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Asset Tag: Bacterial genome

September 22, 2019

Comparative genomic analysis reveals the evolution and environmental adaptation strategies of vibrios.

Vibrios are among the most diverse and ecologically important marine bacteria, which have evolved many characteristics and lifestyles to occupy various niches. The relationship between genome features and environmental adaptation strategies is an essential part for understanding the ecological functions of vibrios in the marine system. The advent of complete genome sequencing technology has provided an important method of examining the genetic characteristics of vibrios on the genomic level.Two Vibrio genomes were sequenced and found to occupy many unique orthologues families which absent from the previously genes pool of the complete genomes of vibrios. Comparative genomics analysis found vibrios encompass a steady core-genome and tremendous pan-genome with substantial gene gain and horizontal gene transfer events in the evolutionary history. Evolutionary analysis based on the core-genome tree suggested that V. fischeri emerged ~?385 million years ago, along with the occurrence of cephalopods and the flourish of fish. The relatively large genomes, the high number of 16S rRNA gene copies, and the presence of R-M systems and CRISPR system help vibrios live in various marine environments. Chitin-degrading related genes are carried in nearly all the Vibrio genomes. The number of chitinase genes in vibrios has been extremely expanded compared to which in the most recent ancestor of the genus. The chitinase A genes were estimated to have evolved along with the genus, and have undergone significant purifying selective force to conserve the ancestral state.Vibrios have experienced extremely genome expansion events during their evolutionary history, allowing them to develop various functions to spread globally. Despite their close phylogenetic relationships, vibrios were found to have a tremendous pan-genome with a steady core-genome, which indicates the highly plastic genome of the genus. Additionally, the existence of various chitin-degrading related genes and the expansion of chitinase A in the genus demonstrate the importance of the chitin utilization for vibrios. Defensive systems in the Vibrio genomes may protect them from the invasion of external DNA. These genomic features investigated here provide a better knowledge of how the evolutionary process has forged Vibrio genomes to occupy various niches.

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September 22, 2019

Characterization and heterologous expression of the neoabyssomicin/abyssomicin biosynthetic gene cluster from Streptomyces koyangensis SCSIO 5802.

The deep-sea-derived microbe Streptomyces koyangensis SCSIO 5802 produces neoabyssomicins A-B (1-2) and abyssomicins 2 (3) and 4 (4). Neoabyssomicin A (1) augments human immunodeficiency virus-1 (HIV-1) replication whereas abyssomicin 2 (3) selectively reactivates latent HIV and is also active against Gram-positive pathogens including methicillin-resistant Staphylococcus aureus (MRSA). Structurally, neoabyssomicins A-B constitute a new subtype within the abyssomicin family and feature unique structural traits characteristic of extremely interesting biosynthetic transformations.In this work, the biosynthetic gene cluster (BGC) for the neoabyssomicins and abyssomicins, composed of 28 opening reading frames, was identified in S. koyangensis SCSIO 5802, and its role in neoabyssomicin/abyssomicin biosynthesis was confirmed via gene inactivation and heterologous expression experiments. Bioinformatics and genomics analyses enabled us to propose a biosynthetic pathway for neoabyssomicin/abyssomicin biosynthesis. Similarly, a protective export system by which both types of compounds are secreted from the S. koyangensis producer was identified, as was a four-component ABC transporter-based import system central to neoabyssomicin/abyssomicin biosynthesis. Furthermore, two regulatory genes, abmI and abmH, were unambiguously shown to be positive regulators of neoabyssomicin/abyssomicin biosynthesis. Consistent with their roles as positive regulatory genes, the overexpression of abmI and abmH (independent of each other) was shown to improve neoabyssomicin/abyssomicin titers.These studies provide new insight into the biosynthesis of the abyssomicin class of natural products, and highlight important exploitable features of its BGC for future efforts. Elucidation of the neoabyssomicin/abyssomicin BGC now enables combinatorial biosynthetic initiatives aimed at improving both the titers and pharmaceutical properties of these important natural products-based drug leads.

