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75100.8973Global transcriptomics and targeted metabolite analysis reveal the involvement of the AcrAB efflux pump in physiological functions by exporting signaling molecules in Photorhabdus laumondii. In Gram-negative bacteria, resistance-nodulation-division (RND)-type efflux pumps, particularly AcrAB-TolC, play a critical role in mediating resistance to antimicrobial agents and toxic metabolites, contributing to multidrug resistance. Photorhabdus laumondii is an entomopathogenic bacterium that has garnered significant interest due to its production of bioactive specialized metabolites with anti-inflammatory, antimicrobial, and scavenger deterrent properties. In previous work, we demonstrated that AcrAB confers self-resistance to stilbenes in P. laumondii TT01. Here, we explore the pleiotropic effects of AcrAB in this bacterium. RNA sequencing of ∆acrA compared to wild type revealed growth-phase-specific gene regulation, with stationary-phase cultures showing significant downregulation of genes involved in stilbene, fatty acid, and anthraquinone pigment biosynthesis, as well as genes related to cellular clumping and fimbrial pilin formation. Genes encoding putative LuxR regulators, type VI secretion systems, two-partner secretion systems, and contact-dependent growth inhibition systems were upregulated in ∆acrA. Additionally, exponential-phase cultures revealed reduced expression of genes related to motility in ∆acrA. The observed transcriptional changes were consistent with phenotypic assays, demonstrating that the ∆acrA mutant had altered bioluminescence and defective orange pigmentation due to disrupted anthraquinone production. These findings confirm the role of stilbenes as signaling molecules involved in gene expression, thereby shaping these phenotypes. Furthermore, we showed that AcrAB contributes to swarming and swimming motilities independently of stilbenes. Collectively, these results highlight that disrupting acrAB causes transcriptional and metabolic dysregulation in P. laumondii, likely by impeding the export of key signaling molecules such as stilbenes, which may serve as a ligand for global transcriptional regulators.IMPORTANCERecent discoveries have highlighted Photorhabdus laumondii as a promising source of novel anti-infective compounds, including non-ribosomal peptides and polyketides. One key player in the self-resistance of this bacterium to stilbene derivatives is the AcrAB-TolC complex, which is also a well-known contributor to multidrug resistance. Here, we demonstrate the pleiotropic effects of the AcrAB efflux pump in P. laumondii TT01, impacting secondary metabolite biosynthesis, motility, and bioluminescence. These effects are evident at transcriptional, metabolic, and phenotypic levels and are likely mediated by the efflux of signaling molecules such as stilbenes. These findings shed light on the multifaceted roles of efflux pumps and open avenues to better explore the complexity of resistance-nodulation-division (RND) pump-mediated signaling pathways in bacteria, thereby aiding in combating multidrug-resistant infections.202540920493
847210.8907Genetic architecture of resistance to plant secondary metabolites in Photorhabdus entomopathogenic bacteria. BACKGROUND: Entomopathogenic nematodes of the genus Heterorhabditis establish a symbiotic association with Photorhabdus bacteria. Together, they colonize and rapidly kill insects, making them important biological control agents against agricultural pests. Improving their biocontrol traits by engineering resistance to plant secondary metabolites (benzoxazinoids) in Photorhabdus symbiotic bacteria through experimental evolution has been shown to increase their lethality towards benzoxazinoid-defended larvae of the western corn rootworm, a serious crop pest of maize, and it is therefore a promising approach to develop more efficient biocontrol agents to manage this pest. To enhance our understanding of the genetic bases of benzoxazinoid resistance in Photorhabdus bacteria, we conducted an experimental evolution experiment with a phylogenetically diverse collection of Photorhabdus strains from different geographic origins. We cultured 27 different strains in medium containing 6-methoxy-2-benzoxazolinone (MBOA), a highly active benzoxazinoid breakdown product, for 35 24 h-cycles to select for benzoxazinoid-resistant strains. Then, we carried out genome-wide sequence comparisons to uncover the genetic alterations associated with benzoxazinoid resistance. Lastly, we evaluated the resistance of the newly isolated resistant Photorhabdus strains to eight additional bioactive