Serum Institute of India is among the world’s largest vaccine producers. Here, we report the complete genome sequences for four Bordetella pertussis strains used by Serum Institute of India in the production of whole-cell pertussis vaccines. Copyright © 2016 Weigand et al.
Entomopathogenic fungi such as Beauveria bassiana are promising biological agents for control of malaria mosquitoes. Indeed, infection with B. bassiana reduces the lifespan of mosquitoes in the laboratory and in the field. Natural isolates of B. bassiana show up to 10-fold differences in virulence between the most and the least virulent isolate. In this study, we sequenced the genomes of five isolates representing the extremes of low/high virulence and three RNA libraries, and applied a genome comparison approach to uncover genetic mechanisms underpinning virulence.A high-quality, near-complete genome assembly was achieved for the highly virulent isolate Bb8028, which was compared to the assemblies of the four other isolates. Whole genome analysis showed a high level of genetic diversity between the five isolates (2.85-16.8 SNPs/kb), which grouped into two distinct phylogenetic clusters. Mating type gene analysis revealed the presence of either the MAT1-1-1 or the MAT1-2-1 gene. Moreover, a putative new MAT gene (MAT1-2-8) was detected in the MAT1-2 locus. Comparative genome analysis revealed that Bb8028 contains 163 genes exclusive for this isolate. These unique genes have a tendency to cluster in the genome and to be often located near the telomeres. Among the genes unique to Bb8028 are a Non-Ribosomal Peptide Synthetase (NRPS) secondary metabolite gene cluster, a polyketide synthase (PKS) gene, and five genes with homology to bacterial toxins. A survey of candidate virulence genes for B. bassiana is presented.Our results indicate several genes and molecular processes that may underpin virulence towards mosquitoes. Thus, the genome sequences of five isolates of B. bassiana provide a better understanding of the natural variation in virulence and will offer a major resource for future research on this important biological control agent.
Enterococcus faecalis, the type strain of the genus Enterococcus, is not only a commensal bacterium in the gastrointestinal tract in vertebrates and invertebrates, but also causes serious disease as an opportunistic pathogen. To date, genome sequences have been published for over four hundred E. faecalis strains; however, pathogenicity of these microbes remains complicated. To increase our knowledge of E. faecalis virulence factors, we isolated strain CBA7120 from the feces of an 81-year-old female from the Republic of Korea and performed a comparative genomic analysis.The genome sequence of E. faecalis CBA7120 is 3,134,087 bp in length, with a G + C content of 37.35 mol%, and is comprised of four contigs with an N50 value of 2,922,046 bp. The genome showed high similarity with other strains of E. faecalis, including OG1RF, T13, 12107 and T20, based on OrthoANI values. Strain CBA7120 contains 374 pan-genome orthologous groups (POGs) as singletons, including “Phages, Prophages, Transposable elements, Plasmids,” “Carbohydrates,” “DNA metabolism,” and “Virulence, Disease and Defense” subsystems. Genes related to multidrug resistance efflux pumps were annotated in the genome.The comparative genomic analysis of E. faecalis strains presented in this study was performed using a variety of analysis methods and will facilitate future identification of hypothetical proteins.
How the pathogen Clostridium difficile might survive, evolve and be transferred between reservoirs within the natural environment is poorly understood. Some ribotypes are found both in clinical and environmental settings. Whether these strains are distinct from each another and evolve in the specific environments is not established. The possession of a highly mobile genome has contributed to the genetic diversity and ongoing evolution of C. difficile. Interpretations of genetic diversity have been limited by fragmented assemblies resulting from short-read length sequencing approaches and by a limited understanding of epigenetic regulation of diversity. To address this, single molecule real time (SMRT) sequencing was used in this study as it produces high quality genome sequences, with resolution of repeat regions (including those found in mobile elements) and can generate data to determine methylation modifications across the sequence (the methylome).Chromosomal rearrangements and ribosomal operon duplications were observed in both genomes. The rearrangements occurred at insertion sites within two mobile genetic elements (MGEs), Tn6164 and Tn6293, present only in the M120 and CD105HS27 genomes, respectively. The gene content of these two transposons differ considerably which could impact upon horizontal gene transfer; differences include CDSs encoding methylases and a conjugative prophage only in Tn6164. To investigate mechanisms which could affect MGE transfer, the methylome, restriction modification (RM) and the CRISPR/Cas systems were characterised for each strain. Notably, the environmental isolate, CD105HS27, does not share a consensus motif for (m4)C methylation, but has one additional spacer when compared to the clinical isolate M120.These findings show key differences between the two strains in terms of their genetic capacity for MGE transfer. The carriage of horizontally transferred genes appear to have genome wide effects based on two different methylation patterns. The CRISPR/Cas system appears active although perhaps slow to evolve. Data suggests that both mechanisms are functional and impact upon horizontal gene transfer and genome evolution within C. difficile.
