Many mycoplasma genes contain internal UGA (opal) codons because of their use as tryptophan coding codons. This results in a lack of expression of many cloned mycoplasma antigenic epitopes in Escherichia coli. It has been shown that opal suppressors can be used to enhance expression of defined mycoplasma gene sequences, but no studies have been published using E. coli suppressor strains to screen mycoplasma gene libraries for immunoreactive epitopes. The E. coli suppressor strain EM612 was used to screen a ~Uycoplasma hyopneumoniue Lambda gene library. This strain contained an inducible opal suppressor, trpT, as well as the release factor 2 mutation prjB3. Strain ISM612 was shown to enhance antibody recognition of cloned mycoplasmal gene sequences.

Membrane nucleases of mycoplasmas are believed to play important roles in growth and pathogenesis, although no clear evidence for their importance has yet been obtained. As a first step in defining the function of this unusual membrane activity, studies were undertaken to clone and analyze one of the membrane nuclease genes fromMycoplasma pulmonis. A novel screening strategy was used to identify a recombinant lambda phage expressing nuclease activity, and its cloned fragment was analyzed. Transposon mutagenesis was used to identify an open reading frame of 1,410 bp, which coded for nuclease activity in Escherichia coli. This gene coded for a 470-amino-acid polypeptide of 53,739 Da and was designatedmnuA (for “membrane nuclease”). The MnuA protein contained a prolipoprotein signal peptidase II recognition sequence along with an extensive hydrophobic region near the amino terminus, suggesting that the protein may be lipid modified or that it is anchored in the membrane by this membrane-spanning region. Antisera raised against two MnuA peptide sequences identified an M. pulmonis membrane protein of approximately 42 kDa by immunoblotting, which corresponded to a trypsin-sensitive nucleolytic band of the same size. Maxicell experiments with E. coli confirmed that mnuA coded for a nuclease of unknown specificity. Hybridization studies showed that mnuAsequences are found in few Mycoplasma species, suggesting that mycoplasma membrane nucleases display significant sequence variation within the genus Mycoplasma.

Many mycoplasma genes contain internal UGA (opal) codons because of their use as tryptophan coding codons. This results in a lack of expression of many cloned mycoplasma antigenic epitopes in Escherichia coli. It has been shown that opal suppressors can be used to enhance expression of defined mycoplasma gene sequences, but no studies have been published using E. coli suppressor strains to screen mycoplasma gene libraries for immunoreactive epitopes. The E. coli suppressor strain ISM612 was used to screen a Mycoplasma hyopneumoniae Lambda gene library. This strain contained an inducible opal suppressor, trpT, as well as the release factor 2 mutation prfB3. Strain ISM612 was shown to enhance antibody recognition of cloned mycoplasmal gene sequences.