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Asset Tag: Bacterial genome

September 22, 2019

Characterization of a novel multidrug resistance plasmid pSGB23 isolated from Salmonella enterica subspecies enterica serovar Saintpaul.

Salmonella enterica subspecies enterica serovar Saintpaul (S. Saintpaul) is an important gut pathogen which causes salmonellosis worldwide. Although intestinal salmonellosis is usually self-limiting, it can be life-threatening in children, the elderlies and immunocompromised patients. Appropriate antibiotic treatment is therefore required for these patients. However, the efficacy of many antibiotics on S. enterica infections has been greatly compromised due to spreading of multidrug resistance (MDR) plasmids, which poses serious threats on public health and needs to be closely monitored. In this study, we sequenced and fully characterized an S. enterica MDR plasmid pSGB23 isolated from chicken.Complete genome sequence analysis revealed that S. Saintpaul strain SGB23 harbored a 254 kb megaplasmid pSGB23, which carries 11 antibiotic resistance genes responsible for resistance to 9 classes of antibiotics and quaternary ammonium compounds that are commonly used to disinfect food processing facilities. Furthermore, we found that pSGB23 carries multiple conjugative systems, which allow it to spread into other Enterobacteriaceae spp. by self-conjugation. It also harbors multiple types of replicons and plasmid maintenance and addictive systems, which explains its broad host range and stable inheritance.We report here a novel MDR plasmid pSGB23 harboured by S. enterica. To our knowledge, it carried the greatest number of antibiotic resistance genes with the broadest range of resistance spectrum among S. enterica MDR plasmids identified so far. The isolation of pSGB23 from food sources is worrisome, while surveillance on its further spreading will be carried out based on the findings reported in this study.

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September 22, 2019

Transcriptional regulation of cysteine and methionine metabolism in Lactobacillus paracasei FAM18149.

Lactobacillus paracasei is common in the non-starter lactic acid bacteria (LAB) community of raw milk cheeses. This species can significantly contribute to flavor formation through amino acid metabolism. In this study, the DNA and RNA of L. paracasei FAM18149 were sequenced using next-generation sequencing technologies to reconstruct the metabolism of the sulfur-containing amino acids cysteine and methionine. Twenty-three genes were found to be involved in cysteine biosynthesis, the conversion of cysteine to methionine and vice versa, the S-adenosylmethionine recycling pathway, and the transport of sulfur-containing amino acids. Additionally, six methionine-specific T-boxes and one cysteine-specific T-box were found. Five of these were located upstream of genes encoding transporter functions. RNA-seq analysis and reverse-transcription quantitative polymerase reaction assays showed that expression of genes located downstream of these T-boxes was affected by the absence of either cysteine or methionine. Remarkably, the cysK2-ctl1-cysE2 operon, which is associated with te methionine-to-cysteine conversion and is upregulated in the absence of cysteine, showed high read coverage in the 5′-untranslated region and an antisense-RNA in the 3′-untranslated region. This indicates that this operon is regulated by the combination of cis- and antisense-mediated regulation mechanisms. The results of this study may help in the selection of L. paracasei strains to control sulfuric flavor formation in cheese.

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September 22, 2019

Co-culture of soil biofilm isolates enables the discovery of novel antibiotics

Bacterial natural products (NPs) are considered to be a promising source of drug discovery. However, the biosynthesis gene clusters (BGCs) of NP are not often expressed, making it difficult to identify them. Recently, the study of biofilm community showed bacteria may gain competitive advantages by the secretion of antibiotics, implying a possible way to screen antibiotic by evaluating the social behavior of bacteria. In this study, we have described an efficient workflow for novel antibiotic discovery by employing the bacterial social interaction strategy with biofilm cultivation, co-culture, transcriptomic and genomic methods. We showed that a biofilm dominant species, i.e. Pseudomonas sp. G7, which was isolated from cultivated soil biofilm community, was highly competitive in four-species biofilm communities, as the synergistic combinations preferred to exclude this strain while the antagonistic combinations did not. Through the analysis of transcriptomic changes in four-species co-culture and the complete genome of Pseudomonas sp. G7, we finally discovered two novel non-ribosomal polypeptide synthetic (NRPS) BGCs, whose products were predicted to have seven and six amino acid components, respectively. Furthermore, we provide evidence showing that only when Pseudomonas sp. G7 was co-cultivated with at least two or three other bacterial species can these BGC genes be induced, suggesting that the co-culture of the soil biofilm isolates is critical to the discovery of novel antibiotics. As a conclusion, we set a model of applying microbial interaction to the discovery of new antibiotics.