compounds, including 2-benzoxazolinone (BOA), nicotine, caffeine, 6-chloroacetyl-2-benzoxazolinone (CABOA), digitoxin, fenitrothion, ampicillin, and kanamycin. RESULTS: We found that benzoxazinoid resistance evolves rapidly in Photorhabdus in a strain-specific manner. Across the different Photorhabdus strains, a total of nineteen nonsynonymous point mutations, two stop codon gains, and one frameshift were associated with higher benzoxazinoid resistance. The different genetic alterations were polygenic and occurred in genes coding for the EnvZ/OmpR two-component regulatory system, the different subunits of the DNA-directed RNA polymerase, and the AcrABZ-TolC multidrug efflux pump. Apart from increasing MBOA resistance, the different mutations were also associated with cross-resistance to 2-benzoxazolinone (BOA), nicotine, caffeine, and 6-chloroacetyl-2-benzoxazolinone (CABOA) and with collateral sensitivity to fenitrothion, ampicillin, and kanamycin. Targeted mutagenesis will provide a deeper mechanistic understanding, including the relative contribution of the different mutation types. CONCLUSIONS: Our study reveals several genomic features that are associated with resistance to xenobiotics in this important group of biological control agents and enhances the availability of molecular tools to develop better biological control agents, which is essential for more sustainable and ecologically friendly agricultural practices.202541168779
80620.8891A two-component small multidrug resistance pump functions as a metabolic valve during nicotine catabolism by Arthrobacter nicotinovorans. The genes nepAB of a small multidrug resistance (SMR) pump were identified as part of the pAO1-encoded nicotine regulon responsible for nicotine catabolism in Arthrobacter nicotinovorans. When [(14)C]nicotine was added to the growth medium the bacteria exported the (14)C-labelled end product of nicotine catabolism, methylamine. In the presence of the proton-motive force inhibitors 2,4-dinitrophenol (DNP), carbonyl cyanide m-chlorophenylhydrazone (CCCP) or the proton ionophore nigericin, export of methylamine was inhibited and radioactivity accumulated inside the bacteria. Efflux of [(14)C]nicotine-derived radioactivity from bacteria was also inhibited in a pmfR : cmx strain with downregulated nepAB expression. Because of low amine oxidase levels in the pmfR : cmx strain, gamma-N-methylaminobutyrate, the methylamine precursor, accumulated. Complementation of this strain with the nepAB genes, carried on a plasmid, restored the efflux of nicotine breakdown products. Both NepA and NepB were required for full export activity, indicating that they form a two-component efflux pump. NepAB may function as a metabolic valve by exporting methylamine, the end product of nicotine catabolism, and, in conditions under which it accumulates, the intermediate gamma-N-methylaminobutyrate.200717464069
2130.8889miR159a modulates poplar resistance against different fungi and bacteria. Trees are inevitably attacked by different kinds of pathogens in their life. However, little is known about the regulatory factors in poplar response to different pathogen infections. MicroRNA159 (miR159) is a highly conserved microRNA (miRNA) in plants and regulates plant development and stress responses. Here, transgenic poplar overexpressing pto-miR159a (OX-159) showed antagonistic regulation mode to poplar stem disease caused by fungi Cytospora chrysosperma and bacteria Lonsdalea populi. OX-159 lines exhibited a higher susceptibility after inoculation with bacterium L. populi, whereas enhanced disease resistance to necrotrophic fungi C. chrysosperma compared with wild-type (WT) poplars. Intriguingly, further disease assay found that OX159 line rendered the poplar susceptible to hemi-biotrophic fungi Colletotrichum gloeosporioide, exhibiting larger necrosis and lower ROS accumulation than WT lines. Transcriptome analyses revealed that more down-regulated differentially expressed genes with disease-resistant domains in OX-159 line compared with WT line. Moreover, the central mediator NPR1 of salicylic acid (SA) pathway showed a decrease in expression level, while jasmonic acid/ethylene (JA/ET) signal pathway marker genes ERF, as well as PR3, MPK3, and MPK6 genes showed an increase level in OX159-2 and OX159-5 compared with WT lines. Further spatio-temporal expression analysis revealed JA/ET signaling was involved in the dynamic response process to C. gloeosporioides in WT and OX159 lines. These results demonstrate that overexpression of pto-miR159a resulted in the crosstalk changes of the downstream hub genes, thereby controlling the disease resistance of poplars, which provides clues for understanding pto-miR159a role in coordinating poplar-pathogen interactions.202337494825
