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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.


July 7, 2019

Genomics of Helicobacter pylori

As Helicobacter pylori infects half the world’s population and displays an extensive intraspecies diversity, genomics is a powerful tool to understand evolution and disease, to identify factors that confer higher risk of severe sequelae, and to find new approaches for therapy both among bacterial and host targets. In line with these objectives, this review article summarizes the major findings in Helicobacter genomics in papers published between April 2016 and March 2017.


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.


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.


July 7, 2019

Evaluation of nine popular de novo assemblers in microbial genome assembly.

Next generation sequencing (NGS) technologies are revolutionizing biology, with Illumina being the most popular NGS platform. Short read assembly is a critical part of most genome studies using NGS. Hence, in this study, the performance of nine well-known assemblers was evaluated in the assembly of seven different microbial genomes. Effect of different read coverage and k-mer parameters on the quality of the assembly were also evaluated on both simulated and actual read datasets. Our results show that the performance of assemblers on real and simulated datasets could be significantly different, mainly because of coverage bias. According to outputs on actual read datasets, for all studied read coverages (of 7×, 25× and 100×), SPAdes and IDBA-UD clearly outperformed other assemblers based on NGA50 and accuracy metrics. Velvet is the most conservative assembler with the lowest NGA50 and error rate. Copyright © 2017. Published by Elsevier B.V.


July 7, 2019

Whole-genome sequencing identification of a multidrug-resistant Salmonella enterica serovar Typhimurium strain carrying blaNDM-5 from Guangdong, China.

A carbapenem-resistant Salmonella enterica serovar Typhimurium (sequence type 34 [ST34]) strain was isolated from a fecal specimen from a child with acute diarrhea. Whole-genome sequencing revealed that the 84.5-kb IncFII plasmid pST41-NDM carrying the NDM-5 carbapenemase gene possesses a structure identical to that of the IncFII-type plasmid backbone. However, the blaNDM-5 flanking sequence found in this plasmid is identical to the blaNDM-5-positive IncX3 plasmids carried by 10 strains of Enterobacteriaceae identified in the same hospital. Copyright © 2017 Elsevier B.V. All rights reserved.


July 7, 2019

The complete genome sequence of Streptomyces albolongus YIM 101047, the producer of novel bafilomycins and odoriferous sesquiterpenoids.

Streptomyces albolongus YIM 101047 produces novel bafilomycins and odoriferous sesquiterpenoids with cytotoxic and antimicrobial activities. Here, we report the complete genome sequence of S. albolongus YIM 101047, which consists of an 8,027,788bp linear chromosome. Forty-six putative biosynthetic gene clusters of secondary metabolites were found. The sesquiterpenoid gene cluster was on the left arm (0.09-0.10Mb), and the bafilomycin biosynthetic gene cluster was on the right arm (7.46-7.64Mb) of the chromosome. Twenty-two putative gene clusters with high or moderate similarity to important antibiotic biosynthetic gene clusters were found, including the antitumor agents bafilomycin, epothilone and hedamycin; the antibacterial/antifungal agents clavulanic acid, collismycin A, frontalamides, kanamycin, streptomycin and streptothricin; the protein phosphatase inhibitor RK-682; and the acute iron poisoning medication desferrioxamine B. The genome sequence reported here will enable us to study the biosynthetic mechanism of these important antibiotics and will facilitate the discovery of novel secondary metabolites with potential applications to human health. Copyright © 2017 Elsevier B.V. All rights reserved.


July 7, 2019

Complete genome sequence of endophyte Bacillus flexus KLBMP 4941 reveals its plant growth promotion mechanism and genetic basis for salt tolerance.

Bacillus flexus KLBMP 4941 is a halotolerant endophyte isolated from the halophyte Limonium sinense. This strain can improve host seedling growth under salt stress conditions. We here report the complete genome information of endophyte KLBMP 4941. It has a circular chromosome and two plasmids for a total genome 4,104,242 bp in size with a G+C content of 38.09%. Genes related to plant growth promotion (PGP), such as those associated with nitrogen fixation, siderophore, spermidine, and acetoin synthesis were found in the KLBMP 4941 genome. Some genes responsible for high salinity tolerance, like genes associated with the Na(+)/H(+) antiporter, glycine betaine transporter, and betaine-aldehyde dehydrogenase were also found in the KLBMP 4941 genome. The genome analysis will provide better understanding of the mechanisms underlying the promotion of plant growth in strain KLBMP 4941 under salt stress conditions and its ability to adapt to coastal salt marsh habitats, and provide a basis for its further biotechnological applications in agriculture. Copyright © 2017 Elsevier B.V. All rights reserved.


July 7, 2019

Comparative whole genome analysis of three consecutive Salmonella diarizonae isolates.

