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872100.8563Chromium metabolism characteristics of coexpression of ChrA and ChrT gene. OBJECTIVE: Serratia sp. S2 is a wild strain with chromium resistance and reduction ability. Chromium(VI) metabolic-protein-coding gene ChrA and ChrT were cloned from Serratia sp. S2, and ligated with prokaryotic expression vectors pET-28a (+) and transformed into E. coli BL21 to construct ChrA, ChrT and ChrAT engineered bacteria. By studying the characteristics of Cr(VI) metabolism in engineered bacteria, the function and mechanism of the sole expression and coexpression of ChrA and ChrT genes were studied. METHODS: Using Serratia sp. S2 genome as template, ChrA and ChrT genes were amplified by PCR, and prokaryotic expression vectors was ligated to form the recombinant plasmid pET-28a (+)-ChrA, pET-28a (+)-ChrT and pET-28a (+)-ChrAT, and transformed into E. coli BL21 to construct ChrA, ChrT, ChrAT engineered bacteria. The growth curve, tolerance, and reduction of Cr(VI), the distribution of intracellular and extracellular Cr, activity of chromium reductase and intracellular oxidative stress in engineered bacteria were measured to explore the metabolic characteristics of Cr(VI) in ChrA, ChrT, ChrAT engineered bacteria. RESULTS: ChrA, ChrT and ChrAT engineered bacteria were successfully constructed by gene recombination technology. The tolerance to Cr(VI) was Serratia sp. S2 > ChrAT ≈ ChrA > ChrT > Control (P < 0.05), and the reduction ability to Cr(VI) was Serratia sp. S2 > ChrAT ≈ ChrT > ChrA (P < 0.05). The chromium distribution experiments confirmed that Cr(VI) and Cr(III) were the main valence states. Effect of electron donors on chromium reductase activity was NADPH > NADH > non-NAD(P)H (P < 0.05). The activity of chromium reductase increased significantly with NAD(P)H (P < 0.05). The Glutathione and NPSH (Non-protein Sulfhydryl) levels of ChrA, ChrAT engineered bacteria increased significantly (P < 0.05) under the condition of Cr(VI), but there was no significant difference in the indexes of ChrT engineered bacteria (P > 0.05). CONCLUSION: ChrAT engineered bacteria possesses resistance and reduction abilities of Cr(VI). ChrA protein endows the strain with the ability to resist Cr(VI). ChrT protein reduces Cr(VI) to Cr(III) by using NAD(P)H as electronic donor. The reduction process promotes the production of GSH, GSSG and NPSH to maintain the intracellular reduction state, which further improves the Cr(VI) tolerance and reduction ability of ChrAT engineered bacteria.202032768747
872010.8547Chromium resistance characteristics of Cr(VI) resistance genes ChrA and ChrB in Serratia sp. S2. OBJECTIVE: To find an efficient chromium (VI) resistance system, with a highly efficient, economical, safe, and environmentally friendly chromium-removing strain, ChrA, ChrB, and ChrAB fragments of the chromium (VI) resistance gene in Serratia sp. S2 were cloned, and their prokaryotic expression vectors were constructed and transformed into E. coli BL21. The anti-chromium (VI) capacity and characteristics of engineered bacteria, role of ChrA and ChrB genes in the anti-chromium (VI) processes, and the mechanism of chromium metabolism, were explored. METHODS: The PCR technique was used to amplify ChrA, ChrB, and ChrAB genes from the Serratia sp. S2 genome. ChrA, ChrB, and ChrAB genes were connected to the prokaryotic expression vector pET-28a and transferred into E. coli BL21 for prokaryotic expression. Cr-absorption and Cr-efflux ability of the engineered strains were determined. The effects of respiratory inhibitors and oxygenated anions on Cr-efflux of ChrA and ChrB engineered strains were explored. RESULTS: ChrA, ChrB, and ChrAB engineered strains were constructed successfully; there was no significant difference between the control strain and the ChrB engineered strain for Cr-metabolism (P > 0.05). Cr-absorption and Cr-efflux of ChrA and ChrAB engineered strains were significantly stronger than the control strain (P < 0.05). Oxyanions (sulfate and molybdate) and inhibitors (valinomycin and CN(-)) could significantly inhibit the Cr-efflux capacities of ChrA and ChrAB engineered strains (P < 0.05), while NADPH could significantly promote such capacities (P < 0.05). CONCLUSION: The Cr-transporter, encoded by ChrA gene, confer the ability to pump out intracellular Cr on ChrA and ChrAB engineered strains. The ChrB gene plays a positive regulatory role in ChrA gene regulation. The Cr-metabolism ability of the ChrAB engineered strain is stronger than the ChrA engineered strain. ChrA and ChrAB genes in the Cr-resistance system may involve a variety of mechanisms, such as sulfate ion channel and respiratory chain electron transfer.201829655157
