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1 Department of General Microbiology, Georg-August-University, 37077 Göttingen, Germany
2 Department of Biomedical Sciences, University of Maryland Dental School, Baltimore, MD 21201, USA
3 Department of Microbiology, Cornell University, Ithaca, NY 14853-8101, USA
Correspondence
Thorsten Mascher
tmasche{at}gwdg.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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, 2006; Walsh, 2003). Its integrity is closely monitored to detect and counteract threats before their action can lead to irreversible damages. The LiaRS two-component system (TCS) is part of the complex regulatory network that orchestrates the cell-envelope stress response in Bacillus subtilis (Mascher et al., 2003). It responds strongly to the external presence of cell wall antibiotics that interfere with the lipid II cycle, such as bacitracin, ramoplanin, vancomycin or cationic antimicrobial peptides (Mascher et al., 2004; Pietiäinen et al., 2005). It is also induced by alkaline shock, detergents, ethanol, phenol, organic solvents and secretion stress, albeit to a lesser extent (Hyyryläinen et al., 2005; Mascher et al., 2004; Petersohn et al., 2001; Pietiäinen et al., 2005; Tam le et al., 2006; Wiegert et al., 2001).
The LiaRS TCS is functionally and genetically linked to a third protein, LiaF, which acts as a strong inhibitor of LiaR-dependent gene expression (Jordan et al., 2006). The LiaRS–LiaF three-component system is conserved by sequence and genomic context in Gram-positive bacteria with a low G+C content (Firmicutes) (Jordan et al., 2006; Mascher, 2006), and LiaRS-homologous TCSs are also involved in responding to cell envelope stress in Bacillus licheniformis, Streptococcus pneumoniae and Staphylococcus aureus (Haas et al., 2005; Kuroda et al., 2003; Wecke et al., 2006). It is interesting to note that membrane-anchored inhibitory proteins, working together with a classical TCS, have also been described for the cell-wall-related, essential TCS YycFG; the YycH and YycI proteins both inhibit the YycG kinase (Szurmant et al., 2007).
In B. subtilis, only two promoters are known to be regulated by the LiaRS TCS: the liaI promoter (PliaI) and the yhcY promoter (Jordan et al., 2006), with PliaI being the primary target. In contrast to PliaI, a LiaR-dependent PyhcY activity was only observed in a liaF mutant, i.e. in the absence of the LiaRS-inhibitor protein (Jordan et al., 2006; Mascher et al., 2004). PliaI is tightly regulated: in the absence of a stimulus, it is virtually switched off, while addition of bacitracin results in about 200-fold induction (Mascher et al., 2003, 2004).
The lia locus consists of six genes, liaIH–liaGFSR. A basal expression level of the last four genes, liaGFSR, encoding the three-component system (liaFSR) and a putative membrane-anchored hypothetical protein (liaG), is ensured by a weak constitutive promoter upstream of liaG. In contrast, expression of the liaIH operon from PliaI is completely LiaR-dependent (Jordan et al., 2006). LiaI is a small hydrophobic protein of unknown function with two putative transmembrane helices. LiaH is a member of the phage-shock protein family (see below). While the strong induction of liaIH (and to a lesser degree also liaGFSR) by cell envelope stress is well documented (see above), mutational analysis of the lia locus has so far failed to identify strong phenotypes associated with these genes. Deletion of lia genes did not alter the sensitivity of the corresponding mutants to the known inducers of the Lia system. Moreover, none of the complex differentiation processes of B. subtilis (i.e. sporulation, competence for genetic transformation, motility and pellicle and fruiting body formation) was affected in lia mutants. So far, the only phenotype that could be linked to the Lia system is delayed spore germination in a liaH mutant (D. Hoyer & T. Mascher, unpublished). Moreover, LiaH seems to negatively affect the expression of the yhcYZ operon by a currently unknown mechanism. It is weakly inducible by bacitracin only in a liaH mutant, but not in the wild-type (Mascher et al., 2003).
