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847100.9900Effects of Klebsiella michiganensis LDS17 on Codonopsis pilosula growth, rhizosphere soil enzyme activities, and microflora, and genome-wide analysis of plant growth-promoting genes. Codonopsis pilosula is a perennial herbaceous liana with medicinal value. It is critical to promote Codonopsis pilosula growth through effective and sustainable methods, and the use of plant growth-promoting bacteria (PGPB) is a promising candidate. In this study, we isolated a PGPB, Klebsiella michiganensis LDS17, that produced a highly active 1-aminocyclopropane-1-carboxylate deaminase from the Codonopsis pilosula rhizosphere. The strain exhibited multiple plant growth-promoting properties. The antagonistic activity of strain LDS17 against eight phytopathogenic fungi was investigated, and the results showed that strain LDS17 had obvious antagonistic effects on Rhizoctonia solani, Colletotrichum camelliae, Cytospora chrysosperma, and Phomopsis macrospore with growth inhibition rates of 54.22%, 49.41%, 48.89%, and 41.11%, respectively. Inoculation of strain LDS17 not only significantly increased the growth of Codonopsis pilosula seedlings but also increased the invertase and urease activities, the number of culturable bacteria, actinomycetes, and fungi, as well as the functional diversity of microbial communities in the rhizosphere soil of the seedlings. Heavy metal (HM) resistance tests showed that LDS17 is resistant to copper, zinc, and nickel. Whole-genome analysis of strain LDS17 revealed the genes involved in IAA production, siderophore synthesis, nitrogen fixation, P solubilization, and HM resistance. We further identified a gene (koyR) encoding a plant-responsive LuxR solo in the LDS17 genome. Klebsiella michiganensis LDS17 may therefore be useful in microbial fertilizers for Codonopsis pilosula. The identification of genes related to plant growth and HM resistance provides an important foundation for future analyses of the molecular mechanisms underlying the plant growth promotion and HM resistance of LDS17. IMPORTANCE: We comprehensively evaluated the plant growth-promoting characteristics and heavy metal (HM) resistance ability of the LDS17 strain, as well as the effects of strain LDS17 inoculation on the Codonopsis pilosula seedling growth and the soil qualities in the Codonopsis pilosula rhizosphere. We conducted whole-genome analysis and identified lots of genes and gene clusters contributing to plant-beneficial functions and HM resistance, which is critical for further elucidating the plant growth-promoting mechanism of strain LDS17 and expanding its application in the development of plant growth-promoting agents used in the environment under HM stress.202438563743
1810.9898Antivirulence effects of cell-free culture supernatant of endophytic bacteria against grapevine crown gall agent, Agrobacterium tumefaciens, and induction of defense responses in plantlets via intact bacterial cells. BACKGROUND: Crown gall disease caused by Agrobacterium tumefaciens is a very destructive affliction that affects grapevines. Endophytic bacteria have been discovered to control plant diseases via the use of several mechanisms. This research examined the potential for controlling crown gall by three endophytic bacteria that were previously isolated from healthy cultivated and wild grapevines including Pseudomonas kilonensis Ba35, Pseudomonas chlororaphis Ba47, and Serratia liquefaciens Ou55. RESULT: At various degrees, three endophytic bacteria suppressed the populations of A. tumefaciens Gh1 and greatly decreased the symptoms of crown gall. Furthermore, biofilm production and motility behaviors of A. tumefaciens Gh1were greatly inhibited by the Cell-free Culture Supernatant (CFCS) of endophytic bacteria. According to our findings, CFCS may reduce the adhesion of A. tumefaciens Gh1 cells to grapevine cv. Rashe root tissues as well as their chemotaxis motility toward the extract of the roots. When compared to the untreated control, statistical analysis showed that CFCS significantly reduced the swimming, twitching, and swarming motility of A. tumefaciens Gh1. The findings demonstrated that the endophytic bacteria effectively stimulated the production of plant defensive enzymes including superoxide dismutase (SOD), polyphenol oxidase (PPO), peroxidase (POD), phenylalanine ammonia