81020.8433Draft genome sequencing and functional annotation and characterization of biofilm-producing bacterium Bacillus novalis PD1 isolated from rhizospheric soil. Biofilm forming bacterium Bacillus novalis PD1 was isolated from the rhizospheric soil of a paddy field. B. novalis PD1 is a Gram-positive, facultatively anaerobic, motile, slightly curved, round-ended, and spore-forming bacteria. The isolate B. novalis PD1 shares 98.45% similarity with B. novalis KB27B. B. vireti LMG21834 and B. drentensis NBRC 102,427 are the closest phylogenetic neighbours for B. novalis PD1. The draft genome RAST annotation showed a linear chromosome with 4,569,088 bp, encoding 6139 coding sequences, 70 transfer RNA (tRNA), and 11 ribosomal RNA (rRNA) genes. The genomic annotation of biofilm forming B. novalis PD1(> 3.6@OD(595nm)) showed the presence of exopolysaccharide-forming genes (ALG, PSL, and PEL) as well as other biofilm-related genes (comER, Spo0A, codY, sinR, TasA, sipW, degS, and degU). Antibiotic inactivation gene clusters (ANT (6)-I, APH (3')-I, CatA15/A16 family), efflux pumps conferring antibiotic resistance genes (BceA, BceB, MdtABC-OMF, MdtABC-TolC, and MexCD-OprJ), and secondary metabolites linked to phenazine, terpene, and beta lactone gene clusters are part of the genome.202134537868
80630.8431A 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
54040.8421Effect of ogt expression on mutation induction by methyl-, ethyl- and propylmethanesulphonate in Escherichia coli K12 strains. We have previously reported the isolation of an Escherichia coli K12 mutant that is extremely sensitive to mutagenesis by low doses of ethylating agents. We now show by Southern analysis that the mutation involves a gross deletion covering at least the ogt and fnr genes and that no O6-alkylguanine-DNA-alkyltransferase activity is present in cell-free extracts of an ada::Tn10 derivative of these bacteria. Confirmation that sensitisation to ethylation-induced mutagenesis was attributable to ogt and not to any other loci covered by the deletion was obtained by constructing derivatives. Thus an ogt::kanr disruption mutation was introduced into the parental ogt+ bacteria, and the ogt::kanr mutation was then eliminated by cotransduction of ogt+ with the closely linked Tetr marker (zcj::Tn10). The delta(ogt-fnr) deletion or ogt::kanr disruption mutants were highly sensitive to ethyl methanesulphonate-induced mutagenesis, as measured by the induction of forward mutations to L-arabinose resistance (Arar). Furthermore, the number of Arar mutants increased linearly with dose, unlike the case in ogt+ bacteria, which had a threshold dose below which no mutants accumulated. Differences in mutability were even greater with propyl methanesulphonate. Overproduction of the ogt alkyltransferase from a multicopy plasmid reduced ethylmethanesulphonate-induced mutagenesis in the ogt- mutant strains and also methylmethanesulphonate mutagenesis in ada- bacteria. A sample of AB1157 obtained from the E. coli K12 genetic stock centre also had a deletion covering the ogt and fnr genes. Since such deletions greatly influence the mutagenic responses to alkylating agents, a survey of the presence of the ogt gene in the E. coli K12 strain being used is advisable.19948152424
32850.8419Multiresistance genes of Rhizobium etli CFN42. Multidrug efflux pumps of bacteria are involved in the resistance to various antibiotics and toxic compounds. In Rhizobium etli, a mutualistic symbiont of Phaseolus vulgaris (bean), genes resembling multidrug efflux pump genes were identified and designated rmrA and rmrB. rmrA was obtained after the screening of transposon-generated fusions that are inducible by bean-root released flavonoids. The predicted gene products of rmrAB shared significant homology to membrane fusion and major facilitator proteins, respectively. Mutants of rmrA formed on average 40% less nodules in bean, while mutants of rmrA and rmrB had enhanced sensitivity to phytoalexins, flavonoids, and salicylic acid, compared with the wild-type strain. Multidrug resistance genes emrAB from Escherichia coli complemented an rmrA mutant from R. etli for resistance to high concentrations of naringenin.200010796024
