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Bacillus subtilis uses the SigM signaling pathway to prioritize the use of its lipid carrier for cell wall synthesis [1]

['Ian J. Roney', 'Department Of Microbiology', 'Harvard Medical School', 'Boston', 'Massachusetts', 'United States Of America', 'David Z. Rudner']

Date: 2024-05

Peptidoglycan (PG) and most surface glycopolymers and their modifications are built in the cytoplasm on the lipid carrier undecaprenyl phosphate (UndP). These lipid-linked precursors are then flipped across the membrane and polymerized or directly transferred to surface polymers, lipids, or proteins. Despite its essential role in envelope biogenesis, UndP is maintained at low levels in the cytoplasmic membrane. The mechanisms by which bacteria distribute this limited resource among competing pathways is currently unknown. Here, we report that the Bacillus subtilis transcription factor SigM and its membrane-anchored anti-sigma factor respond to UndP levels and prioritize its use for the synthesis of the only essential surface polymer, the cell wall. Antibiotics that target virtually every step in PG synthesis activate SigM-directed gene expression, confounding identification of the signal and the logic of this stress-response pathway. Through systematic analyses, we discovered 2 distinct responses to these antibiotics. Drugs that trap UndP, UndP-linked intermediates, or precursors trigger SigM release from the membrane in <2 min, rapidly activating transcription. By contrasts, antibiotics that inhibited cell wall synthesis without directly affecting UndP induce SigM more slowly. We show that activation in the latter case can be explained by the accumulation of UndP-linked wall teichoic acids precursors that cannot be transferred to the PG due to the block in its synthesis. Furthermore, we report that reduction in UndP synthesis rapidly induces SigM, while increasing UndP production can dampen the SigM response. Finally, we show that SigM becomes essential for viability when the availability of UndP is restricted. Altogether, our data support a model in which the SigM pathway functions to homeostatically control UndP usage. When UndP levels are sufficiently high, the anti-sigma factor complex holds SigM inactive. When levels of UndP are reduced, SigM activates genes that increase flux through the PG synthesis pathway, boost UndP recycling, and liberate the lipid carrier from nonessential surface polymer pathways. Analogous homeostatic pathways that prioritize UndP usage are likely to be common in bacteria.

The sigM gene resides in an operon with yhdL and yhdK that encode integral membrane proteins that hold SigM inactive at the membrane [ 9 , 20 ]. Unlike many membrane anchored anti-sigma factors, the release of SigM is not controlled by regulated proteolysis of its anti-sigma factors [ 21 ]. Instead, the YhdL-YhdK (YhdLK) complex is thought to be controlled allosterically. Despite years of study, the signal sensed by the YhdLK complex that triggers release of SigM has remained unclear [ 13 ]. Here, we report that antibiotics that trap UndP or UndP-linked intermediates rapidly deplete the UndP pool and trigger SigM release from the membrane within minutes. By contrasts, antibiotics that inhibited cell wall synthesis without directly affecting UndP deplete the carrier lipid more slowly and induce a slower and weaker SigM response. We show this slow response can be explained by sequestration of the carrier lipid in UndP-linked secondary wall polymers. In a complementary set of experiments, we show that depletion of enzymes involved in PG biogenesis mimic the responses observed with antibiotic inhibition. In addition, reduction in UndP synthesis rapidly induces SigM, while increasing UndP production suppresses the SigM response. Importantly, our analysis indicates that UndP-linked precursors do not function as proxies for UndP levels and instead point to UndP as the signal sense by the YhdLK complex. Finally, we show that sigM becomes essential when the availability of UndP is limiting. Altogether, our data support a model in which the YhdLK-SigM pathway functions to prioritize UndP usage for cell wall synthesis. When the levels of free UndP are sufficiently high, the anti-sigma factor complex holds SigM inactive. When levels of the carrier lipid are reduced, SigM is released from the membrane and activates genes that increase PG synthesis, boost UndP recycling, and liberate the lipid carrier from nonessential surface polymer pathways.

SigM was first described as a transcription factor required for the outgrowth of B. subtilis spores [ 9 ]. When spores lacking SigM were induced to germinate in medium containing high salt, the outgrowing cells displayed morphological defects prior to lysis, suggesting they were impaired in cell wall synthesis. SigM mutant cells were subsequently shown to have increased sensitivities to antibiotics that inhibit PG biogenesis [ 10 , 11 ]. Transcriptional profiling experiments revealed that many of the genes in the SigM regulon are involved in cell wall biogenesis [ 12 , 13 ]. Specifically, SigM controls several genes involved in the synthesis and transport of PG precursors as well as PG polymerization and crosslinking. SigM also regulates genes involved in recycling UndP, its de novo synthesis, and liberation of the carrier lipid from UndPP-WTA precursors [ 14 , 15 ]. Work from several groups that spans 2 decades revealed that antibiotics that target virtually every step in PG biogenesis activate SigM-direct gene expression [ 12 , 16 – 18 ]. A SigM-responsive transcriptional reporter has even been used to screen for small molecules that impair PG synthesis [ 17 , 19 ]. Although responsive to these exogenous stresses, SigM is active at low levels during unperturbed exponential growth, suggesting that its principal function is homeostatic [ 9 ].

