Summary Bacteria respond to physical and chemical stresses that affect the integrity of the cell wall and membrane by activating an intricate cell envelope stress response. The ability of cells to regulate the biophysical properties of the membrane by adjusting fatty acid composition is known as homeoviscous adaptation. Here, we identify a homeoviscous adaptation mechanism in Bacillussubtilis regulated by the extracytoplasmic function σ factor σW. Cell envelope active compounds, including detergents, activate a sense-oriented, σW-dependent promoter within the first gene of the fabHa fabF operon. Activation leads to a decrease in the amount of FabHa coupled with an increase in FabF, the initiation and elongation condensing enzymes of fatty acid biosynthesis, respectively. Down-regulation of FabHa results in an increased reliance on the FabHb paralog leading to a greater proportion of straight chain fatty acids in the membrane, and the up-regulation of FabF increases the average fatty acid chain length. The net effect is to reduce membrane fluidity. The inactivation of the σW-dependent promoter within fabHa increased sensitivity to detergents and to antimicrobial compounds produced by other Bacillus spp. Thus, the σW stress response provides a mechanism to conditionally decrease membrane fluidity through the opposed regulation of FabHa and FabF.

Summary In Bacillus subtilis, the extracytoplasmic function (ECF) σ factors σM, σW, and σX all contribute to resistance against lantibiotics. Nisin, a model lantibiotic, has a dual mode of action: it inhibits cell wall synthesis by binding lipid II, and this complex also forms pores in the cytoplasmic membrane. These activities can be separated in a nisin hinge-region variant (N20P M21P) that binds lipid II, but no longer permeabilizes membranes. The major contribution of σM to nisin resistance is expression of ltaSa, encoding a stress-activated lipoteichoic acid synthase, and σX functions primarily by activation of the dlt operon controlling D-alanylation of teichoic acids. Together, σM and σX regulate cell envelope structure to decrease access of nisin to its lipid II target. In contrast, σW is principally involved in protection against membrane permeabilization as it provides little protection against the nisin hinge region variant. σW contributes to nisin resistance by regulation of a signal peptide peptidase (SppA), phage shock proteins (PspA and YvlC, a PspC homolog), and tellurite resistance related proteins (YceGHI). These defensive mechanisms are also effective against other lantibiotics such as mersacidin, gallidermin, and subtilin and comprise an important subset of the intrinsic antibiotic resistome of B. subtilis.

Heptaprenyl diphosphate (C35-PP) is an isoprenoid intermediate in the synthesis of both menaquinone and the sesquarterpenoids. We demonstrate that inactivation of ytpB, encoding a C35-PP utilizing enzyme required for sesquarterpenoid synthesis, leads to an increased sensitivity to bacitracin, an antibiotic that binds undecaprenyl pyrophosphate (C55-PP), a key intermediate in cell wall synthesis. Genetic studies indicate that bacitracin sensitivity is due to accumulation of C35-PP, rather than the absence of sesquarterpenoids. Sensitivity is accentuated in a ytpB menA double mutant, lacking both known C35-PP consuming enzymes, and in a ytpB strain overexpressing the HepST enzyme that synthesizes C35-PP. Conversely, sensitivity in the ytpB background is suppressed by mutation of hepST or by supplementation with 1,4-dihydroxy-2-naphthoate, a co-substrate with C35-PP for MenA. Bacitracin sensitivity results from impairment of the BceAB and BcrC resistance mechanisms by C35-PP: in a bceAB bcrC double mutant disruption of ytpB no longer increases bacitracin sensitivity. These results suggest that C35-PP inhibits both BcrC (a C55-PP phosphatase) and BceAB (an ABC transporter that confers bacitracin resistance). These findings lead to a model in which BceAB protects against bacitracin by transfer of the target, C55-PP, rather than the antibiotic across the membrane.

