April 21, 2020  |  

Defining transgene insertion sites and off-target effects of homology-based gene silencing informs the use of functional genomics tools in Phytophthora infestans.

DNA transformation and homology-based transcriptional silencing are frequently used to assess gene function in Phytophthora. Since unplanned side-effects of these tools are not well-characterized, we used P. infestans to study plasmid integration sites and whether knockdowns caused by homology-dependent silencing spreads to other genes. Insertions occurred both in gene-dense and gene-sparse regions but disproportionately near the 5′ ends of genes, which disrupted native coding sequences. Microhomology at the recombination site between plasmid and chromosome was common. Studies of transformants silenced for twelve different gene targets indicated that neighbors within 500-nt were often co-silenced, regardless of whether hairpin or sense constructs were employed and the direction of transcription of the target. However, cis-spreading of silencing did not occur in all transformants obtained with the same plasmid. Genome-wide studies indicated that unlinked genes with partial complementarity with the silencing-inducing transgene were not usually down-regulated. We learned that hairpin or sense transgenes were not co-silenced with the target in all transformants, which informs how screens for silencing should be performed. We conclude that transformation and gene silencing can be reliable tools for functional genomics in Phytophthora but must be used carefully, especially by testing for the spread of silencing to genes flanking the target.


April 21, 2020  |  

Advantage of the F2:A1:B- IncF Pandemic Plasmid over IncC Plasmids in In Vitro Acquisition and Evolution of blaCTX-M Gene-Bearing Plasmids in Escherichia coli.

Despite a fitness cost imposed on bacterial hosts, large conjugative plasmids play a key role in the diffusion of resistance determinants, such as CTX-M extended-spectrum ß-lactamases. Among the large conjugative plasmids, IncF plasmids are the most predominant group, and an F2:A1:B- IncF-type plasmid encoding a CTX-M-15 variant was recently described as being strongly associated with the emerging worldwide Escherichia coli sequence type 131 (ST131)-O25b:H4 H30Rx/C2 sublineage. In this context, we investigated the fitness cost of narrow-range F-type plasmids, including the F2:A1:B- IncF-type CTX-M-15 plasmid, and of broad-range C-type plasmids in the K-12-like J53-2 E. coli strain. Although all plasmids imposed a significant fitness cost to the bacterial host immediately after conjugation, we show, using an experimental-evolution approach, that a negative impact on the fitness of the host strain was maintained throughout 1,120 generations with the IncC-IncR plasmid, regardless of the presence or absence of cefotaxime, in contrast to the F2:A1:B- IncF plasmid, whose cost was alleviated. Many chromosomal and plasmid rearrangements were detected after conjugation in transconjugants carrying the IncC plasmids but not in transconjugants carrying the F2:A1:B- IncF plasmid, except for insertion sequence (IS) mobilization from the fliM gene leading to the restoration of motility of the recipient strains. Only a few mutations occurred on the chromosome of each transconjugant throughout the experimental-evolution assay. Our findings indicate that the F2:A1:B- IncF CTX-M-15 plasmid is well adapted to the E. coli strain studied, contrary to the IncC-IncR CTX-M-15 plasmid, and that such plasmid-host adaptation could participate in the evolutionary success of the CTX-M-15-producing pandemic E. coli ST131-O25b:H4 lineage.Copyright © 2019 Mahérault et al.


April 21, 2020  |  

Spreading Patterns of NDM-Producing Enterobacteriaceae in Clinical and Environmental Settings in Yangon, Myanmar.

