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Asset Tag: Bacterial

April 21, 2020

Metaepigenomic analysis reveals the unexplored diversity of DNA methylation in an environmental prokaryotic community.

DNA methylation plays important roles in prokaryotes, and their genomic landscapes-prokaryotic epigenomes-have recently begun to be disclosed. However, our knowledge of prokaryotic methylation systems is focused on those of culturable microbes, which are rare in nature. Here, we used single-molecule real-time and circular consensus sequencing techniques to reveal the ‘metaepigenomes’ of a microbial community in the largest lake in Japan, Lake Biwa. We reconstructed 19 draft genomes from diverse bacterial and archaeal groups, most of which are yet to be cultured. The analysis of DNA chemical modifications in those genomes revealed 22 methylated motifs, nine of which were novel. We identified methyltransferase genes likely responsible for methylation of the novel motifs, and confirmed the catalytic specificities of four of them via transformation experiments using synthetic genes. Our study highlights metaepigenomics as a powerful approach for identification of the vast unexplored variety of prokaryotic DNA methylation systems in nature.

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April 21, 2020

Assignment of virus and antimicrobial resistance genes to microbial hosts in a complex microbial community by combined long-read assembly and proximity ligation.

We describe a method that adds long-read sequencing to a mix of technologies used to assemble a highly complex cattle rumen microbial community, and provide a comparison to short read-based methods. Long-read alignments and Hi-C linkage between contigs support the identification of 188 novel virus-host associations and the determination of phage life cycle states in the rumen microbial community. The long-read assembly also identifies 94 antimicrobial resistance genes, compared to only seven alleles in the short-read assembly. We demonstrate novel techniques that work synergistically to improve characterization of biological features in a highly complex rumen microbial community.

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April 21, 2020

Complete genome sequence of 3-chlorobenzoate-degrading bacterium Cupriavidus necator NH9 and reclassification of the strains of the genera Cupriavidus and Ralstonia based on phylogenetic and whole-genome sequence analyses.

Cupriavidus necator NH9, a 3-chlorobenzoate (3-CB)-degrading bacterium, was isolated from soil in Japan. In this study, the complete genome sequence of NH9 was obtained via PacBio long-read sequencing to better understand the genetic components contributing to the strain’s ability to degrade aromatic compounds, including 3-CB. The genome of NH9 comprised two circular chromosomes (4.3 and 3.4 Mb) and two circular plasmids (427 and 77 kb) containing 7,290 coding sequences, 15 rRNA and 68 tRNA genes. Kyoto Encyclopedia of Genes and Genomes pathway analysis of the protein-coding sequences in NH9 revealed a capacity to completely degrade benzoate, 2-, 3-, or 4-hydroxybenzoate, 2,3-, 2,5-, or 3,4-dihydroxybenzoate, benzoylformate, and benzonitrile. To validate the identification of NH9, phylogenetic analyses (16S rRNA sequence-based tree and multilocus sequence analysis) and whole-genome sequence analyses (average nucleotide identity, percentage of conserved proteins, and tetra-nucleotide analyses) were performed, confirming that NH9 is a C. necator strain. Over the course of our investigation, we noticed inconsistencies in the classification of several strains that were supposed to belong to the two closely-related genera Cupriavidus and Ralstonia. As a result of whole-genome sequence analysis of 46 Cupriavidus strains and 104 Ralstonia strains, we propose that the taxonomic classification of 41 of the 150 strains should be changed. Our results provide a clear delineation of the two genera based on genome sequences, thus allowing taxonomic identification of strains belonging to these two genera.

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April 21, 2020

Whole-Genome Sequences of Five Acinetobacter baumannii Strains From a Child With Leukemia M2.

