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618900.9776Characterization 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
634910.9774High-level chromate resistance in Arthrobacter sp. strain FB24 requires previously uncharacterized accessory genes. BACKGROUND: The genome of Arthrobacter sp. strain FB24 contains a chromate resistance determinant (CRD), consisting of a cluster of 8 genes located on a 10.6 kb fragment of a 96 kb plasmid. The CRD includes chrA, which encodes a putative chromate efflux protein, and three genes with amino acid similarities to the amino and carboxy termini of ChrB, a putative regulatory protein. There are also three novel genes that have not been previously associated with chromate resistance in other bacteria; they encode an oxidoreductase (most similar to malate:quinone oxidoreductase), a functionally unknown protein with a WD40 repeat domain and a lipoprotein. To delineate the contribution of the CRD genes to the FB24 chromate [Cr(VI)] response, we evaluated the growth of mutant strains bearing regions of the CRD and transcript expression levels in response to Cr(VI) challenge. RESULTS: A chromate-sensitive mutant (strain D11) was generated by curing FB24 of its 96-kb plasmid. Elemental analysis indicated that chromate-exposed cells of strain D11 accumulated three times more chromium than strain FB24. Introduction of the CRD into strain D11 conferred chromate resistance comparable to wild-type levels, whereas deletion of specific regions of the CRD led to decreased resistance. Using real-time reverse transcriptase PCR, we show that expression of each gene within the CRD is specifically induced in response to chromate but not by lead, hydrogen peroxide or arsenate. Higher levels of chrA expression were achieved when the chrB orthologs and the WD40 repeat domain genes were present, suggesting their possible regulatory roles. CONCLUSION: Our findings indicate that chromate resistance in Arthrobacter sp. strain FB24 is due to chromate efflux through the ChrA transport protein. More importantly, new genes have been identified as having significant roles in chromate resistance. Collectively, the functional predictions of these additional genes suggest the involvement of a signal transduction system in the regulation of chromate efflux and warrants further study.200919758450
522120.9773Molecular cloning of the DNA gyrase genes from Methylovorus sp. strain SS1 and the mechanism of intrinsic quinolone resistance in methylotrophic bacteria. The genes encoding the DNA gyrase A (GyrA) and B subunits (GyrB) of Methylovorus sp. strain SS1 were cloned and sequenced. gyrA and gyrB coded for proteins of 846 and 799 amino acids with calculated molecular weights of 94,328 and 88,714, respectively, and complemented Escherichia coli gyrA and gyrB temperature sensitive (ts) mutants. To analyze the role of type II topoisomerases in the intrinsic quinolone resistance of methylotrophic bacteria, the sequences of the quinolone resistance-determining regions (QRDRs) in the A subunit of DNA gyrase and the C subunit (ParC) of topoisomerase IV (Topo IV) of Methylovorus sp. strain SS1, Methylobacterium extorquens AM1 NCIB 9133, Methylobacillus sp, strain SK1 DSM 8269, and Methylophilus methylotrophus NCIB 10515 were determined. The deduced amino acid sequences of the QRDRs of the ParCs in the four methylotrophic bacteria were identical to that of E. coli ParC. The sequences of the QRDR in GyrA were also identical to those in E. coli GyrA except for the amino acids at positions 83, 87, or 95. The Ser83 to Thr substitution in Methylovorus sp. strain SS1, and the Ser83 to Leu and Asp87 to Asn substitutions in the three other methylotrophs, agreed well with the minimal inhibitory concentrations of quinolones in the four bacteria, suggesting that these residues play a role in the intrinsic susceptibility of methylotrophic bacteria to quinolones.200516404155
39930.9771Identification of genes conferring arsenic resistance to Escherichia coli from an effluent treatment plant sludge metagenomic library. The majority of bacteria elude culture in the laboratory. A metagenomic approach provides culture-independent access to the gene pool of the whole bacterial community. A metagenomic library was constructed from an industrial effluent treatment plant sludge containing about 1.25 Gb of microbial community DNA. Two arsenic-resistant clones were selected from the metagenomic library. Clones MT3 and MT6 had eight- and 18-fold higher resistance to sodium arsenate in comparison with the parent strain, respectively. The clones also showed increased resistance to arsenite but not to antimony. Sequence analysis of the clones revealed genes encoding for putative arsenate reductases and arsenite efflux pumps. A novel arsenate resistance gene (arsN) encoding a protein with similarity to acetyltransferases was identified from clone MT6. ArsN homologues were found to be closely associated with arsenic resistance genes in many bacterial genomes. ArsN homologues were found fused to putative arsenate reductases in Methylibium petroleiphilum PM1 and Anaeromyxobacter dehalogenans 2CP-C and with a putative arsenite chaperone in Burkholderia vietnamiensis G4. ArsN alone resulted in an approximately sixfold higher resistance to sodium arsenate in wild-type Escherichia coli W3110.200919016868