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September 22, 2019

Origin of the plasmid-mediated fosfomycin resistance gene fosA3.

fosA3 is the most commonly reported plasmid-mediated fosfomycin resistance gene among Enterobacteriaceae.To identify the origin of fosA3.The chromosome of Kluyvera georgiana clinical strain YDC799 was fully sequenced with single-molecule real-time sequencing. Comparative genetic analysis was performed for K. georgiana YDC799, K. georgiana type strain ATCC 51603 and representative fosA3-carrying plasmids. fosA genes were cloned in Escherichia coli to confirm function.K. georgiana YDC799 harboured fosA (designated fosAKG) and blaCTX-M-8 on the chromosome. The genetic environments surrounding fosA3 and bounded by IS26 were nearly identical with the corresponding regions of K. georgiana YDC799 and ATCC 51603. The amino acid sequence of FosAKG from YDC799 and K. georgiana ATCC 51603 shared 99% and 94% identity with FosA3, respectively. Cloned FosAKG conferred fosfomycin resistance with an MIC of?>1024 mg/L for E. coli.The plasmid-mediated fosA3 gene was likely mobilized from the chromosome of K. georgiana by an IS26-mediated event.© The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

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September 22, 2019

Insights on a founder effect: the case of Xylella fastidiosa in the Salento area of Apulia, Italy

Xylella fastidiosa causing disease on different plant species has been reported in several European countries, since 2013. Based on multilocus sequence typing (MLST) results, there is evidence of repeated introductions of the pathogen in Spain and France. In contrast, in the Salento area of Apulia (Puglia) in Southern Italy, the existence of a unique Apulian MLST genotype of X. fastidiosa, causing the olive quick decline syndrome (OQDS; also referred to as “CoDiRO” or “ST53”) was proven, and this was tentatively ascribed to X. fastidiosa subsp. pauca. In order to acquire information on intra population diversity European Food Safety Authority (EFSA) has strongly called for the characterization of X. fastidiosa isolates from Apulia to produce the necessary data to better understand strain diversity and evolution. In this work, for the first time the existence of sub-variants within a set of 14 “ST53” isolates of X. fastidiosa collected from different locations was searched using DNA typing methods targeting the whole pathogen genome. Invariably, VNTR, RAPD and rep-PCR (ERIC and BOX motifs) analyses indicated that all tested isolates possessed the same genomic fingerprint, supporting the existence of predominant epidemiological strain in Apulia. To further explore the degree of clonality within this population, two isolates from two different Salento areas (Taviano and Ugento) were completely sequenced using PacBio SMRT technology. The whole genome map and sequence comparisons revealed that both isolates are nearly identical, showing less than 0.001% nucleotide diversity. However, the complete and circularized Salento-1 and Salento-2 genome sequences were different, in genome and plasmid size, from the reference strain 9a5c of X. fastidiosa subsp. pauca (from citrus), and showed a PCR-proved large genome inversion of about 1.7 Mb. Genome-wide indices ANIm and dDDH indicated that the three isolates of X. fastidiosa from Salento (Apulia, Italy), namely Salento-1, Salento-2, and De Donno, whose complete genome sequence has been recently released, share a very recent common ancestor. This highlights the importance of continuous and extensive monitoring of molecular variation of this invasive pathogen to understand evolution of adaptive traits, and the necessity for adoption of all possible measures to reduce the risk of new introductions that may augment pathogen diversity.

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September 22, 2019

Using experimental evolution to identify druggable targets that could inhibit the evolution of antimicrobial resistance.

