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61800.9756A novel chemical inducer of Streptococcus quorum sensing acts by inhibiting the pheromone-degrading endopeptidase PepO. Bacteria produce chemical signals (pheromones) to coordinate behaviors across a population in a process termed quorum sensing (QS). QS systems comprising peptide pheromones and their corresponding Rgg receptors are widespread among Firmicutes and may be useful targets for manipulating microbial behaviors, like suppressing virulence. The Rgg2/3 QS circuit of the human pathogen Streptococcus pyogenes controls genes affecting resistance to host lysozyme in response to short hydrophobic pheromones (SHPs). Considering that artificial activation of a QS pathway may be as useful in the objective of manipulating bacteria as inhibiting it, we sought to identify small-molecule inducers of the Rgg2/3 QS system. We report the identification of a small molecule, P516-0475, that specifically induced expression of Rgg2/3-regulated genes in the presence of SHP pheromones at concentrations lower than typically required for QS induction. In searching for the mode of action of P516-0475, we discovered that an S. pyogenes mutant deficient in pepO, a neprilysin-like metalloendopeptidase that degrades SHP pheromones, was unresponsive to the compound. P516-0475 directly inhibited recombinant PepO in vitro as an uncompetitive inhibitor. We conclude that this compound induces QS by stabilizing SHP pheromones in culture. Our study indicates the usefulness of cell-based screens that modulate pathway activities to identify unanticipated therapeutic targets contributing to QS signaling.201829203527
823910.9754Surviving bacterial sibling rivalry: inducible and reversible phenotypic switching in Paenibacillus dendritiformis. Natural habitats vary in available nutrients and room for bacteria to grow, but successful colonization can lead to overcrowding and stress. Here we show that competing sibling colonies of Paenibacillus dendritiformis bacteria survive overcrowding by switching between two distinct vegetative phenotypes, motile rods and immotile cocci. Growing colonies of the rod-shaped bacteria produce a toxic protein, Slf, which kills cells of encroaching sibling colonies. However, sublethal concentrations of Slf induce some of the rods to switch to Slf-resistant cocci, which have distinct metabolic and resistance profiles, including resistance to cell wall antibiotics. Unlike dormant spores of P. dendritiformis, the cocci replicate. If cocci encounter conditions that favor rods, they secrete a signaling molecule that induces a switch to rods. Thus, in contrast to persister cells, P. dendritiformis bacteria adapt to changing environmental conditions by inducible and reversible phenotypic switching. IMPORTANCE: In favorable environments, species may face space and nutrient limits due to overcrowding. Bacteria provide an excellent model for analyzing principles underlying overcrowding and regulation of density in nature, since their population dynamics can be easily and accurately assessed under controlled conditions. We describe a newly discovered mechanism for survival of a bacterial population during overcrowding. When competing with sibling colonies, Paenibacillus dendritiformis produces a lethal protein (Slf) that kills cells at the interface of encroaching colonies. Slf also induces a small proportion of the cells to switch from motile, rod-shaped cells to nonmotile, Slf-resistant, vegetative cocci. When crowding is reduced and nutrients are no longer limiting, the bacteria produce a signal that induces cocci to switch back to motile rods, allowing the population to spread. Genes encoding components of this phenotypic switching pathway are widespread among bacterial species, suggesting that this survival mechanism is not unique to P. dendritiformis.201121628502
53820.9754The biochemical and genetic basis for high frequency thiomethyl galactoside resistance in lambda,lambdadg lysogens of Escherichia coli. In a culture of Escherichia coli K12 gal (lambdadg), cells which form large colonies on agar plates containing galactose and thiomethyl beta-D-galactoside (TMG) appear at high frequency. These clones are resistant to growth inhibition by TMG on galactose minimal medium. Biochemical studies of the steady-state levels of galactokinase and UDPgalactose 4-epimerase suggest that the resistant clones have extra copies of the genes for the galactose-metabolizing enzymes. The mutation for TMG resistance is not located in either the bacterial or the bacteriophage genome, but is probably due to an aberrant association between cell and prophage DNA. Mapping the TMG-resistant characteristic by phage P1 indicates that TMG-resistant bacteria posses at least two GAL+ OPERONS, ONE OF WHICH IS COTRANSDUCIBLe with bio+. In addition, TMG-resistant bacteria behave like lambdadg polylysogens when challenged with the phage lambdaI90c17. From these genetic experiments we conclude that TMG-resistant bacteria arise by duplication of the lambdadg prophage. Finally, gal+ bacteria which carry a single, additional, lambdadg prophage are TMG-resistant. TMG resistance is probably a gal+ gene dosage effect.1978344832
