June 1, 2021  |  

Comparative genome analysis of Clavibacter michiganensis subsp. michiganensis strains provides insights into genetic diversity and virulence.

Clavibacter michiganensis subsp. michiganensis (Cmm) is a gram positive actinomycete, causing bacterial canker of tomato (Solanum lycopersicum) a disease that can cause significant losses in tomato production. In this study, we determined the complete genome sequence of 13 California Cmm strains and one saprophytic Clavibacter strain using a combination of Ilumina and PacBio sequencing. The California Cmm strains have genome size (3.2 -3.3 mb) similar to the reference strain NCPPB382 (3.3 mb) with =98% sequence identity. Cmm strains from California share =92% genes (8-10% are noble genes) with the reference Cmm strain NCPPB382. Despite this similarity, we detected significant alternatives in California strains with respect to plasmid number, plasmid composition, and genomic island presence indicating acquisition of unique mechanisms controlling virulence. Plasmids pCM1 and pCM2, that were previously demonstrated to be required for NCPPB382 virulence, also differ in their presence and gene content across Cmm strains. pCM2 is absent in some Cmm strains and that still retain virulence in tomato. Saprophytic Clavibacter possess a novel plasmid, pSCM, and lacks the majority of characterized virulence factors. Genome sequence information was also used to design specific and sensitive primer pairs for Cmm detection. A mechanistic understanding of how genomic changes have impacted Cmm virulence and survival across diverse strains will be necessary for developing a robust disease control strategies for bacterial canker of tomato.


June 1, 2021  |  

Complete microbial genomes, epigenomes, and transcriptomes using long-read PacBio Sequencing.

For comprehensive metabolic reconstructions and a resulting understanding of the pathways leading to natural products, it is desirable to obtain complete information about the genetic blueprint of the organisms used. Traditional Sanger and next-generation, short-read sequencing technologies have shortcomings with respect to read lengths and DNA-sequence context bias, leading to fragmented and incomplete genome information. The development of long-read, single molecule, real-time (SMRT) DNA sequencing from Pacific Biosciences, with >10,000 bp average read lengths and a lack of sequence context bias, now allows for the generation of complete genomes in a fully automated workflow. In addition to the genome sequence, DNA methylation is characterized in the process of sequencing. PacBio® sequencing has also been applied to microbial transcriptomes. Long reads enable sequencing of full-length cDNAs allowing for identification of complete gene and operon sequences without the need for transcript assembly. We will highlight several examples where these capabilities have been leveraged in the areas of industrial microbiology, including biocommodities, biofuels, bioremediation, new bacteria with potential commercial applications, antibiotic discovery, and livestock/plant microbiome interactions.


April 21, 2020  |  

Chlorella vulgaris genome assembly and annotation reveals the molecular basis for metabolic acclimation to high light conditions.

Chlorella vulgaris is a fast-growing fresh-water microalga cultivated at the industrial scale for applications ranging from food to biofuel production. To advance our understanding of its biology and to establish genetics tools for biotechnological manipulation, we sequenced the nuclear and organelle genomes of Chlorella vulgaris 211/11P by combining next generation sequencing and optical mapping of isolated DNA molecules. This hybrid approach allowed to assemble the nuclear genome in 14 pseudo-molecules with an N50 of 2.8 Mb and 98.9% of scaffolded genome. The integration of RNA-seq data obtained at two different irradiances of growth (high light-HL versus low light -LL) enabled to identify 10,724 nuclear genes, coding for 11,082 transcripts. Moreover 121 and 48 genes were respectively found in the chloroplast and mitochondrial genome. Functional annotation and expression analysis of nuclear, chloroplast and mitochondrial genome sequences revealed peculiar features of Chlorella vulgaris. Evidence of horizontal gene transfers from chloroplast to mitochondrial genome was observed. Furthermore, comparative transcriptomic analyses of LL vs HL provide insights into the molecular basis for metabolic rearrangement in HL vs. LL conditions leading to enhanced de novo fatty acid biosynthesis and triacylglycerol accumulation. The occurrence of a cytosolic fatty acid biosynthetic pathway can be predicted and its upregulation upon HL exposure is observed, consistent with increased lipid amount under HL. These data provide a rich genetic resource for future genome editing studies, and potential targets for biotechnological manipulation of Chlorella vulgaris or other microalgae species to improve biomass and lipid productivity.This article is protected by copyright. All rights reserved.


April 21, 2020  |  

Potent LpxC Inhibitors with In Vitro Activity Against Multi-Drug Resistant Pseudomonas aeruginosa.