80540.8884LexR Positively Regulates the LexABC Efflux Pump Involved in Self-Resistance to the Antimicrobial Di-N-Oxide Phenazine in Lysobacter antibioticus. Myxin, a di-N-oxide phenazine isolated from the soil bacterium Lysobacter antibioticus, exhibits potent activity against various microorganisms and has the potential to be developed as an agrochemical. Antibiotic-producing microorganisms have developed self-resistance mechanisms to protect themselves from autotoxicity. Antibiotic efflux is vital for such protection. Recently, we identified a resistance-nodulation-division (RND) efflux pump, LexABC, involved in self-resistance against myxin in L. antibioticus. Expression of its genes, lexABC, was induced by myxin and was positively regulated by the LysR family transcriptional regulator LexR. The molecular mechanisms, however, have not been clear. Here, LexR was found to bind to the lexABC promoter region to directly regulate expression. Moreover, myxin enhanced this binding. Molecular docking and surface plasmon resonance analysis showed that myxin bound LexR with valine and lysine residues at positions 146 (V146) and 195 (K195), respectively. Furthermore, mutation of K195 in vivo led to downregulation of the gene lexA. These results indicated that LexR sensed and bound with myxin, thereby directly activating the expression of the LexABC efflux pump and increasing L. antibioticus resistance against myxin. IMPORTANCE Antibiotic-producing bacteria exhibit various sophisticated mechanisms for self-protection against their own secondary metabolites. RND efflux pumps that eliminate antibiotics from cells are ubiquitous in Gram-negative bacteria. Myxin is a heterocyclic N-oxide phenazine with potent antimicrobial and antitumor activities produced by the soil bacterium L. antibioticus. The RND pump LexABC contributes to the self-resistance of L. antibioticus against myxin. Herein, we report a mechanism involving the LysR family regulator LexR that binds to myxin and directly activates the LexABC pump. Further study on self-resistance mechanisms could help the investigation of strategies to deal with increasing bacterial antibiotic resistance and enable the discovery of novel natural products with resistance genes as selective markers.202337166326
57950.8866Control 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
998960.8866Molecular Insights into Fungal Innate Immunity Using the Neurospora crassa - Pseudomonas syringae Model. Recent comparative genomics and mechanistic analyses support the existence of a fungal immune system. Fungi encode genes with features similar to non-self recognition systems in plants, animals, and bacteria. However, limited functional or mechanistic evidence exists for the surveillance-system recognition of heterologous microbes in fungi. We found that Neurospora species coexist with Pseudomonas in their natural environment. We leveraged two model organisms, Neurospora crassa and Pseudomonas syringae DC3000 (PSTDC3000) to observe immediate fungal responses to bacteria. PSTDC3000 preferentially surrounds N. crassa cells on a solid surface, causing environmental dependent growth responses, bacterial proliferation and varying fungal fitness. Specifically, the Type III secretion system (T3SS) ΔhrcC mutant of PSTDC3000 colonized N. crassa hyphae less well. To dissect initial cellular signaling events within the population of germinated asexual spores (germlings), we performed transcriptomics on N. crassa after PSTDC3000 inoculation. Upon contact with live bacteria, a subpopulation of fungal germlings initiate a response as early as ten minutes post-contact revealing transcriptional differentiation of Reactive Oxygen Species (ROS) mechanisms, trace metal warfare, cell wall remodeling dynamics, multidrug-efflux transporters, secondary metabolite synthesis, and excretion. We dissected mutants of plausible receptors, signaling pathways, and responses that N. crassa uses to detect and mount a defense against PSTDC3000 and found seven genes that influence resistant and susceptibility phenotypes of N. crassa to bacterial colonization. Mutants in genes encoding a ctr copper transporter ( tcu-1 ), ferric reductase ( fer-1 ), superoxide reductase ( sod-2 ), multidrug resistance transporter ( mdr-6 ), a secreted lysozyme-Glycoside hydrolase ( lyz ) and the Woronin body tether leashin (NCU02793, lah-1 and lah-2 ) showed a significant reduction of growth in the presence of bacteria, allowing the bacteria to fully take over the fungal mycelium faster than wildtype. In this study we provide a bacterial-fungal model system within Dikarya that allows us to begin to dissect signaling pathways of the putative fungal immune system.202539896647
