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September 22, 2019

Emergence of an extensively drug-resistant Salmonella enterica serovar Typhi clone harboring a promiscuous plasmid encoding resistance to fluoroquinolones and third-generation cephalosporins.

Antibiotic resistance is a major problem in Salmonella enterica serovar Typhi, the causative agent of typhoid. Multidrug-resistant (MDR) isolates are prevalent in parts of Asia and Africa and are often associated with the dominant H58 haplotype. Reduced susceptibility to fluoroquinolones is also widespread, and sporadic cases of resistance to third-generation cephalosporins or azithromycin have also been reported. Here, we report the first large-scale emergence and spread of a novel S. Typhi clone harboring resistance to three first-line drugs (chloramphenicol, ampicillin, and trimethoprim-sulfamethoxazole) as well as fluoroquinolones and third-generation cephalosporins in Sindh, Pakistan, which we classify as extensively drug resistant (XDR). Over 300 XDR typhoid cases have emerged in Sindh, Pakistan, since November 2016. Additionally, a single case of travel-associated XDR typhoid has recently been identified in the United Kingdom. Whole-genome sequencing of over 80 of the XDR isolates revealed remarkable genetic clonality and sequence conservation, identified a large number of resistance determinants, and showed that these isolates were of haplotype H58. The XDR S. Typhi clone encodes a chromosomally located resistance region and harbors a plasmid encoding additional resistance elements, including the blaCTX-M-15 extended-spectrum ß-lactamase, and carrying the qnrS fluoroquinolone resistance gene. This antibiotic resistance-associated IncY plasmid exhibited high sequence identity to plasmids found in other enteric bacteria isolated from widely distributed geographic locations. This study highlights three concerning problems: the receding antibiotic arsenal for typhoid treatment, the ability of S. Typhi to transform from MDR to XDR in a single step by acquisition of a plasmid, and the ability of XDR clones to spread globally. IMPORTANCE Typhoid fever is a severe disease caused by the Gram-negative bacterium Salmonella enterica serovar Typhi. Antibiotic-resistant S. Typhi strains have become increasingly common. Here, we report the first large-scale emergence and spread of a novel extensively drug-resistant (XDR) S. Typhi clone in Sindh, Pakistan. The XDR S. Typhi is resistant to the majority of drugs available for the treatment of typhoid fever. This study highlights the evolving threat of antibiotic resistance in S. Typhi and the value of antibiotic susceptibility testing and whole-genome sequencing in understanding emerging infectious diseases. We genetically characterized the XDR S. Typhi to investigate the phylogenetic relationship between these isolates and a global collection of S. Typhi isolates and to identify multiple genes linked to antibiotic resistance. This S. Typhi clone harbored a promiscuous antibiotic resistance plasmid previously identified in other enteric bacteria. The increasing antibiotic resistance in S. Typhi observed here adds urgency to the need for typhoid prevention measures.

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September 22, 2019

Construction and characterization of bacterial artificial chromosomes harboring the full-length genome of a highly attenuated vaccinia virus LC16m8.

LC16m8 (m8), a highly attenuated vaccinia virus (VAC) strain, was developed as a smallpox vaccine, and its safety and immunogenicity have been confirmed. Here, we aimed to develop a system that recovers infectious m8 from a bacterial artificial chromosome (BAC) that retains the full-length viral genomic DNA (m8-BAC system). The infectious virus was successfully recovered from a VAC-BAC plasmid, named pLC16m8-BAC. Furthermore, the bacterial replicon-free virus was generated by intramolecular homologous recombination and was successfully recovered from a modified VAC-BAC plasmid, named pLC16m8.8S-BAC. Also, the growth of the recovered virus was indistinguishable from that of authentic m8. The full genome sequence of the plasmid, which harbors identical inverted terminal repeats (ITR) to that of authentic m8, was determined by long-read next-generation sequencing (NGS). The ITR contains x 18 to 32 of the 70 and x 30 to 45 of 54 base pair tandem repeats, and the number of tandem repeats was different between the ITR left and right. Since the virus recovered from pLC16m8.8S-BAC was expected to retain the identical viral genome to that of m8, including the ITR, a reference-based alignment following a short-read NGS was performed to validate the sequence of the recovered virus. Based on the pattern of coverage depth in the ITR, no remarkable differences were observed between the virus and m8, and the other region was confirmed to be identical as well. In summary, this new system can recover the virus, which is geno- and phenotypically indistinguishable from authentic m8.