67430.9752Interplay between Two-Component Regulatory Systems Is Involved in Control of Cupriavidus metallidurans Metal Resistance Genes. Metal resistance of Cupriavidus metallidurans is based on determinants that were acquired in the past by horizontal gene transfer during evolution. Some of these determinants encode transmembrane metal efflux systems. Expression of most of the respective genes is controlled by two-component regulatory systems composed of a membrane-bound sensor/sensory histidine kinase (HK) and a cytoplasmic, DNA-binding response regulator (RR). Here, we investigated the interplay between the three closely related two-component regulatory systems CzcRS, CzcR(2)S(2), and AgrRS. All three systems regulate the response regulator CzcR, while the RRs AgrR and CzcR(2) were not involved in czc regulation. Target promoters were czcNp and czcPp for genes upstream and downstream of the central czc gene region. The two systems together repressed CzcRS-dependent upregulation of czcP-lacZ at low zinc concentrations in the presence of CzcS but activated this signal transmission at higher zinc concentrations. AgrRS and CzcR(2)S(2) interacted to quench CzcRS-mediated expression of czcNp-lacZ and czcPp-lacZ. Together, cross talk between the three two-component regulatory systems enhanced the capabilities of the Czc systems by controlling expression of the additional genes czcN and czcP. IMPORTANCE Bacteria are able to acquire genes encoding resistance to metals and antibiotics by horizontal gene transfer. To bestow an evolutionary advantage on their host cell, new genes must be expressed, and their expression should be regulated so that resistance-mediating proteins are produced only when needed. Newly acquired regulators may interfere with those already present in a host cell. Such an event was studied here in the metal-resistant bacterium Cupriavidus metallidurans. The results demonstrate how regulation by the acquired genes interacts with the host's extant regulatory network. This leads to emergence of a new system level of complexity that optimizes the response of the cell to periplasmic signals.202336892288
616940.9747The effect of mating on immunity can be masked by experimental piercing in female Drosophila melanogaster. Mating and immunity are two major components of fitness and links between them have been demonstrated in a number of recent investigations. In Drosophila melanogaster, a seminal fluid protein, sex-peptide (SP), up-regulates a number of antimicrobial peptide (AMP) genes in females after mating but the resulting effect on pathogen resistance is unclear. In this study, we tested (1) whether SP-induced changes in gene expression affect the ability of females to kill injected non-pathogenic bacteria and (2) how the injection process per se affects the expression of AMP genes relative to SP. The ability of virgin females and females mated to SP lacking or control males to clear bacteria was assayed using an established technique in which Escherichia coli are injected directly into the fly body and the rate of clearance of the injected bacteria is determined. We found no repeatable differences in clearance rates between virgin females and females mated to SP producing or SP lacking males. However, we found that the piercing of the integument, as occurs during injection, up-regulates AMP gene expression much more strongly than SP. Thus, assays that involve piercing, which are commonly used in immunity studies, can mask more subtle and biologically relevant changes in immunity, such as those induced by mating.200818068720
