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September 22, 2019

Identification of the DNA methyltransferases establishing the methylome of the cyanobacterium Synechocystis sp. PCC 6803.

DNA methylation in bacteria is important for defense against foreign DNA, but is also involved in DNA repair, replication, chromosome partitioning, and regulatory processes. Thus, characterization of the underlying DNA methyltransferases in genetically tractable bacteria is of paramount importance. Here, we characterized the methylome and orphan methyltransferases in the model cyanobacterium Synechocystis sp. PCC 6803. Single molecule real-time (SMRT) sequencing revealed four DNA methylation recognition sequences in addition to the previously known motif m5CGATCG, which is recognized by M.Ssp6803I. For three of the new recognition sequences, we identified the responsible methyltransferases. M.Ssp6803II, encoded by the sll0729 gene, modifies GGm4CC, M.Ssp6803III, encoded by slr1803, represents the cyanobacterial dam-like methyltransferase modifying Gm6ATC, and M.Ssp6803V, encoded by slr6095 on plasmid pSYSX, transfers methyl groups to the bipartite motif GGm6AN7TTGG/CCAm6AN7TCC. The remaining methylation recognition sequence GAm6AGGC is probably recognized by methyltransferase M.Ssp6803IV encoded by slr6050. M.Ssp6803III and M.Ssp6803IV were essential for the viability of Synechocystis, while the strains lacking M.Ssp6803I and M.Ssp6803V showed growth similar to the wild type. In contrast, growth was strongly diminished of the ?sll0729 mutant lacking M.Ssp6803II. These data provide the basis for systematic studies on the molecular mechanisms impacted by these methyltransferases.

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September 22, 2019

Sequencing of pT5282-CTXM, p13190-KPC and p30860-NR, and comparative genomics analysis of IncX8 plasmids.

This study proposes a replicon-based scheme for typing IncX plasmids into nine separately clustering subgroups, including IncX1a, IncX1ß and IncX2-8. The complete nucleotide sequences of three IncX8 plasmids, namely pT5282-CTXM and p30860-NR from Enterobacter cloacae and p13190-KPC from Klebsiella pneumoniae, were determined and were compared with two other previously sequenced IncX8 plasmids (pCAV1043-58 and pCAV1741-16). These five plasmids possessed conserved IncX8 backbones with limited genetic variation with respect to gene content and organisation, and each of them carried one or three accessory modules that harboured resistance markers and metabolic gene clusters as well as transposons, insertion sequence (IS)-based transposition units and miniature inverted repeat transposable elements (MITEs), indicating that the relatively small IncX8 backbones were able to integrate various foreign genetic contents. The resistance genes blaCTX-M-3 and blaTEM-1 (ß-lactam resistance), blaKPC-2 (carbapenem resistance) and ?blaTEM-1, and tet(A) (tetracycline resistance) and mph(E) (macrolide resistance) were found in pT5282-CTXM, p13190-KPC and pCAV1741-16, respectively, whilst p30860-NR and pCAV1043-58 carried no resistance genes. The data presented here provide an insight into the diversification and evolution history of IncX8 plasmids. Copyright © 2018 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

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September 22, 2019

Identification and characterization of conjugative plasmids that encode ciprofloxacin resistance in Salmonella.

This study aimed to characterize novel conjugative plasmids that encode transferrable ciprofloxacin resistance in Salmonella In this study, 157 non-duplicated Salmonella isolates were recovered from food products, 55 out of which were found to be resistant to ciprofloxacin. Interestingly, 37 out of the 55 (67%) CipRSalmonella isolates did not harbor any mutations in the Quinolone resistance determine regions (QRDR). Interestingly, six Salmonella isolates were shown to carry two novel types of conjugative plasmids that could transfer ciprofloxacin resistance phenotype to E. coli J53 (AziR). The first type belonged to the ~110kb IncFIB type conjugative plasmid carrying qnrB-bearing and aac(6′)-Ib-cr-bearing mobile elements. Transfer of the plasmid between E. coli or Salmonella could confer CIP MIC to 1 to 2µg/ml. The second type of conjugative plasmid belonged to ~240kb IncH1/IncF plasmids carrying a single PMQR gene, qnrS Importantly, this type of conjugative ciprofloxacin resistance plasmids could be detected in clinical isolates of Salmonella Dissemination of these conjugative plasmids that confer ciprofloxaicn resistance poses serious public health impact and Salmonella infection control. Copyright © 2018 American Society for Microbiology.