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September 22, 2019

Analyzing AbrB-knockout effects through genome and transcriptome sequencing of Bacillus licheniformis DW2.

As an industrial bacterium, Bacillus licheniformis DW2 produces bacitracin which is an important antibiotic for many pathogenic microorganisms. Our previous study showed AbrB-knockout could significantly increase the production of bacitracin. Accordingly, it was meaningful to understand its genome features, expression differences between wild and AbrB-knockout (?AbrB) strains, and the regulation of bacitracin biosynthesis. Here, we sequenced, de novo assembled and annotated its genome, and also sequenced the transcriptomes in three growth phases. The genome of DW2 contained a DNA molecule of 4,468,952 bp with 45.93% GC content and 4,717 protein coding genes. The transcriptome reads were mapped to the assembled genome, and obtained 4,102~4,536 expressed genes from different samples. We investigated transcription changes in B. licheniformis DW2 and showed that ?AbrB caused hundreds of genes up-regulation and down-regulation in different growth phases. We identified a complete bacitracin synthetase gene cluster, including the location and length of bacABC, bcrABC, and bacT, as well as their arrangement. The gene cluster bcrABC were significantly up-regulated in ?AbrB strain, which supported the hypothesis in previous study of bcrABC transporting bacitracin out of the cell to avoid self-intoxication, and was consistent with the previous experimental result that ?AbrB could yield more bacitracin. This study provided a high quality reference genome for B. licheniformis DW2, and the transcriptome data depicted global alterations across two strains and three phases offered an understanding of AbrB regulation and bacitracin biosynthesis through gene expression.

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September 22, 2019

Characterizing the DNA methyltransferases of Haloferax volcanii via bioinformatics, gene deletion, and SMRT Sequencing.

DNA methyltransferases (MTases), which catalyze the methylation of adenine and cytosine bases in DNA, can occur in bacteria and archaea alongside cognate restriction endonucleases (REases) in restriction-modification (RM) systems or independently as orphan MTases. Although DNA methylation and MTases have been well-characterized in bacteria, research into archaeal MTases has been limited. A previous study examined the genomic DNA methylation patterns (methylome) of the halophilic archaeonHaloferax volcanii, a model archaeal system which can be easily manipulated in laboratory settings, via single-molecule real-time (SMRT) sequencing and deletion of a putative MTase gene (HVO_A0006). In this follow-up study, we deleted other putative MTase genes inH. volcaniiand sequenced the methylomes of the resulting deletion mutants via SMRT sequencing to characterize the genes responsible for DNA methylation. The results indicate that deletion of putative RM genesHVO_0794,HVO_A0006, andHVO_A0237in a single strain abolished methylation of the sole cytosine motif in the genome (Cm4TAG). Amino acid alignments demonstrated thatHVO_0794shares homology with characterized cytosine CTAG MTases in other organisms, indicating that this MTase is responsible for Cm4TAG methylation inH. volcanii. The CTAG motif has high density at only one of the origins of replication, and there is no relative increase in CTAG motif frequency in the genome ofH. volcanii, indicating that CTAG methylation might not have effectively taken over the role of regulating DNA replication and mismatch repair in the organism as previously predicted. Deletion of the putative Type I RM operonrmeRMS(HVO_2269-2271) resulted in abolished methylation of the adenine motif in the genome (GCAm6BN6VTGC). Alignments of the MTase (HVO_2270) and site specificity subunit (HVO_2271) demonstrate homology with other characterized Type I MTases and site specificity subunits, indicating that thermeRMSoperon is responsible for adenine methylation inH. volcanii. Together with HVO_0794, these genes appear to be responsible for all detected methylation inH. volcanii, even though other putative MTases (HVO_C0040,HVO_A0079) share homology with characterized MTases in other organisms. We also report the construction of a multi-RM deletion mutant (?RM), with multiple RM genes deleted and with no methylation detected via SMRT sequencing, which we anticipate will be useful for future studies on DNA methylation inH. volcanii.