904660.8418Burkholderia pseudomallei resistance to antibiotics in biofilm-induced conditions is related to efflux pumps. Burkholderia pseudomallei, the causative agent of melioidosis, has been found to increase its resistance to antibiotics when growing as a biofilm. The resistance is related to several mechanisms. One of the possible mechanisms is the efflux pump. Using bioinformatics analysis, it was found that BPSL1661, BPSL1664 and BPSL1665 were orthologous genes of the efflux transporter encoding genes for biofilm-related antibiotic resistance, PA1874-PA1877 genes in Pseudomonas aeruginosa strain PAO1. Expression of selected encoding genes for the efflux transporter system during biofilm formation were investigated. Real-time reverse transcriptase PCR expression of amrB, cytoplasmic membrane protein of AmrAB-OprA efflux transporter encoding gene, was slightly increased, while BPSL1665 was significantly increased during growth of bacteria in biofilm formation. Minimum biofilm inhibition concentration and minimum biofilm eradication concentration (MBEC) of ceftazidime (CTZ), doxycycline (DOX) and imipenem were found to be 2- to 1024-times increased when compared to their MICs for of planktonic cells. Inhibition of the efflux transporter by adding phenylalanine arginine β-napthylamide (PAβN), a universal efflux inhibitor, decreased 2 to 16 times as much as MBEC in B. pseudomallei biofilms with CTZ and DOX. When the intracellular accumulation of antibiotics was tested to reveal the pump inhibition, only the concentrations of CTZ and DOX increased in PAβN treated biofilm. Taken together, these results indicated that BPSL1665, a putative precursor of the efflux pump gene, might be related to the adaptation of B. pseudomallei in biofilm conditions. Inhibition of efflux pumps may lead to a decrease of resistance to CTZ and DOX in biofilm cells.201627702426
80470.8415Cloning, mutagenesis, and characterization of the microalga Parietochloris incisa acetohydroxyacid synthase, and its possible use as an endogenous selection marker. Parietochloris incisa is an oleaginous fresh water green microalga that accumulates an unusually high content of the valuable long-chain polyunsaturated fatty acid (LC-PUFA) arachidonic acid within triacylglycerols in cytoplasmic lipid bodies. Here, we describe cloning and mutagenesis of the P. incisa acetohydroxyacid synthase (PiAHAS) gene for use as an herbicide resistance selection marker for transformation. Use of an endogenous gene circumvents the risks and regulatory difficulties of cultivating antibiotic-resistant organisms. AHAS is present in plants and microorganisms where it catalyzes the first essential step in the synthesis of branched-chain amino acids. It is the target enzyme of the herbicide sulfometuron methyl (SMM), which effectively inhibits growth of bacteria and plants. Several point mutations of AHAS are known to confer herbicide resistance. We cloned the cDNA that encodes PiAHAS and introduced a W605S point mutation (PimAHAS). Catalytic activity and herbicide resistance of the wild-type and mutant proteins were characterized in the AHAS-deficient E. coli, BUM1 strain. Cloned PiAHAS wild-type and mutant genes complemented AHAS-deficient bacterial growth. Furthermore, bacteria expressing the mutant PiAHAS exhibited high resistance to SMM. Purified PiAHAS wild-type and mutant proteins were assayed for enzymatic activity and herbicide resistance. The W605S mutation was shown to cause a twofold decrease in enzymatic activity and in affinity for the Pyruvate substrate. However, the mutant exhibited 7 orders of magnitude higher resistance to the SMM herbicide than that of the wild type.201222488216