With multi-drug and pan-drug-resistant bacteria becoming increasingly common in hospitals, antibiotic resistance has threatened to return us to a pre-antibiotic era that would completely undermine modern medicine. There is an urgent need to develop new antibiotics and strategies to combat resistance that are substantially different from earlier drug discovery efforts. One such strategy that would complement current and future antibiotics would be a class of co-drugs that target the evolution of resistance and thereby extend the efficacy of specific classes of antibiotics. A critical step in the development of such strategies lies in understanding the critical evolutionary trajectories responsible for resistance and which proteins or biochemical pathways within those trajectories would be good candidates for co-drug discovery. We identify the most important steps in the evolution of resistance for a specific pathogen and antibiotic combination by evolving highly polymorphic populations of pathogens to resistance in a novel bioreactor that favors biofilm development. As the populations evolve to increasing drug concentrations, we use deep sequencing to elucidate the network of genetic changes responsible for resistance and subsequent in vitro biochemistry and often structure determination to determine how the adaptive mutations produce resistance. Importantly, the identification of the molecular steps, their frequency within the populations and their chronology within the evolutionary trajectory toward resistance is critical to assessing their relative importance. In this work, we discuss findings from the evolution of the ESKAPE pathogen, Pseudomonas aeruginosa to the drug of last resort, colistin to illustrate the power of this approach.

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September 22, 2019

Cultivation-independent and cultivation-dependent analysis of microbes in the shallow-sea hydrothermal system off Kueishantao island, Taiwan: Unmasking heterotrophic bacterial diversity and functional capacity.

Shallow-sea hydrothermal systems experience continuous fluctuations of physicochemical conditions due to seawater influx which generates variable habitats, affecting the phylogenetic composition and metabolic potential of microbial communities. Until recently, studies of submarine hydrothermal communities have focused primarily on chemolithoautotrophic organisms, however, there have been limited studies on heterotrophic bacteria. Here, fluorescence in situ hybridization, high throughput 16S rRNA gene amplicon sequencing, and functional metagenomes were used to assess microbial communities from the shallow-sea hydrothermal system off Kueishantao Island, Taiwan. The results showed that the shallow-sea hydrothermal system harbored not only autotrophic bacteria but abundant heterotrophic bacteria. The potential for marker genes sulfur oxidation and carbon fixation were detected in the metagenome datasets, suggesting a role for sulfur and carbon cycling in the shallow-sea hydrothermal system. Furthermore, the presence of diverse genes that encode transporters, glycoside hydrolases, and peptidase indicates the genetic potential for heterotrophic utilization of organic substrates. A total of 408 cultivable heterotrophic bacteria were isolated, in which the taxonomic families typically associated with oligotrophy, copiotrophy, and phototrophy were frequently found. The cultivation-independent and -dependent analyses performed herein show that Alphaproteobacteria and Gammaproteobacteria represent the dominant heterotrophs in the investigated shallow-sea hydrothermal system. Genomic and physiological characterization of a novel strain P5 obtained in this study, belonging to the genus Rhodovulum within Alphaproteobacteria, provides an example of heterotrophic bacteria with major functional capacity presented in the metagenome datasets. Collectively, in addition to autotrophic bacteria, the shallow-sea hydrothermal system also harbors many heterotrophic bacteria with versatile genetic potential to adapt to the unique environmental conditions.

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September 22, 2019

Comparative genome and methylome analysis reveals restriction/modification system diversity in the gut commensal Bifidobacterium breve.

Bifidobacterium breve represents one of the most abundant bifidobacterial species in the gastro-intestinal tract of breast-fed infants, where their presence is believed to exert beneficial effects. In the present study whole genome sequencing, employing the PacBio Single Molecule, Real-Time (SMRT) sequencing platform, combined with comparative genome analysis allowed the most extensive genetic investigation of this taxon. Our findings demonstrate that genes encoding Restriction/Modification (R/M) systems constitute a substantial part of the B. breve variable gene content (or variome). Using the methylome data generated by SMRT sequencing, combined with targeted Illumina bisulfite sequencing (BS-seq) and comparative genome analysis, we were able to detect methylation recognition motifs and assign these to identified B. breve R/M systems, where in several cases such assignments were confirmed by restriction analysis. Furthermore, we show that R/M systems typically impose a very significant barrier to genetic accessibility of B. breve strains, and that cloning of a methyltransferase-encoding gene may overcome such a barrier, thus allowing future functional investigations of members of this species.