While the physiological role of LiaI and LiaH remains obscure, we noted some similarities between LiaH and phage-shock protein A (PspA) of Escherichia coli. The latter is induced by various stress conditions such as filamentous phage infection (hence the name), heat shock, osmotic shock and exposure to organic solvents and proton ionophores as well as prolonged incubation under alkaline conditions (Brissette et al., 1990; Kobayashi et al., 1998; Weiner & Model, 1994). This inducer spectrum shows some overlap with the known inducers of liaIH expression, which include organic solvents and alkaline shock (Mascher et al., 2004; Wiegert et al., 2001). PspA exhibits a dual function that is linked to two different cellular locations (Brissette et al., 1990; Kleerebezem & Tommassen, 1993). Peripherally bound to the inner surface of the cytoplasmic membrane (through protein–protein interactions), PspA is somehow involved in the maintenance of cell membrane integrity (Darwin, 2005; Kleerebezem et al., 1996). As a free cytosolic protein it inhibits the AAA+ enhancer protein PspF, also through protein–protein interactions (Adams et al., 2003; Bordes et al., 2003; Dworkin et al., 2000). Based on the strong induction of liaH by cell envelope stress and its co-transcription with liaI, which encodes a small putative membrane protein, we speculate that LiaI serves as a membrane anchor for LiaH, thereby facilitating an activity that might somehow be linked to envelope integrity.
Here, we investigated the intrinsic activity and regulation of PliaI in the absence of exogenous stimuli. We show that PliaI is induced at the onset of stationary phase. This time point in the B. subtilis life cycle is characterized by the initiation of a complex regulatory cascade that allows the bacterium to adapt to worsening living conditions, which can ultimately lead to the formation of dormant endospores (Errington, 2003; Msadek, 1999; Phillips & Strauch, 2002). We demonstrate that PliaI is directly repressed by binding of the transition-state regulator AbrB within the promoter sequence, thereby acting as a roadblock to prevent premature PliaI activity during exponential growth. AbrB repression is released during the transition state by Spo0A, the master regulator of sporulation, and PliaI is induced by an unidentified endogenous stimulus, resulting in the expression of the liaIH operon. While AbrB binding is sufficient to inhibit the endogenous growth-dependent induction of PliaI, it can be bypassed completely by exogenous induction with cell wall antibiotics.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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. Kanamycin (10 µg ml–1), chloramphenicol (5 µg ml–1), spectinomycin (100 µg ml–1), tetracycline (10 µg ml–1), and erythromycin (1 µg ml–1) plus lincomycin (25 µg ml–1) for macrolide-lincosamide-streptogramin (MLS) resistance were used for the selection of the B. subtilis mutants used in this study. Transformation was carried out as described previously (Harwood & Cutting, 1990).
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) and was performed as described previously (Mascher et al., 2003). In brief: resistance cassettes were amplified from a suitable vector as template (Guerout-Fleury et al., 1995; Youngman, 1990). Two primer pairs were designed to amplify 
1000 bp DNA fragments flanking the region to be deleted at its 5'- and 3'-ends. The resulting fragments are here called ‘up' and ‘do' fragments. The 3'-end of the up-fragment and the 5'-end of the do-fragment extended into the gene(s) to be deleted in a way that all expression signals of genes up- and downstream of the targeted genes remained intact. Extensions of 25 nucleotides were added to the 5'-end of the ‘up-reverse' and the ‘do-forward' primers that were complementary (opposite strand and inverted sequence) to the 5'- and 3'-ends of the amplified resistance cassette. All fragments obtained were purified using the PCR-purification kit from Qiagen. In a second PCR 100–150 ng of the up- and do-fragments and 250–300 ng of the resistance cassette were used together with the specific up-forward and do-reverse primers at standard concentrations. In this reaction the three fragments were joined by the 25 nucleotide overlapping complementary ends and simultaneously amplified by normal primer annealing. The PCR products were directly used to transform B. subtilis. Transformants were screened by colony-PCR, using the up-forward primer with a reverse-check primer annealing inside the resistance cassette (Table 2). The integrity of the regions flanking the integrated resistance cassettes was verified by sequencing PCR products of 1000 bp amplified from chromosomal DNA of the resulting mutants. Sequencing was performed in-house by the GenoMIK centre. All PCRs were done in a total volume of 50 µl (10 µl for colony PCR) using the HotStar DNA-Polymerase Mastermix (Qiagen) or TripleMaster Polymerase Mix (Eppendorf) according to the manufacturers' procedures. The constructed strains are listed in Table 1
. The primers used are listed in Table 2.
