<i>psm-mec</i> RNA inhibits <i>agrA</i> translation.

Abstract

<p>(<b>A</b>) Cell extracts of overnight cultures of Newman strain (WT) and the <i>agr-</i>null mutant (Δ<i>agr</i>) were electrophoresed in sodium dodecyl sulfate-polyacrylamide gels. One gel was stained with Coomassie Brilliant Blue (Left panel). Proteins in another gel were transferred to a membrane and used for Western blotting by anti-AgrA IgG (Right panel). (<b>B</b>) Cell extracts of 24 h-cultures of Newman strains transformed with empty vector (pND50), a plasmid carrying wild-type <i>psm-mec</i> (pF), a plasmid carrying <i>psm-mec</i> with a stop-codon (pC1), and a plasmid carrying <i>psm-mec</i> with the -7T>C promoter mutation (pM1) were subjected to Western blotting by anti-AgrA IgG. Each lane contains 3.5 µg proteins of cell extracts. (<b>C</b>) Cell extracts of 24 h-cultures of Newman, MW2 (USA400), and FRP3757 (USA300) strains that were transformed with pF carrying <i>psm-mec</i> (multi-copy), or integrated with <i>psm-mec</i> into the chromosome (single-copy) were subjected to Western blotting by anti-AgrA IgG (Upper panel). Each lane contains 3 µg proteins of cell extracts. Band intensities of AgrA were measured and are presented in the lower graph. The vertical axis represents the relative value against the AgrA band intensity of the parent strain in each Newman, MW2, and FRP3757 genetic background. Means ± standard deviations from four independent experiments are presented. Student t-test P-values between the parent strain and the <i>psm-mec</i>-introduced strain in each genetic background are presented. (<b>D</b>) The <i>agr</i> null mutant of Newman transformed with pMNS-agrBDCA carrying IPTG-inducible <i>agrBDCA</i> and pKE516 (empty vector), or pMNS-agrBDCA and pKE516-F carrying wild-type <i>psm-mec</i> was cultured in the presence or absence of IPTG. Cell extracts of 24-h cultures were subjected to Western blotting by anti-AgrA IgG. Each lane contains 6 µg proteins of cell extracts. (<b>E</b>) Schematic representation of <i>luc-</i>fusions of the <i>recF</i> promoter, <i>agrA</i> SD, the <i>agrA</i> ORF, and the <i>luc</i> ORF. Bold gray lines represent the plasmid construct. Horizontal dotted lines represent the regions deleted from the plasmids. Putative binding region means the region predicted to bind to the <i>psm-mec</i> RNA by <i>in silico</i> analysis. SD means Shine-Dalgarno sequence of <i>agrA</i>. (<b>F</b>) Luciferase activities of Newman strains that were transformed with the <i>luc-</i>fusion plasmids with <i>psm-mec</i> (+F) or without <i>psm-mec</i> (−F) were measured. The vertical axis represents the luciferase activity. Student t-test P-values between +F and −F are presented. NS, P>0.05. (<b>G</b>) Newman strain, which was integrated with <i>psm-mec</i> or without <i>psm-mec</i>, was transformed with the <i>luc-</i>fusion plasmids. Luciferase activities of the strains were measured. The vertical axis represents the relative luciferase activity of the <i>psm-mec</i>-integrated Newman [+F (single-copy)] against that of the Newman strain (−F). Student t-test P-values between +F and −F are presented. NS, P>0.05.</p