Genetic and functional characterization of the Escherichia coli BarA-UvrY two-component system: point mutations in the HAMP linker of the BarA sensor give a dominant-negative phenotype.
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 site-directed 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.
Tomenius
Henrik
H
Microbiology and Tumorbiology Center, Karolinska Institutet, SE-171 77 Stockholm, Sweden. ojar.melefors@mtc.ki.se.
Pernestig
Anna-Karin
AK
Méndez-Catalá
Claudia F
CF
Georgellis
Dimitris
D
Normark
Staffan
S
Melefors
Ojar
O
D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics* / Q000502:physiology; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000502:physiology; D050939:Gene Fusion; D005799:Genes, Dominant; D017930:Genes, Reporter; D005816:Genetic Complementation Test; D008565:Membrane Proteins / Q000235:genetics* / Q000502:physiology; D020125:Mutation, Missense; D010641:Phenotype; D010749:Phosphoprotein Phosphatases / Q000235:genetics; D010770:Phosphotransferases / Q000235:genetics* / Q000502:physiology; D017354:Point Mutation; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology; D014157:Transcription Factors / Q000235:genetics* / Q000502:physiology; D001616:beta-Galactosidase / Q000032:analysis / Q000235:genetics
D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D014157:Transcription Factors; C419603:UvrY protein, E coli; C074276:barA protein, E coli; D010770:Phosphotransferases; D010749:Phosphoprotein Phosphatases; D001616:beta-Galactosidase