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September 22, 2019

Genome sequence and metabolic analysis of a fluoranthene-degrading strain Pseudomonas aeruginosa DN1.

Pseudomonas aeruginosa DN1, isolated from petroleum-contaminated soil, showed excellent degradation ability toward diverse polycyclic aromatic hydrocarbons (PAHs). Many studies have been done to improve its degradation ability. However, the molecular mechanisms of PAHs degradation in DN1 strain are unclear. In this study, the whole genome of DN1 strain was sequenced and analyzed. Its genome contains 6,641,902 bp and encodes 6,684 putative open reading frames (ORFs), which has the largest genome in almost all the comparative Pseudomonas strains. Results of gene annotation showed that this strain harbored over 100 candidate genes involved in PAHs degradation, including those encoding 25 dioxygenases, four ring-hydroxylating dioxygenases, five ring-cleaving dioxygenases, and various catabolic enzymes, transcriptional regulators, and transporters in the degradation pathways. In addition, gene knockout experiments revealed that the disruption of some key PAHs degradation genes in DN1 strain, such as catA, pcaG, pcaH, and rhdA, did not completely inhibit fluoranthene degradation, even though their degradative rate reduced to some extent. Three intermediate metabolites, including 9-hydroxyfluorene, 1-acenaphthenone, and 1, 8-naphthalic anhydride, were identified as the dominating intermediates in presence of 50 µg/mL fluoranthene as the sole carbon source according to gas chromatography mass spectrometry analysis. Taken together, the genomic and metabolic analysis indicated that the fluoranthene degradation by DN1 strain was initiated by dioxygenation at the C-1, 2-, C-2, 3-, and C-7, 8- positions. These results provide new insights into the genomic plasticity and environmental adaptation of DN1 strain.


September 22, 2019

Eco-friendly Management of Karnal Bunt (Neovossia indica) of Wheat

Karnal bunt incited by Neovossia indica is one of the most important disease of wheat crop. To develop an eco-friendly management practice against Karnal bunt of wheat, integration of fungicidal seed treatment with foliar sprays of phytoextracts, bio-control agent and fungicide revealed. Uses of Thiram 75DS or Kavach 75WP @2g/Kg, Dithane M-45 or Captan [email protected]/Kg, Vitavax [email protected]/Kg, Tilt 25EC or Raxil 2DS@1mL/Kg or Pseudomonas fluorescens@5 mL/Kg or Trichoderma viride (Ecoderma) or T. harzianum@5 mL/Kg seed treatment for eliminating primary inoculum (teliospores). Seed soaking in Lantana (L. camara) or Eucalyptus (E. globulus) or Akh (Calotropis procera) or Kali basuti (Eupatorium adenophorum) @ 250 mL/L for 60 min and dry in shad are effective in eradicating the seed infection also. Application foliar spray of Baycor 25WP or Bavistin 50WP or F-100 or Moximate [email protected]/Kg, Tilt 25EC or Folicur 25EC or Contaf 25EC@1mL/Kg at boot leaf stage and 50% emergence flowering heads against the secondary air-borne inoculum (Allantoides sporidia). This is concerning integration of fungicide seed treatment with foliar spray of bio- control agent and phyto-extract. It is cheaper and eco-friendly practice for the control of Karnal bunt of wheat.


September 22, 2019

Antimicrobial resistance profile of mcr-1 positive clinical isolates of Escherichia coli in China From 2013 to 2016.

