Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, Kerklaan 30, 9751 NN Haren, The Netherlands
Correspondence
Sierd Bron
S.Bron{at}biol.rug.nl
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ABSTRACT |
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Abbreviations: Cm, chloramphenicol; Opp, oligopeptide permease; Phr, phosphate regulator; Rap, regulator aspartate phosphatase; Sp, spectinomycin
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INTRODUCTION |
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Most B. subtilis 168 rap genes are transcribed in operons with genes that encode their antagonistic Phr peptides (Kunst et al., 1997). While the Rap proteins remain in the cytoplasm, Phr peptides contain an amino-terminal signal peptide and have the potential to be exported as pro-peptides, most likely via the Sec pathway (Tjalsma et al., 2000
). Further extracellular processing results in active Phr pentapeptides with a weakly conserved XRXXT sequence (Meijer et al., 1998
; Perego, 1997
). After reimport via the oligopeptide permease (Opp) system, Phr peptides specifically inhibit the activity of their cognate Rap protein (Perego, 1997
, 1998
; Meijer et al., 1998
). However, four of the eleven known rap genes are not followed by functional phr genes, suggesting that these are not subject to quorum sensing. Interestingly, rapphr operons were also found on several endogenous rolling-circle plasmids from B. subtilis, but no experiments were carried out to find target genes that can be regulated by this class of extra-chromosomally encoded RapPhr systems (Uozumi et al., 1980
; Meijer et al., 1998
).
The chromosomal-encoded RapPhr systems orchestrate the sequential programs of global gene expression in a growing culture of B. subtilis cells. During exponential growth (low cell densities), RapC dephosphorylates ComA~P, thereby inhibiting competence development. At the end of the exponential growth phase, RapC is inactivated by the accumulation of the PhrC peptide and competence development is stimulated (Mueller et al., 1992; Roggiani & Dubnau, 1993
; Lazazzera et al., 1997
, 1999
). During the transition phase between exponential and post-exponential growth, sporulation is repressed, due to the stimulation of rapA and rapE expression via ComA~P. This results in high cellular levels of RapA and RapE that together dephosphorylate Spo0F (Jiang et al., 2000b
; Perego et al., 1996
). During the post-exponential growth phase, when nutrients become limiting, RapA and RapE are antagonized by PhrA and PhrE, respectively, and sporulation will be initiated via Spo0F~P. Although the production of degradative enzymes is known to be increased during high cell densities via the DegSDegU two-component system (Msadek et al., 1990
), no involvement of Rap proteins in the repression of protein secretion at low cell densities has been documented thus far. The phosphorylation state of the DegU response regulator, mediated by the sensor DegS, acts as a molecular switch allowing either the development of genetic competence or the production of degradative enzymes (Dahl et al., 1992
; Kunst et al., 1994
; Kunst & Rapoport, 1995
).
In this study, we show that the pTA1060-encoded phosphatase Rap60 can actually repress the production of proteolytic enzymes in B. subtilis 168. Expression of aprE, encoding the major extracellular proteolytic enzyme subtilisin, is strongly repressed in cells containing Rap60. However, co-expression of Phr60 or the addition of synthetic Phr60 peptide restored aprE transcription under these conditions. The regulatory cascade affected by Rap60 comprises, most likely, components of the phosphorelay (Hoch, 1993) and the transition-state regulator AbrB (Strauch & Hoch, 1993
). These data show for the first time that endogenous plasmids of B. subtilis contain RapPhr systems that have a function in the cell-density-controlled production of at least one secreted protease. This may be of particular importance for the fitness of B. subtilis that can secrete large amounts of proteins into the medium as an adaptive response to environmental fluctuations.
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METHODS |
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Construction of xylose-inducible rap60 and/or phr60 genes.
Functional analysis of the rap60phr60 operon was performed with three constructs in which rap60, phr60 or both genes were placed under control of the PxylA' promoter on the integration plasmid pX (Kim et al., 1996). To this purpose, PCR reactions with pTA1060 as DNA template were performed with the following primer sets (mismatches with the original sequence of pTA1060 are indicated in lower case; restriction sites are underlined). First, prRap1 (5'-gctctagaTTA AAT TAG GGG AGG AG-3') and prRap2 (5'-cgggatccGGA CCG CTG AAA AAA GACC-3') were used to amplify rap60 (1174 bp). Second, prRap3 (5'-gctctagaGAG ATG GTG TGC GCT CAA AG-3') and prRap4 (5'-cgggatccCAT GGT CGC ACA CAA AG-3') were used to amplify phr60 (246 bp). Third, prRap1 and prRap4 was used to amplify the rap60phr60 operon (1420 bp). After digestion of these fragments with XbaI and BamHI, these were ligated into the SpeI and BamHI sites of pX, resulting in pXR (rap60) and pXP (phr60) pXRP (rap60phr60). Next, B. subtilis 168 was transformed with these plasmids, resulting in the integration of these constructs in the amyE locus by homologous gene replacement (Table 1
, Fig. 1
). Integration into the amyE locus of transformants was confirmed by the lack of halo formation upon growth on plates containing 1 % starch.
