Highlights d The majority of Bacillus subtilis cells die upon oxygen depletion d Surfactin production depolarizes cells to maintain viability upon oxygen depletion d Surfactin promotes growth in early stationary phase by enhancing oxygen diffusion d The autolytic enzyme LytC and surfactin mediate lysis upon oxygen depletion

Background Salicylaldehyde isonicotinoyl hydrazone (SIH) is an iron chelator of the aroylhydrazone class that displays antioxidant or prooxidant effects in different mammalian cell lines. Because the liver is the major site of iron storage, elucidating the effect of SIH on hepatic oxidative metabolism is critical for designing effective hepatic antioxidant therapies. Methods Hepatocyte-like HepG2 cells were exposed to SIH or to analogs showing greater stability, such as N′-[1-(2-Hydroxyphenyl)ethyliden]isonicotinoyl hydrazide (HAPI), or devoid of iron chelating properties, such as benzaldehyde isonicotinoyl hydrazone (BIH), and toxicity, oxidative stress and antioxidant (glutathione) metabolism were evaluated. Results Autoxidation of Fe2+ in vitro increased in the presence of SIH or HAPI (but not BIH), an effect partially blocked by Fe2+ chelation. Incubation of HepG2 cells with SIH or HAPI (but not BIH) was non-toxic and increased reactive oxygen species (ROS) levels, activated the transcription factor Nrf2, induced the catalytic subunit of γ-glutamate cysteine ligase (Gclc), and increased glutathione concentration. Fe2+ chelation decreased ROS and inhibited Nrf2 activation, and Nrf2 knock-down inhibited the induction of Gclc in the presence of HAPI. Inhibition of γ-glutamate cysteine ligase enzymatic activity inhibited the increase in glutathione caused by HAPI, and increased oxidative stress. Conclusions SIH iron chelators display both prooxidant (increasing the autoxidation rate of Fe2+) and antioxidant (activating Nrf2 signaling) effects. General significance Activation by SIH iron chelators of a hormetic antioxidant response contributes to its antioxidant properties and modulates the anti- and pro-oxidant balance.

LytM-domain containing proteins are LAS peptidases (lysostaphin-type enzymes, D-Ala-D-Ala metallopeptidases, and sonic hedgehog) and are known to play diverse roles throughout the bacterial cell cycle through direct or indirect hydrolysis of the bacterial cell wall. A subset of the LytM factors are catalytically inactive but regulate the activity of other cell wall hydrolases and are classically described as cell separation factors NlpD and EnvC. Here, we explore the function of four LytM factors in the alphaproteobacterial plant pathogen Agrobacterium tumefaciens. An LmdC ortholog (Atu1832) and a MepM ortholog (Atu4178) are predicted to be catalytically active. While Atu1832 does not have an obvious function in cell growth or division, Atu4178 is essential for polar growth and likely functions as a space-making endopeptidase that cleaves amide bonds in the peptidoglycan cell wall during elongation. The remaining LytM factors are degenerate EnvC and NlpD orthologs. Absence of these proteins results in striking phenotypes indicative of misregulation of cell division and growth pole establishment. The deletion of an amidase, AmiC, closely phenocopies the deletion of envC suggesting that EnvC might regulate AmiC activity. The NlpD ortholog DipM is unprecedently essential for viability and depletion results in the misregulation of early stages of cell division, contrasting with the canonical view of DipM as a cell separation factor. Finally, we make the surprising observation that absence of AmiC relieves the toxicity induced by dipM overexpression. Together, these results suggest EnvC and DipM may function as regulatory hubs with multiple partners to promote proper cell division and establishment of polarity.