818650.9746Tumor-infiltrating bacteria disrupt cancer epithelial cell interactions and induce cell-cycle arrest. Tumor-infiltrating bacteria are increasingly recognized as modulators of cancer progression and therapy resistance. We describe a mechanism by which extracellular intratumoral bacteria, including Fusobacterium, modulate cancer epithelial cell behavior. Spatial imaging and single-cell spatial transcriptomics show that these bacteria predominantly localize extracellularly within tumor microniches of colorectal and oral cancers, characterized by reduced cell density, transcriptional activity, and proliferation. In vitro, Fusobacterium nucleatum disrupts epithelial contacts, inducing G0-G1 arrest and transcriptional quiescence. This state confers 5-fluorouracil resistance and remodels the tumor microenvironment. Findings were validated by live-cell imaging, spatial profiling, mouse models, and a 52-patient colorectal cancer cohort. Transcriptomics reveals downregulation of cell cycle, transcription, and antigen presentation genes in bacteria-enriched regions, consistent with a quiescent, immune-evasive phenotype. In an independent rectal cancer cohort, high Fusobacterium burden correlates with reduced therapy response. These results link extracellular bacteria to cancer cell quiescence and chemoresistance, highlighting microbial-tumor interactions as therapeutic targets.202541106380
873560.9745The Effect of Ice-Nucleation-Active Bacteria on Metabolic Regulation in Evergestis extimalis (Scopoli) (Lepidoptera: Pyralidae) Overwintering Larvae on the Qinghai-Tibet Plateau. Evergestis extimalis (Scopoli) is a significant pest of spring oilseed rape in the Qinghai-Tibet Plateau. It has developed resistance to many commonly used insecticides. Therefore, biopesticides should be used to replace the chemical pesticides in pest control. In this study, the effects of ice-nucleation-active (INA) microbes (Pseudomonas syringae 1.7277, P. syringae 1.3200, and Erwinia pyrifoliae 1.3333) on E. extimalis were evaluated. The supercooling points (SCP) were markedly increased due to the INA bacteria application when they were compared to those of the untreated samples. Specifically, the SCP of E. extimalis after its exposure to a high concentration of INA bacteria in February were -10.72 °C, -13.73 °C, and -14.04 °C. Our findings have demonstrated that the trehalase (Tre) genes were up-regulated by the application of the INA bacteria, thereby resulting in an increased trehalase activity. Overall, the INA bacteria could act as effective heterogeneous ice nuclei which could lower the hardiness of E. extimalis to the cold and then freeze them to death in an extremely cold winter. Therefore, the control of insect pests with INA bacteria goes without doubt, in theory.202236292857
58970.9744Insulin Signaling and Insulin Resistance Facilitate Trained Immunity in Macrophages Through Metabolic and Epigenetic Changes. Adaptation of the innate immune system has been recently acknowledged, explaining sustained changes of innate immune responses. Such adaptation is termed trained immunity. Trained immunity is initiated by extracellular signals that trigger a cascade of events affecting cell metabolism and mediating chromatin changes on genes that control innate immune responses. Factors demonstrated to facilitate trained immunity are pathogenic signals (fungi, bacteria, viruses) as well non-pathogenic signals such as insulin, cytokines, adipokines or hormones. These signals initiate intracellular signaling cascades that include AKT kinases and mTOR as well as histone methylases and demethylases, resulting in metabolic changes and histone modifications. In the context of insulin resistance, AKT signaling is affected resulting in sustained activation of mTORC1 and enhanced glycolysis. In macrophages elevated glycolysis readily impacts responses to pathogens (bacteria, fungi) or danger signals (TLR-driven signals of tissue damage), partly explaining insulin resistance-related pathologies. Thus, macrophages lacking insulin signaling exhibit reduced responses to pathogens and altered metabolism, suggesting that insulin resistance is a state of trained immunity. Evidence from Insulin Receptor as well as IGF1Receptor deficient macrophages support the contribution of insulin signaling in macrophage responses. In addition, clinical evidence highlights altered macrophage responses to pathogens or metabolic products in patients with systemic insulin resistance, being in concert with cell culture and animal model studies. Herein, we review the current knowledge that supports the impact of insulin signaling and other insulin resistance related signals as modulators of trained immunity.201931244863
