- University of Bristol
- Start date
- End date
- Escherichia coli CreBC is a regulator of the expression of genes whose products are involved in intermediary metabolism. It controls cre regulon gene expression directly, and may also be able to regulate the expression of non-cre regulon genes through transcriptional networking.
One example of transcriptional networking is that CreBC regulates the expression of cpdA, encoding a cAMP phosphodiesterase. Thus, when CreBC become active cAMP levels in the cell fall, and this invokes catabolite repression, potentially affecting the expression of a separate regulon, which is controlled by CRP.
We will determine the extent to which CreBC influence gene expression through (1) global transcriptome experiments using mutants of known CreBC activity; (2) we will repeat these transcriptome experiments in cpdA or crp knockout backgrounds.
There seem to be two signals that mediate control of cre regulon gene expression by CreBC. One is mimicked by a naladixic acid resistant phenotype and the other is growth in minimal media with gluconeogenic carbon sources such as pyruvate. Using classical genetic approaches, we will (3) define why naladixic acid resistance permits carbon source regulation of the cre regulon by CreBC and will (4) attempt to find the environmental signal that is mimicked by naladixic acid resistance. We will also (5) learn more about the CreC signal by identifying disrupted loci in mutants where CreC is constitutively active. CreBC homologues are rare in the bacterial kingdom, but are present in a number of enterobacteriaceae, including salmonella typhimurium, and in other Gram-negative organisms like pseudomonas aeruginosa and xanthomonas campestris. In all cases, a homologue of creD is found downstream of creBC.
To determine if the role of CreBC in s. typhimurium is similar to that in E coli, we will (6) determine the extent of the s. typhimurium LT2 cre regulon using microarray transcriptome analysis of CreBC over-active mutants versus wild-type LT2. We will also generate creD over-expressing mutants of P. aeruginosa and X. campestris for future studies. Methods to be employed will be RT-PCR for measurements of specific gene expression, microarray transcriptome analysis (performed at the University of Birmingham for E. coli, and at the Wellcome Trust Sanger Institute for S. typhimurium). As well as basic molecular biology techniques, gene disruptions will be performed using the suicide vector pEXTc to deliver a frameshifted allele by homologous recombination into the chromosome, or Tn5-induced random insertion mutagenesis to insert a kanamycin resistance gene into the chromosome, and so look at loss of function mutations.
Various PCR approaches will be employed, either to introduce random point mutations in the creBC, or to determine the site of insertion of the Tn5 transposon. MBA has hands-on experience of all of these techniques except the use of microarrays, which will be performed under close supervision from experts in the collaborating organisation outlined in the case for support.
- Funding Source
- Biotechnology and Biological Sciences Research Council
- Project number
- Escherichia coli