8740.9769Involvement of specific calmodulin isoforms in salicylic acid-independent activation of plant disease resistance responses. The Ca2+ signal is essential for the activation of plant defense responses, but downstream components of the signaling pathway are still poorly defined. Here we demonstrate that specific calmodulin (CaM) isoforms are activated by infection or pathogen-derived elicitors and participate in Ca2+-mediated induction of plant disease resistance responses. Soybean CaM (SCaM)-4 and SCaM-5 genes, which encode for divergent CaM isoforms, were induced within 30 min by a fungal elicitor or pathogen, whereas other SCaM genes encoding highly conserved CaM isoforms did not show such response. This pathogen-triggered induction of these genes specifically depended on the increase of intracellular Ca2+ level. Constitutive expression of SCaM-4 and SCaM-5 in transgenic tobacco plants triggered spontaneous induction of lesions and induces an array of systemic acquired resistance (SAR)-associated genes. Surprisingly, these transgenic plants have normal levels of endogenous salicylic acid (SA). Furthermore, coexpression of nahG gene did not block the induction of SAR-associated genes in these transgenic plants, indicating that SA is not involved in the SAR gene induction mediated by SCaM-4 or SCaM-5. The transgenic plants exhibit enhanced resistance to a wide spectrum of virulent and avirulent pathogens, including bacteria, fungi, and virus. These results suggest that specific CaM isoforms are components of a SA-independent signal transduction chain leading to disease resistance.19999892708
623350.9769Exploring the response of Escherichia coli O157:H7 EDL933 within Acanthamoeba castellanii by genome-wide transcriptional profiling. Free-living protozoa, such as Acanthamoeba castellanii, are environmental hosts for pathogenic bacteria. Protozoa have been implicated in harboring pathogenic bacteria and enhancing virulence factors and antibiotic resistance. To better understand this relationship with Escherichia coli O157:H7, we characterized its transcriptome within A. castellanii compared with broth-grown organisms using two-color microarrays. Statistical analysis indicated that 969 genes were differentially expressed at P<0.018, with a false discovery rate of 1.9% and a fold change cutoff of 1.3 or greater. There were 655 upregulated transcripts that include 40 genes associated with virulence, of which 32 are encoded on O-islands, and include shiga toxin genes (stx1A, stx1B stx2A) and 14 genes involved in Type III secretion system components. Also included are SOS response genes such as lexA and recA, genes involved in or predicted to be involved in antibiotic resistance (rarD, macAB, marABR, mdtK, yojI, yhgN), the quorum-sensing operon lsrACDB, and the efe and feo iron-acquisition systems. There were 314 downregulated transcripts that included 19 transcripts associated with virulence, seven of which are encoded on O-islands. Our results demonstrate that a significant portion of the E. coli O157:H7 genome was differentially expressed as a result of the protozoan intracellular environment.201020831595
615860.9768Nitric oxide stress resistance in Porphyromonas gingivalis is mediated by a putative hydroxylamine reductase. Porphyromonas gingivalis, the causative agent of adult periodontitis, must maintain nitric oxide (NO) homeostasis and surmount nitric oxide stress from host immune responses or other oral bacteria to survive in the periodontal pocket. To determine the involvement of a putative hydroxylamine reductase (PG0893) and a putative nitrite reductase-related protein (PG2213) in P. gingivalis W83 NO stress resistance, genes encoding those proteins were inactivated by allelic exchange mutagenesis. The isogenic mutants P. gingivalis FLL455 (PG0893ermF) and FLL456 (PG2213ermF) were black pigmented and showed growth rates and gingipain and hemolytic activities similar to those of the wild-type