The Lly protein is essential for p-hydroxyphenylpyruvate dioxygenase activity in Legionella pneumophila.
The lly locus confers fluorescence, haemolysis, brown pigmentation and an increased resistance to light in Legionella pneumophila. In this study, we correlated the pigment production of two lly-positive L. pneumophila isolates and a recombinant lly-positive Escherichia coli strain with the presence of homogentisic acid (HGA) in the culture supernatant. The detection of HGA by high performance liquid chromatography and the data analysis of the deduced amino acid sequence of the lly gene indicate that the lly locus codes for a p-hydroxyphenylpyruvate dioxygenase (HPPD). This enzyme catalyses the transformation of p-hydroxyphenylpyruvate into HGA, which subsequently oxidises and polymerises into a melanin-like pigment. One open reading frame (ORF 1) in the lly region exhibited homologies with genes of Synechocystis sp., Petroselium crispum and Streptomyces mycarofaciens that code for methyltransferases. By screening a genomic library of L. pneumophila (serogroup 1) strain Corby with a monoclonal antibody against the legiolysin (lly), we identified two recombinant E. coli clones that did not produce the brown pigment and showed no haemolysis and fluorescence. DNA sequencing revealed that both clones contained 874 nucleotides of the N-terminal part of the lly gene. The recombinant strains expressed truncated legiolysin proteins of 39.5 and 35.7 kDa and did not produce HGA. Considering the highly conserved structure of legiolysin-like HPPD genes from other organisms, we suggest that the C-terminus of the legiolysin may be essential for the enzymatic activity that conferred pigmentation via HGA polymerisation, haemolysis and fluorescence.
Steinert
M
M
Institut für Molekulare Infektionsbiologie, Universität Würzburg, Röntgenring 11, D-97070 Würzburg, Germany. michael.steinert@mail.uni-wuerzburg.de
Flügel
M
M
Schuppler
M
M
Helbig
J H
JH
Supriyono
A
A
Proksch
P
P
Lück
P C
PC
D015111:4-Hydroxyphenylpyruvate Dioxygenase / Q000378:metabolism*; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000235:genetics; D006713:Homogentisic Acid / Q000302:isolation & purification / Q000378:metabolism; D016952:Legionella pneumophila / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010667:Phenylpyruvic Acids / Q000378:metabolism; D010860:Pigments, Biological / Q000302:isolation & purification / Q000378:metabolism; D014169:Transformation, Bacterial
D001426:Bacterial Proteins; C094283:Lly protein, Legionella pneumophila; D010667:Phenylpyruvic Acids; D010860:Pigments, Biological; C010590:4-hydroxyphenylpyruvic acid; D015111:4-Hydroxyphenylpyruvate Dioxygenase; D006713:Homogentisic Acid