998870.8852Genome-wide fitness profiling reveals molecular mechanisms that bacteria use to interact with Trichoderma atroviride exometabolites. Trichoderma spp. are ubiquitous rhizosphere fungi capable of producing several classes of secondary metabolites that can modify the dynamics of the plant-associated microbiome. However, the bacterial-fungal mechanisms that mediate these interactions have not been fully characterized. Here, a random barcode transposon-site sequencing (RB-TnSeq) approach was employed to identify bacterial genes important for fitness in the presence of Trichoderma atroviride exudates. We selected three rhizosphere bacteria with RB-TnSeq mutant libraries that can promote plant growth: the nitrogen fixers Klebsiella michiganensis M5aI and Herbaspirillum seropedicae SmR1, and Pseudomonas simiae WCS417. As a non-rhizosphere species, Pseudomonas putida KT2440 was also included. From the RB-TnSeq data, nitrogen-fixing bacteria competed mainly for iron and required the siderophore transport system TonB/ExbB for optimal fitness in the presence of T. atroviride exudates. In contrast, P. simiae and P. putida were highly dependent on mechanisms associated with membrane lipid modification that are required for resistance to cationic antimicrobial peptides (CAMPs). A mutant in the Hog1-MAP kinase (Δtmk3) gene of T. atroviride showed altered expression patterns of many nonribosomal peptide synthetase (NRPS) biosynthetic gene clusters with potential antibiotic activity. In contrast to exudates from wild-type T. atroviride, bacterial mutants containing lesions in genes associated with resistance to antibiotics did not show fitness defects when RB-TnSeq libraries were exposed to exudates from the Δtmk3 mutant. Unexpectedly, exudates from wild-type T. atroviride and the Δtmk3 mutant rescued purine auxotrophic mutants of H. seropedicae, K. michiganensis and P. simiae. Metabolomic analysis on exudates from wild-type T. atroviride and the Δtmk3 mutant showed that both strains excrete purines and complex metabolites; functional Tmk3 is required to produce some of these metabolites. This study highlights the complex interplay between Trichoderma-metabolites and soil bacteria, revealing both beneficial and antagonistic effects, and underscoring the intricate and multifaceted nature of this relationship.202337651474
819380.8852Sinorhizobium 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
12890.8851Identification of a novel system for boron transport: Atr1 is a main boron exporter in yeast. Boron is a micronutrient in plants and animals, but its specific roles in cellular processes are not known. To understand boron transport and functions, we screened a yeast genomic DNA library for genes that confer resistance to the element in Saccharomyces cerevisiae. Thirty boron-resistant transformants were isolated, and they all contained the ATR1 (YML116w) gene. Atr1 is a multidrug resistance transport protein belonging to the major facilitator superfamily. C-terminal green fluorescent protein-tagged Atr1 localized to the cell membrane and vacuole, and ATR1 gene expression was upregulated by boron and several stress conditions. We found that atr1Delta mutants were highly sensitive to boron treatment, whereas cells overexpressing ATR1 were boron resistant. In addition, atr1Delta cells accumulated boron, whereas ATR1-overexpressing cells had low intracellular levels of the element. Furthermore, atr1Delta cells showed stronger boron-dependent phenotypes than mutants deficient in genes previously reported to be implicated in boron metabolism. ATR1 is widely distributed in bacteria, archaea, and lower eukaryotes. Our data suggest that Atr1 functions as a boron efflux pump and is required for boron tolerance.200919414602
9037100.8850Assessment of three Resistance-Nodulation-Cell Division drug efflux transporters of Burkholderia cenocepacia in intrinsic antibiotic resistance. BACKGROUND: Burkholderia cenocepacia are opportunistic Gram-negative bacteria that can cause chronic pulmonary infections in patients with cystic fibrosis. These bacteria demonstrate a high-level of intrinsic antibiotic resistance to most clinically useful antibiotics complicating treatment. We previously identified 14 genes encoding putative Resistance-Nodulation-Cell Division (RND) efflux pumps in the genome of B. cenocepacia J2315, but the contribution of these pumps to the intrinsic drug resistance of this bacterium remains unclear. RESULTS: To investigate the contribution of efflux