Acinetobacter baumannii is an opportunistic pathogen and is one of the primary etiological agents of healthcare-associated infections (HAIs). A. baumannii infections are difficult to treat due to the intrinsic and acquired antibiotic resistance of strains of this bacterium, which frequently limits therapeutic options. In this study, five A. baumannii strains (810CP, 433H, 434H, 483H, and A-2), all of which were isolated from a child with leukemia M2, were characterized through antibiotic susceptibility profiling, the detection of genes encoding carbapenem hydrolyzing oxacillinases, pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), adherence and invasion assays toward the A549 cell line, and the whole-genome sequence (WGS). The five strains showed Multidrug resistant (MDR) profiles and amplification of the blaOXA-23 gene, belonging to ST758 and grouped into two PFGE clusters. WGS of 810CP revealed the presence of a circular chromosome and two small plasmids, pAba810CPa and pAba810CPb. Both plasmids carried genes encoding the Sp1TA system, although resistance genes were not identified. A gene-by-gene comparison analysis was performed among the A. baumannii strains isolated in this study and others A. baumannii ST758 strains (HIMFG and INCan), showing that 86% of genes were present in all analyzed strains. Interestingly, the 433H, 434H, and 483H strains varied by 8-10 single-nucleotide variants (SNVs), while the A2 and 810CP strains varied by 46 SNVs. Subsequently, an analysis using BacWGSTdb showed that all of our strains had the same resistance genes and were ST758. However, some variations were observed in relation to virulence genes, mainly in the 810CP strain. The genes involved in the synthesis of hepta-acylated lipooligosaccharides, the pgaABCD locus encoding poly-ß-1-6-N-acetylglucosamine, the ompA gene, Csu pili, bap, the two-component system bfms/bfmR, a member of the phospholipase D family, and two iron-uptake systems were identified in our A. baumannii strains genome. The five A. baumannii strains isolated from the child were genetically different and showed important characteristics that promote survival in a hospital environment. The elucidation of their genomic sequences provides important information for understanding their epidemiology, antibiotic resistance, and putative virulence factors.

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April 21, 2020

Valinomycin, produced by Streptomyces sp. S8, a key antifungal metabolite in large patch disease suppressiveness.

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.

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April 21, 2020

Complete genome sequence of Raoultella sp. strain X13, a promising cell factory for the synthesis of CdS quantum dots.

A novel cadmium-resistant bacterium, Raoultella sp. strain X13, recently isolated from heavy metal-contaminated soil, and this strain can synthesize CdS quantum dots using cadmium nitrate [Cd(NO4)2] and l-cysteine. Biomineralization of CdS by strain X13 can efficiently remove cadmium from aqueous solution. To illuminate the molecular mechanisms for the biosynthesis of CdS nanoparticle, the complete genome of Raoultella sp. strain X13 was sequenced. The whole genome sequence comprises a circular chromosome and a circular plasmid. Cysteine desulfhydrase smCSE has been previously found to be associated with the synthesis of CdS quantum dots. Bioinformatics analysis indicated that the genome of Raoultella sp. strain X13 encodes five putative cysteine desulfhydrases and all of them are located in the chromosome. The genome information may help us to determine the molecular mechanisms of the synthesis of CdS quantum dots and potentially enable us to engineer this microorganism for applications in biotechnology.

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April 21, 2020

New genetic context of lnu(B) composed of two multi-resistance gene clusters in clinical Streptococcus agalactiae ST-19 strains.

Clindamycin is a lincosamide antibiotic used to treat staphylococcal and streptococcal infections. Reports of clinical Streptococcus agalactiae isolates with the rare lincosamide resistance/macrolide susceptibility (LR/MS) phenotype are increasing worldwide. In this study, we characterised three clinical S. agalactiae strains with the unusual L phenotype from China.Three clinical S. agalactiae strains, Sag3, Sag27 and Sag4104, with the L phenotype were identified from 186 isolates collected from 2016 to 2018 in Shanghai, China. The MICs of clindamycin, erythromycin, tetracycline, levofloxacin, and penicillin were determined using Etest. PCR for the lnu(B) gene was conducted. Whole genome sequencing and sequence analysis were carried out to investigate the genetic context of lnu(B). Efforts to transfer lincomycin resistance by conjugation and to identify the circular form by inverse PCR were made.Sag3, Sag27, and Sag4104 were susceptible to erythromycin (MIC =0.25?mg/L) but resistant to clindamycin (MIC =1?mg/L). lnu(B) was found to be responsible for the L phenotype. lnu(B) in Sag3 and Sag27 were chromosomally located in an aadE-spw-lsa(E)-lnu(B) resistance gene cluster adjacent to an upstream 7-kb tet(L)-cat resistance gene cluster. Two resistance gene clusters were flanked by the IS6-like element, IS1216. Sag4104 only contained partial genes of aadE-spw-lsa(E)-lnu(B) resistance gene cluster and was also flanked by IS1216.These results established the presence of the L phenotype associated with lnu(B) in clinical S. agalactiae isolates in China. The lnu(B)-containing multi-resistance gene cluster possibly acts as a composite transposon flanked by IS1216 and as a vehicle for the dissemination of multidrug resistance among S. agalactiae.

