EXPRESSION OF FOREIGN GENES AND SELECTION OF PROMOTER SEQUENCES IN ACHOLEPLASMA-LAIDLAWII

The stable maintenance and expression of foreign genes in mollicutes ( mycoplasmas) have been difficult to achieve due to the lack of suitabl e vectors. In this paper we show for the first time that a replicating vector can been used to express foreign genes other than antibiotic r esistance genes in Acholeplasma laidlawii. Plasmids derived from the l actococcal vector pNZ18 could introduce and maintain four different ge nes for many generations in A. laidlawii. One of these, encoding the d ominant membrane lipoprotein spiralin from the mollicute Spiroplasma c itri, was expressed; however, expression was weak, the signal peptide of spiralin was not cleaved and the protein was not covalently modifie d by fatty acids. This resulted in a hydrophilic character of spiralin and its cytoplasmic localization in A. laidlawii. To increase the exp ression of foreign genes, random A. laidlawii DNA fragments were clone d into a pNZ18-related plasmid and expression signals were selected us ing the Bacillus licheniformis alpha-amylase gene as a probe. Selectio n was done in Escherichia coil as well as directly in A. laidlawii. Ac tive recombinants from E. coli were also able to express alpha-amylase activities and an enzyme of native size in A. laidlawii. The highest activity was obtained from a recombinant selected directly in A. laidl awii. This is the first: example of a promoter sequence selected in a mollicute. Analysis of the putative promoters in seven clones revealed similar -10 and -35 regions, and similar spacer distances in A. laidl awii, Acholeplasma oculi, Lactococcus and E. coli. Vectors related to pNZ18 should be useful for the genetic analysis of specific A. laidlaw ii proteins and functions.