Multidrug-resistant (MDR) Escherichia coli poses a great challenge for public health in recent decades. Polymyxins have been reconsidered as a valuable therapeutic option for the treatment of infections caused by MDR E. coli. A plasmid-encoded colistin resistance gene mcr-1 encoding phosphoethanolamine transferase has been recently described in Enterobacteriaceae. In this study, a total of 123 E. coli isolates obtained from patients with diarrheal diseases in China were used for the genetic analysis of colistin resistance in clinical isolates. Antimicrobial resistance profile of polymyxin B (PB) and 11 commonly used antimicrobial agents were determined. Among the 123 E. coli isolates, 9 isolates (7.3%) were resistant to PB and PCR screening showed that seven (5.7%) isolates carried the mcr-1 gene. A hybrid sequencing analysis using single-molecule, real-time (SMRT) sequencing and Illumina sequencing was then performed to resolve the genomes of the seven mcr-1 positive isolates. These seven isolates harbored multiple plasmids and are MDR, with six isolates carrying one mcr-1 positive plasmid and one isolate (14EC033) carrying two mcr-1 positive plasmids. These eight mcr-1 positive plasmids belonged to the IncX4, IncI2, and IncP1 types. In addition, the mcr-1 gene was the solo antibiotic resistance gene identified in the mcr-1 positive plasmids, while the rest of the antibiotic resistance genes were mostly clustered into one or two plasmids. Interestingly, one mcr-1 positive isolate (14EC047) was susceptible to PB, and we showed that the activity of MCR-1-mediated colistin resistance was not phenotypically expressed in 14EC047 host strain. Furthermore, three isolates exhibited resistance to PB but did not carry previously reported mcr-related genes. Multilocus sequence typing (MLST) showed that these mcr-1 positive E. coli isolates belonged to five different STs, and three isolates belonged to ST301 which carried multiple virulence factors related to diarrhea. Additionally, the mcr-1 positive isolates were all susceptible to imipenem (IMP), suggesting that IMP could be used to treat infection caused by mcr-1 positive E. coli isolates. Collectively, this study showed a high occurrence of mcr-1 positive plasmids in patients with diarrheal diseases of Guangzhou in China and the abolishment of the MCR-1 mediated colistin resistance in one E. coli isolate.


September 22, 2019

The Butanol Producing Microbe Clostridium beijerinckii NCIMB 14988 Manipulated Using Forward and Reverse Genetic Tools.

The solventogenic anaerobe Clostridium beijerinckii has potential for use in the sustainable bioconversion of plant-derived carbohydrates into solvents, such as butanol or acetone. However, relatively few strains have been extensively characterised either at the genomic level or through exemplification of a complete genetic toolkit. To remedy this situation, a new strain of C. beijerinckii, NCIMB 14988, is selected from among a total of 55 new clostridial isolates capable of growth on hexose and pentose sugars. Chosen on the basis of its favorable properties, the complete genome sequence of NCIMB 14988 is determined and a high-efficiency plasmid transformation protocol devised. The developed DNA transfer procedure allowed demonstration in NCIMB 14988 of the forward and reverse genetic techniques of transposon mutagenesis and gene knockout, respectively. The latter is accomplished through the successful deployment of both group II intron retargeting (ClosTron) and allelic exchange. In addition to gene inactivation, the developed allelic exchange procedure is used to create point mutations in the chromosome, allowing for the effect of amino acid changes in enzymes involved in primary metabolism to be characterized. ClosTron mediated disruption of the currently unannotated non-coding region between genes LF65_05915 and LF65_05920 is found to result in a non-sporulating phenotype.© 2018 The Authors. Biotechnology Journal Published by Wiley-VCH Verlag GmbH & Co. KGaA.


September 22, 2019

Functional genomic analysis of phthalate acid ester (PAE) catabolism genes in the versatile PAE-mineralising bacterium Rhodococcus sp. 2G.

Microbial degradation is considered the most promising method for removing phthalate acid esters (PAEs) from polluted environments; however, a comprehensive genomic understanding of the entire PAE catabolic process is still lacking. In this study, the repertoire of PAE catabolism genes in the metabolically versatile bacterium Rhodococcus sp. 2G was examined using genomic, metabolic, and bioinformatic analyses. A total of 4930 coding genes were identified from the 5.6?Mb genome of the 2G strain, including 337 esterase/hydrolase genes and 48 transferase and decarboxylase genes that were involved in hydrolysing PAEs into phthalate acid (PA) and decarboxylating PA into benzoic acid (BA). One gene cluster (xyl) responsible for transforming BA into catechol and two catechol-catabolism gene clusters controlling the ortho (cat) and meta (xyl &mhp) cleavage pathways were also identified. The proposed PAE catabolism pathway and some key degradation genes were validated by intermediate-utilising tests and real-time quantitative polymerase chain reaction. Our results provide novel insight into the mechanisms of PAE biodegradation at the molecular level and useful information on gene resources for future studies. Copyright © 2018 Elsevier B.V. All rights reserved.


