Genome-Wide Screening of Genes Whose Enhanced Expression Affects Glycogen Accumulation in Escherichia coli

Using a systematic and comprehensive gene expression library (the ASKA library), we have carried out a genome-wide screening of the genes whose increased plasmid-directed expression affected glycogen metabolism in Escherichia coli. Of the 4123 clones of the collection, 28 displayed a glycogen-excess phenotype, whereas 58 displayed a glycogen-deficient phenotype. The genes whose enhanced expression affected glycogen accumulation were classified into various functional categories including carbon sensing, transport and metabolism, general stress and stringent responses, factors determining intercellular communication, aggregative and social behaviour, nitrogen metabolism and energy status. Noteworthy, one-third of them were genes about which little or nothing is known. We propose an integrated metabolic model wherein E. coli glycogen metabolism is highly interconnected with a wide variety of cellular processes and is tightly adjusted to the nutritional and energetic status of the cell. Furthermore, we provide clues about possible biological roles of genes of still unknown functions

A connector of two-component regulatory systems promotes signal amplification and persistence of expression

Organisms rely on a variety of regulatory architectures to control gene transcription. Whereas the functional characteristics of particular architectures are well understood, the properties of newly discovered regulatory designs cannot be easily predicted. One emerging design depends on small proteins that connect two-component regulatory systems, which constitute the dominant form of bacterial signal transduction. These connectors enable one system to respond to the signal perceived by a different system. To understand the functional properties of such connector-mediated architectures, we investigated the pathway controlled by the PhoP-dependent connector protein PmrD of Salmonella enterica and contrasted it to the circuit in which genes are regulated directly by the transcription factor PhoP. The PmrD-mediated pathway displayed both signal amplification and persistence of expression when compared with the direct pathway. Mathematical modeling of the two pathways allowed us to identify critical factors responsible for signal amplification

High stimulus unmasks positive feedback in an autoregulated bacterial signaling circuit

We examined the effect of positive autoregulation on the steady-state behavior of the PhoQ/PhoP two-component signaling system in Escherichia coli. We found that autoregulation has no effect on the steady-state output for a large range of input stimulus, which was modulated by varying the concentration of magnesium in the growth medium. We provide an explanation for this finding with a simple model of the PhoQ/PhoP circuit. The model predicts that even when autoregulation is manifest across a range of stimulus levels, the effects of positive feedback on the steady-state output emerge only in the limit that the system is strongly stimulated. Consistent with this prediction, amplification associated with autoregulation was observed in growth-limiting levels of magnesium, a condition that strongly activates PhoQ/PhoP. In a further test of the model, we found that strains harboring a phosphatase-defective PhoQ showed strong positive feedback and considerable cell-to-cell variability under growth conditions where the wild-type circuit did not show this behavior. Our results demonstrate a simple and general mechanism for regulating the positive feedback associated with autoregulation within a bacterial signaling circuit to boost response range and maintain a relatively uniform and graded output

Stimulus-dependent differential regulation in the Escherichia coli PhoQ–PhoP system

In Escherichia coli, Salmonella, and related bacteria, the PhoQ–PhoP system regulates the expression of a large collection of genes in response to conditions of low magnesium or to the presence of certain antimicrobial peptides. We measured transcription of four PhoP-regulated promoters in E. coli that have significantly different PhoP-binding sites. Surprisingly, three promoters show identical responses to magnesium concentrations that range over four orders of magnitude. By analyzing and testing a simple model of transcriptional regulation, we find an explanation for this puzzle and show that these promoters are indeed differentially regulated at sufficiently high levels of stimulus. We then use this analysis to infer an effective level of phosphorylated PhoP as a function of magnesium stimulus. Our results demonstrate that differential regulation generally depends on the strength of the stimulus and highlight how quantitative analysis of stimulus–response curves can be used to infer properties of cell regulatory circuits that cannot be easily obtained from in vitro measurements

Phenotypic differences between Salmonella and Escherichia coli resulting from the disparate regulation of homologous genes

Phenotypic differences among closely related bacteria have been largely ascribed to species-specific genes, such as those residing in pathogenicity islands. However, we now report that the differential regulation of homologous genes is the mechanism responsible for the divergence of the enteric bacteria Salmonella enterica and Escherichia coli in their ability to make LPS modifications mediating resistance to the antibiotic polymyxin B. In S. enterica serovar Typhimurium, the PmrA/PmrB two-component system governing polymyxin B resistance is induced in low Mg(2+) in a process that requires the PmrD protein and by Fe(3+) in a PmrD-independent fashion. We establish that E. coli K-12 induces PmrA-activated gene transcription and polymyxin B resistance in response to Fe(3+), but that it is blind to the low Mg(2+) signal. The highly divergent PmrD protein is responsible for this phenotype as replacement of the E. coli pmrD gene by its Salmonella counterpart resulted in an E. coli strain that transcribed PmrA-activated genes and displayed polymyxin B resistance under the same conditions as Salmonella. Molecular analysis of natural isolates of E. coli and Salmonella revealed that the PmrD proteins are conserved within each genus and that selection might have driven the divergence between the Salmonella and E. coli PmrD proteins. Investigation of PmrD function demonstrated statistically different distributions for the Salmonella and E. coli isolates in PmrD-dependent transcription occurring in low Mg(2+). Our results suggest that the differential regulation of conserved genes may have ecological consequences, determining the range of niches a microorganism can occupy

A dual-signal regulatory circuit activates transcription of a set of divergent operons in Salmonella typhimurium

We present a molecular mechanism for signal transduction that activates transcription of the SlyA regulon in Salmonella typhimurium. We demonstrate that SlyA mediates transcriptional activation in response to guanosine tetraphosphate, ppGpp, according to the following observations: (i) in vivo transcription of SlyA-dependent genes is repressed when ppGpp is absent; this transcription can be restored by overproducing SlyA; (ii) in vivo dimerization and binding of SlyA to the target promoter are facilitated in the presence of ppGpp; and (iii) in vitro SlyA binding to the target promoter is enhanced when ppGpp is supplemented. Thus, ppGpp must be the cytoplasmic component that stimulates SlyA regulatory function by interacting directly with this regulator in Salmonella. This signaling domain, integrated by the PhoP/PhoQ 2-component system that activates slyA transcription by sensing Mg2+, forms feedforward loops that regulate chromosomal loci identified through a motif search over the S. typhimurium genome. Many such loci are divergent operons, each formed by 2 neighboring genes in which transcription of these 2 loci proceeds in opposite directions. Both genes, however, are controlled by PhoP and SlyA through a single shared PhoP box and SlyA box present in their intergenic regions. A substitution in either box sequence causes a simultaneous cessation of transcription of a divergent operon, pagD-pagC, equivalent to the phenotype in a phoP or slyA mutant. We also identified several chromosomal loci that possess pagC-type genes without the cognate pagD-type genes. Therefore, our results provide a molecular basis for the understanding of SlyA-dependent phenotypes associated with Salmonella virulence