The whitefly Bemisia tabaci (Hemiptera: Aleyrodidae) is among the 100 worst invasive species in the world. As one of the most important crop pests and virus vectors, B. tabaci causes substantial crop losses and poses a serious threat to global food security. We report the 615-Mb high-quality genome sequence of B. tabaci Middle East-Asia Minor 1 (MEAM1), the first genome sequence in the Aleyrodidae family, which contains 15,664 protein-coding genes. The B. tabaci genome is highly divergent from other sequenced hemipteran genomes, sharing no detectable synteny. A number of known detoxification gene families, including cytochrome P450s and UDP-glucuronosyltransferases, are significantly expanded in B. tabaci. Other expanded gene families, including cathepsins, large clusters of tandemly duplicated B. tabaci-specific genes, and phosphatidylethanolamine-binding proteins (PEBPs), were found to be associated with virus acquisition and transmission and/or insecticide resistance, likely contributing to the global invasiveness and efficient virus transmission capacity of B. tabaci. The presence of 142 horizontally transferred genes from bacteria or fungi in the B. tabaci genome, including genes encoding hopanoid/sterol synthesis and xenobiotic detoxification enzymes that are not present in other insects, offers novel insights into the unique biological adaptations of this insect such as polyphagy and insecticide resistance. Interestingly, two adjacent bacterial pantothenate biosynthesis genes, panB and panC, have been co-transferred into B. tabaci and fused into a single gene that has acquired introns during its evolution.The B. tabaci genome contains numerous genetic novelties, including expansions in gene families associated with insecticide resistance, detoxification and virus transmission, as well as numerous horizontally transferred genes from bacteria and fungi. We believe these novelties likely have shaped B. tabaci as a highly invasive polyphagous crop pest and efficient vector of plant viruses. The genome serves as a reference for resolving the B. tabaci cryptic species complex, understanding fundamental biological novelties, and providing valuable genetic information to assist the development of novel strategies for controlling whiteflies and the viruses they transmit.
Escherichia coli serotype O25b:H4 is involved in human urinary tract infections.In this study, we sequenced and analyzed E. coli O25b:H4 isolated from a patient sufferingfrom recurring UTI infections in an intensive care unit at Hera General Hospital inMakkah, Saudi Arabia. We aimed to determine the virulence genes for pathogenesis anddrug resistance of this isolate compared to other E. coli strains. We sequenced and analyzedthe E. coli O25b:H4 Saudi strain clinical isolate using next generation sequencing. Usingthe ERGO genome analysis platform, we performed annotations and identified virulenceand antibiotic resistance determinants of this clinical isolate. The E. coli O25b:H4 genomewas assembled into four contigs representing a total chromosome size of 5.28 Mb, andthree contigs were identified, including a 130.9 kb (virulence plasmid) contig bearing thebla-CTX gene and 32 kb and 29 kb contigs. In comparing this genome to otheruropathogenic E. coli genomes, we identified unique drug resistance and pathogenicityfactors. In this work, whole-genome sequencing and targeted comparative analysis of aclinical isolate of uropathogenic Escherichia coli O25b:H4 was performed. This strainencodes virulence genes linked with extraintestinal pathogenic E. coli (ExPEC) that areexpressed constitutively in E. coli ST131. We identified the genes responsible forpathogenesis and drug resistance and performed comparative analyses of the virulenceand antibiotic resistance determinants with those of other E. coli UPEC isolates. This isthe first report of genome sequencing and analysis of a UPEC strain from Saudi Arabia.
With next-generation sequencing (NGS) technologies, the life sciences face a deluge of raw data. Classical analysis processes for such data often begin with an assembly step, needing large amounts of computing resources, and potentially removing or modifying parts of the biological information contained in the data. Our approach proposes to focus directly on biological questions, by considering raw unassembled NGS data, through a suite of six command-line tools.