Infections of very young children or immunocompromised people with Salmonella of higher subspecies are a well-known phenomenon often associated with contact to cold-blooded animals. We describe the molecular characterization of three S. enterica subsp. diarizonae strains, isolated consecutively over a period of several months from a hospital patient suffering from diarrhea and sepsis with fatal outcome. With the initial isolate the first complete genome sequence of a member of subsp. diarizonae is provided and based on this reference we revealed the genomic differences between the three isolates by use of next-generation sequencing and confirmed by phenotypical tests. Genome comparisons revealed mutations within gpt, hfq and purK in the first isolate as a sign of clonal variation rather than host-directed evolution. Furthermore, our work demonstrates that S. enterica subsp. diarizonae possess, besides a conserved set of known Salmonella Pathogenicity Islands, a variable portfolio of additional genomic islands of unknown function. Copyright © 2017 Elsevier GmbH. All rights reserved.


July 7, 2019

Genomic insights of Pannonibacter phragmitetus strain 31801 isolated from a patient with a liver abscess.

Pannonibacter phragmitetus is a bioremediation reagent for the detoxification of heavy metals and polycyclic aromatic compounds (PAHs) while it rarely infects healthy populations. However, infection by the opportunistic pathogen P. phragmitetus complicates diagnosis and treatments, and poses a serious threat to immunocompromised patients owing to its multidrug resistance. Unfortunately, genome features, antimicrobial resistance, and virulence potentials in P. phragmitetus have not been reported before. A predominant colony (31801) was isolated from a liver abscess patient, indicating that it accounted for the infection. To investigate its infection mechanism(s) in depth, we sequenced this bacterial genome and tested its antimicrobial resistance. Average nucleotide identity (ANI) analysis assigned the bacterium to the species P. phragmitetus (ANI, >95%). Comparative genomics analyses among Pannonibacter spp. representing the different living niches were used to describe the Pannonibacter pan-genomes and to examine virulence factors, prophages, CRISPR arrays, and genomic islands. Pannonibacter phragmitetus 31801 consisted of one chromosome and one plasmid, while the plasmid was absent in other Pannonibacter isolates. Pannonibacter phragmitetus 31801 may have a great infection potential because a lot of genes encoding toxins, flagellum formation, iron uptake, and virulence factor secretion systems in its genome. Moreover, the genome has 24 genomic islands and 2 prophages. A combination of antimicrobial susceptibility tests and the detailed antibiotic resistance gene analysis provide useful information about the drug resistance mechanisms and therefore can be used to guide the treatment strategy for the bacterial infection.© 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.


July 7, 2019

Simultaneous production of Anabaenopeptins and Namalides by the cyanobacterium Nostoc sp. CENA543.

Anabaenopeptins are a diverse group of cyclic peptides, which contain an unusual ureido linkage. Namalides are shorter structural homologues of anabaenopeptins, which also contain an ureido linkage. The biosynthetic origins of namalides are unknown despite a strong resemblance to anabaenopeptins. Here, we show the cyanobacterium Nostoc sp. CENA543 strain producing new (nostamide B-E (2, 4, 5, and 6)) and known variants of anabaenopeptins (schizopeptin 791 (1) and anabaenopeptin 807 (3)). Surprisingly, Nostoc sp. CENA543 also produced namalide B (8) and the new namalides D (7), E (9), and F (10) in similar amounts to anabaenopeptins. Analysis of the complete Nostoc sp. CENA543 genome sequence indicates that both anabaenopeptins and namalides are produced by the same biosynthetic pathway through module skipping during biosynthesis. This unique process involves the skipping of two modules present in different nonribosomal peptide synthetases during the namalide biosynthesis. This skipping is an efficient mechanism since both anabaenopeptins and namalides are synthesized in similar amounts by Nostoc sp. CENA543. Consequently, gene skipping may be used to increase and possibly broaden the chemical diversity of related peptides produced by a single biosynthetic gene cluster. Genome mining demonstrated that the anabaenopeptin gene clusters are widespread in cyanobacteria and can also be found in tectomicrobia bacteria.


July 7, 2019

Complete genetic analysis of a Salmonella enterica serovar Indiana isolate accompanying four plasmids carrying mcr-1, ESBL and other resistance genes in China

One mcr-1-carrying Salmonella enterica serovar Indiana strain D90, was identified from 1320 Salmonella enterica isolates from poultry slaughterhouse in 2012 in China. The objective of this study was to verify the transferability of the mcr-1 gene and also completely characterize the sequence of the strain at the whole-genome level. Broth matting assays were carried out to detect the transferability and whole-genome sequencing (WGS) of S. enterica serovar Indiana D90 was performed using the PacBio RS II system. Open reading frames were assigned using Rapid Annotation using Subsystem Technology (RAST) and analysed by BLASTn and BLASTp. Salmonella Pathogenisity Islands (SPIs) were annotated by SPIFinder platform. The complete genome sequence of S. enterica serovar Indiana D90 contained a circular 4,779,514-bp chromosome and four plasmids. Genome analysis and sequencing revealed that 24 multi-drug resistance (MDR) genes were located on plasmids. The largest plasmid pD90-1, was found to be of an IncHI2/HI2A/Q1/N type that encoded a blaCTX-M-65 gene along with 20 additional antimicrobial resistance genes. A 60.5-kbp IncI2 plasmid pD90-2 contained a nikA-nikB-mcr-1 genetic structure, that can be successfully transferred to E. coli and S. enterica serovar Typhimurium at low transfer rates. Interestingly, comparative sequence analysis revealed the plasmids pD90-1 and pD90-2 showed considerable nucleotide similarity to pHNSHP45-2 and pHNSHP45, respectively. Moreover, the genome and the plasmid pD90-2 also showed high similarity to one carbapenem resistant S. enterica serovar Indiana strain, C629 and its plasmid pC629, respectively. This is the first report of the complete nucleotide sequence of one mcr-1-carrying MDR S. enterica serovar Indiana strain.


