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July 19, 2019  |  

Whole genome complete resequencing of Bacillus subtilis natto by combining long reads with high-quality short reads.

De novo microbial genome sequencing reached a turning point with third-generation sequencing (TGS) platforms, and several microbial genomes have been improved by TGS long reads. Bacillus subtilis natto is closely related to the laboratory standard strain B. subtilis Marburg 168, and it has a function in the production of the traditional Japanese fermented food “natto.” The B. subtilis natto BEST195 genome was previously sequenced with short reads, but it included some incomplete regions. We resequenced the BEST195 genome using a PacBio RS sequencer, and we successfully obtained a complete genome sequence from one scaffold without any gaps, and we also applied Illumina MiSeq short reads to enhance quality. Compared with the previous BEST195 draft genome and Marburg 168 genome, we found that incomplete regions in the previous genome sequence were attributed to GC-bias and repetitive sequences, and we also identified some novel genes that are found only in the new genome.

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July 19, 2019  |  

Selections that isolate recombinant mitochondrial genomes in animals.

Homologous recombination is widespread and catalyzes evolution. Nonetheless, its existence in animal mitochondrial DNA is questioned. We designed selections for recombination between co-resident mitochondrial genomes in various heteroplasmic Drosophila lines. In four experimental settings, recombinant genomes became the sole or dominant genome in the progeny. Thus, selection uncovers occurrence of homologous recombination in Drosophila mtDNA and documents its functional benefit. Double-strand breaks enhanced recombination in the germ line and revealed somatic recombination. When the recombination partner was a diverged D. melanogaster genome or a genome from a different species such as D. yakuba, sequencing revealed long continuous stretches of exchange. In addition, the distribution of sequence polymorphisms in recombinants allowed us to map a selected trait to a particular region in the Drosophila mitochondrial genome. Thus, recombination can be harnessed to dissect function and evolution of mitochondrial genome.

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July 19, 2019  |  

Mitotic intragenic recombination: A mechanism of survival for several congenital disorders of glycosylation.

Congenital disorders of glycosylation (CDGs) are disorders of abnormal protein glycosylation that affect multiple organ systems. Because most CDGs have been described in only a few individuals, our understanding of the associated phenotypes and the mechanisms of individual survival are limited. In the process of studying two siblings, aged 6 and 11 years, with MOGS-CDG and biallelic MOGS (mannosyl-oligosaccharide glucosidase) mutations (GenBank: NM_006302.2; c.[65C>A; 329G>A] p.[Ala22Glu; Arg110His]; c.[370C>T] p.[Gln124(*)]), we noted that their survival was much longer than the previous report of MOGS-CDG, in a child who died at 74 days of age. Upon mutation analysis, we detected multiple MOGS genotypes including wild-type alleles in their cultured fibroblast and peripheral blood DNA. Further analysis of DNA from cultured fibroblasts of six individuals with compound heterozygous mutations of PMM2 (PMM2-CDG), MPI (MPI-CDG), ALG3 (ALG3-CDG), ALG12 (ALG12-CDG), DPAGT1 (DPAGT1-CDG), and ALG1 (ALG1-CDG) also identified multiple genotypes including wild-type alleles for each. Droplet digital PCR showed a ratio of nearly 1:1 wild-type to mutant alleles for most, but not all, mutations. This suggests that mitotic recombination contributes to the survival and the variable expressivity of individuals with compound heterozygous CDGs. This also provides an explanation for prior observations of a reduced frequency of homozygous mutations and might contribute to increased levels of residual enzyme activity in cultured fibroblasts of individuals with MPI- and PMM2-CDGs. Copyright © 2016 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

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July 19, 2019  |  

Single-molecule sequencing reveals complex genomic variation of hepatitis B virus during 15 years of chronic infection following liver transplantation.

