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July 7, 2019

Exception to the rule: Genomic characterization of naturally occurring unusual Vibrio cholerae strains with a single chromosome.

The genetic make-up of most bacteria is encoded in a single chromosome while about 10% have more than one chromosome. Among these, Vibrio cholerae, with two chromosomes, has served as a model system to study various aspects of chromosome maintenance, mainly replication, and faithful partitioning of multipartite genomes. Here, we describe the genomic characterization of strains that are an exception to the two chromosome rules: naturally occurring single-chromosome V. cholerae. Whole genome sequence analyses of NSCV1 and NSCV2 (natural single-chromosome vibrio) revealed that the Chr1 and Chr2 fusion junctions contain prophages, IS elements, and direct repeats, in addition to large-scale chromosomal rearrangements such as inversions, insertions, and long tandem repeats elsewhere in the chromosome compared to prototypical two chromosome V. cholerae genomes. Many of the known cholera virulence factors are absent. The two origins of replication and associated genes are generally intact with synonymous mutations in some genes, as are recA and mismatch repair (MMR) genes dam, mutH, and mutL; MutS function is probably impaired in NSCV2. These strains are ideal tools for studying mechanistic aspects of maintenance of chromosomes with multiple origins and other rearrangements and the biological, functional, and evolutionary significance of multipartite genome architecture in general.

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July 7, 2019

Complete circular genome sequence and temperature independent adaptation to anaerobiosis of Listeria weihenstephanensis DSM 24698.

The aim of this study was to analyze the adaptation of the environmental Listeria weihenstephanensis DSM 24698 to anaerobiosis. The complete circular genome sequence of this species is reported and the adaptation of L. weihenstephanensis DSM 24698 to oxygen availability was investigated by global transcriptional analyses via RNAseq at 18 and 34°C. A list of operons was created based on the transcriptional data. Forty-two genes were upregulated anaerobically and 62 genes were downregulated anaerobically. The oxygen dependent gene expression of selected genes was further validated via qPCR. Many of the differentially regulated genes encode metabolic enzymes indicating broad metabolic adaptations with respect to oxygen availability. Genes showing the strongest oxygen-dependent adaption encoded nitrate (narGHJI) and nitrite (nirBD) reductases. Together with the observation that nitrate supported anaerobic growth, these data indicate that L. weihenstephanensis DSM 24698 performs anaerobic nitrate respiration. The wide overlap between the oxygen-dependent transcriptional regulation at 18 and 34°C suggest that temperature does not play a key role in the oxygen-dependent transcriptional regulation of L. weihenstephanensis DSM 24698.

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July 7, 2019

Adaptation of surface-associated bacteria to the open ocean: A genomically distinct subpopulation of Phaeobacter gallaeciensis Ccolonizes Pacific mesozooplankton.

The marine Roseobacter group encompasses numerous species which occupy a large variety of ecological niches. However, members of the genus Phaeobacter are specifically adapted to a surface-associated lifestyle and have so far been found nearly exclusively in disjunct, man-made environments including shellfish and fish aquacultures, as well as harbors. Therefore, the possible natural habitats, dispersal and evolution of Phaeobacter spp. have largely remained obscure. Applying a high-throughput cultivation strategy along a longitudinal Pacific transect, the present study revealed for the first time a widespread natural occurrence of Phaeobacter in the marine pelagial. These bacteria were found to be specifically associated to mesoplankton where they constitute a small but detectable proportion of the bacterial community. The 16S rRNA gene sequences of 18 isolated strains were identical to that of Phaeobacter gallaeciensis DSM26640(T) but sequences of internal transcribed spacer and selected genomes revealed that the strains form a distinct clade within P. gallaeciensis. The genomes of the Pacific and the aquaculture strains were highly conserved and had a fraction of the core genome of 89.6%, 80 synteny breakpoints, and differed 2.2% in their nucleotide sequences. Diversification likely occurred through neutral mutations. However, the Pacific strains exclusively contained two active Type I restriction modification systems which is commensurate with a reduced acquisition of mobile elements in the Pacific clade. The Pacific clade of P. gallaeciensis also acquired a second, homolog phosphonate transport system compared to all other P. gallaeciensis. Our data indicate that a previously unknown, distinct clade of P. gallaeciensis acquired a limited number of clade-specific genes that were relevant for its association with mesozooplankton and for colonization of the marine pelagial. The divergence of the Pacific clade most likely was driven by the adaptation to this novel ecological niche rather than by geographic isolation.

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July 7, 2019

Complete genome sequence and comparative analysis of Staphylococcus condimenti DSM 11674, a potential starter culture isolated from soy sauce mash.