lyase (PAL), and total soluble phenols at different time intervals in grapevine inoculated with A. tumefaciens Gh1. The Ba47 strain markedly increased the expression levels of defense genes associated with plant resistance. The up-regulation of PR1, PR2, VvACO1, and GAD1 genes in grapevine leaves indicates the activation of SA and JA pathways, which play a role in enhancing resistance to pathogen invasion. The results showed that treating grapevine with Ba47 increased antioxidant defense activities and defense-related gene expression, which reduced oxidative damage caused by A. tumefaciens and decreased the incidence of crown gall disease. CONCLUSION: This is the first study on how A. tumefaciens, the grapevine crown gall agent, is affected by CFCS generated by endophytic bacteria in terms of growth and virulence features. To create safer plant disease management techniques, knowledge of the biocontrol processes mediated by CFCS during microbial interactions is crucial.202438336608
607920.9897Genomic and metabonomic methods reveal the probiotic functions of swine-derived Ligilactobacillus salivarius. BACKGROUND: As substitutes for antibiotics, probiotic bacteria protect against digestive infections caused by pathogenic bacteria. Ligilactobacillus salivarius is a species of native lactobacillus found in both humans and animals. Herein, a swine-derived Ligilactobacillus salivarius was isolated and shown to colonize the ileal mucous membrane, thereby promoting nutritional digestion, absorption, and immunity. To evaluate its probiotic role, the entire genome was sequenced, the genetic information was annotated, and the metabolic information was analyzed. RESULTS: The phylogenetic relationship indicated that the bacteria was closer to L. salivarius MT573555.1 and MT585431.1. Functional genes included transporters, membrane proteins, enzymes, heavy metal resistance proteins, and putative proteins; metabolism-related genes were the most abundant. The six types of metabolic pathways secreted by L. salivarius were mainly composed of secretory transmembrane proteins and peptides. The secretory proteins of L. salivarius were digestive enzymes, functional proteins that regulate apoptosis, antibodies, and hormones. Non-targeted metabolomic analysis of L. salivarius metabolites suggested that ceramide, pyrrolidone- 5- carboxylic acid, N2-acetyl-L-ornithine, 2-ethyl-2-hydroxybutyric acid, N-lactoyl-phenylalanine, and 12 others were involved in antioxidation, repair of the cellular membrane, anticonvulsant, hypnosis, and appetite inhibition. Metabolites of clavaminic acid, antibiotic X14889C, and five other types of bacteriocins were identified, namely phenyllactic acid, janthitrem G, 13-demethyl tacrolimus, medinoside E, and tertonasin. The adherence and antioxidation of L. salivarius were also predicted. No virulence genes were found. CONCLUSION: The main probiotic properties of L. salivarius were identified using genomic, metabonomic, and biochemical assays, which are beneficial for porcine feeding. Our results provided deeper insights into the probiotic effects of L. salivarius.202337648978
608830.9897Complete Genome of Achromobacter xylosoxidans, a Nitrogen-Fixing Bacteria from the Rhizosphere of Cowpea (Vigna unguiculata [L.] Walp) Tolerant to Cucumber Mosaic Virus Infection. Achromobacter xylosoxidans is one of the nitrogen-fixing bacteria associated with cowpea rhizosphere across Africa. Although its role in improving soil fertility and inducing systemic resistance in plants against pathogens has been documented, there is limited information on its complete genomic characteristics from cowpea roots. Here, we report the complete genome sequence of A. xylosoxidans strain DDA01 isolated from the topsoil of a field where cowpea plants tolerant to cucumber mosaic virus (CMV) were grown in Ibadan, Nigeria. The genome of DDA01 was sequenced via Illumina MiSeq and contained 6,930,067 nucleotides with 67.55% G + C content, 73 RNAs, 59 tRNAs, and 6421 protein-coding genes, including those associated with nitrogen fixation, phosphate solubilization, Indole3-acetic acid production, and siderophore activity. Eleven genetic clusters for secondary metabolites, including alcaligin, were identified. The potential of DDA01 as a plant growth-promoting bacteria with genetic capabilities to enhance soil fertility for resilience against CMV infection in cowpea is discussed. To our knowledge, this is the first complete genome of diazotrophic bacteria obtained from cowpea rhizosphere in sub-Saharan Africa, with potential implications for improved soil fertility, plant disease resistance, and food security.202439278894
