essential for competence, or for antibiotic production or degradative enzyme synthesis, may affect sporula-
tion. (A consequence of these interrelationships is that we are faced with somewhat of a nomenclatural nightmare, since genes are named for the phenotypes with which they were first associated). A picture begins to emerge, then, of a signal transduction network, rather than of a series of dedicated pathways. An attractive model is that the network serves to integrate combinatorial signals; each combination of signals eficits a particular set of transcriptional responses. To fully comprehend regulatory networks such as this, new methods and new ways of thinking will be required. In a general sense, the analytical phase of molecular biology, in which individual steps in synthetic or regulatory pathways are dissected, must give way to a synthetic phase, in which we begin to piece together a more holistic picture of the cell and the organism. However, since this review concerns only the competence response, we will analyse regulatory interactions using the paradigm of a pathway, although at the risk of considerable oversimplification.
Competence Competence, the ability of the cell to bind and internalize exogenous high molecular weight DNA, is a naturally expressed physiological state in B. subtilis (reviewed in Dubnau, 1993). When grown in minimal salts media containing glucose and amino acids, a set of novel gene products, encoded by the competence genes, are elaborated beginning at the time of transition to stationary phase (To). This growth stage-regulated response is also nutritionally governed, since glucose is required for competencespecific transcription, and glutamine represses the development of competence. In addition, amino acids repress competence expression during exponential growth, but at T o this repression is overridden (Dubnau et al., 1991). Since competence is expressed in
12th F O R U M I N M I C R O B I O L O G Y only a distinct subfraction of the population, comprising 10-20 o70 of the cells (Hadden and Nester, 1968; Haseltine-Cahn and Fox, 1968), a cell-typespecific form of regulation must also exist, which is not understood. These three modes of regulation, growth-stage-specific, nutritional and cell-typespecific, depend on the transduction of information from a partially described set of environmental and possibly cell-cycle-related signals. When the proper signals are present, a signal transduction cascade relays information to the comK gene, which specifies a competence transcription factor (Mohan and Dubnau, 1990; van Sinderen et al., 1990; van Sinderen, L. Hamoen, A. Luttinger, G. Venema and D. Dubnau, unpubfished). A sharp increase in ComK production results, causing an increase in the transcription of the late competence genes. The increase in comK transcription is mediated by the Mec system (Dubnau and Roggiani, 1990; Kong et al., 1993; Roggiani et al., 1990). The latter consists of two protein, MecA and B, which negatively regulate comK, probably by protein-protein intel-action (Kong and Dubnau, 1994 submitted; Msadek et al., 1994). Since ComK positively regulates its own transcription (D. van Sinderen and G. Venema, personal communication), the interaction of Mec with ComK probably represses the expression of comK. The signal transduction system operating upstream of Mec serves to lift the repression of comK, since mecA or mecB loss-of-function mutations bypass the requirements for the upstream regulatory genes (Roggiani et al., 1990). Here we will describe what i q knnwn nhrmt th,~ early signal transduction steps that elicit the competenoe response, in particular the parts played by three distinct two-component systems in the regulation of comK expression; the comP-comA, degU and spoOA systems. As their names imply, these genes were first identified in relationship to the competence, degradative enzyme and sporulation responses. The degS-degU two-component regulators are discussed in greater detail by Kunst et ai. (1994) in this Forum. The roles of ComP and Coma in the regulation of post-exponential responses
A key player in the competence regulatory pathway is the srfA operon (Nakano et al., 1991a; Nakano and Zuber, 1990; van Sinderen et al., 1990). This locus specifies several proteins required for the post-exponential production of the heptapeptide antibiotic surfactin. It is required for the expression of competence, and appears to play a role, albeit a subtie one, in the regulation of sporulation. The precise part played by srfA in competence development has received a fair amount of attention but is still poorly understood (D'Souza et al., 1993; Nakano et al.,
1991a; Nakano and Zuber, 1993). It suffices for our purpose to suggest that it participates in generating a signal for lifting Mec-mediated repression, srfA transcription, in glucose minimal salts medium, is sharply induced at T_ 1, and this induction is required for the expression of competence. Four loci have been shown to function in the activation of srfA transcription. Two of these, comP and coma are clearly histidine kinase and response regulator members of the two-component regulator family 0Veinrauch et al., 1989; Weinrauch et al., 1990). Analysis of the ComP sequence leads to the prediction that it is an integral membrane protein, with eight transmembrane segments. We have raised antiserum against ComP and shown that it indeed behaves like an integral membrane protein (unpublished). In addition to the conserved N-terminal domain characteristic of response regulators, ComA possesses a C-terminal domain with striking similarity to several DNA-binding proteins that contain helix-turn-helix motifs (Dubnau, 1991). It is noteworthy that DegU and ComA, in particular, share sequence similarity over their entire lengths. ComA, when phosphorylated in vitro by reaction with acetyl-PO4, binds to at least two sites upstream from the srfA promoter (Roggiani and Dubnau, 1993). These sites had previously been predicted to be ComA binding sites (Nakano et al., 1991b), based on mutagenesis studies and by comparison of srfA sequences with those upstream from other genes also regulated by ComA; degQ (Msadek et al., 1991), and gsiA (Mueller et al., 1992). It has proven difficult to demonstrate direct phosphorylation of C o m a by ComP in vitro (unpublished). However, overexpression of ComA bypasses the competence requirement for ComP 0Veinrauch et al., 1990), suggesting that the normal role of the latter protein is to activate ComA. In addition, purified ComA is readily phosphorylated by acetyl-PO4 (Roggiani and Dubnau, 1993) and can also be phosphorylated by purified NRII, kindly provided by A. Ninfa (unpublished). In vitro gel shift and footprinting experiments have demonstrated that the phosphorylated form of ComA has a higher binding affinity for the C o m a boxes upstream from the srfA promoter than does the unphosphorylated form (Roggiani and Dubnau, 1993). We propose therefore, that in response to some signal, ComP and ComA interact so as to increase the concentration of ComA-PO 4, resulting in the transcriptional activation of srfA (fig. 1). Mutational studies on ComA are consistent with this model. Asp residue 55 is predicted to be the residue that accepts a phosphate from ComP. Mutations which convert D55 to either E (D55E) or to N (D55N) fail to complement null mutations in ComA when present in single copy at an ectopic chromosomal location (unpublished). The wild type comA fully complements when located at the same
BACTERIAL SENSOR KINASE/RESPONSE REGULATOR SYSTEMS
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405
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ectopic site. It is interesting that the D55N mutation when present on a multicopy comA plasmid, can complement a deletion of both comP and comA. This suggests that the bypass of a comP mutation by overexpression of ComA mentioned above, can be ascribed to limited function of the unphosphorylated response regulator protein, rather than to phosphorylation by cross-talk. In accordance with this interpretation, in vitro DNA-binding experiments reveal that although the specific binding of ComAP O 4 t o DNA is tighter than that of ComA, the latter does possess a tte~.ec_*able affinity for the C o m a boxes upstream from srfA. Nakano and Zuber (1993) have analysed the C o m a boxes associated with srfA. They have shown that the upstream ComA box is dispensable when the downstream box is altered so as to conform to the ComA box consensus. This was interpreted in terms of a looping model for ComA activity, in which the upstream box serves to raise the local concentration of ComA, facilitating binding of ComA to the downstream box. Our in vitro footprinting studies are consistent with this model; several phosphodiester bonds located between the two C o m a boxes become hypersensitive to DNAse when ComA binding occurs, suggesting that loop formation has occurred (Roggiani and Dubnau, 1993). In addition to comP and comA, two other loci are required for srfA induction; comQ (Weinrauch et al., 1991) and spoOK (Grossman et al., 1991; Hahn and Dubnau, 1991 ; Nakano and Zuber, 1991). spoOK is an operon that specifies the B. subtilis oligopeptide permease (Perego et al., 1991 ; Rudner et al., 1991). The organization of this operon, and
the amino acid sequences of its five predicted protein products, show that this is a typical bacterial ATP-driven permease. The predicted sequence of the ComQ protein, on the other hand, does not resemble that of any member of the GenBank database. The requirements for both comQ and spoOK for competence, as well as the comP requirement (Weinrauch et al., 1990; unpublished), are completely bypas~d when Com~. is overexpress~, sugg~fang that these genes act upstream of ComA in the signal transduction pathway. A multicopy plasmid that overexpresses ComP from the relatively weak Pspac promoter, results in the partial bypass of comQ and spoOK (unpublished). This is consistent with the idea that they act upstream from ComP. Grossman et al. (1991) have shown that B. subtilis secretes a soluble pheromone that acts to turn on srfA transcription. The peptide moiety of this factor is encoded by a small open reading frame (comX) which overlaps the 3' end of comQ (fig. 2) (Magnuson et al., 1994). Inactivation of comQ produces a competence negative pi~enotype, which can be rescued by the addition of exogenous factor (Magnuson et al., 1993, submitte.d). Mutational polarity was excluded as an explanation for this competence deftciency, and it appears, therefore, that ComQ may be required for the processing or secretion of the competence pheromone (fig. 1). Since spoOK specifies an oligopeptide permease, it is attractive to consider that the pheromone, which is at least partly peptide in nature, is detected by cellsurface-localized SpoOK proteins (fig. 1). This has led to the hypothesis that the permease informs ComP, also residing on the cell membrane, about the
406
12th F O R U M I N M I C R O B I O L O G Y
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Fig. 2. Map of the B. subtiiis chromosomal region containing comQ, comX, comP and comA. The location of the comX gene (which overlaps the 3' end of comQ) is from Magnuson et al. (Magnuson et al., 1994). The locations of known and suspected promoters and terminators are indicated by arrows 0Veinrauch et al., 1989; Weinrauch et al., 1991; Weinrauch et al., 1990).