All organisms use polyprenyl-phosphate lipids to transport sugars across membranes [ 1 – 3 ]. In bacteria, the 55-carbon isoprenoid, undecaprenyl phosphate (UndP), ferries a diverse set of sugars and glycopolymers across the cytoplasmic membrane. The most prominent among these is the monomeric building block of the cell wall peptidoglycan (PG). The PG precursor, a disaccharide pentapeptide, is built on UndP in the cytoplasm [ 4 , 5 ]. The lipid-linked muropeptide, called lipid II, is then flipped across the cytoplasmic membrane where it is polymerized and crosslinked into the existing cell wall meshwork. The byproduct of this assembly reaction is undecaprenyl pyrophosphate (UndPP), which is dephosphorylated by membrane phosphatases, and then UndP is flipped back across the cytoplasmic membrane for reuse [ 6 , 7 ]. In addition to PG precursors, UndP transports O-antigen, capsule, exopolysaccharide, and secondary cell wall polymers like wall teichoic acids (WTAs) across the cytoplasmic membrane. The carrier lipid is also used to ferry sugars across the membrane that are used to glycosylate proteins, lipids, and surface polymers. Despite its essential role in these diverse envelope biogenesis and modification pathways, UndP is maintained at low levels in the cytoplasmic membrane (approximately 10 5 UndP molecules/cell; approximately 0.1% of all membrane lipids) [ 8 ]. How cells distribute this limited resource among competing pathways remains an outstanding question in all bacteria. Here, we report that in Bacillus subtilis, the alternative sigma factor, SigM, and its anti-sigma factor complex, YhdL-YhdK, function to prioritize UndP for cell wall synthesis.

Results

Depletion of cell wall synthesis factors that trap UndP activate SigM Our data support a model in which SigM is activated by a drop in the pools of UndP and not a general block to cell wall synthesis. To further test this model, we complemented the chemical genetic experiments described above with depletions of enzymes involved in distinct steps in PG biogenesis [4,5]. We generated IPTG-regulated alleles of 3 genes involved in precursor synthesis (murAA, murB, and mraY) that do not directly reduce UndP pools and IPTG-regulated alleles of 3 genes (murG, murJ, and bcrC) that trap UndP-linked intermediates and reduce the pool of the carrier lipid. MurAA and MurB are required for the synthesis of UDP-N-acetyl muramic acid (UDP-MurNAc), while MraY attaches the UDP-MurNAc-pentapeptide onto UndP to generate lipid I (Fig 1A). Depletion of MurAA, MurB, or MraY should initially have no impact on UndP or possibly increase the pool of the carrier lipid. MurG catalyzes the conversion of Lipid I to Lipid II and its depletion results in accumulation of lipid I (Fig 1A). MurJ and Amj are functionally redundant lipid II flippases that transport the UndP-linked PG precursors from the inner to the outer leaflet of the cytoplasmic membrane [22,33]. Depletion of MurJ in a Δamj strain should accumulate inward-facing lipid II. BcrC and UppP are functionally redundant UndPP phosphatases that regenerate UndP enabling its transport to the inner leaflet of the membrane (Fig 1A) [34,35]. Depletion of BcrC in a ΔuppP mutant should accumulate outward-facing UndPP. All strains were precultured in CH medium in the presence of IPTG and then washed and inoculated in medium lacking inducer at low optical density (OD600 = 0.02). Growth was monitored after removal of IPTG and SigM-dependent gene expression was analyzed at the earliest time point when the strains exhibited a reduction in mass doubling (Fig 3C). Importantly, at the time points analyzed a similar percentage of cells had membrane permeability defects as assayed by propidium iodide (Fig F in S1 Text), consistent with a similar degree of cell wall synthesis inhibition. In accordance with our findings with acute exposure to antibiotics, SigM activation upon enzyme depletion fell into 2 classes. Depletion of the enzymes (MurAA, MurB, MraY) that do not directly reduce UndP pools had very weak or undetectable SigM activation, while depletion of the factors (MurG, MurJ, BcrC) that trap UndP strongly activated SigM (>10-fold) (Fig 3A and 3B). Importantly all 6 strains had similarly low SigM activity when grown in the presence of IPTG (Fig G in S1 Text). PPT PowerPoint slide

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TIFF original image Download: Fig 3. Depletion of cell wall synthesis enzymes that cause accumulation of UndP-linked precursors activates SigM. (A) Representative fluorescence images of the indicated B. subtilis depletion strains harboring a σM-responsive reporter (P(amj)-yfp) after removal of IPTG. Scale bar indicates 2 μm. (B) Quantification of fluorescence intensity from images in (A). Bar represents median. (C) Growth curves of depletion strains grown in the presence (squares) or absence (circles) of IPTG. Red arrow indicates the time point at which samples were removed for microscopy in (A). IPTG concentrations were murAA (100 μM), murB (25 μM), mraY (25 μM), murG (12.5 μM), murJ (25 μM), and bcrC (25 μM). The data underlying B and C are provided in S1 Data. https://doi.org/10.1371/journal.pbio.3002589.g003 Altogether, the experiments described above indicate that YhdLK-SigM signaling is activated by the accumulation of UndP-linked cell wall precursors and/or a drop in UndP pools rather than a general inhibition of cell wall synthesis. This model is further supported by previous studies showing that depletion of enzymes involved in WTA synthesis that trap UndP-linked WTA intermediates activate SigM [22]. We observed similar results using our transcriptional reporter under the same assay conditions described above. Depletion of the UndP-WTA transporter, TagG, activated SigM to high levels, whereas depletion of the initiating glycosyltransferase, TagO, which does not trap UndP did not (Fig H in S1 Text).