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September 22, 2019

The plant growth-promoting rhizobacterium Variovorax boronicumulans CGMCC 4969 regulates the level of indole-3-acetic acid synthesized from indole-3-acetonitrile.

Variovorax is a metabolically diverse genus of plant growth-promoting rhizobacteria (PGPR) that engages in mutually beneficial interactions between plants and microbes. Unlike most PGPR, Variovorax cannot synthesize the phytohormone indole-3-acetic acid (IAA) via tryptophan. However, we found that V. boronicumulans strain CGMCC 4969 could produce IAA using indole-3-acetonitrile (IAN) as the precursor. Thus, in the present study, the IAA synthesis mechanism of V. boronicumulans CGMCC 4969 was investigated. V. boronicumulans CGMCC 4969 metabolized IAN to IAA through both a nitrilase-dependent pathway and a nitrile hydratase (NHase) and amidase-dependent pathway. Cobalt enhanced the metabolic flux via the NHase/amidase, by which IAN was rapidly converted to indole-3-acetamide (IAM) and in turn to IAA. IAN stimulated the metabolic flux via the nitrilase, by which IAN was rapidly converted to IAA. Subsequently, the IAA was degraded. V. boronicumulans CGMCC 4969 could use IAN as the sole carbon and nitrogen source for growth. Genome sequencing confirmed the IAA synthesis pathways. Gene cloning and overexpression in Escherichia coli indicated that NitA has the nitrilase activity, and IamA has the amidase activity to respectively transform IAN and IAM to IAA. Interestingly, NitA showed a close genetic relationship with the nitrilase of the phytopathogen Pseudomonas syringae Quantitative PCR analysis indicated that the NHase/amidase system is constitutively expressed, whereas the nitrilase is inducible. The present study helps our understanding of the versatile functions of Variovorax nitrile-converting enzymes that mediate IAA synthesis and the interactions between plants and these bacteria.IMPORTANCE We demonstrated that Variovorax boronicumulans CGMCC 4969 has two enzymatic systems-nitrilase and nitrile hydratase/amidase-that convert indole-3-acetonitrile (IAN) to the important plant hormone indole-3-acetic acid (IAA). The two IAA synthesis systems have very different regulatory mechanisms, affecting the IAA synthesis rate and duration. The nitrilase was induced by IAN, which was rapidly converted to IAA; subsequently IAA was rapidly consumed for cell growth. The NHase and amidase system was constitutively expressed and slowly but continuously synthesized IAA. In addition to synthesizing IAA from IAN, CGMCC 4969 has a rapid IAA degradation system, which would be helpful for a host plant to eliminate redundant IAA. This study indicates that the plant growth-promoting rhizobacterium V. boronicumulans CGMCC 4969 has the potential to be used by host plants to regulate the IAA level. Copyright © 2018 American Society for Microbiology.

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September 22, 2019

Emergence of an XDR and carbapenemase-producing hypervirulent Klebsiella pneumoniae strain in Taiwan.

Carbapenemase-producing Klebsiella pneumoniae causes high mortality owing to the limited therapeutic options available. Here, we investigated an emergent carbapenem-resistant K. pneumoniae strain with hypervirulence found among KPC-2-producing strains in Taiwan.KPC-producing K. pneumoniae strains were collected consecutively from clinical specimens at the Taipei Veterans General Hospital between January 2012 and December 2014. Capsular types and the presence of rmpA/rmpA2 were analysed, and PFGE and MLST performed using these strains. The strain positive for rmpA/rmpA2 was tested in an in vivo mouse lethality study to verify its virulence and subjected to WGS to delineate its genomic features.A total of 62 KPC-2-producing K. pneumoniae strains were identified; all of these belonged to ST11 and capsular genotype K47. One strain isolated from a fatal case with intra-abdominal abscess (TVGHCRE225) harboured rmpA and rmpA2 genes. This strain was resistant to tigecycline and colistin, in addition to carbapenems, and did not belong to the major cluster in PFGE. TVGHCRE225 exhibited high in vivo virulence in the mouse lethality experiment. WGS showed that TVGHCRE225 acquired a novel hybrid virulence plasmid harbouring a set of virulence genes (iroBCDN, iucABCD, rmpA and rmpA2, and iutA) compared with the classic ST11 KPC-2-producing strain.We identified an XDR ST11 KPC-2-producing K. pneumoniae strain carrying a hybrid virulent plasmid in Taiwan. Active surveillance focusing on carbapenem-resistant hypervirulent K. pneumoniae strains is necessary, as the threat to human health is imminent.

