Identification of mycoplasma membrane proteins by systematic Tn phoA mutagenesis of a recombinant library.
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.
Cleavinger
C M
CM
Department of Molecular Microbiology and Immunology, University of Missouri-Columbia 65212, USA.
Kim
M F
MF
Im
J H
JH
Wise
K S
KS
D000469:Alkaline Phosphatase / Q000032:analysis / Q000235:genetics; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000032:analysis*; D001483:Base Sequence; D004251:DNA Transposable Elements / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D015723:Gene Library; D005822:Genetic Vectors; D008565:Membrane Proteins / Q000032:analysis* / Q000235:genetics; D008967:Molecular Biology / Q000379:methods; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016858:Mycoplasma fermentans / Q000166:cytology / Q000235:genetics*; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000032:analysis; D014170:Transformation, Genetic
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D008565:Membrane Proteins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D000469:Alkaline Phosphatase