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). The genomic regions 1 kb upstream and downstream of liaS were amplified using primers listed in Table 2 [fragments liaS(clean) up, and liaS(clean) down], thereby introducing a 26 bp extension to the 3'-end of the up-fragment, which is complementary to the 5'-end of the down-fragment. The two fragments were fused in a second, joining, PCR, and the resulting fragment was cloned into pMAD via BamHI and EcoRI, generating pMM101. Generation of the clean deletion basically followed the established procedure (Arnaud et al., 2004). In brief: B. subtilis W168 was transformed with pMM101 and incubated at 30 °C with MLS selection on LB agar plates supplemented with X-Gal. Blue colonies were picked and incubated for 6–8 h at 42 °C in LB medium with MLS selection, resulting in the integration of pMM101 into the chromosome. Again, blue colonies were picked from LB (X-Gal) plates and incubated for 6 h in LB medium without selection. Subsequently, the liquid culture was shifted to 30 °C for 3 h, and the cells were then plated on LB (X-Gal) plates, this time without selective pressure. White colonies that had lost the plasmid were picked and checked for MLS sensitivity. Those harbouring a clean deletion of liaS ( 50 % of the white clones) were identified by PCR.
Measurement of induction by β-galactosidase assay.
For time-course experiments on PliaI induction, the respective reporter strains (listed in Table 1) were inoculated in LB medium from a fresh mid-exponential pre-culture to OD6000.1 and incubated at 37 °C with aeration. Samples (2 ml) were taken every hour and OD600 was monitored to follow growth of the cultures. The pellets were resuspended in 1 ml working buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4, 20 mM β-mercaptoethanol) and suitable dilutions were assayed for β-galactosidase activity, with normalization to cell density (Miller, 1972). For induction experiments, the cells were inoculated from fresh overnight cultures and grown in LB medium at 37 °C with aeration until they reached OD6000.6. The culture was split, adding bacitracin (50 µg ml–1 final concentration) to one half (induced sample) and leaving the other half untreated (uninduced control). After incubation for an additional 30 min at 37 °C with aeration, 2 ml of each culture was harvested and the cell pellets were frozen and kept at –20 °C. The pellets were resuspended in 1 ml working buffer and assayed for β-galactosidase activity as described with normalization to cell density (Miller, 1972).
Western blotting.
Total cytoplasmic proteins were prepared from 15 ml culture per time point by using a French press. Proteins (20 µg per lane) were separated by SDS-PAGE, according to standard procedure (Sambrook & Russell, 2001). After electrophoresis the gels were equilibrated in transfer buffer [15.2 g Tris; 72.1 g glycine; 750 ml methanol (100 %) in a final volume of 5 l with deionized water] for 30 s. A PVDF membrane was activated with methanol (100 %) and subsequently incubated in transfer buffer for 5 min. The proteins were blotted to this membrane using a semi-dry blot apparatus. After transfer (1 h at 0.8 mA cm–2) the membrane was incubated in blotto [1xTBS (50 mM Tris, 150 mM NaCl, pH 7.6), 2.5 % skim milk] overnight to prevent non-specific binding. The LiaH antibody [polyclonal rabbit antisera raised against purified His10–LiaH (F. Kalamorz & T. Mascher, unpublished) at SEQLAB (Göttingen, Germany)] was diluted 1 : 20 000 in blotto. After incubation for 3 h, the membrane was washed three times for 30 min with blotto. The secondary antibody [anti rabbit IgG, coupled with alkaline phosphatase (Roche Diagnostics)] was diluted 1 : 100 000 and the membrane was incubated for 30 min. After three more 20 min washing steps, the membrane was washed with deionized water and incubated in buffer III (0.1 M Tris, 0.1 M NaCl; pH 9.5) for 5 min to adjust the pH. For LiaH detection, 10 µl CDP-Star chemiluminescence substrate (Roche Diagnostics) in 1 ml buffer III was used. The signal was documented with a ChemiSmart LumiImager (peqlab).