New drugs with novel mechanisms of resistance are desperately needed to address both community and nosocomial infections due to Gram-negative bacteria. One such potential target is LpxC, an essential enzyme that catalyzes the first committed step of Lipid A biosynthesis. Achaogen conducted an extensive research campaign to discover novel LpxC inhibitors with activity against Pseudomonas aeruginosa We report here the in vitro antibacterial activity and pharmacodynamics of ACHN-975, the only molecule from these efforts and the first ever LpxC inhibitor to be evaluated in Phase 1 clinical trials. In addition, we describe the profile of three additional LpxC inhibitors that were identified as potential lead molecules. These efforts did not produce an additional development candidate with a sufficiently large therapeutic window and the program was subsequently terminated.Copyright © 2019 American Society for Microbiology.


April 21, 2020  |  

Genomic analysis of Marinobacter sp. NP-4 and NP-6 isolated from the deep-sea oceanic crust on the western flank of the Mid-Atlantic Ridge

Two Marinobacter sp. NP-4 and NP-6 were isolated from a deep oceanic basaltic crust at North Pond, located at the western flank of the Mid-Atlantic Ridge. These two strains are capable of using multiple carbon sources such as acetate, succinate, glucose and sucrose while take oxygen as a primary electron acceptor. The strain NP-4 is also able to grow anaerobically under 20?MPa, with nitrate as the electron acceptor, thus represents a piezotolerant. To explore the metabolic potentials of Marinobacter sp. NP-4 and NP-6, the complete genome of NP-4 and close-to-complete genome of NP-6 were sequenced. The genome of NP-4 contains one chromosome and two plasmids with the size of 4.6?Mb in total, and with average GC content of 57.0%. The genome of NP-6 is 4.5?Mb and consists of 6 scaffolds, with an average GC content of 57.1%. Complete glycolysis, citrate cycle and aromatics compounds degradation pathways are identified in genomes of these two strains, suggesting that they possess a heterotrophic life style. Additionally, one plasmid of NP-4 contains genes for alkane degradation, phosphonate ABC transporter and cation efflux system, enabling NP-4 extra surviving abilities. In total, genomic information of these two strains provide insights into the physiological features and adaptation strategies of Marinobacter spp. in the deep oceanic crust biosphere.


April 21, 2020  |  

Complete genome sequence provides insights into the quorum sensing-related spoilage potential of Shewanella baltica 128 isolated from spoiled shrimp.

Shewanella baltica 128 is a specific spoilage organism (SSO) isolated from the refrigerated shrimp that results in shrimp spoilage. This study reported the complete genome sequencing of this strain, with the primary annotations associated with amino acid transport and metabolism (8.66%), indicating that S. baltica 128 has good potential for degrading proteins. In vitro experiments revealed Shewanella baltica 128 could adapt to the stress conditions by regulating its growth and biofilm formation. Genes that related to the spoilage-related metabolic pathways, including trimethylamine metabolism (torT), sulfur metabolism (cysM), putrescine metabolism (speC), biofilm formation (rpoS) and serine protease production (degS), were identified. Genes (LuxS, pfs, LuxR and qseC) that related to the specific QS system were also identified. Complete genome sequence of S. baltica 128 provide insights into the QS-related spoilage potential, which might provide novel information for the development of new approaches for spoilage detection and prevention based on QS target.Copyright © 2019. Published by Elsevier Inc.


April 21, 2020  |  

Genome sequence analysis of 91 Salmonella Enteritidis isolates from mice caught on poultry farms in the mid 1990s.

A total of 91 draft genome sequences were used to analyze isolates of Salmonella enterica serovar Enteritidis obtained from feral mice caught on poultry farms in Pennsylvania. One objective was to find mutations disrupting open reading frames (ORFs) and another was to determine if ORF-disruptive mutations were present in isolates obtained from other sources. A total of 83 mice were obtained between 1995-1998. Isolates separated into two genomic clades and 12 subgroups due to 742 mutations. Nineteen ORF-disruptive mutations were found, and in addition, bigA had exceptional heterogeneity requiring additional evaluation. The TRAMS algorithm detected only 6 ORF disruptions. The sefD mutation was the most frequently encountered mutation and it was prevalent in human, poultry, environmental and mouse isolates. These results confirm previous assessments of the mouse as a rich source of Salmonella enterica serovar Enteritidis that varies in genotype and phenotype. Copyright © 2019. Published by Elsevier Inc.


April 21, 2020  |  

Rapid transcriptional responses to serum exposure are associated with sensitivity and resistance to antibody-mediated complement killing in invasive Salmonella Typhimurium ST313

Background: Salmonella Typhimurium ST313 exhibits signatures of adaptation to invasive human infection, including higher resistance to humoral immune responses than gastrointestinal isolates. Full resistance to antibody-mediated complement killing (serum resistance) among nontyphoidal Salmonellae is uncommon, but selection of highly resistant strains could compromise vaccine-induced antibody immunity. Here, we address the hypothesis that serum resistance is due to a distinct genotype or transcriptome response in S. Typhimurium ST313.


