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Sunday, July 7, 2019

Development of an orthogonal fatty acid biosynthesis system in E. coli for oleochemical production.

Here we report recombinant expression and activity of several type I fatty acid synthases that can function in parallel with the native Escherichia coli fatty acid synthase. Corynebacterium glutamicum FAS1A was the most active in E. coli and this fatty acid synthase was leveraged to produce oleochemicals including fatty alcohols and methyl ketones. Coexpression of FAS1A with the ACP/CoA-reductase Maqu2220 from Marinobacter aquaeolei shifted the chain length distribution of fatty alcohols produced. Coexpression of FAS1A with FadM, FadB, and an acyl-CoA-oxidase from Micrococcus luteus resulted in the production of methyl ketones, although at a lower level than cells using the…

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Sunday, July 7, 2019

Covalent modification of bacteriophage T4 DNA inhibits CRISPR-Cas9.

The genomic DNAs of tailed bacteriophages are commonly modified by the attachment of chemical groups. Some forms of DNA modification are known to protect phage DNA from cleavage by restriction enzymes, but others are of unknown function. Recently, the CRISPR-Cas nuclease complexes were shown to mediate bacterial adaptive immunity by RNA-guided target recognition, raising the question of whether phage DNA modifications may also block attack by CRISPR-Cas9. We investigated phage T4 as a model system, where cytosine is replaced with glucosyl-hydroxymethylcytosine (glc-HMC). We first quantified the extent and distribution of covalent modifications in T4 DNA by single-molecule DNA sequencing and…

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Sunday, July 7, 2019

A multidrug resistance plasmid contains the molecular switch for type VI secretion in Acinetobacter baumannii.

Infections with Acinetobacter baumannii, one of the most troublesome and least studied multidrug-resistant superbugs, are increasing at alarming rates. A. baumannii encodes a type VI secretion system (T6SS), an antibacterial apparatus of Gram-negative bacteria used to kill competitors. Expression of the T6SS varies among different strains of A. baumannii, for which the regulatory mechanisms are unknown. Here, we show that several multidrug-resistant strains of A. baumannii harbor a large, self-transmissible resistance plasmid that carries the negative regulators for T6SS. T6SS activity is silenced in plasmid-containing, antibiotic-resistant cells, while part of the population undergoes frequent plasmid loss and activation of the…

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Sunday, July 7, 2019

Sequencing of plasmids pAMBL1 and pAMBL2 from Pseudomonas aeruginosa reveals a blaVIM-1 amplification causing high-level carbapenem resistance.

Carbapenemases are a major concern for the treatment of infectious diseases caused by Gram-negative bacteria. Although plasmids are responsible for the spread of resistance genes among these pathogens, there is limited information on the nature of the mobile genetic elements carrying carbapenemases in Pseudomonas aeruginosa.We combined data from two different next-generation sequencing platforms, Illumina HiSeq2000 and PacBio RSII, to obtain the complete nucleotide sequences of two blaVIM-1-carrying plasmids (pAMBL1 and pAMBL2) isolated from P. aeruginosa clinical isolates.Plasmid pAMBL1 has 26?440 bp and carries a RepA_C family replication protein. pAMBL1 is similar to plasmids pNOR-2000 and pKLC102 from P. aeruginosa and…

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Sunday, July 7, 2019

Retrohoming of a mobile group II intron in human cells suggests how eukaryotes limit group II intron proliferation.

Mobile bacterial group II introns are evolutionary ancestors of spliceosomal introns and retroelements in eukaryotes. They consist of an autocatalytic intron RNA (a “ribozyme”) and an intron-encoded reverse transcriptase, which function together to promote intron integration into new DNA sites by a mechanism termed “retrohoming”. Although mobile group II introns splice and retrohome efficiently in bacteria, all examined thus far function inefficiently in eukaryotes, where their ribozyme activity is limited by low Mg2+ concentrations, and intron-containing transcripts are subject to nonsense-mediated decay (NMD) and translational repression. Here, by using RNA polymerase II to express a humanized group II intron reverse…

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Sunday, July 7, 2019

First detection of Klebsiella variicola producing OXA-181 carbapenemase in fresh vegetable imported from Asia to Switzerland.