pumps to intrinsic drug resistance of B. cenocepacia J2315, we deleted 3 operons encoding the putative RND transporters RND-1, RND-3, and RND-4 containing the genes BCAS0591-BCAS0593, BCAL1674-BCAL1676, and BCAL2822-BCAL2820. Each deletion included the genes encoding the RND transporter itself and those encoding predicted periplasmic proteins and outer membrane pores. In addition, the deletion of rnd-3 also included BCAL1672, encoding a putative TetR regulator. The B. cenocepacia rnd-3 and rnd-4 mutants demonstrated increased sensitivity to inhibitory compounds, suggesting an involvement of these proteins in drug resistance. Moreover, the rnd-3 and rnd-4 mutants demonstrated reduced accumulation of N-acyl homoserine lactones in the growth medium. In contrast, deletion of the rnd-1 operon had no detectable phenotypes under the conditions assayed. CONCLUSION: Two of the three inactivated RND efflux pumps in B. cenocepacia J2315 contribute to the high level of intrinsic resistance of this strain to some antibiotics and other inhibitory compounds. Furthermore, these efflux systems also mediate accumulation in the growth medium of quorum sensing molecules that have been shown to contribute to infection. A systematic study of RND efflux systems in B. cenocepacia is required to provide a full picture of intrinsic antibiotic resistance in this opportunistic bacterium.200919761586
807110.8850Transcriptomic analysis of Saccharomyces cerevisiae upon honokiol treatment. Honokiol (HNK), one of the main medicinal components in Magnolia officinalis, possesses antimicrobial activity against a variety of pathogenic bacteria and fungi. However, little is known of the molecular mechanisms underpinning the antimicrobial activity. To explore the molecular mechanism of its antifungal activity, we determined the effects of HNK on the mRNA expression profile of Saccharomyces cerevisiae using a DNA microarray approach. HNK markedly induced the expression of genes related to iron uptake and homeostasis. Conversely, genes associated with respiratory electron transport were downregulated, mirroring the effects of iron starvation. Meanwhile, HNK-induced growth deficiency was partly rescued by iron supplementation and HNK reacted with iron, producing iron complexes that depleted iron. These results suggest that HNK treatment induced iron starvation. Additionally, HNK treatment resulted in the upregulation of genes involved in protein synthesis and drug resistance networks. Furthermore, the deletion of PDR5, a gene encoding the plasma membrane ATP binding cassette (ABC) transporter, conferred sensitivity to HNK. Overexpression of PDR5 enhanced resistance of WT and pdr5Δ strains to HNK. Taken together, these findings suggest that HNK, which can be excluded by overexpression of Pdr5, functions in multiple cellular processes in S. cerevisiae, particularly in inducing iron starvation to inhibit cell growth.201728499955
673120.8846CarRS Two-Component System Essential for Polymyxin B Resistance of Vibrio vulnificus Responds to Multiple Host Environmental Signals. Enteropathogenic bacteria express two-component systems (TCSs) to sense and respond to host environments, developing resistance to host innate immune systems like cationic antimicrobial peptides (CAMPs). Although an opportunistic human pathogen Vibrio vulnificus shows intrinsic resistance to the CAMP-like polymyxin B (PMB), its TCSs responsible for resistance have barely been investigated. Here, a mutant exhibiting a reduced growth rate in the presence of PMB was screened from a random transposon mutant library of V. vulnificus, and response regulator CarR of the CarRS TCS was identified as essential for its PMB resistance. Transcriptome analysis revealed that CarR strongly activates the expression of the eptA, tolCV2, and carRS operons. In particular, the eptA operon plays a major role in developing the CarR-mediated PMB resistance. Phosphorylation of CarR by the sensor kinase CarS is required for the regulation of its downstream genes, leading to the PMB resistance. Nevertheless, CarR directly binds to specific sequences in the upstream regions of the eptA and carRS operons, regardless of its phosphorylation. Notably, the CarRS TCS alters its own activation state by responding to several environmental stresses, including PMB, divalent cations, bile salts, and pH change. Furthermore, CarR modulates the resistance of V. vulnificus to bile salts and acidic pH among the stresses, as well as PMB. Altogether, this study suggests that the CarRS TCS, in responding to multiple host environmental signals, could provide V. vulnificus with the benefit of surviving within the host by