== Acetyl-P, but not BarA, is responsible for UvrY phosphorylation in acsrAmutant

== Acetyl-P, but not BarA, is responsible for UvrY phosphorylation in acsrAmutant. system (TCS) ofEscherichia coliconsists of the membrane-bound sensor kinase BarA and its cognate response regulator UvrY (1). BarA, which belongs to the subfamily of tripartite sensor kinases (2, 3), senses and responds to the presence of formate and acetate but also to that of other short-chain fatty acids (4). Phosphorylated BarA catalyzes the transphosphorylation of UvrY (1), a typical response regulator of the FixJ family (1, 5), which in turn activates expression of the noncoding RNAs of thecarbonstorageregulation (Csr) system, CsrB and CsrC. These small regulatory RNAs possess repeated sequence elements that allow them to interact with multiple copies of the RNA binding protein CsrA and thereby prevent its regulatory interaction with its mRNA targets (6). CsrA is a small , dimeric RNA binding protein that coordinates gene expression by positively or negatively regulating the translation, stability, and/or elongation of its target transcripts (7, 8). CsrA directly interacts with the 5 untranslated leaders of target mRNAs at sites characterized by a GGA sequence that is often located within the loop of a short stem-loop structure (911). In this way, CsrA Tyrphostin A1 activates exponential-phase processes while it represses several stationary-phase functions (12). CsrA is widely distributed among eubacteria (13) and regulates expression of genes for virulence factors (14, 15), quorum sensing (16, 17), motility (18, 19), carbon metabolism (20, 21), biofilm formation (22, 23), cyclic di-GMP synthesis (24), and peptide uptake (10). Curiously, activation ofcsrBtranscription, which depends directly on UvrY-P, does not take place in acsrAmutant strain (25). Therefore , it has been suggested that CsrA has a positive effect on the BarA/UvrY TCS. In this study, we confirmed and extended these results by examining the effects of CsrA on either the expression or the activity of BarA and UvrY. Our results demonstrate that CsrA, apparently indirectly, is required for properuvrYexpression and also for activation of the BarA kinase activity. == MATERIALS AND METHODS == == Tyrphostin A1 Bacterial strains, plasmids, and growth conditions. == The strains and plasmids used in this work are listed inTable 1 . Strains IFC5010 (csrA:: KanrcsrB-lacZ) and IFC5016 (uvrY:: CamrcsrA:: KanrcsrB-lacZ) were constructed by P1virtransduction of thecsrA:: Kanrallele from strain TR1-5 (25) into strains KSB837 (csrB-lacZ) (26) and UYKSB837 (uvrY:: CamrcsrB-lacZ) (25), respectively. Strain IFC5015 (ackA:: tetR:: pta csrA:: KanrcsrB-lacZ) was constructed by P1virtransduction of theackA:: tetR:: ptaallele from strain ECL5336 (27) into strain IFC5010. For strain IFC5017 (barA:: CamrcsrA:: KanrcsrB-lacZ) construction, thebarAgene was deleted by homologous recombination using the lambda Red recombinase system (28). Briefly, a fragment amplified by PCR, using primers barAdel-Fw (5-ATTTAACAGTGTGACCTTAATTGTCCCATAACGGAACTCCGTGTAGGCTGGAGCTGCTTC-3) and barAdel-Rv (5-CATAAACACAGGCACTTTGTCACCAATCTGAAACCAGCGTATGAATATCCTCCTTAGTTCC-3) and plasmid pKD3 (28) as the template, was used to replace thebarAallele with a chloramphenicol cassette in strain IFC5010. == TABLE 1 . == E. colistrains and plasmids To construct Ampr-linkedlacZoperon fusions, plasmid pAH125-bla was first generated by replacing the kanamycin resistance cassette of pAH125 (29) with an ampicillin resistance cassette. To this end, ablaPCR product was generated, using primers Amp-Prom-Fw (5-GCGGCGCCTTCAAATATGTATCCGCTCATG-3) and Amp-Rv (5-GCGCGGCCGCGGTCTGACAGTTACCAATGC-3) and plasmid pUC18 as the template, and cloned into the NarI-NotI sites of pAH125. Helper plasmid pINT-cat was constructed by replacing the ampicillin Tyrphostin A1 resistance cassette of plasmid pINT-ts (29) with the chloramphenicol resistance cassette. A 1. 1-kb DNA fragment containing thecatgene, obtained from plasmid pKD3 (28) by HindIII digestion, was blunt ended and cloned into blunt-ended XmnI-BsaI sites of pINT-ts. Plasmid pAH-uvrY, containing anuvrY-lacZoperon fusion, was constructed by cloning a PCR-amplified fragment containing the upstream noncoding region through the first 4 codons (nucleotides [nt] 409 to+12 relative to the start of translation) (using primers uvrYP-fw-Pst [5-AACTGCAGGGCGGCGGAGTATACCATAAG-3] and uvrYP-Rv-BamHI [5-CGGGATCCAGAACGTTGATCAAAGGAATATC-3]) into the PstI-BamHI sites of pAH125-bla. Plasmid pUV-uvrY22, containing a translationaluvrY-lacZfusion under the control of thelacUV5promoter, was constructed by cloning the region from nt 47 to Tlr4 +66 relative to the start ofuvrYtranslation, amplified by PCR using primers uvrY-lead-Fw (5-GGAATTCAATGACTAACTATCAGTAGC-3) and uvrY-lead22-Rv (5-CGGGATCCTCTTCCAGAATGCGTCG-3), into the EcoRI-BamHI sites of plasmid pUV5 (30). All fusions were integrated into the CF7789 and TR1-5CF7789 chromosomes,.