Cloning and temperature-dependent expression in Escherichia coli of a Legionella pneumophila gene coding for a genus-common 60-kilodalton antigen.
All Legionella species express a 60-kilodalton (kDa) protein which contains a genus-specific epitope recognized by murine monoclonal antibody GW2X4B8B2H6. A genomic cosmid library of Legionella pneumophila chromosomal DNA was constructed in pHC79 and screened for 60-kDa antigen-expressing clones with the monoclonal antibody. A 3.2-kilobase EcoRI fragment from cosmid 14B11 expressing a 60-kDa protein was subcloned into pUC19 (pSH16), and deletion of a 1.2-kilobase HindIII fragment (pSH16A) generated a 33-kDa truncated polypeptide no longer reactive with the monoclonal antibody. Southern blot analysis of chromosomal DNA from selected Legionella species restricted with EcoRI and probed with the 1.2-kilobase fragment coding for the carboxyl region of the protein revealed DNA homology which was not observed with DNA from Escherichia coli. Maxicell analysis of pSH16 identified a second polypeptide of approximately 15 kDa expressed from a gene (htpA) upstream of the gene coding the 60-kDa protein (htpB). Both proteins were preferentially synthesized by L. pneumophila following heat shock (temperature shift from 25 to 42 degrees C), and under steady-state growth conditions the relative level of 60-kDa protein was unaffected by temperature. In E. coli, expression of a 60-kDa protein from pSH16 also increased following heat shock (25 to 42 degrees C), but under steady-state conditions expression was temperature dependent. Temperature-dependent expression from pSH16 was not observed in an rpoH (htpR) mutant strain of E. coli. The Legionella 60-kDa protein appears to be a heat shock protein which shares cross-reactive epitopes with the GroEL homolog of E. coli. In addition, a region of htpB encoding the 27-kDa carboxyl portion of the protein containing the monoclonal antibody-reactive epitope also contains DNA sequences unique to and conserved within the genus.
Hoffman
P S
PS
Department of Microbiology and Immunology, University of Tennessee, Memphis 38163.
Butler
C A
CA
Quinn
F D
FD
D000942:Antigens, Bacterial / Q000235:genetics* / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification; D015139:Blotting, Southern; D018834:Chaperonin 60; D003001:Cloning, Molecular; D003360:Cosmids; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial; D006360:Heat-Shock Proteins / Q000235:genetics / Q000302:isolation & purification; D007875:Legionella / Q000235:genetics*; D008970:Molecular Weight; D009693:Nucleic Acid Hybridization; D015183:Restriction Mapping; D013696:Temperature
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D018834:Chaperonin 60; D006360:Heat-Shock Proteins