aureushas a peptidic nature and is not affected by the lactonase activity of YtnP (36,38,49). == Fig 1. carried out in the model organismBacillus subtilisNCIB3610 led to the recognition of YtnP, a protein that shares a similar deduced website to proteins described as quorum-quenching lactonases (Fig. 1A). Using ClustalW, a comparative positioning of the following proteins was performed: YtnP and AiiA fromBacillussp. (1012); AiiA homologues fromStaphylococcus aureusandListeria innocua(6); AttM, AiiB, and AiiC fromAgrobacterium tumefaciens(6,17,29); AhlD fromArthrobactersp. (39); and glyoxalase II (YcbL) fromSalmonella enterica(45). AiiM fromMicrobacterium testaceum(52) and AidH fromOchrobactrumsp. (33) were excluded from this comparative analysis because of their nature of unusual lactonases. Results of the alignment confirmed that the important residues in the active sites of metallohydrolases are present in YtnP (Fig. 1B) (12,24,26,27). Subsequently, overexpression of a 6His-tagged version of the YtnP protein was carried out withEscherichia coli. 6His-YtnP was purified using Ni+2exchange chromatography (observe Fig. S1 in the supplemental material). Inhibition of the quorum-sensing activity of YtnP was assayed based on the ability ofPseudomonas aeruginosato form a biofilm in a quorum-sensing-dependent manner (3,8,32). Small concentrations of purified YtnP added to the biofilm formation assay of the cultures ofP. aeruginosainhibited biofilm formation, while comparable concentrations of YtnP did not affect biofilm formation in cultures ofS. aureus(Fig. 1D). This might be because the quorum-sensing molecule that controls biofilm formation inS. aureushas a peptidic nature and is not affected by the lactonase activity of YtnP (36,38,49). == Fig 1. == YtnP is usually a lactonase-homologous protein. (A) Amino acid sequence of YtnP fromB. subtilisstrain 3610. Conserved motifs present in other metallolactamases are marked as Box 1 and Box 2 and are underlined and strong. Also an additional serine (Ser36) important for YtnP activity is usually marked as Ser-P. (B) Alignment of the conserved motifs of the active site of the following: YtnP and other metallolactamases; AiiA fromBacillussp. and homologues fromS. aureusandL. innocua; AttM, AiiB, and AiiC fromA. tumefaciens; AhlD fromArthrobactersp.; and glyoxalase II (YcbL) fromS. enterica. The last lane of the alignment shows the consensus sequence. (C) Addition of YtnP to the biofilm formation assays inhibited biofilm Kira8 Hydrochloride formation inP. aeruginosabut not inS. aureus. Biofilm formation is observed as a pellicle attached to the submerged surfaces (at the bottom of the well plate). Crystal violet staining was used in the assay for better visualization, according to the protocol explained by O’Toole and Kolter (37). For the quantification of biofilm formation, the crystal violet associated with biofilms was dissolved in acetic acid (33%) and was measured spectrophotometrically with an optical density at 595 nm (37). To determine whether the expression ofytnPinB. subtilisserves as a defensive strategy against competing bacteria, an experiment was FLJ16239 carried out to test the activation ofytnPexpression in the presence of several acylhomoserine lactone (AHL) Kira8 Hydrochloride molecules. To do this,B. subtilisstrain 3610 was labeled with a transcriptional fusion, PytnP-yfp(yfpencodes theyellowfluorescenceprotein [YFP]), and the emission of fluorescence was monitored when small concentrations of AHL (purchased from Sigma-Aldrich) were added to cultures ofB. subtilisgrowing in MSgg (2). We did not observe any switch inytnPexpression associated with the addition of AHL molecules to the cultures ofB. subtilis. Then, other natural products were tested for their abilities to induceytnPexpression inB. subtilis. This idea was based on the results published in a previous report that explains an increase in the expression of a protein much like YtnP inS. aureuswhen certain antimicrobials were added to the cultures (1). Consequently, a battery of antibiotics was collected, and they were tested for their abilities to induce the expression Kira8 Hydrochloride of PytnP-YFP when added at sub-growth-inhibitory concentrations (<50 nM) (observe Table S1 in the supplemental material). We.
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