14440.9896Genome analysis of Bacillus amyloliquefaciens Subsp. plantarum UCMB5113: a rhizobacterium that improves plant growth and stress management. The Bacillus amyloliquefaciens subsp. plantarum strain UCMB5113 is a Gram-positive rhizobacterium that can colonize plant roots and stimulate plant growth and defense based on unknown mechanisms. This reinforcement of plants may provide protection to various forms of biotic and abiotic stress. To determine the genetic traits involved in the mechanism of plant-bacteria association, the genome sequence of UCMB5113 was obtained by assembling paired-end Illumina reads. The assembled chromosome of 3,889,532 bp was predicted to encode 3,656 proteins. Genes that potentially contribute to plant growth promotion such as indole-3-acetic acid (IAA) biosynthesis, acetoin synthesis and siderophore production were identified. Moreover, annotation identified putative genes responsible for non-ribosomal synthesis of secondary metabolites and genes supporting environment fitness of UCMB5113 including drug and metal resistance. A large number of genes encoding a diverse set of secretory proteins, enzymes of primary and secondary metabolism and carbohydrate active enzymes were found which reflect a high capacity to degrade various rhizosphere macromolecules. Additionally, many predicted membrane transporters provides the bacterium with efficient uptake capabilities of several nutrients. Although, UCMB5113 has the possibility to produce antibiotics and biosurfactants, the protective effect of plants to pathogens seems to be indirect and due to priming of plant induced systemic resistance. The availability of the genome enables identification of genes and their function underpinning beneficial interactions of UCMB5113 with plants.201425119988
879750.9896Presence of quorum-sensing systems associated with multidrug resistance and biofilm formation in Bacteroides fragilis. Bacteroides fragilis constitutes 1-2% of the natural microbiota of the human digestive tract and is the predominant anaerobic opportunistic pathogen in gastrointestinal infections. Most bacteria use quorum sensing (QS) to monitor cell density in relation to other cells and their environment. In Gram-negative bacteria, the LuxRI system is common. The luxR gene encodes a transcriptional activator inducible by type I acyl-homoserine lactone autoinducers (e.g., N-[3-oxohexanoyl] homoserine lactone and hexanoyl homoserine lactone [C6-HSL]). This study investigated the presence of QS system(s) in B. fragilis. The genome of American-type culture collection strain no. ATCC25285 was searched for QS genes. The strain was grown to late exponential phase in the presence or absence of synthetic C6-HSL and C8-HSL or natural homoserine lactones from cell-free supernatants from spent growth cultures of other bacteria. Growth, susceptibility to antimicrobial agents, efflux pump gene (bmeB) expression, and biofilm formation were measured. Nine luxR and no luxI orthologues were found. C6-HSL and supernatants from Yersinia enterocolitica, Vibrio cholerae, and Pseudomonas aeruginosa caused a significant (1) reduction in cellular density and (2) increases in expression of four putative luxR genes, bmeB3, bmeB6, bmeB7, and bmeB10, resistance to various antibiotics, which was reduced by carbonyl cyanide-m-chlorophenyl hydrazone (CCCP, an uncoupler that dissipates the transmembrane proton gradient, which is also the driving force of resistance nodulation division efflux pumps) and (3) increase in biofilm formation. Susceptibility of ATCC25285 to C6-HSL was also reduced by CCCP. These data suggest that (1) B. fragilis contains putative luxR orthologues, which could respond to exogenous homoserine lactones and modulate biofilm formation, bmeB efflux pump expression, and susceptibility to antibiotics, and (2) BmeB efflux pumps could transport homoserine lactones.200818188535
