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1.
Water Res ; 168: 115160, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31614233

RESUMO

Aquatic ecosystems have been increasingly threatened by anthropogenic activities, e.g., wastewater discharge and farm operation. Several methods are adopted to evaluate the effects of anthropogenic activities on biological risk in the environment, such as qPCR and amplicon next-generation sequencing. However, these methods fall short of providing genomic information of target species, which is vital for risk assessment from genomic aspect. Here, we developed a novel approach integrating metagenomic analysis and flow cytometry to identify and quantify potential pathogenic antibiotic resistant bacteria (PARB; carrying both antibiotic resistance genes (ARGs) and virulence factor genes (VFGs)) in the environment, which are of particular concern due to their infection ability and antibiotic resistance. Based on the abundance/density of PARB, we evaluated microbiological risk in a river impacted by both municipal drainage and agriculture runoff. We collected samples upstream (mountainous area) as the control. Results showed that 81.8% of dominant PARB (33) recovered using our approach were related to known pathogenic taxa. In addition, intragenomic ARGs-VFGs coexistence patterns in the dominant Pseudomonas genomes (20 out of 71 PARB) showed high similarity with the most closely related Pseudomonas genomes from the NCBI RefSeq database. These results reflect acceptable reliability of the approach for (potential) pathogen identification in environmental samples. According to the PARB density, microbiological risk in samples from the agricultural area was significantly higher than in samples from the urban area. We speculated that this was due to the higher antibiotic usage in agriculture as well as intragenomic ARGs-VFGs co-evolution under antibiotic selective pressure. This study provides an alternative approach for the identification and quantification of PARB in aquatic environments, which can be applied for microbiological risk assessment.


Assuntos
Antibacterianos , Ecossistema , Resistência Microbiana a Medicamentos , Genes Bacterianos , Genoma Bacteriano , Reprodutibilidade dos Testes , Medição de Risco , Fatores de Virulência
2.
Microb Cell Fact ; 18(1): 162, 2019 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-31581942

RESUMO

BACKGROUND: Efficient and convenient genome-editing toolkits can expedite genomic research and strain improvement for desirable phenotypes. Zymomonas mobilis is a highly efficient ethanol-producing bacterium with a small genome size and desirable industrial characteristics, which makes it a promising chassis for biorefinery and synthetic biology studies. While classical techniques for genetic manipulation are available for Z. mobilis, efficient genetic engineering toolkits enabling rapidly systematic and high-throughput genome editing in Z. mobilis are still lacking. RESULTS: Using Cas12a (Cpf1) from Francisella novicida, a recombinant strain with inducible cas12a expression for genome editing was constructed in Z. mobilis ZM4, which can be used to mediate RNA-guided DNA cleavage at targeted genomic loci. gRNAs were then designed targeting the replicons of native plasmids of ZM4 with about 100% curing efficiency for three native plasmids. In addition, CRISPR-Cas12a recombineering was used to promote gene deletion and insertion in one step efficiently and precisely with efficiency up to 90%. Combined with single-stranded DNA (ssDNA), CRISPR-Cas12a system was also applied to introduce minor nucleotide modification precisely into the genome with high fidelity. Furthermore, the CRISPR-Cas12a system was employed to introduce a heterologous lactate dehydrogenase into Z. mobilis with a recombinant lactate-producing strain constructed. CONCLUSIONS: This study applied CRISPR-Cas12a in Z. mobilis and established a genome editing tool for efficient and convenient genome engineering in Z. mobilis including plasmid curing, gene deletion and insertion, as well as nucleotide substitution, which can also be employed for metabolic engineering to help divert the carbon flux from ethanol production to other products such as lactate demonstrated in this work. The CRISPR-Cas12a system established in this study thus provides a versatile and powerful genome-editing tool in Z. mobilis for functional genomic research, strain improvement, as well as synthetic microbial chassis development for economic biochemical production.


Assuntos
Edição de Genes/métodos , Genoma Bacteriano , Zymomonas/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Endonucleases/metabolismo , Francisella/enzimologia , Plasmídeos/genética , Plasmídeos/metabolismo , RNA Guia/genética , RNA Guia/metabolismo , Zymomonas/metabolismo
3.
An Acad Bras Cienc ; 91(3): e20180568, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31596388

RESUMO

Cyclodextrin glycosyltransferases (CGTases) are important enzymes in the biotechnology field because they catalyze starch conversion into cyclodextrins and linear oligosaccharides, which are used in food, pharmaceutical and cosmetic industries. The CGTases are classified according to their product specificity in α-, ß-, α/ß- and γ-CGTases. As molecular markers are the preferred tool for bacterial identification, we employed six molecular markers (16S rRNA, dnaK, gyrB, recA, rpoB and tufA) to test the identification of a CGTase-producing bacterial strain (DF 9R) in a phylogenetic context. In addition, we assessed the phylogenetic relationship of CGTases along bacterial evolution. The results obtained here allowed us to identify the strain DF 9R as Paenibacillus barengoltzii, and to unveil a complex origin for CGTase types during archaeal and bacterial evolution. We postulate that the α-CGTase activity represents the ancestral type, and that the γ-activity may have derived from ß-CGTases.