The spread of carbapenemase-producing Enterobacteriaceae (CPE), contributing to widespread carbapenem resistance, has become a global concern. However, the specific dissemination patterns of carbapenemase genes have not been intensively investigated in developing countries, including Myanmar, where NDM-type carbapenemases are spreading in clinical settings. In the present study, we phenotypically and genetically characterized 91 CPE isolates obtained from clinical (n = 77) and environmental (n = 14) samples in Yangon, Myanmar. We determined the dissemination of plasmids harboring genes encoding NDM-1 and its variants using whole-genome sequencing and plasmid analysis. IncFII plasmids harboring blaNDM-5 and IncX3 plasmids harboring blaNDM-4 or blaNDM-7 were the most prevalent plasmid types identified among the isolates. The IncFII plasmids were predominantly carried by clinical isolates of Escherichia coli, and their clonal expansion was observed within the same ward of a hospital. In contrast, the IncX3 plasmids were found in phylogenetically divergent isolates from clinical and environmental samples classified into nine species, suggesting widespread dissemination of plasmids via horizontal transfer. Half of the environmental isolates were found to possess IncX3 plasmids, and this type of plasmid was confirmed to transfer more effectively to recipient organisms at a relatively low temperature (25°C) compared to the IncFII plasmid. Moreover, various other plasmid types were identified harboring blaNDM-1, including IncFIB, IncFII, IncL/M, and IncA/C2, among clinical isolates of Klebsiella pneumoniae or Enterobacter cloacae complex. Overall, our results highlight three distinct patterns of the dissemination of blaNDM-harboring plasmids among CPE isolates in Myanmar, contributing to a better understanding of their molecular epidemiology and dissemination in a setting of endemicity.Copyright © 2019 American Society for Microbiology.


April 21, 2020  |  

Complete genome sequence of the novel agarolytic Catenovulum-like strain CCB-QB4

Members of the genus Catenovulum are recognized for their ability to degrade algal biomass. Here we report the complete genome of Cantenovulum–like strain CCB-QB4, an agarolytic bacterium isolated from the coastal area of Penang, Malaysia. The sequenced genome is composed of a 5,663,044?bp circular chromosome and a 208,085?bp circular plasmid. It contained 4409 protein coding and 83 RNA genes, including 62 tRNAs and 21 rRNAs. The genome of CCB-QB4 contains many agarases, which correlate with the high capacity of the strain to degrade agar. Genome sequencing of CCB-QB4 reveals gene candidates of potential interest in enzymatic industries or applications in the field of polysaccharides degradation.


April 21, 2020  |  

Complete genome sequence of Pseudomonas frederiksbergensis ERDD5:01 revealed genetic bases for survivability at high altitude ecosystem and bioprospection potential.

Pseudomonas frederiksbergensis ERDD5:01 is a psychrotrophic bacteria isolated from the glacial stream flowing from East Rathong glacier in Sikkim Himalaya. The strain showed survivability at high altitude stress conditions like freezing, frequent freeze-thaw cycles, and UV-C radiations. The complete genome of 5,746,824?bp circular chromosome and a plasmid of 371,027?bp was sequenced to understand the genetic basis of its survival strategy. Multiple copies of cold-associated genes encoding cold active chaperons, general stress response, osmotic stress, oxidative stress, membrane/cell wall alteration, carbon storage/starvation and, DNA repair mechanisms supported its survivability at extreme cold and radiations corroborating with the bacterial physiological findings. The molecular cold adaptation analysis in comparison with the genome of 15 mesophilic Pseudomonas species revealed functional insight into the strategies of cold adaptation. The genomic data also revealed the presence of industrially important enzymes.Copyright © 2018 Elsevier Inc. All rights reserved.


April 21, 2020  |  

Genome-Scale Sequence Disruption Following Biolistic Transformation in Rice and Maize.

Biolistic transformation delivers nucleic acids into plant cells by bombarding the cells with microprojectiles, which are micron-scale, typically gold particles. Despite the wide use of this technique, little is known about its effect on the cell’s genome. We biolistically transformed linear 48-kb phage lambda and two different circular plasmids into rice (Oryza sativa) and maize (Zea mays) and analyzed the results by whole genome sequencing and optical mapping. Although some transgenic events showed simple insertions, others showed extreme genome damage in the form of chromosome truncations, large deletions, partial trisomy, and evidence of chromothripsis and breakage-fusion bridge cycling. Several transgenic events contained megabase-scale arrays of introduced DNA mixed with genomic fragments assembled by nonhomologous or microhomology-mediated joining. Damaged regions of the genome, assayed by the presence of small fragments displaced elsewhere, were often repaired without a trace, presumably by homology-dependent repair (HDR). The results suggest a model whereby successful biolistic transformation relies on a combination of end joining to insert foreign DNA and HDR to repair collateral damage caused by the microprojectiles. The differing levels of genome damage observed among transgenic events may reflect the stage of the cell cycle and the availability of templates for HDR. © 2019 American Society of Plant Biologists. All rights reserved.