74880.9743Contact-dependent growth inhibition toxins exploit multiple independent cell-entry pathways. Contact-dependent growth inhibition (CDI) systems function to deliver toxins into neighboring bacterial cells. CDI+ bacteria export filamentous CdiA effector proteins, which extend from the inhibitor-cell surface to interact with receptors on neighboring target bacteria. Upon binding its receptor, CdiA delivers a toxin derived from its C-terminal region. CdiA C-terminal (CdiA-CT) sequences are highly variable between bacteria, reflecting the multitude of CDI toxin activities. Here, we show that several CdiA-CT regions are composed of two domains, each with a distinct function during CDI. The C-terminal domain typically possesses toxic nuclease activity, whereas the N-terminal domain appears to control toxin transport into target bacteria. Using genetic approaches, we identified ptsG, metI, rbsC, gltK/gltJ, yciB, and ftsH mutations that confer resistance to specific CdiA-CTs. The resistance mutations all disrupt expression of inner-membrane proteins, suggesting that these proteins are exploited for toxin entry into target cells. Moreover, each mutation only protects against inhibition by a subset of CdiA-CTs that share similar N-terminal domains. We propose that, following delivery of CdiA-CTs into the periplasm, the N-terminal domains bind specific inner-membrane receptors for subsequent translocation into the cytoplasm. In accord with this model, we find that CDI nuclease domains are modular payloads that can be redirected through different import pathways when fused to heterologous N-terminal "translocation domains." These results highlight the plasticity of CDI toxin delivery and suggest that the underlying translocation mechanisms could be harnessed to deliver other antimicrobial agents into Gram-negative bacteria.201526305955
61390.97424-Hydroxy-2-nonenal antimicrobial toxicity is neutralized by an intracellular pathogen. Pathogens encounter numerous antimicrobial responses during infection, including the reactive oxygen species (ROS) burst. ROS-mediated oxidation of host membrane poly-unsaturated fatty acids (PUFAs) generates the toxic alpha-beta carbonyl 4-hydroxy-2-nonenal (4-HNE). Although studied extensively in the context of sterile inflammation, research into 4-HNE's role during infection remains limited. Here, we found that 4-HNE is generated during bacterial infection, that it impacts growth and survival in a range of bacteria, and that the intracellular pathogen Listeria monocytogenes induces many genes in response to 4-HNE exposure. A component of the L. monocytogenes 4-HNE response is the expression of the genes lmo0103 and lmo0613, deemed rha1 and rha2 (reductase of host alkenals), respectively, which code for two NADPH-dependent oxidoreductases that convert 4-HNE to the product 4-hydroxynonanal (4-HNA). Loss of these genes had no impact on L. monocytogenes bacterial burdens during murine or tissue culture infection. However, heterologous expression of rha1/2 in Bacillus subtilis significantly increased bacterial resistance to 4-HNE in vitro and promoted bacterial survival following phagocytosis by murine macrophages in an ROS-dependent manner. Thus, Rha1 and Rha2 are not necessary for 4-HNE resistance in L. monocytogenes but are sufficient to confer resistance to an otherwise sensitive organism in vitro and in host cells. Our work demonstrates that 4-HNE is a previously unappreciated component of ROS-mediated toxicity encountered by bacteria within eukaryotic hosts.202133955352
730100.9742How intracellular bacteria survive: surface modifications that promote resistance to host innate immune responses. Bacterial pathogens regulate the expression of virulence factors in response to environmental signals. In the case of salmonellae, many virulence factors are regulated via PhoP/PhoQ, a two-component signal transduction system that is repressed by magnesium and calcium in vitro. PhoP/PhoQ-activated genes promote intracellular survival within macrophages, whereas PhoP-repressed genes promote entrance into epithelial cells and macrophages by macropinocytosis and stimulate epithelial cell cytokine production. PhoP-activated genes include those that alter the cell envelope through structural alterations of lipopolysaccharide and lipid A, the bioactive component of lipopolysaccharide. PhoP-activated changes in the bacterial envelope likely promote intracellular survival by increasing resistance to host cationic antimicrobial peptides and decreasing host cell cytokine production.199910081503
