Lysogenic conversion of staphylococcal lipase is caused by insertion of the bacteriophage L54a genome into the lipase structural gene

Abstract

Staphylococcus aureus PS54 manifests no lipase (geh) activity. This is due to the insertion of bacteriophage L54a DNA into the geh structural gene. The nucleotide sequence of this 2,968-base-pair DNA fragment was determined. Lipase deduced from the nucleotide sequence is a polypeptide of 690 amino acids which extends from nucleotide 706 to 2776.Many strains of staphylococci produce a true lipase or glycerol ester hydrolase (EC 3.1.1.3). The activity of the staphylococcal lipase gene is negatively regulated by bacteriophage lysogenization, also known as lysogenic conversion (3, 21 385(1 x SSC is 0.15 M NaCI and 15 mM sodium citrate [pH 7.0]) containing 0.1% sodium dodecyl sulfate. The membrane was dried at 80°C for 10 min and then autoradiographed as we described previously (4).DNA sequence analysis. Various restriction endonuclease fragments of the geh element were cloned into the M13 bacteriophage derivatives mpl8 or mpl9 (33) and propagated in Escherichia coli JM103. DNA sequencing was carried out by the dideoxy chain termination method of Sanger et al. (22). A computer-assisted sequence analysis was carried out with Seqaid (19), a software package kindly provided by Donald J. Roufa, Kansas State University.Deletion mutagenesis. Plasmids pLI210 and pLI211 containing the 2.9-kilobase (kb) insert with the lipase gene from S. aureus PS54C (11) were linearized by endonuclease digestion at the unique BamHI site and further digested with BAL 31 exonuclease to obtain various-length deletions from either end of the 2.9-kb geh insert. The digests were then phenol extracted, ethanol precipitated, ligated with T4 DNA ligase, and transformed into competent E. coli LE392 (11). Transformants were selected on L-broth plates containing 10 ,ug of chloramphenicol per ml. A panel of plasmids with various size deletions in the geh fragment was obtained from the transformants. Size estimates were made by agarose gel electrophoresis of minilysates of clones after linearization of the plasmids by restriction enzyme digestion. Alternatively, plasmids pLI210 or pLI211 were digested with restriction enzymes to delete specific sections of DNA and then religated. RESULTSDeletion mutagenesis. We reported earlier (11) that plasmids pLI210 and pLI211 carry a 2,968-base-pair (bp) DNA fragment containing the lipase gene (geh) of S. aureus PS54C which expressed lipase activity both in E. coli and S. aureus. To further localize the geh gene, various plasmids containing deletions at either end of the 2.9-kb fragment were generated. These deletions are schematically shown in Fig. 1 along with an indication of the effect of the deletion of lipase activity. Up to 500 bp could be removed from the left end of the fragment and up to 80 bp could be removed from the right end without influencing activity. Larger deletions at either end of the insert resulted in loss of enzymatic activity.ONA sequence of the geh gene. The strategy used for nucleotide sequencing is shown in Fig. 2. Each restriction fragment was subcloned into bacteriophages M13 mpl8 or ...

Purification and properties of extracellular lipase from Pseudomonas aeruginosa EF2

“…which have also been investigated (Dring & Fox, 1983;Bozoglu et al, 1984;Kugimiya et al, 1986;Stuer et al, 1986;Lee & Iandolo, 1986;Yamamoto & Fujiwara, 1988;Aoyama et al, 1988;van Oort et al, 1989).…”

“…aeruginosa EF2 lipase is strikingly similar to that of the other Pseudomonas lipases which have recently been sequenced, either by direct analysis of the purified enzymes or from the DNA sequences of the lipase genes (Kugimiya et , 1986), it must be concluded that these two enzymes contain a non Nterminal deletion and addition, respectively. It should be noted that none of the Pseudomonas lipases show any Nterminal sequence similarity to the lipases from S. aureus and S. hyicus (Lee & Iandolo, 1986 ;van Oort et al, 1989).…”

“…aeruginosa EF2 lipase, like all of these other bacterial lipases, is composed of a single type of subunit which can apparently undergo a variable degree of aggregation. The subunit M , (29000) is very similar to that of several other Pseudomonas lipases (29000 to 35 000), but substantially different from those reported for the Ps.fragi AFO 3458 (14600), Ps.Jluorescens MC 50 (55 000), Staphylococcus aureus (70 000) and Staphylococcus hyicus (86000) enzymes (BozoQlu et al, 1984;Kugimiya et al, 1986;Lee & Iandolo, 1986;van Oort et al, 1989).…”

Analysis of Genetic Elements ControllingStaphylococcus aureus lrgABExpression: Potential Role of DNA Topology in SarA Regulation

“…The integrated loci were transferred into RN6390 by 11-mediated transduction, producing strains designated KB700, KB707, KB706, and KB705 that were used for reporter gene assays (Table 1). Proper integration of the promoter fusion constructs was confirmed by Southern blot analysis and/or PCR analysis using the 5Ј primer employed for the production of the promoter fragment along with a second oligomer (5Ј-GTTCTGCACCTTTACGTTG-3Ј) that is complementary to a DNA sequence downstream of the attB integration site within the geh locus (19).…”

Physical and Genetic Mapping of the Protein A Gene in the Chromosome of Staphylococcus aureus 8325-4

“…The enterotoxin A gene which is associated with a converting bacteriophage (Betley & Mekalanos, 1985) maps in interval 6 (Pattee & Glatz, 1980) but can occupy other chromosomal sites (Mallonee et al, 1982). The a-toxin (hly) and lipase (geh) genes were inactivated with drug resistance insertion mutations isolated by allele replacement (O'Reilly et al, 1986) and plasmid integration (Lee & Iandolo, 1986), respectively. The integrated drug resistance determinants served as selective markers for the virulence factor loci in mapping experiments (Pattee, 1986(Pattee, , 1987.…”