Coagulase-negative staphylococci (CNS) are key players in the majority of food fermentation ecosystems, which are commonly found in the production of fermented meat and milk products (Blaiotta et al., 2005; Resch et al., 2008). Strains of CNS have been implicated in exerting desirable effects as components of a fermentation flora, such as color formation, aroma development, and shelf-life enhancement, and may therefore have the potential for future application as starter cultures (Zell et al., 2008). Staphylococcus condimenti is one of the most prominent species and has the potential for use in starter cultures for the production of fermented sausage and cured ham (Zell et al., 2008). S. condimenti DSM 11674 was originally isolated from fermenting soy sauce mash and suggested to be a new species in 1998 (Probst et al., 1998). However, S. condimenti has been found in a few clinical samples (Argemi et al., 2015; Misawa et al., 2015). Therefore, some concerns have been raised with regard to the safety of this species for use in food production (Zell et al., 2008; Seitter et al., 2011a,b). To further understand the biochemical and genetic characteristics of DSM 11674 and advance the potential biotechnological applications of this strain, we constructed the complete genome sequence of S. condimenti DSM 11674.

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July 7, 2019

Whole-genome sequence of Streptococcus tigurinus strain osk_001, isolated from postmortem material.

Streptococcus tigurinus was recently described as a novel species, and some strains are highly virulent. We detected S. tigurinus in infected tissue sampled by necropsy. In order to characterize and confirm the virulence of this species, whole-genome sequencing of the pure cultured bacterium was performed. We found that the strain has specific and unique genetic elements contained in highly virulent strains of S. tigurinus. Copyright © 2017 Yoshizawa et al.

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July 7, 2019

Rapid and affordable size-selected PacBio single-molecule real-time sequencing template library construction using the bead-beating DNA extraction method

This study demonstrated that bead-beating method facilitates a simple and rapid protocol for genomic DNA isolation for Pacific BioSciences (PacBio) sequencing with library construction of sufficient length. The protocol may also be beneficial for inactivating pathogens by simultaneous and instant DNA fragmentation, with no special equipment required to obtain large DNA fragments. This protocol was comparable in terms of quality to the standard protocol suggested by PacBioand represents an alternative, rapid shortcut for performing accurate PacBio sequencing.

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July 7, 2019

Complete genome analysis of Lactobacillus fermentum SK152 from kimchi reveals genes associated with its antimicrobial activity.

Research findings on probiotics highlight their importance in repressing harmful bacteria, leading to more extensive research on their potential applications. We analysed the genome of Lactobacillus fermentum SK152, which was isolated from the Korean traditional fermented vegetable dish kimchi, to determine the genetic makeup and genetic factors responsible for the antimicrobial activity of L. fermentum SK152 and performed a comparative genome analysis with other L. fermentum strains. The genome of L. fermentum SK152 was found to comprise a complete circular chromosome of 2092 273 bp, with an estimated GC content of 51.9% and 2184 open reading frames. It consisted of 2038 protein-coding genes and 73 RNA-coding genes. Moreover, a gene encoding a putative endolysin was found. A comparative genome analysis with other L. fermentum strains showed that SK152 is closely related to L. fermentum 3872 and F-6. An evolutionary analysis identified five positively selected genes that encode proteins associated with transport, survival and stress resistance. These positively selected genes may be essential for L. fermentum to colonise and survive in the stringent environment of the human gut and exert its beneficial effects. Our findings highlight the potential benefits of SK152.© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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July 7, 2019

Complete mitochondrial genome sequence of Aspergillus oryzae RIB 127 and its comparative analysis with related species

Here, we determined the complete sequence and annotation of the mitochondrial genome of A. oryzae (strain RIB 127). The complete mitochondrial genome is 29,202 base pairs (bp), with low GC content of 26.2%. Conserved genes identified include 26 transfer RNAs, the small and large ribosomal RNA subunits, and 14 protein-coding genes. Phylogenetic analysis based on the complete mitochondrial genome revealed that RIB 127 formed a single clade with two other A. oryzae species.

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July 7, 2019

Quantitative proteomics for the comprehensive analysis of stress responses of Lactobacillus paracasei subsp. paracasei F19.