19260.9893N-Succinyltransferase Encoded by a Cryptic Siderophore Biosynthesis Gene Cluster in Streptomyces Modifies Structurally Distinct Antibiotics. The antibiotic desertomycin A and its previously undescribed inactive N-succinylated analogue, desertomycin X, were isolated from Streptomyces sp. strain YIM 121038. Genome sequencing and analysis readily identified the desertomycin biosynthetic gene cluster (BGC), which lacked genes encoding acyltransferases that would account for desertomycin X formation. Scouting the genome for putative N-acyltransferase genes led to the identification of a candidate within a cryptic siderophore BGC (csb) encoding a putative homologue of the N6'-hydroxylysine acetyltransferase IucB. Expression of the codon-optimized gene designated csbC in Escherichia coli yielded the recombinant protein that was able to N-succinylate desertomycin A as well as several other structurally distinct antibiotics harboring amino groups. Some antibiotics were rendered antibiotically inactive due to the CsbC-catalyzed succinylation in vitro. Unlike many known N-acyltransferases involved in antibiotic resistance, CsbC could not efficiently acetylate the same antibiotics. When expressed in E. coli, CsbC provided low-level resistance to kanamycin and ampicillin, suggesting that it may play a role in antibiotic resistance in natural habitats, where the concentration of antibiotics is usually low. IMPORTANCE In their natural habitats, bacteria encounter a plethora of organic compounds, some of which may be represented by antibiotics produced by certain members of the microbial community. A number of antibiotic resistance mechanisms have been described, including those specified by distinct genes encoding proteins that degrade, modify, or expel antibiotics. In this study, we report identification and characterization of an enzyme apparently involved in the biosynthesis of a siderophore, but also having the ability of modify and thereby inactivate a wide variety of structurally diverse antibiotics. This discovery sheds light on additional capabilities of bacteria to withstand antibiotic treatment and suggests that enzymes involved in secondary metabolism may have an additional function in the natural environment.202236040031
902770.9893Scorpion Venom Antimicrobial Peptides Induce Siderophore Biosynthesis and Oxidative Stress Responses in Escherichia coli. The increasing development of microbial resistance to classical antimicrobial agents has led to the search for novel antimicrobials. Antimicrobial peptides (AMPs) derived from scorpion and snake venoms offer an attractive source for the development of novel therapeutics. Smp24 (24 amino acids [aa]) and Smp43 (43 aa) are broad-spectrum AMPs that have been identified from the venom gland of the Egyptian scorpion Scorpio mauruspalmatus and subsequently characterized. Using a DNA microarray approach, we examined the transcriptomic responses of Escherichia coli to subinhibitory concentrations of Smp24 and Smp43 peptides following 5 h of incubation. Seventy-two genes were downregulated by Smp24, and 79 genes were downregulated by Smp43. Of these genes, 14 genes were downregulated in common and were associated with bacterial respiration. Fifty-two genes were specifically upregulated by Smp24. These genes were predominantly related to cation transport, particularly iron transport. Three diverse genes were independently upregulated by Smp43. Strains with knockouts of differentially regulated genes were screened to assess the effect on susceptibility to Smp peptides. Ten mutants in the knockout library had increased levels of resistance to Smp24. These genes were predominantly associated with cation transport and binding. Two mutants increased resistance to Smp43. There was no cross-resistance in mutants resistant to Smp24 or Smp43. Five mutants showed increased susceptibility to Smp24, and seven mutants showed increased susceptibility to Smp43. Of these mutants, formate dehydrogenase knockout (fdnG) resulted in increased susceptibility to both peptides. While the electrostatic association between pore-forming AMPs and bacterial membranes followed by integration of the peptide into the membrane is the initial starting point, it is clear that there are numerous subsequent additional intracellular mechanisms that contribute to their overall antimicrobial effect.IMPORTANCE The development of life-threatening resistance of pathogenic bacteria to the antibiotics typically in use in hospitals and the community today has led to an urgent need to discover novel antimicrobial agents with different mechanisms of action. As an ancient host defense mechanism of the innate immune system, antimicrobial peptides (AMPs) are attractive candidates to fill that role. Scorpion venoms have proven to be a rich source of AMPs. Smp24 and Smp43 are new AMPs that have been identified from the venom gland of the Egyptian scorpion Scorpio maurus palmatus, and these peptides can kill a wide range of bacterial pathogens. By better understanding how these AMPs affect bacterial cells, we can modify their structure to make better drugs in the future.202133980680
