Genetic and Functional Characterization of theEscherichia coliBarA-UvrY Two-Component System: Point Mutations in the HAMP Linker of the BarA Sensor Give a Dominant-Negative Phenotype

Abstract

The BarA-UvrY two-component system family is strongly associated with virulence but is poorly understood at the molecular level. During our attempts to complement a barA deletion mutant, we consistently generated various mutated BarA proteins. We reasoned that characterization of the mutants would help us to better understand the signal transduction mechanism in tripartite sensors. This was aided by the demonstrated ability to activate the UvrY regulator with acetyl phosphate independently of the BarA sensor. Many of the mutated BarA proteins had poor complementation activity but could counteract the activity of the wild-type sensor in a dominant-negative fashion. These proteins carried point mutations in or near the recently identified HAMP linker, previously implicated in signal transduction between the periplasm and cytoplasm. This created sensor proteins with an impaired kinase activity and a net dephosphorylating activity. Using further sitedirected mutagenesis of a HAMP linker-mutated protein, we could demonstrate that the phosphoaccepting aspartate 718 and histidine 861 are crucial for the dephosphorylating activity. Additional analysis of the HAMP linker-mutated BarA sensors demonstrated that a dephosphorylating activity can operate via phosphotransfer within a tripartite sensor dimer in vivo. This also means that a tripartite sensor can be arranged as a dimer even in the dephosphorylating mode.

Regulation of Bacterial Virulence by Csr (Rsm) Systems

“…The SCFA acetate is abundant in the intestinal tracts of mammalian hosts, where it may serve as a potent activator of BarA during enteric colonization or infection (68). Alternatively, BarA-independent phosphorylation of UvrY has been observed to occur (both in vitro and in vivo) via acetylphosphate, a high-energy intermediate in the synthesis of acetate (68,69). Though phosphorylation of UvrY and other response regulators by acetyl-phosphate has been observed in various bacterial species, the physiological relevance/significance of this observation is poorly understood (70)(71)(72).…”

pH-Dependent Activation of the BarA-UvrY Two-Component System inEscherichia coli

“…We found that neither the pH nor the growth stage had a significant effect on the transcription of the two reporters. The efficiency of translation of the two genes was examined by using strain KSY009, which carries a chromosomal ⌽(uvrYЈ-lacZ) translational fusion (18), and by Western blot analysis using BarAspecific antibodies (21) and crude extracts of cells grown under the conditions described above. No differences in translation of the uvrY reporter (Fig.…”

“…Samples were subjected to electrophoresis in a sodium dodecyl sulfate-10% polyacrylamide gel, and the resolved proteins were electrotransferred to a Hybond-ECL filter (Amersham). Immunoblot analyses were subsequently performed, using BarA polyclonal antibodies as previously described (21). exp, exponential growth phase; stat, stationary growth phase.…”

The Virulence of aDickeya dadantii3937 Mutant Devoid of Osmoregulated Periplasmic Glucans Is Restored by Inactivation of the RcsCD-RcsB Phosphorelay

“…Three dominant mutations were isolated in the barA gene encoding the sensor protein of the BarA-UvrY phosphorelay system of E. coli. Like the rcsC2 mutation, these mutations lie within the linker domain of BarA located just upstream of the histidine kinase domain and harbor constitutive phosphatase activity (38). Constitutive kinase activity-dominant mutations were isolated in the same region of the rcsC gene of S. enterica (20).…”