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IraD and IraP trigger a conformational change in RssB. (A) The domain structure of RssB was analyzed by partial proteolysis using thermolysin in the absence (top panel) or presence (lower panel) of IraD. As a control IraD (alone) was also analyzed in the presence of thermolysin (middle panel). (B) The core domain of IraD (IraDcore) is stabilized by RssBN (from partial proteolysis with thermolysin). (C) The domain structure of RssB was monitored in the presence of IraP, using thermolysin. (A–C) Proteins were separated by SDS-PAGE and stained with CBB. (D) SigmaS (σS) is unable to protect the RssB linker region from cleavage by thermolysin. The domain structure of RssB was analyzed by partial proteolysis with thermolysin in the absence (top panel) or presence (bottom panel) of σS. Following partial proteolysis in the presence of thermolysin (lanes 2–6), the protein fragments were separated by SDS-PAGE, transferred to PVDF membrane and immunodecorated with α-RssB antisera, to specifically detect the RssB fragments.
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In Escherichia coli, σS is the master regulator of the general stress response. The level of σS changes in response to multiple stress conditions and it is regulated at many levels including protein turnover. In the absence of stress, σS is rapidly degraded by the AAA+ protease, ClpXP in a regulated manner that depends on the adaptor protein RssB....
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... these data sug- gest that IraP docks to the C-terminal domain of RssB, how- ever its binding appears be stabilized by the phosphorylation state of the N-terminal domain. Next, to confirm if the stabi- lized binding of IraP to RssB C was driven by a conformational change in the C-terminal domain of RssB induced by phospho- rylation of the N-domain we examined if binding of RssB C (or indeed RssB N ) could be enhanced in trans ( Figure S5). To do so, we monitored the binding of IraP to each RssB domain, either alone or together ( Figure S5). ...
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... to confirm if the stabi- lized binding of IraP to RssB C was driven by a conformational change in the C-terminal domain of RssB induced by phospho- rylation of the N-domain we examined if binding of RssB C (or indeed RssB N ) could be enhanced in trans ( Figure S5). To do so, we monitored the binding of IraP to each RssB domain, either alone or together ( Figure S5). Consistent with the idea that phosphorylation of the receiver domain drives a conforma- tional change in the effector domain, which triggers improved interaction with IraP, the addition of RssB N in trans did not enhance the recovery of RssB C . ...
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... with the idea that phosphorylation of the receiver domain drives a conforma- tional change in the effector domain, which triggers improved interaction with IraP, the addition of RssB N in trans did not enhance the recovery of RssB C . In contrast, the recovery of RssB N was reduced in the presence of RssB C , confirming that the N-domain alone interacts only weakly with IraP ( Figure S5, lane 5). ...
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... do so we compared the binding of each domain to IraM, either alone or in the presence of the other domain ( Figure S7). Unexpectedly, and in contrast to IraP (Fig- ure S5), we observed an improved recovery of RssB N (when incu- bated in the presence of RssB C ). These data suggest that the C- terminal domain of RssB can promote binding of RssB N to IraM, by stabilizing RssB N (or IraM) in a conformation that is permis- sive for interaction with the other component. ...
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... alternative possibility is that anti-adaptor binding triggers a conformational change in RssB that promotes release of σ S from a second site. To gain further insight into the mode of action of the RssB anti-adaptors we repeated the partial proteolysis experiments, in the absence or presence of either IraD (Figure 5A) or IraP ( Figure 5C). Consistent with our previous data (Figure 1A), in the absence of anti-adaptor, RssB was rapidly cleaved into two stable domains upon addition of thermolysin ( Figure 5A, top panel). ...
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... alternative possibility is that anti-adaptor binding triggers a conformational change in RssB that promotes release of σ S from a second site. To gain further insight into the mode of action of the RssB anti-adaptors we repeated the partial proteolysis experiments, in the absence or presence of either IraD (Figure 5A) or IraP ( Figure 5C). Consistent with our previous data (Figure 1A), in the absence of anti-adaptor, RssB was rapidly cleaved into two stable domains upon addition of thermolysin ( Figure 5A, top panel). ...