11680.9892The 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
847290.9892Genetic 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
751100.9892Global 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
9040110.9891Gene expression changes linked to antimicrobial resistance, oxidative stress, iron depletion and retained motility are observed when Burkholderia cenocepacia grows in cystic fibrosis sputum. BACKGROUND: Bacteria from the Burkholderia cepacia complex (Bcc) are the only group of cystic fibrosis (CF) respiratory pathogens that may cause death by an invasive infection known as cepacia syndrome. Their large genome (> 7000 genes) and multiple pathways encoding the same putative functions make virulence factor identification difficult in these bacteria. METHODS: A novel microarray was designed to the genome of Burkholderia cenocepacia J2315 and transcriptomics used to identify genes that were differentially regulated when the pathogen was grown in a CF sputum-based infection model. Sputum samples from CF individuals infected with the same B. cenocepacia strain as genome isolate were used, hence, other than a dilution into a minimal growth medium (used as the control condition), no further treatment of the sputum was carried out. RESULTS: A total of 723 coding sequences were significantly altered, with 287 upregulated and 436 downregulated; the microarray-observed expression was validated by quantitative PCR on five selected genes. B. cenocepacia genes with putative functions in antimicrobial resistance, iron uptake, protection against reactive oxygen and nitrogen species, secretion and motility were among the most altered in sputum. Novel upregulated genes included: a transmembrane ferric reductase (BCAL0270) implicated in iron metabolism, a novel protease (BCAL0849) that may play a role in host tissue destruction, an organic hydroperoxide resistance gene (BCAM2753), an oxidoreductase (BCAL1107) and a nitrite/sulfite reductase (BCAM1676) that may play roles in resistance to the host defenses. The assumptions of growth under iron-depletion and oxidative stress formulated from the microarray data were tested and confirmed by independent growth of B. cenocepacia under each respective environmental condition. CONCLUSION: Overall, our first full transcriptomic analysis of B. cenocepacia demonstrated the pathogen alters expression of over 10% of the 7176 genes within its genome when it grows in CF sputum. Novel genetic pathways involved in responses to antimicrobial resistance, oxidative stress, and iron metabolism were revealed by the microarray analysis. Virulence factors such as the cable pilus and Cenocepacia Pathogenicity Island were unaltered in expression. However, B. cenocepacia sustained or increased expression of motility-associated genes in sputum, maintaining a potentially invasive phenotype associated with cepacia syndrome.200818801206
5170120.9891Synergistic effect of imp/ostA and msbA in hydrophobic drug resistance of Helicobacter pylori. BACKGROUND: Contamination of endoscopy equipment by Helicobacter pylori (H. pylori) frequently occurs after endoscopic examination of H. pylori-infected patients. In the hospital, manual pre-cleaning and soaking in glutaraldehyde is an important process to disinfect endoscopes. However, this might not be sufficient to remove H. pylori completely, and some glutaraldehyde-resistant bacteria might survive and be passed to the next patient undergoing endoscopic examination through unidentified mechanisms. We identified an Imp/OstA protein associated with glutaraldehyde resistance in a clinical strain, NTUH-C1, from our previous study. To better understand and manage the problem of glutaraldehyde resistance, we further investigated its mechanism. RESULTS: The minimal inhibitory concentrations (MICs) of glutaraldehyde