enhancing its optimal fitness during infection. IMPORTANCE Enteropathogenic bacteria have evolved multiple TCSs to recognize and appropriately respond to host environments. CAMP is one of the inherent host barriers that the pathogens encounter during the course of infection. In this study, the CarRS TCS of V. vulnificus was found to develop resistance to PMB, a CAMP-like antimicrobial peptide, by directly activating the expression of the eptA operon. Although CarR binds to the upstream regions of the eptA and carRS operons regardless of phosphorylation, phosphorylation of CarR is required for the regulation of the operons, resulting in the PMB resistance. Furthermore, the CarRS TCS determines the resistance of V. vulnificus to bile salts and acidic pH by differentially regulating its own activation state in response to these environmental stresses. Altogether, the CarRS TCS responds to multiple host-related signals, and thus could enhance the survival of V. vulnificus within the host, leading to successful infection.202337289068
649130.8841The VirAB ABC Transporter Is Required for VirR Regulation of Listeria monocytogenes Virulence and Resistance to Nisin. Listeria monocytogenes is a Gram-positive intracellular pathogen that causes a severe invasive disease. Upon infecting a host cell, L. monocytogenes upregulates the transcription of numerous factors necessary for productive infection. VirR is the response regulator component of a two-component regulatory system in L. monocytogenes In this report, we have identified the putative ABC transporter encoded by genes lmo1746-lmo1747 as necessary for VirR function. We have designated lmo1746-lmo1747 virAB We constructed an in-frame deletion of virAB and determined that the ΔvirAB mutant exhibited reduced transcription of VirR-regulated genes. The ΔvirAB mutant also showed defects in in vitro plaque formation and in vivo virulence that were similar to those of a ΔvirR deletion mutant. Since VirR is important for innate resistance to antimicrobial agents, we determined the MICs of nisin and bacitracin for ΔvirAB bacteria. We found that VirAB expression was necessary for nisin resistance but was dispensable for resistance to bacitracin. This result suggested a VirAB-independent mechanism of VirR regulation in response to bacitracin. Lastly, we found that the ΔvirR and ΔvirAB mutants had no deficiency in growth in broth culture, intracellular replication, or production of the ActA surface protein, which facilitates actin-based motility and cell-to-cell spread. However, the ΔvirR and ΔvirAB mutants produced shorter actin tails during intracellular infection, which suggested that these mutants have a reduced ability to move and spread via actin-based motility. These findings have demonstrated that L. monocytogenes VirAB functions in a pathway with VirR to regulate the expression of genes necessary for virulence and resistance to antimicrobial agents.201829263107
518140.8840Bacitracin and nisin resistance in Staphylococcus aureus: a novel pathway involving the BraS/BraR two-component system (SA2417/SA2418) and both the BraD/BraE and VraD/VraE ABC transporters. Two-component systems (TCSs) are key regulatory pathways allowing bacteria to adapt their genetic expression to environmental changes. Bacitracin, a cyclic dodecylpeptide antibiotic, binds to undecaprenyl pyrophosphate, the lipid carrier for cell wall precursors, effectively inhibiting peptidoglycan biosynthesis. We have identified a novel and previously uncharacterized TCS in the major human pathogen Staphylococcus aureus that we show to be essential for bacitracin and nisin resistance: the BraS/BraR system (Bacitracin resistance associated; SA2417/SA2418). The braRS genes are located immediately upstream from genes encoding an ABC transporter, accordingly designated BraDE. We have shown that the BraSR/BraDE module is a key bacitracin and nisin resistance determinant in S. aureus. In the presence of low antibiotic concentrations, BraSR activate transcription of two operons encoding ABC transporters: braDE and vraDE. We identified a highly conserved imperfect palindromic sequence upstream from the braDE and vraDE promoter sequences, essential for their transcriptional activation by BraSR, suggesting it is the likely BraR binding site. We demonstrated that the two ABC transporters play distinct and original roles in antibiotic resistance: BraDE is involved in bacitracin sensing and signalling through BraSR, whereas VraDE acts specifically as a detoxification module and is sufficient to confer bacitracin and nisin resistance when produced on its own. We show that these processes require functional BraD and VraD nucleotide-binding domain proteins, and that the large extracellular loop of VraE confers its specificity in bacitracin resistance. This is the first example of a TCS associated with two ABC transporters playing separate roles in signal transduction and antibiotic resistance.201121696458