level of soluble factor present in the extracellular space (Dubnau, 1991; Grossman et ai., 1991). This in turn leads to the accumulation of ComA-PO4, and the activation of srfA transcription. Precedent exists in the Pho regulon (Rao and Torriani, 1990) for the interaction of another permease (psO with two-component regulators (PhoB and PhoR). pst, which encodes a high affinity phosphate uptake system, and the associated gene phoU, serve to reduce the accumulation of the activated response regulator PhoB-PO 4, and therefore to repress expression of the ~ o regulon. Mutations in pst consequently lead to constitutive Pho expression. In contrast, the role of the spoOK operon in competence must be positive, since loss-of-function spoOK mutations lead to loss of competence. It is not known whether signalling requires the internalization of competence pheromone, or whether true transmembrane signalling occurs, as is likely to be the case with Pho (Rao and Ton-iani, 1990). A truncated form of ComP, lacking the hydrophobic N-terminal domain, can complement a total deletion of comP residing on the chromosome (unpublished). Interestingly, this truncated ComP protein is still membrane-localized, although in preliminary expe~ments it behaves like a peripherally attached membrane protein. It is tempting to speculate that the truncated protein attaches to the membrane by protein-protein interaction, perhaps to SpoOK. In the case of intact ComP, in addition to this protein-~rotein interaction, the membrane spanning don~a~:~ presumably associates with the hydrophobic c~r¢ of ~h¢ membraae bilayer. The concentration of soluble competence factor probably serves as a measure of cell density. When grown in a confined space, as in a shaker flask, the
pheromone will accumulate as the cells approach stationary phase, causing induction of srfA transcription at about T 1- Consistent with this hypothesis are experiments in which srfA was placed under control of the IPTG-inducible Pspac promoter (Hahn and Dubnau, 1991; Nakano and Zuber, 1991). Growth in the presence of IPTG resulted in the constitutive expression of competence, although only to about 10 % of the wild type level. The observed incomplete competence induction may be due to the relative weakness of the Pspac promoter. It may also indicate that competence is subject to additional control, which cannot be bypassed by the induction of srfA. In fact it appears that repression by AbrB may be at least part of the explanation for this additional control (see below). Expression of srfA under Pspac control also bypasses the competence requirements for comQ, spoOK, comP and comA. This suggests that the major roles for these four loci in the development of competence is to induce srfA. If the ComPComA system is responding to crowding as opposed to a signal directly indicating growth stage, it may be proper to consider the response to population density to be a fourth mode of competence regulation, in addition to growth stage, cell-type-specific and nutritional signalling. It was mentioned above that competence develops in glucose-containing media. Substitution of glycerol for glucose or the addition of glutamine lowers competence expression markedly, without having a measurable effect on growth rate (Albano et al., 1987; Weinrauch et al., 1990). When ComA is overexpressed, bypassing the spoOK, comQ and comP requirements, glucose is no longer needed for competence development and glutamine no longer inhibits (Weinrauch et al., 1990). It appears that the
BA CTERIAL SENSOR KINASE/RESPONSE REGULATOR S YSTFMS ComP-ComA signal transduction system responds not only to the competence pheromone (via spoOK), but also to the presence or absence of nutritional factors (fig. 1). A final aspect of the ComP-ComA system deserves mention. Using a iacZ fusion we have reported that comA transcription increases 2-3 fold during growth (Weinrauch et al., 1990). Transcription mapping by primer extension revealed that whereas a promoter is located immediately upstream from comA, additional transcriptional readthrough occurs from further upstream. This readthrough transcription appears to increase during growth, probably accounting for the observed increase in comA expression. The location of the Ul~tream promoter is not known. Recent work using antiComP antiserum has suggested that ComP expression also increases during growth (unpublished). A lacZ fusion to comQ, on the other hand, shows that expression of comQ decreases during growth, and primer extension mapping has located a promoter just upstream from this gene 0Veinrauch et al., 1991). In a comQ null mutant, the comA-lacZ fusion is overexpressed (Weinrauch et al., 1991), and use of anti-ComP antiserum suggests that the same is true of ComP expression (unpublished). Perhaps the decrease in comQ expression during growth in competence medium is part of a normal mechanism, causing an increase in ComP and ComA expression, possibly from a promoter located upstream of comP (fig. 2). We do not know how comQ expression regulates the expression of comP-comA. If this is a trans effect exerted by ComQ, the latter may play a more complex role than simply to process or secrete the competence factor.
407
(1990) have constructed a mutation (degU146) in which the Asp residue which receives a phosphate from DegS is altered to Asn. In their hands, this mutation has no effect on competence. In the derivatives of strain IS75 used in our laboratory, however, the degU!46 mutation decreases competence severalhundred-fold (unpublished). This strain difference is not understood, but inspires caution in concluding that unphosphorylated DegU is sufficient for competence under all conditions. Dahl et al. (1991, 1992) have described mutations in DegS and in DegU that result in the accumulation of excess DegU-PO 4. These degS h and degUh mutations have been characterized genetically and biochemically, and result in overexpression of degradative enzymes and the nearly complete suppression of competence. It seems certain that excessive levels of DegU-PO 4 are deleterions for the development of competence, and act at least in part to repress the expression of srfA. The C-ternfinal domains of DegU and of ComA exhibit substantial amino acid sequence similarity (Dubnau, 1991), and it is possible that DegU-PO 4 may repress by competing with ComA-PO 4 for binding to the ComA boxes upstream from srfA. Kunst et al. 0993) have observed that when Na glutamate is substituted for (NH4)2SO4 in competence medium, hi~ther levels of competence are achieved and the degt.ff32 mutation no longer leads to a loss of competence. If, in this medium, a high concentration of ComA-PO 4 accumulates, the latter may overcome the repressive effect of DegU-PO 4. In fact, we have observed that overexpression of C o m a completely eliminates the downregulation of competence due to a degLrh mutation (unpublished). The roles of SpoOA and AbrB in competence
Role of the DegS-DegU system in competence DegS and DegU are, respectively, histidine kinase and response regulator members of a two-component regulatory system (Kunst et al., 1994). They were originally identified as proteins required for the transcriptional activation of a series of post-exponentially expressed genes that specify degradative enzymes. However, null mutations in degU result in the almost total inactivation (about 10*-fold) of competence development (Tanaka and Kawata, 1988), and of comK expression (unpublished). This is in contrast to the 300 to 500-fold effects of comQ, spoOK, comP, comA and srfA inactivation. Despite this dramatic and important effect of degU on competenc e, its targets are not known. It is notable that null mutations in the cognate histidine kinase gene degS have little if any effect on competence (Msadek et ai., 1990). Therefore, either phosphorylation of DegU is not needed for competence or DegU can be phosphorylated by some other pathway. Msadek et al.