strain. P. gingivalis FLL455 was more sensitive to NO than the wild type. Complementation of P. gingivalis FLL455 with the wild-type gene restored the level of NO sensitivity to a level similar to that of the parent strain. P. gingivalis FLL455 and FLL456 showed sensitivity to oxidative stress similar to that of the wild-type strain. DNA microarray analysis showed that PG0893 and PG2213 were upregulated 1.4- and 2-fold, respectively, in cells exposed to NO. In addition, 178 genes were upregulated and 201 genes downregulated more than 2-fold. The majority of these modulated genes were hypothetical or of unknown function. PG1181, predicted to encode a transcriptional regulator, was upregulated 76-fold. Transcriptome in silico analysis of the microarray data showed major metabolomic variations in key pathways. Collectively, these findings indicate that PG0893 and several other genes may play an important role in P. gingivalis NO stress resistance.201222247513
595970.9768High incidence of multiple antibiotic resistant cells in cultures of in enterohemorrhagic Escherichia coli O157:H7. The spontaneous incidence of chloramphenicol (Cam) resistant mutant bacteria is at least ten-fold higher in cultures of enterohemorrhagic Escherichia coli O157:H7 strain EDL933 than in E. coli K-12. It is at least 100-fold higher in the dam (DNA adenine methyltransferase) derivative of EDL933, compared to the dam strain of E. coli K-12, thereby preventing the use of Cam resistance as a marker in gene replacement technology. Genome sequencing of Cam-resistant isolates of EDL933 and its dam derivatives showed that the marR (multiple antibiotic resistance) gene was mutated in every case but not in the Cam-sensitive parental strains. As expected from mutation in the marR gene, the Cam-resistant bacteria were also found to be resistant to tetracycline and nalidixic acid. The marR gene in strain EDL933 is annotated as a shorter open reading frame than that in E. coli K-12 but the longer marR(+) open reading frame was more efficient at complementing the marR antibiotic-resistance phenotype of strain EDL933. Beta-lactamase-tolerant derivatives were present at frequencies 10-100 times greater in cultures of marR derivatives of strain EDL933 than the parent strain. Spontaneous mutation frequency to rifampicin, spectinomycin and streptomycin resistance was the same in E. coli O157:H7 and E. coli K-12 strains.201424361397
635280.9768Cloning and characterization of grpE in Acetobacter pasteurianus NBRC 3283. The grpE gene in Acetobacter pasteurianus NBRC 3283 was cloned and characterized, to elucidate the mechanism underlying the resistance of acetic acid bacteria to the stressors existing during acetic acid fermentation. This gene was found to be located in tandem with two related genes, appearing on the genome in the order grpE-dnaK-dnaJ. A sigma(32)-type promoter sequence was found in the upstream region of grpE. The relative transcription levels of grpE, dnaK, and dnaJ mRNA were in the ratio of approximately 1:2:0.1, and the genes were transcribed as grpE-dnaK, dnaK, and dnaJ. The transcription level of grpE was elevated by heat shock and treatment with ethanol. Co-overexpression of GrpE with DnaK/J in cells resulted in improved growth compared to the single overexpression of DnaK/J in high temperature or ethanol-containing conditions, suggesting that GrpE acts cooperatively with DnaK/J for expressing resistance to those stressors considered to exist during acetic acid fermentation. Our findings indicate that GrpE is closely associated with adaptation to stressors in A. pasteurianus and may play an important role in acetic acid fermentation.201020129077
619490.9767Salmonella typhimurium encodes an SdiA homolog, a putative quorum sensor of the LuxR family, that regulates genes on the virulence plasmid. Quorum sensing is a phenomenon in which bacteria sense and respond to their own population density by releasing and sensing pheromones. In gram-negative bacteria, quorum sensing is often performed by the LuxR family of transcriptional regulators, which affect phenotypes as diverse as conjugation, bioluminescence, and virulence gene expression. The gene encoding one LuxR family member, named sdiA (suppressor of cell division inhibition), is present in the Escherichia coli genome. In this report, we have cloned the Salmonella typhimurium homolog of SdiA and performed a systematic screen for sdiA-regulated genes. A 4.4-kb fragment encoding the S. typhimurium sdiA gene was sequenced and found to encode the 3' end of YecC (homologous to amino acid transporters of the ABC family), all of SdiA and SirA (Salmonella invasion regulator), and the 