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

Abstract

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.

Evidence in the Legionella pneumophila genome for exploitation of host cell functions and high genome plasticity

“…Notably, the lvh region of L. pneumophila Paris is encoded in a 36-kb region that is either integrated in the chromosome or excised as a multicopy plasmid (data not shown). This pattern is similar to that described for the 30-kb unstable element of strain Olda, which is possibly derived from phage and is involved in phase variation 38 . The lvh region has a G+C content (43%) that differs from that of the rest of the chromosome (38%), and it contains some phage-related genes, suggesting a possible phage origin.…”

Multigenome analysis identifies a worldwide distributed epidemic Legionella pneumophila clone that emerged within a highly diverse species

“…Furthermore, plasmid excision and integration appear to constitute a particular mechanism of genome plasticity in Legionella. For example, the Lvh T4ASS can be present in an integrated and excised form Chien et al 2004;Doleans et al 2006), similar to a 30-kb element specific for strain Olda associated with loss of virulence (Luneberg et al 2001) and the two recently reported mobile elements carrying T4ASS in strain Corby (Glöckner et al 2007). In addition, our results suggest that the 130-kb plasmid of strain Paris may integrate into the genome associated with loss of the genes necessary for plasmid replication.…”

“…In the strains where pLpp is present only in part, a 85-kb region carrying the twocomponent system lrpR-lskS implicated in the virulence of L. longbeachae (Doyle and Heuzenroeder 2002) was present, whereas the 45-kb region (plpp0001-plpp0045) bordered by an integrase (plpp0140) and coding a homolog of CsrA and for plasmid-related functions like partition and replication proteins and the conjugation transfer proteins Tra, is missing. As integration and circularization of plasmids have been shown for Legionella (Luneberg et al 2001;Doleans et al 2006), it is tempting to assume that parts of pLpp may, under certain conditions, integrate into the chromosome. …”

“…This represented 324, 242, and 227 PCR probes specific for genes uniquely present in strain Paris, Lens, or Philadelphia, respectively; 145 probes each present in only two of the three sequenced strains and 142 probes corresponding to known and putative virulence factors, 31 genes forming the LPS cluster of Sg1 (Luneberg et al 2000), 30 genes of an unstable genetic element responsible for phase variation of LPS in strain Olda (Luneberg et al 2001) and 14 control genes (Supplemental Tables S2A, S2B, S2C; lpp, lpg, lpl numbers indicate genes of L. pneumophila strains Paris, Philadelphia-1, and Lens. The respective genes and its annotations can be accessed at http:// genolist.pasteur.fr/LegioList/).…”

Bacterial Genome Instability

“…Therefore, the frequency of excision and insertion of IS492 directly regulates the production of these molecules, which are important in biofilm formation. Similarly, the formation of a specific lipopolysaccharide in Legionella pneumophila depends on the presence of a 30-kb unstable genetic element in its chromosome (413). This element probably originates from a phage and can excise as a replicating plasmid, consequently stopping its host's production of lipopolysaccharide.…”