SpoOA is a response regulator protein that was initially characterized as a key player in the regulation of sporulation, and which is also required for the expression of competence genes (Albano et al., 1987; Piggot and Coote, 1976; Sadaie and Kada, 1983; Spizizen, 1965). Null mutations in spoOA have a relatively minor negative effect (2-3-fold) on the expression of srfA (Hahn and Dubnau, 1991). However, they dramatically depress the expression of comK and therefore of the late competence genes (unpublished). Unlike all of the regulatory genes so far mentioned except for comK, loss-of-function mutations in mecA or mecB do not bypass the spoOA requirement for competence (Roggiani et al., 1990), suggesting that spoOA acts downstream from Mec. Certain phenotypes resulting from spoOA ina~ivation are known to be suppressed by mutations in abrB. The latter gene specifies a DNA binding protein that usually acts negatively, but can also have a positive effect on the transcription of certain genes
12th FORUM IN MICROBIOLOGY (Strauch, 1993). The suppression is due to the fact that one important role for SpoOA is to downregulate the expression of AbrB (Perego et al., 1988). Loss-of-function abrB mutations therefore suppress some of the phenotypes resulting from the absence of SpoOA. In this regard, our situation is complex, since AbrB plays a positive role in competence (Aibano et al., 1987) and specifically in the activation of comK expression (unpublished). Several observations however, provide clues concerning the role of spoOA in competence. A knockout of SpoOA depresses competence about 1,000-fold, and a null mutation in abrB depresses competence only about 40- to 50-fold. A double spoOA abrB mutant exhibits the same competence level as does the single abrB mutant, suggesting that the major role for SpoOA in competence is to prevent the excessive accumulation of AbrB (Albano et al., 1987). Since abrB lossof-function mutations are bypassed by mec mutations (Rog~aril et al., 1990), the positive role of AbrB must be exerted upstream from Mec. Since deletions in spoOA are not bypassed by mec mutations, the negative role of AbrB must be exerted downstream of Mec. We believe that AbrB acts by binding directly to the comK promoter as a rcpressor. Consistent with this is our observation that lacZ fusions to comG, a late competence gene, are overexpressed in a spoOA abrB mecA mutant (unpublished). The positive role of AbrB is bypassed by mecA, and spoOA is no longer needed in the abrB background. Ordinarily, SpoOA is phosphorylated at about T 0, and it appears likely that this phosphorylation is required for competence. Green et al. (1991) have constructed a SpoOA derivative in which the Asp residue normally targeted for phosphoryiation is converted to a Gin (D56Q). This mutation results in a sporulation-negative phenotype (Green et ai., 1991) and also in a severe decrease in competence (B.D. Green and P. Youngman, personal communication). However, the level of competence observed in this mutant background is about 10-fold higher than in a spoOA null mutant, suggesting that the unphosphorylated protein may retain some activity, presumably preventing excessive accumulation of AbrB. One caveat is that the stability of the mutant protein may be reduced and the observed effect may be due to this instability rather than to the absence of phosphorylation. Another is that the mutation in SpoOA may compromise some function of the protein in addition to its ability to be phosphorylated. However, Green and Youngman have combined the D56Q mutation with a small deletion in SpoOA that results in phosphorylation-independent sporulation (Green et al., 1991). The double mutant exhibits nearly normal competence, strongly suggesting that phosphorylation of SpoOA plays a role in competence signalling. SpoOA can be phosphorylated via an unusual
phosphorelay (Burbulys et al., 1991 ; Hoch, 1993) in which a histidine kinase (SpoIIJ/KinA) donates a phosphate to another response regulator (SpoOF). The phosphate is then transferred to SpoOB and finally to SpoOA. SpoOA-PO 4 is responsible for the transcriptional induction or repression of several do~.nstream genes. Although SpoOA is required for competence, null mutations in kinA (unpublished), spoOB or spoOF (Albano et al., 1987) have minor effects on transformability, suggesting that the KinA/SpoIIJ pathway may not be utilised in competence-specific signalling. SpcOA can also be phosphorylated by at least one other pathway, which utilizes KinB, another histidine kinase (Burbulys et al., 1991). It is not clear whether KinB signalling plays a role in competence.
The competence pathway and the signal transducing roles of ComA, SpoOA and DegU The information supplied above can be summarized in the form of a pathway rather than a network, since only a single outcome (competence) is considered. The scheme shown in figure 3 includes the genes discussed above as well as sinR (Smith, 1993) which is a DNA binding protein that acts negatively on sporulation but is somehow required for the activation of comK transcription. The three response regulators (ComA, DegU and SpoOA) intersect the pathway at distinct points. Each represents a potential control point, although the signals involved are still incompletely understood. The ComA branch of the pathway, as noted ~ L I v ,JV~,,.,
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nutritional signals (fig. 1, fig. 3). ComA, ComP and ComQ are also required for the transcriptional induction of degQ, a regulator of degradative enzyme expression, degQ transcription is additionally dependent on degU and degS. The ComQ dependency implies that degQ expression may respond to crowding via the competence pheromone and it would be interesting to determine whether spoOK null mutations affect degQ expression. Distinct regions upstream from the degQ promoter respond to degU and to comA. The comA but not the degU-dependent response can be induced by amino acid starvation. Thus, several lines of evidence suggest that ComP and ComA respond to a variety of nutritional and population density signals, p k s X is a polyketide synthase locus of unknown function (Scotti et al., 1993) that like degQ, requires comQ, comP, comA, degS and degU for its full expression (A. Albertini, C. Scotti and A. Galizzi, personal communication). gsiA is a glucose starvation-induced gene, which requires comP and comA, but neither comQ nor spoOK for its induction (Mueller et al., 1992). It therefore appears that ComA and ComP can function independently of the spoOK proteins in the regu-
BACTERIAL SENSOR KINASE/RESPONSE REGULATOR SYSTEMS
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Fig. 3. Summary of the competence regulatory cascade as described in the text. Arrowheads and heavy perpendicular lines indicate positive and negative effect, respectively. Lines with solid dots indicate transcriptional regulation and those with empty dots indicate protein-protein interaction or post-translational protein modification.
lation of at least gsiA. As in the case of degQ, the comQ dependency leads to the prediction that pksX expression will also require spoOK. The SpoOA branch probably also senses growth stage, n e r h a n • m n r o c l i r o e t l y t h a n ¢. .;.m. . n l .w, I j a s a .~. A. . .~ . ~ ure of crowding, although it also responds to population density and nutritional signals in the regulation of sporulation (Ireton et aL, 1993). For instance, Grossman and his co-workers have shown that SpoOA can transduce DNA replication signals to the sporulation system (Ireton and Grossman, 1992). Like ComA therefore, SpoOA is clearly able to transduce disparate signals, and can additionally interact with at least two phosphate donors. The SpoOA signalling pathway responsible for the regulation of competence is not understood. Since AbrB plays both negative and positive roles in the control of competence, and can bind to DNA cooperatively to control transcription (Strauch, 1993), it is likely that the activation of SpoOA must be closely regulated so as to maintain the AbrB concentration within narrow limits. In fact, the use of a construct in which abrB transcription is driven by the IPTG-regulatable Pspac promoter has shown that competence requires a narrow range of IPTG concentrations, above and below which transformability is inhibited (unpublished). The competence signal(s) detected by DegU are not understood, and the nature of the signals that .
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trigger DegU phosphorylation by DegS are likewise obscure. Since excessive DegU-PO 4 inhibits competence development, some control probably limits the extent of phosphorylation. On the other hand, as discussed above, in the IS75 strain background, it is possible that some phosphorylation of DegU must occur for competence indu~ion. This presents a situation similar to that with SpoOA and AbrB; perhaps DegU phosphorylation must be maintained within narro~ ~ limits for competence expression. The work described from our laboratory was supported by NIH grants AII0311 and GM43756. We thank the members of our laboratory and that of I. Smith for many useful discussions, as well as T. Msadek, F. Kunst, G. Rapoport, R. Magnuson, J. Solomon, A.D. Grossman, A. AIbertini, C. Scotti and A. Galizzi for further discussions and for providing information prior to pubfication.