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Sporulation assay.
The efficiency of sporulation was determined by overnight growth of cells in SSM medium, killing of the cells with 0·1 vol. chloroform and subsequent plating.
ß-Galactosidase assays.
Overnight cultures were diluted to an OD600 of 0·1 in fresh TY medium, supplemented with Phr60 pentapeptide when desired. Samples for OD600 readings and ß-galactosidase activity determinations were taken at hourly intervals. The assay and the calculation of ß-galactosidase activity (expressed as units ß-galactosidase activity per OD600 unit) of the samples were performed as described by Miller (1982). Experiments were repeated at least three times and the relative effects within one experiment were reproducible.
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RESULTS |
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Synthetic Phr60 pentapeptide is imported by the Opp system and inhibits Rap60
Although the partial restoration of halo formation on skimmed milk plates by the co-expression of Phr60 suggests that this peptide inhibits the activity of Rap60 (Fig. 2), the export and reimport of this peptide from and back into the cell remained unproven. To investigate whether the Phr60 peptide could be imported and antagonize Rap60 activity, the deduced active Phr60 pentapeptide (SRNAT; see Meijer et al., 1998
) was synthetically produced. Next, transcription of aprElacZ was monitored in B. subtilis ALR (xylose-inducible Rap60 expression) grown in TY medium with xylose to which 1, 5 or 10 µM Phr60 pentapeptide was added. As shown in Fig. 4
(a), the addition of Phr60 caused a concentration-dependent increase in aprElacZ transcription under these conditions. In contrast, the addition of 1 µM Phr60 had no effect on the aprElacZ transcription level in the parental strain 168 (AL). These observations show that extracellularly added Phr60 pentapeptides can fulfil an intracellular role in the inhibition of Rap60.
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Rap60 acts on a component of the phosphorelay
Previous studies showed that mutations in spo0 genes resulted in a drastic decrease in aprE transcription, while a suppressor mutation in the abrB gene restored high-level transcription of aprE in these strains (Ferrari et al., 1988). As overproduction of Rap60 causes a decrease in aprE transcription, like spo0 mutations, possible targets for Rap60 are Spo0F~P, Spo0B~P and Spo0A~P, which sequentially form the phosphate-transfer chain of the phosphorelay (Hoch, 1993
). To investigate whether Rap60 acts on a phosphorylated component of the phosphorelay, the abrB mutation was introduced into the B. subtilis strain ALR (aprElacZ; xylose-inducible Rap60 expression). The rationale of this experiment is that, in the absence of AbrB, decreased levels of Spo0A~P would not have an effect on aprE transcription, thereby creating a phosphorelay bypass' (Ferrari et al., 1988
). As shown in Fig. 5
(a), the expression of Rap60 in cells grown in TY medium with 1 % xylose had no effect on the level of aprElacZ transcription in the abrB genetic background. Taken together, these observations show that by creating a phosphorelay bypass, the inhibitory effect of Rap60 on aprE transcription is diminished, indicating that Rap60 might indeed dephosphorylate one or more components of the phosphorelay.
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DISCUSSION |
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Our present studies suggest that the presence of a plasmid-borne RapPhr system from B. subtilis provides this organism with another mechanism to regulate its patterns of gene expression. However, instead of SPase overproduction, under conditions of high-level protein secretion, this Rap60Phr60 system can actually regulate the production of secreted proteases. As indicated by the results obtained from the experiments on the uptake of Phr60 pentapeptide, this regulation is cell-density controlled and repression of protease production occurs under conditions of low cell densities. This property of the Rap60Phr60 system may be important for the fitness of B. subtilis under natural conditions, as it prevents protease production under nutrient-rich conditions, thereby making more efficient use of the available energy sources. Even though these considerations are based on experiments with B. subtilis 168-derived strains, we believe that they are also relevant for B. subtilis strains carrying plasmids pTA1015, pTA1040 or pTA1060. The latter B. subtilis strains produce capsular poly--glutamate and are used for the fermentation of soy beans to produce natto, a traditional Japanese food product. These B. subtilis (natto) strains and B. subtilis 168 are regarded as different isolates of the same organism and are genetically closely related (Priest, 1993
). In this respect, it should be noted that in contrast to B. subtilis (natto) strains, B. subtilis 168 contains a mutation in the degQ promoter causing low DegQ production (Yang et al., 1986
; Msadek et al., 1991
). As AprE production is to a certain extent regulated by the ComPComA quorum-sensing system via DegQ, the latter mutation causes reduced levels of aprE transcription in B. subtilis 168. Finally, the presence of several other endogenous plasmids with genes for RapPhr systems found in natural B. subtilis soil isolates (our unpublished observations), suggests a general importance of plasmid-borne RapPhr systems in the natural habitat(s) of this organism.