13280.8414Chromium resistance strategies and toxicity: what makes Ochrobactrum tritici 5bvl1 a strain highly resistant. Large-scale industrial use of chromium (Cr) resulted in widespread environmental contamination with hexavalent chromium (Cr(VI)). The ability of microorganisms to survive in these environments and detoxify chromate requires the presence of specific resistance systems. Several Cr(VI) resistant species, belonging to a variety of genera, have been isolated in recent years. Ochrobactrum tritici strain 5bvl1 is a model for a highly Cr(VI)-resistant and reducing microorganism, with different strategies to cope with chromium. The strain contains the transposon-located (TnOtChr) chromate resistance genes chrB, chrA, chrC, chrF. The chrB and chrA genes were found to be essential for the establishment of high resistance but not chrC or chrF genes. Other mechanisms involved in chromium resistance in this strain were related to strategies such as specific or unspecific Cr(VI) reduction, free-radical detoxifying activities, and repairing DNA damage. Expression of the chrB, chrC or chrF genes was related to increased resistance to superoxide-generating agents. Genetic analyses also showed that, the ruvB gene is related to chromium resistance in O. tritici 5bvl1. The RuvABC complex probably does not form when ruvB gene is interrupted, and the repair of DNA damage induced by chromium is prevented. Aerobic or anaerobic chromate reductase activity and other unspecific mechanisms for chromium reduction have been identified in different bacteria. In the strain O. tritici 5bvl1, several unspecific mechanisms were found. Dichromate and chromate have different effects on the physiology of the chromium resistant strains and dichromate seems to be more toxic. Toxicity of Cr(VI) was evaluated by following growth, reduction, respiration, glucose uptake assays and by comparing cell morphology.201121472416
873790.8411Role of Biosynthetic Gene Cluster BGC3 in the Cariogenic Virulence of Streptococcus mutans. OBJECTIVE: To investigate the role of the biosynthetic gene cluster BGC3 of Streptococcus mutans (S. mutans) in the process of dental caries. METHODS: BGC3 and ∆BGC3 S. mutans strains were constructed and their growth curves were evaluated. Acid production capacity was assessed by evaluating pH reduction levels over identical culture periods. The survival of bacteria in phosphate citrate buffer solution (pH 3.0) was quantified. The expression levels of virulence genes (atpF, gtfC, gtfD, spaP, vicR and ftf) were analysed using the qPCR. Co-culture experiments were conducted to evaluate bacterial adaptability. Bacterial viability was determined by microscopical examination of live/dead staining. RESULTS: Deletion of BGC3 did not significantly impact S. mutans growth or acid production in biofilms. The ∆BGC3 strain exhibited enhanced acid resistance and higher expression levels of virulence genes compared to the wild type. In addition, ∆BGC3 exhibited superior bacterial viability in the co-culture system. CONCLUSION: BGC3 affected the acid resistance and expression of caries-related genes in S. mutans. The BGC3 knockout strain exhibited a more robust survival capability than the wild-type strain.202540162656
8798100.8408Estrogen mimics induce genes encoding chemical efflux proteins in gram-negative bacteria. Escherichia coli and Pseudomonas aeruginosa are gram-negative bacteria found in wastewater and biosolids. Spanning the inner and outer membrane are resistance-nodulation-cell division superfamily (RND) efflux pumps responsible for detoxification of the cell, typically in response to antibiotics and other toxicity inducing substrates. Here, we show that estrogenic endocrine disruptors, common wastewater pollutants, induce genes encoding chemical efflux proteins. Bacteria were exposed to environmental concentrations of the synthetic estrogen 17α-ethynylestradiol, the surfactant nonylphenol, and the plasticizer bisphenol-A, and analyzed for RND gene expression via q-PCR. Results showed that the genes acrB and yhiV were over-expressed in response to the three chemicals in E. coli, and support previous findings that these two transporters export hormones. P. aeruginosa contains 12 RND efflux pumps, which were differentially expressed in response to the three chemicals: 17α-ethynylestradiol, bisphenol-A, and nonylphenol up-regulated mexD and mexF, while nonylphenol and bisphenol-A positively affected transcription of mexK, mexW, and triC. Gene expression via q-PCR of RND genes may be used to predict the interaction of estrogen mimics with RND genes. One bacterial response to estrogen mimic exposure is to induce gene expression of chemical efflux proteins, which leads to the expulsion of the contaminant from the cell.201525754012