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September 22, 2019

Biodegradation of decabromodiphenyl ether (BDE 209) by a newly isolated bacterium from an e-waste recycling area.

Polybrominated diphenyl ethers (PBDEs) have become widespread environmental pollutants all over the world. A newly isolated bacterium from an e-waste recycling area, Stenotrophomonas sp. strain WZN-1, can degrade decabromodiphenyl ether (BDE 209) effectively under aerobic conditions. Orthogonal test results showed that the optimum conditions for BDE 209 biodegradation were pH 5, 25 °C, 0.5% salinity, 150 mL minimal salt medium volume. Under the optimized condition, strain WZN-1 could degrade 55.15% of 65 µg/L BDE 209 under aerobic condition within 30 day incubation. Moreover, BDE 209 degradation kinetics was fitted to a first-order kinetics model. The biodegradation mechanism of BDE 209 by strain WZN-1 were supposed to be three possible metabolic pathways: debromination, hydroxylation, and ring opening processes. Four BDE 209 degradation genes, including one hydrolase, one dioxygenase and two dehalogenases, were identified based on the complete genome sequencing of strain WZN-1. The real-time qPCR demonstrated that the expression level of four identified genes were significantly induced by BDE 209, and they played an important role in the degradation process. This study is the first to demonstrate that the newly isolated Stenotrophomonas strain has an efficient BDE 209 degradation ability and would provide new insights for the microbial degradation of PBDEs.

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September 22, 2019

Genome analysis of clinical multilocus sequence Type 11 Klebsiella pneumoniae from China.

The increasing prevalence of KPC-producing Klebsiella pneumoniae strains in clinical settings has been largely attributed to dissemination of organisms of specific multilocus sequence types, such as ST258 and ST11. Compared with the ST258 clone, which is prevalent in North America and Europe, ST11 is common in China but information regarding its genetic features remains scarce. In this study, we performed detailed genetic characterization of ST11 K. pneumoniae strains by analyzing whole-genome sequences of 58 clinical strains collected from diverse geographic locations in China. The ST11 genomes were found to be highly heterogeneous and clustered into at least three major lineages based on the patterns of single-nucleotide polymorphisms. Exhibiting five different capsular types, these ST11 strains were found to harbor multiple resistance and virulence determinants such as the blaKPC-2 gene, which encodes carbapenemase, and the yersiniabactin-associated virulence genes irp, ybt and fyu. Moreover, genes encoding the virulence factor aerobactin and the regulator of the mucoid phenotype (rmpA) were detectable in six genomes, whereas genes encoding salmochelin were found in three genomes. In conclusion, our data indicated that carriage of a wide range of resistance and virulence genes constitutes the underlying basis of the high level of prevalence of ST11 in clinical settings. Such findings provide insight into the development of novel strategies for prevention, diagnosis and treatment of K. pneumoniae infections.

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September 22, 2019

Comparative genomics and identification of an enterotoxin-bearing pathogenicity island, SEPI-1/SECI-1, in Staphylococcus epidermidis pathogenic strains.