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September 22, 2019

Analysis of the complete genome sequence of Bacillus atrophaeus GQJK17 reveals its biocontrol characteristics as a plant growth-promoting rhizobacterium

Bacillus atrophaeus GQJK17 was isolated from the rhizosphere of Lycium barbarum L. in China, which was shown to be a plant growth-promoting rhizobacterium as a new biological agent against pathogenic fungi and gram-positive bacteria. We present its biological characteristics and complete genome sequence, which contains a 4,325,818 bp circular chromosome with 4,181 coding DNA sequences and a G+C content of 43.3%. A genome analysis revealed a total of 8 candidate gene clusters for producing antimicrobial secondary metabolites, including surfactin, bacillaene, fengycin, and bacillibactin. Some other antimicrobial and plant growth-promoting genes were also discovered. Our results provide insights into the genetic and biological basis of B. atrophaeus strains as a biocontrol agent for application in agriculture.

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September 22, 2019

Clinical Staphylococcus argenteus develops to small colony variants to promote persistent infection.

Staphylococcus argenteus is a novel staphylococcal species (also considered as a part of Staphylococcus aureus complex) that is infrequently reported on, and clinical S. argenteus infections are largely unstudied. Here, we report a persistent and recurrent hip joint infection case in which a S. argenteus strain and its small colony variants (SCVs) strain were successively isolated. We present features of the two S. argenteus strains and case details of their pathogenicity, explore factors that induce S. argenteus SCVs formation in the course of anti-infection therapy, and reveal potential genetic mechanisms for S. argenteus SCVs formation. S. argenteus strains were identified using phenotypic and genotypic methods. The S. argenteus strain XNO62 and SCV strain XNO106 were characterized using different models. S. argenteus SCVs were induced by the administration of amikacin and by chronic infection course based on the clinical case details. The genomes of both strains were sequenced and aligned in a pair-wise fashion using Mauve. The case details gave us important insights on the characteristics and therapeutic strategies for infections caused by S. argenteus and its SCVs. We found that strain XNO62 and SCV strain XNO106 are genetically-related sequential clones, the SCV strain exhibits reduced virulence but enhanced intracellular persistence compared to strain XNO62, thus promoting persistent infection. The induction experiments for S. argenteus SCVs demonstrated that high concentrations of amikacin greatly induce S. argenteus XNO62 to form SCVs, while a chronic infection of S. argenteus XNO62 slightly induces SCVs formation. Potential genetic mechanisms for S. argenteus SCVs formation were revealed and discussed based on genomic alignments. In conclusion, we report the first case of infection caused by S. argenteus and its SCVs strain. More attention should be paid to infections caused by S. argenteus and its SCVs, as they constitute a challenge to current therapeutic strategies. The problem of S. argenteus SCVs should be noticed, in particular when amikacin is used or in the case of a chronic S. argenteus infection.

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September 22, 2019

Raising the stakes: Loss of efflux pump regulation decreases meropenem susceptibility in Burkholderia pseudomallei

Burkholderia pseudomallei, the causative agent of the high-mortality disease melioidosis, is a gram-negative bacterium that is naturally resistant to many antibiotics. There is no vaccine for melioidosis, and effective eradication is reliant on biphasic and prolonged antibiotic administration. The carbapenem drug meropenem is the current gold standard option for treating severe melioidosis. Intrinsic B. pseudomallei resistance toward meropenem has not yet been documented; however, resistance could conceivably develop over the course of infection, leading to prolonged sepsis and treatment failure.We examined our 30-year clinical collection of melioidosis cases to identify B. pseudomallei isolates with reduced meropenem susceptibility. Isolates were subjected to minimum inhibitory concentration (MIC) testing toward meropenem. Paired isolates from patients who had evolved decreased susceptibility were subjected to whole-genome sequencing. Select agent-compliant genetic manipulation was carried out to confirm the molecular mechanisms conferring resistance.We identified 11 melioidosis cases where B. pseudomallei isolates developed decreased susceptibility toward meropenem during treatment, including 2 cases not treated with this antibiotic. Meropenem MICs increased from 0.5-0.75 µg/mL to 3-8 µg/mL. Comparative genomics identified multiple mutations affecting multidrug resistance-nodulation-division (RND) efflux pump regulators, with concomitant overexpression of their corresponding pumps. All cases were refractory to treatment despite aggressive, targeted therapy, and 2 were associated with a fatal outcome.This study confirms the role of RND efflux pumps in decreased meropenem susceptibility in B. pseudomallei. These findings have important ramifications for the diagnosis, treatment, and management of life-threatening melioidosis cases.