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... gain further insight into the mode of action of the RssB anti-adaptors we repeated the partial proteolysis experiments, in the absence or presence of either IraD (Figure 5A) or IraP ( Figure 5C). Consistent with our previous data (Figure 1A), in the absence of anti-adaptor, RssB was rapidly cleaved into two stable domains upon addition of thermolysin ( Figure 5A, top panel). In contrast to RssB, IraD was rapidly and completely degraded following the addition of thermolysin (Figure 5A, middle panel). ...
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... with our previous data (Figure 1A), in the absence of anti-adaptor, RssB was rapidly cleaved into two stable domains upon addition of thermolysin ( Figure 5A, top panel). In contrast to RssB, IraD was rapidly and completely degraded following the addition of thermolysin (Figure 5A, middle panel). However, when IraD was incubated with RssB, both proteins were clearly protected from cleavage by thermolysin ( Figure 5A, lower panel). ...
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... contrast to RssB, IraD was rapidly and completely degraded following the addition of thermolysin (Figure 5A, middle panel). However, when IraD was incubated with RssB, both proteins were clearly protected from cleavage by thermolysin ( Figure 5A, lower panel). In the case of RssB, the entire protein was completely stable while in the case of IraD only a fragment of the protein (here termed IraD core ) remained sta- ble throughout the experiment. ...
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... the case of RssB, the entire protein was completely stable while in the case of IraD only a fragment of the protein (here termed IraD core ) remained sta- ble throughout the experiment. To determine if stabilization of the IraD core fragment was solely due to its interaction with the N-terminal domain of RssB, we repeated the IraD partial prote- olysis experiment in the presence of RssB N ( Figure 5B). These data clearly show, that RssB N is not only sufficient to stabilize the core fragment of IraD, but also verifies that IraD core migrates The domain structure of RssB was analyzed by partial proteolysis using thermolysin in the absence (top panel) or presence (lower panel) of IraD. ...
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... we examined whether IraP also protected RssB in the limited proteolysis assay, in the same manner as IraD. Consistent with the effect of IraD, IraP also stabi- lized full-length RssB, however in contrast to IraD, a concomitant stabilization of the anti-adaptor was not observed (Figure 5C). These data suggest that only a short fragment of IraP is required for binding to RssB, and that this fragment is sufficient to sta- bilize the protease-protected or inactive conformation of RssB. ...
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... data suggest that only a short fragment of IraP is required for binding to RssB, and that this fragment is sufficient to sta- bilize the protease-protected or inactive conformation of RssB. Importantly, in contrast to both IraD and IraP, σ S did not pro- tect RssB from cleavage by thermolysin ( Figure 5D). These data suggest that in comparison to anti-adaptor docking, substrate binding to RssB either occurs through a different site on RssB or alternatively does not trigger the same conformational change. ...
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... in contrast to Gottesman and colleagues (Battesti et al., 2013), we find that IraP interacts primarily with the C-terminal domain (Figure 3) although the interaction appears to be modulated by phosphorylation of the N-domain of RssB. Importantly, our data suggests that anti-adaptor docking, to either domain, triggers a conformation change in RssB (Figure 5), which results in sub- strate release. Specifically, we propose that IraD binding, to the dimerization interface of RssB, stabilizes an inactive conforma- tion of RssB, which triggers release of σ S from the C-terminal effector domain. ...
Citations
... Cluster II (Thr13, Cys14, Phe18, Leu75, and Trp30 in Fig. 3, inset) interacts with RssB CTD through the signaling helix α8. We note that the IraMRssB CTD interface (607 Å 2 ) is larger than IraMRssB NTD one (404 Å 2 ), consistent with IraM binding to both truncated RssB CTD and RssB NTD in vitro (34) and to RssB CTD only in a bacterial two hybrid assay (13). This previous work also emphasized the importance of the SHL, which when fused to RssB NTD or RssB CTD enhances the interaction (13). ...