Assuntos
Bacillus/genética , Bacillus/isolamento & purificação , Glucosiltransferases/genética , Filogenia , Bacillus/classificação , Genes Essenciais , Marcadores Genéticos , Genoma Bacteriano , Glucosiltransferases/classificação , Glucosiltransferases/isolamento & purificação , RNA Ribossômico 16S/genética , Análise de Sequência
4.
BMC Genomics ; 20(1): 768, 2019 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-31646960

RESUMO

BACKGROUND: The Hafnia genus is an opportunistic pathogen that has been implicated in both nosocomial and community-acquired infections. Although Hafnia is fairly often isolated from clinical material, its taxonomy has remained an unsolved riddle, and the involvement and importance of Hafnia in human disease is also uncertain. Here, we used comparative genomic analysis to define the taxonomy of Hafnia, identify species-specific genes that may be the result of ecological and pathogenic specialization, and reveal virulence-related genetic profiles that may contribute to pathogenesis. RESULTS: One complete genome sequence and 19 draft genome sequences for Hafnia strains were generated and combined with 27 publicly available genomes. We provided high-resolution typing methods by constructing phylogeny and population structure based on single-copy core genes in combination with whole genome average nucleotide identity to identify two distant Hafnia species (alvei and paralvei) and one mislabeled strain. The open pan-genome and the presence of numerous mobile genetic elements reveal that Hafnia has undergone massive gene rearrangements. Presence of species-specific core genomes associated with metabolism and transport suggests the putative niche differentiation between alvei and paralvei. We also identified possession of diverse virulence-related profiles in both Hafnia species., including the macromolecular secretion system, virulence, and antimicrobial resistance. In the macromolecular system, T1SS, Flagellum 1, Tad pilus and T6SS-1 were conserved in Hafnia, whereas T4SS, T5SS, and other T6SSs exhibited the evolution of diversity. The virulence factors in Hafnia are related to adherence, toxin, iron uptake, stress adaptation, and efflux pump. The identified resistance genes are associated with aminoglycoside, beta-lactam, bacitracin, cationic antimicrobial peptide, fluoroquinolone, and rifampin. These virulence-related profiles identified at the genomic level provide insights into Hafnia pathogenesis and the differentiation between alvei and paralvei. CONCLUSIONS: Our research using core genome phylogeny and comparative genomics analysis of a larger collection of strains provides a comprehensive view of the taxonomy and species-specific traits between Hafnia species. Deciphering the genome of Hafnia strains possessing a reservoir of macromolecular secretion systems, virulence factors, and resistance genes related to pathogenicity may provide insights into addressing its numerous infections and devising strategies to combat the pathogen.


Assuntos
Genoma Bacteriano , Hafnia/classificação , Hafnia/patogenicidade , Virulência , Sistemas de Secreção Bacterianos/genética , Hibridização Genômica Comparativa , Farmacorresistência Bacteriana/genética , Genótipo , Filogenia , Especificidade da Espécie , Fatores de Virulência/genética
5.
Int J Syst Evol Microbiol ; 69(9): 2735-2738, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31483242

RESUMO

Early characterization of strains designated into the genera Entomoplasma and Mesoplasma was based upon biological and chemical characteristics. With the advent of 16S rRNA gene sequence analysis as an added taxonomic character, it became clear that the two genera did not form distinct and separate monophyletic clusters. A genome-level analysis of all 17 validly published species within the family Entomoplasmataceae has recently been performed. Phylogenetic analyses, comparisons of gene content, and the lack of genus-specific genes supported that species from the two genera are intermixed and should not be taxonomically separated. This level of analysis clearly reveals the necessity to revise the taxonomy of this family by merging the two genera into one, Entomoplasma. Additionally, it was definitively determined that the strain originally designated as Acholeplasma multilocale resides in this cluster and should be formally renamed as Entomoplasma multilocale. Merging Mesoplasma and Entomoplasma yields a paraphyletic genus, but is supported by cell morphology and ecology to be distinguished from the genera Spiroplasma and Mycoplasma.


