bBacteria within biofilms are protected from multiple stresses, including immune responses and antimicrobial agents. The biofilm-forming ability of bacterial pathogens has been associated with increased antibiotic resistance and chronic recurrent infections. Although biofilms have been well studied for several gut pathogens, little is known about biofilm formation by anaerobic gut species. The obligate anaerobe Clostridium difficile causes C. difficile infection (CDI), a major health care-associated problem primarily due to the high incidence of recurring infections. C. difficile colonizes the gut when the normal intestinal microflora is disrupted by antimicrobial agents; however, the factors or processes involved in gut colonization during infection remain unclear. We demonstrate that clinical C. difficile strains, i.e., strain 630 and the hypervirulent strain R20291, form structured biofilms in vitro, with R20291 accumulating substantially more biofilm. Microscopic and biochemical analyses show multiple layers of bacteria encased in a biofilm matrix containing proteins, DNA, and polysaccharide. Employing isogenic mutants, we show that virulence-associated proteins, Cwp84, flagella, and a putative quorum-sensing regulator, LuxS, are all required for maximal biofilm formation by C. difficile. Interestingly, a mutant in Spo0A, a transcription factor that controls spore formation, was defective for biofilm formation, indicating a possible link between sporulation and biofilm formation. Furthermore, we demonstrate that bacteria in clostridial biofilms are more resistant to high concentrations of vancomycin, a drug commonly used for treatment of CDI. Our data suggest that biofilm formation by C. difficile is a complex multifactorial process and may be a crucial mechanism for clostridial persistence in the host.

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Mitomycin C (MMC) is a genotoxic agent that induces DNA cross-links, DNA alkylation, and the production of reactive oxygen species (ROS). MMC induces the SOS response and RpoS regulons in Escherichia coli. SOS-encoded functions are required for DNA repair, whereas the RpoS regulon is typically induced by metabolic stresses that slow growth. Thus, induction of the RpoS regulon by MMC may be coincidental, because DNA damage slows growth; alternatively, the RpoS regulon may be an adaptive response contributing to cell survival. In this study, we show that the RpoS regulon is primarily induced by MMC-induced ROS production. We also show that RpoS regulon induction is required for the survival of MMC-treated growing cells. The major contributor to RpoS-dependent resistance to MMC treatment is DNA polymerase Pol II, which is encoded by the polB gene belonging to the SOS regulon. The observation that polB gene expression is controlled by the two major stress response regulons that are required to maximize survival and fitness further emphasizes the key role of this DNA polymerase as an important factor in genome stability.

We found elevated DNA replication errors in isogenic E. coli subpopulations experiencing endogenous stresses.

Quick growth restart after upon encountering favourable environmental conditions is a major fitness contributor in natural environment. It is widely assumed that the time required to restart growth after nutritional upshift is determined by how long it takes for cells to synthesize enough ribosomes to produce the proteins required to reinitiate growth. Here we show that a reduction in the capacity to synthesize ribosomes by reducing number of ribosomal RNA (rRNA) operons (rrn) causes a longer transition from stationary phase to growth of Escherichia coli primarily due to high mortality rates. Cell death results from DNA replication blockage and massive DNA breakage at the sites of the remaining rrn operons that become overloaded with RNA polymerases (RNAPs). Mortality rates and growth restart duration can be reduced by preventing R-loop formation and improving DNA repair capacity. The same molecular mechanisms determine the duration of the recovery phase after ribosome-damaging stresses, such as antibiotics, exposure to bile salts or high temperature. Our study therefore suggests that a major function of rrn operon multiplicity is to ensure that individual rrn operons are not saturated by RNAPs, which can result in catastrophic chromosome replication failure and cell death during adaptation to environmental fluctuations.

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The anaerobic pathogen Clostridioides difficile, which is a primary cause of antibiotic-associated diarrhoea, faces a variety of stresses in the environment and in the mammalian gut. To cope with environmental stresses, it uses the alternative sigma factor B (σB) to modulate gene transcription, which is regulated by an anti-sigma factor, RsbW. To understand the role of RsbW in C. difficile physiology, a rsbW mutant (ΔrsbW) where σB is always on, was generated. ΔrsbW did not have deleterious fitness defects but tolerated acidic environments and detoxified reactive oxygen and nitrogen species better. ΔrsbW was defective in spore and biofilm formation, adhered better to human gut epithelia and was less virulent in a Galleria mellonella infection model. A transcriptomic analysis to understand this unique phenotype showed a change in expression of some σB-controlled genes along with several non-σB controlled genes. Interestingly, the sinRR locus that encodes a pleiotropic regulator, was highly upregulated in ΔrsbW indicating a potential indirect role for σB or RsbW in control of sinRR. Furthermore, the unexpected lower intracellular levels of σB observed suggest post translational control mechanisms. Our study thus provides new insight into the regulatory role of RsbW and the complexity of regulatory networks in C. difficile.