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Elucidation of the CodY target genes in Staphylococcus aureus USA300 using ChIPexo

ABSTRACT: CodY is a highly conserved global transcriptional factor that regulates the expression of dozens of genes related to metabolism and virulence in Staphylococcus aureus. The S. aureus CodY regulon has been studied in vitro. However, in vivo CodY DNA-binding activity and the identity of the corresponding target genes remain unknown due to lack of a ChIP-grade monoclonal antibody. Using a novel CodY monoclonal antibody that we developed in established ChIP-exo protocols, we report in vivo target genes of CodY in two S. aureus USA300 clinical strains (TCH1516 and LAC). The total number of CodY-binding sites exceeded 110, but their location varied between the two strains. The majority of the identified binding sites were located within the promoter regions. Based on the sequences of the CodY-binding sites, a model of CodY interaction with DNA is proposed. Furthermore, S. aureus CodY protein is highly conserved across a G+C Gram-positive species, including Bacillus subtilis and Listeria monocytogenes, and thus this study paves the way for exploration of the differential binding of CodY among a range of gram-positive species, including pathogenic ones

ORGANISM(S): Staphylococcus aureus

PROVIDER: GSE159856 | GEO | 2021/08/17

REPOSITORIES: GEO

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Elucidating the CodY regulon in Staphylococcus aureus USA300 lineage using ChIPexo

Project description:CodY is a conserved broad acting transcriptional factor that regulates the expression of genes related to amino acid metabolism and virulence in Staphylococcus aureus. CodY target genes have been studied by using in vitro DNA affinity purification and deep sequencing (IDAP-Seq). In this study we performed the first in vivo determination of CodY target genes using a novel CodY monoclonal antibody in established ChIP-exo protocols. Our results showed, 1) The same 165 CodY target genes in both strains; 2) That the differential binding intensity for the same target genes under the same conditions were due to sequence differences in the same CodY binding site in the two strains; 3) Based on transcriptomic data, a CodY regulon comprising 72 target genes that revealed that CodY is mainly involved in amino acid transport and metabolism, inorganic ion transport and metabolism, and cellular transcription and translation; and 4) CodY systematically regulated central metabolic flux to generate branched-chain amino acids (BCAAs) by mapping the CodY regulon onto a genome-scale metabolic model of S. aureus. Our study performed the first system-level analysis of CodY in two closely related dominant USA300 TCH1516 and LAC strains, thus expanding the size of the known CodY regulon, and giving new insights into the similarities and differences of CodY regulatory roles between closely related strains.

2022-08-29 | GSE163312 | GEO

CodY in Staphylococcus aureus: regulatory link between metabolism and virulence gene expression

Project description:CodY is a conserved regulator in gram-positive organisms described to repress metabolic genes mainly involved in nitrogen metabolism but also to control the expression of virulence genes in pathogens. We constructed codY gene-replacement mutants in three unrelated S. aureus strains (Newman, UAMS-1, RN1HG). codY mutants grew slower in a chemically defined medium compared to the wild type strains. However, only codY mutants were able to grow in medium lacking threonine. Excess of isoleucine resulted in growth inhibition in the wild type but not in the codY mutants indicating a role of isoleucine for CodY dependent repression of metabolic genes. The prototypic CodY-repressed operon ilvDBCleuABCilvA is preceded by a CodY-binding motif and repressed after up-shift with isoleucine and to a lesser extent guanidine. Transcription of the quorum sensing system agr followed a similar expression pattern. The codY dependent repression of agr is in line with the concomitant influence of CodY on typical agr regulated genes such as cap, spa fnbA and coa. However, transcriptional analysis revealed that most of these virulence genes (e.g. cap, fnbA, spa hla) are also regulated by CodY in an agr negative background. Microarray analysis revealed the large majority of codY-repressed genes are involved in amino-acid transport and metabolism, genes showing codY dependent activation were mainly involved in nucleotide transport and metabolism or virulence. In summary, CodY in S. aureus not only acts as repressor for genes involved in nitrogen metabolism but also contributes to virulence gene regulation by supporting as well as substituting agr function.

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Project description:More than 200 direct CodY target genes in Staphylococcus aureus were identified by genome-wide analysis of in vitro DNA binding. This analysis, which was confirmed for some genes by DNase I footprinting assays, revealed that CodY is a direct regulator of numerous transcription units associated with amino acid biosynthesis, transport of macromolecules and virulence. The virulence genes regulated by CodY fell into three groups. One group was dependent on the Agr system for its expression; these genes were indirectly regulated by CodY through its repression of the agr locus. A second group was regulated directly by CodY. The third group, which includes genes for alpha-toxin and capsule synthesis, was regulated by CodY in two ways, i.e., by direct repression and by repression of the agr locus. Since S. aureus CodY was activated in vitro by the branched chain amino acids and GTP, CodY appears to link changes in intracellular metabolite pools with the induction of numerous adaptive responses, including virulence. Affymetrix GeneChips were used to compare the transcript titers of S. aureus strains UAMS-1 (wild type) and corresponding agr- (strain CM18), codY- (strain MS1), and agr- codY- (strain CM19) isogenic mutant strains during exponential and stationary phase growth. At least two biological replicates were assessed for each strain and each growth phase.

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