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April 21, 2020

Development of a metabolic pathway transfer and genomic integration system for the syngas-fermenting bacterium Clostridium ljungdahlii.

Clostridium spp. can synthesize valuable chemicals and fuels by utilizing diverse waste-stream substrates, including starchy biomass, lignocellulose, and industrial waste gases. However, metabolic engineering in Clostridium spp. is challenging due to the low efficiency of gene transfer and genomic integration of entire biosynthetic pathways.We have developed a reliable gene transfer and genomic integration system for the syngas-fermenting bacterium Clostridium ljungdahlii based on the conjugal transfer of donor plasmids containing large transgene cassettes (>?5 kb) followed by the inducible activation of Himar1 transposase to promote integration. We established a conjugation protocol for the efficient generation of transconjugants using the Gram-positive origins of replication repL and repH. We also investigated the impact of DNA methylation on conjugation efficiency by testing donor constructs with all possible combinations of Dam and Dcm methylation patterns, and used bisulfite conversion and PacBio sequencing to determine the DNA methylation profile of the C. ljungdahlii genome, resulting in the detection of four sequence motifs with N6-methyladenosine. As proof of concept, we demonstrated the transfer and genomic integration of a heterologous acetone biosynthesis pathway using a Himar1 transposase system regulated by a xylose-inducible promoter. The functionality of the integrated pathway was confirmed by detecting enzyme proteotypic peptides and the formation of acetone and isopropanol by C. ljungdahlii cultures utilizing syngas as a carbon and energy source.The developed multi-gene delivery system offers a versatile tool to integrate and stably express large biosynthetic pathways in the industrial promising syngas-fermenting microorganism C. ljungdahlii. The simple transfer and stable integration of large gene clusters (like entire biosynthetic pathways) is expanding the range of possible fermentation products of heterologously expressing recombinant strains. We also believe that the developed gene delivery system can be adapted to other clostridial strains as well.

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April 21, 2020

The bile salt glycocholate induces global changes in gene and protein expression and activates virulence in enterotoxigenic Escherichia coli.

Pathogenic bacteria use specific host factors to modulate virulence and stress responses during infection. We found previously that the host factor bile and the bile component glyco-conjugated cholate (NaGCH, sodium glycocholate) upregulate the colonization factor CS5 in enterotoxigenic Escherichia coli (ETEC). To further understand the global regulatory effects of bile and NaGCH, we performed Illumina RNA-Seq and found that crude bile and NaGCH altered the expression of 61 genes in CS5?+?CS6 ETEC isolates. The most striking finding was high induction of the CS5 operon (csfA-F), its putative transcription factor csvR, and the putative ETEC virulence factor cexE. iTRAQ-coupled LC-MS/MS proteomic analyses verified induction of the plasmid-borne virulence proteins CS5 and CexE and also showed that NaGCH affected the expression of bacterial membrane proteins. Furthermore, NaGCH induced bacteria to aggregate, increased their adherence to epithelial cells, and reduced their motility. Our results indicate that CS5?+?CS6 ETEC use NaGCH present in the small intestine as a signal to initiate colonization of the epithelium.

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April 21, 2020

Survival Mechanisms of Campylobacter hepaticus Identified by Genomic Analysis and Comparative Transcriptomic Analysis of in vivo and in vitro Derived Bacteria.

Chickens infected with Campylobacter jejuni or Campylobacter coli are largely asymptomatic, however, infection with the closely related species, Campylobacter hepaticus, can result in Spotty Liver Disease (SLD). C. hepaticus has been detected in the liver, bile, small intestine and caecum of SLD affected chickens. The survival and colonization mechanisms that C. hepaticus uses to colonize chickens remain unknown. In this study, we compared the genome sequences of 14 newly sequenced Australian isolates of C. hepaticus, isolates from outbreaks in the United Kingdom, and reference strains of C. jejuni and C. coli, with the aim of identifying virulence genes associated with SLD. We also carried out global comparative transcriptomic analysis between C. hepaticus recovered from the bile of SLD infected chickens and C. hepaticus grown in vitro. This revealed how the bacteria adapt to proliferate in the challenging host environment in which they are found. Additionally, biochemical experiments confirmed some in silico metabolic predictions. We found that, unlike other Campylobacter sp., C. hepaticus encodes glucose and polyhydroxybutyrate metabolism pathways. This study demonstrated the metabolic plasticity of C. hepaticus, which may contribute to survival in the competitive, nutrient and energy-limited environment of the chicken. Transcriptomic analysis indicated that gene clusters associated with glucose utilization, stress response, hydrogen metabolism, and sialic acid modification may play an important role in the pathogenicity of C. hepaticus. An understanding of the survival and virulence mechanisms that C. hepaticus uses will help to direct the development of effective intervention methods to protect birds from the debilitating effects of SLD.