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September 22, 2019

Comparative genomics of Pseudomonas syringae reveals convergent gene gain and loss associated with specialization onto cherry (Prunus avium).

Genome-wide analyses of the effector- and toxin-encoding genes were used to examine the phylogenetics and evolution of pathogenicity amongst diverse strains of Pseudomonas syringae causing bacterial canker of cherry (Prunus avium), including pathovars P. syringae pv morsprunorum (Psm) races 1 and 2, P. syringae pv syringae (Pss) and P. syringae pv avii. Phylogenetic analyses revealed Psm races and P. syringae pv avii clades were distinct and were each monophyletic, whereas cherry-pathogenic strains of Pss were interspersed amongst strains from other host species. A maximum likelihood approach was used to predict effectors associated with pathogenicity on cherry. Pss possesses a smaller repertoire of type III effectors but has more toxin biosynthesis clusters than Psm and P. syringae pv avii. Evolution of cherry pathogenicity was correlated with gain of genes such as hopAR1 and hopBB1 through putative phage transfer and horizontal transfer respectively. By contrast, loss of the avrPto/hopAB redundant effector group was observed in cherry-pathogenic clades. Ectopic expression of hopAB and hopC1 triggered the hypersensitive reaction in cherry leaves, confirming computational predictions. Cherry canker provides a fascinating example of convergent evolution of pathogenicity that is explained by the mix of effector and toxin repertoires acting on a common host.© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.

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September 22, 2019

Comparative genomics of Spiraeoideae-infecting Erwinia amylovora strains provides novel insight to genetic diversity and identifies the genetic basis of a low-virulence strain.

Erwinia amylovora is the causal agent of fire blight, one of the most devastating diseases of apple and pear. Erwinia amylovora is thought to have originated in North America and has now spread to at least 50 countries worldwide. An understanding of the diversity of the pathogen population and the transmission to different geographical regions is important for the future mitigation of this disease. In this research, we performed an expanded comparative genomic study of the Spiraeoideae-infecting (SI) E. amylovora population in North America and Europe. We discovered that, although still highly homogeneous, the genetic diversity of 30 E. amylovora genomes examined was about 30 times higher than previously determined. These isolates belong to four distinct clades, three of which display geographical clustering and one of which contains strains from various geographical locations (‘Widely Prevalent’ clade). Furthermore, we revealed that strains from the Widely Prevalent clade displayed a higher level of recombination with strains from a clade strictly from the eastern USA, which suggests that the Widely Prevalent clade probably originated from the eastern USA before it spread to other locations. Finally, we detected variations in virulence in the SI E. amylovora strains on immature pear, and identified the genetic basis of one of the low-virulence strains as being caused by a single nucleotide polymorphism in hfq, a gene encoding an important virulence regulator. Our results provide insights into the population structure, distribution and evolution of SI E. amylovora in North America and Europe.© 2017 BSPP AND JOHN WILEY & SONS LTD.

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September 22, 2019

Characterization and high-quality draft genome sequence of Herbivorax saccincola A7, an anaerobic, alkaliphilic, thermophilic, cellulolytic, and xylanolytic bacterium.

An anaerobic, cellulolytic-xylanolytic bacterium, designated strain A7, was isolated from a cellulose-degrading bacterial community inhabiting bovine manure compost on Ishigaki Island, Japan, by enrichment culture using unpretreated corn stover as the sole carbon source. The strain was Gram-positive, non-endospore forming, non-motile, and formed orange colonies on solid medium. Strain A7 was identified as Herbivorax saccincola by DNA-DNA hybridization, and phylogenetic analysis based on 16S rRNA gene sequences showed that it was closely related to H. saccincola GGR1 (= DSM 101079T). H. saccincola A7 (= JCM 31827=DSM 104321) had quite similar phenotypic characteristics to those of strain GGR1. However, the optimum growth of A7 was at alkaline pH (9.0) and 55°C, compared to pH 7.0 at 60°C for GGR1, and the fatty acid profile of A7 contained 1.7-times more C17:0 iso than GGR1. The draft genome sequence revealed that H. saccincola A7 possessed a cellulosome-like extracellular macromolecular complex, which has also been found for Clostridium thermocellum and C. clariflavum. H. saccincola A7 contained more glycoside hydrolases (GHs) belonging to GH families-11 and -2, and more diversity of xylanolytic enzymes, than C. thermocellum and C. clariflavum. H. saccincola A7 could grow on xylan because it encoded essential genes for xylose metabolism, such as a xylose transporter, xylose isomerase, xylulokinase, and ribulose-phosphate 3-epimerase, which are absent from C. thermocellum. These results indicated that H. saccincola A7 has great potential as a microorganism that can effectively degrade lignocellulosic biomass. Copyright © 2018 Elsevier GmbH. All rights reserved.

