Inflammatory diseases of the gut are associated with increased intestinal oxygen concentrations and high levels of inflammatory oxidants, including hydrogen peroxide (H2O2) and hypochlorous acid (HOCl), which are antimicrobial compounds produced by the innate immune system. This contributes to dysbiotic changes in the gut microbiome, including increased populations of proinflammatory enterobacteria (Escherichia coli and related species) and decreased levels of health-associated anaerobic Firmicutes and Bacteroidetes. The pathways for H2O2 and HOCl resistance in E. coli have been well studied, but little is known about how commensal and probiotic bacteria respond to inflammatory oxidants. In this work, we have characterized the transcriptomic response of the anti-inflammatory, gut-colonizing Gram-positive probiotic Lactobacillus reuteri to both H2O2 and HOCl. L. reuteri mounts distinct but overlapping responses to each of these stressors, and both gene expression and survival were strongly affected by the presence or absence of oxygen. Oxidative stress response in L. reuteri required several factors not found in enterobacteria, including the small heat shock protein Lo18, polyphosphate kinase 2, and RsiR, an L. reuteri-specific regulator of anti-inflammatory mechanisms. IMPORTANCE Reactive oxidants, including hydrogen peroxide and hypochlorous acid, are antimicrobial compounds produced by the immune system during inflammation. Little is known, however, about how many important types of bacteria present in the human microbiome respond to these oxidants, especially commensal and other health-associated species. We have now mapped the stress response to both H2O2 and HOCl in the intestinal lactic acid bacterium Lactobacillus reuteri.

Inorganic polyphosphate (polyP) is synthesized by bacteria under stressful environmental conditions and acts by a variety of mechanisms to promote cell survival. While the kinase that synthesizes polyP (PPK, encoded by the ppk gene) is well known, ppk transcription is not activated by environmental stress and little is understood about how environmental stress signals lead to polyP accumulation. Previous work has shown that the transcriptional regulators DksA, RpoN (σ54) and RpoE (σ24) positively regulate polyP production, but not ppk transcription, in Escherichia coli . In this work, we examine the role of the alternative sigma factor RpoN and nitrogen starvation stress response pathways in controlling polyP synthesis. We show that the RpoN enhancer binding proteins GlnG and GlrR impact polyP production, and uncover a new role for the nitrogen phosphotransferase regulator PtsN (EIIANtr) as a positive regulator of polyP production, acting upstream of DksA, downstream of RpoN and apparently independently of RpoE. However, neither these regulatory proteins nor common nitrogen metabolites appear to act directly on PPK, and the precise mechanism(s) by which polyP production is modulated after stress remain(s) unclear. Unexpectedly, we also found that the genes that impact polyP production vary depending on the composition of the rich media in which the cells were grown before exposure to polyP-inducing stress. These results constitute progress towards deciphering the regulatory networks driving polyP production under stress, and highlight the remarkable complexity of this regulation and its connections to a broad range of stress-sensing pathways.

Inorganic polyphosphate (polyP) is synthesized by bacteria under stressful environmental conditions and acts by a variety of mechanisms to promote cell survival. While the kinase that synthesizes polyP (PPK, enocoded by the ppk gene) is well known, little is understood about how environmental stress signals lead to activation of this enzyme. Previous work has shown that the transcriptional regulators DksA, RpoN (σ54), and RpoE (σ24) positively regulate polyP production, but not ppk transcription, in Escherichia coli. In this work, we set out to examine the role of the alternative sigma factor RpoN and nitrogen starvation stress response pathways in controlling polyP synthesis in more detail. In the course of these experiments, we identified GlnG, GlrR, PhoP, PhoQ, RapZ, and GlmS as proteins that affect polyP production, and uncovered a central role for the nitrogen phosphotransferase regulator PtsN (EIIANtr) in a polyP regulatory pathway, acting upstream of DksA, downstream of RpoN, and apparently independently of RpoE. However, none of these regulators appears to act directly on PPK, and the mechanism(s) by which they modulate polyP production remain unclear. Unexpectedly, we also found that the pathways that regulate polyP production vary depending not only on the stress condition applied, but also on the composition of the media in which the cells were grown before exposure to polyP-inducing stress. These results constitute substantial progress towards deciphering the regulatory networks driving polyP production under stress, but highlight the remarkable complexity of this regulation and its connections to a broad range of stress-sensing pathways.