Computer-assisted chromosome mapping in Staphylococcus aureus by protoplast fusion

The genes responsible for utilization of lactose in Staphylococcus aureus are organized as an inducible operon, with galactose 6-phosphate being the intraceflular inducer. To clone the repressor gene of this operon, we constructed an integration vehicle carrying 1.9 kilobases (kb) of DNA sequences from a region upstream of the structural genes of the operon. Through integration and subsequent rescue of this plasmid, we were able to clone approximately 7 kb of staphylococcal chromosomal DNA. We have shown that the plasmid insert complemented lac constitutive mutants. This repressor activity was localized to a 1.8-kb DNA fragment and, through maxicell analysis, was shown to correlate with the presence of a polypeptide with an apparent molecular weight of 32,000. Furthermore, a region between the repressor gene and the other genes of the operon was identified which, when carried on multicopy plasmids, resulted in expression of the operon in the absence of any exogenous induction. This'region may represent an operator-type element capable of titrating repressor molecules away from chromosomal operator, allowing transcription of the operon in the absence of induction.Metabolism of lactose by Staphylococcus aureus is initiated with the uptake of the disaccharide by the phosphoenolpyruvate sugar:phosphotransferase system (20,(28)(29)(30). The intracellular lactose-phosphate is then cleaved into glucose and galactose 6-phosphate by the enzyme phospho--galactosidase (7, 20, 28). The two P-galactoside-specific components of the phosphotransferase system, enzyme IIlac and factor Ii1ac, as well as phospho-p-galactosidase, compose part of a lactose-inducible operon for which galactose 6-phosphate is the actual intracellular inducer (20). Our laboratory has reported the molecular cloning of lacG, the gene for phospho-p-galactosidase (7), and the genes for factor Ill1ac (lacF) and enzyme Iliac (lacE) have also been identified (F. Breidt, W. Hengstenberg, U. Finkeldei, and G. C. Stewart, J. Biol. Chem., in press). The genetic arrangement of these genes is as follows: 5'-lacF-lacE-lacG-3'.The expression of the lac genes has been shown to be induced with the addition of lactose or galactose to the culture medium. The system is also subject to catabolite repression, although cyclic AMP, at least in physiologically significant concentrations, has not been detected in S. aureus (6; unpublished data).The lactose operon appears to be analogous to other catabolic operons by being negatively controlled. The presence of a repressor molecule for the operon has been alluded to by other investigators (17,20). Mutations resulting in a lactose constitutive phenotype have been shown to be tightly linked to the lac structural genes (7,20

mentioning

confidence: 99%

Cloning and characterization of the repressor gene of the Staphylococcus aureus lactose operon

Oskouian

Stewart

1987

“…Protoplast fusions, transformations with high-molecularweight transforming DNA, and analysis of recombinants were performed as described by Stahl and Pattee (35). Sensitivity or resistance to MNNG (the Ngr phenotype) was scored on MNNG agar.…”

Section: Methodsmentioning

confidence: 99%

“…The strains of S. aureus, Bacillus subtilis, and E. coli used in this study are described in Table 1. All cultures were maintained as described previously (35).…”

Section: Methodsmentioning

confidence: 99%

“…AP endonuclease activity was also assayed by measuring the release of acidsoluble material from depurinated DNA as described by Verly and Rassart (41,42 protoplast fusion (35) were used in attempts to determine the approximate chromosomal location of the Rec-determinant. Protoplast fusions between strains ISP1185 and ISP1171 (Table 1) showed that the Ngr phenotype preferentially segregated with the Thr phenotype, suggesting that the Ngrs determinant was located near thrB.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Characterization and genetic mapping of a mutation affecting apurinic endonuclease activity in Staphylococcus aureus