April 21, 2020  |  

Characterization of NDM-5- and CTX-M-55-coproducing Escherichia coli GSH8M-2 isolated from the effluent of a wastewater treatment plant in Tokyo Bay.

New Delhi metallo-ß-lactamase (NDM)-5-producing Enterobacteriaceae have been detected in rivers, sewage, and effluents from wastewater treatment plants (WWTPs). Environmental contamination due to discharged effluents is of particular concern as NDM variants may be released into waterways, thereby posing a risk to humans. In this study, we collected effluent samples from a WWTP discharged into a canal in Tokyo Bay, Japan.Testing included the complete genome sequencing of Escherichia coli GSH8M-2 isolated from the effluent as well as a gene network analysis.The complete genome sequencing of GSH8M-2 revealed that it was an NDM-5-producing E. coli strain sequence type ST542, which carries multiple antimicrobial resistance genes for ß-lactams, quinolone, tetracycline, trimethoprim-sulfamethoxazole, florfenicol/chloramphenicol, kanamycin, and fosfomycin. The blaNDM-5 gene was found in the IncX3 replicon plasmid pGSH8M-2-4. Gene network analysis using 142 IncX3 plasmid sequences suggested that pGSH8M-2-4 is related to both clinical isolates of  E. coli and Klebsiella species in Eastern Asia. GSH8M-2 also carries the blaCTX-M-55 gene in IncX1 plasmid pGSH8M-2-3.This is the first report of environmental NDM-5-producing E. coli isolated from a WWTP in Japan. NDM-5 detection is markedly increasing in veterinary and clinical settings, suggesting that dual ß-lactamases, such as NDM-5 and CTX-M-55, might be acquired through multiple steps in environment settings. Environmental contamination through WWTP effluents that contain producers of NDM variants could be an emerging potential health hazard. Thus, regular monitoring of WWTP effluents is important for the detection of antimicrobial-resistant bacteria that may be released into the waterways and nearby communities.


April 21, 2020  |  

Characterization of vanM carrying clinical Enterococcus isolates and diversity of the suppressed vanM gene cluster.

Here we report the prevalence of the suppressed vanM gene cluster as a reservoir of vancomycin resistance genes. Among 1284 clinical isolates of enterococci from four hospitals in Hangzhou, China, 55 isolates of Enterococcus faecium and one isolate of Enterococcus faecalis were screened positive for the vanM genotype. Antimicrobial susceptibility testing showed that 55 of the 56 vanM-positive isolates were susceptible to vancomycin and teicoplanin. Most of them (54/56) belonged to the main epidemic lineage CC17, mostly the ST78 type. The vanM gene clusters in the 55 vancomycin-susceptible isolates showed sequence diversity owing to different insertion locations of IS1216E. The vanM transposons could be classified into five types and they all carried two or more IS1216E elements, leading to complete or partial deletions of vanR, vanS, or vanX. Quantitative reverse transcription polymerase chain reaction showed that the expression level of vanM was significantly lower in the vancomycin-susceptible isolates than in the vancomycin-resistant isolate. Considering the prevalence of the vanM genotype and the potential for conversion to a resistant phenotype, vanM might act as an important determinant of glycopeptide resistance in the future. It is essential to strengthen the surveillance of vanM-containing enterococci to control the dissemination of vancomycin resistance. Copyright © 2018. Published by Elsevier B.V.


April 21, 2020  |  

Complete Assembly of the Genome of an Acidovorax citrulli Strain Reveals a Naturally Occurring Plasmid in This Species.