Upon Mg²⁺ starvation, a condition often associated with virulence, enterobacteria inhibit the ClpXP-dependent proteolysis of the master transcriptional regulator, σs, via IraM, a poorly understood antiadaptor that prevents RssB-dependent loading of σs onto ClpXP. This inhibition results in σs accumulation and expression of stress resistance genes. Here, we report on the structural analysis of RssB bound to IraM, which reveals that IraM induces two folding transitions within RssB, amplified via a segmented helical linker. These conformational changes result in an open, yet inhibited RssB structure in which IraM associates with both the C-terminal and N-terminal domains of RssB and prevents binding of σs to the 4-5-5 face of the N-terminal receiver domain. This work highlights the remarkable structural plasticity of RssB and reveals how a stress-specific RssB antagonist modulates a core stress response pathway that could be leveraged to control biofilm formation, virulence, and the development of antibiotic resistance.
... σ s as the phosphoaspartyl moiety would presumably clash with K173 (35), a σ s residue critical for the interaction (24). This model is, however, at odds with reports that beryllofluoride and AcP enhance σ s :RssB interactions (27,29,30,48,49) (also shown here in Fig. 1). Micevski et al demonstrated that σ s docks to both the RssB NTD truncation and also to an SHL-RssB CTD fusion, albeit with lower affinity (30). ...
... RssB oligomerization has been detected by bacterial two-hybrid assay in clpX and clpXP-deleted strains (32), consistent with the model above. However, in vitro, dimerization and higher order oligomerization has only been detected by chemical crosslinking of full-length RssB (34) and by gel filtration and X-ray crystallography of isolated RssB NTD , but not with the full-length protein (49). The contacts holding the RssB NTD dimer together in crystallo are different from those seen in other response regulators (45), do not involve the 4-5-5 face, and cannot be ruled out as a crystallization artifact. ...
In enterobacteria such as Escherichia coli, the general stress response is mediated by σs, the stationary phase dissociable promoter specificity subunit of RNA polymerase. σs is degraded by ClpXP during active growth in a process dependent on the RssB adaptor, which is thought to be stimulated by the phosphorylation of a conserved aspartate in its N-terminal receiver domain. Here we present the crystal structure of full-length RssB bound to a beryllofluoride phosphomimic. Compared to the structure of RssB bound to the IraD anti-adaptor, our new RssB structure with bound beryllofluoride reveals conformational differences and coil-to-helix transitions in the C-terminal region of the RssB receiver domain and in the interdomain segmented helical linker. These are accompanied by masking of the α4-β5-α5 (4-5-5) “signaling” face of the RssB receiver domain by its C-terminal domain. Critically, using hydrogen-deuterium exchange mass spectrometry, we identify σs-binding determinants on the 4-5-5 face, implying that this surface needs to be unmasked to effect an interdomain interface switch and enable full σs engagement and hand-off to ClpXP. In activated receiver domains, the 4-5-5 face is often the locus of intermolecular interactions, but its masking by intramolecular contacts upon phosphorylation is unusual, emphasizing that RssB is a response regulator that undergoes atypical regulation.
... Regulation of RssB activity in E. coli is carried out by IraP, IraM and IraD; three small proteins called anti-adaptors, induced by phosphate and magnesium starvation and DNA damage, respectively (Bougdour et al., 2006;Bougdour et al., 2008). These proteins use disting binding modes to interact with RssB, resulting in an impediment of the adaptor to deliver RpoS to ClpXP protease (Battesti et al., 2013;Micevski et al., 2015). The genomes of both A. vinelandii F I G U R E 9 Genomic context of rssBC in several species belonging to the Pseudomonadaceae family. ...
