Sequence and TnphoA analysis of a Mycoplasma hyorhinis protein with membrane export function.
Proteins translocated across the single plasma membrane of mycoplasmas (class Mollicutes) represent important components likely to affect several interactions of these wall-less microbes with their respective hosts. However, identification and functional analysis of such proteins is hampered by the lack of mutational systems in mycoplasmas and by a perceived limitation in translating recombinant mycoplasma genes containing UGA (Trp) codons in other eubacteria. Here we directly analyze a gene encoding a Mycoplasma hyorhinis protein capable of promoting its membrane translocation. It was initially detected by screening a recombinant phage genomic library with antibody from a host with M. hyorhinis-induced arthritis and was localized by Tn5 and deletion mutations affecting expression of antigenic translational products. Sequence analysis of the isolated gene predicted a hydrophilic protein, P101, containing three UGA codons and a putative signal peptide with an uncharacteristic cluster of positively charged amino acids near its C terminus. Nevertheless, lambda::TnphoA transposon mutagenesis of an Escherichia coli plasmid bearing the p101 gene resulted in p101::TnphoA fusions expressing products that could translocate as much as 48 kDa of the P101 sequence (up to the first UGA codon) across the E. coli plasma membrane. Fusion proteins containing mature P101 sequences expressed mycoplasma epitopes and were found by cell fractionation and detergent phase partitioning to be integral membrane proteins in E. coli, suggesting a lack of signal peptide cleavage in this system. Importantly, identification of P101 by direct analysis of its export function relied neither on prior identification of the mycoplasmal product nor on complete expression of the product from the cloned mycoplasma gene.
Yogev
D
D
Department of Molecular Microbiology and Immunology, School of Medicine, University of Missouri, Columbia 65212.
Watson-McKown
R
R
McIntosh
M A
MA
Wise
K S
KS
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001692:Biological Transport; D002451:Cell Compartmentation; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D004251:DNA Transposable Elements; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009174:Mycoplasma / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015183:Restriction Mapping; D012995:Solubility
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; C067817:P101 protein, Mycoplasma hyorhinis