Genome assembly depends critically on read length. Two recent technologies, from Pacific Biosciences (PacBio) and Oxford Nanopore, produce read lengths >20 kb, which yield de novo genome assemblies with vastly greater contiguity than those based on Sanger, Illumina, or other technologies. However, the very high error rates of these two new technologies (~15% per base) makes assembly imprecise at repeats longer than the read length and computationally expensive. Here we show that the contiguity and quality of the assembly of these noisy long reads can be significantly improved at a minimal cost, by leveraging on the low error rate and low cost of Illumina short reads. Namely, k-mers from the PacBio raw reads that are not present in Illumina reads (which account for ~95% of the distinct k-mers) are deemed sequencing errors and ignored at the seed alignment step. By focusing on the ~5% of k-mers that are error free, read overlap sensitivity is dramatically increased. Of equal importance, the validation procedure can be extended to exclude repetitive k-mers, which prevents read miscorrection at repeats and further improves the resulting assemblies. We tested the k-mer validation procedure using one long-read technology (PacBio) and one assembler (MHAP/Celera Assembler), but it is very likely to yield analogous improvements with alternative long-read technologies and assemblers, such as Oxford Nanopore and BLASR/DALIGNER/Falcon, respectively.© 2016 Carvalho et al.; Published by Cold Spring Harbor Laboratory Press.
The human microbiota and epigenetic processes have both been shown to play a crucial role in health and disease. However, there is extremely scarce information on epigenetic modulation of microbiota members except for a few pathogens. Mainly DNA adenine methylation has been described extensively in modulating the virulence of pathogenic bacteria in particular. It would thus appear likely that such mechanisms are widespread for most bacterial members of the microbiota. This review will present briefly the current knowledge on epigenetic processes in bacteria, give examples of known methylation processes in microbial members of the human microbiota and summarize the knowledge on regulation of host epigenetic processes by the human microbiota.
To investigate the potential evolutionary obstacles in the sustainable therapeutic use of plasmid-dependent phages to control the clinically important conjugative plasmid-mediated dissemination of antibiotic resistance genes to pathogenic bacteria.The lytic plasmid-dependent phage PRD1 and the multiresistance conferring plasmid RP4 in an Escherichia coli host were utilized to assess the genetic and phenotypic changes induced by combined phage and antibiotic selection.Resistance to PRD1 was always coupled with either completely lost or greatly reduced conjugation ability. Reversion to full conjugation efficiency was found to be rare, and it also restored the susceptibility to plasmid-dependent phages. Consequently, plasmid-dependent phages constitute an interesting candidate for development of sustainable anticonjugation/antiresistance therapeutic applications.
Protection against enteric infections, also termed colonization resistance, results from mutualistic interactions of the host and its indigenous microbes. The gut microbiota of humans and mice is highly diverse and it is therefore challenging to assign specific properties to its individual members. Here, we have used a collection of murine bacterial strains and a modular design approach to create a minimal bacterial community that, once established in germ-free mice, provided colonization resistance against the human enteric pathogen Salmonella enterica serovar Typhimurium (S. Tm). Initially, a community of 12 strains, termed Oligo-Mouse-Microbiota (Oligo-MM(12)), representing members of the major bacterial phyla in the murine gut, was selected. This community was stable over consecutive mouse generations and provided colonization resistance against S. Tm infection, albeit not to the degree of a conventional complex microbiota. Comparative (meta)genome analyses identified functions represented in a conventional microbiome but absent from the Oligo-MM(12). By genome-informed design, we created an improved version of the Oligo-MM community harbouring three facultative anaerobic bacteria from the mouse intestinal bacterial collection (miBC) that provided conventional-like colonization resistance. In conclusion, we have established a highly versatile experimental system that showed efficacy in an enteric infection model. Thus, in combination with exhaustive bacterial strain collections and systems-based approaches, genome-guided design can be used to generate insights into microbe-microbe and microbe-host interactions for the investigation of ecological and disease-relevant mechanisms in the intestine.