The emergence and worldwide spread of carbapenemase-producing Enterobacteriaceae is of great concern to public health services. The aim of this study was to investigate the occurrence of carbapenemase-producing Enterobacteriaceae in fresh vegetables and spices imported from Asia to Switzerland.Twenty-two different fresh vegetable samples were purchased in March 2015 from different retail shops specializing in Asian food. The vegetables included basil leaves, bergamont leaves, coriander, curry leaves, eggplant and okra (marrow). Samples had been imported from Thailand, the Socialist Republic of Vietnam and India. After an initial enrichment-step, carbapenemase-producing Enterobacteriaceae were isolated from two carbapenem-containing selective media (SUPERCARBA II and Brilliance…

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Sunday, July 7, 2019

Fosfomycin resistance in Escherichia coli, Pennsylvania, USA.

Fosfomycin resistance in Escherichia coli is rare in the United States. An extended-spectrum ß-lactamase-producing E. coli clinical strain identified in Pennsylvania, USA, showed high-level fosfomycin resistance caused by the fosA3 gene. The IncFII plasmid carrying this gene had a structure similar to those found in China, where fosfomycin resistance is commonly described.

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Sunday, July 7, 2019

Genomic epidemiology of an endoscope-associated outbreak of Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae.

Increased incidence of infections due to Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae (KPC-Kp) was noted among patients undergoing endoscopic retrograde cholangiopancreatography (ERCP) at a single hospital. An epidemiologic investigation identified KPC-Kp and non-KPC-producing, extended-spectrum ß-lactamase (ESBL)-producing Kp in cultures from 2 endoscopes. Genotyping was performed on patient and endoscope isolates to characterize the microbial genomics of the outbreak. Genetic similarity of 51 Kp isolates from 37 patients and 3 endoscopes was assessed by pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). Five patient and 2 endoscope isolates underwent whole genome sequencing (WGS). Two KPC-encoding plasmids were characterized by single…

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Sunday, July 7, 2019

IncI1 plasmids encoding various blaCTX-Ms contributed to ceftriaxone resistance in Salmonella Enteritidis in China.

Resistance to extended spectrum ß-lactams in Salmonella, in particular serotypes such as S. Enteritidis that are frequently associated with clinical infections, is a serious public health concern. In this study, phenotypic characterization of 433 clinical S. Enteritidis strains obtained from a nationwide collection of China CDC during the period of 2005~2010 depicted an increasing trend of resistance to ceftriaxone from 2008 onwards. Seventeen (4%) of the strains were found to be resistant to ceftriaxone, 7% to ciprofloxacin and 0.7% to both ciprofloxacin and ceftriaxone. Most of the ceftriaxone-resistant S. Enteritidis strains (15/17) were genetically unrelated, and originated from Henan province.…

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Sunday, July 7, 2019

Molecular characterization using next generation sequencing of plasmids containing blaNDM-7 in Enterobacteriaceae from Calgary, Canada.

Enterobacteriaceae with blaNDM-7 is relatively uncommon and had previously been described in Europe, India, USA and Japan. This study describes the characteristics of Enterobacteriaceae [Klebsiella pneumoniae (n=2), Escherichia coli (n=2), Serratia marcescens (n=1), Enterobacter hormaechei (n=1)] with blaNDM-7 obtained in 4 patients from Calgary, Canada during 2013-4. The 46,161 bp IncX3 plasmids with blaNDM-7 are highly similar to other blaNDM-harboring IncX3 plasmids and interestingly, showed identical structures within the different isolates. This finding may indicate horizontal transmission within our health region or may indicate contact with individuals from endemic areas within the hospital setting. Patients infected or colonized with bacteria…