References Albano, M., Hahn, J. & Dubnau, D. (1987), Expression of competence genes in Bacillussubtilis. J. Bacteriol., 169, 3110-3117. Burbulys, D., Trach, K.A. & Hoch, J.A. (1991), Initiation of spondation in B. subtil/s is controlled by a multicomponent phosphorelay. Cell, 64, 545-552. D'Souza, C., Nakano, M.M., Corbell, N. & Zuber, P. (1993), Amino-acylation site mutations in amino acid
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acdvati~hg domains of surfactin synthetase: effects on surfactin production and competence development in Bacillus subtilis. J. Bacteriol., 175, 3502-3510. Dahl, M.K., Msadek, T., Kunst, F. & Rapopon, G. (1992). The phosphorylation state of the DegU response regulator acts as a molecular switch allowing eithe, degradative enzyme synthesis or expression of gen.~-ac competence in Bacillus subtilis. J. Biol. Chem. 267, 14509-14514. Dubnau, D. (1991), Genetic com~tence in Bacilh:s subtiiis. Microbiol. Revs., 55, 395-424. Dubnau, D. (1993), Genetic exchange arid homologous recombination, in "Genetic excharge and homologous recombination" (A.L. Sonen.~nein, R. Losick & J.A. Hoch) (pp. 555-584). American Society for Microbiology, Washington, DC. Dahl, M.K., Msadek, T., Kunst, F. & Rapoport, G. (1991), Mutational analysis of the Bacillus subtilis DegU regulator and its phosphorylation by the DegS protein kinase. J. Bacteriol., 173, _339-2547. Dubnau, D., Hahn, J., Kong, L., Roggiani, M. & Weinrauch, Y. (1991), Genetic competence as a postexponential global response. Semin. Dee. Biol., 2, 3-12. Dubnau, D. & Roggiani, M. (1990), Growth mediumindependent genetic competence mutants of Bacillus subtilis. J. Bacteriol., 172, 4048-4055. Green, B.D., Bramucci, M.G. & Youngman, P. (1991), Mutant forms of SpoOA that affect sporulation initiation: a general model for phosphorylationmediated activation of bacterial signal transduction proteins. Semin. Dee. Biol., 2, 21-29. Grossman, A.D., Ireton, K., Hoff, E.F., LeDeaux, J.R., Rudner, D.Z., Magnuson, R. & Hicks, K.A. (1991), Signal transduction and the initiation of sporulation in Bacillus subtilis. Semin. Dee. Biol., 2, 31-36. Hadden, C. & Nester, E.W. (1968), Purification of competent cells in the Bacillus subtilis transformation sysLI.IaZ..i.
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~.J,
olcJ-ooJ.
Hahn, J. & Dubnau, D. (1991), Growth stagesignal transduction and the requirements for srfA induction in the development of competence. J. BacterioL, 173, 7275-7282. Hascltinc-Ca~, F. & Fox, M.S. (1968), Fracfionation of transformable bacteria from competent cultures of Bacillus subtilis on renograffin gradients. J. BacteHol., 95, 867-875. Hoch, J.A. (1993),spoO genes, the phosphorelay,and the initiation of sporulation, in "Bacillus subtilis and other Gram-positive bacteria. Biochemistry, physiology and molecular genetics" (A.L. Sonenshein,J.A. Hoch & R. Losick) (pp. 747-755). American Society for Microbiology, Washington, DC. lreton, K. & Grossman, A.D. (1992), Coupling between gene expression and DNA synthesis early during development in Bacillus subtilis. Proc. Natl. Acad. Sci. (USA), 89, 8808-8812. Ireton, K., Rudner, D.Z., Jaacks-Siranosian, K. & Grossman, A.D. (1993), Integration of multiple developmental signals in Bacillus subtilis through the SpoOA transcription factor. Genes & Dev., 7, 283-294. Kong, L., Siranosian, K.J., Grossman, A.D. & Dubnau, D. (1993), Sequence and properties of mecA, a negative regulator of genetic competence in Bacillus subtilis. Molec. Microbiol., 9, 365-373.
Kt,r.st, i'., Msadek, T. & Rapoport, G. (1994), Signal transduction network controlling degradative enzyme synthesis and competence in Bacillus subtilis, in "Regulation of bacterial differentiation" (P.J. Piggot, C.P. Moran & P. Youngman) (pp. 1-19). American Society for Microbiology, Washington, DC. Kunst, F., Msadek, T., Bignon, J. & Rapoport, G. (1994), The DegS/DegU and ComP/ComA two-component systems are part of a network controlling degradative enzyme synthesis and competence in Bacillus subtilisin. Res. Microbiol. (in press). Mohan, S. & Dubnau, D. (1990), Transcriptional regulation of comC: evidence for a competence-specific factor in Bacillus subtilis. J. Bacteriol., 172, 4064-4071. Msadek, T., Kunst, F., Henner, D., Klier, A., Rapoport, G. & Dedonder, R. (1990), Signal transduction pathway controlling the synthesis of a class of degradative enzymes in Bacillus subtilis: expression of the regulatory genes and analysis of mutations in degS and degU. J. Bacteriol., 172, 824-834. Msadek, T., Kunst, F., Klier, A. & Rapoport, G. (1991), DegS-DegU and ComP-ComA modulator-effector pairs control expression of the Bacillus subtilis pleiotropic regulatory gene degQ. J. Bacteriol., 173, 2366-2377. Msadek, T., Kunst, F. & Rapoport, G. (1994), MecB of Bacillus subtilis is a pleiotropic regulator of the CIpC ATPase family, controlling competence gene expression and survival at high temperature. Proc. Nat. Acad. Sci. (Wash.) (in press). Mueller, J.P., Bukusoglu, G. & Sonenshein, A.L. (1992), Transcriptional regulation of Bacillus subtilis glucose starvation-inducible genes: control of gsiA by the ComP-ComA signal transduction system. J. Bacteriol., 174, 4361-4373. Nakano, M.M., Magnusson, R., Myers, A., Curry, J., Grossman, A.D. & Zuber, P. (1991a), srfA is an operon required for surfactin production, competence de¥1g,l O l . , C l l l l g ~ l l L,
ggllqJI.
