High levels of the general bacterial second messenger cyclic di-GMP (c-di-GMP)

High levels of the general bacterial second messenger cyclic di-GMP (c-di-GMP) promote the establishment of surface-attached growth in lots of bacteria. research we Malol utilized peptide arrays to discover the c-di-GMP binding site of the proteins (PA3740) that was isolated within a chemical substance proteomics strategy. PA3740 was proven to bind c-di-GMP with a higher affinity and peptide arrays uncovered LKKALKKQTNLR to be always a putative c-di-GMP binding theme. Most interestingly not the same as the previously discovered c-di-GMP binding theme from the PilZ domains (RXXXR) or the I site of diguanylate cyclases (RXXD) two leucine residues and a Malol glutamine residue rather than the charged proteins provided the main element residues from the binding series. Those three proteins are extremely conserved across PA3740 homologs and their singular exchange to alanine reduced c-di-GMP binding within the full-length protein. IMPORTANCE In many bacterial pathogens the common bacterial second messenger c-di-GMP governs the switch from your planktonic motile mode of growth to the sessile biofilm mode of growth. Bacteria adapt their intracellular c-di-GMP levels to a variety of environmental difficulties. Several classes of c-di-GMP binding proteins have been structurally characterized and varied c-di-GMP binding domains have been recognized. Nevertheless for a number of c-di-GMP receptors the binding motif remains to be determined. Here we display that the use of a synthetic peptide array allowed the recognition of a Malol c-di-GMP binding motif of a putative c-di-GMP receptor protein in the opportunistic pathogen (37). With this study we coupled chemically unmodified c-di-GMP to a Sepharose matrix and recognized two not previously recognized c-di-GMP binding proteins. One of those PA3740 was confirmed to bind c-di-GMP by the use of surface plasmon resonance (SPR). To uncover the amino acid motif that binds its target c-di-GMP we applied a peptide array approach (38) with a series of tiled overlapping peptides derived from the full amino acid sequence of PA3740. We recognized a LKKALKKQTNLR peptide sequence to be a novel c-di-GMP binding motif. Interestingly the leucine residues and the glutamate residue proved to be essential for c-di-GMP binding to the peptide as well as to the purified full-length PA3740 protein. MATERIALS AND METHODS Malol Bacterial strains plasmids and press. PAO1 and strains were cultured in lysogeny broth (LB) medium at 37°C Malol and 180 rpm. If required 100 μg/ml ampicillin was added. For those cloning steps strain DH5α was used. A mutant harboring a transposon insertion within PA3740 was selected from your PAO1 mini-Tntransposon mutant library (of Robert E. W. Hancock [39]). Overexpression of PA3740 was recognized by using pJN105::PA3740 by which the PA3740 gene was amplified with PA3740-specific ahead primer fPr6 (5′-GATCGAATTCTAAGAAGGAGATATAATGACCATGTCCAATCAACAAC-3′) and PA3740-specific reverse primer rPr6 (5′-GATCTCTAGAGATCAGGGCCGCAGGCTGA-3′) and put between the EcoRI and XbaI restriction sites. The mutant and the PA3740-overexpressing strain were utilized for phenotypic assays. For protein purification purposes PA3740 was PCR amplified from the polymerase with the gene-specific primers PA3740fPr (5′-AAAACATATGATGACCATGTCCAATCAACAAC-3′) and PA3740rPr2 (5′-GATCAAGCTTGGGCCGCAGGCTGAT-3′). The PCR product was digested with the restriction enzymes NdeI and HindIII and cloned into pET21a+ (Novagen) which comprises a hexahistidine tag after the multiple-cloning site. The producing plasmids were verified by sequencing. Alanine mutants of PA3740-His6 were generated by introducing respective point mutations into the pET21a+::PA3740 plasmid with the help of mutagenic primers and a QuikChange II site-directed mutagenesis kit (Agilent Systems). The CHEK2 sequences of the mutagenic primers are available upon request. Swimming and swarming motility. Swimming and swarming assays were performed as previously explained (56) with a slight modification: instead of 0.5% (wt/vol) Casamino Acids only 0.1% (wt/vol) Casamino Acids was added to the swarm agar plates. To the motility plates 0.5% arabinose and 50 μg/ml gentamicin were added. Bacteria were cultivated for 6 h at 37°C in LB medium supplemented with 50 μg/ml gentamicin at 180 rpm. Cells were harvested by centrifugation resuspended in phosphate-buffered saline (PBS) and modified in respect to cell number. Each motility dish was inoculated with 1 μl bacterial suspension system. The plates had been incubated at 33°C within a humid atmosphere for 17 h..