Operon structure and expression of the genes of the Type 3 cluster in <i>Mmm</i>.

<p><b>A</b>. RT-PCR experiments were carried out on intergenic regions to demonstrate the co-transcription of the <i>MSC_0618</i> to <i>MSC_0627</i> genes. The region of the genome region surrounding the Type 3 cluster in <i>Mmm</i> is shown. Gene mnemonics and numbers are shown for the Type 3 cluster. The site of transposon insertion in the <i>MSC_0619</i> disrupted mutant (Δ619) is indicated by an arrow. Expected sizes of the putative transcripts are indicated. Amplification products of the expected sizes were obtained with primers binding within and upstream from the cluster (+) but not downstream from the cluster (−). <b>B</b>. Immunodetection of proteins from the Type 3 cluster of T1/44 and Δ619. A control for membrane protein detection was included, in the form of an antibody against a membrane protein, LppQ (anti-LppQ serum kindly supplied by Prof. J. Frey). <b>C</b>. Nano-LC-MS/MS detection of Type 3 ATPase proteins. Numbers of scans and distinct peptides detected are indicated. <b>D</b>. Evaluation of the sensitivity of Protein 5 and β-like subunit to trypsin degradation. Intact and lysed cells of <i>Mmm</i> T1/44 were incubated with (+) or without (−) trypsin enzyme coated on beads for six hours. Protection against hydrolysis was assessed by immunodetection with antibodies raised against Protein 5 (MSC_0620) and β-like subunit (MSC_0618).