Ig~l l l l t . , l l g ; l l L
~I.YUI UIdLIUII
111
Dggg-illl~,.~
subtilis. J. Bacteriol., 173, 1770-1778. Nakano, M.M., Xia, L. & Zuber, P. (1991b), Transcription initiation region of the srfA operon which is controlled by the comP-comA signal transduction system in Bacillus subtilis. J. BacterioL, 173, 548%5493. Nakano, M.M. & Zuber, P. (1990), Identification of genes required for the biosynthesis of the lipopeptide antibiotic surfactin in Bacillus subtilis, in "Genetics and Biotecbnology of Bacilli, vol. 3" (M.M. Zukowski, A.T. Ganesan & J.A. Hoch) (pp. 397-405). Academic Press, London, New York. Nakano, M.M. & Zuber, P. (1993), Mutational analysis of the regulatory region of the srfA operon in Bacillus subtilis. J. Bacteriol., 175, 3188-3191. Nakano, M.N. & Zuber, P. (1991), The primary role of C o m a in establishment of the competent state in Bacillus subtilis is to activate the expression of srfA. J. Bacteriol., 173, 7269-7274. Ordal, G., Kirsch, M.L., Carpenter, P.B. & Hanlon, D.W. (1994), Novel aspects of chemotaxis in Bacillus subtilis. Res. Microbiol. (in press). Perego, M., Higgins, C.F., Pearce, S.R., Gallagher, M.P. & Hoch, J.A. (1991), The oligopeptide transport system of Bacillus subtilis plays a role in the initiation of sporulation. Mol. MicrobioL, 5, 173-185. Perego, M., Spiegelman, G.B. & Hoch, J.A. (1988), Struc-
BACTERIAL
SENSOR KINASE/RESPONSE
ture of the gene for the transition state regulator abrB: regulator synthesis is controlled by the spoOA sporulation gene in Bacillus subtilis. Mol. Microbiol., 2, 689-699. Piggot, P. & Coote, J.G. (1976), Genetic aspects of bacterial endospore formation. Bacteriol. Rev., 40, 908-962. Rao, N.N. & Torriani, A. (1990), Molecular aspects of phosphate transport in Escherichia coli. Mol. MicrobioL, 4, 1083-1090. Roggiani, M. & Dubnau, D. (1993), ComA, a phosphorylated response regulator protein of Bacillus subtilis, binds to the promoter region of srfA. J. BacterioL, 175, 3182-3187. Roggiani, M., Hahn, J. & Dubnau, D. (1990), Suppression of early competence mutations in Bacillus subtiiis by mec mutations. J. BacterioL, 172, 4056-4063. Rudner, D.Z., LeDeaux, J.R., Ireton, K. & Grossman, A.D. (1991), The spoOK locus of Bacillus subtilis is homologous to the oligopeptide permease locus and is required for sporulation and competence. J. BacterioL, 173, 1388-1398. Sadaie, Y. & Kada, T. (1983), Formation of competent Bacillus subtilis cells. J. BacterioL, 153, 813-821. Scotti, C., Piatti, M., Cuzzoni, A., Perani, P., Tognoni, A., Grandi, G., Galizzi, A. & Albertini, A. (1993), A Bacillus subtilis large ORF coding for a polypeptide highly similar to polyketide synthases. Gene, 130, 65-71. Smith, I. (1993), Regulatory proteins that control lategrowth development, in "Bacillus subtilis and other Gram-positive bacteria. Biochemistry, physiology and molecular genetics" (A.L. Sonenshein, J.A. Hoch & R. Losick) (pp. 785-800). American Society for Microbiology, Washington, DC.
REGULATOR
SYSTEMS
411
Spizizen, J. (1965), Analysis of asporogenic mutants in Bacillus subtilis by genetic transformation, in "Spores III" (L.L. Campbell & H.O. Halvorsen) (pp. 125-137). American Society for Microbiology, Washington, DC. Strauch, M. (1993), AbrB, a transition state regulator, in "Bacillus subtilis and other Gram-positive bacteria. Biochemistry, physiology, and molecular genetics" (A.L. Sonenshein, J.A. Hoch & R. Losick) (pp. 757-764). American Society for Microbiology, Washington, DC. Tanaka, T. & Kawata, M. (1988), Cloning and characterization of Bacillus subtilis iep, which has positive and negative effects on production of extracellular proteases. J. Bacteriol., 170, 3593-3600. Van Sinderen, D., Withoff, S., Boels, H. & Venema, G. (1990), Isolation and characterization of comL, a transcription unit involved in competence developmerit of Bacillus subtilis. Mol. Gen. Genet., 224, 396-404. Weinrauch, Y., Guillen, N. & Dubnau, D. (1989), Sequence and transcription mapping of Bacillus subtilis competence genes comb and comA, one of which is related to a family of bacterial regulatory determinants. J. BacterioL, 171, 5362-5375. Weinrauch, Y., Msadek, T., Kunst, F., Rapoport, G. & Dubnau, D. (1991), Sequence and properties of comQ, a new competence gene of Bacillus subtilis. J. Bacterioi., 173, 5685-5693. Weinrauch, Y., Penchev, R., Dubnau, E., Smith, I. & Dubnau, D. (1990), A Bacillus subtilis regulatory gene product for genetic competence and sporulation resembles sensor protein members of the bacterial two-component signal-transduction systems. Genes & Dev., 4, 860-872.
403
Two-component regulators and genetic competence in Bacillus subtilis D. D u b n a u 0), j . H a h n
(1)
M. Roggiani (2), F. Piazza 0) and Y. Weinrauch O)
I1) Department o f Microbiology, Public Health Research Institute, 455 First Ave., New York, N Y 10016 (USA), ~2~Department o f Microbiology, Medical School, University o f Minnesota, 420 Delaware Street, SE-Box 196, Minneapolis, M N 55455-039 (USA), and (3~Department o f Microbiology, N Y U School o f Medicine, 550 First Ave., New York, N Y 10016 (USA)
Introduction Microorganisms adapt to external and intracellular conditions by the induction of appropriate transcriptional responses. These responses often include changes in the transcription of many genes which may or may not be genetically linked to one another, comprising so-called global regulatory responses. Bacillus subtilis is a Gram-positive soil organism which can undergo varying developmental responses during the transition from exponential growth. These responses include sporulation, the elaboration of degradative enzymes, increased motility, antibiotic production, induction of the SOS response and the expression of genetic competence. Some of these responses are mutually exclusive" sporuiation, for instance, clearly precludes the other responses. In general however, the relationship among the various possible post-exponential outcomes has not been adequately described as either sequential, mutually exclusive or simultaneous. The regulatory systems governing degradative enzyme production, motility and competence, are discussed by papers appearing in this Forum (Ordal et al., 1994; Kunst et al., 1994). One important generalization to emerge from these and other studies is that postexponential global regulons are controlled by overlapping signal transduction pathways, constituting a network of protein-protein and proteinDNA interactions which culminate in a finely tuned set of transcriptional responses. Another important generalization, which is a corollary of the first, is that few, if any, of the gene products involved in the sensing and transduction of environmental information, are specific for a single form of global response. Thus, genes originally defined as required for the initiation of sporulation may also be required for competence, and alleles of genes identified as essential for competence, or for antibiotic production or degradative enzyme synthesis, may affect sporula-
tion. (A consequence of these interrelationships is that we are faced with somewhat of a nomenclatural nightmare, since genes are named for the phenotypes with which they were first associated). A picture begins to emerge, then, of a signal transduction network, rather than of a series of dedicated pathways. An attractive model is that the network serves to integrate combinatorial signals; each combination of signals eficits a particular set of transcriptional responses. To fully comprehend regulatory networks such as this, new methods and new ways of thinking will be required. In a general sense, the analytical phase of molecular biology, in which individual steps in synthetic or regulatory pathways are dissected, must give way to a synthetic phase, in which we begin to piece together a more holistic picture of the cell and the organism. However, since this review concerns only the competence response, we will analyse regulatory interactions using the paradigm of a pathway, although at the risk of considerable oversimplification.