6005110.8408Antimicrobial activity of Pediococcus pentosaceus strains against diarrheal pathogens isolated from pigs and effect on paracellular permeability of HT-29 cells. This study aimed to investigate lactic acid bacteria with antimicrobial activities against infectious diarrheal pathogens in pigs and their genetic characteristics. Acid-resistant lactic acid bacteria were examined for bile resistance, pancreatic enzyme resistance, gelatinase and urease activities, and antibiotic resistance. Subsequently, selected isolates were examined for antimicrobial activities against Campylobacter coli, Clostridium perfringens, Escherichia coli, and Salmonella Typhimurium, and their effects on paracellular permeability and the expression of tight junction protein-encoding genes in HT-29 cells were assessed. Whole genome sequencing was performed to identify the genes related to safety and antibacterial activity. Of the 51 isolates examined, 12 were resistant to bile and pancreatin and did not produce gelatinase and urease. Of these 12, isolates 19, 20, 30, 36, and 67 showed tetracycline resistance and isolates 15, 19, and 38W showed antimicrobial activity against infectious diarrheal bacteria. Treatment with isolate 38W significantly reduced the paracellular permeability induced by E. coli in HT-29 cells and alleviated the expression of tight junction protein-encoding genes (claudin-1, occludin, and ZO-1) induced by E. coli inoculation. Isolates 15, 19, and 38W were named as Pediococcus pentosaceus SMFM2016-NK1, SMFM2016-YK1, and SMFM2016-WK1, respectively. Bacteriocin-related genes were YheH, ytrF, BceA, BceB, and MccF in SMFM2016-NK1; YheH, ytrF, BceA, BceB, entK, lcnA, MccF, and skgD in SMFM2016-YK1; and YheH, ytrF, BceA, BceB, and MccF in SMFM2016-WK1. SMFM2016-YK1 harbored the tetM gene. These results indicate that P. pentosaceus SMFM2016-WK1 might control diarrheal pathogens isolated from pigs. However, a further study is necessary because the results were obtained only from in vitro experiment.202540873998
126120.8407Single-gene knockout of a novel regulatory element confers ethionine resistance and elevates methionine production in Corynebacterium glutamicum. Despite the availability of genome data and recent advances in methionine regulation in Corynebacterium glutamicum, sulfur metabolism and its underlying molecular mechanisms are still poorly characterized in this organism. Here, we describe the identification of an ORF coding for a putative regulatory protein that controls the expression of genes involved in sulfur reduction dependent on extracellular methionine levels. C. glutamicum was randomly mutagenized by transposon mutagenesis and 7,000 mutants were screened for rapid growth on agar plates containing the methionine antimetabolite D,L-ethionine. In all obtained mutants, the site of insertion was located in the ORF NCgl2640 of unknown function that has several homologues in other bacteria. All mutants exhibited similar ethionine resistance and this phenotype could be transferred to another strain by the defined deletion of the NCgl2640 gene. Moreover, inactivation of NCgl2640 resulted in significantly increased methionine production. Using promoter lacZ-fusions of genes involved in sulfur metabolism, we demonstrated the relief of L-methionine repression in the NCgl2640 mutant for cysteine synthase, o-acetylhomoserine sulfhydrolase (metY) and sulfite reductase. Complementation of the mutant strain with plasmid-borne NCgl2640 restored the wild-type phenotype for metY and sulfite reductase.200515668756
6366130.8401Fluorinated Beta-diketo Phosphorus Ylides Are Novel Efflux Pump Inhibitors in Bacteria. BACKGROUND: One of the most important resistance mechanisms in bacteria is the increased expression of multidrug efflux pumps. To combat efflux-related resistance, the development of new efflux pump inhibitors is essential. MATERIALS AND METHODS: Ten phosphorus ylides were compared based on their MDR-reverting activity in multidrug efflux pump system consisting of the subunits acridine-resistance proteins A and B (AcrA and AcrB) and the multidrug efflux pump outer membrane factor TolC (TolC) of Escherichia coli K-12 AG100 strain and its AcrAB-TolC-deleted strain. Efflux inhibition was assessed by real-time fluorimetry and the inhibition of quorum sensing (QS) was also investigated. The relative gene expression of efflux QS genes was determined by real-time reverse transcriptase quantitative polymerase chain reaction. RESULTS: The most potent derivative was Ph(3)P=C(COC(2)F(5))CHO and its effect was more pronounced on the AcrAB-TolC-expressing E. coli strain, furthermore the most active compounds, Ph(3)P=C(COCF(3))OMe, Ph(3)P=C(COC(2)F(5))CHO and Ph(3)P=C(COCF(3))COMe, reduced the expression of efflux pump and QS genes. CONCLUSION: Phosphorus ylides might be valuable EPI compounds to reverse efflux related MDR in bacteria.201627815466