July 7, 2019

Fluorescent CRISPR Adaptation Reporter for rapid quantification of spacer acquisition.

CRISPR-Cas systems are adaptive prokaryotic immune systems protecting against horizontally transferred DNA or RNA such as viruses and other mobile genetic elements. Memory of past invaders is stored as spacers in CRISPR loci in a process called adaptation. Here we developed a novel assay where spacer integration results in fluorescence, enabling detection of memory formation in single cells and quantification of as few as 0.05% cells with expanded CRISPR arrays in a bacterial population. Using this fluorescent CRISPR Adaptation Reporter (f-CAR), we quantified adaptation of the two CRISPR arrays of the type I-E CRISPR-Cas system in Escherichia coli, and confirmed that more integration events are targeted to CRISPR-II than to CRISPR-I. The f-CAR conveniently analyzes and compares many samples, allowing new insights into adaptation. For instance, we show that in an E. coli culture the majority of acquisition events occur in late exponential phase.


July 7, 2019

Methylation-dependent DNA discrimination in natural transformation of Campylobacter jejuni.

Campylobacter jejuni, a leading cause of bacterial gastroenteritis, is naturally competent. Like many competent organisms, C. jejuni restricts the DNA that can be used for transformation to minimize undesirable changes in the chromosome. Although C. jejuni can be transformed by C. jejuni-derived DNA, it is poorly transformed by the same DNA propagated in Escherichia coli or produced with PCR. Our work indicates that methylation plays an important role in marking DNA for transformation. We have identified a highly conserved DNA methyltransferase, which we term Campylobacter transformation system methyltransferase (ctsM), which methylates an overrepresented 6-bp sequence in the chromosome. DNA derived from a ctsM mutant transforms C. jejuni significantly less well than DNA derived from ctsM(+) (parental) cells. The ctsM mutation itself does not affect transformation efficiency when parental DNA is used, suggesting that CtsM is important for marking transforming DNA, but not for transformation itself. The mutant has no growth defect, arguing against ongoing restriction of its own DNA. We further show that E. coli plasmid and PCR-derived DNA can efficiently transform C. jejuni when only a subset of the CtsM sites are methylated in vitro. A single methylation event 1 kb upstream of the DNA involved in homologous recombination is sufficient to transform C. jejuni, whereas otherwise identical unmethylated DNA is not. Methylation influences DNA uptake, with a slight effect also seen on DNA binding. This mechanism of DNA discrimination in C. jejuni is distinct from the DNA discrimination described in other competent bacteria.


July 7, 2019

Genetic characterization of blaNDM-harboring plasmids in carbapenem-resistant Escherichia coli from Myanmar.

The bacterial enzyme New Delhi metallo-ß-lactamase hydrolyzes almost all ß-lactam antibiotics, including carbapenems, which are drugs of last resort for severe bacterial infections. The spread of carbapenem-resistant Enterobacteriaceae that carry the New Delhi metallo-ß-lactamase gene, blaNDM, poses a serious threat to public health. In this study, we genetically characterized eight carbapenem-resistant Escherichia coli isolates from a tertiary care hospital in Yangon, Myanmar. The eight isolates belonged to five multilocus-sequence types and harbored multiple antimicrobial-resistance genes, resulting in resistance against nearly all of the antimicrobial agents tested, except colistin and fosfomycin. Nine plasmids harboring blaNDM genes were identified from these isolates. Multiple blaNDM genes were found in the distinct Inc-replicon types of the following plasmids: an IncA/C2 plasmid harboring blaNDM-1 (n = 1), IncX3 plasmids harboring blaNDM-4 (n = 2) or blaNDM-7 (n = 1), IncFII plasmids harboring blaNDM-4 (n = 1) or blaNDM-5 (n = 3), and a multireplicon F plasmid harboring blaNDM-5 (n = 1). Comparative analysis highlighted the diversity of the blaNDM-harboring plasmids and their distinct characteristics, which depended on plasmid replicon types. The results indicate circulation of phylogenetically distinct strains of carbapenem-resistant E. coli with various plasmids harboring blaNDM genes in the hospital.


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