5' end of UvrC. This gene organization is conserved between E. coli and S. typhimurium. We determined that the S. typhimurium sdiA gene was able to weakly complement the E. coli sdiA gene for activation of ftsQAZ at promoter 2 and for suppression of filamentation caused by an ftsZ(Ts) allele. To better understand the function of sdiA in S. typhimurium, we screened 10,000 random lacZY transcriptional fusions (MudJ transposon mutations) for regulation by sdiA. Ten positively regulated fusions were isolated. Seven of the fusions were within an apparent operon containing ORF8, ORF9, rck (resistance to complement killing), and ORF11 of the S. typhimurium virulence plasmid. The three ORFs have now been named srgA, srgB, and srgC (for sdiA-regulated gene), respectively. The DNA sequence adjacent to the remaining three fusions shared no similarity with previously described genes.19989495757
128100.9767Identification 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
551110.9767virK and mig-14 constitute a PhoP-dependent operon and contribute to the intracellular survival and polymyxin B resistance of Salmonella Typhi. In bacteria, adjacent and functionally similar genes are typically transcribed as operons. The virulence genes virK and mig-14 are acquired through horizontal gene transfer in Salmonella. Previous studies have reported that these two genes have similar functions in terms of bacterial survival within macrophages and resistance to antimicrobial peptides. Nevertheless, the specific expression characteristics of the two genes remain unclear. This study revealed that virK and mig-14 were transcribed as a single operon in Salmonella Typhi. The virK-mig-14 operon was found to be activated under conditions of early hyperosmotic stress and polymyxin B stimulation, and its activation was dependent on the presence of the regulator PhoP. The luminescence assay demonstrated that the activity of the virK promoter was markedly elevated in an environment conducive to operon activation, whereas the mig-14 promoter exhibited no discernible change. This suggests that mig-14 is predominantly transcribed as a component of the operon. In the PhoP activation environment, which has a mildly acidic pH, low Mg(2+) levels, and intracellular macrophages, the virK-mig-14 operon exhibited significant activation. The absence of virK or mig-14 resulted in the impaired survival of Salmonella Typhi within macrophages and decreased its tolerance to polymyxin B. Collectively, this study shows that virK and mig-14 constitute an operon whose activation depends on PhoP and that it promotes S. Typhi's survival in macrophages and resistance to polymyxin B.202540345346
6160120.9767Comparative transcriptional profiling of tildipirosin-resistant and sensitive Haemophilus parasuis. Numerous studies have been conducted to examine the molecular mechanism of Haemophilus parasuis resistance to antibiotic, but rarely to tildipirosin. In the current study, transcriptional profiling was applied to analyse the variation in gene expression of JS0135 and tildipirosin-resistant JS32. The growth curves showed that JS32 had a higher growth rate but fewer bacteria than JS0135. The cell membranes of JS32 and a resistant clinical isolate (HB32) were observed to be smoother than those of JS0135. From the comparative gene expression profile 349 up- and 113 downregulated genes were observed, covering 37 GO and 63 KEGG pathways which are involved in biological processes (11), cellular components (17), molecular function (9), cellular processes (1), environmental information processing (4), genetic information processing (9) and metabolism (49) affected in JS32. In addition, the relative overexpression of genes of the metabolism pathway (HAPS_RS09315, HAPS_RS09320), ribosomes (HAPS_RS07815) and ABC transporters (HAPS_RS10945) was detected, particularly the metabolism pathway, and verified with RT-qPCR. Collectively, the gene expression profile in connection with tildipirosin resistance factors revealed unique and highly resistant determinants of H. parasuis to macrolides that warrant further attention due to the significant threat of bacterial resistance.201728790420
