Chromosomal insertion and excision of a 30 kb unstable genetic element is responsible for phase variation of lipopolysaccharide and other virulence determinants in Legionella pneumophila.
We recently described the phase-variable expression of a virulence-associated lipopolysaccharide (LPS) epitope in Legionella pneumophila. In this study, the molecular mechanism for phase variation was investigated. We identified a 30 kb unstable genetic element as the molecular origin for LPS phase variation. Thirty putative genes were encoded on the 30 kb sequence, organized in two putative opposite transcription units. Some of the open reading frames (ORFs) shared homologies with bacteriophage genes, suggesting that the 30 kb element was of phage origin. In the virulent wild-type strain, the 30 kb element was located on the chromosome, whereas excision from the chromosome and replication as a high-copy plasmid resulted in the mutant phenotype, which is characterized by alteration of an LPS epitope and loss of virulence. Mapping and sequencing of the insertion site in the genome revealed that the chromosomal attachment site was located in an intergenic region flanked by genes of unknown function. As phage release could not be induced by mitomycin C, it is conceivable that the 30 kb element is a non-functional phage remnant. The protein encoded by ORF T on the 30 kb plasmid could be isolated by an outer membrane preparation, indicating that the genes encoded on the 30 kb element are expressed in the mutant phenotype. Therefore, it is conceivable that the phenotypic alterations seen in the mutant depend on high-copy replication of the 30 kb element and expression of the encoded genes. Excision of the 30 kb element from the chromosome was found to occur in a RecA-independent pathway, presumably by the involvement of RecE, RecT and RusA homologues that are encoded on the 30 kb element.
Institut für Hygiene und Mikrobiologie, Universität Würzburg, Josef-Schneider-Str. 2, D-97080 Würzburg, Germany. berg@hygiene.uni-wuerzberg.de
E
E
Lüneberg
B
B
Mayer
N
N
Daryab
O
O
Kooistra
U
U
Zähringer
M
M
Rohde
J
J
Swanson
M
M
Frosch
D015139:Blotting, Southern; D002876:Chromosomes, Bacterial / Q000235:genetics*; D003001:Cloning, Molecular; D004251:DNA Transposable Elements / Q000235:genetics*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D018922:HL-60 Cells; D006801:Humans; D016952:Legionella pneumophila / Q000235:genetics / Q000254:growth & development* / Q000472:pathogenicity* / Q000821:virology; D007877:Legionnaires' Disease / Q000382:microbiology; D008070:Lipopolysaccharides / Q000378:metabolism*; D016685:Mitomycin / Q000494:pharmacology; D016366:Open Reading Frames / Q000235:genetics / Q000502:physiology; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic; D017422:Sequence Analysis, DNA; D014774:Virulence
D004251:DNA Transposable Elements; D008070:Lipopolysaccharides; D016685:Mitomycin; D015252:Deoxyribonucleases, Type II Site-Specific; C024400:GTYRAC-specific type II deoxyribonucleases