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September 22, 2019

Complete genome of Cobetia marina JCM 21022T and phylogenomic analysis of the family Halomonadaceae

Cobetia marina is a model proteobacteria in researches on marine biofouling. Its taxonomic nomenclature has been revised many times over the past few decades. To better understand the role of the surface-associated lifestyle of C. marina and the phylogeny of the family Halomonadaceae, we sequenced the entire genome of C. marina JCM 21022T using single molecule real-time sequencing technology (SMRT) and performed comparative genomics and phylogenomics analyses. The circular chromosome was 4 176 300 bp with an average GC content of 62.44% and contained 3 611 predicted coding sequences, 72 tRNA genes, and 21 rRNA genes. The C. marina JCM 21022T genome contained a set of crucial genes involved in surface colonization processes. The comparative genome analysis indicated the significant diff erences between C. marina JCM 21022T and Cobetia amphilecti KMM 296 (formerly named C. marina KMM 296) resulted from sequence insertions or deletions and chromosomal recombination. Despite these diff erences, pan and core genome analysis showed similar gene functions between the two strains. The phylogenomic study of the family Halomonadaceae is reported here for the first time. We found that the relationships were well resolved among every genera tested, including Chromohalobacter, Halomonas, Cobetia, Kushneria, Zymobacter, and Halotalea.

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September 22, 2019

Biosynthesis of antibiotic chuangxinmycin from Actinoplanes tsinanensis.

Chuangxinmycin is an antibiotic isolated from Actinoplanes tsinanensis CPCC 200056 in the 1970s with a novel indole-dihydrothiopyran heterocyclic skeleton. Chuangxinmycin showed in vitro antibacterial activity and in vivo efficacy in mouse infection models as well as preliminary clinical trials. But the biosynthetic pathway of chuangxinmycin has been obscure since its discovery. Herein, we report the identification of a stretch of DNA from the genome of A. tsinanensis CPCC 200056 that encodes genes for biosynthesis of chuangxinmycin by bioinformatics analysis. The designated cxn cluster was then confirmed to be responsible for chuangxinmycin biosynthesis by direct cloning and heterologous expressing in Streptomyces coelicolor M1146. The cytochrome P450 CxnD was verified to be involved in the dihydrothiopyran ring closure reaction by the identification of seco-chuangxinmycin in S. coelicolor M1146 harboring the cxn gene cluster with an inactivated cxnD. Based on these results, a plausible biosynthetic pathway for chuangxinmycin biosynthesis was proposed, by hijacking the primary sulfur transfer system for sulfur incorporation. The identification of the biosynthetic gene cluster of chuangxinmycin paves the way for elucidating the detail biochemical machinery for chuangxinmycin biosynthesis, and provides the basis for the generation of novel chuangxinmycin derivatives by means of combinatorial biosynthesis and synthetic biology.

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September 22, 2019

A combinatorial approach to synthetic transcription factor-promoter combinations for yeast strain engineering.