6192130.9766Quantitative RT-PCR analysis of multiple genes encoding putative metronidazole nitroreductases from Helicobacter pylori. Metronidazole (Mtz), a pro-drug, requires reductive activation by ferredoxin-like electron carrier proteins to kill bacteria and Mtz resistance is associated with a decrease or deficiency of Mtz nitroreductase activities in a target cell. Several genes encoding ferredoxin-like or -linked proteins such as pyruvate oxidoreductase (POR), ferredoxin oxidoreductase (FOR), ferredoxin (FdxA), ferredoxin-like protein (FdxB), flavodoxin (FldA) and oxygen insensitive nitroreductase (RdxA) have been identified from the complete genomic sequence of Helicobacter pylori. To understand the roles of these genes in H. pylori Mtz resistance, the gene expression for the proteins was examined using a method optimized for quantitative reverse transcription polymerase chain reaction (RT-PCR). The RT-PCR products of FOR and RdxA were significantly decreased in the total RNA prepared from H. pylori cultured in the presence of Mtz as compared to the total RNA prepared from H. pylori cultured without Mtz in the media. A slight decrease, however, in band intensity of the RT-PCR products of the POR and, to a lesser extent, FdxB was obtained in the presence of Mtz. In contrast, the RT-PCR products of the FdxA, FldA, and GalE (UDP-galactose 4-epimerase; a control gene) were unchanged in total RNA prepared from H. pylori cultured with or without Mtz in the culture media. These results suggest that Mtz resistance may also be acquired by decreasing the transcription of some genes involved in Mtz reductive activation, in addition to the mutation in some individual genes such as rdxA.200010856674
8887140.9764RpoE is a Putative Antibiotic Resistance Regulator of Salmonella enteric Serovar Typhi. Bacterial antimicrobial resistance has been associated with the up regulation of genes encoding efflux pumps and the down regulation of genes encoding outer membrane proteins (OMPs). Gene expression in bacteria is primarily initiated by sigma factors (σ factors) such as RpoE, which plays an important role in responding to many environmental stresses. Here, we report the first observation that RpoE serves as an antibiotic resistance regulator in Salmonella enteric serovar Typhi (S. Typhi). In this study, we found that the rpoE mutant (ΔrpoE) of S. Typhi GIFU10007 has elevated resistance to several antimicrobial agents, including β-lactams, quinolones, and aminoglycosides. Genomic DNA microarray analysis was used to investigate the differential gene expression profiles between a wild type and rpoE mutant in response to ampicillin. The results showed that a total of 57 genes displayed differential expression (two-fold increase or decrease) in ΔrpoE versus the wild-type strain. The expressions of two outer membrane protein genes, ompF and ompC, were significantly down-regulated in ΔrpoE (six and seven-fold lower in comparison to wild-type strain) and RamA, a member of the efflux pump AraC/XylS family, was up-regulated about four-fold in the ΔrpoE. Our results suggest RpoE is a potential antimicrobial regulator in S. Typhi, controlling both the down regulation of the OMP genes and up-regulating the efflux system.201626742769
9033150.9764Stress 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
573160.9763Termination factor Rho and its cofactors NusA and NusG silence foreign DNA in E. coli. Transcription of the bacterial genome by the RNA polymerase must terminate at specific points. Transcription can be terminated by Rho factor, an essential protein in enterobacteria. We used the antibiotic bicyclomycin, which inhibits Rho, to assess its role on a genome-wide scale. Rho is revealed as a global regulator of gene expression that matches Escherichia coli transcription to translational needs. We also found that genes in E. coli that are most repressed by Rho are prophages and other horizontally acquired portions of the genome. Elimination of these foreign DNA elements increases resistance to bicyclomycin. Although rho remains essential, such reduced-genome bacteria no longer require Rho cofactors NusA and NusG. Deletion of the cryptic rac prophage in wild-type E. coli increases bicyclomycin resistance and permits deletion of nusG. Thus, Rho termination, supported by NusA and NusG, is required to suppress the toxic activity of foreign genes.200818487194
717170.9762Sodium regulates Escherichia coli acid resistance, and influences GadX- and GadW-dependent activation of gadE. Enteric bacteria must survive the extreme acid of the stomach (pH 2 or less) before entering the intestine where they can colonize and cause disease. Escherichia coli is superior to most other Enterobacteriaceae in surviving pH 2 acid stress because it has four known acid-resistance systems, the most studied of which depends on glutamic acid. Glutamate-dependent acid resistance requires glutamate decarboxylase isozymes GadA and GadB, as well as a glutamate/gamma-aminobutyric acid antiporter encoded by gadC. The regulatory protein GadE is the essential activator of the gadA and gadBC genes. The transcription of gadE, however, is