RESUMO
TraR of Agrobacterium tumefaciens is a LuxR-type transcription factor that regulates genes required for replication and conjugation of the tumour-inducing plasmid. TraR binds the pheromone 3-oxo-octanoylhomoserine lactone (OOHL) and requires this molecule for folding into a protease-resistant, soluble conformation. Even after binding to OOHL, TraR is degraded at readily detectable rates. Here we show that the N-terminal domain of TraR, which binds OOHL, is more resistant to degradation than the full length protein, suggesting that sites on the C-terminal DNA binding domain [TraR(170-234)] enhance protein turnover. A fusion between GFP and TraR(170-234) was poorly fluorescent, and truncations of this fusion protein allowed us to identify residues in this domain that contribute to protein degradation. TraR activity was previously shown to be inhibited by the antiactivator TraM. These proteins form 2:2 complexes that fail to bind DNA sequences. Here we show that TraM sharply decreased the accumulation of TraR in whole cells, indicating that TraM facilitates proteolysis of TraR. The TraM component of these complexes is spared from proteolysis, and could therefore act catalytically.
Assuntos
Acil-Butirolactonas/metabolismo , Agrobacterium tumefaciens/metabolismo , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Percepção de Quorum , Agrobacterium tumefaciens/genética , Ligação Proteica , Estabilidade Proteica , ProteóliseRESUMO
Vegetative replication and partitioning of many plasmids and some chromosomes of alphaproteobacteria are directed by their repABC operons. RepA and RepB proteins direct the partitioning of replicons to daughter cells, while RepC proteins are replication initiators, although they do not resemble any characterized replication initiation protein. Here we show that the replication origin of an Agrobacterium tumefaciens Ti plasmid resides fully within its repC gene. Purified RepC bound to a site within repC with moderate affinity, high specificity and with twofold cooperativity. The binding site was localized to an AT-rich region that contains a large number of GANTC sites, which have been implicated in replication regulation in related organisms. A fragment of RepC containing residues 26-158 was sufficient to bind DNA, although with limited sequence specificity. This portion of RepC is predicted to have structural homology to members of the MarR family of transcription factors. Overexpression of RepC in A. tumefaciens caused large increases in copy number in cis but did not change the copy number of plasmids containing the same oriV sequence in trans, confirming other observations that RepC functions only in cis.
Assuntos
Agrobacterium tumefaciens/genética , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Plasmídeos Indutores de Tumores em Plantas/metabolismo , Origem de Replicação , Transativadores/metabolismo , Agrobacterium tumefaciens/metabolismo , Sítios de Ligação , DNA Helicases/isolamento & purificação , Análise Mutacional de DNA , Replicação do DNA , Proteínas de Ligação a DNA/isolamento & purificação , Modelos Moleculares , Ligação Proteica , Estrutura Terciária de Proteína , Deleção de Sequência , Transativadores/isolamento & purificaçãoRESUMO
The proteolytic activity of Pseudomonas fluorescens 07A was investigated, and was optimal on tryptone-calcium medium. N-acyl-homoserine lactones (AHLs) were not detected on supernatants of late-exponential and stationary-phase culture broths. Synthetic AHLs or bacterial cell extracts added to the medium did not influence growth or proteolytic activity suggesting that quorum sensing might not regulate protease production in this strain.