andexpression of imp/ostA RNA in 11 clinical isolates from the National Taiwan University Hospital were determined. After glutaraldehyde treatment, RNA expression in the strains with the MICs of 4-10 microg/ml was higher than that in strains with the MICs of 1-3 microg/ml. We examined the full-genome expression of strain NTUH-S1 after glutaraldehyde treatment using a microarray and found that 40 genes were upregulated and 31 genes were downregulated. Among the upregulated genes, imp/ostA and msbA, two putative lipopolysaccharide biogenesis genes, were selected for further characterization. The sensitivity to glutaraldehyde or hydrophobic drugs increased in both of imp/ostA and msbA single mutants. The imp/ostA and msbA double mutant was also hypersensitive to these chemicals. The lipopolysaccharide contents decreased in individual imp/ostA and msbA mutants and dramatically reduced in the imp/ostA and msbA double mutant. Outer membrane permeability assay demonstrated that the imp/ostA and msbA double mutation resulted in the increase of outer membrane permeability. Ethidium bromide accumulation assay demonstrated that MsbA was involved in efflux of hydrophobic drugs. CONCLUSION: The expression levels of imp/ostA and msbA were correlated with glutaraldehyde resistance in clinical isolates after glutaraldehyde treatment. Imp/OstA and MsbA play a synergistic role in hydrophobic drugs resistance and lipopolysaccharide biogenesis in H. pylori.200919594901
8193130.9891Sinorhizobium 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
9988140.9890Genome-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
9033150.9890Stress Resistance Development and Genome-Wide Transcriptional Response of Escherichia coli O157:H7 Adapted to Sublethal Thymol, Carvacrol, and trans-Cinnamaldehyde. Thymol, carvacrol, and trans-cinnamaldehyde are essential oil (EO) compounds with broad-spectrum antimicrobial activities against foodborne pathogens, including Escherichia coli O157:H7. However, little is known regarding direct resistance and cross-resistance development in E. coli O157:H7 after adaptation to sublethal levels of these compounds, and information is scarce on microbial adaptive responses at a molecular level. The present study demonstrated that E. coli O157:H7 was able to grow in the presence of sublethal thymol (1/2T), carvacrol (1/2C), or trans-cinnamaldehyde (1/2TC), displaying an extended lag phase duration and a lower maximum growth rate. EO-adapted cells developed direct resistance against lethal EO treatments and cross-resistance against heat (58°C) and oxidative (50 mM H(2)O(2)) stresses. However, no induction of acid resistance (simulated gastric fluid, pH 1.5) was observed. RNA sequencing revealed a large number (310 to 338) of differentially expressed (adjusted P value [P(adj) ], <0.05; fold change, ≥5) genes in 1/2T and 1/2C cells, while 1/2TC cells only showed 27 genes with altered expression. In accordance with resistance phenotypes, the genes related to membrane, heat, and oxidative stress responses and genes related to iron uptake and metabolism were upregulated. Conversely, virulence genes associated with motility, biofilm formation, and efflux pumps were repressed. This study demonstrated the development of direct resistance and cross-resistance and characterized whole-genome transcriptional responses in E. coli O157:H7 adapted to sublethal thymol, carvacrol, or trans-cinnamaldehyde. The data suggested that caution should be exercised when using EO compounds as food antimicrobials, due to the potential stress resistance development in E. coli O157:H7.IMPORTANCE The present study was designed to understand transcriptomic changes and the potential development of direct and cross-resistance in essential oil (EO)-adapted Escherichia coli O157:H7. The results demonstrated altered growth behaviors of E. coli O157:H7 during adaptation in sublethal thymol, carvacrol, and trans-cinnamaldehyde. Generally, EO-adapted bacteria showed enhanced resistance against subsequent lethal EO, heat, and oxidative stresses, with no induction of acid resistance in simulated gastric fluid. A transcriptomic analysis revealed the upregulation of related stress resistance genes and a downregulation of various virulence genes in EO-adapted cells. This study provides new insights into microbial EO adaptation behaviors and highlights the risk of resistance development in adapted bacteria.201830217837