Chronic hepatitis B (CHB) is prevalent worldwide. The infectious agent, hepatitis B virus (HBV) replicates via an RNA intermediate and is error-prone, leading to rapid generation of closely related but not identical viral variants, including those that can escape host immune responses and antiviral treatments. The complexity of CHB can be further enhanced by the presence of HBV variants with large deletions in the genome, generated via splicing (spHBV). Although spHBV variants are incapable of autonomous replication, their replication is rescued by wild-type HBV. SpHBV variants have been shown to enhance wild-type virus replication, and their prevalence increases with liver disease progression. Single-molecule deep sequencing was performed on whole HBV genomes extracted from longitudinal samples of a post-liver transplant CHB subject, collected over a 15-year period that included the liver explant. By employing novel bioinformatics methods, this analysis showed a complex dynamics of the viral population across a period of changing treatment regimens. The spHBV detected in the liver explant remained present post-transplantation, along with emergence of a highly diverse novel spHBV population as well as variants with multiple deletions in the preS genes. The identification of novel mutations outside the HBV reverse transcriptase gene that co-occur with known drug resistant mutations, highlight the relevance of using full genome deep sequencing and support the hypothesis that drug resistance involves interactions across the full-length HBV genome.Single-molecule sequencing allowed characterising, in unprecedented detail, the evolution of HBV populations and offered unique insights into the dynamics of defective and spHBV variants following liver transplantation and complex treatment regimes. This analysis also showed rapid adaptation of HBV populations to treatment regimens with evolving drug resistance phenotypes and evidence of purifying selection across the whole genome. Finally, the new open source bioinformatics tools are freely available, with the capacity to easily identify potential spliced variants from deep sequencing data. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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July 19, 2019  |  

Separate F-type plasmids have shaped the evolution of the H30 subclone of Escherichia coli sequence type 131.

The extraintestinal pathogenic Escherichia coli (ExPEC) H30 subclone of sequence type 131 (ST131-H30) has emerged abruptly as a dominant lineage of ExPEC responsible for human disease. The ST131-H30 lineage has been well described phylogenetically, yet its plasmid complement is not fully understood. Here, single-molecule, real-time sequencing was used to generate the complete plasmid sequences of ST131-H30 isolates and those belonging to other ST131 clades. Comparative analyses revealed separate F-type plasmids that have shaped the evolution of the main fluoroquinolone-resistant ST131-H30 clades. Specifically, an F1:A2:B20 plasmid is strongly associated with the H30R/C1 clade, whereas an F2:A1:B- plasmid is associated with the H30Rx/C2 clade. A series of plasmid gene losses, gains, and rearrangements involving IS26 likely led to the current plasmid complements within each ST131-H30 sublineage, which contain several overlapping gene clusters with putative functions in virulence and fitness, suggesting plasmid-mediated convergent evolution. Evidence suggests that the H30Rx/C2-associated F2:A1:B- plasmid type was present in strains ancestral to the acquisition of fluoroquinolone resistance and prior to the introduction of a multidrug resistance-encoding gene cassette harboring bla CTX-M-15. In vitro experiments indicated a host strain-independent low frequency of plasmid transfer, differential levels of plasmid stability even between closely related ST131-H30 strains, and possible epistasis for carriage of these plasmids within the H30R/Rx lineages. IMPORTANCE A clonal lineage of Escherichia coli known as ST131 has emerged as a dominating strain type causing extraintestinal infections in humans. The evolutionary history of ST131 E. coli is now well understood. However, the role of plasmids in ST131’s evolutionary history is poorly defined. This study utilized real-time, single-molecule sequencing to compare plasmids from various current and historical lineages of ST131. From this work, it was determined that a series of plasmid gains, losses, and recombinational events has led to the currently circulating plasmids of ST131 strains. These plasmids appear to have evolved to acquire similar gene clusters on multiple occasions, suggesting possible plasmid-mediated convergent evolution leading to evolutionary success. These plasmids also appear to be better suited to exist in specific strains of ST131 due to coadaptive mutations. Overall, a series of events has enabled the evolution of ST131 plasmids, possibly contributing to the lineage’s success.

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July 7, 2019  |  

Accumulation-associated protein enhances Staphylococcus epidermidis biofilm formation under dynamic conditions and is required for infection in a rat catheter model.