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September 22, 2019

Two ancestral genes shaped the Xanthomonas campestris TAL effector gene repertoire.

Xanthomonas transcription activator-like effectors (TALEs) are injected inside plant cells to promote host susceptibility by enhancing transcription of host susceptibility genes. TALE-encoding (tal) genes were thought to be absent from Brassicaceae-infecting Xanthomonas campestris (Xc) genomes based on four reference genomic sequences. We discovered tal genes in 26 of 49 Xc strains isolated worldwide and used a combination of single molecule real time (SMRT) and tal amplicon sequencing to yield a near-complete description of the TALEs found in Xc (Xc TALome). The 53 sequenced tal genes encode 21 distinct DNA binding domains that sort into seven major DNA binding specificities. In silico analysis of the Brassica rapa promoterome identified a repertoire of predicted TALE targets, five of which were experimentally validated using quantitative reverse transcription polymerase chain reaction. The Xc TALome shows multiple signs of DNA rearrangements that probably drove its evolution from two ancestral tal genes. We discovered that Tal12a and Tal15a of Xcc strain Xca5 contribute together in the development of disease symptoms on susceptible B. oleracea var. botrytis cv Clovis. This large and polymorphic repertoire of TALEs opens novel perspectives for elucidating TALE-mediated susceptibility of Brassicaceae to black rot disease and for understanding the molecular processes underlying TALE evolution.© 2018 The Authors New Phytologist © 2018 New Phytologist Trust.

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September 22, 2019

Complete genome sequence provides insights into the biodrying-related microbial function of Bacillus thermoamylovorans isolated from sewage sludge biodrying material.

To enable the development of microbial agents and identify suitable candidate used for biodrying, the existence and function of Bacillus thermoamylovorans during sewage sludge biodrying merits investigation. This study isolated a strain of B. thermoamylovorans during sludge biodrying, submitted it for complete genome sequencing and analyzed its potential microbial functions. After biodrying, the moisture content of the biodrying material decreased from 66.33% to 50.18%, and B. thermoamylovorans was the ecologically dominant Bacillus, with the primary annotations associated with amino acid transport and metabolism (9.53%) and carbohydrate transport and metabolism (8.14%). It contains 96 carbohydrate-active- enzyme-encoding gene counts, mainly distributed in glycoside hydrolases (33.3%) and glycosyl transferases (27.1%). The virulence factors are mainly associated with biosynthesis of capsule and polysaccharide capsule. This work indicates that among the biodrying microorganisms, B. thermoamylovorans has good potential for degrading recalcitrant and readily degradable components, thus being a potential microbial agent used to improve biodrying. Copyright © 2018 Elsevier Ltd. All rights reserved.

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September 22, 2019

Whole-genome sequence and genome annotation of Xanthomonas citri pv. mangiferaeindicae, causal agent of bacterial black spot on Mangifera indica.

A newly isolated strain XC01 was identified as Xanthomonas citri pv. mangiferaeindicae, isolated from an infected mango fruit in Guangxi, China. The complete genome sequence of XC01 was carried out using the PacBio RSII platform. The genome contains a circular chromosome with 3,865,165 bp, 3442 protein-coding genes, 53 tRNAs, and 2 rRNA operons. Phylogenetic analysis revealed that this pathogen is very close to the soybeans bacterial pustule pathogen X. citri pv. glycines CFBP 2526, with a completely different host range. The genome sequence of XC01 may shed a highlight genes with a demonstrated or proposed role in on the pathogenesis.

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September 22, 2019

Whole-genome comparison of high and low virulent Staphylococcus aureus isolates inducing implant-associated bone infections.