Our data indicate that the Rap60Phr60 system is analogous to the known chromosomal-encoded systems, as schematically depicted in Fig. 6. Cells containing pTA1060 express Rap60 which remains in the cytoplasm and Phr60 which is secreted into the medium. Rap60 is likely to be involved in the dephosphorylation of one of the components of the phosphorelay (Spo0FSpo0BSpo0A), thereby repressing aprE transcription. Previous studies have shown that an abrB mutation restored aprE transcription in spo0 mutants (Ferrari et al., 1988
). This indicated that AbrB is a preventer of aprE transcription when Spo0A~P is not available (Strauch & Hoch, 1993
). Surprisingly, our results suggest that AbrB is directly, or indirectly, also an activator of aprE transcription, as the expression of aprE decreases when abrB is disrupted (Fig. 5a
). As the transcription of aprE is regulated by complex systems involving at least ten known regulators (Kallio et al., 1991
; Smith, 1993
; Strauch & Hoch, 1993
; Bolhuis et al., 2000
), the exact role of AbrB in aprE regulation is not completely clear at the moment. Nevertheless, expression of Rap60 did not affect the transcription of aprE in an abrB mutant background (Fig. 5a
). In contrast, expression of Rap60 did affect the transcription of spoIIA (Fig. 5b
), a well-known target gene of the phosphorelay (Trach et al., 1991
). Together, these observations strongly suggest the existence of a Rap60phosphorelayAbrB regulatory cascade involved in aprE transcription. At high cell densities, the extracellular Phr60 concentration increases and is internalized by the Opp system. Finally, these high intracellular Phr60 concentrations cause inhibition of Rap60, more phosphorylated Spo0A, less AbrB and eventually increased AprE production.
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The reason for presence of RapPhr systems on the B. subtilis plasmids pTA1015, pTA1040 and pTA1060 (Meijer et al., 1998) is presently not clear. The identification of these systems on endogenous plasmids of B. subtilis strains producing poly-
-glutamate suggests that RapPhr systems may have a role during the fermentation of soy beans for the production of natto. Therefore, these plasmid-borne rapphr operons might be involved in the cell-density controlled regulation of secreted proteases necessary for producing poly-
-glutamate (Birrer et al., 1994
; Nagai et al., 2000
). Alternatively, these plasmid-encoded RapPhr systems might be important for the temporal repression of protease production during natto production. Finally, this system may be important for the, until now unidentified, regulation of other plasmid-borne or chromosomal genes.
At least one further issue that needs to be addressed is whether only plasmid-encoded RapPhr systems are involved in cell-density controlled protease production. As Rap60 seems to be acting on a component of the phosphorelay, other chromosomal-encoded Raps that have been shown to be involved in the phosphorelay might be involved in protease production as well. The latter idea is corroborated by some lines of evidence. First, the most closely related chromosomal-encoded homologue of Rap60 is RapE (our unpublished observations), which is involved in the cell-density controlled dephosphorylation of Spo0F~P (Jiang et al., 2000b). Second, high Phr60 concentrations result in aprE transcription levels that exceed those of the parental strain (our unpublished observations), suggesting that other chromosomal-encoded Raps can be inhibited by the Phr60 pentapeptide. As the Rap60 active pentapeptide (SRNAT) is quite similar to PhrE (SRNVT), a likely candidate for being subject to Phr60 inhibition is RapE. Furthermore, the inhibitory effect of Phr60 on Rap60 can be mimicked by the PhrE peptide, albeit with a much lower efficiency (our unpublished observations). These data indicate that cross-talk may exist between the plasmid- and chromosomal-encoded RapPhr systems. Taken together, these findings are important to increase our insight into how B. subtilis can exploit (plasmid-borne) RapPhr systems for the modulation of extracellular protease production during highly fluctuating environmental conditions.
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ACKNOWLEDGEMENTS |
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Received 15 May 2002;
revised 24 September 2002;
accepted 24 September 2002.
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