200110.9742Drosophila Toll is activated by Gram-positive bacteria through a circulating peptidoglycan recognition protein. Microbial infection activates two distinct intracellular signalling cascades in the immune-responsive fat body of Drosophila. Gram-positive bacteria and fungi predominantly induce the Toll signalling pathway, whereas Gram-negative bacteria activate the Imd pathway. Loss-of-function mutants in either pathway reduce the resistance to corresponding infections. Genetic screens have identified a range of genes involved in these intracellular signalling cascades, but how they are activated by microbial infection is largely unknown. Activation of the transmembrane receptor Toll requires a proteolytically cleaved form of an extracellular cytokine-like polypeptide, Spätzle, suggesting that Toll does not itself function as a bona fide recognition receptor of microbial patterns. This is in apparent contrast with the mammalian Toll-like receptors and raises the question of which host molecules actually recognize microbial patterns to activate Toll through Spätzle. Here we present a mutation that blocks Toll activation by Gram-positive bacteria and significantly decreases resistance to this type of infection. The mutation semmelweis (seml) inactivates the gene encoding a peptidoglycan recognition protein (PGRP-SA). Interestingly, seml does not affect Toll activation by fungal infection, indicating the existence of a distinct recognition system for fungi to activate the Toll pathway.200111742401
579120.9741Control of expression of a periplasmic nickel efflux pump by periplasmic nickel concentrations. There is accumulating evidence that transenvelope efflux pumps of the resistance, nodulation, cell division protein family (RND) are excreting toxic substances from the periplasm across the outer membrane directly to the outside. This would mean that resistance of Gram-negative bacteria to organic toxins and heavy metals is in fact a two-step process: one set of resistance factors control the concentration of a toxic substance in the periplasm, another one that in the cytoplasm. Efficient periplasmic detoxification requires periplasmic toxin sensing and transduction of this signal into the cytoplasm to control expression of the periplasmic detoxification system. Such a signal transduction system was analyzed using the Cnr nickel resistance system from Cupriavidus (Wautersia, Ralstonia, Alcaligenes) metallidurans strain CH34. Resistance is based on nickel efflux mediated by the CnrCBA efflux pump encoded by the cnrYHXCBAT metal resistance determinant. The products of the three genes cnrYXH transcriptionally regulate expression of cnr. CnrY and CnrX are membrane-bound proteins probably functioning as anti sigma factors while CnrH is a cnr-specific extracytoplasmic functions (ECF) sigma factors. Experimental data provided here indicate a signal transduction chain leading from nickel in the periplasm to transcription initiation at the cnr promoters cnrYp and cnrCp, which control synthesis of the nickel efflux pump CnrCBA.200516158236
729130.9740Extracellular DNA-induced antimicrobial peptide resistance mechanisms in Pseudomonas aeruginosa. Extracellular DNA (eDNA) is in the environment, bodily fluids, in the matrix of biofilms, and accumulates at infection sites. eDNA can function as a nutrient source, a universal biofilm matrix component, and an innate immune effector in eDNA traps. In biofilms, eDNA is required for attachment, aggregation, and stabilization of microcolonies. We have recently shown that eDNA can sequester divalent metal cations, which has interesting implications on antibiotic resistance. eDNA binds metal cations and thus activates the Mg(2+)-responsive PhoPQ and PmrAB two-component systems. In Pseudomonas aeruginosa and many other Gram-negative bacteria, the PhoPQ/PmrAB systems control various genes required for virulence and resisting killing by antimicrobial peptides (APs), including the pmr genes (PA3552-PA3559) that are responsible for the addition of aminoarabinose to lipid A. The PA4773-PA4775 genes are a second DNA-induced cluster and are required for the production of spermidine on the outer surface, which protects the outer membrane from AP treatment. Both modifications mask the negative surface charges and limit membrane damage by APs. DNA-enriched biofilms or planktonic cultures have increased antibiotic resistance phenotypes to APs and aminoglycosides. These dual antibiotic resistance and immune evasion strategies may be expressed in DNA-rich environments and contribute to long-term survival.201323419933
603140.9740Transcriptomic Analysis Reveals Adaptive Responses of an Enterobacteriaceae Strain LSJC7 to Arsenic Exposure. Arsenic (As) resistance determinant ars operon is present in many bacteria and has been demonstrated to enhance As(V) resistance of bacteria. However, whole molecular mechanism adaptations of bacteria in response to As(V) stress remain largely unknown. In this study, transcriptional profiles of Enterobacteriaceae strain LSJC7 responding to As(V) stress were analyzed using RNA-seq and qRT-PCR. As expected, genes involved in As(V) uptake were down-regulated, those involved in As(V) reduction and As(III) efflux were up-regulated, which avoided cellular As accumulation. Reactive oxygen species and nitric oxide (NO) were induced, which caused cellular damages including DNA, protein, and Fe-S cluster damage in LSJC7. The expression of specific genes encoding transcriptional regulators, such as nsrR and soxRS were also induced. NsrR and SoxRS modulated many critical metabolic activities in As(V) stressed LSJC7 cells, including reactive species scavenging and repairing damaged DNA, proteins, and Fe-S clusters. Therefore, besides As uptake, reduction, and efflux; oxidative stress defense and damage repair were the main cellular adaptive responses of LSJC7 to As(V) stress.201627199962