8194150.8838Role of the phenazine-inducing protein Pip in stress resistance of Pseudomonas chlororaphis. The triggering of antibiotic production by various environmental stress molecules can be interpreted as bacteria's response to obtain increased fitness to putative danger, whereas the opposite situation - inhibition of antibiotic production - is more complicated to understand. Phenazines enable Pseudomonas species to eliminate competitors for rhizosphere colonization and are typical virulence factors used for model studies. In the present work, we have investigated the negative effect of subinhibitory concentrations of NaCl, fusaric acid and two antibiotics on quorum-sensing-controlled phenazine production by Pseudomonas chlororaphis. The selected stress factors inhibit phenazine synthesis despite sufficient cell density. Subsequently, we have identified connections between known genes of the phenazine-inducing cascade, including PsrA (Pseudomonas sigma regulator), RpoS (alternative sigma factor), Pip (phenazine inducing protein) and PhzI/PhzR (quorum-sensing system). Under all tested conditions, overexpression of Pip or PhzR restored phenazine production while overexpression of PsrA or RpoS did not. This forced restoration of phenazine production in strains overexpressing regulatory genes pip and phzR significantly impairs growth and stress resistance; this is particularly severe with pip overexpression. We suggest a novel physiological explanation for the inhibition of phenazine virulence factors in pseudomonas species responding to toxic compounds. We propose that switching off phenazine-1-carboxamide (PCN) synthesis by attenuating pip expression would favour processes required for survival. In our model, this 'decision' point for promoting PCN production or stress resistance is located downstream of rpoS and just above pip. However, a test with the stress factor rifampicin shows no significant inhibition of Pip production, suggesting that stress factors may also target other and so far unknown protagonists of the PCN signalling cascade.201121030433
754160.8837Resistance to Bipyridyls Mediated by the TtgABC Efflux System in Pseudomonas putida KT2440. Resistance-nodulation-division (RND) transporters are involved in antibiotic resistance and have a broad substrate specificity. However, the physiological significance of these efflux pumps is not fully understood. Here, we have investigated the role of the RND system TtgABC in resistance to metal ion chelators in the soil bacterium Pseudomonas putida KT2440. We observed that the combined action of an RND inhibitor and the chelator 2,2'-bipyridyl inhibited bacterial growth. In addition, the deletion of ttgB made the strain susceptible to 2,2'-bipyridyl and natural bipyridyl derivatives such as caerulomycin A, indicating that TtgABC is required for detoxification of compounds of the bipyridyl family. Searching for the basis of growth inhibition by bipyridyls, we found reduced adenosine triphosphate (ATP) levels in the ttgB mutant compared to the wild type. Furthermore, the expression of genes related to iron acquisition and the synthesis of the siderophore pyoverdine were reduced in the mutant compared to the wild type. Investigating the possibility that 2,2'-bipyridyl in the ttgB mutant mediates iron accumulation in cells (which would cause the upregulation of genes involved in oxidative stress via the Fenton reaction), we measured the expression of genes coding for proteins involved in intracellular iron storage and the response to oxidative stress. However, none of the genes was significantly upregulated. In a further search for a possible link between 2,2'-bipyridyl and the observed phenotypes, we considered the possibility that the ion chelator limits the intracellular availability of metabolically important metal ions. In this context, we found that the addition of copper restores the growth of the ttgB mutant and the production of pyoverdine, suggesting a relationship between copper availability and iron acquisition. Taken together, the results suggest that detoxification of metal chelating compounds of the bipyridyl family produced by other bacteria or higher ordered organisms is one of the native functions of the RND efflux pump TtgABC. Without the efflux pump, these compounds may interfere with cell ion homeostasis with adverse effects on cell metabolism, including siderophore production. Finally, our results suggest that TtgABC is involved in resistance to bile salts and deoxycholate.202032973714
