Proteins secreted via the TAT system are often, but not limited to, proteins that bind cofactors in the cytoplasm prior to transport, such as those involved in respiration and electron transport, and proteins that bind catalytic metal ions [59–62]. The TAT system has also been shown to secrete several factors important for bacterial pathogenesis including iron acquisition, flagella synthesis, toxins, phospholipases, and beta-lactamases
[59, 62–74]. In this study, we identified genes encoding a TAT system in M. catarrhalis SGC-CBP30 in vivo and mutated these genes in order to elucidate the role of this translocase in the secretion of proteins that may be important for pathogenesis. Results and discussion Identification Thiazovivin research buy of tatA,
tatB and tatC genes in M. catarrhalis Analysis of the patented genomic sequence of M. catarrhalis strain ATCC43617 using NCBI’s tblastn service (http://blast.ncbi.nlm.nih.gov/Blast.cgi) identified an ORF (nucleotides 267,266 to 266,526 of GenBank accession number AX06766.1) that encodes a protein similar to the tatC gene product of Pseudomonas stutzeri (expect value of 7e-56). TatC is the most highly-conserved component of the TAT system among organisms known (or predicted) to utilize this particular secretion apparatus [59–62]. TatC is selleck compound located in the cytoplasmic membrane, typically contains 6 membrane-spanning regions, and plays a key role in recognizing the twin-arginine Methane monooxygenase motif in the signal sequence of molecules secreted by the TAT system. The M. catarrhalis ATCC43617 tatC-like ORF specifies a 27-kDa protein of 247 amino acids,
and analysis using the TMPred server (http://www.ch.embnet.org/software/TMPRED_form.html) revealed that it contains 6 potential membrane-spanning domains (data not shown). Sequence analysis upstream of the M. catarrhalis tatC ortholog identified gene products similar to other conserved components of the TAT system, TatA and TatB (Figure 1). The ORF immediately upstream encodes a 178-residue protein with a molecular weight of 20-kDa that resembles TatB of Providencia stuartii  (expect value of 3e-8). Upstream of the M. catarrhalis tatB-like gene, we identified an ORF specifying a 9-kDa protein of 77 aa that is most similar to TatA of Xanthomonas oryzae  (expect value of 2e-5). TatA and TatB are cytoplasmic proteins anchored to the cytoplasmic membrane via hydrophobic N-termini. TatB forms a complex with TatC often referred to as the twin-arginine motif recognition module, while TatA oligomerizes and forms a channel that is used to secrete TAT substrates [59–62]. Both M. catarrhalis ATCC43617 TatA (aa 4–21) and TatB (aa 5–21) orthologs are predicted to contain hydrophobic membrane-spanning domains in their N-termini using TMPred (data not shown).