g., nutrients, temperature, pH, toxins or oxidative stress) [20]. The second protein of a TCS is a response regulator, onto which a phosphoryl group is transferred from the phosphorylated HPK, and which functions as a phosphorylation-activated Selleck PF-6463922 switch
that regulates output responses within the cell causing changes in the expression of target genes [21, 22]. BaeSR is a TCS that responds cell envelope damages in E. coli[23]. The small core regulon of BaeSR find more includes the RND-type transporters AcrD and MdtABC and the periplasmic chaperone Spy [24]. The presence of a homologous BaeSR system in E. amylovora, prompted us to analyze the impact of the response regulator BaeR on the expression levels of acrD. Herein, we report that overexpression of the RND pump AcrD in an acrB-deficient mutant leads to increased resistance to two substrates, clotrimazole and luteolin, previously not described as substrates of AcrD in other enterobacteria. In order to determine the promoter activity in vitro, we utilized a transcriptional fusion of the promoter regions of acrAB and acrD, respectively, with the reporter gene egfp. We demonstrate that the response regulator BaeR is able to bind to the upstream region of acrD in E. amylovora Ea1189 and to induce acrD expression. Furthermore, we show that the inactivation of the RND pump AcrD did not result in reduction of virulence of E. amylovora
on host plants. Results Identification of an acrD homologue in E. clonidine amylovora Ea1189 A search with the BLASTP program (NCBI) using the amino acid sequence of AcrD from E. coli K-12 as the query (accession number P24177) identified Repotrectinib mw a homologous sequence in the genome of E. amylovora CFBP1430 (GenBank:EAMY_2508, annotated as cmeB). The annotated protein EAMY_2508 is 18 amino acids shorter at the N-terminus than the AcrD protein of E.
coli. Comparison of the acrD gene sequences from E. coli and E. amylovora suggests that the EAMY_2508 gene of E. amylovora CFBP1430 has been annotated with a wrong start codon. Sequence analysis revealed an alternative ATG start codon 54 bp upstream of the annotated EAMY_2508 gene. The 18 amino acids, encoded by the 54 additional nucleotides, are 100% identical to the N-terminal region of AcrD from E. coli. We used the genome sequence of E. amylovora CFBP1430 to design primers to PCR amplify the respective region from the genomic DNA of our model strain E. amylovora Ea1189 whose genome sequence is not yet available. The nucleotide sequence of acrD and its upstream region from E. amylovora Ea1189 show 100% identity to EAMY_2508 and its upstream region from E. amylovora CFBP1430 based on our sequencing results. AcrD is a member of the RND superfamily of transporters belonging to the Hydrophobe/Amphiphile Efflux-1 (HAE1) family (Transporter Classification Database TC #2.A.6.2.7). A BLASTP search (NCBI) of the deduced AcrD sequence from E.