Genetic and functional studies of the Mip protein of Legionella

R Ratcliff, J A Lanser, P A Manning, M W Heuzenroeder

The nucleotide sequence of the mip genes, and their inferred amino acid sequences were determined from 35 Legionella species, and compared with the published sequences for L. pneumophila, L. micdadei and L. longbeachae. The sequences were 69-97 per cent conserved at the nucleotide level and 82-99 per cent at the amino acid level, with total conservation of amino acids determined to be associated with sites known to be involved in peptidyl prolyl cis - trans isomerase activity. No apparent difference could be determined in the arrangement of amino acids which would predict a functional difference in Mip from species associated with disease, and Mip in species isolated only from the environment. Additionally, a phylogenetic comparison of the sequences with published 16S rRNA sequences, using both genetic distance and maximum parsimony methods was performed. Few relationships were apparent which were well supported by both data sets, the most robust being a clade comprising (((cincinnatiensis, longbeachae, sainthelensi, santicrucis) gratiana) (moravica, quateirensis, shakespearei, worsleiensis) anisa, bozemanii, cherrii, dumoffii, gormanii, jordanis, parisiensis, pneumophila, steigerwaltii, tucsonensis, and wadsworthii).

A sequence based identification system for Legionella utilising the mip gene has also been developed, current identification requires complex methods, the most successful being chromatographic fingerprinting of cellular ubiquinones and fatty acids. This method has become less reliable as new species have been identified, and is also subject to isolate and extraction variation. A comparison of sequence derived from the conserved mip gene enabled most isolates to be clearly identified to species level. Strain variation was found for some species, usually conforming to a clonal subtype. Some isolates produced unique sequence indicating that they are probably as yet uncharacterised species. In summary the ‘digital’ sequence scheme was specific and robust, and likely to be more applicable to most diagnostic laboratories than 'analogue' chromatographic methods. One publication has arisen from this work and another is in preparation.