Assuntos
Entomoplasmataceae/classificação , Genoma Bacteriano , Filogenia , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , RNA Ribossômico 16S/genética , Análise de Sequência de DNA
6.
World J Microbiol Biotechnol ; 35(8): 128, 2019 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-31375920

RESUMO

Large patch disease, caused by Rhizoctonia solani AG2-2, is the most devastating disease in Zoysiagrass (Zoysia japonica). Current large patch disease control strategies rely primarily upon the use of chemical pesticides. Streptomyces sp. S8 is known to possess exceptional antagonistic properties that could potentially suppress the large patch pathogen found at turfgrass plantations. This study aims to demonstrate the feasibility of using the strain as a biological control mechanism. Sequencing of the S8 strain genome revealed a valinomycin biosynthesis gene cluster. This cluster is composed of the vlm1 and vlm2 genes, which are known to produce antifungal compounds. In order to verify this finding for the large patch pathogen, a valinomycin biosynthesis knockout mutant was created via the CRISPR/Cas9 system. The mutant lost antifungal activity against the large patch pathogen. Consequently, it is anticipated that eco-friendly microbial preparations derived from the S8 strain can be utilized to biologically control large patch disease.


Assuntos
Antifúngicos/metabolismo , Antifúngicos/farmacologia , Rhizoctonia/efeitos dos fármacos , Streptomyces/metabolismo , Valinomicina/metabolismo , Valinomicina/farmacologia , Vias Biossintéticas/genética , Técnicas de Inativação de Genes , Genoma Bacteriano , Família Multigênica , Controle Biológico de Vetores/métodos , Doenças das Plantas/microbiologia , Doenças das Plantas/prevenção & controle , Poaceae/microbiologia , Rhizoctonia/crescimento & desenvolvimento , Análise de Sequência de DNA , Streptomyces/genética
7.
World J Microbiol Biotechnol ; 35(9): 133, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31432254

RESUMO

There is a significant increase in the discovery of new antimicrobial compounds in recent past to combat drug resistant pathogens. Members of the genus Bacillus and related genera have been screened extensively due to their ability to produce wide range of antimicrobial compounds. In this study, we have isolated and characterized a new antimicrobial peptide from a marine bacterium identified as Virgibacillus species. The low molecular mass and stability of the antimicrobial substance pointed towards the bacteriocinogenic nature of the compound. The RAST analysis of genome sequence showed presence of a putative bacteriocin biosynthetic cluster containing genes necessary for synthesis of a lanthipeptide. Translated amino acid sequence of mature C-terminal propeptide showed identity with salivaricin A (52.2%) and lacticin A (33.3%). Accordingly, the mass (2417 Da) obtained by MALDI analysis was in agreement with posttranslational modifications of the leader peptide to yield three methyl lanthionine rings and a disulfide bond between two free cysteine residues. The lanthipeptide was named as virgicin, which selectively inhibited the growth of Gram-positive bacteria and biofilm formation by Enterococcus faecalis. Inhibition of biofilm formation by E. faecalis was also observed in in vitro model experiments using hydroxyapatite discs. Thus, virgicin appears to be a promising new bacteriocin to control oral biofilm formation by selective pathogens.


Assuntos
Bacteriocinas/isolamento & purificação , Bacteriocinas/farmacologia , Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecalis/crescimento & desenvolvimento , Peptídeos/isolamento & purificação , Peptídeos/farmacologia , Virgibacillus/metabolismo , Bacteriocinas/química , Bacteriocinas/genética , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Vias Biossintéticas/genética , Genoma Bacteriano , Peso Molecular , Família Multigênica , Peptídeos/química , Peptídeos/genética , Água do Mar/microbiologia , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Virgibacillus/classificação , Virgibacillus/isolamento & purificação
8.
Implement Sci ; 14(1): 79, 2019 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-31409417

