Cloning of mnuA, a membrane nuclease gene of Mycoplasma pulmonis, and analysis of its expression in Escherichia coli

Membrane nucleases of mycoplasmas are believed to play important roles in g rowth and pathogenesis, although no clear evidence for their importance has yet been obtained. As a first step in defining the function of this unusua l membrane activity, studies were undertaken to clone and analyze one of th e membrane nuclease genes from Mycoplasma pulmonis. A novel screening strat egy was used to identify a recombinant lambda phage expressing nuclease act ivity, and its cloned fragment was analyzed. Transposon mutagenesis was use d to identify an open reading frame of 1,410 bp, which coded for nuclease a ctivity in Escherichia coli. This gene coded for a 470-amino-acid polypepti de of 53,739 Da and was designated mnuA (for "membrane nuclease"), The MnuA protein contained a prolipoprotein signal peptidase II recognition sequenc e along with an extensive hydrophobic region near the amino terminus, sugge sting 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 approximat ely 42 kDa by immunoblotting, which corresponded to a trypsin-sensitive nuc leolytic band of the same size. Maxicell experiments with E, coli confirmed that mnuA coded for a nuclease of unknown specificity. Hybridization studi es showed that mnuA sequences are found in few Mycoplasma species, suggesti ng that mycoplasma membrane nucleases display significant sequence variatio n,within the genus Mycoplasma.