Project description:Total transcript amplification (TTA) from single eukaryotic cells for transcriptome analysis is established, but TTA from a single prokaryotic cell presents additional challenges with much less starting material and lack of poly(A)-tails. We described, here, a novel method for single bacterium TTA, using a Burkholderia thailandensis model exposed to subinhibitory concentration of the antibacterial agent, glyphosate. Utilizing B. thialandensis microarray to assess the TTA method showed little gene bias (< 2 fold-change) and absence (~5-6%) when compared to the larger scale non-amplified RNA samples. B. thailandensis genes important to possibly recuperate and balance the intracellular amino acid pool were induced (or repressed) by the aromatic amino acid biosynthesis inhibitor, glyphosate. We validated our single-cell microarray data at the multi-cells and single-cell levels with lacZ and gfp reporter-gene fusions, respectively. This novel method will rejuvenate and expand the prokaryotic transcriptomic field. Two identical cultures of B. thailandensis wildtype strain E264 were grown in 1x M9 minimal medium supplemented with 1% Brij-58 and 20 mM glucose (MG) to mid-log phase. One culture was induced by final concentration of 0.01% (w/v) glyphosate for 30 minutes. Total RNA was then purified from B. thailandensis uninduced and induced samples, converted to cDNA and hybridized onto B. thailandensis 70mer triplicate array
Project description:A total of 27 Listeria isolates that could not be classified to the species level were obtained from soil samples from different locations in the contiguous United States and an agricultural water sample from New York. Whole-genome sequence-based average nucleotide identity blast (ANIb) showed that the 27 isolates form five distinct clusters; for each cluster, all draft genomes showed ANI values of <95 % similarity to each other and any currently described Listeria species, indicating that each cluster represents a novel species. Of the five novel species, three cluster with the Listeria sensu stricto clade and two cluster with sensu lato. One of the novel sensu stricto species, designated L. cossartiae sp. nov., contains two subclusters with an average ANI similarity of 94.9%, which were designated as subspecies. The proposed three novel sensu stricto species (including two subspecies) are Listeria farberi sp. nov. (type strain FSL L7-0091T=CCUG 74668T=LMG 31917T; maximum ANI 91.9 % to L. innocua), Listeria immobilis sp. nov. (type strain FSL L7-1519T=CCUG 74666T=LMG 31920T; maximum ANI 87.4 % to L. ivanovii subsp. londoniensis) and Listeria cossartiae sp. nov. [subsp. cossartiae (type strain FSL L7-1447T=CCUG 74667T=LMG 31919T; maximum ANI 93.4 % to L. marthii) and subsp. cayugensis (type strain FSL L7-0993T=CCUG 74670T=LMG 31918T; maximum ANI 94.7 % to L. marthii). The two proposed novel sensu lato species are Listeria portnoyi sp. nov. (type strain FSL L7-1582T=CCUG 74671T=LMG 31921T; maximum ANI value of 88.9 % to L. cornellensis and 89.2 % to L. newyorkensis) and Listeria rustica sp. nov. (type strain FSL W9-0585T=CCUG 74665T=LMG 31922T; maximum ANI value of 88.7 % to L. cornellensis and 88.9 % to L. newyorkensis). L. immobilis is the first sensu stricto species isolated to date that is non-motile. All five of the novel species are non-haemolytic and negative for phosphatidylinositol-specific phospholipase C activity; the draft genomes lack the virulence genes found in Listeria pathogenicity island 1 (LIPI-1), and the internalin genes inlA and inlB, indicating that they are non-pathogenic.
Project description:Burkholderia thailandensis is a soil-dwelling bacterium that shares many metabolic pathways with the ecologically similar, but evolutionarily distant, Pseudomonas aeruginosa. Among the diverse nutrients it can utilize is choline, which can be converted into the osmoprotectant glycine betaine and further catabolized as a source of carbon and nitrogen, similar to P. aeruginosa. Orthologs of genes in the choline catabolic pathway in these two bacteria showed distinct differences in gene arrangement as well as an additional orthologous transcriptional regulator in B. thailandensis. In this study, we showed that multiple glutamine amidotransferase1 (GATase1)-containing AraC-family transcription regulators (GATRs) are involved in regulation of the B. thailandensis choline catabolic pathway (gbdR1, gbdR2, souR). Using genetic analyses and sequencing the transcriptome in the presence and absence of choline, we identified the likely regulons of gbdR1 (BTH_II1869) and gbdR2 (BTH_II0968). We also identified a functional ortholog for P. aeruginosa souR, a GATR that regulates the metabolism of sarcosine to glycine. GbdR1 is absolutely required for expression of the choline catabolic locus, similar to P. aeruginosa GbdR, while GbdR2 is important to increase expression of the catabolic locus. Additionally, the B. thailandensis SouR ortholog (BTH_II0994) is required for catabolism of choline and its metabolites as carbon sources, whereas in P. aeruginosa, SouR function can by bypassed by GbdR. The strategy employed by B. thailandensis represents a distinct regulatory solution to control choline catabolism and thus provides both an evolutionary counterpoint and an experimental system to compare the acquisition and regulation of this pathway during environmental growth and infection.