Biofilm formation is the primary virulence factor of Staphylococcus epidermidis. S. epidermidis biofilms preferentially form on abiotic surfaces and may contain multiple matrix components, including proteins such as accumulation-associated protein (Aap). Following proteolytic cleavage of the A domain, which has been shown to enhance binding to host cells, B domain homotypic interactions support cell accumulation and biofilm formation. To further define the contribution of Aap to biofilm formation and infection, we constructed an aap allelic replacement mutant and an icaADBC aap double mutant. When subjected to fluid shear, strains deficient in Aap production produced significantly less biofilm than Aap-positive strains. To examine the in vivo relevance of our findings, we modified our previously described rat jugular catheter model and validated the importance of immunosuppression and the presence of a foreign body to the establishment of infection. The use of our allelic replacement mutants in the model revealed a significant decrease in bacterial recovery from the catheter and the blood in the absence of Aap, regardless of the production of polysaccharide intercellular adhesin (PIA), a well-characterized, robust matrix molecule. Complementation of the aap mutant with full-length Aap (containing the A domain), but not the B domain alone, increased initial attachment to microtiter plates, as did in trans expression of the A domain in adhesion-deficient Staphylococcus carnosus. These results demonstrate Aap contributes to S. epidermidis infection, which may in part be due to A domain-mediated attachment to abiotic surfaces. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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July 7, 2019  |  

Genomic analysis of 495 vancomycin-resistant Enterococcus faecium reveals broad dissemination of a vanA plasmid in more than 19 clones from Copenhagen, Denmark.

From 2012 to 2014, there has been a huge increase in vancomycin-resistant (vanA) Enterococcus faecium (VREfm) in Copenhagen, Denmark, with 602 patients infected or colonized with VREfm in 2014 compared with just 22 in 2012. The objective of this study was to describe the genetic epidemiology of VREfm to assess the contribution of clonal spread and horizontal transfer of the vanA transposon (Tn1546) and plasmid in the dissemination of VREfm in hospitals.VREfm from Copenhagen, Denmark (2012-14) were whole-genome sequenced. The clonal structure was determined and the structure of Tn1546-like transposons was characterized. One VREfm isolate belonging to the largest clonal group was sequenced using long-read technology to close a 37 kb vanA plasmid.Phylogeny revealed a polyclonal structure where 495 VREfm isolates were divided into 13 main groups and 7 small groups. The majority of the isolates were located in three groups (n?=?44, 100 and 218) and clonal spread of VREfm between wards and hospitals was identified. Five Tn1546-like transposon types were identified. A dominant truncated transposon (type 4, 92%) was spread across all but one VREfm group. The closed vanA plasmid was highly covered by reads from isolates containing the type 4 transposon.This study suggests that it was the dissemination of the type 4 Tn1546-like transposon and plasmid via horizontal transfer to multiple populations of E. faecium, followed by clonal spread of new VREfm clones, that contributed to the increase in and diversity of VREfm in Danish hospitals.© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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July 7, 2019  |  

Assessment of bacterial profiles in aged, home-made Sichuan paocai brine with varying titratable acidity by PacBio SMRT Sequencing technology

Sichuan paocai, a traditional Chinese fermented vegetable, is rife with lactic acid bacteria (LAB). However, the precise bacterial profiles of home-made Sichuan paocai brine (HSPB) remain unclear. In this study, the bacterial compositions of 38 aged HSPB samples with varying titratable acidity (TA) were determined by SMRT sequencing of the full-length 16S rRNA gene. The lactic and acetic acids of HSPBs were also measured to determine any relevance with the bacterial profiles. The SMRT sequencing results reveal that the HSPB bacterial communities were comprised of numerous phylogenetic taxa, including 35 phyla, 371 genera, and 593 species; the bacterial diversity decreased as HSPB acidity increased. Lactobacillus acetotolerans, which was positively correlated to HSPB acidity, was the most dominant species followed by Lactobacillus brevis, which was positively related to acetic acid in the samples. A few opportunistic pathogens (e.g. Serratia marcescens and Stenotrophomonas maltophilia) were also detected. Sample groups with lower acidity had higher bacterial diversity and more Lactobacillus species with relative abundance >1% and opportunistics than higher-acidity samples. The results presented here report the comprehensive bacterial profiles of home-made Sichuan paocai for the first time via SMRT sequencing technology and the correlation between TA and bacterial compositions. It is necessary to further investigate the opportunistics detected in this work as they relate to the safety and quality of paocai.