RESUMO

BACKGROUND: Next-generation sequencing (NGS) is increasingly being translated into routine public health practice, affecting the surveillance and control of many pathogens. The purpose of this scoping review is to identify and characterize the recent literature concerning the application of bacterial pathogen genomics for public health practice and to assess the added value, challenges, and needs related to its implementation from an epidemiologist's perspective. METHODS: In this scoping review, a systematic PubMed search with forward and backward snowballing was performed to identify manuscripts in English published between January 2015 and September 2018. Included studies had to describe the application of NGS on bacterial isolates within a public health setting. The studied pathogen, year of publication, country, number of isolates, sampling fraction, setting, public health application, study aim, level of implementation, time orientation of the NGS analyses, and key findings were extracted from each study. Due to a large heterogeneity of settings, applications, pathogens, and study measurements, a descriptive narrative synthesis of the eligible studies was performed. RESULTS: Out of the 275 included articles, 164 were outbreak investigations, 70 focused on strategy-oriented surveillance, and 41 on control-oriented surveillance. Main applications included the use of whole-genome sequencing (WGS) data for (1) source tracing, (2) early outbreak detection, (3) unraveling transmission dynamics, (4) monitoring drug resistance, (5) detecting cross-border transmission events, (6) identifying the emergence of strains with enhanced virulence or zoonotic potential, and (7) assessing the impact of prevention and control programs. The superior resolution over conventional typing methods to infer transmission routes was reported as an added value, as well as the ability to simultaneously characterize the resistome and virulome of the studied pathogen. However, the full potential of pathogen genomics can only be reached through its integration with high-quality contextual data. CONCLUSIONS: For several pathogens, it is time for a shift from proof-of-concept studies to routine use of WGS during outbreak investigations and surveillance activities. However, some implementation challenges from the epidemiologist's perspective remain, such as data integration, quality of contextual data, sampling strategies, and meaningful interpretations. Interdisciplinary, inter-sectoral, and international collaborations are key for an appropriate genomics-informed surveillance.


Assuntos
Genoma Bacteriano , Genômica/métodos , Prática de Saúde Pública , Humanos , Sequenciamento Completo do Genoma
9.
Mol Biol (Mosk) ; 53(4): 600-612, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31397434

RESUMO

A new plasmid, pSM22, was isolated from Serratia marcescens and sequenced. Its 43 190-bp sequence with an average GC-content of 58% contains 31 open reading frames (ORFs) which form replication, conjugation, stability, and adaptive modules. The replication module includes a site of initiation of leading-strand synthesis in plasmid replication, a replication termination site (terC), the rep A (=repA1) and repA4 genes, and the copA sequence, which codes for an antisense RNA (asRNA). These structures are functionally integrated in an FII replicon (incompatibility group IncFII). Based on the significant differences between the FII replicon and the canonical sequences of the R plasmids R1 and NR1 (=R100=R222), pSM22 was assigned to a new subtype. The conjugation module includes 13 genes with a high identity to the genes responsible for conjugation of the F plasmid. A comparative genomic analysis showed that the conjugation modules of pSM22 and F are structurally similar. By the conjugation system and the presence of three conserved motifs in relaxase (TraI), pSM22 belongs to the F12 clade of the MOBF type. The stability module includes the resD and parA genes, which are responsible for the resolution of multimeric plasmid forms and their subsequent segregation between daughter cells. The adaptive module contains the microcin H47 (MccH47) secretion/processing and UV resistance genes. The mosaic structure of pSM22 and reductive evolution of its modules suggest high genomic plasticity for the genus Serratia. An analysis of the architecture of the pSM22 modules clarifies the evolutionary relationships among IncF/MOBF12 group plasmids in bacteria of the family Enterobacteriaceae and opens a novel avenue for further comparative genomic studies of Serratia plasmids.


Assuntos
Evolução Molecular , Genômica , Plasmídeos/classificação , Plasmídeos/genética , Replicação do DNA , Genes Bacterianos , Genoma Bacteriano/genética , Replicon/genética , Serratia marcescens/genética
10.
BMC Bioinformatics ; 20(1): 447, 2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31462231

RESUMO

BACKGROUND: Metabolic coupling of product synthesis and microbial growth is a prominent approach for maximizing production performance. Growth-coupling (GC) also helps stabilizing target production and allows the selection of superior production strains by adaptive laboratory evolution. To support the implementation of growth-coupling strain designs, we seek to identify biologically relevant, metabolic principles that enforce strong growth-coupling on the basis of reaction knockouts. RESULTS: We adapted an established bilevel programming framework to maximize the minimally guaranteed production rate at a fixed, medium growth rate. Using this revised formulation, we identified various GC intervention strategies for metabolites of the central carbon metabolism, which were examined for GC generating principles under diverse conditions. Curtailing the metabolism to render product formation an essential carbon drain was identified as one major strategy generating strong coupling of metabolic activity and target synthesis. Impeding the balancing of cofactors and protons in the absence of target production was the underlying principle of all other strategies and further increased the GC strength of the aforementioned strategies. CONCLUSION: Maximizing the minimally guaranteed production rate at a medium growth rate is an attractive principle for the identification of strain designs that couple growth to target metabolite production. Moreover, it allows for controlling the inevitable compromise between growth coupling strength and the retaining of microbial viability. With regard to the corresponding metabolic principles, generating a dependency between the supply of global metabolic cofactors and product synthesis appears to be advantageous in enforcing strong GC for any metabolite. Deriving such strategies manually, is a hard task, due to which we suggest incorporating computational metabolic network analyses in metabolic engineering projects seeking to determine GC strain designs.