Despite the need for inducible promoters in strain development efforts, the majority of engineering in Saccharomyces cerevisiae continues to rely on a few constitutively active or inducible promoters. Building on advances that use the modular nature of both transcription factors and promoter regions, we have built a library of hybrid promoters that are regulated by a synthetic transcription factor. The hybrid promoters consist of native S. cerevisiae promoters, in which the operator regions have been replaced with sequences that are recognized by the bacterial LexA DNA binding protein. Correspondingly, the synthetic transcription factor (TF) consists of the DNA binding domain of the LexA protein, fused with the human estrogen binding domain and the viral activator domain, VP16. The resulting system with a bacterial DNA binding domain avoids the transcription of native S. cerevisiae genes, and the hybrid promoters can be induced using estradiol, a compound with no detectable impact on S. cerevisiae physiology. Using combinations of one, two or three operator sequence repeats and a set of native S. cerevisiae promoters, we obtained a series of hybrid promoters that can be induced to different levels, using the same synthetic TF and a given estradiol. This set of promoters, in combination with our synthetic TF, has the potential to regulate numerous genes or pathways simultaneously, to multiple desired levels, in a single strain.© 2017 The Authors. Yeast published by John Wiley & Sons, Ltd.

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September 22, 2019

Enhancing the adaptability of the deep-sea bacterium Shewanella piezotolerans WP3 to high pressure and low temperature by experimental evolution under H2O2 stress.

Oxidative stresses commonly exist in natural environments, and microbes have developed a variety of defensive systems to counteract such events. Although increasing evidence has shown that high hydrostatic pressure (HHP) and low temperature (LT) induce antioxidant defense responses in cells, there is no direct evidence to prove the connection between antioxidant defense mechanisms and the adaptation of bacteria to HHP and LT. In this study, using the wild-type (WT) strain of a deep-sea bacterium, Shewanella piezotolerans WP3, as an ancestor, we obtained a mutant, OE100, with an enhanced antioxidant defense capacity by experimental evolution under H2O2 stress. Notably, OE100 exhibited better tolerance not only to H2O2 stress but also to HHP and LT (20 MPa and 4°C, respectively). Whole-genome sequencing identified a deletion mutation in the oxyR gene, which encodes the transcription factor that controls the oxidative stress response. Comparative transcriptome analysis showed that the genes associated with oxidative stress defense, anaerobic respiration, DNA repair, and the synthesis of flagella and bacteriophage were differentially expressed in OE100 compared with the WT at 20 MPa and 4°C. Genetic analysis of oxyR and ccpA2 indicated that the OxyR-regulated cytochrome c peroxidase CcpA2 significantly contributed to the adaptation of WP3 to HHP and LT. Taken together, these results confirmed the inherent relationship between antioxidant defense mechanisms and the adaptation of a benthic microorganism to HHP and LT.IMPORTANCE Oxidative stress exists in various niches, including the deep-sea ecosystem, which is an extreme environment with conditions of HHP and predominantly LT. Although previous studies have shown that HHP and LT induce antioxidant defense responses in cells, direct evidence to prove the connection between antioxidant defense mechanisms and the adaptation of bacteria to HHP and LT is lacking. In this work, using the deep-sea bacterium Shewanella piezotolerans WP3 as a model, we proved that enhancement of the adaptability of WP3 to HHP and LT can benefit from its antioxidant defense mechanism, which provided useful insight into the ecological roles of antioxidant genes in a benthic microorganism and contributed to an improved understanding of microbial adaptation strategies in deep-sea environments.

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September 22, 2019

Complete genome sequence of N2-fixing model strain Klebsiella sp. nov. M5al, which produces plant cell wall-degrading enzymes and siderophores.

The bacterial strain M5al is a model strain for studying the molecular genetics of N2-fixation and molecular engineering of microbial production of platform chemicals 1,3-propanediol and 2,3-butanediol. Here, we present the complete genome sequence of the strain M5al, which belongs to a novel species closely related toKlebsiella michiganensis. M5al secretes plant cell wall-degrading enzymes and colonizes rice roots but does not cause soft rot disease. M5al also produces siderophores and contains the gene clusters for synthesis and transport of yersiniabactin which is a critical virulence factor forKlebsiellapathogens in causing human disease. We propose that the model strain M5al can be genetically modified to study bacterial N2-fixation in association with non-legume plants and production of 1,3-propanediol and 2,3-butanediol through degradation of plant cell wall biomass.

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September 22, 2019

2′-O-methylation in mRNA disrupts tRNA decoding during translation elongation.