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July 7, 2019  |  

Complete genome sequence of Streptococcus thermophilus KLDS 3.1003,a strain with high antimicrobial potential against foodborne and vaginal pathogens.

Lactic acid bacteria play increasingly important roles in the food industry. Streptococcus thermophilus KLDS 3.1003 strain was isolated from traditional yogurt in Inner Mongolia, China. It has shown high antimicrobial activity against selected foodborne and vaginal pathogens. In this study, we investigated and analyzed its complete genome sequence. The S. thermophilus KLDS 3.1003 genome comprise of a 1,899,956 bp chromosome with a G+C content of 38.92%, 1,995 genes, and 6 rRNAs. With the exception of S. thermophilus M17TZA496, S. thermophilus KLDS 3.1003 has more tRNAs (amino acid coding genes) compared to some S. thermophilus strains available on the National Centre for Biotechnology Information database. MG-RAST annotation showed that this strain has 317 subsystems with most genes associated with amino acid and carbohydrate metabolism. This strain also has a unique EPS gene cluster containing 23 genes, and may be a mixed dairy starter culture. This information provides more insight into the molecular basis of its potentials for further applications in the dairy and allied industries.

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July 7, 2019  |  

Towards systems metabolic engineering in Pichia pastoris.

The methylotrophic yeast Pichia pastoris is firmly established as a host for the production of recombinant proteins, frequently outperforming other heterologous hosts. Already, a sizeable amount of systems biology knowledge has been acquired for this non-conventional yeast. By applying various omics-technologies, productivity features have been thoroughly analyzed and optimized via genetic engineering. However, challenging clonal variability, limited vector repertoire and insufficient genome annotation have hampered further developments. Yet, in the last few years a reinvigorated effort to establish P. pastoris as a host for both protein and metabolite production is visible. A variety of compounds from terpenoids to polyketides have been synthesized, often exceeding the productivity of other microbial systems. The clonal variability was systematically investigated and strategies formulated to circumvent untargeted events, thereby streamlining the screening procedure. Promoters with novel regulatory properties were discovered or engineered from existing ones. The genetic tractability was increased via the transfer of popular manipulation and assembly techniques, as well as the creation of new ones. A second generation of sequencing projects culminated in the creation of the second best functionally annotated yeast genome. In combination with landmark physiological insights and increased output of omics-data, a good basis for the creation of refined genome-scale metabolic models was created. The first application of model-based metabolic engineering in P. pastoris showcased the potential of this approach. Recent efforts to establish yeast peroxisomes for compartmentalized metabolite synthesis appear to fit ideally with the well-studied high capacity peroxisomal machinery of P. pastoris. Here, these recent developments are collected and reviewed with the aim of supporting the establishment of systems metabolic engineering in P. pastoris. Copyright © 2017. Published by Elsevier Inc.

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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  |  

Rapid genetic and developmental morphological change following extreme celerity

Proximate environmental effects on metamorphosis have been explored in many vertebrate systems, but less attention has been devoted to how the environment affects developmental morphological change in mammals. Understanding proximate environmental effects on mammalian morphological change, particularly changes involving skin replacement, may aid in the design of therapeutic strategies to address severe burn or other debilitating injuries. Here, we specifically explore effects of celerity broadly, and we present results showing rapid change in mammalian morphological development following encountering maximum celerity. Morphological changes were pronounced within 96 hours and included at least partial regeneration of skin and organs as well as an elevated somatic mutation rate. Significantly, this high mutation rate did not result in detectable loss of fertility or viability of offspring. Overall, our findings strongly suggest that extreme celerity, an environmental factor rarely considered, can produce strikingly rapid developmental changes in morphology even in mammalian systems and open the door to future studies on the impact of celerity on genetics and morphology.