The glutamate dehydrogenase RocG of Bacillus subtilis is a bifunctional protein with both enzymatic and regulatory functions. Here we show that the rocG null mutant is sensitive to ␤-lactams, including cefuroxime (CEF), and to fosfomycin but that resistant mutants arise due to gain-of-function mutations in gudB, which encodes an otherwise inactive glutamate dehydrogenase. In the presence of CEF, ⌬rocG ⌬gudB mutant cells exhibit growth arrest when they reach mid-exponential phase. Using microarray-based transcriptional profiling, we found that the W regulon was downregulated in the ⌬rocG ⌬gudB null mutant. A survey of W -controlled genes for effects on CEF resistance identified both the NfeD protein YuaF and the flotillin homologue YuaG (FloT). Notably, overexpression of yuaFG in the rocG null mutant prevents the growth arrest induced by CEF. The YuaG flotillin has been shown previously to localize to defined lipid microdomains, and we show here that the yuaFGI operon contributes to a W -dependent decrease in membrane fluidity. We conclude that glutamate dehydrogenase activity affects the expression of the W regulon, by pathways that are yet unclear, and thereby influences resistance to CEF and other antibiotics. In Bacillus subtilis, a model system for the Gram-positive bacteria (36), the synthesis of glutamate is catalyzed uniquely by the heterodimeric product of the gltAB operon. Glutamate acts as a central metabolite providing the link between carbon and nitrogen metabolism (11,40). The degradation of glutamate is catalyzed by the strictly catabolic glutamate dehydrogenase RocG (2). In addition to rocG, B. subtilis has a second glutamate dehydrogenase gene, gudB, whose product is cryptic due to an insertion of three amino acids close to the active site of this enzyme. However, null mutants of rocG rapidly accumulate spontaneous gain-offunction suppressor mutations in gudB that remove the repeat sequence encoding the three-amino-acid insertion, thereby resulting in the synthesis of active GudB (3,12).Recent studies have shown that RocG has a second activity as a regulatory protein. RocG, if glutamate is available, directly interacts with GltC, the transcription activator of the gltAB operon, thus inhibiting its activity (10, 15). However, whether it has additional functions remains largely unknown. In addition to RocG, several other bacterial enzymes are now known to regulate gene expression. Some act as transcription factors by direct binding to either DNA or RNA, and others modulate the activity of transcription factors either by covalent modification or by protein-protein interactions (9).Cefuroxime (CEF) belongs to the group of broad-spectrum ␤-lactam cephalosporin antibiotics, with antimicrobial activity against both Gram-positive and Gram-negative bacteria (31). The mode of action of CEF is conventional: by binding to specific penicillin-binding proteins (PBPs), it inhibits the third and final stage of bacterial cell wall synthesis. In Gram-negative bacteria such as Escherichia coli, CEF shows high affin...

Bacteria use a variety of DNA-mobilizing enzymes to facilitate environmental niche adaptation via horizontal gene transfer. This has led to real-world problems, like the spread of antibiotic resistance, yet many mobilization proteins remain undefined. In the study described here, we investigated the uncharacterized family of YhgA-like transposase_31 (Pfam PF04754) proteins. Our primary focus was the genetic and biochemical properties of the five Escherichia coli K-12 members of this family, which we designate RpnA to RpnE, where Rpn represents recombination-promoting nuclease. We employed a conjugal system developed by our lab that demanded RecA-independent recombination following transfer of chromosomal DNA. Overexpression of RpnA (YhgA), RpnB (YfcI), RpnC (YadD), and RpnD (YjiP) increased RecA-independent recombination, reduced cell viability, and induced the expression of reporter of DNA damage. For the exemplar of the family, RpnA, mutational changes in proposed catalytic residues reduced or abolished all three phenotypes in concert. In vitro, RpnA displayed magnesium-dependent, calcium-stimulated DNA endonuclease activity with little, if any, sequence specificity and a preference for double-strand cleavage. We propose that Rpn/YhgA-like family nucleases can participate in gene acquisition processes.IMPORTANCE Bacteria adapt to new environments by obtaining new genes from other bacteria. Here, we characterize a set of genes that can promote the acquisition process by a novel mechanism. Genome comparisons had suggested the horizontal spread of the genes for the YhgA-like family of proteins through bacteria. Although annotated as transposase_31, no member of the family has previously been characterized experimentally. We show that four Escherichia coli K-12 paralogs contribute to a novel RecA-independent recombination mechanism in vivo. For RpnA, we demonstrate in vitro action as a magnesium-dependent, calcium-stimulated nonspecific DNA endonuclease. The cleavage products are capable of providing priming sites for DNA polymerase, which can enable DNA joining by primer-template switching.