Acidovorax citrulli is the causal agent of bacterial fruit blotch (BFB), a serious threat to cucurbit crop production worldwide. Based on genetic and phenotypic properties, A. citrulli strains are divided into two major groups: group I strains have been generally isolated from melon and other non-watermelon cucurbits, while group II strains are closely associated with watermelon. In a previous study, we reported the genome of the group I model strain, M6. At that time, the M6 genome was sequenced by MiSeq Illumina technology, with reads assembled into 139 contigs. Here, we report the assembly of the M6 genome following sequencing with PacBio technology. This approach not only allowed full assembly of the M6 genome, but it also revealed the occurrence of a ~53 kb plasmid. The M6 plasmid, named pACM6, was further confirmed by plasmid extraction, Southern-blot analysis of restricted fragments and obtention of M6-derivative cured strains. pACM6 occurs at low copy numbers (average of ~4.1 ± 1.3 chromosome equivalents) in A. citrulli M6 and contains 63 open reading frames (ORFs), most of which (55.6%) encoding hypothetical proteins. The plasmid contains several genes encoding type IV secretion components, and typical plasmid-borne genes involved in plasmid maintenance, replication and transfer. The plasmid also carries an operon encoding homologs of a Fic-VbhA toxin-antitoxin (TA) module. Transcriptome data from A. citrulli M6 revealed that, under the tested conditions, the genes encoding the components of this TA system are among the highest expressed genes in pACM6. Whether this TA module plays a role in pACM6 maintenance is still to be determined. Leaf infiltration and seed transmission assays revealed that, under tested conditions, the loss of pACM6 did not affect the virulence of A. citrulli M6. We also show that pACM6 or similar plasmids are present in several group I strains, but absent in all tested group II strains of A. citrulli.


April 21, 2020  |  

Assignment of virus and antimicrobial resistance genes to microbial hosts in a complex microbial community by combined long-read assembly and proximity ligation.

We describe a method that adds long-read sequencing to a mix of technologies used to assemble a highly complex cattle rumen microbial community, and provide a comparison to short read-based methods. Long-read alignments and Hi-C linkage between contigs support the identification of 188 novel virus-host associations and the determination of phage life cycle states in the rumen microbial community. The long-read assembly also identifies 94 antimicrobial resistance genes, compared to only seven alleles in the short-read assembly. We demonstrate novel techniques that work synergistically to improve characterization of biological features in a highly complex rumen microbial community.


April 21, 2020  |  

Characterization of an NDM-5 carbapenemase-producing Escherichia coli ST156 isolate from a poultry farm in Zhejiang, China.

The emergence of carbapenem-resistant Enterobacteriaceae strains has posed a severe threat to public health in recent years. The mobile elements carrying the New Delhi metallo-ß-lactqtamase (NDM) gene have been regarded as the major mechanism leading to the rapid increase of carbapenem-resistant Enterobacteriaceae strains isolated from clinics and animals.We describe an NDM-5-producing Escherichia coli strain, ECCRA-119 (sequence type 156 [ST156]), isolated from a poultry farm in Zhejiang, China. ECCRA-119 is a multidrug-resistant (MDR) isolate that exhibited resistance to 27 antimicrobial compounds, including imipenem and meropenem, as detected by antimicrobial susceptibility testing (AST). The complete genome sequence of the ECCRA-119 isolate was also obtained using the PacBio RS II platform. Eleven acquired resistance genes were identified in the chromosome; four were detected in plasmid pTB201, while six were detected in plasmid pTB202. Importantly, the carbapenem-resistant gene blaNDM-5 was detected in the IncX3 plasmid pTB203. In addition, seven virulence genes and one metal-resistance gene were also detected. The results of conjugation experiments and the transfer regions identification indicated that the blaNDM-5-harboring plasmid pTB203 could be transferred between E. coli strains.The results reflected the severe bacterial resistance in a poultry farm in Zhejiang province and increased our understanding of the presence and transmission of the blaNDM-5 gene.


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