Identification of mycoplasma membrane proteins by systematic TnphoA mutagenesis of a recombinant library

Abstract

Wall-less prokaryotes in the genus Mycoplasma include over 90 species of infectious agents whose pathogenicity for humans and other animals is currently being assessed. Molecular characterization of surface proteins is critical in this regard but is hampered by the lack of genetic systems in these organisms. We used TnphoA transposition to systematically mutagenize, in Escherichia coli, a genomic plasmid library constructed from Mycoplasma fermentans, a potential human pathogen. The strategy circumvented problems of expressing mycoplasma genes containing UGA (Trp) codons and relied on the construction of the vector pG7ZCW, designed to reduce TnphoA transposition into vector sequences. Functional phoA gene fusions directly identified genes encoding 19 putative membrane-associated proteins of M. fermentans. Sequences of fusion constructs defined three types of export sequence: (1) non-cleavable, membrane-spanning sequences, (2) signal peptides with signal peptidase (SPase) I-like cleavage sites, and (3) signal peptides with SPase II-like lipoprotein-cleavage sites which, like most other mycoplasmal lipoprotein signals analysed to date, differed from those in several Gram-negative and Gram-positive eubacteria in their lack of a Leu residue at the -3 position. Antibodies to synthetic peptides that were deduced from two fusions to predicted lipoproteins, identified corresponding amphiphilic membrane proteins of 57 kDa and 78 kDa expressed in the mycoplasma. The P57 sequence contained a proline-rich N-terminal region analogous to an adhesin of Mycoplasma gallisepticum. The P78 protein was identical to a serologically defined phase-variant surface lipoprotein. TnphoA mutagenesis provides an efficient means of systematically characterizing functionally diverse lipoproteins and other exported proteins in mycoplasmas.

Gene Families Encoding Phase- and Size-Variable Surface Lipoproteins of Mycoplasma hyorhinis

“…The synthetic peptide pepG was prepared on a model 432A peptide synthesizer (Applied Biosystems) by standard 9-fluorenylmethylcarbonyl protection chemistries and purified as previously described (4,22). The amino acid sequence of pepG, C-SGTSSTTETGSTTESSGQA)DSTSGTSTSI, corresponds to the C-terminal 29 amino acid residues of VlpG deduced from the 3Ј region of the vlpG gene sequence determined in this study.…”

Identification of Genes Encoding Exported Mycobacterium tuberculosis Proteins Using a Tn 552′phoA In Vitro Transposition System

“…PhoA (Escherichia coli alkaline phosphatase) protein fusions have been used in many different organisms to identify exported proteins, including ones that are important to bacterial virulence (7,17,26,36,49). Importantly, PhoA has been shown to function as a reporter for secreted proteins in M. smegmatis (60).…”

Localized frameshift mutation generates selective, high-frequency phase variation of a surface lipoprotein encoded by a mycoplasma ABC transporter operon

“…Mouse antisera to synthetic peptides were generated and used as described previously (13). Peptides included NH 2 -CSKSFGVIKANQDINI QIKKGTIHALIGEN-COOH, representing an N-terminal region of P63, and NH 2 -CKKPNEIKESDLKFYSKNMPSFNSE-COOH, representing an N-terminal region of the mature P78 lipoprotein (13). Polyclonal antibodies (PAbs) to whole organisms or membrane proteins isolated by detergent partitioning (see below) have been characterized elsewhere (64).…”

“…MAb F202C21A to P78 has been described elsewhere (64). Mouse antisera to synthetic peptides were generated and used as described previously (13). Peptides included NH 2 -CSKSFGVIKANQDINI QIKKGTIHALIGEN-COOH, representing an N-terminal region of P63, and NH 2 -CKKPNEIKESDLKFYSKNMPSFNSE-COOH, representing an N-terminal region of the mature P78 lipoprotein (13).…”