Multigene families encoding the major hemagglutinins in phylogenetically distinct mycoplasmas

Abstract

Mycoplasma synoviae has two major membrane antigens, MSPA and MSPB, both of which are phase variable and which may be coordinately involved in adhesion of the organism to erythrocytes. A single gene (vlhA) from M. synoviae was characterized, and polypeptides were expressed from nonoverlapping 5' and 3' regions in Escherichia coli. The expression product of the vlhA 5' region reacted with specific reagents against MSPB, while that of the 3' region reacted with specific reagents against MSPA. Analysis of the predicted amino acid sequence showed a characteristic signal peptidase II cleavage site, and the presence of the acylation site was confirmed by identification of a lipid-associated membrane protein, similar in molecular mass to MSPB, in [3H]palmitate-labelled membrane proteins. Further sequence analysis of the vlhA gene revealed a high identity with the Mycoplasma gallisepticum pMGA1.7 gene, a member of a large translated family. The vlhA gene was shown to hybridize to multiple restriction fragments of the M. synoviae genome, suggesting that it was also a member of a multigene family. These findings indicate that coordinate phase variation of the two major surface antigens of M. synoviae WVU may be due to their expression from the same gene and that homologous gene families encode the major hemagglutinins of two phylogenetically distinct mycoplasmas. The presence of homologous multigene families in such phylogenetically distinct species, but not in the genomes of more closely related species, suggests that the families may have been transferred horizontally.

FIG. 1

Truncated nucleotide and deduced amino acid sequences of the vlhA gene (uppercase letters). Two nearly identical tandemly repeated 19-amino-acid regions at the amino-terminal end of the sequence are underlined. The location of the putative signal peptidase II recognition sequence is underlined with asterisks. Lowercase letters indicate the target site of the oligonucleotide primers (PCRF and PCRR) used to amplify the gene by PCR. Numbers to the left show the positions of the nucleotides, relative to the first 5′ nucleotide in the target site of the PCRF oligonucleotide primer.

FIG. 2

(A) The vlhA ORF. The region encoding the putative signal peptide (filled) and regions used to express fusion proteins in E. coli (hatched) are shown on the ORF. The scale on the top indicates amino acid residues encoded by the ORF. (B) Immunostaining of whole-cell proteins of E. coli cells expressing fusion proteins from recombinant plasmid PinPoint Xa1-T containing region I (panel I) or II (panel II) of the vlhA gene. In each panel, immunoblots were probed with a pool of MSPB-specific MAbs (lanes 1) and with rabbit monospecific antisera to MSPA (lanes 2) and MSPB (lanes 3).

FIG. 3

SDS-PAGE analysis of [³H]palmitate-labelled M. synoviae integral membrane proteins. (A) Lane 1, whole-cell proteins of M. synoviae were fractionated by TX-114 phase partitioning, and proteins in the detergent phase were separated by SDS-PAGE and stained with Coomassie brilliant blue. Lane 2, radiolabelled protein molecular mass markers. (B) The gel was autoradiographed as described previously (5). The arrow to the right indicates a lipoprotein similar in molecular mass to MSPB.

FIG. 4

Comparison of the amino acid sequence predicted from vlhA with that for the pMGA1.7 gene from M. gallisepticum. Numbers to the right of the sequences refer to the positions of adjacent residues relative to the first encoded amino acid. Vertical lines indicate identical residues, while colons (:) indicate conservative amino acid substitutions.

FIG. 5

Southern blot of genomic DNAs from M. synoviae WVU (lanes 1, 3, and 5) and M. gallisepticum S6 DNA (lanes 2, 4, and 6) digested with restriction enzymes BglII (lanes 1 and 2), EcoRI (lanes 3 and 4), and HindIII (lanes 5 and 6) and probed with the ³²P-labelled vlhA gene. The blot was washed under low-stringency conditions and autoradiographed. Three fragments of M. gallisepticum digested with each restriction endonuclease (BglII, 7.4, 5.8, and 1.2 kb; EcoRI, 11.2, 6, and 1.1 kb; and HindIII, 4.5, 3.2, and 1.5 kb) hybridized to the vlhA gene probe. Numbers on the left indicate the sizes of the nucleic acid molecular size markers (HindIII-digested λ phage).