The htpAB operon of  Legionella pneumophila  cannot be deleted in the presence of the groE chaperonin operon of  Escherichia coli.
HtpB, the chaperonin of the intracellular bacterial pathogen Legionella pneumophila , displays several virulence-related functions in vitro. To confirm HtpB's role in vivo, host infections with an htpB deletion mutant would be required. However, we previously reported that the htpAB operon (encoding co-chaperonin and chaperonin) is essential. We attempted here to delete htpAB in a L. pneumophila strain carrying the groE operon (encoding the Escherichia coli co-chaperonin and chaperonin). The groE operon was inserted into the chromosome of L. pneumophila Lp02, and then allelic replacement of htpAB with a gentamicin resistance cassette was attempted. Although numerous potential postallelic replacement transformants showed a correct selection phenotype, we still detected htpAB by PCR and full-size HtpB by immunoblot. Southern blot and PCR analysis indicated that the gentamicin resistance cassette had apparently integrated in a duplicated htpAB region. However, we showed by Southern blot that strain Lp02, and the Lp02 derivative carrying the groE operon, have only one copy of htpAB. These results confirmed that the htpAB operon cannot be deleted, not even in the presence of the groE operon, and suggested that attempts to delete htpAB under strong phenotypic selection result in aberrant genetic recombinations that could involve duplication of the htpAB locus.
Department of Microbiology and Immunology, Dalhousie University, Halifax, NS, Canada.
Gheyath K
GK
Nasrallah
Elizabeth
E
Gagnon
Dennis J
DJ
Orton
Rafael A
RA
Garduño
D018833:Chaperonins / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics*; D018628:Gene Dosage; D005798:Genes, Bacterial / Q000235:genetics; D020043:Genes, Essential / Q000235:genetics; D006360:Heat-Shock Proteins / Q000235:genetics*; D007613:Kanamycin Resistance / Q000235:genetics; D016952:Legionella pneumophila / Q000235:genetics*; D009876:Operon / Q000235:genetics*
D029968:Escherichia coli Proteins; C028715:GroE protein, E coli; D006360:Heat-Shock Proteins; D018833:Chaperonins