Assuntos
Escherichia coli/crescimento & desenvolvimento , Genoma Bacteriano , Engenharia Metabólica , Redes e Vias Metabólicas , Modelos Biológicos , Simulação por Computador , Escherichia coli/genética , Escherichia coli/metabolismo
11.
Nat Biotechnol ; 37(8): 953-961, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31375809

RESUMO

Ruminants provide essential nutrition for billions of people worldwide. The rumen is a specialized stomach that is adapted to the breakdown of plant-derived complex polysaccharides. The genomes of the rumen microbiota encode thousands of enzymes adapted to digestion of the plant matter that dominates the ruminant diet. We assembled 4,941 rumen microbial metagenome-assembled genomes (MAGs) using approximately 6.5 terabases of short- and long-read sequence data from 283 ruminant cattle. We present a genome-resolved metagenomics workflow that enabled assembly of bacterial and archaeal genomes that were at least 80% complete. Of note, we obtained three single-contig, whole-chromosome assemblies of rumen bacteria, two of which represent previously unknown rumen species, assembled from long-read data. Using our rumen genome collection we predicted and annotated a large set of rumen proteins. Our set of rumen MAGs increases the rate of mapping of rumen metagenomic sequencing reads from 15% to 50-70%. These genomic and protein resources will enable a better understanding of the structure and functions of the rumen microbiota.


Assuntos
Archaea/genética , Bactérias/genética , Metagenoma , Metagenômica/métodos , Rúmen/microbiologia , Animais , Proteínas Arqueais , Proteínas de Bactérias , Bovinos/microbiologia , Bases de Dados de Proteínas , Genoma Arqueal , Genoma Bacteriano , Filogenia , Ovinos/microbiologia
12.
Microbiol Res ; 228: 126306, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31422233

RESUMO

The mariner transposon family of Himar1 has been widely used for the random mutagenesis of bacteria to generate single insertions into the chromosome. Here, a versatile toolbox of mariner transposon derivatives was generated and applied to the functional genomics investigation of fish pathogen Edwardsiella piscicida. In this study, we combined the merits of the random mutagenesis of mariner transposon and common efficient reporter marker genes or regulatory elements, mCherry, gfp, luxAB, lacZ, sacBR, and PBAD and antibiotic resistance cassettes to construct a series of derivative transposon vectors, pMmch, pMKGR, pMCGR, pMXKGR, pMLKGR, pMSGR, and pMPR, based on the initial transposon pMar2xT7. The function and effectiveness of the modified transposons were verified by introducing them into E. piscicida EIB202. Based on the toolbox, a transposon insertion mutant library containing approximately 3.0 × 105 distinct mutants was constructed to explore the upstream regulators of esrB, the master regulator of the type III and type VI secretion systems (T3/T6SS) in E. piscicida. Following analysis by Con-ARTIST, ETAE_3474, annotated as fabR and involved in fatty acid metabolism, was screened out and identified as a novel regulator mediating T3SS and T6SS expression. In addition, the fabR mutants displayed critical virulence attenuation in turbot. Due to the broad-range host compatibility of mariner transposons, the newly built transposon toolbox can be applied for functional genomics studies in various bacteria.


Assuntos
Proteínas de Bactérias/genética , Elementos de DNA Transponíveis , Edwardsiella/genética , Regulação Bacteriana da Expressão Gênica/genética , Testes Genéticos/métodos , Genoma Bacteriano/genética , Animais , Mapeamento Cromossômico , Farmacorresistência Bacteriana/genética , Ácidos Graxos/metabolismo , Doenças dos Peixes/microbiologia , Biblioteca Gênica , Genes Reporter/genética , Genômica/métodos , Mutagênese Insercional/métodos , Fatores de Transcrição/genética , Sistemas de Secreção Tipo III/genética , Sistemas de Secreção Tipo VI/genética , Virulência , Fatores de Virulência/genética
13.
Mem Inst Oswaldo Cruz ; 114: e190105, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31389522