In several Gram‐negative bacteria, the general stress response is mediated by the alternative sigma factor RpoS, a subunit of RNA polymerase that confers promoter specificity. In Escherichia coli, regulation of protein levels of RpoS involves the adaptor protein RssB, which binds RpoS for presenting it to the ClpXP protease for its degradation. However, in species from the Pseudomonadaceae family, RpoS is also degraded by ClpXP, but an adaptor has not been experimentally demonstrated. Here, we investigated the role of an E. coli RssB‐like protein in two representative Pseudomonadaceae species such as Azotobacter vinelandii and Pseudomonas aeruginosa. In these bacteria, inactivation of the rssB gene increased the levels and stability of RpoS during exponential growth. Downstream of rssB lies a gene that encodes a protein annotated as an anti‐sigma factor antagonist (rssC). However, inactivation of rssC in both A. vinelandii and P. aeruginosa also increased the RpoS protein levels, suggesting that RssB and RssC work together to control RpoS degradation. Furthermore, we identified an in vivo interaction between RssB and RpoS only in the presence of RssC using a bacterial three‐hybrid system. We propose that both RssB and RssC are necessary for the ClpXP‐dependent RpoS degradation during exponential growth in two species of the Pseudomonadaceae family.
... The copyright holder for this preprint this version posted January 8, 2023. ; https://doi.org/10.1101/2023.01.07.523045 doi: bioRxiv preprint 2E-F, insets), consistent with IraM binding to both truncated RssB CTD and RssB NTD in vitro 37 and to RssB CTD only in a bacterial two hybrid assay 20 . This previous work also emphasized the importance of the SHL, which when fused to RssB NTD or RssB CTD enhances the interaction 20 . ...
Upon Mg ²⁺ starvation, a condition often associated with virulence, enterobacteria inhibit the ClpXP-dependent proteolysis of the master transcriptional regulator, σ s , via IraM, a poorly understood anti-adaptor that prevents RssB-dependent loading of σ s onto ClpXP. This inhibition results in σ s accumulation, and expression of stress resistance genes. Here we report on the structural analysis of RssB bound to IraM, which reveals that IraM induces two folding transitions within RssB, which are amplified via a segmental helical linker. This work highlights the remarkable structural plasticity of RssB and reveals how a stress-specific RssB antagonist modulates a core stress response pathway that could be leveraged to control biofilm formation, virulence, and the development of antibiotic resistance.
... regulators can be accessed spontaneously by conformational breathing, in the absence of phosphorylation have also been reported [38][39][40] . In the case of RssB, most of our understanding of phosphorylation-dependent activation comes from studies employing AcP to stimulate formation of an s s :RssB:ClpX assembly 27 and promote s s degradation without being an absolute requirement for it 27,32,34,41 . We first used our high-quality preparations of pure RssB to verify RssB activity by performing in vitro phosphorylation assays coupled to Phos-tag electrophoresis. ...
... ; https://doi.org/10.1101/2022.12.15.520641 doi: bioRxiv preprint pronounced functional defect caused by substitution of K108 30,32 , a highly conserved residue critical for general signaling by receiver domains 46 . The mechanistic underpinnings of the defects seen in the K108 variants remain unknown, but since phosphorylation is not an absolute requirement for s s degradation 24,27,34,36,41 , it appears likely that K108 might play roles in addition to signaling, such as in protein-protein interactions with either s s or ClpX. Substitutions in the polyglutamate motif (E135A; ...
In γ-proteobacteria such as Escherichia coli , the general stress response is mediated by σ s , the stationary phase dissociable promoter specificity subunit of RNA polymerase. σ s is degraded by ClpXP during active growth in a process dependent on the RssB adaptor, which acts catalytically and is thought to be stimulated by phosphorylation of a conserved aspartate in its N-terminal receiver domain. Here we present the crystal structure of full-length RssB bound to a beryllofluoride phosphomimic. Compared to the inhibited IraD anti-adaptor-bound RssB structure, our study reveals movements and coil-to-helix transitions in the C-terminal region of the RssB receiver domain and in the inter-domain segmented helical linker, accompanied by packing of the C-terminal effector domain onto the [alpha]4-β5-α5 (4-5-5) signaling face of the RssB receiver domain. This face is often the locus of protein-protein interactions in unphosphorylated receiver domains, but its masking is unusual in their phosphorylated forms. Our structure emphasizes the remarkable plasticity that underpins regulatory strategies within the large family of response regulators.