Project description:Introduction. Burkholderia thailandensis is a clinically rare opportunistic pathogen in the genus Burkholderia, and the genomic features and virulence characteristics of B. thailandensis strains that cause human infection remain unclear.Gap Statement. B. thailandensis strains with different virulence induce different host innate immune responses in vitro.Aim. This work aimed to understand the sequence diversity, phylogenetic relationship, and virulence of B. thailandensis BPM causing human infection.Methodology. The comparative molecular and genomic analyses, and mouse infection studies were applied to analyse the virulence and genomic features of B. thailandensis BPM originating from China.Results. The whole genome sequence analysis showed that the genomes of BPM and other avirulent B. thailandensis strains were broadly similar, comprising two highly syntenic chromosomes with comparable numbers of coding regions (CDs), protein family distributions, and horizontally acquired genomic islands. By examining species-specific genomic regions, we obtained molecular explanations for previously known differences in virulence and discovered the potential specific virulence-associated genes of BPM, which likely work together to confer the virulence of BPM. Significantly reduced LD50 and survival rates during mouse infection experiments were found in BPM compared to the avirulent B. thailandensis E264 (BtE264).Conclusion. Taken together, the results of this study provide basic information on the genomic features and virulence characteristics of the virulent B. thailandensis strain BPM, which is helpful for understanding its evolution as it relates to pathogenesis and environmental adaptability.
Project description:Innate immunity responds to pathogens by producing alarm signals and activating pathways that make host cells inhospitable for pathogen replication. The intracellular bacterium Burkholderia thailandensis invades the cytosol, hijacks host actin, and induces cell fusion to spread to adjacent cells, forming multinucleated giant cells (MNGCs) which promotes bacterial replication. We show that type I interferon (IFN) restricts macrophage MNGC formation during B. thailandensis infection. Guanylate-binding proteins (GBPs) expressed downstream of type I IFN were required to restrict MNGC formation through inhibition of Arp2/3-dependent actin motility during infection. GTPase activity and the CAAX prenylation domain were required for GBP2 recruitment to B. thailandensis, which restricted bacterial actin polymerization required for MNGC formation. Consistent with in vitro macrophages, Gbp2-/- Gbp5-/-, GbpChr3-KO mice were more susceptible to intranasal infection with B. thailandensis than wildtype mice. Our findings reveal that IFN and GBPs play a critical role in restricting cell-cell fusion during infection
Project description:Four Gram-stain-negative bacterial strains, CS20T, AUT15.5T, XENO-11T, and CCN3.3T, isolated from Steinernema entomopathogenic nematodes, were found to represent novel species within the genus Xenorhabdus (Gammaproteobacteria, Morganellaceae). In this study, we described these new species using whole-genome phylogenomic reconstructions, sequence identity values from core genome sequences, and phenotypic characterization. Phylogenetic reconstructions based on 16S rRNA gene sequences showed that: (i) strain CS20T is closely related to X. stockiae DSM 17904T, (ii) strain AUT15.5T is closely related to X. budapestensis DSM 16342T, (iii) strain XENO-11T is closely related to X. khoisanae DSM 25463T, and (iv) strain CCN3.3T is closely related to X. griffiniae DSM 17911T. The 16S rRNA gene sequence similarity value between strain CS20T and X. stockiae DSM 17904T is 97.8%, between strain AUT15.5T and X. budapestensis DSM 16342T is 98.1%, between strain XENO-11T and X. khoisanae DSM 25463T is 97.8%, and between strain CCN3.3T and X. griffiniae DSM 17911T is 98.6%. Phylogenomic reconstructions using whole-genome sequences showed that: (i) strain CS20T is closely related to X. stockiae DSM 17904T and X. innexi DSM 16336T, (ii) strain AUT15.5T is closely related to X. indica DSM 17382T, (iii) strain XENO-11T is closely related to X. khoisanae DSM 25463T, and (iv) strain CCN3.3T is closely related to X. griffiniae DSM 17911T. The digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values between strains CS20T, AUT15.5T, XENO-11T, and CCN3.3T and the type strains of their more closely related species are below the 70% and the 95-96% divergence thresholds, respectively, used for prokaryotic species delineation. Hence, we propose the following four new species: Xenorhabdus bharatensis sp. nov. (the type strain is CS20T=CCM 9320T=CCOS 2070T), X. entomophaga sp. nov. (the type strain is XENO-11T=CCM 9389T=CCOS 2111T), X. siamensis sp. nov. (the type strain is AUT15.5T=CCM 9405T=CCOS 2116T), and X. thailandensis sp. nov. (the type strain is CCN3.3T=CCM 9406T=CCOS 2115T). The following biochemical tests may be useful for differentiating the novel species from their more closely related taxa: acetoin production, arginine dihydrolase, citrate utilization, gelatinase, glucose oxidation, indole production, and tryptophan deaminase. Our study contributes to a better understanding of the biodiversity and phylogenetic relationships of entomopathogenic bacteria associated with insect parasitic nematodes.