Staphylococcus epidermidis is a leading cause of nosocomial infections, majorly resistant to beta-lactam antibiotics, and may transfer several mobile genetic elements among the members of its own species, as well as to Staphylococcus aureus; however, a genetic exchange from S. aureus to S. epidermidis remains controversial. We recently identified two pathogenic clinical strains of S. epidermidis that produce a staphylococcal enterotoxin C3-like (SEC) similar to that by S. aureus pathogenicity islands. This study aimed to determine the genetic environment of the SEC-coding sequence and to identify the mobile genetic elements. Whole-genome sequencing and annotation of the S. epidermidis strains were performed using Illumina technology and a bioinformatics pipeline for assembly, which provided evidence that the SEC-coding sequences were located in a composite pathogenicity island that was previously described in the S. epidermidis strain FRI909, called SePI-1/SeCI-1, with 83.8-89.7% nucleotide similarity. Various other plasmids were identified, particularly p_3_95 and p_4_95, which carry antibiotic resistance genes (hsrA and dfrG, respectively), and share homologies with SAP085A and pUSA04-2-SUR11, two plasmids described in S. aureus. Eventually, one complete prophage was identified, FSE90, sharing 30 out of 52 coding sequences with the Acinetobacter phage vB_AbaM_IME200. Thus, the SePI-1/SeCI-1 pathogenicity island was identified in two pathogenic strains of S. epidermidis that produced a SEC enterotoxin causing septic shock. These findings suggest the existence of in vivo genetic exchange from S. aureus to S. epidermidis.

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September 22, 2019

Induced salt tolerance of perennial ryegrass by a novel bacterium strain from the rhizosphere of a desert shrub Haloxylon ammodendron.

Drought and soil salinity reduce agricultural output worldwide. Plant-growth-promoting rhizobacteria (PGPR) can enhance plant growth and augment plant tolerance to biotic and abiotic stresses.Haloxylon ammodendron, a C4 perennial succulent xerohalophyte shrub with excellent drought and salt tolerance, is naturally distributed in the desert area of northwest China. In our previous work, a bacterium strain numbered as M30-35 was isolated from the rhizosphere ofH. ammodendronin Tengger desert, Gansu province, northwest China. In current work, the effects of M30-35 inoculation on salt tolerance of perennial ryegrass were evaluated and its genome was sequenced to identify genes associated with plant growth promotion. Results showed that M30-35 significantly enhanced growth and salt tolerance of perennial ryegrass by increasing shoot fresh and dry weights, chlorophyll content, root volume, root activity, leaf catalase activity, soluble sugar and proline contents that contributed to reduced osmotic potential, tissue K? content and K?/Na? ratio, while decreasing malondialdehyde (MDA) content and relative electric conductivity (REC), especially under higher salinity. The genome of M30-35 contains 4421 protein encoding genes, 12 rRNA, 63 tRNA-encoding genes and four rRNA operons. M30-35 was initially classified as a new species inPseudomonasand named asPseudomonassp. M30-35. Thirty-four genes showing homology to genes associated with PGPR traits and abiotic stress tolerance were identified inPseudomonassp. M30-35 genome, including 12 related to insoluble phosphorus solubilization, four to auxin biosynthesis, four to other process of growth promotion, seven to oxidative stress alleviation, four to salt and drought tolerance and three to cold and heat tolerance. Further study is needed to clarify the correlation between these genes from M30-35 and the salt stress alleviation of inoculated plants under salt stress. Overall, our research indicated that desert shrubs appear rich in PGPRs that can help important crops tolerate abiotic stress.

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September 22, 2019

High genetic plasticity in multidrug-resistant sequence type 3-IncHI2 plasmids revealed by sequence comparison and phylogenetic analysis.

We report a novel fusion plasmid, pP2-3T, cointegrating sequence type 3 (ST3)-IncHI2 with an IncFII plasmid backbone mediating multidrug resistance (MDR) and virulence. Phylogenetic analysis and comparative genomics revealed that pP2-3T and other MDR ST3-IncHI2 plasmids clustered together, representing a unique IncHI2 lineage that exhibited high conservation in backbones of plasmids but possessed highly genetic plasticity in various regions by acquiring numerous antibiotic resistance genes and fusing with other plasmids. Surveillance studies should be performed to monitor multiresistance IncHI2 plasmids among Enterobacteriaceae. Copyright © 2018 American Society for Microbiology.

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September 22, 2019

Genetic separation of Listeria monocytogenes causing central nervous system infections in animals.