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April 21, 2020

Genome expansion of an obligate parthenogenesis-associated Wolbachia poses an exception to the symbiont reduction model.

Theory predicts that dependency within host-endosymbiont interactions results in endosymbiont genome size reduction. Unexpectedly, the largest Wolbachia genome was found in the obligate, parthenogenesis-associated wFol. In this study, we investigate possible processes underlying this genome expansion by comparing a re-annotated wFol genome to other Wolbachia genomes. In addition, we also search for candidate genes related to parthenogenesis induction (PI).Within wFol, we found five phage WO regions representing 25.4% of the complete genome, few pseudogenized genes, and an expansion of DNA-repair genes in comparison to other Wolbachia. These signs of genome conservation were mirrored in the wFol host, the springtail F. candida, which also had an expanded DNA-repair gene family and many horizontally transferred genes. Across all Wolbachia genomes, there was a strong correlation between gene numbers of Wolbachia strains and their hosts. In order to identify genes with a potential link to PI, we assembled the genome of an additional PI strain, wLcla. Comparisons between four PI Wolbachia, including wFol and wLcla, and fourteen non-PI Wolbachia yielded a small set of potential candidate genes for further investigation.The strong similarities in genome content of wFol and its host, as well as the correlation between host and Wolbachia gene numbers suggest that there may be some form of convergent evolution between endosymbiont and host genomes. If such convergent evolution would be strong enough to overcome the evolutionary forces causing genome reduction, it would enable expanded genomes within long-term obligate endosymbionts.

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April 21, 2020

Adaptive Strategies in a Poly-Extreme Environment: Differentiation of Vegetative Cells in Serratia ureilytica and Resistance to Extreme Conditions.

Poly-extreme terrestrial habitats are often used as analogs to extra-terrestrial environments. Understanding the adaptive strategies allowing bacteria to thrive and survive under these conditions could help in our quest for extra-terrestrial planets suitable for life and understanding how life evolved in the harsh early earth conditions. A prime example of such a survival strategy is the modification of vegetative cells into resistant resting structures. These differentiated cells are often observed in response to harsh environmental conditions. The environmental strain (strain Lr5/4) belonging to Serratia ureilytica was isolated from a geothermal spring in Lirima, Atacama Desert, Chile. The Atacama Desert is the driest habitat on Earth and furthermore, due to its high altitude, it is exposed to an increased amount of UV radiation. The geothermal spring from which the strain was isolated is oligotrophic and the temperature of 54°C exceeds mesophilic conditions (15 to 45°C). Although the vegetative cells were tolerant to various environmental insults (desiccation, extreme pH, glycerol), a modified cell type was formed in response to nutrient deprivation, UV radiation and thermal shock. Scanning (SEM) and Transmission Electron Microscopy (TEM) analyses of vegetative cells and the modified cell structures were performed. In SEM, a change toward a circular shape with reduced size was observed. These circular cells possessed what appears as extra coating layers under TEM. The resistance of the modified cells was also investigated, they were resistant to wet heat, UV radiation and desiccation, while vegetative cells did not withstand any of those conditions. A phylogenomic analysis was undertaken to investigate the presence of known genes involved in dormancy in other bacterial clades. Genes related to spore-formation in Myxococcus and Firmicutes were found in S. ureilytica Lr5/4 genome; however, these genes were not enough for a full sporulation pathway that resembles either group. Although, the molecular pathway of cell differentiation in S. ureilytica Lr5/4 is not fully defined, the identified genes may contribute to the modified phenotype in the Serratia genus. Here, we show that a modified cell structure can occur as a response to extremity in a species that was previously not known to deploy this strategy. This strategy may be widely spread in bacteria, but only expressed under poly-extreme environmental conditions.

