Thermonuclease gene as a target nucleotide sequence for specific recognition of Staphylococcus aureus.
DNA fragments, 450 bp in length, were amplified by polymerase chain reaction (PCR) from the thermonuclease gene (nuc) carried by seven epidemiologically independent Staphylococcus aureus isolates. Sequencing of the PCR products led us to characterize 210 bp strictly conserved. A 186 bp piece from within this conserved region was cloned into pUC18. The resulting recombinant plasmid, pIP1608, was used as a probe against the cellular DNA of 360 staphylococcal isolates belonging to 28 species. Only the 146 S. aureus isolates, including four which were not thermonuclease producers, had DNA that hybridized with pIP1608. Among the 214 non-S. aureus staphylococci, 55 exhibited a thermonuclease activity. For 32 of these, the enzymatic activity was inhibited by a commercially available polyclonal antiserum directed against the thermonuclease of an S. aureus strain. These results are in favour of the use of pIP1608 as a probe to specifically recognize S. aureus. Furthermore, we propose a method based on colony blot hybridization and potentially useful to enumerate S. aureus cells in biological samples.
Chesneau
O
O
National Reference Centre for Staphylococci, Institut Pasteur, Paris, France.
Allignet
J
J
el Solh
N
N
D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D015342:DNA Probes; D004269:DNA, Bacterial / Q000235:genetics; D019793:Fluorescein; D005452:Fluoresceins; D005798:Genes, Bacterial; D008836:Micrococcal Nuclease / Q000235:genetics*; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D017422:Sequence Analysis, DNA; D013045:Species Specificity; D013211:Staphylococcus aureus / Q000201:enzymology / Q000235:genetics / Q000302:isolation & purification*
D017931:DNA Primers; D015342:DNA Probes; D004269:DNA, Bacterial; D005452:Fluoresceins; D008836:Micrococcal Nuclease; D019793:Fluorescein