8831160.9890Search for biocontrol agents among endophytic lipopeptide-synthesizing bacteria Bacillus spp. to protect wheat plants against Greenbug aphid (Schizaphis graminum). Beneficial endophytic bacteria can suppress the development of insect pests through direct antagonism, with the help of metabolites, or indirectly by the induction of systemic resistance through the regulation of hormonal signaling pathways. Lipopeptides are bacterial metabolites that exhibit direct antagonistic activity against many organisms, including insects. Also, lipopeptides are able to trigger induced systemic resistance (ISR) in plants against harmful organisms, but the physiological mechanisms of their action are just beginning to be studied. In this work, we studied ten strains of bacteria isolated from the tissues of wheat and potatoes. Sequencing of the 16S rRNA gene showed that all isolates belong to the genus Bacillus and to two species, B. subtilis and B. velezensis. The genes for lipopeptide synthetase - surfactin synthetase (Bs_srf ), iturin synthetase (Bs_ituA, Bs_ituB) and fengycin synthetase (Bs_fenD) - were identified in all bacterial isolates using PCR. All strains had high aphicidal activity against the Greenbug aphid (Schizaphis graminum Rond.) due to the synthesis of lipopeptides, which was proven using lipopeptide-rich fractions (LRFs) isolated from the strains. Endophytic lipopeptide-synthesizing strains of Bacillus spp. indirectly affected the viability of aphids, the endurance of plants against aphids and triggered ISR in plants, which manifested itself in the regulation of oxidative metabolism and the accumulation of transcripts of the Pr1, Pr2, Pr3, Pr6 and Pr9 genes due to the synthesis of lipopeptides, which was proven using LRF isolated from three strains: B. subtilis 26D, B. subtilis 11VM, and B. thuringiensis B-6066. We have for the first time demonstrated the aphicidal effect of fengycin and the ability of the fengycin-synthesizing strains and isolates, B. subtilis Ttl2, Bacillus sp. Stl7 and B. thuringiensis B-6066, to regulate components of the pro-/antioxidant system of aphid-infested plants. In addition, this work is the first to demonstrate an elicitor role of fengycin in triggering a systemic resistance to S. graminum in wheat plants. We have discovered new promising strains and isolates of endophytes of the genus Bacillus, which may be included in the composition of new biocontrol agents against aphids. One of the criteria for searching for new bacteria active against phloem-feeding insects can be the presence of lipopeptide synthetase genes in the bacterial genome.202438952706
8373170.9890Weak Acid Resistance A (WarA), a Novel Transcription Factor Required for Regulation of Weak-Acid Resistance and Spore-Spore Heterogeneity in Aspergillus niger. Propionic, sorbic, and benzoic acids are organic weak acids that are widely used as food preservatives, where they play a critical role in preventing microbial growth. In this study, we uncovered new mechanisms of weak-acid resistance in molds. By screening a library of 401 transcription factor deletion strains in Aspergillus fumigatus for sorbic acid hypersensitivity, a previously uncharacterized transcription factor was identified and named weak acid resistance A (WarA). The orthologous gene in the spoilage mold Aspergillus niger was identified and deleted. WarA was required for resistance to a range of weak acids, including sorbic, propionic, and benzoic acids. A transcriptomic analysis was performed to characterize genes regulated by WarA during sorbic acid treatment in A. niger Several genes were significantly upregulated in the wild type compared with a ΔwarA mutant, including genes encoding putative weak-acid detoxification enzymes and transporter proteins. Among these was An14g03570, a putative ABC-type transporter which we found to be required for weak-acid resistance in A. niger We also show that An14g03570 is a functional homologue of the Saccharomyces cerevisiae protein Pdr12p and we therefore name it PdrA. Last, resistance to sorbic acid was found to be highly heterogeneous within genetically uniform populations of ungerminated A. niger conidia, and we demonstrate that pdrA is a determinant of this heteroresistance. This study has identified novel mechanisms of weak-acid resistance in