The marine environment harbors a large proportion of the total biodiversity on this planet, including the majority of the earths’ different phyla and classes. Studying the genomes of marine organisms can bring interesting insights into genome evolution. Today, almost all marine organismal groups are understudied with respect to their genomes. One potential reason is that extraction of high-quality DNA in sufficient amounts is challenging for many marine species. This is due to high polysaccharide content, polyphenols and other secondary metabolites that will inhibit downstream DNA library preparations. Consequently, protocols developed for vertebrates and plants do not always perform well for invertebrates and algae. In addition, many marine species have large population sizes and, as a consequence, highly variable genomes. Thus, to facilitate the sequence read assembly process during genome sequencing, it is desirable to obtain enough DNA from a single individual, which is a challenge in many species of invertebrates and algae. Here, we present DNA extraction protocols for seven marine species (four invertebrates, two algae, and a marine yeast), optimized to provide sufficient DNA quality and yield for de novo genome sequencing projects.
Efficient conversion of hexoses and pentoses into value-added chemicals represents one core step for establishing economically feasible biorefineries from lignocellulosic material. While extensive research efforts have recently provided advances in the overall process performance, the quest for new microbial cell factories and novel enzymes sources is still open. As demonstrated recently the yeast Sugiyamaella lignohabitans (formerly Candida lignohabitans) represents a promising microbial cell factory for the production of organic acids from lignocellulosic hydrolysates. We report here the de novo genome assembly of S. lignohabitans using the Single Molecule Real-Time platform, with gene prediction refined by using RNA-seq. The sequencing revealed a 15.98 Mb genome, subdivided into four chromosomes. By phylogenetic analysis, Blastobotrys (Arxula) adeninivorans and Yarrowia lipolytica were found to be close relatives of S. lignohabitans Differential gene expression was evaluated in typical growth conditions on glucose and xylose and allowed a first insight into the transcriptional response of S. lignohabitans to different carbon sources and different oxygenation conditions. Novel sequences for enzymes and transporters involved in the central carbon metabolism, and therefore of potential biotechnological interest, were identified. These data open the way for a better understanding of the metabolism of S. lignohabitans and provide resources for further metabolic engineering.© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
An ammonia-oxidizing bacterium, strain D1FHS, was enriched into pure culture from a sediment sample retrieved in Jiaozhou Bay, a hyper-eutrophic semi-closed water body hosting the metropolitan area of Qingdao, China. Based on initial 16S rRNA gene sequence analysis, strain D1FHS was classified in the genus Nitrosococcus, family Chromatiaceae, order Chromatiales, class Gammaproteobacteria; the 16S rRNA gene sequence with highest level of identity to that of D1FHS was obtained from Nitrosococcus halophilus Nc4(T). The average nucleotide identity between the genomes of strain D1FHS and N. halophilus strain Nc4 is 89.5%. Known species in the genus Nitrosococcus are obligate aerobic chemolithotrophic ammonia-oxidizing bacteria adapted to and restricted to marine environments. The optimum growth (maximum nitrite production) conditions for D1FHS in a minimal salts medium are: 50 mM ammonium and 700 mM NaCl at pH of 7.5 to 8.0 and at 37°C in dark. Because pertinent conditions for other studied Nitrosococcus spp. are 100-200 mM ammonium and <700 mM NaCl at pH of 7.5 to 8.0 and at 28-32°C, D1FHS is physiologically distinct from other Nitrosococcus spp. in terms of substrate, salt, and thermal tolerance.
Pedobacter steynii DX4, isolated from Qinghai-Tibet plateau, exhibited capability to effectively degrade crude oil at low temperature. In order to illustrate its biodegradation mechanism, whole genome and transcriptome sequencing were performed. It is the first genome of crude oil degrading strain in Pedobacter genus. The P. steynii DX4 genome consists of a single circular chromosome of 6,581,659 bp with an average G+C content of 41.31% and encodes 5464 genes in all. GIs were predicted and comparison analysis was performed between relative species. Genome annotation predicted several hydrocarbon oxygenases, chemotaxis proteins and biosurfactant synthetases. The transcriptional sequences profiled a lot of differently expressed genes when cells respectively grown on crude oil and pyruvate mediums. Crude oil significantly stimulated the expression of the genes related to the hydrocarbon oxidation and resparitory chain. Genomic and transcriptomic analysis of P. steynii DX4 have revealed the machenism of the crude oil degradation in Pedobacter steynii DX4 and provided us with valuable knowledge base to make effective strategy to mitigate the ecological damage caused by crude oil pollution.
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