Genetic analysis of the Moraxella lacunata pilin gene inversion region.

Abstract

This work describes experiments to study the molecular genetics of Moraxella lacunata pilin. A cloned Moraxella bovis pilin gene was used as a hybridization probe to screen a M. lacunata genomic DNA library. A plasmid clone, pMxL1 was isolated that contained one complete and one partial type 4 pilin gene (tfpQ and tfpI) on a 2-kb segment that inverts in E. coli. The M. lacunata tfpI pilin gene contains a perfect 19 bp tandem repeat that begins in the inversion junction site and fails to produce pilin on immunoblots. A putative trans-active invertase gene piv, was identified that is probably not related to the Din (Hin family) invertases. Several differences between the M. lacunata and M. bovis pilin gene regions include a 15 bp insert in the putative M. bovis recombinational enhancer, a truncated M. lacunata tfpB gene (function unknown), and the lack of a transcriptional terminator in the M. lacunata tfpB/tfpQ intercistronic region. These differences were confirmed by sequencing PCR amplified M. lacunata genomic DNA. The ability of pMxL1 to invert in E. coli host mutants deficient in various DNA binding proteins IHF, Fis, and HU was tested in vivo. The HU mutants were found to reduce, but not eliminate inversion of pMxL1, while the other mutants had no effect on inversion. The role of Fis in Moraxellae inversion is still uncertain since Fis binding sites may be different in M. lacunata and M. bovis. The Fis binding sites in M. lacunata may be defective, lowering the inversion frequency relative to M. bovis. Inversion reduces the stability of pMxL1 and increased inversion frequency may explain why the intact M. bovis pilin gene region has not been cloned. An additional gene (tfpC) was identified that encodes a potential chaperone protein. Deletions in tfpC produce pilin of greater electrophoretic mobility on immunoblots, which is reversible when a functional tfpC gene is provided on a compatible plasmid in trans. The functional relationships of the different genes of the M. lacunata and M. bovis pilin gene regions and possible similarities to the Din invertase families are discussed.