September 22, 2019

Detection of mcr-1 plasmids in Enterobacteriaceae isolates from human specimens: Comparison with those in Escherichia coli isolates from livestock in Korea.

The emerging mobile colistin resistance gene, mcr-1, is an ongoing worldwide concern and an evaluation of clinical isolates harboring this gene is required in Korea. We investigated mcr-1-possessing Enterobacteriaceae among Enterobacteriaceae strains isolated in Korea, and compared the genetic details of the plasmids with those in Escherichia coli isolates from livestock.Among 9,396 Enterobacteriaceae clinical isolates collected between 2010 and 2015, 1,347 (14.3%) strains were resistant to colistin and those were screened for mcr-1 by PCR. Colistin minimum inhibitory concentrations (MICs) were determined by microdilution, and conjugal transfer of the mcr-1-harboring plasmids was assessed by direct mating. Whole genomes of three mcr-1-positive Enterobacteriaceae clinical isolates and 11 livestock-origin mcr-1-positive E. coli isolates were sequenced.Two E. coli and one Enterobacter aerogenes clinical isolates carried carried IncI2 plasmids harboring mcr-1, which conferred colistin resistance (E. coli MIC, 4 mg/L; E. aerogenes MIC, 32 mg/L). The strains possessed the complete conjugal machinery except for E. aerogenes harboring a truncated prepilin peptidase. The E. coli plasmid transferred more efficiently to E. coli than to Klebsiella pneumoniae or Enterobacter cloacae recipients. Among the three bacterial hosts, the colistin MIC was the highest for E. coli owing to the higher mcr-1-plasmid copy number and mcr-1 expression levels. Ten mcr-1-positive chicken-origin E. coli strains also possessed mcr-1-harboring IncI2 plasmids closely related to that in the clinical E. aerogenes isolate, and the remaining one porcine-origin E. coli possessed an mcr-1-harboring IncX4 plasmid.mcr-1-harboring IncI2 plasmids were identified in clinical Enterobacteriaceae isolates. These plasmids were closely associated with those in chicken-origin E. coli strains in Korea, supporting the concept of mcr-1 dissemination between humans and livestock.© The Korean Society for Laboratory Medicine.


September 22, 2019

Isolation, characterization, genomic sequencing, and GFP-marked insertional mutagenesis of a high-performance nitrogen-fixing bacterium, Kosakonia radicincitans GXGL-4A and visualization of bacterial colonization on cucumber roots.

A gram-negative bacterium GXGL-4A was originally isolated from maize roots. It displayed nitrogen-fixing (NF) ability under nitrogen-free culture condition, and had a significant promotion effect on cucumber growth in the pot inoculation test. The preliminary physiological and biochemical traits of GXGL-4A were characterized. Furthermore, a phylogenetic tree was constructed based on 16S ribosomal DNA (rDNA) sequences of genetically related species. To determine the taxonomic status of GXGL-4A and further utilize its nitrogen-fixing potential, genome sequence was obtained using PacBio RS II technology. The analyses of average nucleotide identity based on BLAST+ (ANIb) and correlation indexes of tetra-nucleotide signatures (Tetra) showed that the NF isolate GXGL-4A is closely related to the Kosakonia radicincitans type strain DSM 16656. Therefore, the isolate GXGL-4A was eventually classified into the species of Kosakonia radicincitans and designated K. radicincitans GXGL-4A. A high consistency in composition and gene arrangement of nitrogen-fixing gene cluster I (nif cluster I) was found between K. radicincitans GXGL-4A and other Kosakonia NF strains. The mutants tagged with green fluorescence protein (GFP) were obtained by transposon Tn5 mutagenesis, and then, the colonization of gfp-marked K. radicincitans GXGL-4A cells on cucumber seedling root were observed under fluorescence microscopy. The preferential sites of the labeled GXGL-4A cell population were the lateral root junctions, the differentiation zone, and the elongation zone. All these results should benefit for the deep exploration of nitrogen fixation mechanism of K. radicincitans GXGL-4A and will definitely facilitate the genetic modification process of this NF bacterium in sustainable agriculture.


