A defining characteristic of Gram-negative bacteria is the presence of an outer membrane, which functions as an additional barrier inhibiting the penetration of toxic chemicals, such as antibiotics. Porins are outer membrane proteins associated with the modulation of cellular permeability and antibiotic resistance. Although there are numerous studies regarding porins, a systematic approach about the roles of porins in bacterial physiology and antibiotic resistance does not exist yet. In this study, we constructed mutants of all porins in Escherichia coli and examined the effect of porins on antibiotic resistance and membrane integrity. The OmpF-defective mutant was resistant to several antibiotics including β-lactams, suggesting that OmpF functions as the main route of outer membrane penetration for many antibiotics. In contrast, OmpA was strongly associated with the maintenance of membrane integrity, which resulted in the increased susceptibility of the ompA mutant to many antibiotics. Notably, OmpC was involved in both the roles. Additionally, our systematic analyses revealed that other porins were not involved in the maintenance of membrane integrity, but several porins played a major or minor role in the outer membrane penetration for a few antibiotics. Collectively, these results show that each porin plays a distinct role in antibiotic resistance and membrane integrity, which could improve our understanding of the physiological function and clinical importance of porins.

The maintenance of ionic homeostasis in response to changes in the environment is essential for all living cells. Although there are still many important questions concerning the role of the major monovalent cation K ؉ , cytoplasmic K ؉ in bacteria is required for diverse processes. Here, we show that enzyme IIA Ntr (EIIA Ntr ) of the nitrogen-metabolic phosphotransferase system interacts with and regulates the Escherichia coli K ؉ transporter TrkA. Previously we reported that an E. coli K-12 mutant in the ptsN gene encoding EIIA Ntr was extremely sensitive to growth inhibition by leucine or leucine-containing peptides (LCPs). This sensitivity was due to the requirement of the dephosphorylated form of EIIA Ntr for the derepression of ilvBN expression. Whereas the ptsN mutant is extremely sensitive to LCPs, a ptsN trkA double mutant is as resistant as WT. Furthermore, the sensitivity of the ptsN mutant to LCPs decreases as the K ؉ level in culture media is lowered. We demonstrate that dephosphorylated EIIA Ntr , but not its phosphorylated form, forms a tight complex with TrkA that inhibits the accumulation of high intracellular concentrations of K ؉ . High cellular K ؉ levels in a ptsN mutant promote the sensitivity of E. coli K-12 to leucine or LCPs by inhibiting both the expression of ilvBN and the activity of its gene products. Here, we delineate the similarity of regulatory mechanisms for the paralogous carbon and nitrogen phosphotransferase systems. Dephosphorylated EIIA Glc regulates a variety of transport systems for carbon sources, whereas dephosphorylated EIIA Ntr regulates the transport system for K ؉ , which has global effects related to nitrogen metabolism. leucine toxicity ͉ nitrogen-metabolic phosphotransferase system (PTS) ͉ potassium transporter TrkA ͉ protein-protein interaction ͉ signal transduction T he well defined phosphotransferase system (PTS) is composed of two general cytoplasmic proteins, enzyme I (EI) and histidine phosphocarrier protein (HPr), and some sugarspecific components collectively known as enzymes II (1). The carbohydrate PTS, especially for glucose uptake, occupies a central position in bacterial physiology as a result of the identification of multiple regulatory functions superimposed on the transport functions, such as regulation of chemotaxis by EI (2); regulation of glycogen breakdown by HPr (3, 4); regulation of the global repressor Mlc by the membrane-bound glucose transporter EIICB Glc (5-7); and regulation of carbohydrate transport and metabolism (1,8,9), the metabolic flux between fermentation and respiration (10), and adenylyl cyclase activity (11) by EIIA Glc . These regulatory functions of the carbohydrate PTS depend on the phosphorylation state of the involved components, which have been shown to increase in the absence and decrease in the presence of a PTS sugar substrate.The nitrogen-metabolic PTS consists of enzyme I Ntr (EI Ntr , an

Widespread antimicrobial use and concomitant resistance have led to a significant threat to public health. Because inappropriate use and overuse of antibiotics based on insufficient knowledge are one of the major drivers of antibiotic resistance, education about prudent antibiotic use aimed at both the prescribers and the public is important. This review investigates recent studies on the effect of interventions for promoting prudent antibiotics prescribing. Up to now, most educational efforts have been targeted to medical professionals, and many studies showed that these educational efforts are significantly effective in reducing antibiotic prescribing. Recently, the development of educational programs to reduce antibiotic use is expanding into other groups, such as the adult public and children. The investigation of the contents of educational programs for prescribers and the public demonstrates that it is important to develop effective educational programs suitable for each group. In particular, it seems now to be crucial to develop appropriate curricula for teaching medical and nonmedical (pharmacy, dentistry, nursing, veterinary medicine, and midwifery) undergraduate students about general medicine, microbial virulence, mechanism of antibiotic resistance, and judicious antibiotic prescribing.

SsrA/SsrB is a primary two-component system that mediates the survival and replication of Salmonella within host cells. When activated, the SsrB response regulator directly promotes the transcription of multiple genes within Salmonella pathogenicity island 2 (SPI-2). As expression of the SsrB protein is promoted by several transcription factors, including SsrB itself, the expression of SPI-2 genes can increase to undesirable levels under activating conditions. Here, we report that Salmonella can avoid the hyperactivation of SPI-2 genes by using ptsN-encoded EIIA Ntr , a component of the nitrogen-metabolic phosphotransferase system. Under SPI-2-inducing conditions, the levels of SsrB-regulated gene transcription increased abnormally in a ptsN deletion mutant, whereas they decreased in a strain overexpressing EIIA Ntr . We found that EIIA Ntr controls SPI-2 genes by acting on the SsrB protein at the posttranscriptional level. EIIA Ntr interacted directly with SsrB, which prevented the SsrB protein from binding to its target promoter. Finally, the Salmonella strain, either lacking the ptsN gene or overexpressing EIIA Ntr , was unable to replicate within macrophages, and the ptsN deletion mutant was attenuated for virulence in mice. These results indicated that normal SPI-2 gene expression maintained by an EIIA Ntr -SsrB interaction is another determinant of Salmonella virulence.virulence gene regulation | nitrogen-metabolic phosphotransferase system (PTS) | protein-protein interaction

SummaryWhile the proteins of the phosphoenolpyruvate:carbohydrate phosphotransferase system (carbohydrate PTS) have been shown to regulate numerous targets, little such information is available for the nitrogenmetabolic phosphotransferase system (nitrogenmetabolic PTS). To elucidate the physiological role of the nitrogen-metabolic PTS, we carried out phenotype microarray (PM) analysis with Escherichia coli K-12 strain MG1655 deleted for the ptsP gene encoding the first enzyme of the nitrogen-metabolic PTS. Together with the PM data, growth studies revealed that a ptsN (encoding enzyme IIA Ntr ) mutant became extremely sensitive to leucine-containing peptides (LCPs), while both ptsP (encoding enzyme I Ntr ) and ptsO (encoding NPr) mutants were more resistant than wild type. The toxicity of LCPs was found to be due to leucine and the dephospho-form of enzyme IIA Ntr was found to be necessary to neutralize leucine toxicity. Further studies showed that the dephospho-form of enzyme IIA Ntr is required for derepression of the ilvBN operon encoding acetohydroxy acid synthase I catalysing the first step common to the biosynthesis of the branched-chain amino acids.