Chemical modifications of mRNA may regulate many aspects of mRNA processing and protein synthesis. Recently, 2′-O-methylation of nucleotides was identified as a frequent modification in translated regions of human mRNA, showing enrichment in codons for certain amino acids. Here, using single-molecule, bulk kinetics and structural methods, we show that 2′-O-methylation within coding regions of mRNA disrupts key steps in codon reading during cognate tRNA selection. Our results suggest that 2′-O-methylation sterically perturbs interactions of ribosomal-monitoring bases (G530, A1492 and A1493) with cognate codon-anticodon helices, thereby inhibiting downstream GTP hydrolysis by elongation factor Tu (EF-Tu) and A-site tRNA accommodation, leading to excessive rejection of cognate aminoacylated tRNAs in initial selection and proofreading. Our current and prior findings highlight how chemical modifications of mRNA tune the dynamics of protein synthesis at different steps of translation elongation.

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September 22, 2019

Xanthomonas citri jumbo phage XacN1 exhibits a wide host range and high complement of tRNA genes.

Xanthomonas virus (phage) XacN1 is a novel jumbo myovirus infecting Xanthomonas citri, the causative agent of Asian citrus canker. Its linear 384,670?bp double-stranded DNA genome encodes 592 proteins and presents the longest (66?kbp) direct terminal repeats (DTRs) among sequenced viral genomes. The DTRs harbor 56 tRNA genes, which correspond to all 20 amino acids and represent the largest number of tRNA genes reported in a viral genome. Codon usage analysis revealed a propensity for the phage encoded tRNAs to target codons that are highly used by the phage but less frequently by its host. The existence of these tRNA genes and seven additional translation-related genes as well as a chaperonin gene found in the XacN1 genome suggests a relative independence of phage replication on host molecular machinery, leading to a prediction of a wide host range for this jumbo phage. We confirmed the prediction by showing a wider host range of XacN1 than other X. citri phages in an infection test against a panel of host strains. Phylogenetic analyses revealed a clade of phages composed of XacN1 and ten other jumbo phages, indicating an evolutionary stable large genome size for this group of phages.

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September 22, 2019

Enterobacter bugandensis: a novel enterobacterial species associated with severe clinical infection.

Nosocomial pathogens can cause life-threatening infections in neonates and immunocompromised patients. E. bugandensis (EB-247) is a recently described species of Enterobacter, associated with neonatal sepsis. Here we demonstrate that the extended spectrum ß-lactam (ESBL) producing isolate EB-247 is highly virulent in both Galleria mellonella and mouse models of infection. Infection studies in a streptomycin-treated mouse model showed that EB-247 is as efficient as Salmonella Typhimurium in inducing systemic infection and release of proinflammatory cytokines. Sequencing and analysis of the complete genome and plasmid revealed that virulence properties are associated with the chromosome, while antibiotic-resistance genes are exclusively present on a 299?kb IncHI plasmid. EB-247 grew in high concentrations of human serum indicating septicemic potential. Using whole genome-based transcriptome analysis we found 7% of the genome was mobilized for growth in serum. Upregulated genes include those involved in the iron uptake and storage as well as metabolism. The lasso peptide microcin J25 (MccJ25), an inhibitor of iron-uptake and RNA polymerase activity, inhibited EB-247 growth. Our studies indicate that Enterobacter bugandensis is a highly pathogenic species of the genus Enterobacter. Further studies on the colonization and virulence potential of E. bugandensis and its association with septicemic infection is now warranted.

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September 22, 2019

Biodegradation of di-n-butyl phthalate (DBP) by a novel endophytic Bacillus megaterium strain YJB3.