RESUMO

BACKGROUND: Healthcare-associated infections caused by bacteria such as Pseudomonas aeruginosa are a major public health problem worldwide. Gene regulatory networks (GRN) computationally represent interactions among regulatory genes and their targets. They are an important approach to help understand bacterial behaviour and to provide novel ways of overcoming scientific challenges, including the identification of potential therapeutic targets and the development of new drugs. OBJECTIVES: The goal of this study was to reconstruct the multidrug-resistant (MDR) P. aeruginosa GRN and to analyse its topological properties. METHODS: The methodology used in this study was based on gene orthology inference using the reciprocal best hit method. We used the genome of P. aeruginosa CCBH4851 as the basis of the reconstruction process. This MDR strain is representative of the sequence type 277, which was involved in an endemic outbreak in Brazil. FINDINGS: We obtained a network with a larger number of regulatory genes, target genes and interactions as compared to the previously reported network. Topological analysis results are in accordance with the complex network representation of biological processes. MAIN CONCLUSIONS: The properties of the network were consistent with the biological features of P. aeruginosa. To the best of our knowledge, the P. aeruginosa GRN presented here is the most complete version available to date.


Assuntos
Farmacorresistência Bacteriana Múltipla/genética , Redes Reguladoras de Genes , Pseudomonas aeruginosa/genética , Genoma Bacteriano , Família Multigênica , Infecções por Pseudomonas/genética , Valores de Referência
14.
Genome Biol ; 20(1): 158, 2019 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-31391098

RESUMO

BACKGROUND: Several genome-scale metabolic reconstruction software platforms have been developed and are being continuously updated. These tools have been widely applied to reconstruct metabolic models for hundreds of microorganisms ranging from important human pathogens to species of industrial relevance. However, these platforms, as yet, have not been systematically evaluated with respect to software quality, best potential uses and intrinsic capacity to generate high-quality, genome-scale metabolic models. It is therefore unclear for potential users which tool best fits the purpose of their research. RESULTS: In this work, we perform a systematic assessment of current genome-scale reconstruction software platforms. To meet our goal, we first define a list of features for assessing software quality related to genome-scale reconstruction. Subsequently, we use the feature list to evaluate the performance of each tool. To assess the similarity of the draft reconstructions to high-quality models, we compare each tool's output networks with that of the high-quality, manually curated, models of Lactobacillus plantarum and Bordetella pertussis, representatives of gram-positive and gram-negative bacteria, respectively. We additionally compare draft reconstructions with a model of Pseudomonas putida to further confirm our findings. We show that none of the tools outperforms the others in all the defined features. CONCLUSIONS: Model builders should carefully choose a tool (or combinations of tools) depending on the intended use of the metabolic model. They can use this benchmark study as a guide to select the best tool for their research. Finally, developers can also benefit from this evaluation by getting feedback to improve their software.


Assuntos
Bactérias/metabolismo , Genoma Bacteriano , Redes e Vias Metabólicas/genética , Software , Bordetella pertussis/genética , Bordetella pertussis/metabolismo , Genes Bacterianos , Lactobacillus plantarum/genética , Lactobacillus plantarum/metabolismo , Pseudomonas putida/genética , Pseudomonas putida/metabolismo
15.
Genome Biol ; 20(1): 163, 2019 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-31405375

RESUMO

BACKGROUND: Like many bacteria, Vibrio cholerae deploys a harpoon-like type VI secretion system (T6SS) to compete against other microbes in environmental and host settings. The T6SS punctures adjacent cells and delivers toxic effector proteins that are harmless to bacteria carrying cognate immunity factors. Only four effector/immunity pairs encoded on one large and three auxiliary gene clusters have been characterized from largely clonal, patient-derived strains of V. cholerae. RESULTS: We sequence two dozen V. cholerae strain genomes from diverse sources and develop a novel and adaptable bioinformatics tool based on hidden Markov models. We identify two new T6SS auxiliary gene clusters and describe Aux 5 here. Four Aux 5 loci are present in the host strain, each with an atypical effector/immunity gene organization. Structural prediction of the putative effector indicates it is a lipase, which we name TleV1 (type VI lipase effector Vibrio). Ectopic TleV1 expression induces toxicity in Escherichia coli, which is rescued by co-expression of the TliV1a immunity factor. A clinical V. cholerae reference strain expressing the Aux 5 cluster uses TleV1 to lyse its parental strain upon contact via its T6SS but is unable to kill parental cells expressing the TliV1a immunity factor. CONCLUSION: We develop a novel bioinformatics method and identify new T6SS gene clusters in V. cholerae. We also show the TleV1 toxin is delivered in a T6SS manner by V. cholerae and can lyse other bacterial cells. Our web-based tool can be modified to identify additional novel T6SS genomic loci in diverse bacterial species.


