monocytogenes EGD-e rpoN (σL) mutant [22] (Table 2), supporting their negative regulation by σL. Overall, the 56 proteins identified here as negatively regulated by σL represented 13 role categories (e.g., SHP099 chemical structure energy metabolism, transport and binding

proteins, central intermediary metabolism), including 31 proteins Momelotinib in the energy metabolism role category; statistical analyses showed overrepresentation of the role category “energy metabolism” (p < 0.01; Odds Ratio = 5.6) among these 56 proteins. Specific proteins identified as negatively regulated by σL included flagellin (FlaA), chemotaxis protein CheA, and a glutamate-γ-aminobutyric acid (GABA) antiporter (Lmo2362, GadC, GadT2), which have known roles in stress adaptation or virulence in

L. monocytogenes[1, 27]. σC regulates a small number of proteins Previous studies indicated a role for σC in L. monocytogenes thermal adaptive response as well as in cold adaptation [3, 13], however only a few genes have been identified as part of the σC regulon [7]. Similarly, we were only able to identify one protein, Lmo0096, that showed higher protein levels (FC ≥ 1.5; p c < 0.05) in the presence of σC (i.e., the comparison between the ΔBHL and the ΔBCHL strain; Table 3). Lmo0096 has been previously reported to be induced under cold stress in L. monocytogenes[28], supporting a role of σC in response to temperature stress in the bacterium. By comparison, the transcriptomic study by Chaturongakul et al., 2011 only identified lmo0422, which is in the same operon as sigC (lmo0423), as positively regulated by σC[7]. Table 3 Proteins found Fedratinib to be differentially regulated by σ C , as determined by a proteomic comparison between L. monocytogenes 10403S Δ BHL and Δ BCHL Proteina Fold change ΔBHL/ΔBCHL Description Gene name Role categoryb Sub-Role categoryb Proteins GPX6 with positive fold change ( > 1.5) and p < 0.05 (indicating positive regulation by σ C ) Lmo0096c 3.19 mannose-specific PTS system IIAB component ManL mptA Energy metabolism Pyruvate dehydrogenase         Amino acid biosynthesis Aromatic amino acid family         Transport and binding proteins Carbohydrates, organic alcohols,

and acids Proteins with negative fold change ( < -1.5) and p < 0.05 (indicating negative regulation by σ C ) Lmo2094 −1.82 hypothetical protein lmo2094 Energy metabolism Sugars Lmo1902 −1.61 3-methyl-2-oxobutanoate hydroxymethyltransferase panB Biosynthesis of cofactors, prosthetic groups, and carriers Pantothenate and coenzyme A aProtein names are based on the L. monocytogenes EGD-e locus. bRole Categories and Sub-Role categories are based on JCVI classification [26]. cPreceded by a putative σL promoter; tggcacagaacttgca; -12 and -24 regions are underlined. We also identified two proteins, Lmo2094 and Lmo1902, that showed higher protein levels in the absence of σC, suggesting negative regulation of these proteins by σC (Table 3).

Active inward transport of protons by cytoplasmic BMS345541 molecular weight membrane cation/H+ antiporters is crucial to the latter strategy and often plays a dominant role in alkaline pH homeostasis in bacteria [6, 7]. The transportomes of most free-living

bacteria contain numerous integral membrane secondary active cation/H+ antiporters that can couple the inward movement of protons to the outward movement of either Na+ or K+ ions in a process driven by the proton motive force (PMF) [7]. To date, only a few of the transporters likely to be involved in alkaline pH homeostasis by neutralophilic bacteria have been identified and characterised. Nevertheless, studies of specific sodium/proton (Na+/H+) and potassium/proton (K+/H+) antiporters have helped illuminate selleck inhibitor their individual contributions to this process. In E. coli alkaline pH homeostasis is realised by the combined and partially overlapping functions of at least three such transporters: the paradigm Na+/H+ antiporter NhaA [8]; MdfA, a well-characterised

Na+/(K+)/H+ antiporter that was first identified as a multidrug-resistance transporter [9] belonging to the ubiquitous, large and diverse major facilitator superfamily (MFS)[10, 11]; and the K+/(Na+)(Ca2+) /H+ antiporter ChaA [12]. NhaA is dominant in the alkaline pH range of up to pH 9, and it confers alkalitolerance to cells only in the presence of externally added Na+[13]. Furthermore, nhaA deletion mutants can only grow at alkaline Astemizole pH in the absence of external Na+ ions [14]. MdfA overexpressed from a multicopy plasmid extends the alkalitolerance of E. coli cells up

to pH 10 when Na+ or K+ is added to the external growth medium, and MdfA can take over from NhaA when the latter is deleted or dysfunctional [9]. Finally, ChaA is active at pH values above 8.0 in the presence of external K+ and it supports alkaline pH homeostasis by coupling the efflux of intracellular K+ to the uptake of protons [12]. The role of MdfA in alkaline pH homeostasis is of particular interest considering its contribution to multidrug resistance in E. coli[15]. Like MdfA, other multidrug transporters of the MFS are polyspecific with respect to substrate recognition profile, and they can efflux a remarkably diverse range of learn more substrates from bacterial cells [16]. Interest in these proteins is further compounded by the recent shift in perception that they function not merely as part of a defensive response to drugs, but as vital components of other fundamental physiological processes in bacteria [17–20]; despite this, a function independent of multidrug efflux has been described for very few of them [9, 21–23]. Working from this perspective, we hypothesised that multidrug efflux proteins other than MdfA could play a role in pH homeostasis in E. coli. One candidate is the 12-transmembrane spanning segment drug/H+ antiporter MdtM, a recently characterised member of the MFS that contributes to intrinsic resistance of E.