725150.9740The Bacillus subtilis extracytoplasmic function σ factor σ(V) is induced by lysozyme and provides resistance to lysozyme. Bacteria encounter numerous environmental stresses which can delay or inhibit their growth. Many bacteria utilize alternative σ factors to regulate subsets of genes required to overcome different extracellular assaults. The largest group of these alternative σ factors are the extracytoplasmic function (ECF) σ factors. In this paper, we demonstrate that the expression of the ECF σ factor σ(V) in Bacillus subtilis is induced specifically by lysozyme but not other cell wall-damaging agents. A mutation in sigV results in increased sensitivity to lysozyme killing, suggesting that σ(V) is required for lysozyme resistance. Using reverse transcription (RT)-PCR, we show that the previously uncharacterized gene yrhL (here referred to as oatA for O-acetyltransferase) is in a four-gene operon which includes sigV and rsiV. In quantitative RT-PCR experiments, the expression of oatA is induced by lysozyme stress. Lysozyme induction of oatA is dependent upon σ(V). Overexpression of oatA in a sigV mutant restores lysozyme resistance to wild-type levels. This suggests that OatA is required for σ(V)-dependent resistance to lysozyme. We also tested the ability of lysozyme to induce the other ECF σ factors and found that only the expression of sigV is lysozyme inducible. However, we found that the other ECF σ factors contributed to lysozyme resistance. We found that sigX and sigM mutations alone had very little effect on lysozyme resistance but when combined with a sigV mutation resulted in significantly greater lysozyme sensitivity than the sigV mutation alone. This suggests that sigV, sigX, and sigM may act synergistically to control lysozyme resistance. In addition, we show that two ECF σ factor-regulated genes, dltA and pbpX, are required for lysozyme resistance. Thus, we have identified three independent mechanisms which B. subtilis utilizes to avoid killing by lysozyme.201121856855
726160.9740Regulation of antimicrobial resistance by extracytoplasmic function (ECF) sigma factors. Extracytoplasmic function (ECF) sigma factors are a subfamily of σ(70) sigma factors that activate genes involved in stress-response functions. In many bacteria, ECF sigma factors regulate resistance to antimicrobial compounds. This review will summarize the ECF sigma factors that regulate antimicrobial resistance in model organisms and clinically relevant pathogens.201728153747
9065170.9740Gut Bacteria Promote Phosphine Susceptibility of Tribolium castaneum by Aggravating Oxidative Stress and Fitness Costs. Knowledge about resistance mechanisms can provide ideas for pesticide resistance management. Although several studies have unveiled the positive or negative impacts of gut microbes on host pesticide resistance, minimal research is available regarding the association between gut microbes and host phosphine resistance. To explore the influence of gut bacteria on host phosphine susceptibility and its molecular basis, mortality, fitness, redox responses, and immune responses of adult Tribolium castaneum were determined when it was challenged by phosphine exposure and/or gut bacteria inoculation. Five cultivable gut bacteria were excised from a population of phosphine-resistant T. castaneum. Among them, only Enterococcus sp. inoculation significantly promoted host susceptibility to phosphine, while inoculation of any other gut bacteria had no significant effect on host phosphine susceptibility. Furthermore, when T. castaneum was exposed to phosphine, Enterococcus sp. inoculation decreased the female fecundity, promoted host oxidative stress, and suppressed the expression and activity of host superoxide dismutase, catalase, and peroxidase. In the absence of phosphine, Enterococcus sp. inoculation also elicited overactive immune responses in T. castaneum, including the immune deficiency and Toll signaling pathways and the dual oxidase-reactive oxygen species system. These results indicate that Enterococcus sp. likely promotes host phosphine susceptibility by aggravating oxidative stress and fitness costs.202337887827
