Bacterial persisters are able to tolerate high levels of antibiotics and give rise to new populations. Persister tolerance is generally attributed to minimally active cellular processes that prevent ...antibiotic-induced damage, which has led to the supposition that persister offspring give rise to antibiotic-resistant mutants at comparable rates to normal cells. Using time-lapse microscopy to monitor Escherichia coli populations following ofloxacin treatment, we find that persisters filament extensively and induce impressive SOS responses before returning to a normal appearance. Further, populations derived from fluoroquinolone persisters contain significantly greater quantities of antibiotic-resistant mutants than those from untreated controls. We confirm that resistance is heritable and that the enhancement requires RecA, SOS induction, an opportunity to recover from treatment, and the involvement of error-prone DNA polymerase V (UmuDC). These findings show that fluoroquinolones damage DNA in persisters and that the ensuing SOS response accelerates the development of antibiotic resistance from these survivors.
Microbial biocontainment is an essential goal for engineering safe, next-generation living therapeutics. However, the genetic stability of biocontainment circuits, including kill switches, is a ...challenge that must be addressed. Kill switches are among the most difficult circuits to maintain due to the strong selection pressure they impart, leading to high potential for evolution of escape mutant populations. Here we engineer two CRISPR-based kill switches in the probiotic Escherichia coli Nissle 1917, a single-input chemical-responsive switch and a 2-input chemical- and temperature-responsive switch. We employ parallel strategies to address kill switch stability, including functional redundancy within the circuit, modulation of the SOS response, antibiotic-independent plasmid maintenance, and provision of intra-niche competition by a closely related strain. We demonstrate that strains harboring either kill switch can be selectively and efficiently killed inside the murine gut, while strains harboring the 2-input switch are additionally killed upon excretion. Leveraging redundant strategies, we demonstrate robust biocontainment of our kill switch strains and provide a template for future kill switch development.
Bacteria induce stress responses that protect the cell from lethal factors such as DNA-damaging agents. Bacterial populations also form persisters, dormant cells that are highly tolerant to ...antibiotics and play an important role in recalcitrance of biofilm infections. Stress response and dormancy appear to represent alternative strategies of cell survival. The mechanism of persister formation is unknown, but isolated persisters show increased levels of toxin/antitoxin (TA) transcripts. We have found previously that one or more components of the SOS response induce persister formation after exposure to a DNA-damaging antibiotic. The SOS response induces several TA genes in Escherichia coli. Here, we show that a knockout of a particular SOS-TA locus, tisAB/istR, had a sharply decreased level of persisters tolerant to ciprofloxacin, an antibiotic that causes DNA damage. Step-wise administration of ciprofloxacin induced persister formation in a tisAB-dependent manner, and cells producing TisB toxin were tolerant to multiple antibiotics. TisB is a membrane peptide that was shown to decrease proton motive force and ATP levels, consistent with its role in forming dormant cells. These results suggest that a DNA damage-induced toxin controls production of multidrug tolerant cells and thus provide a model of persister formation.
Summary
Inhibition of cell division is critical for viability under DNA‐damaging conditions. DNA damage induces the SOS response that in bacteria inhibits cell division while repairs are being made. ...In coccoids, such as the human pathogen, Staphylococcus aureus, this process remains poorly studied. Here, we identify SosA as the staphylococcal SOS‐induced cell division inhibitor. Overproduction of SosA inhibits cell division, while sosA inactivation sensitizes cells to genotoxic stress. SosA is a small, predicted membrane protein with an extracellular C‐terminal domain in which point mutation of residues that are conserved in staphylococci and major truncations abolished the inhibitory activity. In contrast, a minor truncation led to SosA accumulation and a strong cell division inhibitory activity, phenotypically similar to expression of wild‐type SosA in a CtpA membrane protease mutant. This suggests that the extracellular C‐terminus of SosA is required both for cell division inhibition and for turnover of the protein. Microscopy analysis revealed that SosA halts cell division and synchronizes the cell population at a point where division proteins such as FtsZ and EzrA are localized at midcell, and the septum formation is initiated but unable to progress to closure. Thus, our findings show that SosA is central in cell division regulation in staphylococci.
Staphylococcus aureus is a human pathogen and a model organism for cell division in spherical bacteria. We show that SosA is the DNA‐damage‐inducible cell‐division inhibitor in S. aureus responsible for cessation of the cell cycle prior to division plate completion. The extracellular domain of SosA appears essential for activity but is also a likely target for regulation by the CtpA protease. This represents the first description of a cell division inhibition process in coccoid bacteria.
Colibactin is a chemically unstable small-molecule genotoxin that is produced by several different bacteria, including members of the human gut microbiome
. Although the biological activity of ...colibactin has been extensively investigated in mammalian systems
, little is known about its effects on other microorganisms. Here we show that colibactin targets bacteria that contain prophages, and induces lytic development through the bacterial SOS response. DNA, added exogenously, protects bacteria from colibactin, as does expressing a colibactin resistance protein (ClbS) in non-colibactin-producing cells. The prophage-inducing effects that we observe apply broadly across different phage-bacteria systems and in complex communities. Finally, we identify bacteria that have colibactin resistance genes but lack colibactin biosynthetic genes. Many of these bacteria are infected with predicted prophages, and we show that the expression of their ClbS homologues provides immunity from colibactin-triggered induction. Our study reveals a mechanism by which colibactin production could affect microbiomes and highlights a role for microbial natural products in influencing population-level events such as phage outbreaks.