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September 22, 2019

Emergence of XDR Escherichia coli carrying both blaNDM and mcr-1 genes in chickens at slaughter and the characterization of two novel blaNDM-bearing plasmids.

The emergence and spread of carbapenem-resistant isolates, especially New Delhi MBL (NDM)-producing Enterobacteriaceae, has become a global concern. Although NDM-producing Enterobacteriaceae have been mostly observed in clinical cases, they have also been identified in food-producing animals and wildlife. Recently, XDR bacteria harbouring both blaNDMand mcr-1 genes were observed in isolates from animals, posing a potential threat to public health. However, reports on the coexistence of blaNDMand mcr-1 in bacteria isolated from animals at slaughter remains sporadic. Here, we report two Escherichia coli strains, SD133 and SD138, co-producing NDM and MCR-1, isolated from chickens at slaughter in July 2015 in China.

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September 22, 2019

Transcriptome analysis of Neisseria gonorrhoeae during natural infection reveals differential expression of antibiotic resistance determinants between men and women.

Neisseria gonorrhoeae is a bacterial pathogen responsible for the sexually transmitted infection gonorrhea. Emergence of antimicrobial resistance (AMR) of N. gonorrhoeae worldwide has resulted in limited therapeutic choices for this infection. Men who seek treatment often have symptomatic urethritis; in contrast, gonococcal cervicitis in women is usually minimally symptomatic, but may progress to pelvic inflammatory disease. Previously, we reported the first analysis of gonococcal transcriptome expression determined in secretions from women with cervical infection. Here, we defined gonococcal global transcriptional responses in urethral specimens from men with symptomatic urethritis and compared these with transcriptional responses in specimens obtained from women with cervical infections and in vitro-grown N. gonorrhoeae isolates. This is the first comprehensive comparison of gonococcal gene expression in infected men and women. RNA sequencing analysis revealed that 9.4% of gonococcal genes showed increased expression exclusively in men and included genes involved in host immune cell interactions, while 4.3% showed increased expression exclusively in women and included phage-associated genes. Infected men and women displayed comparable antibiotic-resistant genotypes and in vitro phenotypes, but a 4-fold higher expression of the Mtr efflux pump-related genes was observed in men. These results suggest that expression of AMR genes is programed genotypically and also driven by sex-specific environments. Collectively, our results indicate that distinct N. gonorrhoeae gene expression signatures are detected during genital infection in men and women. We propose that therapeutic strategies could target sex-specific differences in expression of antibiotic resistance genes.IMPORTANCE Recent emergence of antimicrobial resistance of Neisseria gonorrhoeae worldwide has resulted in limited therapeutic choices for treatment of infections caused by this organism. We performed global transcriptomic analysis of N. gonorrhoeae in subjects with gonorrhea who attended a Nanjing, China, sexually transmitted infection (STI) clinic, where antimicrobial resistance of N. gonorrhoeae is high and increasing. We found that N. gonorrhoeae transcriptional responses to infection differed in genital specimens taken from men and women, particularly antibiotic resistance gene expression, which was increased in men. These sex-specific findings may provide a new approach to guide therapeutic interventions and preventive measures that are also sex specific while providing additional insight to address antimicrobial resistance of N. gonorrhoeae. Copyright © 2018 Nudel et al.

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September 22, 2019

Chromosomally encoded mcr-5 in colistin non-susceptible Pseudomonas aeruginosa.

Whole genome sequencing (WGS) of historical Pseudomonas aeruginosa clinical isolates identified a chromosomal copy of mcr-5 within a Tn3-like transposon in P. aeruginosa MRSN 12280. The isolate was non-susceptible to colistin by broth microdilution and genome analysis revealed no mutations known to confer colistin resistance. To the best of our knowledge, this is the first report of mcr in colistin non-susceptible P. aeruginosa.

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September 22, 2019

Analysis of the Gli-D2 locus identifies a genetic target for simultaneously improving the breadmaking and health-related traits of common wheat.