579140.8399Control 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
6190150.8395Identifying Escherichia coli genes involved in intrinsic multidrug resistance. Multidrug resistance is a major cause of clinical failure in treating bacterial infections. Increasing evidence suggests that bacteria can resist multiple antibiotics through intrinsic mechanisms that rely on gene products such as efflux pumps that expel antibiotics and special membrane proteins that block the penetration of drug molecules. In this study, Escherichia coli was used as a model system to explore the genetic basis of intrinsic multidrug resistance. A random mutant library was constructed in E. coli EC100 using transposon mutagenesis. The library was screened by growth measurement to identify the mutants with enhanced or reduced resistance to chloramphenicol (Cm). Out of the 4,000 mutants screened, six mutants were found to be more sensitive to Cm and seven were more resistant compared to the wild-type EC100. Mutations in 12 out of the 13 mutants were identified by inverse polymerase chain reaction. Mutants of the genes rob, garP, bipA, insK, and yhhX were more sensitive to Cm compared to the wild-type EC100, while the mutation of rhaB, yejM, dsdX, nagA, yccE, atpF, or htrB led to higher resistance. Overexpression of rob was found to increase the resistance of E. coli biofilms to tobramycin (Tob) by 2.7-fold, while overexpression of nagA, rhaB, and yccE significantly enhanced the susceptibility of biofilms by 2.2-, 2.5-, and 2.1-fold respectively.200818807027
9999160.8394Assessment of competitiveness of rhizobia infecting Galega orientalis on the basis of plant yield, nodulation, and strain identification by antibiotic resistance and PCR. Competition between effective and ineffective Rhizobium galegae strains nodulating Galega orientalis was examined on the basis of plant growth, nodulation, antibiotic resistance, and PCR results. In a preliminary experiment in Leonard's jars, ineffective R. galegae strains HAMBI 1207 and HAMBI 1209 competed in similar manners with the effective strain R. galegae HAMBI 1174. In a pot experiment, soil was inoculated with 0 to 10(5) HAMBI 1207 cells per g before G. orientalis was sown. Seeds of G. orientalis were surface inoculated with 2 x 10(4) and 2 x 10(5) cells of HAMBI 1174 per seed (which represent half and fivefold the commercially recommended amount of inoculant, respectively). Plant yield and nodulation by the effective strain were significantly reduced, with as few as 10(2) ineffective rhizobia per g of soil, and the inoculation response was not improved by the 10-fold greater dose of the inoculant. Bacteria occupying the nodules were identified by antibiotic resistance and PCR with primers specific for R. galegae HAMBI 1174, R. galegae, and genes coding for bacterial 16S rRNA (bacterial 16S rDNA). Sixty-two large nodules examined were occupied by the effective strain HAMBI 1174, as proven by antibiotic resistance and amplification of the strain-specific fragment. From 20 small nodules, only the species-specific fragment could be amplified, and isolated bacteria had the same antibiotic resistance and 16S PCR restriction pattern as strain HAMBI 1207. PCR with our strain-specific and species-specific primers provides a powerful tool for strain identification of R. galegae directly from nodules without genetic modification of the bacteria.19968593053