controlled by numerous proteins. Two of these proteins, GadX and GadW, are AraC-family regulators whose sensory input signals are not known. Since Na(+) and K(+) play important roles in pH homeostasis, the contribution of these ions toward the regulation of this acid-resistance system was examined. The results indicated that a decrease in Na(+), but not K(+), concentration coincided with diminished acid resistance, and decreased expression of the gadE, gadA and gadBC genes. However, Na(+)-dependent regulation of these genes dissipated in the absence of GadX and GadW. Since Na(+) levels did not regulate gadX or gadW transcription, it is proposed that GadX and GadW sense intracellular Na(+) concentration or some consequence of altered Na(+) levels.200717768258
6118180.9762Integrated genomics and transcriptomics reveal the extreme heavy metal tolerance and adsorption potentiality of Staphylococcus equorum. In this study, we successfully isolated 11 species of cadmium-tolerant bacterium from Pu-erh rhizosphere soil, of which Staphylococcus equorum PU1 showed the highest cadmium tolerance, with a minimum inhibitory concentration (MIC) value of 500 mg/L. The cadmium removal efficiency of PU1 in 400 mg/L cadmium medium reached 58.7 %. Based on the Nanopore PromethION and Illumina NovaSeq platforms, we successfully obtained the complete PU1 genome with a size of 2,705,540 bp, which encoded 2729 genes. We further detected 82 and 44 indel mutations in the PU1 genome compared with the KS1039 and KM1031 genomes from the database. Transcriptional analysis showed that the expression of 11 genes in PU1 increased with increasing cadmium concentrations (from 0 to 200, then to 400 mg/L), which encoded cadmium resistance, cadmium transport, and mercury resistance genes. In addition, some genes showed differential expression patterns with changes in cadmium concentration, including quinone oxidoreductase-like protein, ferrous iron transport protein, and flavohemoprotein. Gene Ontology (GO) functions, including oxidation reduction process and oxidoreductase activity functions, and KEGG pathways, including glycolysis/gluconeogenesis and biosynthesis of secondary metals, were also considered closely related to the extreme cadmium tolerance of PU1. This study provides novel insight into the cadmium tolerance mechanism of bacteria.202336592848
8446190.9762Genome-wide association study for resistance to Pseudomonas syringae pv. garcae in Coffea arabica. Bacteria halo blight (BHB), a coffee plant disease caused by Pseudomonas syringae pv. garcae, has been gaining importance in producing mountain regions and mild temperatures areas as well as in coffee nurseries. Most Coffea arabica cultivars are susceptible to this disease. In contrast, a great source of genetic diversity and resistance to BHB are found in C. arabica Ethiopian accessions. Aiming to identify quantitative trait nucleotides (QTNs) associated with resistance to BHB and the influence of these genomic regions during the domestication of C. arabica, we conducted an analysis of population structure and a Genome-Wide Association Study (GWAS). For this, we used genotyping by sequencing (GBS) and phenotyping for resistance to BHB of a panel with 120 C. arabica Ethiopian accessions from a historical FAO collection, 11 C. arabica cultivars, and the BA-10 genotype. Population structure analysis based on single-nucleotide polymorphisms (SNPs) markers showed that the 132 accessions are divided into 3 clusters: most wild Ethiopian accessions, domesticated Ethiopian accessions, and cultivars. GWAS, using the single-locus model MLM and the multi-locus models mrMLM, FASTmrMLM, FASTmrEMMA, and ISIS EM-BLASSO, identified 11 QTNs associated with resistance to BHB. Among these QTNs, the four with the highest values of association for resistance to BHB are linked to g000 (Chr_0_434_435) and g010741 genes, which are predicted to encode a serine/threonine-kinase protein and a nucleotide binding site leucine-rich repeat (NBS-LRR), respectively. These genes displayed a similar transcriptional downregulation profile in a C. arabica susceptible cultivar and in a C. arabica cultivar with quantitative resistance, when infected with P. syringae pv. garcae. However, peaks of upregulation were observed in a C. arabica cultivar with qualitative resistance, for both genes. Our results provide SNPs that have potential for application in Marker Assisted Selection (MAS) and expand our understanding about the complex genetic control of the resistance to BHB in C. arabica. In addition, the findings contribute to increasing understanding of the C. arabica domestication history.202236330243