Gliadins are a major component of wheat seed proteins. However, the complex homoeologous Gli-2 loci (Gli-A2, -B2 and -D2) that encode the a-gliadins in commercial wheat are still poorly understood. Here we analyzed the Gli-D2 locus of Xiaoyan 81 (Xy81), a winter wheat cultivar. A total of 421.091 kb of the Gli-D2 sequence was assembled from sequencing multiple bacterial artificial clones, and 10 a-gliadin genes were annotated. Comparative genomic analysis showed that Xy81 carried only eight of the a-gliadin genes of the D genome donor Aegilops tauschii, with two of them each experiencing a tandem duplication. A mutant line lacking Gli-D2 (DLGliD2) consistently exhibited better breadmaking quality and dough functionalities than its progenitor Xy81, but without penalties in other agronomic traits. It also had an elevated lysine content in the grains. Transcriptome analysis verified the lack of Gli-D2 a-gliadin gene expression in DLGliD2. Furthermore, the transcript and protein levels of protein disulfide isomerase were both upregulated in DLGliD2 grains. Consistent with this finding, DLGliD2 had increased disulfide content in the flour. Our work sheds light on the structure and function of Gli-D2 in commercial wheat, and suggests that the removal of Gli-D2 and the gliadins specified by it is likely to be useful for simultaneously enhancing the end-use and health-related traits of common wheat. Because gliadins and homologous proteins are widely present in grass species, the strategy and information reported here may be broadly useful for improving the quality traits of diverse cereal crops.© 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.

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September 22, 2019

Characterization of LE3 and LE4, the only lytic phages known to infect the spirochete Leptospira.

Leptospira is a phylogenetically unique group of bacteria, and includes the causative agents of leptospirosis, the most globally prevalent zoonosis. Bacteriophages in Leptospira are largely unexplored. To date, a genomic sequence is available for only one temperate leptophage called LE1. Here, we sequenced and analysed the first genomes of the lytic phages LE3 and LE4 that can infect the saprophyte Leptospira biflexa using the lipopolysaccharide O-antigen as receptor. Bioinformatics analysis showed that the 48-kb LE3 and LE4 genomes are similar and contain 62% genes whose function cannot be predicted. Mass spectrometry led to the identification of 21 and 23 phage proteins in LE3 and LE4, respectively. However we did not identify significant similarities with other phage genomes. A search for prophages close to LE4 in the Leptospira genomes allowed for the identification of a related plasmid in L. interrogans and a prophage-like region in the draft genome of a clinical isolate of L. mayottensis. Long-read whole genome sequencing of the L. mayottensis revealed that the genome contained a LE4 phage-like circular plasmid. Further isolation and genomic comparison of leptophages should reveal their role in the genetic evolution of Leptospira.

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September 22, 2019

Citrobacter freundii fitness during bloodstream infection.

Sepsis resulting from microbial colonization of the bloodstream is a serious health concern associated with high mortality rates. The objective of this study was to define the physiologic requirements of Citrobacter freundii in the bloodstream as a model for bacteremia caused by opportunistic Gram-negative pathogens. A genetic screen in a murine host identified 177 genes that contributed significantly to fitness, the majority of which were broadly classified as having metabolic or cellular maintenance functions. Among the pathways examined, the Tat protein secretion system conferred the single largest fitness contribution during competition infections and a putative Tat-secreted protein, SufI, was also identified as a fitness factor. Additional work was focused on identifying relevant metabolic pathways for bacteria in the bloodstream environment. Mutations that eliminated the use of glucose or mannitol as carbon sources in vitro resulted in loss of fitness in the murine model and similar results were obtained upon disruption of the cysteine biosynthetic pathway. Finally, the conservation of identified fitness factors was compared within a cohort of Citrobacter bloodstream isolates and between Citrobacter and Serratia marcescens, the results of which suggest the presence of conserved strategies for bacterial survival and replication in the bloodstream environment.

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September 22, 2019

Whole genome sequencing, de novo assembly and phenotypic profiling for the new budding yeast species Saccharomyces jurei.

Saccharomyces sensu stricto complex consist of yeast species, which are not only important in the fermentation industry but are also model systems for genomic and ecological analysis. Here, we present the complete genome assemblies of Saccharomyces jurei, a newly discovered Saccharomyces sensu stricto species from high altitude oaks. Phylogenetic and phenotypic analysis revealed that S. jurei is more closely related to S. mikatae, than S. cerevisiae, and S. paradoxus The karyotype of S. jurei presents two reciprocal chromosomal translocations between chromosome VI/VII and I/XIII when compared to the S. cerevisiae genome. Interestingly, while the rearrangement I/XIII is unique to S. jurei, the other is in common with S. mikatae strain IFO1815, suggesting shared evolutionary history of this species after the split between S. cerevisiae and S. mikatae The number of Ty elements differed in the new species, with a higher number of Ty elements present in S. jurei than in S. cerevisiae Phenotypically, the S. jurei strain NCYC 3962 has relatively higher fitness than the other strain NCYC 3947T under most of the environmental stress conditions tested and showed remarkably increased fitness in higher concentration of acetic acid compared to the other sensu stricto species. Both strains were found to be better adapted to lower temperatures compared to S. cerevisiae. Copyright © 2018 Naseeb et al.