April 21, 2020  |  

Complete Genome Sequence of Desulfovibrio desulfuricans IC1, a Sulfonate-Respiring Anaerobe.

We report the complete genome sequence of the anaerobic, sulfonate-respiring, sulfate-reducing bacterium Desulfovibrio desulfuricans IC1. The genome was assembled into a single 3.25-Mb circular chromosome with 2,680 protein-coding genes identified. Sequencing of sulfonate-metabolizing anaerobes is key for understanding sulfonate degradation and its role in the sulfur cycle.Copyright © 2019 Day et al.


April 21, 2020  |  

A draft nuclear-genome assembly of the acoel flatworm Praesagittifera naikaiensis.

Acoels are primitive bilaterians with very simple soft bodies, in which many organs, including the gut, are not developed. They provide platforms for studying molecular and developmental mechanisms involved in the formation of the basic bilaterian body plan, whole-body regeneration, and symbiosis with photosynthetic microalgae. Because genomic information is essential for future research on acoel biology, we sequenced and assembled the nuclear genome of an acoel, Praesagittifera naikaiensis.To avoid sequence contamination derived from symbiotic microalgae, DNA was extracted from embryos that were free of algae. More than 290x sequencing coverage was achieved using a combination of Illumina (paired-end and mate-pair libraries) and PacBio sequencing. RNA sequencing and Iso-Seq data from embryos, larvae, and adults were also obtained. First, a preliminary ~17-kilobase pair (kb) mitochondrial genome was assembled, which was deleted from the nuclear sequence assembly. As a result, a draft nuclear genome assembly was ~656 Mb in length, with a scaffold N50 of 117 kb and a contig N50 of 57 kb. Although ~70% of the assembled sequences were likely composed of repetitive sequences that include DNA transposons and retrotransposons, the draft genome was estimated to contain 22,143 protein-coding genes, ~99% of which were substantiated by corresponding transcripts. We could not find horizontally transferred microalgal genes in the acoel genome. Benchmarking Universal Single-Copy Orthologs analyses indicated that 77% of the conserved single-copy genes were complete. Pfam domain analyses provided a basic set of gene families for transcription factors and signaling molecules.Our present sequencing and assembly of the P. naikaiensis nuclear genome are comparable to those of other metazoan genomes, providing basic information for future studies of genic and genomic attributes of this animal group. Such studies may shed light on the origins and evolution of simple bilaterians. © The Author(s) 2019. Published by Oxford University Press.


April 21, 2020  |  

Genomic and transcriptomic characterization of Pseudomonas aeruginosa small colony variants derived from a chronic infection model.

Phenotypic change is a hallmark of bacterial adaptation during chronic infection. In the case of chronic Pseudomonas aeruginosa lung infection in patients with cystic fibrosis, well-characterized phenotypic variants include mucoid and small colony variants (SCVs). It has previously been shown that SCVs can be reproducibly isolated from the murine lung following the establishment of chronic infection with mucoid P. aeruginosa strain NH57388A. Using a combination of single-molecule real-time (PacBio) and Illumina sequencing we identify a large genomic inversion in the SCV through recombination between homologous regions of two rRNA operons and an associated truncation of one of the 16S rRNA genes and suggest this may be the genetic switch for conversion to the SCV phenotype. This phenotypic conversion is associated with large-scale transcriptional changes distributed throughout the genome. This global rewiring of the cellular transcriptomic output results in changes to normally differentially regulated genes that modulate resistance to oxidative stress, central metabolism and virulence. These changes are of clinical relevance because the appearance of SCVs during chronic infection is associated with declining lung function.


April 21, 2020  |  

DNA methylation from a Type I restriction modification system influences gene expression and virulence in Streptococcus pyogenes.

DNA methylation is pervasive across all domains of life. In bacteria, the presence of N6-methyladenosine (m6A) has been detected among diverse species, yet the contribution of m6A to the regulation of gene expression is unclear in many organisms. Here we investigated the impact of DNA methylation on gene expression and virulence within the human pathogen Streptococcus pyogenes, or Group A Streptococcus. Single Molecule Real-Time sequencing and subsequent methylation analysis identified 412 putative m6A sites throughout the 1.8 Mb genome. Deletion of the Restriction, Specificity, and Methylation gene subunits (?RSM strain) of a putative Type I restriction modification system lost all detectable m6A at the recognition sites and failed to prevent transformation with foreign-methylated DNA. RNA-sequencing identified 20 genes out of 1,895 predicted coding regions with significantly different gene expression. All of the differentially expressed genes were down regulated in the ?RSM strain relative to the parent strain. Importantly, we found that the presence of m6A DNA modifications affected expression of Mga, a master transcriptional regulator for multiple virulence genes, surface adhesins, and immune-evasion factors in S. pyogenes. Using a murine subcutaneous infection model, mice infected with the ?RSM strain exhibited an enhanced host immune response with larger skin lesions and increased levels of pro-inflammatory cytokines compared to mice infected with the parent or complemented mutant strains, suggesting alterations in m6A methylation influence virulence. Further, we found that the ?RSM strain showed poor survival within human neutrophils and reduced adherence to human epithelial cells. These results demonstrate that, in addition to restriction of foreign DNA, gram-positive bacteria also use restriction modification systems to regulate the expression of gene networks important for virulence.