Assuntos
Genoma Bacteriano , Sistemas de Secreção Tipo VI/genética , Vibrio cholerae/genética , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Genes Bacterianos , Variação Genética , Software , Vibrio cholerae/isolamento & purificação
16.
Medicine (Baltimore) ; 98(33): e16749, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31415371

RESUMO

This study is to analyze the functional genes and metabolic pathways of dexamethasone degradation in Burkholderia through genome sequencing.A new Burkholderia sp. CQQ001 (B. CQ001) with dexamethasone degrading activity was isolated from the hospital wastewater and sequenced using Illumina Hiseq4000 combined with the third-generation sequencing technology. The genomes were assembled, annotated, and genomically mapped. Compared with six Burkholderia strains with typical features and four Burkholderia strains with special metabolic ability, the functional genes and metabolic pathways of dexamethasone degradation were analyzed and confirmed by RT-qPCR.Genome of B. CQ001 was 7,660,596 bp long with 6 ring chromosomes. The genes related to material metabolism accounted for 80.15%. These metabolism related genes could participate in 117 metabolic pathways and cover various microbial metabolic pathways in different environments and decomposition pathways of secondary metabolites, especially the degradation of aromatic compounds. The steroidal metabolic pathway containing 1 ABC transporter and 9 key metabolic enzymes related genes were scattered in the genome. Among them, the ABC transporter, KshA, and KshB increased significantly under the culture conditions of dexamethasone sodium phosphate as carbon source.B. CQ001 is a bacterium with strong metabolic function and rich metabolic pathways. It has the potential to degrade aromatics and other exogenous chemicals and contains genes for steroid metabolism. Our study enriches the genetic information of Burkholderia and provides information for the application of Burkholderia in bioremediation and steroid medicine production.


Assuntos
Anti-Inflamatórios/metabolismo , Burkholderia/metabolismo , Dexametasona/metabolismo , Águas Residuárias , Microbiologia da Água , Burkholderia/genética , Genoma Bacteriano , Humanos , Águas Residuárias/microbiologia , Sequenciamento Completo do Genoma
17.
DNA Cell Biol ; 38(9): 922-932, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31329463

RESUMO

The present study aims to identify the species and strains of Mycoplasma hyopneumoniae isolated from Tibetan pigs (Mh TB1) at the genetic level for understanding the basis of its pathogenicity. Mh TB1 was isolated from the consolidated lungs of Tibetan pigs by liquid culture and agar plate colony method. Polymerase chain reaction (PCR) amplification of the 16S recombinant DNA (rDNA) conservative sequence and a species-specific gene (P36) of Mh provided species confirmation. PCR products were imaged on gels and shotgun sequencing was performed. DNA sequences were compared for assessing genetic similarity between Mh TB1 and Mh reference strains in the GenBank database. The isolated strains were >98% similar to the Mh reference strains. Genomic analysis revealed significant sequence conservation between Mh TB1 and the reference strains; however, differential genes were more prevalent in Mh TB1 than in other reported strains. Therefore, we concluded that Mh is a major pathogen of Tibetan pigs that cause enzootic pneumonia. The Mh TB1 strain harbors more genes and specific virulence factors, consistent with its plateau-related adaptability to hypoxia and virulence. Differential gene analysis revealed gene variations in the inclement plateau environment, enriched gene pool, and plateau adaptability of the Mh TB1 strain, which will be important for vaccine development.


Assuntos
Mycoplasma hyopneumoniae/genética , Pneumonia Suína Micoplasmática/microbiologia , Animais , Genoma Bacteriano , Pulmão/microbiologia , Mycoplasma hyopneumoniae/patogenicidade , RNA Ribossômico 16S/genética , Suínos
18.
Nat Commun ; 10(1): 2931, 2019 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-31270321

RESUMO

The virulence of Pseudomonas aeruginosa, a Gram-negative opportunistic pathogen, is regulated by many transcriptional factors (TFs) that control the expression of quorum sensing and protein secretion systems. Here, we report a genome-wide, network-based approach to dissect the crosstalk between 20 key virulence-related TFs. Using chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq), as well as RNA-seq, we identify 1200 TF-bound genes and 4775 differentially expressed genes. We experimentally validate 347 of these genes as functional target genes, and describe the regulatory relationships of the 20 TFs with their targets in a network that we call 'Pseudomonas aeruginosa genomic regulatory network' (PAGnet). Analysis of the network led to the identification of novel functions for two TFs (ExsA and GacA) in quorum sensing and nitrogen metabolism. Furthermore, we present an online platform and R package based on PAGnet to facilitate updating and user-customised analyses.