Competence Competence, the ability of the cell to bind and internalize exogenous high molecular weight DNA, is a naturally expressed physiological state in B. subtilis (reviewed in Dubnau, 1993). When grown in minimal salts media containing glucose and amino acids, a set of novel gene products, encoded by the competence genes, are elaborated beginning at the time of transition to stationary phase (To). This growth stage-regulated response is also nutritionally governed, since glucose is required for competencespecific transcription, and glutamine represses the development of competence. In addition, amino acids repress competence expression during exponential growth, but at T o this repression is overridden (Dubnau et al., 1991). Since competence is expressed in
12th F O R U M I N M I C R O B I O L O G Y only a distinct subfraction of the population, comprising 10-20 o70 of the cells (Hadden and Nester, 1968; Haseltine-Cahn and Fox, 1968), a cell-typespecific form of regulation must also exist, which is not understood. These three modes of regulation, growth-stage-specific, nutritional and cell-typespecific, depend on the transduction of information from a partially described set of environmental and possibly cell-cycle-related signals. When the proper signals are present, a signal transduction cascade relays information to the comK gene, which specifies a competence transcription factor (Mohan and Dubnau, 1990; van Sinderen et al., 1990; van Sinderen, L. Hamoen, A. Luttinger, G. Venema and D. Dubnau, unpubfished). A sharp increase in ComK production results, causing an increase in the transcription of the late competence genes. The increase in comK transcription is mediated by the Mec system (Dubnau and Roggiani, 1990; Kong et al., 1993; Roggiani et al., 1990). The latter consists of two protein, MecA and B, which negatively regulate comK, probably by protein-protein intel-action (Kong and Dubnau, 1994 submitted; Msadek et al., 1994). Since ComK positively regulates its own transcription (D. van Sinderen and G. Venema, personal communication), the interaction of Mec with ComK probably represses the expression of comK. The signal transduction system operating upstream of Mec serves to lift the repression of comK, since mecA or mecB loss-of-function mutations bypass the requirements for the upstream regulatory genes (Roggiani et al., 1990). Here we will describe what i q knnwn nhrmt th,~ early signal transduction steps that elicit the competenoe response, in particular the parts played by three distinct two-component systems in the regulation of comK expression; the comP-comA, degU and spoOA systems. As their names imply, these genes were first identified in relationship to the competence, degradative enzyme and sporulation responses. The degS-degU two-component regulators are discussed in greater detail by Kunst et ai. (1994) in this Forum. The roles of ComP and Coma in the regulation of post-exponential responses
A key player in the competence regulatory pathway is the srfA operon (Nakano et al., 1991a; Nakano and Zuber, 1990; van Sinderen et al., 1990). This locus specifies several proteins required for the post-exponential production of the heptapeptide antibiotic surfactin. It is required for the expression of competence, and appears to play a role, albeit a subtie one, in the regulation of sporulation. The precise part played by srfA in competence development has received a fair amount of attention but is still poorly understood (D'Souza et al., 1993; Nakano et al.,
1991a; Nakano and Zuber, 1993). It suffices for our purpose to suggest that it participates in generating a signal for lifting Mec-mediated repression, srfA transcription, in glucose minimal salts medium, is sharply induced at T_ 1, and this induction is required for the expression of competence. Four loci have been shown to function in the activation of srfA transcription. Two of these, comP and coma are clearly histidine kinase and response regulator members of the two-component regulator family 0Veinrauch et al., 1989; Weinrauch et al., 1990). Analysis of the ComP sequence leads to the prediction that it is an integral membrane protein, with eight transmembrane segments. We have raised antiserum against ComP and shown that it indeed behaves like an integral membrane protein (unpublished). In addition to the conserved N-terminal domain characteristic of response regulators, ComA possesses a C-terminal domain with striking similarity to several DNA-binding proteins that contain helix-turn-helix motifs (Dubnau, 1991). It is noteworthy that DegU and ComA, in particular, share sequence similarity over their entire lengths. ComA, when phosphorylated in vitro by reaction with acetyl-PO4, binds to at least two sites upstream from the srfA promoter (Roggiani and Dubnau, 1993). These sites had previously been predicted to be ComA binding sites (Nakano et al., 1991b), based on mutagenesis studies and by comparison of srfA sequences with those upstream from other genes also regulated by ComA; degQ (Msadek et al., 1991), and gsiA (Mueller et al., 1992). It has proven difficult to demonstrate direct phosphorylation of C o m a by ComP in vitro (unpublished). However, overexpression of ComA bypasses the competence requirement for ComP 0Veinrauch et al., 1990), suggesting that the normal role of the latter protein is to activate ComA. In addition, purified ComA is readily phosphorylated by acetyl-PO4 (Roggiani and Dubnau, 1993) and can also be phosphorylated by purified NRII, kindly provided by A. Ninfa (unpublished). In vitro gel shift and footprinting experiments have demonstrated that the phosphorylated form of ComA has a higher binding affinity for the C o m a boxes upstream from the srfA promoter than does the unphosphorylated form (Roggiani and Dubnau, 1993). We propose therefore, that in response to some signal, ComP and ComA interact so as to increase the concentration of ComA-PO 4, resulting in the transcriptional activation of srfA (fig. 1). Mutational studies on ComA are consistent with this model. Asp residue 55 is predicted to be the residue that accepts a phosphate from ComP. Mutations which convert D55 to either E (D55E) or to N (D55N) fail to complement null mutations in ComA when present in single copy at an ectopic chromosomal location (unpublished). The wild type comA fully complements when located at the same
BACTERIAL SENSOR KINASE/RESPONSE REGULATOR SYSTEMS
ADP~ ATP
ComP-PO 4 -~
405
ComA
ComP ~/' ~ ComA-PO4-l~srfA SpoOK Nutritional signals Extracellular
Factor
~ComQ ComX Fig. 1. The ComA signal transduction cascade as presently understood. comQ and comX apparently generate a competence pheromone (Magnuson et al., 1994) which interacts with SpoOK proteins, ultimately resulting in the enhanced phosphorylation of the transcription factor ComA.
ectopic site. It is interesting that the D55N mutation when present on a multicopy comA plasmid, can complement a deletion of both comP and comA. This suggests that the bypass of a comP mutation by overexpression of ComA mentioned above, can be ascribed to limited function of the unphosphorylated response regulator protein, rather than to phosphorylation by cross-talk. In accordance with this interpretation, in vitro DNA-binding experiments reveal that although the specific binding of ComAP O 4 t o DNA is tighter than that of ComA, the latter does possess a tte~.ec_*able affinity for the C o m a boxes upstream from srfA. Nakano and Zuber (1993) have analysed the C o m a boxes associated with srfA. They have shown that the upstream ComA box is dispensable when the downstream box is altered so as to conform to the ComA box consensus. This was interpreted in terms of a looping model for ComA activity, in which the upstream box serves to raise the local concentration of ComA, facilitating binding of ComA to the downstream box. Our in vitro footprinting studies are consistent with this model; several phosphodiester bonds located between the two C o m a boxes become hypersensitive to DNAse when ComA binding occurs, suggesting that loop formation has occurred (Roggiani and Dubnau, 1993). In addition to comP and comA, two other loci are required for srfA induction; comQ (Weinrauch et al., 1991) and spoOK (Grossman et al., 1991; Hahn and Dubnau, 1991 ; Nakano and Zuber, 1991). spoOK is an operon that specifies the B. subtilis oligopeptide permease (Perego et al., 1991 ; Rudner et al., 1991). The organization of this operon, and
the amino acid sequences of its five predicted protein products, show that this is a typical bacterial ATP-driven permease. The predicted sequence of the ComQ protein, on the other hand, does not resemble that of any member of the GenBank database. The requirements for both comQ and spoOK for competence, as well as the comP requirement (Weinrauch et al., 1990; unpublished), are completely bypas~d when Com~. is overexpress~, sugg~fang that these genes act upstream of ComA in the signal transduction pathway. A multicopy plasmid that overexpresses ComP from the relatively weak Pspac promoter, results in the partial bypass of comQ and spoOK (unpublished). This is consistent with the idea that they act upstream from ComP. Grossman et al. (1991) have shown that B. subtilis secretes a soluble pheromone that acts to turn on srfA transcription. The peptide moiety of this factor is encoded by a small open reading frame (comX) which overlaps the 3' end of comQ (fig. 2) (Magnuson et al., 1994). Inactivation of comQ produces a competence negative pi~enotype, which can be rescued by the addition of exogenous factor (Magnuson et al., 1993, submitte.d). Mutational polarity was excluded as an explanation for this competence deftciency, and it appears, therefore, that ComQ may be required for the processing or secretion of the competence pheromone (fig. 1). Since spoOK specifies an oligopeptide permease, it is attractive to consider that the pheromone, which is at least partly peptide in nature, is detected by cellsurface-localized SpoOK proteins (fig. 1). This has led to the hypothesis that the permease informs ComP, also residing on the cell membrane, about the
406
12th F O R U M I N M I C R O B I O L O G Y
P--I~ P---I~
Ter
Ter
Ter
I,
I,
,I,
0
2
4
6kb
I
I
I
I
Fig. 2. Map of the B. subtiiis chromosomal region containing comQ, comX, comP and comA. The location of the comX gene (which overlaps the 3' end of comQ) is from Magnuson et al. (Magnuson et al., 1994). The locations of known and suspected promoters and terminators are indicated by arrows 0Veinrauch et al., 1989; Weinrauch et al., 1991; Weinrauch et al., 1990).