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Sunday, July 7, 2019

Complete sequence of a conjugative IncN plasmid harboring blakpc-2, blashv-12, and qnrS1 from an Escherichia coli sequence type 648 strain

We sequenced a novel conjugative blaKPC-2-harboring IncN plasmid, pYD626E, from an Escherichia coli sequence type 648 strain previously identified in Pittsburgh, Pennsylvania. pYD626E was 72,800 bp long and carried four ß-lactamase genes, blaKPC-2, blaSHV-12, blaLAP-1, and blaTEM-1. In addition, it harbored qnrS1 (fluoroquinolone resistance) and dfrA14 (trimethoprim resistance). The plasmid profile and clinical history supported the in vivo transfer of this plasmid between Klebsiella pneumoniae and Escherichia coli. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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Sunday, July 7, 2019

Phylogenomically guided identification of industrially relevant GH1 ß-glucosidases through DNA synthesis and nanostructure-initiator mass spectrometry.

Harnessing the biotechnological potential of the large number of proteins available in sequence databases requires scalable methods for functional characterization. Here we propose a workflow to address this challenge by combining phylogenomic guided DNA synthesis with high-throughput mass spectrometry and apply it to the systematic characterization of GH1 ß-glucosidases, a family of enzymes necessary for biomass hydrolysis, an important step in the conversion of lignocellulosic feedstocks to fuels and chemicals. We synthesized and expressed 175 GH1s, selected from over 2000 candidate sequences to cover maximum sequence diversity. These enzymes were functionally characterized over a range of temperatures and pHs using…

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Sunday, July 7, 2019

The Mycobacterium avium ssp. paratuberculosis specific mptD gene is required for maintenance of the metabolic homeostasis necessary for full virulence in mouse infections.

Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne’s disease, a chronic granulomatous enteritis in ruminants. Furthermore, infections of humans with MAP have been reported and a possible association with Crohn’s disease and diabetes type I is currently discussed. MAP owns large sequence polymorphisms (LSPs) that were exclusively found in this mycobacteria species. The relevance of these LSPs in the pathobiology of MAP is still unclear. The mptD gene (MAP3733c) of MAP belongs to a small group of functionally uncharacterized genes, which are not present in any other sequenced mycobacteria species. mptD is part of a predicted operon (mptABCDEF), encoding a putative…

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Sunday, July 7, 2019

Comparative genomics of extrachromosomal elements in Bacillus thuringiensis subsp. israelensis.

Bacillus thuringiensis subsp. israelensis is one of the most important microorganisms used against mosquitoes. It was intensively studied following its discovery and became a model bacterium of the B. thuringiensis species. Those studies focused on toxin genes, aggregation-associated conjugation, linear genome phages, etc. Recent announcements of genomic sequences of different strains have not been explicitly related to the biological properties studied. We report data on plasmid content analysis of four strains using ultra-high-throughput sequencing. The strains were commercial product isolates, with their putative ancestor and type B. thuringiensis subsp. israelensis strain sequenced earlier. The assembled contigs corresponding to published and…

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Sunday, July 7, 2019

Prevalence of mcr-1 in Escherichia coli and Klebsiella pneumoniae recovered from bloodstream infections in China: a multicentre longitudinal study.

Polymyxin antibiotics are used as last-resort therapies to treat infections caused by multidrug-resistant Gram-negative bacteria. The plasmid-mediated colistin resistance determinant MCR-1 has been identified in Enterobacteriaceae in China. We did this study to investigate the prevalence of the mcr-1 gene in clinical isolates from patients with bloodstream infections in China.Clinical isolates of Escherichia coli and Klebsiella pneumoniae were collected from patients with bloodstream infections at 28 hospitals in China, then screened for colistin resistance by broth microdilution and for the presence of the mcr-1 gene by PCR amplification. We subjected mcr-1-positive isolates to genotyping, susceptibility testing, and clinical data analysis.…

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