... 92 On the other hand, IraP is an antiadaptor protein, which stabilizes the levels of RpoS by preventing the RssB-mediated degradation of RpoS. 93 Moreover, MiaA plays a significant role via RpoS in the expression of multiple genes associated with synthesis of integral components such as flagella, fimbriae (Figure 1), and curli in E. coli biofilms ( Figure 8A). Earlier work on P. aeruginosa has also shown that the absence of MiaA leads to smaller cellular aggregates with coli under low pH conditions. ...
Flux balance analysis (FBA) and ordinary differential equation models have been instrumental in depicting the metabolic functioning of a cell. Nevertheless, they demonstrate a population’s average behavior (summation of individuals), thereby portraying homogeneity. However, living organisms such as Escherichia coli contain more biochemical reactions than engaging metabolites, making them an underdetermined and degenerate system. This results in a heterogeneous population with varying metabolic patterns. We have formulated a population systems biology model that predicts this degeneracy by emulating a diverse metabolic makeup with unique biochemical signatures. The model mimics the universally accepted experimental view that a subpopulation of bacteria, even under normal growth conditions, renders a unique biochemical state, leading to the synthesis of metabolites and persister progenitors of antibiotic resistance and biofilms. We validate the platform’s predictions by producing commercially important heterologous (isobutanol) and homologous (shikimate) metabolites. The predicted fluxes are tested in vitro resulting in 32- and 42-fold increased product of isobutanol and shikimate, respectively. Moreover, we authenticate the platform by mimicking a bacterial population in the presence of glyphosate, a metabolic pathway inhibitor. Here, we observe a fraction of subsisting persisters despite inhibition, thus affirming the signature of a heterogeneous populace. The platform has multiple uses based on the disposition of the user.
... 92 On the other hand, IraP is an antiadaptor protein, which stabilizes the levels of RpoS by preventing the RssB-mediated degradation of RpoS. 93 Moreover, MiaA plays a significant role via RpoS in the expression of multiple genes associated with synthesis of integral components such as flagella, fimbriae (Figure 1), and curli in E. coli biofilms ( Figure 8A). Earlier work on P. aeruginosa has also shown that the absence of MiaA leads to smaller cellular aggregates with coli under low pH conditions. ...
Using Escherichia coli as the representative biofilm former, we report here the development of an in silico model built by simulating events that transform a free-living bacterial entity into self-encased multicellular biofilms. Published literature on ∼300 genes associated with pathways involved in biofilm formation was curated, static maps were created, and suitably interconnected with their respective metabolites using ordinary differential equations. Precise interplay of genetic networks that regulate the transitory switching of bacterial growth pattern in response to environmental changes and the resultant multicomponent synthesis of the extracellular matrix were appropriately represented. Subsequently, the in silico model was analyzed by simulating time-dependent changes in the concentration of components by using the R and python environment. The model was validated by simulating and verifying the impact of key gene knockouts (KOs) and systematic knockdowns on biofilm formation, thus ensuring the outcomes were comparable with the reported literature. Similarly, specific gene KOs in laboratory and pathogenic E. coli were constructed and assessed. MiaA, YdeO, and YgiV were found to be crucial in biofilm development. Furthermore, qRT-PCR confirmed the elevation of expression in biofilm-forming clinical isolates. Findings reported in this study offer opportunities for identifying biofilm inhibitors with applications in multiple industries. The application of this model can be extended to the health care sector specifically to develop novel adjunct therapies that prevent biofilms in medical implants and reduce emergence of biofilm-associated resistant polymicrobial-chronic infections. The in silico framework reported here is open source and accessible for further enhancements.