level of soluble factor present in the extracellular space (Dubnau, 1991; Grossman et ai., 1991). This in turn leads to the accumulation of ComA-PO4, and the activation of srfA transcription. Precedent exists in the Pho regulon (Rao and Torriani, 1990) for the interaction of another permease (psO with two-component regulators (PhoB and PhoR). pst, which encodes a high affinity phosphate uptake system, and the associated gene phoU, serve to reduce the accumulation of the activated response regulator PhoB-PO 4, and therefore to repress expression of the ~ o regulon. Mutations in pst consequently lead to constitutive Pho expression. In contrast, the role of the spoOK operon in competence must be positive, since loss-of-function spoOK mutations lead to loss of competence. It is not known whether signalling requires the internalization of competence pheromone, or whether true transmembrane signalling occurs, as is likely to be the case with Pho (Rao and Ton-iani, 1990). A truncated form of ComP, lacking the hydrophobic N-terminal domain, can complement a total deletion of comP residing on the chromosome (unpublished). Interestingly, this truncated ComP protein is still membrane-localized, although in preliminary expe~ments it behaves like a peripherally attached membrane protein. It is tempting to speculate that the truncated protein attaches to the membrane by protein-protein interaction, perhaps to SpoOK. In the case of intact ComP, in addition to this protein-~rotein interaction, the membrane spanning don~a~:~ presumably associates with the hydrophobic c~r¢ of ~h¢ membraae bilayer. The concentration of soluble competence factor probably serves as a measure of cell density. When grown in a confined space, as in a shaker flask, the
pheromone will accumulate as the cells approach stationary phase, causing induction of srfA transcription at about T 1- Consistent with this hypothesis are experiments in which srfA was placed under control of the IPTG-inducible Pspac promoter (Hahn and Dubnau, 1991; Nakano and Zuber, 1991). Growth in the presence of IPTG resulted in the constitutive expression of competence, although only to about 10 % of the wild type level. The observed incomplete competence induction may be due to the relative weakness of the Pspac promoter. It may also indicate that competence is subject to additional control, which cannot be bypassed by the induction of srfA. In fact it appears that repression by AbrB may be at least part of the explanation for this additional control (see below). Expression of srfA under Pspac control also bypasses the competence requirements for comQ, spoOK, comP and comA. This suggests that the major roles for these four loci in the development of competence is to induce srfA. If the ComPComA system is responding to crowding as opposed to a signal directly indicating growth stage, it may be proper to consider the response to population density to be a fourth mode of competence regulation, in addition to growth stage, cell-type-specific and nutritional signalling. It was mentioned above that competence develops in glucose-containing media. Substitution of glycerol for glucose or the addition of glutamine lowers competence expression markedly, without having a measurable effect on growth rate (Albano et al., 1987; Weinrauch et al., 1990). When ComA is overexpressed, bypassing the spoOK, comQ and comP requirements, glucose is no longer needed for competence development and glutamine no longer inhibits (Weinrauch et al., 1990). It appears that the
BA CTERIAL SENSOR KINASE/RESPONSE REGULATOR S YSTFMS ComP-ComA signal transduction system responds not only to the competence pheromone (via spoOK), but also to the presence or absence of nutritional factors (fig. 1). A final aspect of the ComP-ComA system deserves mention. Using a iacZ fusion we have reported that comA transcription increases 2-3 fold during growth (Weinrauch et al., 1990). Transcription mapping by primer extension revealed that whereas a promoter is located immediately upstream from comA, additional transcriptional readthrough occurs from further upstream. This readthrough transcription appears to increase during growth, probably accounting for the observed increase in comA expression. The location of the Ul~tream promoter is not known. Recent work using antiComP antiserum has suggested that ComP expression also increases during growth (unpublished). A lacZ fusion to comQ, on the other hand, shows that expression of comQ decreases during growth, and primer extension mapping has located a promoter just upstream from this gene 0Veinrauch et al., 1991). In a comQ null mutant, the comA-lacZ fusion is overexpressed (Weinrauch et al., 1991), and use of anti-ComP antiserum suggests that the same is true of ComP expression (unpublished). Perhaps the decrease in comQ expression during growth in competence medium is part of a normal mechanism, causing an increase in ComP and ComA expression, possibly from a promoter located upstream of comP (fig. 2). We do not know how comQ expression regulates the expression of comP-comA. If this is a trans effect exerted by ComQ, the latter may play a more complex role than simply to process or secrete the competence factor.
407
(1990) have constructed a mutation (degU146) in which the Asp residue which receives a phosphate from DegS is altered to Asn. In their hands, this mutation has no effect on competence. In the derivatives of strain IS75 used in our laboratory, however, the degU!46 mutation decreases competence severalhundred-fold (unpublished). This strain difference is not understood, but inspires caution in concluding that unphosphorylated DegU is sufficient for competence under all conditions. Dahl et al. (1991, 1992) have described mutations in DegS and in DegU that result in the accumulation of excess DegU-PO 4. These degS h and degUh mutations have been characterized genetically and biochemically, and result in overexpression of degradative enzymes and the nearly complete suppression of competence. It seems certain that excessive levels of DegU-PO 4 are deleterions for the development of competence, and act at least in part to repress the expression of srfA. The C-ternfinal domains of DegU and of ComA exhibit substantial amino acid sequence similarity (Dubnau, 1991), and it is possible that DegU-PO 4 may repress by competing with ComA-PO 4 for binding to the ComA boxes upstream from srfA. Kunst et al. 0993) have observed that when Na glutamate is substituted for (NH4)2SO4 in competence medium, hi~ther levels of competence are achieved and the degt.ff32 mutation no longer leads to a loss of competence. If, in this medium, a high concentration of ComA-PO 4 accumulates, the latter may overcome the repressive effect of DegU-PO 4. In fact, we have observed that overexpression of C o m a completely eliminates the downregulation of competence due to a degLrh mutation (unpublished). The roles of SpoOA and AbrB in competence
Role of the DegS-DegU system in competence DegS and DegU are, respectively, histidine kinase and response regulator members of a two-component regulatory system (Kunst et al., 1994). They were originally identified as proteins required for the transcriptional activation of a series of post-exponentially expressed genes that specify degradative enzymes. However, null mutations in degU result in the almost total inactivation (about 10*-fold) of competence development (Tanaka and Kawata, 1988), and of comK expression (unpublished). This is in contrast to the 300 to 500-fold effects of comQ, spoOK, comP, comA and srfA inactivation. Despite this dramatic and important effect of degU on competenc e, its targets are not known. It is notable that null mutations in the cognate histidine kinase gene degS have little if any effect on competence (Msadek et ai., 1990). Therefore, either phosphorylation of DegU is not needed for competence or DegU can be phosphorylated by some other pathway. Msadek et al.
