Dataset Information

A Functional Tricarboxylic Acid Cycle Operates during Growth of Bordetella pertussis on Amino Acid Mixtures as Sole Carbon Substrates.

ABSTRACT: It has been claimed that citrate synthase, aconitase and isocitrate dehydrogenase activities are non-functional in Bordetella pertussis and that this might explain why this bacterium's growth is sometimes associated with accumulation of polyhydroxybutyrate (PHB) and/or free fatty acids. However, the sequenced genome includes the entire citric acid pathway genes. Furthermore, these genes were expressed and the corresponding enzyme activities detected at high levels for the pathway when grown on a defined medium imitating the amino acid content of complex media often used for growth of this pathogenic microorganism. In addition, no significant PHB or fatty acids could be detected. Analysis of the carbon balance and stoichiometric flux analysis based on specific rates of amino acid consumption, and estimated biomass requirements coherent with the observed growth rate, clearly indicate that a fully functional tricarboxylic acid cycle operates in contrast to previous reports.

SUBMITTER: Izac M

PROVIDER: S-EPMC4684311 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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A Functional Tricarboxylic Acid Cycle Operates during Growth of Bordetella pertussis on Amino Acid Mixtures as Sole Carbon Substrates.

Izac Marie M Garnier Dominique D Speck Denis D Lindley Nic D ND

PloS one 20151218 12

It has been claimed that citrate synthase, aconitase and isocitrate dehydrogenase activities are non-functional in Bordetella pertussis and that this might explain why this bacterium's growth is sometimes associated with accumulation of polyhydroxybutyrate (PHB) and/or free fatty acids. However, the sequenced genome includes the entire citric acid pathway genes. Furthermore, these genes were expressed and the corresponding enzyme activities detected at high levels for the pathway when grown on a ...[more]

PMID: 26684737

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Project description:Purpose: Within the past 20 years, outbreaks of whooping cough, caused by Bordetella pertussis, have led to respiratory disease and infant mortalities, despite good vaccination coverage. This is due at least in part to the introduction of a less effective acellular vaccine in the 1990’s. It is crucial then to understand the molecular basis of B. pertussis growth and infection. The two-component system BvgA/BvgS is the B. pertussis master regulator that regulates the expression of various virulence genes. Previous genome wide studies to identify the bvgAS regulon in B. pertussis have been limited to microarray analyses. The goal of this study is to define the bvgAS regulon using NGS based RNA-seq analysis and RT-qPCR and to compare the differences with the previous microarray data. Method: To define the bvgAS regulon, transcriptomes were generated using B. pertussis Tohama I BP536 (WT) and a ΔbvgAS derivative in the presence and absence of phosphorylated BvgA. Bacteria were grown either under modulating (50 mM MgSO4) conditions, in which phosphorylated BvgA is undetectable (Bvg- mode), or non-modulating (no MgSO4) conditions in which BvgA is phosphorylated (Bvg+ mode). After total RNA extraction and rRNA removal, strand-specific DNA libraries were prepared for Illumina sequencing using the ScriptSeq 2.0 kit (Illumina) in duplicates. Libraries were sequenced using a HiSeq 2000 sequencer (Illumina; University of Buffalo Next Generation Sequencing Core Facility). Sequence reads were mapped to the reference genome (NC_002929.2) and normalized against total reads. Differential expression analyses were performed using CLC Biomedical Genomic Workbench version 3.5.4 with default parameters. Differential gene expression profiles were obtained after Empirical DGE statistical test with FDR p-value. RT–qPCR validation was performed using SYBR Green assays. Result: Using CLC Biomedical Genomic Workbench version 3.5.4, ~ 20-25 million reads per sample were mapped to the reference B. pertussis genome (NC_002929.2). RNA-seq analyses identified >550 genes (15% of genome) that were regulated in a bvgAS-dependent manner with a fold difference ≥ 1.6 and FDR p-value < 0.05. RT-qPCR was used for confirmatory analyses for 80 genes. Overall, we identified 245 genes that were positively regulated and 326 genes that were negatively regulated by bvgAS. 362 members of the bvgAS regulon were newly identified by our study. Most importantly, our analyses indicated that the expression of dozens of transcriptional regulators increases, while the expression of multiple metabolic genes decreases in the presence of BvgA~P. Conclusion: We report here the first RNA-seq analysis of the bvgAS regulon in B. pertussis, revealing that > 550 genes are regulated by bvgAS. Our results suggest that metabolic changes in the Bvg- mode (absence of BvgA phosphorylation) may be participating in bacterial survival, transmission, and/or persistence and identify over 200 new genes negatively regulated by bvgAS that can be tested for function.

Dataset Information

A Functional Tricarboxylic Acid Cycle Operates during Growth of Bordetella pertussis on Amino Acid Mixtures as Sole Carbon Substrates.

Publications

A Functional Tricarboxylic Acid Cycle Operates during Growth of Bordetella pertussis on Amino Acid Mixtures as Sole Carbon Substrates.

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