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July 7, 2019  |  

Development of Streptomyces sp. FR-008 as an emerging chassis

Microbial-derived natural products are important in both the pharmaceutical industry and academic research. As the metabolic potential of original producer especially Streptomyces is often limited by slow growth rate, complicated cultivation profile, and unfeasible genetic manipulation, so exploring a Streptomyces as a super industrial chassis is valuable and urgent. Streptomyces sp. FR-008 is a fast-growing microorganism and can also produce a considerable amount of macrolide candicidin via modular polyketide synthase. In this study, we evaluated Streptomyces sp. FR-008 as a potential industrial-production chassis. First, PacBio sequencing and transcriptome analyses indicated that the Streptomyces sp. FR-008 genome size is 7.26 Mb, which represents one of the smallest of currently sequenced Streptomyces genomes. In addition, we simplified the conjugation procedure without heat-shock and pre-germination treatments but with high conjugation efficiency, suggesting it is inherently capable of accepting heterologous DNA. In addition, a series of promoters selected from literatures was assessed based on GusA activity in Streptomyces sp. FR-008. Compared with the common used promoter ermE*-p, the strength of these promoters comprise a library with a constitutive range of 60–860%, thus providing the useful regulatory elements for future genetic engineering purpose. In order to minimum the genome, we also target deleted three endogenous polyketide synthase (PKS) gene clusters to generate a mutant LQ3. LQ3 is thus an “updated” version of Streptomyces sp. FR-008, producing fewer secondary metabolites profiles than Streptomyces sp. FR-008. We believe this work could facilitate further development of Streptomyces sp. FR-008 for use in biotechnological applications.

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July 7, 2019  |  

Dissection of exopolysaccharide biosynthesis in Kozakia baliensis.

Acetic acid bacteria (AAB) are well known producers of commercially used exopolysaccharides, such as cellulose and levan. Kozakia (K.) baliensis is a relatively new member of AAB, which produces ultra-high molecular weight levan from sucrose. Throughout cultivation of two K. baliensis strains (DSM 14400, NBRC 16680) on sucrose-deficient media, we found that both strains still produce high amounts of mucous, water-soluble substances from mannitol and glycerol as (main) carbon sources. This indicated that both Kozakia strains additionally produce new classes of so far not characterized EPS.By whole genome sequencing of both strains, circularized genomes could be established and typical EPS forming clusters were identified. As expected, complete ORFs coding for levansucrases could be detected in both Kozakia strains. In K. baliensis DSM 14400 plasmid encoded cellulose synthase genes and fragments of truncated levansucrase operons could be assigned in contrast to K. baliensis NBRC 16680. Additionally, both K. baliensis strains harbor identical gum-like clusters, which are related to the well characterized gum cluster coding for xanthan synthesis in Xanthomanas campestris and show highest similarity with gum-like heteropolysaccharide (HePS) clusters from other acetic acid bacteria such as Gluconacetobacter diazotrophicus and Komagataeibacter xylinus. A mutant strain of K. baliensis NBRC 16680 lacking EPS production on sucrose-deficient media exhibited a transposon insertion in front of the gumD gene of its gum-like cluster in contrast to the wildtype strain, which indicated the essential role of gumD and of the associated gum genes for production of these new EPS. The EPS secreted by K. baliensis are composed of glucose, galactose and mannose, respectively, which is in agreement with the predicted sugar monomer composition derived from in silico genome analysis of the respective gum-like clusters.By comparative sugar monomer and genome analysis, the polymeric substances secreted by K. baliensis can be considered as unique HePS. Via genome sequencing of K. baliensis DSM 14400 + NBRC 16680 we got first insights into the biosynthesis of these novel HePS, which is related to xanthan and acetan biosynthesis. Consequently, the present study provides the basis for establishment of K. baliensis strains as novel microbial cell factories for biotechnologically relevant, unique polysaccharides.

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