Listeria monocytogenes is a foodborne pathogen that causes abortion, septicemia, gastroenteritis and central nervous system (CNS) infections in ruminants and humans. L. monocytogenes strains mainly belong to two distinct phylogenetic groups, named lineages I and II. In general, clinical cases in humans and animals, in particular CNS infections, are caused by lineage I strains, while most of the environmental and food strains belong to lineage II. Little is known about why lineage I is more virulent than lineage II, even though various molecular factors and mechanisms associated with pathogenesis are known. In this study, we have used a variety of whole genome sequence analyses and comparative genomic tools in order to find characteristics that distinguish lineage I from lineage II strains and CNS infection strains from non-CNS strains. We analyzed 225 strains and identified single nucleotide variants between lineages I and II, as well as differences in the gene content. Using a novel approach based on Reads Per Kilobase per Million Mapped (RPKM), we identified 167 genes predominantly absent in lineage II but present in lineage I. These genes are mostly encoding for membrane-associated proteins. Additionally, we found 77 genes that are largely absent in the non-CNS associated strains, while 39 genes are especially lacking in our defined “non-clinical” group. Based on the RPKM analysis and the metadata linked to the L. monocytogenes strains, we identified 6 genes potentially associated with CNS cases, which include a transcriptional regulator, an ABC transporter and a non-coding RNA. Although there is not a clear separation between pathogenic and non-pathogenic strains based on phylogenetic lineages, the presence of the genes identified in our study reveals potential pathogenesis traits in ruminant L. monocytogenes strains. Ultimately, the differences that we have found in our study will help steer future studies in understanding the virulence mechanisms of the most pathogenic L. monocytogenes strains.

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September 22, 2019

The DNA methylome of the hyperthermoacidophilic crenarchaeon Sulfolobus acidocaldarius.

DNA methylation is the most common epigenetic modification observed in the genomic DNA (gDNA) of prokaryotes and eukaryotes. Methylated nucleobases, N6-methyl-adenine (m6A), N4-methyl-cytosine (m4C), and 5-methyl-cytosine (m5C), detected on gDNA represent the discrimination mark between self and non-self DNA when they are part of restriction-modification systems in prokaryotes (Bacteria and Archaea). In addition, m5C in Eukaryotes and m6A in Bacteria play an important role in the regulation of key cellular processes. Although archaeal genomes present modified bases as in the two other domains of life, the significance of DNA methylations as regulatory mechanisms remains largely uncharacterized in Archaea. Here, we began by investigating the DNA methylome of Sulfolobus acidocaldarius. The strategy behind this initial study entailed the use of combined digestion assays, dot blots, and genome resequencing, which utilizes specific restriction enzymes, antibodies specifically raised against m6A and m5C and single-molecule real-time (SMRT) sequencing, respectively, to identify DNA methylations occurring in exponentially growing cells. The previously identified restriction-modification system, specific of S. acidocaldarius, was confirmed by digestion assay and SMRT sequencing while, the presence of m6A was revealed by dot blot and identified on the characteristic Dam motif by SMRT sequencing. No m5C was detected by dot blot under the conditions tested. Furthermore, by comparing the distribution of both detected methylations along the genome and, by analyzing DNA methylation profiles in synchronized cells, we investigated in which cellular pathways, in particular the cell cycle, this m6A methylation could be a key player. The analysis of sequencing data rejected a role for m6A methylation in another defense system and also raised new questions about a potential involvement of this modification in the regulation of other biological functions in S. acidocaldarius.

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September 22, 2019

Complete genome sequence and genomic characterization of Lactobacillus acidophilus LA1 (11869BP).

Our body has natural defense systems to protect against potentially harmful microbes, including the physical and chemical barriers of the intestinal epithelium (Corfield et al., 2000). The physical barrier of the intestinal epithelium protects the host against pathogenic microbes (Anderson et al., 1993), and the intestinal mucosa coated with mucus excretes pathogens from the intestinal tract (Corfield et al., 2000).

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