A. niger which could help inform and improve future food spoilage prevention strategies.IMPORTANCE Weak acids are widely used as food preservatives, as they are very effective at preventing the growth of most species of bacteria and fungi. However, some species of molds can survive and grow in the concentrations of weak acid employed in food and drink products, thereby causing spoilage with resultant risks for food security and health. Current knowledge of weak-acid resistance mechanisms in these fungi is limited, especially in comparison to that in yeasts. We characterized gene functions in the spoilage mold species Aspergillus niger which are important for survival and growth in the presence of weak-acid preservatives. Such identification of weak-acid resistance mechanisms in spoilage molds will help in the design of new strategies to reduce food spoilage in the future.202031915214
8790180.9889Bacillus circulans GN03 Alters the Microbiota, Promotes Cotton Seedling Growth and Disease Resistance, and Increases the Expression of Phytohormone Synthesis and Disease Resistance-Related Genes. Plant growth-promoting bacteria (PGPB) are components of the plant rhizosphere that promote plant growth and/or inhibit pathogen activity. To explore the cotton seedlings response to Bacillus circulans GN03 with high efficiency of plant growth promotion and disease resistance, a pot experiment was carried out, in which inoculations levels of GN03 were set at 10(4) and 10(8) cfu(⋅)mL(-1). The results showed that GN03 inoculation remarkably enhanced growth promotion as well as disease resistance of cotton seedlings. GN03 inoculation altered the microbiota in and around the plant roots, led to a significant accumulation of growth-related hormones (indole acetic acid, gibberellic acid, and brassinosteroid) and disease resistance-related hormones (salicylic acid and jasmonic acid) in cotton seedlings, as determined with ELISA, up-regulated the expression of phytohormone synthesis-related genes (EDS1, AOC1, BES1, and GA20ox), auxin transporter gene (Aux1), and disease-resistance genes (NPR1 and PR1). Comparative genomic analyses was performed between GN03 and four similar species, with regards to phenotype, biochemical characteristics, and gene function. This study provides valuable information for applying the PGPB alternative, GN03, as a plant growth and disease-resistance promoting fertilizer.202133936131
8729190.9889Protein S-Acyl Transferase GhPAT27 Was Associated with Verticillium wilt Resistance in Cotton. Protein palmitoylation is an ability of the frame of the cell marker protein is one of the most notable reversible changes after translation. However, studies on protein palmitoylation in cotton have not yet been performed. In our current research, the PAT gene family was systematically identified and bioinformatically analyzed in G. arboreum, G. raimondii, G. barbadense and G. hirsutum, and 211 PAT genes were authenticated and classified into six subfamilies. Sixty-nine PAT genes were identified in upland cotton, mainly at the ends of its the 26 chromosomes of upland cotton. The majority of these genes are located in the nucleus of the plant. Gene structure analysis revealed that each member encodes a protein that which contains at least one DHHC structural domain. Cis-acting element analysis indicated that GhPATs genes are mainly involved in hormone production, light response and stress response. Gene expression pattern analysis indicated that most GhPATs genes were differentially expressed upon induction by pathogenic bacteria, drought, salt, hot and cold stresses, and some GhPATs could be induced by multiple abiotic stresses simultaneously. GhPATs genes showed different expression patterns in tissue-specific assays and were found to be preferentially expressed in roots, followed by expression in stems and leaves. Virus-induced gene silencing (VIGS) experiments showed that cotton was significantly less resistant to Verticillium dahliae when GhPAT27 was silenced. We conclude that the GhPAT27 gene, which mediates S-palmitoylation acetylation, may be involved in the regulation of upland cotton resistance to Verticillium wilt (VW). Overall, this work has provided a fundamental framework for understanding the latent capabilities of GhPATs and a solid foundation for molecular breeding and plant pathogen resistance in cotton.202236297782