Identification of a novel nutrient-sensing histidine kinase/response regulator network in Escherichia coli.

When carbon sources become limiting for growth, bacteria must choose which of the remaining nutrients should be used first. We have identified a nutrient-sensing signaling network in Escherichia coli that is activated at the transition to stationary phase. The network is composed of the two histidine kinase/response regulator systems YehU/YehT and YpdA/YpdB and their target proteins, YjiY and YhjX (both of which are membrane-integrated transporters). The peptide/amino acid-responsive YehU/YehT system was found to have a negative effect on expression of the target gene, yhjX, of the pyruvate-responsive YpdA/YpdB system, while the YpdA/YpdB system stimulated expression of yjiY, the target of the YehU/YehT system. These effects were confirmed in mutants lacking any of the genes for the three primary components of either system. Furthermore, an in vivo interaction assay based on bacterial adenylate cyclase detected heteromeric interactions between the membrane-bound components of the two systems, specifically, between the two histidine kinases and the two transporters, which is compatible with the formation of a larger signaling unit. Finally, the carbon storage regulator A (CsrA) was shown to be involved in posttranscriptional regulation of both yjiY and yhjX.

Identification of a target gene and activating stimulus for the YpdA/YpdB histidine kinase/response regulator system in Escherichia coli.

Escherichia coli contains 30 two-component systems (TCSs), each consisting of a histidine kinase and a response regulator. Whereas most TCSs are well characterized in this model organism, little is known about the YpdA/YpdB system. To identify YpdB-regulated genes, we compared the transcriptomes of E. coli cells overproducing either YpdB or a control protein. Expression levels of 15 genes differed by more than 1.9-fold between the two strains. A comprehensive evaluation of these genes identified yhjX as the sole target of YpdB. Electrophoretic mobility shift assays with purified YpdB confirmed its interaction with the yhjX promoter. Specifically, YpdB binds to two direct repeats of the motif GGCATTTCAT separated by an 11-bp spacer in the yhjX promoter. yhjX encodes a cytoplasmic membrane protein of unknown function that belongs to the major facilitator superfamily of transporters. Finally, we characterized the pattern of yhjX expression and identified extracellular pyruvate as a stimulus for the YpdA/YpdB system. It is suggested that YpdA/YpdB contributes to nutrient scavenging before entry into stationary phase.

First insights into the unexplored two-component system YehU/YehT in Escherichia coli.

Two-component systems (TCSs) consisting of a membrane-anchored histidine kinase (HK) and a response regulator (RR) with DNA-binding activity. are major players in signal transduction in prokaryotes. Whereas most TCSs in Escherichia coli are well characterized, almost nothing is known about the LytS-like HK YehU and the corresponding LytTR-like RR YehT. To identify YehT-regulated genes, we compared the transcriptomes of E. coli cells overproducing either YehT or the RR KdpE (control). The expression levels of 32 genes differed more than 8-fold between the two strains. A comprehensive evaluation of these genes identified yjiY as a target of YehT. Electrophoretic mobility shift assays with purified YehT confirmed that YehT interacts directly with the yjiY promoter. Specifically, YehT binds to two direct repeats of the motif ACC(G/A)CT(C/T)A separated by a 13-bp spacer in the yjiY promoter. The target gene yjiY encodes an inner membrane protein belonging to the CstA superfamily of transporters. In E. coli cells growing in media containing peptides or amino acids as a carbon source, yjiY is strongly induced at the onset of the stationary-growth phase. Moreover, expression was found to be dependent on cyclic AMP (cAMP)/cAMP receptor protein (CRP). It is suggested that YehU/YehT participates in the stationary-phase control network.

A comprehensive toolbox for the rapid construction of lacZ fusion reporters.

ß-Galactosidase encoded by lacZ remains a popular reporter enzyme. Here, we present three fast and convenient tools that facilitate rapid construction of reporter lacZ fusions. The first enables the simple generation of lacZ (slacZ)-based chromosomally encoded reporter fusions within the lac operon in Escherichia coli using Red®/ET® recombination. The slacZ tool is based on rpsL counter-selection in combination with homologous recombination catalyzed by the λ Red recombinase, and blue/white screening. This permits construction of transcriptional and translational reporter lacZ fusions within a day. The second tool allows the introduction of lacZ reporter fusions into the chromosome by a single-crossover method. The strategy relies on the γ-origin-based suicide vector pNPTS138-R6KT, which can only replicate in λpir E. coli strains. The third tool comprises four pBBR1-based broad-host-range vectors for transcriptional and translational lacZ fusions. The functionality of our toolbox was confirmed by the K(+)-dependent activation of kdp promoter-lacZ fusions in vivo.

Histidine kinases and response regulators in networks.

Two-component systems, composed of a histidine kinase (HK) and a response regulator (RR), are the major signal transduction devices in bacteria. Originally it was thought that these two components function as linear, phosphorylation-driven stimulus-response system. Here, we will review how accessory proteins are employed by HKs and RRs to mediate signal integration, scaffolding, interconnection and allosteric regulation, and how these two components are embedded in regulatory networks.