April 21, 2020  |  

Genomic characterization of Kerstersia gyiorum SWMUKG01, an isolate from a patient with respiratory infection in China.

The Gram-negative bacterium Kerstersia gyiorum, a potential etiological agent of clinical infections, was isolated from several human patients presenting clinical symptoms. Its significance as a possible pathogen has been previously overlooked as no disease has thus far been definitively associated with this bacterium. To better understand how the organism contributes to the infectious disease, we determined the complete genomic sequence of K. gyiorum SWMUKG01, the first clinical isolate from southwest China.The genomic data obtained displayed a single circular chromosome of 3, 945, 801 base pairs in length, which contains 3, 441 protein-coding genes, 55 tRNA genes and 9 rRNA genes. Analysis on the full spectrum of protein coding genes for cellular structures, two-component regulatory systems and iron uptake pathways that may be important for the success of the bacterial survival, colonization and establishment in the host conferred new insights into the virulence characteristics of K. gyiorum. Phylogenomic comparisons with Alcaligenaceae species indicated that K. gyiorum SWMUKG01 had a close evolutionary relationships with Alcaligenes aquatilis and Alcaligenes faecalis.The comprehensive analysis presented in this work determinates for the first time a complete genome sequence of K. gyiorum, which is expected to provide useful information for subsequent studies on pathogenesis of this species.


April 21, 2020  |  

Integrated transcriptomic and proteomic analysis of pathogenic mycobacteria and their esx-1 mutants reveal secretion-dependent regulation of ESX-1 substrates and WhiB6 as a transcriptional regulator.

The mycobacterial type VII secretion system ESX-1 is responsible for the secretion of a number of proteins that play important roles during host infection. The regulation of the expression of secreted proteins is often essential to establish successful infection. Using transcriptome sequencing, we found that the abrogation of ESX-1 function in Mycobacterium marinum leads to a pronounced increase in gene expression levels of the espA operon during the infection of macrophages. In addition, the disruption of ESX-1-mediated protein secretion also leads to a specific down-regulation of the ESX-1 substrates, but not of the structural components of this system, during growth in culture medium. This effect is observed in both M. marinum and M. tuberculosis. We established that down-regulation of ESX-1 substrates is the result of a regulatory process that is influenced by the putative transcriptional regulator whib6, which is located adjacent to the esx-1 locus. In addition, the overexpression of the ESX-1-associated PE35/PPE68 protein pair resulted in a significantly increased secretion of the ESX-1 substrate EsxA, demonstrating a functional link between these proteins. Taken together, these data show that WhiB6 is required for the secretion-dependent regulation of ESX-1 substrates and that ESX-1 substrates are regulated independently from the structural components, both during infection and as a result of active secretion.


April 21, 2020  |  

Genomic characterization of Nocardia seriolae strains isolated from diseased fish.

Members of the genus Nocardia are widespread in diverse environments; a wide range of Nocardia species are known to cause nocardiosis in several animals, including cat, dog, fish, and humans. Of the pathogenic Nocardia species, N. seriolae is known to cause disease in cultured fish, resulting in major economic loss. We isolated two N. seriolae strains, CK-14008 and EM15050, from diseased fish and sequenced their genomes using the PacBio sequencing platform. To identify their genomic features, we compared their genomes with those of other Nocardia species. Phylogenetic analysis showed that N. seriolae shares a common ancestor with a putative human pathogenic Nocardia species. Moreover, N. seriolae strains were phylogenetically divided into four clusters according to host fish families. Through genome comparison, we observed that the putative pathogenic Nocardia strains had additional genes for iron acquisition. Dozens of antibiotic resistance genes were detected in the genomes of N. seriolae strains; most of the antibiotics were involved in the inhibition of the biosynthesis of proteins or cell walls. Our results demonstrated the virulence features and antibiotic resistance of fish pathogenic N. seriolae strains at the genomic level. These results may be useful to develop strategies for the prevention of fish nocardiosis. © 2018 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.


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