SpoOA is a response regulator protein that was initially characterized as a key player in the regulation of sporulation, and which is also required for the expression of competence genes (Albano et al., 1987; Piggot and Coote, 1976; Sadaie and Kada, 1983; Spizizen, 1965). Null mutations in spoOA have a relatively minor negative effect (2-3-fold) on the expression of srfA (Hahn and Dubnau, 1991). However, they dramatically depress the expression of comK and therefore of the late competence genes (unpublished). Unlike all of the regulatory genes so far mentioned except for comK, loss-of-function mutations in mecA or mecB do not bypass the spoOA requirement for competence (Roggiani et al., 1990), suggesting that spoOA acts downstream from Mec. Certain phenotypes resulting from spoOA ina~ivation are known to be suppressed by mutations in abrB. The latter gene specifies a DNA binding protein that usually acts negatively, but can also have a positive effect on the transcription of certain genes
12th FORUM IN MICROBIOLOGY (Strauch, 1993). The suppression is due to the fact that one important role for SpoOA is to downregulate the expression of AbrB (Perego et al., 1988). Loss-of-function abrB mutations therefore suppress some of the phenotypes resulting from the absence of SpoOA. In this regard, our situation is complex, since AbrB plays a positive role in competence (Aibano et al., 1987) and specifically in the activation of comK expression (unpublished). Several observations however, provide clues concerning the role of spoOA in competence. A knockout of SpoOA depresses competence about 1,000-fold, and a null mutation in abrB depresses competence only about 40- to 50-fold. A double spoOA abrB mutant exhibits the same competence level as does the single abrB mutant, suggesting that the major role for SpoOA in competence is to prevent the excessive accumulation of AbrB (Albano et al., 1987). Since abrB lossof-function mutations are bypassed by mec mutations (Rog~aril et al., 1990), the positive role of AbrB must be exerted upstream from Mec. Since deletions in spoOA are not bypassed by mec mutations, the negative role of AbrB must be exerted downstream of Mec. We believe that AbrB acts by binding directly to the comK promoter as a rcpressor. Consistent with this is our observation that lacZ fusions to comG, a late competence gene, are overexpressed in a spoOA abrB mecA mutant (unpublished). The positive role of AbrB is bypassed by mecA, and spoOA is no longer needed in the abrB background. Ordinarily, SpoOA is phosphorylated at about T 0, and it appears likely that this phosphorylation is required for competence. Green et al. (1991) have constructed a SpoOA derivative in which the Asp residue normally targeted for phosphoryiation is converted to a Gin (D56Q). This mutation results in a sporulation-negative phenotype (Green et ai., 1991) and also in a severe decrease in competence (B.D. Green and P. Youngman, personal communication). However, the level of competence observed in this mutant background is about 10-fold higher than in a spoOA null mutant, suggesting that the unphosphorylated protein may retain some activity, presumably preventing excessive accumulation of AbrB. One caveat is that the stability of the mutant protein may be reduced and the observed effect may be due to this instability rather than to the absence of phosphorylation. Another is that the mutation in SpoOA may compromise some function of the protein in addition to its ability to be phosphorylated. However, Green and Youngman have combined the D56Q mutation with a small deletion in SpoOA that results in phosphorylation-independent sporulation (Green et al., 1991). The double mutant exhibits nearly normal competence, strongly suggesting that phosphorylation of SpoOA plays a role in competence signalling. SpoOA can be phosphorylated via an unusual
phosphorelay (Burbulys et al., 1991 ; Hoch, 1993) in which a histidine kinase (SpoIIJ/KinA) donates a phosphate to another response regulator (SpoOF). The phosphate is then transferred to SpoOB and finally to SpoOA. SpoOA-PO 4 is responsible for the transcriptional induction or repression of several do~.nstream genes. Although SpoOA is required for competence, null mutations in kinA (unpublished), spoOB or spoOF (Albano et al., 1987) have minor effects on transformability, suggesting that the KinA/SpoIIJ pathway may not be utilised in competence-specific signalling. SpcOA can also be phosphorylated by at least one other pathway, which utilizes KinB, another histidine kinase (Burbulys et al., 1991). It is not clear whether KinB signalling plays a role in competence.
The competence pathway and the signal transducing roles of ComA, SpoOA and DegU The information supplied above can be summarized in the form of a pathway rather than a network, since only a single outcome (competence) is considered. The scheme shown in figure 3 includes the genes discussed above as well as sinR (Smith, 1993) which is a DNA binding protein that acts negatively on sporulation but is somehow required for the activation of comK transcription. The three response regulators (ComA, DegU and SpoOA) intersect the pathway at distinct points. Each represents a potential control point, although the signals involved are still incompletely understood. The ComA branch of the pathway, as noted ~ L I v ,JV~,,.,
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nutritional signals (fig. 1, fig. 3). ComA, ComP and ComQ are also required for the transcriptional induction of degQ, a regulator of degradative enzyme expression, degQ transcription is additionally dependent on degU and degS. The ComQ dependency implies that degQ expression may respond to crowding via the competence pheromone and it would be interesting to determine whether spoOK null mutations affect degQ expression. Distinct regions upstream from the degQ promoter respond to degU and to comA. The comA but not the degU-dependent response can be induced by amino acid starvation. Thus, several lines of evidence suggest that ComP and ComA respond to a variety of nutritional and population density signals, p k s X is a polyketide synthase locus of unknown function (Scotti et al., 1993) that like degQ, requires comQ, comP, comA, degS and degU for its full expression (A. Albertini, C. Scotti and A. Galizzi, personal communication). gsiA is a glucose starvation-induced gene, which requires comP and comA, but neither comQ nor spoOK for its induction (Mueller et al., 1992). It therefore appears that ComA and ComP can function independently of the spoOK proteins in the regu-
BACTERIAL SENSOR KINASE/RESPONSE REGULATOR SYSTEMS
409
Crowding (ComQ,ComX), Nutritional Signals
/
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Fig. 3. Summary of the competence regulatory cascade as described in the text. Arrowheads and heavy perpendicular lines indicate positive and negative effect, respectively. Lines with solid dots indicate transcriptional regulation and those with empty dots indicate protein-protein interaction or post-translational protein modification.
lation of at least gsiA. As in the case of degQ, the comQ dependency leads to the prediction that pksX expression will also require spoOK. The SpoOA branch probably also senses growth stage, n e r h a n • m n r o c l i r o e t l y t h a n ¢. .;.m. . n l .w, I j a s a .~. A. . .~ . ~ ure of crowding, although it also responds to population density and nutritional signals in the regulation of sporulation (Ireton et aL, 1993). For instance, Grossman and his co-workers have shown that SpoOA can transduce DNA replication signals to the sporulation system (Ireton and Grossman, 1992). Like ComA therefore, SpoOA is clearly able to transduce disparate signals, and can additionally interact with at least two phosphate donors. The SpoOA signalling pathway responsible for the regulation of competence is not understood. Since AbrB plays both negative and positive roles in the control of competence, and can bind to DNA cooperatively to control transcription (Strauch, 1993), it is likely that the activation of SpoOA must be closely regulated so as to maintain the AbrB concentration within narrow limits. In fact, the use of a construct in which abrB transcription is driven by the IPTG-regulatable Pspac promoter has shown that competence requires a narrow range of IPTG concentrations, above and below which transformability is inhibited (unpublished). The competence signal(s) detected by DegU are not understood, and the nature of the signals that .
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trigger DegU phosphorylation by DegS are likewise obscure. Since excessive DegU-PO 4 inhibits competence development, some control probably limits the extent of phosphorylation. On the other hand, as discussed above, in the IS75 strain background, it is possible that some phosphorylation of DegU must occur for competence indu~ion. This presents a situation similar to that with SpoOA and AbrB; perhaps DegU phosphorylation must be maintained within narro~ ~ limits for competence expression. The work described from our laboratory was supported by NIH grants AII0311 and GM43756. We thank the members of our laboratory and that of I. Smith for many useful discussions, as well as T. Msadek, F. Kunst, G. Rapoport, R. Magnuson, J. Solomon, A.D. Grossman, A. AIbertini, C. Scotti and A. Galizzi for further discussions and for providing information prior to pubfication.
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12th F O R U M I N M;,. ." " R I O L O G Y
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~I.YUI UIdLIUII
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Dggg-illl~,.~
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Spizizen, J. (1965), Analysis of asporogenic mutants in Bacillus subtilis by genetic transformation, in "Spores III" (L.L. Campbell & H.O. Halvorsen) (pp. 125-137). American Society for Microbiology, Washington, DC. Strauch, M. (1993), AbrB, a transition state regulator, in "Bacillus subtilis and other Gram-positive bacteria. Biochemistry, physiology, and molecular genetics" (A.L. Sonenshein, J.A. Hoch & R. Losick) (pp. 757-764). American Society for Microbiology, Washington, DC. Tanaka, T. & Kawata, M. (1988), Cloning and characterization of Bacillus subtilis iep, which has positive and negative effects on production of extracellular proteases. J. Bacteriol., 170, 3593-3600. Van Sinderen, D., Withoff, S., Boels, H. & Venema, G. (1990), Isolation and characterization of comL, a transcription unit involved in competence developmerit of Bacillus subtilis. Mol. Gen. Genet., 224, 396-404. Weinrauch, Y., Guillen, N. & Dubnau, D. (1989), Sequence and transcription mapping of Bacillus subtilis competence genes comb and comA, one of which is related to a family of bacterial regulatory determinants. J. BacterioL, 171, 5362-5375. Weinrauch, Y., Msadek, T., Kunst, F., Rapoport, G. & Dubnau, D. (1991), Sequence and properties of comQ, a new competence gene of Bacillus subtilis. J. Bacterioi., 173, 5685-5693. Weinrauch, Y., Penchev, R., Dubnau, E., Smith, I. & Dubnau, D. (1990), A Bacillus subtilis regulatory gene product for genetic competence and sporulation resembles sensor protein members of the bacterial two-component signal-transduction systems. Genes & Dev., 4, 860-872.