DNA sequence determination and biochemical analysis of the immunogenic protein P36, the lactate dehydrogenase (LDH) of Mycoplasma hyopneumoniae

Abstract

~The DNA sequence of the gene encoding the early and specific immunogenic protein P36 of Mycoplasmu hyopneumoniue has been determined. Comparison of the DNA sequence and the deduced amino acid sequence of P36 with known genes and proteins in data banks indicated that P36 is a L-lactate dehydrogenase (LDH) (EC 1.1.1.27). Biochemical analysis of protein P36 expressed from the cloned gene in Escherichiu coli confirmed that P36 has L-lactate dehydrogenase activity. Protein P36 of M. hyopneumoniae therefore is termed LDH and its gene Cdh. M. hyopneumoniae LDH was shown to contain the typical domains of LDH of other bacterial species. Immunologically however, we have shown that polyclonal antibodies against M. hyopneumoniue LDH do not crossreact with related LDH and show high specificity for M. hyopneunwniue. The ldh gene is preceded by several typical -10 sequences found in promoters of prokaryotes, but lacks the -35 sequence. Sequences rich in A + T, however, precede the -10 boxes, suggesting that factors involved in transcription initiation and their regulation may be different in M. hyopneumoniue compared to other bacterial species, but the putative ribosome binding site seems to be conserved.

Molecular cloning of a 46-kilodalton surface antigen (P46) gene from Mycoplasma hyopneumoniae: direct evidence of CGG codon usage for arginine

“…Upstream of the putative initiation codon (ATG), a consensus sequence for a Ϫ10 (Pribnow) box is found, but the Ϫ35 region is replaced by AT-rich segments. These features are similar to the features of other reported genes of M. hyopneumoniae (1,15,16). A transcription termination sequence (stem-and-loop structure) was found in the 3Ј-flanking region (positions 1506 to 1540 and 1542 to 1600).…”

Characterization and Analysis of a Stable Serotype-Associated Membrane Protein (P30) of Mycoplasma agalactiae

“…Five of these strains were analyzed in detail and found to possess nucleotide substitutions in both possible Ϫ10 boxes of the potential transcription promoter of p30. Although very little is known about transcriptional promoters of mycoplasmal genes or operons, DNA sequence analyses very often reveal repeats of Ϫ10 boxes as potential promoters in these organisms (13). We therefore interpret the substitutions in both possible Ϫ10 boxes of the p30 gene in these strains as the cause of lack of P30 expression, implying that the repeated Ϫ10 boxes found upstream of the RBS of p30 play a major role in transcription of this gene.…”

Reiterated repeat region variability in the ciliary adhesin gene of Mycoplasma hyopneumoniae

“…Mycoplasma membranes are rich in protein, many of which stimulate a humoral immune response in the host (Razin, 1979). Although several immunoreactive M. hyopneumoniae antigens have been characterized and their genes cloned (Klinkert et al, 1985;Haldimann et al, 1993, Fagan et al, 1996, attempts to identify surface components which play a role in early colonization have only recently been reported. The development of microtitre plate adherence and TLC overlay assays has revealed that M. hyopneumoniae binds to sulfated glycolipid receptors present on porcine respiratory tract ciliated cells and that heparin, fucoidan, mucin and chondroitin interact with adhesive molecules on the surface of M. hyopneumoniae and are able to disrupt the adherence of the pathogen to intact ciliated cells (Zhang et al, 1994a, b).…”