September 22, 2019

Genomic characterization reveals significant divergence within Chlorella sorokiniana (Chlorellales, Trebouxiophyceae)

Selection of highly productive algal strains is crucial for establishing economically viable biomass and biopro- duct cultivation systems. Characterization of algal genomes, including understanding strain-specific differences in genome content and architecture is a critical step in this process. Using genomic analyses, we demonstrate significant differences between three strains of Chlorella sorokiniana (strain 1228, UTEX 1230, and DOE1412). We found that unique, strain-specific genes comprise a substantial proportion of each genome, and genomic regions with> 80% local nucleotide identity constitute <15% of each genome among the strains, indicating substantial strain specific evolution. Furthermore, cataloging of meiosis and other sex-related genes in C. sor- okiniana strains suggests strategic breeding could be utilized to improve biomass and bioproduct yields if a sexual cycle can be characterized. Finally, preliminary investigation of epigenetic machinery suggests the pre- sence of potentially unique transcriptional regulation in each strain. Our data demonstrate that these three C. sorokiniana strains represent significantly different genomic content. Based on these findings, we propose in- dividualized assessment of each strain for potential performance in cultivation systems.


September 22, 2019

Plasmid and chromosomal integration of four novel blaIMP-carrying transposons from Pseudomonas aeruginosa, Klebsiella pneumoniae and an Enterobacter sp.

To provide detailed genetic characterization of four novel blaIMP-carrying transposons from Pseudomonas aeruginosa, Klebsiella pneumoniae and an Enterobacter sp.P. aeruginosa 60512, K. pneumoniae 447, P. aeruginosa 12939 and Enterobacter sp. A1137 were subjected to genome sequencing. The complete nucleotide sequences of two plasmids (p60512-IMP from the 60512 isolate and p447-IMP from the 447 isolate) and two chromosomes (the 12939 and A1137 isolates) were determined, then a genomic comparison of p60512-IMP, p447-IMP and four novel blaIMP-carrying transposons (Tn6394, Tn6375, Tn6411 and Tn6397) with related sequences was performed. Transferability of the blaIMP gene and bacterial antimicrobial susceptibility were tested.Tn6394 and Tn6375 were located in p60512-IMP and p447-IMP, respectively, while Tn6411 and Tn6397 were integrated into the 12939 and A1137 chromosomes, respectively. Tn6394 was an ISPa17-based transposition unit that harboured the integron In992 (carrying blaIMP-1). In73 (carrying blaIMP-8), In73 and In992, together with the ISEcp1:IS1R-blaCTX-M-14-IS903D unit, the macAB-tolC region and the truncated aacC2-tmrB region, respectively, were integrated into the prototype transposons Tn1722, Tn1696 and Tn7, respectively, generating the Tn3-family unit transposons, Tn6375 and Tn6378, and the Tn7-family unit transposon Tn6411, respectively. Tn6397 was a large integrative and conjugative element carrying Tn6378.Complex events of transposition and homologous recombination have occurred during the original formation and further plasmid and chromosomal integration of these four transposons, promoting accumulation and spread of antimicrobial resistance genes.


September 22, 2019

Tracing back multidrug-resistant bacteria in fresh herb production: from chive to source through the irrigation water chain.