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September 22, 2019

Conservation genomics of the declining North American bumblebee Bombus terricola reveals inbreeding and selection on immune genes.

The yellow-banded bumblebee Bombus terricola was common in North America but has recently declined and is now on the IUCN Red List of threatened species. The causes of B. terricola’s decline are not well understood. Our objectives were to create a partial genome and then use this to estimate population data of conservation interest, and to determine whether genes showing signs of recent selection suggest a specific cause of decline. First, we generated a draft partial genome (contig set) for B. terricola, sequenced using Pacific Biosciences RS II at an average depth of 35×. Second, we sequenced the individual genomes of 22 bumblebee gynes from Ontario and Quebec using Illumina HiSeq 2500, each at an average depth of 20×, which were used to improve the PacBio genome calls and for population genetic analyses. The latter revealed that several samples had long runs of homozygosity, and individuals had high inbreeding coefficient F, consistent with low effective population size. Our data suggest that B. terricola’s effective population size has decreased orders of magnitude from pre-Holocene levels. We carried out tests of selection to identify genes that may have played a role in ameliorating environmental stressors underlying B. terricola’s decline. Several immune-related genes have signatures of recent positive selection, which is consistent with the pathogen-spillover hypothesis for B. terricola’s decline. The new B. terricola contig set can help solve the mystery of bumblebee decline by enabling functional genomics research to directly assess the health of pollinators and identify the stressors causing declines.

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September 22, 2019

Novel enterobacter lineage as leading cause of nosocomial outbreak involving carbapenemase-producing strains.

We investigated unusual carbapenemase-producing Enterobacter cloacae complex isolates (n = 8) in the novel sequence type (ST) 873, which caused nosocomial infections in 2 hospitals in France. Whole-genome sequence typing showed the 1-year persistence of the epidemic strain, which harbored a blaVIM-4 ST1-IncHI2 plasmid, in 1 health institution and 2 closely related strains harboring blaCTX-M-15 in the other. These isolates formed a new subgroup in the E. hormaechei metacluster, according to their hsp60 sequences and phylogenomic analysis. The average nucleotide identities, specific biochemical properties, and pangenomic and functional investigations of isolates suggested isolates of a novel species that had acquired genes associated with adhesion and mobility. The emergence of this novel Enterobacter phylogenetic lineage within hospitals should be closely monitored because of its ability to persist and spread.

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September 22, 2019

Identification of natural product compounds as quorum sensing inhibitors in Pseudomonas fluorescens P07 through virtual screening.

Pseudomonas fluorescens, a Gram-negative psychrotrophic bacteria, is the main microorganism causing spoilage of chilled raw milk and aquatic products. Quorum sensing (QS) widely exists in bacteria to monitor their population densities and regulate numerous physiological activities, such as the secretion of siderophores, swarming motility and biofilm formation. Thus, searching for quorum sensing inhibitors (QSIs) may be another promising way to control the deterioration of food caused by P. fluorescens. Here, we screened a traditional Chinese medicine (TCM) database to discover potential QSIs with lesser toxicity. The gene sequences of LuxI- and LuxR-type proteins of P. fluorescens P07 were obtained through whole-genome sequencing. In addition, the protein structures built by homology modelling were used as targets to screen for QSIs. Twenty-one compounds with a dock score greater than 6 were purchased and tested by biosensor strains (Chromobacterium violaceum CV026 and Agrobacterium tumefaciens A136). The results showed that 10 of the compounds were determined as hits (hit rate: 66.67%). Benzyl alcohol, rhodinyl formate and houttuynine were effective QSIs. The impact of the most active compound (benzyl alcohol) on the phenotypes of P. fluorescens P07, including swimming and swarming motility, production of extracellular enzymes and siderophores, N-acylhomoserine lactone (AHLs) content and biofilm formation were determined. The inhibitory mechanism of benzyl alcohol on the QS system of P. fluorescens P07 is further discussed. This study reveals the feasibility of searching for novel QSIs through virtual screening. Copyright © 2018 Elsevier Ltd. All rights reserved.

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