Identification and characterization of novel phosphate regulon genes, ecs0540-ecs0544, in Escherichia coli O157:H7.

In response to environmental phosphate limitation, the transcriptional activator PhoB of Escherichia coli (E. coli) activates transcription of the phosphate regulon (pho regulon) genes that are involved in phosphate utilization. At least 31 of pho regulon genes have been identified and well characterized in E. coli by numerous studies using non-pathogenic K-12 derivative strains. In this study, we searched for PhoB-regulated promoters from a lacZ-fused genomic library of the E. coli O157:H7 Sakai in an attempt to find novel pho regulon genes in the strain. A promoter region located upstream of a gene cluster (ecs0540-ecs0544) that mapped within one of the strain-specific chromosomal regions of the E. coli O157:H7 was identified. By further in vivo analysis with various subclones of the 5'-flanking region, it was suggested that the ecs0540 transcription was regulated by at least two promoters, an upstream PhoB-regulated promoter and a downstream constitutive promoter. S1 mapping and footprinting experiments revealed two transcription start sites and a sequence similar to the consensus sequence of PhoB binding, respectively. Bioinformatic analysis of the ecs0540-ecs0544 genes showed that these genes were highly homologous to the Escherichia fergusonii (E. fergusonii) siiCA-DA operon encoding a 718 kDa giant protein (SiiEA) and its cognate type I secretion system. In addition, a highly repetitive region and motifs that are shared among RTX (repeats in toxin) toxin family were found in the amino acid sequence of these giant proteins. Our finding is the first example of a member of the pho regulon identified in the O157:H7 strain-specific chromosomal region.

Expression of Escherichia coli DcuS-R two-component regulatory system is regulated by the secondary internal promoter which is activated by CRP-cAMP.

The DcuS-R two-component system of Escherichia coli senses C4-dicarboxylates of the medium and regulates expression of the genes related to utilization of them. It is known that phospho-DcuR induces expression of genes such as the dcuB-fumB operon, the frdABCD operon, and the dctA gene. We analyzed promoters of the dcuS-R operon to elucidate the transcriptional regulation system. We found a novel internal promoter within the dcuS gene that is regulated by the transcriptional regulator, CRP-cAMP, in both aerobic and anaerobic conditions.

Novel members of the phosphate regulon in Escherichia coli O157:H7 identified using a whole-genome shotgun approach.

Escherichia coli PhoB protein is the transcriptional activator of the phosphate (pho) regulon genes involved in phosphate utilization. To gain further insight into the potential roles of PhoB in the phosphate starvation response, we attempted to identify PhoB-regulated promoters using a random shotgun library of E. coli O157:H7 genomic fragments that were fused to a promoterless lacZ reporter gene on a low-copy-number plasmid. Using this approach, numerous chromosomal regions containing phosphate-starvation-inducible (psi) promoters, including nearly all known pho regulon promoters, were identified. ß-Galactosidase and electrophoretic mobility shift assays showed that transcription from the 22 identified psi promoters was directly regulated by PhoB. PhoB-binding sites within the promoter regions were identified by DNase I footprinting. The genes for yoaI, rpsG, galP, rnr, udp, sstT, ybiM, and vgrE were located downstream of these promoters, indicating that these genes are members of the pho regulon. Surprisingly, the other 14 promoters were located within sense or antisense strands of open reading frames (ORFs), and/or at a distance from ORFs. Our results suggest that PhoB has broader roles in gene regulation and RNA expression in E. coli strains than was previously supposed. Our shotgun-library cloning approach represents a powerful tool for identifying promoters activated or repressed by transcriptional regulators that respond to environmental stimuli.

Identification of PhoB binding sites of the yibD and ytfK promoter regions in Escherichia coli.

By using a lacZ operon fusion genomic library of the Escherichia coli 0157:H7 Sakai, we identified phosphate-starvation-inducible (psi) promoters located upstream of the yibD and ytfK genes. They have been previously proposed to belong to the phosphate regulon (pho regulon) by Beak and Lee (2006), based on the DNA array and in vivo transcriptional experiments. However, the direct interaction of these promoters with the activator protein of the pho regulon, PhoB, has not been determined. We determined the binding regions of PhoB in these promoter regions by DNase I footprinting. Both regions contained two pho boxes similar to the consensus sequence for PhoB binding.

Transcription regulation by feast/famine regulatory proteins, FFRPs, in archaea and eubacteria.

Feast/famine regulatory proteins (FFRPs) comprise a single group of transcription factors systematically distributed throughout archaea and eubacteria. In the eubacterial domain in Escherichia coli, autotrophic pathways are activated and heterotrophic pathways are repressed by an FFRP, the leucine-responsive regulatory protein (Lrp), in some cases in interaction with other transcription factors. By sensing the concentration of leucine, Lrp changes its association state between hexadecamers and octamers to adapt the autotrophic or heterotrophic mode. The lrp gene is regulated so that the concentration of Lrp decreases in the presence of rich nutrition. In the archaeal domain a large part of the metabolism of Pyrococcus OT3 is regulated by another FFRP, FL11. In the presence of rich nutrition, the metabolism is released from repression by FL11; transcription of fl11 is terminated by FL11 forming octamers in interaction with lysine. When the nutrient is depleted, the metabolism is arrested by a high concentration of FL11; FL11 disassembles to dimers in the absence of lysine, and repression of transcription of fl11 is relaxed. Common characteristics of the master regulations by FL11 and Lrp hint at the prototype regulation once achieved in the common ancestor of all extant organisms. Mechanisms of discrimination by FFRPs between DNA sequences and also between co-regulatory molecules, mostly amino acids, and variations of transcription regulations observed with archaea and eubacteria are reviewed.