Staphylococcus aureus can cause wide range of infections from simple soft skin infections to severe endocarditis, bacteremia, osteomyelitis and implant associated bone infections (IABI). The focus of the present investigation was to study virulence properties of S. aureus isolates from acute and chronic IABI by means of their in vivo lethality, in vitro osteoblasts invasion, biofilm formation and subsequently whole genome comparison between high and low virulent strains. Application of insect infection model Galleria mellonella revealed high, intermediate and low virulence phenotypes of these clinical isolates, which showed good correlation with osteoblast invasion and biofilm formation assays. Comparative genomics of selected high (EDCC 5458) and low (EDCC 5464) virulent strains enabled the identification of molecular factors responsible for the development of acute and chronic IABI. Accordingly, the low virulent strain EDCC 5464 harbored point mutations resulting in frame shift mutations in agrC (histidine kinase in agr system), graS (histidine kinase in graSR, a two component system) and efeB (peroxidase in efeOBU operon, an iron acquisition system) genes. Additionally, we found a mobile element (present 11 copies in EDCC 5464) inserted at the end of ß-hemolysin (hlb) and sarU genes, which are involved in the pathogenesis and regulation of virulence gene expression in coordination with quorum sensing system. All these results are in good support with the low virulence behavior of EDCC 5464. From the previous literature, it is well known that agr defective S. aureus clinical strains are isolated from the chronic infections. Similarly, low virulent EDCC 5464 was isolated from chronic implant-associated bone infections infection whereas EDCC 5458 was obtained from acute implant-associated bone infections. Laboratory based in vitro and in vivo results and insights from comparative genomic analysis could be correlated with the clinical conclusion of IABIs and allows evidence-based treatment strategies based on the pathogenesis of the strain to cure life devastating implant-associated infections. Copyright © 2018 Elsevier GmbH. All rights reserved.

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September 22, 2019

T-independent IFN? and B cells cooperate to prevent mortality associated with disseminated Chlamydia muridarum genital tract infection.

CD4 T cells and antibody are required for optimal acquired immunity to C. muridarum genital tract infection, and T cell-mediated IFN? production is necessary to clear infection in the absence of humoral immunity. However, the role of T cell-independent immune responses during primary infection remains unclear. We investigated this question by inoculating wild-type and immune-deficient mice with C. muridarum CM001, a clonal isolate capable of enhanced extragenital replication. Genital inoculation of wild-type mice resulted in transient dissemination to the lungs and spleen that then was rapidly cleared from these organs. However, CM001 genital infection proved lethal for STAT1-/- and IFNG-/- mice, where IFN? signaling is absent, and for Rag1-/- mice that lack T and B cells, but retain innate IFN? signaling. In contrast, B cell-deficient muMT mice that can generate a Th1 response, and T cell-deficient mice with intact B cell and innate IFN? signaling survived. These data collectively indicate that IFN? prevents lethal CM001 dissemination in the absence of T cells and suggests a B cell co-requirement. Adoptive transfer of convalescent immune sera, but not naïve IgM, to Rag1-/- mice infected with CM001 significantly increased survival time, while transfer of naïve B cells completely rescued Rag1-/- mice from CM001 lethality. Protection was associated with a significant reduction in the lung chlamydial burden of genitally infected mice. These data reveal an important cooperation between T-independent B cell responses and innate IFN? in chlamydial host defense, and suggest interactions between T-independent antibody and IFN? are essential for limiting extragenital dissemination. Copyright © 2018 American Society for Microbiology.

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September 22, 2019

Three substrains of the cyanobacterium Anabaena sp. PCC 7120 display divergence in genomic sequences and hetC function.

Anabaena sp. strain PCC 7120 is a model strain for molecular studies of cell differentiation and patterning in heterocyst-forming cyanobacteria. Subtle differences in heterocyst development have been noticed in different laboratories working on the same organism. In this study, 360 mutations, including single nucleotide polymorphisms (SNPs), small insertion/deletions (indels; 1 to 3 bp), fragment deletions, and transpositions, were identified in the genomes of three substrains. Heterogeneous/heterozygous bases were also identified due to the polyploidy nature of the genome and the multicellular morphology but could be completely segregated when plated after filament fragmentation by sonication. hetC is a gene upregulated in developing cells during heterocyst formation in Anabaena sp. strain PCC 7120 and found in approximately half of other heterocyst-forming cyanobacteria. Inactivation of hetC in 3 substrains of Anabaena sp. PCC 7120 led to different phenotypes: the formation of heterocysts, differentiating cells that keep dividing, or the presence of both heterocysts and dividing differentiating cells. The expression of P hetZ -gfp in these hetC mutants also showed different patterns of green fluorescent protein (GFP) fluorescence. Thus, the function of hetC is influenced by the genomic background and epistasis and constitutes an example of evolution under way.IMPORTANCE Our knowledge about the molecular genetics of heterocyst formation, an important cell differentiation process for global N2 fixation, is mostly based on studies with Anabaena sp. strain PCC 7120. Here, we show that rapid microevolution is under way in this strain, leading to phenotypic variations for certain genes related to heterocyst development, such as hetC This study provides an example for ongoing microevolution, marked by multiple heterogeneous/heterozygous single nucleotide polymorphisms (SNPs), in a multicellular multicopy-genome microorganism. Copyright © 2018 American Society for Microbiology.

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