Lytic enzymes play an essential role in the remodeling of bacterial peptidoglycan (PG), an extracellular mesh-like structure that retains the membrane in the context of high internal osmotic pressure. Peptidoglycan (PG) must be unfailingly stable to preserve cell integrity but must also be dynamically remodeled for the cell grow, divide and insert macromolecular machines. The flagellum is one such macromolecular machine that transits the PG and flagellar insertion is aided by localized activity of a dedicated PG lyase in Gram-negative bacteria. To date, there is no known dedicated lyase in Gram-positive bacteria for the insertion of flagella and here we take a reverse-genetic candidate-gene approach and find that cells mutated for the lytic transglycosylase CwlQ exhibited a severe defect in flagellar-dependent swarming motility. We further show that CwlQ was expressed by the motility sigma factor SigD and was secreted by the type III secretion system housed inside the flagellum. Nonetheless, cells mutated for CwlQ remained proficient for flagellar biosynthesis even when mutated in combination with four other lyases related to motility (LytC, LytD, LytF, and CwlO). The PG lyase or lyases essential for flagellar synthesis in B. subtilis, if any, remains unknown. IMPORTANCE Bacteria are surrounded by a wall of peptidoglycan and early work in Bacillus subtilis was the first to suggest that bacteria needed to enzymatically remodel the wall to permit insertion of the flagellum. No PG remodeling enzyme alone or in combination however, has been found to be essential for flagellar assembly in B. subtilis. Here we take a reverse genetic candidate gene approach and find that the PG lytic transglycosylase CwlQ is required for swarming motility. Subsequent characterization determined that while CwlQ was co-expressed with motility genes and is secreted by the flagellar secretion apparatus, it was not required for flagellar synthesis. The PG lyase needed for flagellar assembly in B. subtilis remains unknown.

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The RNA-binding protein CsrA is a post-transcriptional regulator that is encoded in genomes throughout the bacterial phylogeny. In the gamma-proteobacteria, the activity of CsrA is inhibited by small RNAs that competitively sequester CsrA binding. In contrast, the firmicute Bacillus subtilis encodes a protein inhibitor of CsrA called FliW, that non-competitively inhibits CsrA activity but the precise mechanism of antagonism is unclear. Here we take an unbiased genetic approach to identify residues of FliW important for CsrA inhibition that fall into two distinct spatial and functional classes. Most loss-of-function alleles mutated FliW residues that surround the critical regulatory CsrA residue N55 and abolished CsrA interaction. Two loss-of-function alleles however mutated FliW residues near the CsrA core dimerization domain and maintained interaction with CsrA. One of these two alleles reversed charge at what appeared to be a salt bridge with the CsrA core region, charge reversal of the CsrA partner residue phenocopied the FliW allele, and charge reversal of both residues simultaneously restored antagonism. We propose a model in which initial interaction between FliW and CsrA is necessary but not sufficient for antagonism which also requires salt bridge formation with, and deformation of, the CsrA core domain to allosterically abolish RNA binding activity. Importance CsrA is a small dimeric protein that binds RNA and is one of the few known examples of transcript-specific regulators of translation in bacteria. A protein called FliW binds to and antagonizes CsrA to govern flagellin homeostasis and flagellar assembly. Despite having a high-resolution three-dimensional structure of the FliW-CsrA complex, the mechanism of non-competitive inhibition remains unresolved. Here we identify FliW residues required for antagonism and we find that the residues make a linear connection in the complex from initial binding interaction with CsrA to a critical salt bridge near the core of the CsrA dimer. We propose that the salt bridge represents an allosteric contact that distorts the CsrA core to prevent RNA binding.

Hydrolytic enzymes play an essential role in the remodeling of bacterial peptidoglycan (PG), an extracellular mesh-like structure that retains the membrane in the context of high internal osmotic pressure. Peptidoglycan (PG) integrity must be unfailingly stable to preserve cell integrity but must also be dynamically remodeled for the cell grow, divide and insert macromolecular machines. The flagellum is one such macromolecular machine that transits the PG and the insertion of which is aided by localized activity of a dedicated PG hydrolase in Gram-negative bacteria. To date, there is no known dedicated hydrolase in Gram-positive bacteria for insertion of flagella and here we take a reverse-genetic candidate-gene approach to find that cells mutated for the lytic transglycosylase CwlQ exhibited a severe defect in flagellar dependent swarming motility. We show that CwlQ required its active site to promote swarming, was expressed by the motility sigma factor SigD, and was secreted by the type III secretion system housed inside the flagellum. Nonetheless, cells mutated for CwlQ remained proficient for flagellar biosynthesis even when mutated in combination with four other hydrolases related to motility (LytC, LytD, LytF, and CwlO). The PG hydrolase essential for flagellar synthesis in B. subtilis, if any, remains unknown.

No abstract