Environmental antibiotic-resistant bacteria (ARB) can be transferred to humans through foods. Fresh produce in particular is an ideal vector due to frequent raw consumption. A major contamination source of fresh produce is irrigation water. We hypothesized that water quality significantly affects loads of ARB and their diversity on fresh produce despite various other contamination sources present under agricultural practice conditions. Chive irrigated from an open-top reservoir or sterile-filtered water (control) was examined. Heterotrophic plate counts (HPC) and ARB were determined for water and chive with emphasis on Escherichia coli and Enterococcus spp. High HPC of freshly planted chive decreased over time and were significantly lower on control- vs. reservoir-irrigated chive at harvest (1.3 log (CFU/g) lower). Ciprofloxacin- and ceftazidime-resistant bacteria were significantly lower on control-irrigated chive at harvest and end of shelf life (up to 1.8 log (CFU/g) lower). Escherichia coli and Enterococcus spp. repeatedly isolated from water and chive proved resistant to up to six or four antibiotic classes (80% or 49% multidrug-resistant, respectively). Microbial source tracking identified E. coli-ST1056 along the irrigation chain and on chive. Whole-genome sequencing revealed that E. coli-ST1056 from both environments were clonal and carried the same transmissible multidrug-resistance plasmid, proving water as source of chive contamination. These findings emphasize the urgent need for guidelines concerning ARB in irrigation water and development of affordable water disinfection technologies to diminish ARB on irrigated produce.


September 22, 2019

Phenazines in plant-beneficial Pseudomonas spp.: biosynthesis, regulation, function and genomics.

Plant-beneficial phenazine-producing Pseudomonas spp. are proficient biocontrol agents of soil-dwelling plant pathogens. Phenazines are redox-active molecules that display broad-spectrum antibiotic activity toward many fungal, bacterial and oomycete plant pathogens. Phenazine compounds also play a role in the persistence and survival of Pseudomonas spp. in the rhizosphere. This mini-review focuses on plant-beneficial phenazine-producing Pseudomonas spp. from the P. fluorescens species complex, which includes numerous well-known phenazine-producing strains of biocontrol interest. In this review the current knowledge on phenazine biosynthesis and regulation, the role played by phenazines in biocontrol and rhizosphere colonization, as well as exciting new advances in the genomics of plant-beneficial phenazine-producing Pseudomonas spp. will be discussed.© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.


September 22, 2019

Genome mining of Streptomyces xinghaiensis NRRL B-24674T for the discovery of the gene cluster involved in anticomplement activities and detection of novel xiamycin analogs.

Marine actinobacterium Streptomyces xinghaiensis NRRL B-24674T has been characterized as a novel species, but thus far, its biosynthetic potential remains unexplored. In this study, the high-quality genome sequence of S. xinghaiensis NRRL B-24674T was obtained, and the production of anticomplement agents, xiamycin analogs, and siderophores was investigated by genome mining. Anticomplement compounds are valuable for combating numerous diseases caused by the abnormal activation of the human complement system. The biosynthetic gene cluster (BGC) nrps1 resembles that of complestatins, which are potent microbial-derived anticomplement agents. The identification of the nrps1 BGC revealed a core peptide that differed from that in complestatin; thus, we studied the anticomplement activity of this strain. The culture broth of S. xinghaiensis NRRL B-24674T displayed good anticomplement activity. Subsequently, the disruption of the genes in the nrps1 BGC resulted in the loss of anticomplement activity, confirming the involvement of this BGC in the biosynthesis of anticomplement agents. In addition, the mining of the BGC tep5, which resembles that of the antiviral pentacyclic indolosesquiterpene xiamycin, resulted in the discovery of nine xiamycin analogs, including three novel compounds. In addition to the BGCs responsible for desferrioxamine B, neomycin, ectoine, and carotenoid, 18 BGCs present in the genome are predicted to be novel. The results of this study unveil the potential of S. xinghaiensis as a producer of novel anticomplement agents and provide a basis for further exploration of the biosynthetic potential of S. xinghaiensis NRRL B-24674T for the discovery of novel bioactive compounds by genome mining.