Lactic acid bacteria are broadly employed as starter cultures in the manufacture of foods. Upon technological preparation, they are confronted with drying stress that amalgamates numerous stress conditions resulting in losses of fitness and survival. To better understand and differentiate physiological stress responses, discover general and specific markers for the investigated stress conditions, and predict optimal preconditioning for starter cultures, we performed a comprehensive genomic and quantitative proteomic analysis of a commonly used model system, Lactobacillus paracasei subsp. paracasei TMW 1.1434 (isogenic with F19) under 11 typical stress conditions, including among others oxidative, osmotic, pH, and pressure stress. We identified and quantified >1900 proteins in triplicate analyses, representing 65% of all genes encoded in the genome. The identified genes were thoroughly annotated in terms of subcellular localization prediction and biological functions, suggesting unbiased and comprehensive proteome coverage. In total, 427 proteins were significantly differentially expressed in at least one condition. Most notably, our analysis suggests that optimal preconditioning toward drying was predicted to be alkaline and high-pressure stress preconditioning. Taken together, we believe the presented strategy may serve as a prototypic example for the analysis and utility of employing quantitative-mass-spectrometry-based proteomics to study bacterial physiology.

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July 7, 2019

The Tartary buckwheat genome provides insights into rutin biosynthesis and abiotic stress tolerance.

Tartary buckwheat (Fagopyrum tataricum) is an important pseudocereal crop that is strongly adapted to growth in adverse environments. Its gluten-free grain contains complete proteins with a well-balanced composition of essential amino acids and is a rich source of beneficial phytochemicals that provide significant health benefits. Here, we report a high-quality, chromosome-scale Tartary buckwheat genome sequence of 489.3 Mb that is assembled by combining whole-genome shotgun sequencing of both Illumina short reads and single-molecule real-time long reads, sequence tags of a large DNA insert fosmid library, Hi-C sequencing data, and BioNano genome maps. We annotated 33 366 high-confidence protein-coding genes based on expression evidence. Comparisons of the intra-genome with the sugar beet genome revealed an independent whole-genome duplication that occurred in the buckwheat lineage after they diverged from the common ancestor, which was not shared with rosids or asterids. The reference genome facilitated the identification of many new genes predicted to be involved in rutin biosynthesis and regulation, aluminum stress resistance, and in drought and cold stress responses. Our data suggest that Tartary buckwheat’s ability to tolerate high levels of abiotic stress is attributed to the expansion of several gene families involved in signal transduction, gene regulation, and membrane transport. The availability of these genomic resources will facilitate the discovery of agronomically and nutritionally important genes and genetic improvement of Tartary buckwheat. Copyright © 2017 The Author. Published by Elsevier Inc. All rights reserved.

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July 7, 2019

Analysis of resistance genes in pan-resistant Myroides odoratimimus clinical strain PR63039 using whole genome sequencing.

To clarify the antibiotic resistance mechanisms of Myroides odoratimimus, pan-resistant M. odoratimimus strain PR63039 was isolated and its genome sequenced and analyzed. Antimicrobial susceptibility testing was conducted using the Kirby-Bauer disk diffusion method, and the Phoenix-100 Automated Microbiology System with a NMIC/ID-4 panel including aminoglycosides, ß-lactams, polypeptides, quinolones, sulfonamides, chloramphenicols, and tetracyclines. Single-molecule real-time whole genome sequencing was conducted using the PacBio RSII system, and genome annotation was performed using RAST and IMG ER. To characterize the genome features, a number of databases and software programs, including GC-Profile, CG viewer, the VFDB database, ISfinder, RADB, CARD, ResFinder, and PHAST, were used. M. odoratimimus isolate PR63039 was resistant to almost all antibiotics tested, suggesting pan-drug resistance. The genome consisted of a 4,366,950-bp chromosome and a 90,798-bp plasmid (p63039), which contained a large number of resistance genes and virulence factors. The distribution of the resistance genes was distinctive, and a resistance region, designated MY63039-RR, was identified. RAST analysis indicated that 108 of the annotated genes were potentially involved in virulence, disease, and defense, all of which could be associated with resistance and pathogenicity. Prophage analysis also identified two incomplete prophages in the genome of M. odoratimimus PR63039. Multiple antibiotic-resistance genes were identified, including those associated with resistance to tetracycline (tetX), macrolides (ereB, cfrA, lasE), sulfonamides (sul2, sul3), ß-lactams (blaMUS-1, blaTUS-1, blaSFB-1, blaSLB-1, blaOXA-209, blaOXA-347), and chloramphenicol (cat). Further, the presence of 18 antibiotic efflux pump-encoding resistance genes, including acrB, acrD, acrF, adeB, adeG, adeJ, amrB, ceoB, cmeB, mdsB, mexB, mexD, mexF, mtrD, smeE, mdtF, macB, likely accounts for the observed quinolone resistance of strain PR63039. To the best of our knowledge, this is the first report of the presence of the blaSFB-1, blaSLB-1, blaOXA-209, blaOXA-347, and tetX resistance genes in M. odoratimimus. Copyright © 2017 Elsevier Ltd. All rights reserved.

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