DNase I footprinting assays.
AbrB and Abh purification and DNase I footprinting assays were performed essentially as described previously (Bobay et al., 2006; Strauch et al., 1989). The DNA target was a 287 bp fragment containing the liaI promoter region (positions –159 to +128), end-labelled with 32P on the template strand. The binding reactions were performed at 20 °C at pH 8 for AbrB, and pH 7 for Abh (the optimal pHs for each protein's binding) (Bobay et al., 2006).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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; Pietiäinen et al., 2005). To investigate if PliaI is also induced in their absence, we examined expression of a PliaI–lacZ fusion in the reporter strain BFS2470. This strain harbours an insertion of the vector pMUTIN (Vagner et al., 1998) inside the liaI coding sequence, thereby bringing a promoterless lacZ gene under control of the liaI promoter (Mascher et al., 2003, 2004).
To study PliaI activity in the absence of external stimuli, BFS2470 was grown in LB medium with MLS selection over a period of 8 h and samples were taken every hour from mid-exponential to late-stationary growth phase. The cells were harvested and β-galactosidase activity was determined, essentially as described previously (Mascher et al., 2004). The results demonstrate that PliaI is induced 8–10-fold without addition of cell-wall antibiotics during transition to stationary phase (Fig. 1a, grey bars). This induction is completely LiaR-dependent: no PliaI activity was observed in the isogenic liaR mutant strain TMB011 (Fig. 1a, black bars).
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). Total cellular protein was prepared and Western analysis performed with LiaH antibodies. The results are in agreement with the data from the β-galactosidase assays. LiaH expression is induced in stationary phase in the wild-type, but not in the liaR mutant (inset to Fig. 1a). Furthermore, induction of liaIH expression was also observed at the transcript level in a wild-type B. subtilis strain during a chronotranscriptome analysis (see below). While only results from β-galactosidase assays will be shown for subsequent experiments, all key findings were always independently verified by Western analysis.
Transition state adaptation, which enables B. subtilis to gradually adjust to nutrient limitations, is embedded in one of the best-studied bacterial developmental programmes, a complex regulatory cascade that ultimately leads to the formation of highly resistant endospores (Errington, 2003; Msadek, 1999; Phillips & Strauch, 2002). It is well known that sporulation (and other transition-state phenomena such as production of extracellular enzymes, motility and biofilm formation) is subject to carbon catabolite repression (Schaeffer et al., 1965; Shafikhani et al., 2003; Stanley et al., 2003): cells grown with high amounts of glucose enter stationary phase without activating the gene expression cascade associated with sporulation. Therefore, we repeated the experiment shown in Fig. 1 in the presence of increasing glucose concentrations. Addition of glucose to the medium delayed (0.1 % glucose, grey bars) or even abolished (0.5 % glucose, white bars) PliaI induction, without affecting overall growth rate, onset of stationary phase or final cell density (Fig. 1b). High glucose concentrations repress the activation of Spo0A, a key regulator of numerous post-exponential processes including sporulation. The activation of Spo0A results in two major effects on gene expression. First, it leads to the activation of a cascade of sigma factors that ultimately govern the formation of the dormant endospore. Second, Spo0AP represses AbrB, which itself is a repressor of numerous genes associated with antibiotic production and resistance (Errington, 2003; Msadek, 1999; Phillips & Strauch, 2002). While addition of glucose has pleiotropic effects on numerous regulatory pathways, this observation can nevertheless be viewed as an indication of a link between LiaRS-dependent gene expression and transition-state regulation.