725170.8837The 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
6189180.8837Characterization of all RND-type multidrug efflux transporters in Vibrio parahaemolyticus. Resistance nodulation cell division (RND)-type efflux transporters play the main role in intrinsic resistance to various antimicrobial agents in many gram-negative bacteria. Here, we estimated 12 RND-type efflux transporter genes in Vibrio parahaemolyticus. Because VmeAB has already been characterized, we cloned the other 11 RND-type efflux transporter genes and characterized them in Escherichia coli KAM33 cells, a drug hypersusceptible strain. KAM33 expressing either VmeCD, VmeEF, or VmeYZ showed increased minimum inhibitory concentrations (MICs) for several antimicrobial agents. Additional four RND-type transporters were functional as efflux pumps only when co-expressed with VpoC, an outer membrane component in V. parahaemolyticus. Furthermore, VmeCD, VmeEF, and VmeYZ co-expressed with VpoC exhibited a broader substrate specificity and conferred higher resistance than that with TolC of E. coli. Deletion mutants of these transporter genes were constructed in V. parahaemolyticus. TM32 (ΔvmeAB and ΔvmeCD) had significantly decreased MICs for many antimicrobial agents and the number of viable cells after exposure to deoxycholate were markedly reduced. Strains in which 12 operons were all disrupted had very low MICs and much lower fluid accumulation in rabbit ileal loops. These results indicate that resistance nodulation cell division-type efflux transporters contribute not only to intrinsic resistance but also to exerting the virulence of V. parahaemolyticus.201323894076
116190.8837The ADEP Biosynthetic Gene Cluster in Streptomyces hawaiiensis NRRL 15010 Reveals an Accessory clpP Gene as a Novel Antibiotic Resistance Factor. The increasing threat posed by multiresistant bacterial pathogens necessitates the discovery of novel antibacterials with unprecedented modes of action. ADEP1, a natural compound produced by Streptomyces hawaiiensis NRRL 15010, is the prototype for a new class of acyldepsipeptide (ADEP) antibiotics. ADEP antibiotics deregulate the proteolytic core ClpP of the bacterial caseinolytic protease, thereby exhibiting potent antibacterial activity against Gram-positive bacteria, including multiresistant pathogens. ADEP1 and derivatives, here collectively called ADEP, have been previously investigated for their antibiotic potency against different species, structure-activity relationship, and mechanism of action; however, knowledge on the biosynthesis of the natural compound and producer self-resistance have remained elusive. In this study, we identified and analyzed the ADEP biosynthetic gene cluster in S. hawaiiensis NRRL 15010, which comprises two NRPSs, genes necessary for the biosynthesis of (4S,2R)-4-methylproline, and a type II polyketide synthase (PKS) for the assembly of highly reduced polyenes. While no resistance factor could be identified within the gene cluster itself, we discovered an additional clpP homologous gene (named clpP(ADEP)) located further downstream of the biosynthetic genes, separated from the biosynthetic gene cluster by several transposable elements. Heterologous expression of ClpP(ADEP) in three ADEP-sensitive Streptomyces species proved its role in conferring ADEP resistance, thereby revealing a novel type of antibiotic resistance determinant.IMPORTANCE Antibiotic acyldepsipeptides (ADEPs) represent a promising new class of potent antibiotics and, at the same time, are valuable tools to study the molecular functioning of their target, ClpP, the proteolytic core of the bacterial caseinolytic protease. Here, we present a straightforward purification procedure for ADEP1 that yields substantial amounts of the pure compound in a time- and cost-efficient manner, which is a prerequisite to conveniently study the antimicrobial effects of ADEP and the operating mode of bacterial ClpP machineries in diverse bacteria. Identification and characterization of the ADEP biosynthetic gene cluster in Streptomyces hawaiiensis NRRL 15010 enables future bioinformatics screenings for similar gene clusters and/or subclusters to find novel natural compounds with specific substructures. Most strikingly, we identified a cluster-associated clpP homolog (named clpP(ADEP)) as an ADEP resistance gene. ClpP(ADEP) constitutes a novel bacterial resistance factor that alone is necessary and sufficient to confer high-level ADEP resistance to Streptomyces across species.201931399403