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April 21, 2020

Plasmids of Shigella flexneri serotype 1c strain Y394 provide advantages to bacteria in the host.

Shigella flexneri has an extremely complex genome with a significant number of virulence traits acquired by mobile genetic elements including bacteriophages and plasmids. S. flexneri serotype 1c is an emerging etiological agent of bacillary dysentery in developing countries. In this study, the complete nucleotide sequence of two plasmids of S. flexneri serotype 1c strain Y394 was determined and analysed.The plasmid pINV-Y394 is an invasive or virulence plasmid of size 221,293?bp composed of a large number of insertion sequences (IS), virulence genes, regulatory and maintenance genes. Three hundred and twenty-eight open reading frames (ORFs) were identified in pINV-Y394, of which about a half (159 ORFs) were identified as IS elements. Ninety-seven ORFs were related to characterized genes (majority of which are associated with virulence and their regulons), and 72 ORFs were uncharacterized or hypothetical genes. The second plasmid pNV-Y394 is of size 10,866?bp and encodes genes conferring resistance against multiple antibiotics of clinical importance. The multidrug resistance gene cassette consists of tetracycline resistance gene tetA, streptomycin resistance gene strA-strB and sulfonamide-resistant dihydropteroate synthase gene sul2.These two plasmids together play a key role in the fitness of Y394 in the host environment. The findings from this study indicate that the pathogenic S. flexneri is a highly niche adaptive pathogen which is able to co-evolve with its host and respond to the selection pressure in its environment.

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April 21, 2020

Complete genome sequence of acetate-producing Klebsiella pneumoniae L5-2 isolated from infant feces.

Acetate is an important metabolite in infants as it can affect metabolism as well as immune and inflammatory responses. However, there have been no studies on acetate production by Klebsiella pneumoniae isolated from infant feces. In this study, we isolated a K. pneumoniae strain, L5-2, from infant feces, and we found it produces acetate. The genome of L5-2 consisted of a 5,237,123-bp single chromosome and a 139,211-bp single plasmid. The G?+?C content was 57.27%. By whole-genome analysis of K. pneumoniae L5-2, we identified seven genes related to acetate production (poxA, pta, eutD, ackA, eutP, eutQ, and adhE). We confirmed acetate production by K. pneumoniae L5-2 by ion chromatography. The aldehyde/alcohol dehydrogenase (adhE) activity of K. pneumoniae L5-2 was significantly higher than that of the K. pneumoniae subsp. ozaenae ATCC 11296. Thus, the acetate-producing ability of K. pneumoniae L5-2 was influenced by the adhE gene. In addition, K. pneumoniae L5-2 had significantly less virulence factor-encoding genes than other K. pneumoniae strains isolated from humans. In conclusion, K. pneumoniae L5-2 isolated from infant feces has less virulence factors and higher adhE activity than other K. pneumoniae strains.

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April 21, 2020

Characterization of an NDM-5 carbapenemase-producing Escherichia coli ST156 isolate from a poultry farm in Zhejiang, China.

The emergence of carbapenem-resistant Enterobacteriaceae strains has posed a severe threat to public health in recent years. The mobile elements carrying the New Delhi metallo-ß-lactqtamase (NDM) gene have been regarded as the major mechanism leading to the rapid increase of carbapenem-resistant Enterobacteriaceae strains isolated from clinics and animals.We describe an NDM-5-producing Escherichia coli strain, ECCRA-119 (sequence type 156 [ST156]), isolated from a poultry farm in Zhejiang, China. ECCRA-119 is a multidrug-resistant (MDR) isolate that exhibited resistance to 27 antimicrobial compounds, including imipenem and meropenem, as detected by antimicrobial susceptibility testing (AST). The complete genome sequence of the ECCRA-119 isolate was also obtained using the PacBio RS II platform. Eleven acquired resistance genes were identified in the chromosome; four were detected in plasmid pTB201, while six were detected in plasmid pTB202. Importantly, the carbapenem-resistant gene blaNDM-5 was detected in the IncX3 plasmid pTB203. In addition, seven virulence genes and one metal-resistance gene were also detected. The results of conjugation experiments and the transfer regions identification indicated that the blaNDM-5-harboring plasmid pTB203 could be transferred between E. coli strains.The results reflected the severe bacterial resistance in a poultry farm in Zhejiang province and increased our understanding of the presence and transmission of the blaNDM-5 gene.

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