Regulation of competence gene homologs in Salmonella enterica serovar Typhimurium and Escherichia coil
Abstract
Many bacteria have the ability to take up DNA from their environment through a
process termed “natural competence”. Even though there are numerous bacterial species
that are recognized to be naturally competent, competence is undetected or overlooked in
many species such as members of the Enterobacteriaceae. Homologs of competence genes
and the main regulator of natural competence, Sxy, are conserved in Salmonella and
Escherichia coli; however, these genes were found to be silent under all previously-tested
conditions. Thus, I carried out molecular studies to investigate genetic induction and
regulation mechanisms of natural competence in Salmonella Typhimurium, and E. coli.
Despite decades of work and unsuccessful attempts to induce the cryptic
competence genes, our genetic studies in S. Typhimurium show that low temperatures (22
– 25 ᵒC) and growth on semi-solid media to allow for motility induce the expression of
competence genes. Nutrient deprivation is hypothesized to be another inducing signal;
where the expression of competence genes increased under starvation conditions when
using arabinose as a carbon source. We identified Sxy and CRP as positive regulators of
competence gene expression, whereas the nucleoid-associated proteins, FIS and H-NS,
and the alternate sigma factor, RpoS, repress the expression of competence genes.
Bioinformatic analysis of competence gene promoters suggests that DNA supercoiling
regulates competence gene expression. However, experimental relaxation of DNA
supercoiling did not confirm this. Several competence gene homologs have an
evolutionary history of operon and promoter shuffling, nevertheless the transcription of
these genes is coordinated and synchronized in different bacterial lineages through coregulated promoters. Altogether these results demonstrate that several environmental
signals, transcription factors, and regulatory mechanisms are integrated to regulate
competence gene expression in S. Typhimurium.
In Haemophilus influenzae, the poorly characterized competence regulator, Sxy, is
proposed to be stimulated by starvation for purine nucleotides. In S. Typhimurium, we
found that sxy expression is stimulated by temperature, motility on semi-solid media, and
nutrient deprivation signals. Further, we found that sxy expression is activated by CRP and
repressed by FIS, H-NS, and RpoS. Moreover, it was previously proposed that Sxy assists
CRP binding to competence gene promoters. Our phenotypic analysis in E. coli showed
that mutations that alter the Sxy C-terminus abolish CRP-S transcriptional activation, and
remove the toxic effect characteristic of sxy overexpression. A genome-wide analysis of
the Salmonella Sxy regulon using directional RNA-seq revealed that Sxy function is not
restricted to the activation of competence genes. Sxy also plays a role in regulating sugar
metabolism, DNA replication, nucleotide metabolism, and expression of several small
RNAs, transcription factors, and pathogenicity genes.
This project is the first to study the genetics of competence in Salmonella, and it is
pioneering in identifying novel inducing signals and transcription factors that regulate
competence gene expression. The outcomes of this research enhance our understanding of
the role of Sxy in regulating competences and other functions. The body of knowledge
surrounding regulation of competence genes will greatly inform and guide future research
into competence gene regulation and help deduce the ecological role of DNA uptake in
gamma-proteobacteria.