... Each anti-adaptor is induced in response to a specific stress, and currently three anti-adaptor proteins have been identified. Although the precise mechanism of action of these anti-adaptors remains unclear, all are proposed to inhibit σ s turnover via direct interaction with RssB [22][23][24]. ...
... His 6 -tagged ClpX and ClpP were overexpressed in E. coli and purified as described previously [12]. Untagged σ S , RssB (residues 1-337), RssB C (residues 131-337) and RssB N (residues 1-128) (wild type and specific point mutants) were generated using the Ub-fusion system [27] and purified essentially as described previously [24], using a combination of IMAC (immobilized metal ion affinity chromatography) and preparative grade size exclusion chromatography (SEC) using a HiLoad 16/600 Superdex 200 to separate monomeric and dimeric RssB. All columns were pre-equilibrated in chilled GF buffer (20 mM Tris-HCl pH 7.5, 10 mM MgCl 2 , 0.1 mM EDTA, 1 mM DTT, 140 mM NaCl, 5% (v/v) glycerol, 0.005% (v/v) Triton X-100). ...
... To examine the interaction of RssB (full-length or individual domains) with purified ZBD or σ s , in vitro "pull-down" experiments were performed essentially as previously described [24,30]. Briefly, Ni-NTA agarose beads (QIAGEN, Hilden, Germany) (bed volume (BV), 25 µL) were equilibrated with 10 BV of equilibration buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole) followed by 5 BV of Wash Buffer C (20 mM Hepes-KOH pH 7.5, 100 mM KOAc, 10 mM MgOAc, 10% (v/v) glycerol, 10 mM imidazole, 0.5% (v/v) Triton X-100). ...
In Escherichia coli, SigmaS (σS) is the master regulator of the general stress response. The cellular levels of σS are controlled by transcription, translation and protein stability. The turnover of σS, by the AAA+ protease (ClpXP), is tightly regulated by a dedicated adaptor protein, termed RssB (Regulator of Sigma S protein B)—which is an atypical member of the response regulator (RR) family. Currently however, the molecular mechanism of σS recognition and delivery by RssB is only poorly understood. Here we describe the crystal structures of both RssB domains (RssBN and RssBC) and the SAXS analysis of full-length RssB (both free and in complex with σS). Together with our biochemical analysis we propose a model for the recognition and delivery of σS by this essential adaptor protein. Similar to most bacterial RRs, the N-terminal domain of RssB (RssBN) comprises a typical mixed (βα)5-fold. Although phosphorylation of RssBN (at Asp58) is essential for high affinity binding of σS, much of the direct binding to σS occurs via the C-terminal effector domain of RssB (RssBC). In contrast to most RRs the effector domain of RssB forms a β-sandwich fold composed of two sheets surrounded by α-helical protrusions and as such, shares structural homology with serine/threonine phosphatases that exhibit a PPM/PP2C fold. Our biochemical data demonstrate that this domain plays a key role in both substrate interaction and docking to the zinc binding domain (ZBD) of ClpX. We propose that RssB docking to the ZBD of ClpX overlaps with the docking site of another regulator of RssB, the anti-adaptor IraD. Hence, we speculate that docking to ClpX may trigger release of its substrate through activation of a “closed” state (as seen in the RssB-IraD complex), thereby coupling adaptor docking (to ClpX) with substrate release. This competitive docking to RssB would prevent futile interaction of ClpX with the IraD-RssB complex (which lacks a substrate). Finally, substrate recognition by RssB appears to be regulated by a key residue (Arg117) within the α5 helix of the N-terminal domain. Importantly, this residue is not directly involved in σS interaction, as σS binding to the R117A mutant can be restored by phosphorylation. Likewise, R117A retains the ability to interact with and activate ClpX for degradation of σS, both in the presence and absence of acetyl phosphate. Therefore, we propose that this region of RssB (the α5 helix) plays a critical role in driving interaction with σS at a distal site.