Antibiotics can induce mutations that cause antibiotic resistance. Yet, despite their importance, mechanisms of antibiotic-promoted mutagenesis remain elusive. We report that the fluoroquinolone ...antibiotic ciprofloxacin (cipro) induces mutations by triggering transient differentiation of a mutant-generating cell subpopulation, using reactive oxygen species (ROS). Cipro-induced DNA breaks activate the Escherichia coli SOS DNA-damage response and error-prone DNA polymerases in all cells. However, mutagenesis is limited to a cell subpopulation in which electron transfer together with SOS induce ROS, which activate the sigma-S (σS) general-stress response, which allows mutagenic DNA-break repair. When sorted, this small σS-response-“on” subpopulation produces most antibiotic cross-resistant mutants. A U.S. Food and Drug Administration (FDA)-approved drug prevents σS induction, specifically inhibiting antibiotic-promoted mutagenesis. Further, SOS-inhibited cell division, which causes multi-chromosome cells, promotes mutagenesis. The data support a model in which within-cell chromosome cooperation together with development of a “gambler” cell subpopulation promote resistance evolution without risking most cells.
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•Antibiotic-induced mutable cell subpopulation generates resistant mutants•Mitigates risk to most cells; reactive oxygen → σS stress response → gamblers•FDA-approved drug blocks σS response and mutagenesis: anti-evolvability drug•Multiple chromosomes needed: chromosome cooperation can allow rapid adaptation
Bacteria exposed to antibiotic acquire reactive oxygen in a transient “gambler” cell subpopulation that undertakes general stress response-induced mutagenic DNA break repair, evolves resistance to new antibiotics, and is inhibited by an FDA-approved drug that inhibits evolvability.
Antibiotics have a primary mode of actions, and most of them have a common secondary mode of action via reactive species (ROS and RNS) mediated DNA damage. Bacteria have been able to tolerate this ...DNA damage by SOS (Save-Our-Soul) response. RecA is the universal essential key protein of the DNA damage mediated SOS repair in various bacteria including ESKAPE pathogens. In addition, antibiotics also triggers activation of various other bacterial mechanisms such as biofilm formation, host dependent responses, persister subpopulation formation. These supporting the survival of bacteria in unfriendly natural conditions i.e. antibiotic presence. This review highlights the detailed mechanism of RecA mediated SOS response as well as role of RecA-LexA interaction in SOS response. The review also focuses on inter-connection between DNA damage repair pathway (like SOS response) with other survival mechanisms of bacteria such as host mediated RecA induction, persister-SOS interplay, and biofilm-SOS interplay. This understanding of inter-connection of SOS response with different other survival mechanisms will prove beneficial in targeting the SOS response for prevention and development of therapeutics against recalcitrant bacterial infections. The review also covers the significance of RecA as a promising potent therapeutic target for hindering bacterial SOS response in prevailing successful treatments of bacterial infections and enhancing the conventional antibiotic efficiency.
We created a high-throughput modality of photoactivated localization microscopy (PALM) that enables automated 3D PALM imaging of hundreds of synchronized bacteria during all stages of the cell cycle. ...We used high-throughput PALM to investigate the nanoscale organization of the bacterial cell division protein FtsZ in live Caulobacter crescentus. We observed that FtsZ predominantly localizes as a patchy midcell band, and only rarely as a continuous ring, supporting a model of "Z-ring" organization whereby FtsZ protofilaments are randomly distributed within the band and interact only weakly. We found evidence for a previously unidentified period of rapid ring contraction in the final stages of the cell cycle. We also found that DNA damage resulted in production of high-density continuous Z-rings, which may obstruct cytokinesis. Our results provide a detailed quantitative picture of in vivo Z-ring organization.
bacterial LexA transcriptional repressor Butala, M; Žgur-Bertok, D; Busby, S. J. W
Cellular and molecular life sciences : CMLS,
01/2009, Volume:
66, Issue:
1
Journal Article
Peer reviewed
Bacteria respond to DNA damage by mounting a coordinated cellular response, governed by the RecA and LexA proteins. In Escherichia coli, RecA stimulates cleavage of the LexA repressor, inducing more ...than 40 genes that comprise the SOS global regulatory network. The SOS response is widespread among bacteria and exhibits considerable variation in its composition and regulation. In some well-characterised pathogens, induction of the SOS response modulates the evolution and dissemination of drug resistance, as well as synthesis, secretion and dissemination of the virulence. In this review, we discuss the structure of LexA protein, particularly with respect to distinct conformations that enable repression of SOS genes via specific DNA binding or repressor cleavage during the response to DNA damage. These may provide new starting points in the battle against the emergence of bacterial pathogens and the spread of drug resistance among them.
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