A second line of evidence pointing in this direction came from results obtained in a detailed chronotranscriptome study, in which the global gene expression pattern was monitored during a complete growth curve with a resolution of 10 min (Sapolsky et al., 2005). This study not only verified the transition-state induction of liaIH (but not liaGFSR) in a B. subtilis wild-type strain at the transcript level, but also revealed that expression of liaIH coincides with that of only one other gene, aprE (Eugenio Ferrari, personal communication). This observation can be interpreted in two ways: either PliaI is induced as a result of aprE expression, or both loci are subject to the same regulation.
PliaI is repressed by AbrB and activated by Spo0A
To address the first hypothesis, an aprE mutant was constructed by LFH-PCR, and introduced into the PliaI reporter strain, resulting in strain TMB085 (Table 1). No difference of PliaI activity was observed relative to the wild-type reporter strain BFS2470 (data not shown). Therefore, AprE is not involved in PliaI induction.
The aprE promoter is subject to complex regulation: one activator (DegU) and three repressors (ScoC, SinR and AbrB) bind directly to the aprE promoter region. The activity of these proteins is modulated by additional proteins, such as Spo0A, SalA and RapG/PhrG (Ogura et al., 2003, 2004). To analyse a potential role of these proteins in PliaI activity, mutants in abrB, scoC, sinR and degU were constructed and subsequently transferred into the PliaI reporter strain BFS2470 (Table 1). No alterations of PliaI activity were observed in the scoC, sinR and degU mutant backgrounds (data not shown). In contrast, the abrB mutation in strain TMB087 resulted in an about fourfold elevated basal expression level during the exponential growth phase, indicating that AbrB acts as a repressor at PliaI during that time (Fig. 2, black bars). The promoter is still inducible to about wild-type levels. Both the basal promoter activity and the induction of PliaI in the abrB mutant are completely dependent on LiaS-mediated activation of its cognate response regulator, LiaR: a liaS/abrB mutant (TMB330; harbouring a clean liaS deletion to avoid polar effects on liaR expression) behaves similarly to a liaR mutant, i.e. does not show any PliaI activity throughout the growth curve (data not shown).
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H, contributing to the mechanisms governing temporal control of sporulation initiation. Activated Spo0A, on the other hand, represses abrB expression, ultimately releasing transition state functions, and H expression, from AbrB repression at the onset of stationary phase (Msadek, 1999; Phillips & Strauch, 2002). As a consequence, mutations in the two genes usually exhibit converse phenotypes on genes subject to their regulation. This could also be observed for PliaI activity: a spo0A mutant (TMB118, Table 1) behaved very similarly to the liaR mutant in the β-galactosidase assay, i.e. had no detectable PliaI activity (Fig. 2, white bars). These findings were also verified by Western analysis (data not shown). They are in agreement with results from previous transcriptome studies, indicating an indirect Spo0A-dependent induction of liaIH expression (Fawcett et al., 2000; Fujita et al., 2005; Hamon et al., 2004). Therefore, PliaI is subject to AbrB repression and Spo0A activation.
Spo0A activates PliaI indirectly through AbrB
The loss of transition-state induction of PliaI in the spo0A mutant raised the question of how Spo0A affects its promoter activity. The abrB gene is known to be under the direct negative control of Spo0A, and AbrB acts as a repressor of a set of genes that are switched on at the transition state (Strauch et al., 1990, 1989). Therefore, Spo0A activation of PliaI could be an indirect effect due to the lack of Spo0A-dependent repression of abrB. Alternatively, Spo0A itself could be responsible for the expression of genes that ultimately provide the stimulus that is sensed by the LiaRS TCS at the onset of stationary phase. To distinguish between the two possibilities, a mutant lacking both abrB and spo0A was constructed and introduced into BFS2470, resulting in strain TMB209. This mutant showed a PliaI induction pattern comparable to the abrB mutant, i.e. an elevated basal level of PliaI activity during exponential growth, and induction at the onset of stationary phase (Fig. 2). Interestingly, the maximum PliaI activity was reproducibly higher by a factor of two than in the abrB mutant (Fig. 2, grey bars). The reason for this behaviour remains elusive. However, the results clearly demonstrate that Spo0A indirectly modulates PliaI activity by repressing abrB expression.