Phthalic acid esters (PAEs) are a group of recalcitrant and hazardous organic compounds that pose a great threat to both ecosystem and human beings. A novel endophytic strain YJB3 that could utilize a wide range of PAEs as the sole carbon and energy sources for cell growth was isolated from Canna indica root tissue. It was identified as Bacillus megaterium based on morphological characteristics and 16S rDNA sequence homology analysis. The degradation capability of the strain YJB3 was investigated by incubation in mineral salt medium containing di-n-butyl-phthalate (DBP), one of important PAEs under different environmental conditions, showing 82.5% of the DBP removal in 5days of incubation under the optimum conditions (acetate 1.2g·L-1, inocula 1.8%, and temperature 34.2°C) achieved by two-step sequential optimization technologies. The DBP metabolites including mono-butyl phthalate (MBP), phthalic acid (PA), protocatechuic acid (PCA), etc. were determined by GC-MS. The PCA catabolic genes responsible for the aromatic ring cleavage of PCA in the strain YJB3 were excavated by whole-genome sequencing. Thus, a degradation pathway of DBP by the strain YJB3 was proposed that MBP was formed, followed by PA, and then the intermediates were further utilized till complete degradation. To our knowledge, this is the first study to show the biodegradation of PAEs using endophyte. The results in the present study suggest that the strain YJB3 is greatly promising to act as a competent inoculum in removal of PAEs in both soils and crops. Copyright © 2017 Elsevier B.V. All rights reserved.

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September 22, 2019

N4-cytosine DNA methylation regulates transcription and pathogenesis in Helicobacter pylori.

Many bacterial genomes exclusively display an N4-methyl cytosine base (m4C), whose physiological significance is not yet clear. Helicobacter pylori is a carcinogenic bacterium and the leading cause of gastric cancer in humans. Helicobacter pylori strain 26695 harbors a single m4C cytosine methyltransferase, M2.HpyAII which recognizes 5′ TCTTC 3′ sequence and methylates the first cytosine residue. To understand the role of m4C modification, M2.hpyAII deletion strain was constructed. Deletion strain displayed lower adherence to host AGS cells and reduced potential to induce inflammation and apoptosis. M2.hpyAII gene deletion strain exhibited reduced capacity for natural transformation, which was rescued in the complemented strain carrying an active copy of M2.hpyAII gene in the genome. Genome-wide gene expression and proteomic analysis were carried out to discern the possible reasons behind the altered phenotype of the M2.hpyAII gene deletion strain. Upon the loss of m4C modification a total of 102 genes belonging to virulence, ribosome assembly and cellular components were differentially expressed. The present study adds a functional role for the presence of m4C modification in H. pylori and provides the first evidence that m4C signal acts as a global epigenetic regulator in H. pylori.

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September 22, 2019

Rhizospheric microbial communities are driven by Panax ginseng at different growth stages and biocontrol bacteria alleviates replanting mortality

The cultivation of Panax plants is hindered by replanting problems, which may be caused by plant-driven changes in the soil microbial community. Inoculation with microbial antagonists may efficiently alleviate replanting issues. Through high-throughput sequencing, this study revealed that bacterial diversity decreased, whereas fungal diversity increased, in the rhizosphere soils of adult ginseng plants at the root growth stage under different ages. Few microbial community, such as Luteolibacter, Cytophagaceae, Luteibacter, Sphingomonas, Sphingomonadaceae, and Zygomycota, were observed; the relative abundance of microorganisms, namely, Brevundimonas, Enterobacteriaceae, Pandoraea, Cantharellales, Dendryphion, Fusarium, and Chytridiomycota, increased in the soils of adult ginseng plants compared with those in the soils of 2-year-old seedlings. Bacillus subtilis 50-1, a microbial antagonist against the pathogenic Fusarium oxysporum, was isolated through a dual culture technique. These bacteria acted with a biocontrol efficacy of 67.8%. The ginseng death rate and Fusarium abundance decreased by 63.3% and 46.1%, respectively, after inoculation with B. subtilis 50-1. Data revealed that microecological degradation could result from ginseng-driven changes in rhizospheric microbial communities; these changes are associated with the different ages and developmental stages of ginseng plants. Biocontrol using microbial antagonists alleviated the replanting problem.

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