655180.9740Identification of a cell envelope protein (MtrF) involved in hydrophobic antimicrobial resistance in Neisseria gonorrhoeae. The mtrCDE-encoded efflux pump of Neisseria gonorrhoeae provides gonococci with a mechanism to resist structurally diverse antimicrobial hydrophobic agents (HAs). Strains of N. gonorrhoeae that display hypersusceptibility to HAs often contain mutations in the efflux pump genes, mtrCDE. Such strains frequently contain a phenotypically suppressed mutation in mtrR, a gene that encodes a repressor (MtrR) of mtrCDE gene expression, and one that would normally result in HA resistance. We have recently examined HA-hypersusceptible clinical isolates of gonococci that contain such phenotypically suppressed mtrR mutations, in order to determine whether genes other than mtrCDE are involved in HA resistance. These studies led to the discovery of a gene that we have designated mtrF, located downstream of the mtrR gene, that is predicted to encode a 56.1 kDa cytoplasmic membrane protein containing 12 transmembrane domains. Expression of mtrF was enhanced in a strain deficient in MtrR production, indicating that this gene, together with the closely linked mtrCDE operon, is subject to MtrR-dependent transcriptional control. Orthologues of mtrF were identified in a number of diverse bacteria. Except for the AbgT protein of Escherichia coli, their products have been identified as hypothetical proteins with unknown function(s). Genetic evidence is presented that MtrF is important in the expression of high-level detergent resistance by gonococci. We propose that MtrF acts in conjunction with the MtrC-MtrD-MtrE efflux pump, to confer on gonococci high-level resistance to certain HAs.200312493784
8193190.9739Sinorhizobium meliloti Functions Required for Resistance to Antimicrobial NCR Peptides and Bacteroid Differentiation. Legumes of the Medicago genus have a symbiotic relationship with the bacterium Sinorhizobium meliloti and develop root nodules housing large numbers of intracellular symbionts. Members of the nodule-specific cysteine-rich peptide (NCR) family induce the endosymbionts into a terminal differentiated state. Individual cationic NCRs are antimicrobial peptides that have the capacity to kill the symbiont, but the nodule cell environment prevents killing. Moreover, the bacterial broad-specificity peptide uptake transporter BacA and exopolysaccharides contribute to protect the endosymbionts against the toxic activity of NCRs. Here, we show that other S. meliloti functions participate in the protection of the endosymbionts; these include an additional broad-specificity peptide uptake transporter encoded by the yejABEF genes and lipopolysaccharide modifications mediated by lpsB and lpxXL, as well as rpoH1, encoding a stress sigma factor. Strains with mutations in these genes show a strain-specific increased sensitivity profile against a panel of NCRs and form nodules in which bacteroid differentiation is affected. The lpsB mutant nodule bacteria do not differentiate, the lpxXL and rpoH1 mutants form some seemingly fully differentiated bacteroids, although most of the nodule bacteria are undifferentiated, while the yejABEF mutants form hypertrophied but nitrogen-fixing bacteroids. The nodule bacteria of all the mutants have a strongly enhanced membrane permeability, which is dependent on the transport of NCRs to the endosymbionts. Our results suggest that S. meliloti relies on a suite of functions, including peptide transporters, the bacterial envelope structures, and stress response regulators, to resist the aggressive assault of NCR peptides in the nodule cells. IMPORTANCE The nitrogen-fixing symbiosis of legumes with rhizobium bacteria has a predominant ecological role in the nitrogen cycle and has the potential to provide the nitrogen required for plant growth in agriculture. The host plants allow the rhizobia to colonize specific symbiotic organs, the nodules, in large numbers in order to produce sufficient reduced nitrogen for the plants' needs. Some legumes, including Medicago spp., produce massively antimicrobial peptides to keep this large bacterial population in check. These peptides, known as NCRs, have the potential to kill the rhizobia, but in nodules, they rather inhibit the division of the bacteria, which maintain a high nitrogen-fixing activity. In this study, we show that the tempering of the antimicrobial activity of the NCR peptides in the Medicago symbiont Sinorhizobium meliloti is multifactorial and requires the YejABEF peptide transporter, the lipopolysaccharide outer membrane, and the stress response regulator RpoH1.202134311575