AbrB directly binds PliaI
The transition-state regulator AbrB directly regulates (mostly represses) the expression of over 50 genes, with many additional loci being subject to indirect AbrB control (Phillips & Strauch, 2002). Despite in-depth knowledge on numerous AbrB binding sites, no consensus sequence has been identified for chromosomal sites of interaction. It has been hypothesized that AbrB recognizes a conserved three-dimensional DNA structure, rather than specific base pairs, in the promoter regions of its target genes (Bobay et al., 2004; Phillips & Strauch, 2002; Xu & Strauch, 1996). DNase I footprinting analysis of the liaI promoter region demonstrates that AbrB protects a DNA region of about 25 base pairs (from –40 to –14), with weaker protection occurring further downstream (from –11 to about +10) (Fig. 3). In contrast, the AbrB paralogue Abh does not bind the PliaI region under these conditions (Fig. 3). We conclude that AbrB repression of PliaI occurs through direct binding of the repressor within the promoter sequence, thereby preventing transcription initiation.
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). To determine if AbrB or Spo0A are also necessary for PliaI-dependent transcription in the presence of external stimuli, such as bacitracin, we performed β-galactosidase assay and Western analysis in the wild-type, and isogenic liaR, abrB and spo0A mutants from cells harvested mid-exponentially with and without the presence of bacitracin (final concentration 50 µg ml–1). With the exception of the liaR mutant, all strains were inducible to comparable levels, demonstrating that LiaR alone is sufficient for bacitracin-induced PliaI activity (Fig. 4, and data not shown). These experiments also demonstrate that strong inducers such as bacitracin (3000–4000 Miller units from PliaI–lacZ in β-galactosidase assays) can completely overcome AbrB repression, whereas the endogenous transition state induction (100–200 Miller units) can be suppressed by increased cellular levels of AbrB as present in the spo0A mutant. The mechanism by which fully activated LiaR (i.e. in response to bacitracin stress) seemingly ‘overrides’ AbrB control is completely unknown. We can only speculate that the affinity of phosphorylated LiaR for its target promoter somehow is higher than that of AbrB, whereby activated LiaR seems to be able to displace bound AbrB and initiate PliaI-dependent transcription in the presence of strong inducers.
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). To address this question, induction experiments with spent medium were performed. The wild-type reporter strain BFS2470 was grown in LB medium with MLS selection until 2 h after transition state (corresponding to t=6 h in Fig. 1a). A sample of the culture was harvested to check for PliaI induction. The cells were removed from the remaining culture and the spent medium was directly used to resuspend fresh mid-exponential cells (t=2 h) that were incubated in parallel. Additionally, cells were resuspended in fresh prewarmed LB medium, with and without addition of bacitracin (final concentration 50 µg ml–1), as a positive and negative control, respectively. After further incubation at 37 °C for 30 min, the cells were harvested. The cells from the stationary-phase culture showed the expected β-galactosidase activity (150 Miller units). Resuspension of mid-exponential phase cells in fresh LB medium only resulted in the normal background activity of about 10 Miller units, while resuspension in LB medium supplemented with bacitracin gave the typical strong PliaI response (about 2000 Miller units). In contrast, no induction was observed when mid-exponential cells were resuspended in spent medium (data not shown). These results indicate that PliaI is not induced by a secreted compound produced by B. subtilis itself.
However, we cannot rule out the possibility that the inducing antibiotic is not released from the cells in sufficient amounts in the medium to be detectable in our conditioned-medium experiments. For example, a prerequisite for the biological potency of many cationic antimicrobial peptides is their binding to the overall negatively charged cell envelope, and modulating this net charge is an important resistance mechanism of many Gram-positive bacteria against their activity (Kovács et al., 2006; Peschel et al., 1999). Conversely, one could imagine that such an antibiotic, produced by B. subtilis itself, might be retained to a certain degree by the negatively charged cell wall. Therefore, while PliaI induction could then be readily measured in the antibiotic-producing stationary phase culture, this inducer would not necessarily accumulate in the medium in amounts sufficient to activate PliaI in resuspended mid-exponential phase cells.