Reducing UndP synthesis rapidly activates SigM Our data suggest that the YhdLK complex directly monitors UndP levels and activates SigM in response to a drop in the carrier lipid pool. If correct, inhibition of UndP synthesis should strongly activate SigM. A previous study found that a point mutation in the ribosome binding site of uppS, the gene encoding UndPP synthase, caused a modest increase in SigM activity [36]. To more directly test whether a reduction in UndP levels impacts SigM activity, we generated IPTG-regulated alleles of 2 essential genes in the UndP biosynthetic pathway (Fig 4A), uppS and ispH [3], and tested how depletion of these enzymes affected SigM activity. Similar to the experiments described above, we monitored growth after removal of IPTG and analyzed SigM activity at the earliest time point when the strains exhibited a reduction in mass doubling. Depletion of either enzyme strongly increased SigM-directed gene expression (Fig 4 and Fig I in S1 Text). Furthermore, we identified IPTG concentrations for both depletion strains that did not appreciably impair growth rate but activated SigM to high levels (Fig 4 and Fig I in S1 Text). SigM was also strongly activated when UndP synthesis was inhibited by the antibiotic fosmidomycin that targets Dxr [37], an essential enzyme in the isoprenoid biosynthesis pathway (Fig 4 and Fig I in S1 Text). We note that fosmidomycin took longer to impair growth compared to the antibiotics that target cell wall synthesis (Fig I(C) in S1 Text). This finding is consistent with the idea that the recycled lipid carrier can sustain growth for a period without the production of new UndP molecules to maintain the pool size as the cell elongates. PPT PowerPoint slide

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TIFF original image Download: Fig 4. Depletion of UndP synthesis rapidly activates SigM. (A) Schematic diagram of the UndP synthesis pathway. Fosmidomycin that targets Dxr is highlighted in red. Enzymes (UppS and IspH) that were depleted are in bold. (B) Growth curves of UppS depletion strain grown in the presence of 100 μM IPTG (squares), no IPTG (circles), and low (5 μM) IPTG (triangles). Red arrow indicates the time point at which samples were analyzed by fluorescence microscopy. (C) Representative fluorescence and phase-contrast images of the B. subtilis UppS depletion strain harboring the σM-responsive reporter P(amj)-yfp. Wild-type cells with the same reporter were exposed to fosmidomycin and analyzed by fluorescence microscopy 30 min later. Merged images of phase-contrast and propidium iodide fluorescence are shown below. Scale bar indicates 2 μm. (D) Quantification of YFP fluorescence from images like those shown in (C). A similar analysis using an IspH depletion strain is presented in Fig I in S1 Text. Quantification of YFP fluorescence from the IspH depletion strain was included in (D). The data underlying B and D are provided in S1 Data. https://doi.org/10.1371/journal.pbio.3002589.g004

Defects in LTA biogenesis weakly activate SigM and activation is suppressed by UppS overexpression Mutations in the LTA synthesis pathway have been reported to activate SigM [44,45]. LTA synthesis is the only surface polymer in B. subtilis that is not built on UndP [46]. Accordingly, SigM activation in LTA synthesis mutants appears inconsistent with the model that YhdLK responds to changes in UndP [13]. We therefore revisited the LTA synthesis mutants using our SigM reporter and assay conditions. Specifically, we generated deletions of ugtP and ltaS encoding 2 enzymes involved in LTA synthesis [46]. UgtP is required for the synthesis of LTA’s glucolipid anchor, and LtaS is the primary LTA synthase. Mutations in these genes were previously reported to activate SigM. However, under our assay conditions, cells harboring the ΔugtP mutation did not activate SigM while the ΔltaS mutation increased SigM activity approximately 3-fold (Fig M in S1 Text). To investigate whether the differences were due to the growth medium used, we repeated the experiments in LB medium as was used previously [44]. Under these conditions, both the ΔugtP and ΔltaS mutations increased SigM activity by approximately 2-fold. For comparison, perturbations that depleted UndP or trapped UndP-linked precursors induced SigM activity by approximately 10- to 50-fold. We therefore suspected that the absence of UgtP or LtaS was indirectly reducing the carrier lipid pool. To investigate this possibility, we tested whether overexpression of UppS could suppress the SigM activation in the mutants. As can be seen in Fig M in S1 Text, UppS overexpression largely suppressed the increase in SigM activity resulting from defects in LTA synthesis. These findings provide further support for the model that the YhdLK complex is responsive to UndP levels.

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002589

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