8802170.8393The Transcription Factor CsgD Contributes to Engineered Escherichia coli Resistance by Regulating Biofilm Formation and Stress Responses. The high cell density, immobilization and stability of biofilms are ideal characteristics for bacteria in resisting antibiotic therapy. CsgD is a transcription activating factor that regulates the synthesis of curly fimbriae and cellulose in Escherichia coli, thereby enhancing bacterial adhesion and promoting biofilm formation. To investigate the role of CsgD in biofilm formation and stress resistance in bacteria, the csgD deletion mutant ΔcsgD was successfully constructed from the engineered strain E. coli BL21(DE3) using the CRISPR/Cas9 gene-editing system. The results demonstrated that the biofilm of ΔcsgD decreased by 70.07% (p < 0.05). Additionally, the mobility and adhesion of ΔcsgD were inhibited due to the decrease in curly fimbriae and extracellular polymeric substances. Furthermore, ΔcsgD exhibited a significantly decreased resistance to acid, alkali and osmotic stress conditions (p < 0.05). RNA-Seq results revealed 491 differentially expressed genes between the parent strain and ΔcsgD, with enrichment primarily observed in metabolism-related processes as well as cell membrane structure and catalytic activity categories. Moreover, CsgD influenced the expression of biofilm and stress response genes pgaA, motB, fimA, fimC, iraP, ompA, osmC, sufE and elaB, indicating that the CsgD participated in the resistance of E. coli by regulating the expression of biofilm and stress response. In brief, the transcription factor CsgD plays a key role in the stress resistance of E. coli, and is a potential target for treating and controlling biofilm.202337761984
330180.8393A DHA14 drug efflux gene from Xanthomonas albilineans confers high-level albicidin antibiotic resistance in Escherichia coli. AIMS: Identification of a gene for self-protection from the antibiotic-producing plant pathogen Xanthomonas albilineans, and functional testing by heterologous expression. METHODS AND RESULTS: Albicidin antibiotics and phytotoxins are potent inhibitors of prokaryote DNA replication. A resistance gene (albF) isolated by shotgun cloning from the X. albilineans albicidin-biosynthesis region encodes a protein with typical features of DHA14 drug efflux pumps. Low-level expression of albF in Escherichia coli increased the MIC of albicidin 3000-fold, without affecting tsx-mediated albicidin uptake into the periplasm or resistance to other tested antibiotics. Bioinformatic analysis indicates more similarity to proteins involved in self-protection in polyketide-antibiotic-producing actinomycetes than to multi-drug resistance pumps in other gram-negative bacteria. A complex promoter region may co-regulate albF with genes for hydrolases likely to be involved in albicidin activation or self-protection. CONCLUSIONS: AlbF is the first apparent single-component antibiotic-specific efflux pump from a gram-negative antibiotic producer. It shows extraordinary efficiency as measured by resistance level conferred upon heterologous expression. SIGNIFICANCE AND IMPACT OF THE STUDY: Development of the clinical potential of albicidins as potent bactericidial antibiotics against diverse bacteria has been limited because of low yields in culture. Expression of albF with recently described albicidin-biosynthesis genes may enable large-scale production. Because albicidins are X. albilineans pathogenicity factors, interference with AlbF function is also an opportunity for control of the associated plant disease.200616834602
6197190.8392Structure and location of tellurium deposited in Escherichia coli cells harbouring tellurite resistance plasmids. The plasmids RP4Ter and pHH1508a, which belong to the P and HII incompatibility groups, respectively, confer resistance to potassium tellurite (K2TeO3) on Escherichia coli. The genes for tellurite resistance were cloned from each plasmid onto the vector pUC8 to create pDT1366 and pD1364, respectively. Unstained, unfixed bacteria carrying these plasmids contained black intracellular deposits when grown on media containing tellurite. Thin sections of these bacteria fixed with glutaraldehyde were prepared and examined by electron microscopy. The black deposits were located inside the cell and were frequently associated with the inner membrane of the bacterium. Bacteria containing pDT1366 or pDT1364, and therefore a higher gene dosage of the Ter determinant, contained more black deposits, but had a decreased resistance, as measured by the minimum inhibitory concentration using the agar dilution method. Using the technique of electron spectroscopic imaging, the black intracellular deposits were shown to contain predominantly reduced metallic tellurium, and significant amounts of oxygen or carbon, thereby confirming earlier results using X-ray diffraction analysis of whole cells.19883042886