We also attempted to identify potential genetic determinants involved in generating the endogenous stimulus by applying transposon mutagenesis in the PliaI–lacZ reporter strain BFS2470 and screening for blue colonies, indicative of increased PliaI activity. Two independent approaches were used, in vivo transposon mutagenesis, based on the established mini-Tn10 system encoded on plasmid pIC333 (Steinmetz & Richter, 1994), and a newly developed in vitro system, based on Tn7 (Peters & Craig, 2001). The latter also allows gain-of-function mutagenesis screens, due to the presence of an outward-facing, xylose-inducible promoter. We readily isolated mutants with transposon insertions in liaF, the known negative regulator of the LiaRS systems (Jordan et al., 2006). In addition, we recovered insertions in the export pump of a putative bacteriocin, indicating that endogenous peptides produced by B. subtilis can induce the LiaRS system (Butcher and Helmann, unpublished). However, strains lacking the ability to produce this bacteriocin still induce PliaI upon entry into stationary phase (data not shown). Therefore, the nature of this endogenous stimulus remains unknown.
Conclusions
Based on the results of previous studies and those presented herein, the intrinsic induction of liaIH expression at the onset of stationary phase is tightly regulated and delicately balanced by five proteins – LiaR, LiaS, LiaF, AbrB and Spo0A – to allow an appropriate cellular response at the right time. The interactions and hierarchy of these regulators is illustrated in the model in Fig. 5. During exponential growth in the absence of cell-envelope stress, the LiaRS two-component system is kept inactive by the LiaF regulator (Jordan et al., 2006). Furthermore, the transition-state regulator AbrB represses any residual PliaI activity by binding to a DNA fragment that includes the –35 region and reaches the –10 region, thereby serving as a roadblock that efficiently prevents transcription initiation (Fig. 5, right-hand side). At the onset of stationary phase, increasing levels of phosphorylated Spo0A, the master regulator of sporulation, inhibit abrB expression (Strauch et al., 1990), thereby releasing PliaI from its repression. At about the same time, an unidentified stimulus leads to the activation of the histidine kinase LiaS and/or its release from LiaF repression. This, in turn, leads to the activation of the cognate response regulator LiaR, which interacts with its binding site (an imperfect inverted repeat of seven nucleotides with four-nucleotide spacing) (Jordan et al., 2006), ultimately resulting in induction of liaIH expression (Fig. 5, left-hand side).
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), the biological relevance of these observations remains obscure, since the LiaRS system is not involved in mediating resistance against any of these inducers (unpublished results). The induction of PliaI at the onset of stationary phase – while being significantly weaker [about 10–15-fold, Fig. 1a, compared to 50–200-fold in the case of strong inducers (Mascher et al., 2004), Fig. 4] – is therefore an important observation. While a very high dynamic potential and strength of LiaRS-dependent gene expression could be demonstrated by the exogenous addition of cell-wall antibiotics (Mascher et al., 2004), this situation does not necessarily reflect the ‘natural’ condition for the activation of LiaRS-dependent signal transduction. The different sites and modes of action of these known inducers of the LiaRS system, together with the unique domain architecture of the sensor kinase LiaS, argue against a direct binding of these drugs to the input domain of LiaS (Mascher, 2006). Identification of the true sensory input of the LiaRS system, while being a big challenge, is therefore a prerequisite to understanding the difference in LiaR-dependent gene expression observed in this study.
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ACKNOWLEDGEMENTS |
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Edited by: M. Hecker
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Received 9 February 2007;
revised 5 April 2007;
accepted 27 April 2007.
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R) were separated by SDS-PAGE. Western blots were performed using CDP-Star (Roche) for chemiluminescent detection, according to the manufacturer's instructions. See Methods for details. (b) The same experiment as in (a) was performed. Only the wild-type reporter strain BFS2470 was used. The preculture was used to inoculate three different flasks containing 20 ml LB each, without (black symbols/bars), and with the addition of glucose to a final concentration of 0.1 % (grey symbols/bars) or 0.5 % (white symbols/bars).