... One mechanism explaining increasing RpoS levels is via iraD, as it has been shown to be induced by various DNA-damaging agents, including direct DNA breaking agents such as Phleomycin (Merrikh et al., 2009a). IraD functions by inhibiting the proteolysis of sigma factor RpoS from ClpXP, prolonging its lifetime (Bougdour et al., 2008;Merrikh et al., 2009b;Micevski et al., 2015). The mechanism leading to the induction of iraD by DNA damage is not well established, as the regulation of iraD expression is quite complex. ...
The resilience of bacteria depends upon their capacity to proliferate and survive under different conditions, including in the human body where some bacterial infections can be fatal. Many antibiotics used to treat infections cause direct and indirect DNA damage, in particular DNA double-strand breaks, which can lead to bacterial cell death. Bacteria respond to DNA damage by inducing the SOS response, which is an important process in the repair and tolerance of DNA damage. Additional consequences of SOS induction by antibiotic exposure, is the potential increase of mutagenesis, horizontal gene transfer, and tolerance to other antibiotics. Therefore, identifying the factors involved in SOS induction is essential to understanding the dynamics of bacterial infections. Previous studies have indicated that bacterial susceptibility to DNA damaging agents is dependent on growth conditions, but the mechanisms involved are not well understood. Many physiological changes are associated with growth rate, including DNA replication (a major mechanismleading to DNA damage), and reallocation of resources towards growth-limiting processes, which could impair the capacity of cells to induce the SOS response. In addition, previous reports indicate that SOS expression is variable in single cells, and the effect of growth conditions in variability has not been evaluated. In order to evaluate how changes in growth conditions influence the SOS response, we have quantified the levels of SOS induction by DNA damage in single cells using E. coli as a model organism. Our results show that cells with very high levels of SOS expression are more abundant in slow-growing conditions, that is under spontaneous DNA damage, under damage induced by the antibiotic ciprofloxacin, and under replication-dependent chronic double-strand breaks. We explain these observations as a combination of population dynamics, that contributes to enriching for slow dividing cells (high SOS) in slow-growing populations, and an influence of growth conditions in the variability of SOS induction, possibly because of influences in the DNA-repair process via an unknown mechanism. The population dynamics arguments presented here may be relevant to other antibiotics, and argue to the significance of studying the response to antibiotics in single cells. We believe the observations on variability in SOS-expression may open new avenues for understanding the limiting factors for DNA repair.
... Additionally, we noted 12 amino acids near the C-terminal of RssB that show high homology to a 13 amino acid domain of the MgrB conserved region (Fig. S2, Supporting Information). A previous study showed that IraM could dock to the C-terminal of RssB (residues 131-337) to prevent the protein from working (Micevski et al. 2015). We speculated that it is possible that MgrB could also interact with IraM directly to affect its biological function and this might be a possible explanation as to how MgrB regulates the expression of iraM independently of phoP/Q. ...
Although MgrB is established to be a feedback inhibitor of the PhoP/Q system in E. coli, the biological functions of MgrB remain largely unknown. To explore new functions of MgrB, a comparative transcriptome analysis was performed (E. coli K-12 W3110 ΔmgrB vs E. coli K-12 W3110). The results showed that many genes involved in acid stress are upregulated, suggesting that MgrB is related to acid sensitivity in E. coli. The survival rates under acid stress of the ΔmgrB mutant and wild-type showed that deletion of mgrB resulted in acid resistance. According to previous research, we deleted phoP, phoQ, and iraM in the ΔmgrB mutant, and found that further deletion of phoP/phoQ only partially restored acid sensitivity. Additionally, we found that deletion of mgrB resulted in increased accumulation of RpoS during the exponential growth phase, which could be blocked by further deletion of iraM. Mutation of iraM or rpoS completely suppressed the effect of mgrB mutation on acid resistance. Taken together, the data suggest that MgrB affects the acid resistance of E. coli by modulating the expression of iraM, but not completely through PhoP/Q. This indicates that MgrB may have other protein interactors aside from PhoQ, which merits further investigation.