Assuntos
Proteínas de Bactérias/genética , Pseudomonas aeruginosa/genética , Fatores de Transcrição/genética , Proteínas de Bactérias/metabolismo , Imunoprecipitação da Cromatina , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidade , Fatores de Transcrição/metabolismo , Virulência , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
19.
BMC Vet Res ; 15(1): 235, 2019 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-31286947

RESUMO

BACKGROUND: Enterococcus is an important component of normal flora in human and animals, but in recent years, the pathogenicity of Enterococcus has been confirmed in clinical medicine. More and more animal infections have been reported in veterinary clinics. For the last decades, outbreaks of encephalitis in lambs have become much more common in Northern Xinjiang, China. Consequent studies have confirmed that these affected lambs had been commonly infected with E. faecalis. More than 60 E. faecalis were isolated from the brain of infected lambs, A highly virulent strain entitled E. faecalis 2A (XJ05) were selected, sequenced and analyzed. RESULT: Using whole genome sequence and de novo assembly, 18 contigs with NGS and annotation were obtained. It is confirmed that the genome has a size of 2.9 Mb containing 2783 protein-coding genes, as well as 54 tRNA genes and 4 rRNA genes. Some key features of this strain were identified, which included 7 predicted antibiotic resistance genes and 18 candidate virulence factor genes. CONCLUSION: The E. faecalis 2A (XJ05) genome is conspicuous smaller than E.faecalis V583, but not significantly different from other non-pathogenic E. faecalis. It carried 7 resistance genes including 4 kind of antibiotics which were consistent with the results of extensive drug resistance phenotypic, including aminoglycoside, macrolide, phenicol, and tetracycline. 2A (XJ05) also carried 18 new virulence factor genes related to virulence, hemolysin genes (cylA, cylB, cylM, cylL) may play an important role in lamb encephalitis by E. faecalis 2A (XJ05).


Assuntos
Farmacorresistência Bacteriana/genética , Encefalite/veterinária , Enterococcus faecalis/genética , Enterococcus faecalis/patogenicidade , Genoma Bacteriano/genética , Doenças dos Ovinos/microbiologia , Virulência/genética , Animais , Resistência a Múltiplos Medicamentos/genética , Encefalite/microbiologia , Ovinos
20.
J Med Microbiol ; 68(8): 1173-1188, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31268417

RESUMO

PURPOSE: Correct serotype identification of Streptococcus pneumoniae (pneumococcus) is important for monitoring disease epidemiology and assessing the impacts of pneumococcal vaccines. Furthermore, correct identification and differentiation of the pathogenic S. pneumoniae from closely related commensal species of the mitis group of the genus Streptococcus are essential for correct serotype identification. METHODOLOGY: A new protocol for determining the existing 98 serotypes of pneumococcus was developed, applying two PCR amplifications and amplicon sequencing, using newly designed internal primers. The new protocol was validated using S. pneumoniae genome sequences, reference strains with confirmed serotypes and clinical isolates, and comparing the results with those from the traditional Quellung reaction or antiserum panel gel precipitation, in addition to real-time PCR analysis. The taxonomic identifications of 422 publicly available (GenBank) genome sequences of S. pneumoniae, Streptococcus pseudopneumoniae and Streptococcus mitis were assessed by whole-genome sequence average nucleotide identity based on blast (ANIb) analysis. RESULTS: The proposed sequetyping protocol generates a 1017 bp whole cpsB region sequence, increasing resolution for serotype identification in pneumococcus isolates. The identifications of all GenBank genome sequences of S. pneumoniae were confirmed, whereas most of the S. pseudopneumoniae and almost all of the S. mitis genome sequences did not fulfil the ANIb thresholds for species-level identification. The housekeeping biomarker gene, groEL, correctly identified S. pneumoniae but often misclassified S. pseudopneumoniae and S. mitis as S. pneumoniae. CONCLUSIONS: These studies affirm the importance of applying reliable identification protocols for S. pneumoniae before serotyping; our protocols provide reliable diagnostic tools, as well as an improved workflow, for serotype identification of pneumococcus and differentiation of serogroup 6 types.


Assuntos
Cápsulas Bacterianas/genética , Tipagem Molecular/métodos , Streptococcus pneumoniae/classificação , Streptococcus pneumoniae/genética , Proteínas de Bactérias/genética , DNA Bacteriano/genética , Genoma Bacteriano/genética , Humanos , Infecções Pneumocócicas/microbiologia , Proteínas Tirosina Fosfatases/genética , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNA , Sorogrupo , Sorotipagem/normas , Streptococcus/classificação , Streptococcus/genética , Streptococcus/isolamento & purificação , Streptococcus pneumoniae/isolamento & purificação , Fluxo de Trabalho
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