September 22, 2019

Alpha- and beta-mannan utilization by marine Bacteroidetes.

Marine microscopic algae carry out about half of the global carbon dioxide fixation into organic matter. They provide organic substrates for marine microbes such as members of the Bacteroidetes that degrade algal polysaccharides using carbohydrate-active enzymes (CAZymes). In Bacteroidetes genomes CAZyme encoding genes are mostly grouped in distinct regions termed polysaccharide utilization loci (PULs). While some studies have shown involvement of PULs in the degradation of algal polysaccharides, the specific substrates are for the most part still unknown. We investigated four marine Bacteroidetes isolated from the southern North Sea that harbour putative mannan-specific PULs. These PULs are similarly organized as PULs in human gut Bacteroides that digest a- and ß-mannans from yeasts and plants respectively. Using proteomics and defined growth experiments with polysaccharides as sole carbon sources we could show that the investigated marine Bacteroidetes express the predicted functional proteins required for a- and ß-mannan degradation. Our data suggest that algal mannans play an as yet unknown important role in the marine carbon cycle, and that biochemical principles established for gut or terrestrial microbes also apply to marine bacteria, even though their PULs are evolutionarily distant.© 2018 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.


September 22, 2019

Genomic analysis of Picochlorum species reveals how microalgae may adapt to variable environments.

Understanding how microalgae adapt to rapidly changing environments is not only important to science but can help clarify the potential impact of climate change on the biology of primary producers. We sequenced and analyzed the nuclear genome of multiple Picochlorum isolates (Chlorophyta) to elucidate strategies of environmental adaptation. It was previously found that coordinated gene regulation is involved in adaptation to salinity stress, and here we show that gene gain and loss also play key roles in adaptation. We determined the extent of horizontal gene transfer (HGT) from prokaryotes and their role in the origin of novel functions in the Picochlorum clade. HGT is an ongoing and dynamic process in this algal clade with adaptation being driven by transfer, divergence, and loss. One HGT candidate that is differentially expressed under salinity stress is indolepyruvate decarboxylase that is involved in the production of a plant auxin that mediates bacteria-diatom symbiotic interactions. Large differences in levels of heterozygosity were found in diploid haplotypes among Picochlorum isolates. Biallelic divergence was pronounced in P. oklahomensis (salt plains environment) when compared with its closely related sister taxon Picochlorum SENEW3 (brackish water environment), suggesting a role of diverged alleles in response to environmental stress. Our results elucidate how microbial eukaryotes with limited gene inventories expand habitat range from mesophilic to halophilic through allelic diversity, and with minor but important contributions made by HGT. We also explore how the nature and quality of genome data may impact inference of nuclear ploidy.


September 22, 2019

Evaluation of bacterial contamination in goat milk powder using PacBio Single Molecule Real-Time Sequencing and Droplet Digital PCR.

Goat milk powder is a nutritious and easy-to-store product that is highly favored by consumers. However, the presence of contaminating bacteria and their metabolites may significantly affect the flavor, solubility, shelf life, and safety of the product. To comprehensively and accurately understand the sanitary conditions in the goat milk powder production process and potential threats from bacterial contamination, a combination of Pacific Biosciences single molecule real-time sequencing and droplet digital PCR was used to evaluate bacterial contamination in seven goat milk powder samples from three dairies. Ten phyla, 119 genera, and 249 bacterial species were identified. Bacillus, Paenibacillus, Lactococcus, and Cronobacter were the primary genera. Bacillus cereus, Lactococcus lactis, Alkaliphilus oremlandii, and Cronobacter sakazakii were the dominant species. With droplet digital PCR, 6.3 × 104 copies per g of Bacillus cereus and 1.0 × 104 copies per g of Cronobacter spp. were quantified, which may increase the risk of food spoilage and the probability of foodborne illness and should be monitored and controlled. This study offers a new approach for evaluating bacterial contamination in goat milk powder and supplies a reference for the assessment of food safety and control of potential risk, which will be of interest to the dairy industry.


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