Tam¹,

Pattee²

1986

Protoplast fusion between the Rec-mutant RN981 (L. Wyman, R. V. Goering, and R. P. Novick, Genetics 76:681-702, 1974) of Staphylococcus aureus NCTC 8325 and a Rec+ NCTC 8325 derivative yielded Rec+ recombinants that exhibited the increased sensitivity to N-methyl-N'-nitro-N-nitrosoguanidine characteristic of RN981. Transformation analyses identified a specific mutation, designated ngr-374, that was responsible not only for N-methyl-N'-nitro-N-nitrosoguanidine sensitivity, but also sensitivity to methyl methanesulfonate, ethyl methanesulfonate, nitrous acid, and UV irradiation. However, ngr-374-carrying recombinants shotved no significant increase in their sensitivity to mitomycin C or 4-nitroquinoline 1-oxide and were unaffected in recombination proficiency. In vitro assays showed that ngr-374-carrying strains had lower apurinic/apyrimidinic endonuclease activities than the wild type. The chromosomal locus occupied by ngr-374 was shown to exist in the gene order fl(Chr::TnSSl)40-ngr-374-thrBl06. In E. coli, three endonucleases with AP endonuclease activity have been characterized. Exonuclease III is responsible for 90% of the AP endonucleolytic activity in E. coli, and endonucleases III and IV account for the remainder (13,22,44). Generally, exonuclease III-deficient mutants, which carry the xthA mutation, are sensitive to methyl methanesulfonate (MMS) and nitrous acid, but are unchanged in their sensitivity to UV irradiation (4,8,23). Although these mutants lack the major AP endonuclease, they show no increase in their spontaneous mutation rate (49). Apparently, the minor AP endonucleases, endonucleases III and IV, supply sufficient AP endonucleolytic activity to repair the spontaneously occurring AP sites; however, they are unable to manage the increased number of AP sites formed with MMS or nitrous acid treatments (22,44,46).In this report, we describe ngr-374, a mutation impairing AP endonuclease activity and originating in the recombination-deficient (Rec-) mutant RN981 (48) acid. ngr-374-carrying strains were also sensitive to UV irradiation, suggesting that S. aureus may have an AP endonuclease-dependent DNA repair mechanism for UVgenerated DNA lesions. MATERIALS AND METHODSBacterial strains. The strains of S. aureus, Bacillus subtilis, and E. coli used in this study are described in Table 1. All cultures were maintained as described previously (35).Culture media. All commercially available dehydrated culture media were supplemented with thymine (20 ,ug/ml) and adenine, guanine, cytosine, and uracil (5 ,ug/ml each). The composition of complete defined synthetic agar (31) was modified by omitting the appropriate amino acids, purines, and pyrimidines as needed to score and select nutritional markers (2,29,30). N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG) agar was prepared by adding 5 ,ug of MNNG (Sigma Chemical Co.), freshly dissolved in absolute methanol, per ml to autoclaved, cooled (45°C) Trypticase soy agar (TSA; BBL Microbiology Systems) rehydrated in 100 mM KH2PO4, pH 6.0 (10). MNNG agar was used on...

“…Protoplast fusion (46,47), transformation (4,37,39,46,47), and transductional analyses (44) of TnS51 chromosomal insertions have contributed to our understanding of the genomic organization of Staphylococcus aureus NCTC 8325. Diverse chromosomal insertions of TnS51, either as silent insertions or accompanied by the phenotypes of insertionally interrupted genes, have been used to generate a chromosome map.…”

mentioning

confidence: 99%

Transfer of the conjugal tetracycline resistance transposon Tn916 from Streptococcus faecalis to Staphylococcus aureus and identification of some insertion sites in the staphylococcal chromosome

Jones¹,

Yost²,

Pattee³

1987

The Streptococcus faecalis pheromone-dependent conjugative plasmid pAD1::Tn916 and the membrane ifiter-dependent conjugative plasmid pPD5::Tn916 were used to introduce Tn916 into Staphylococcus aureus by intergeneric protoplast fusions and intergeneric membrane-filter matings. In recombinants obtained by protoplast fusion where no plasmid DNA could be detected, tetracycline resistance resulted from transposition of Tn916 from pADi to the S. aureus chromosome. Transformation analyses showed that S. aureus Tn916 chromosomal insertions occurred near pig, ilv, uraA, tyrB, fus, ala, and the trp operon. DNA hybridization analyses of EcoRI-and HindIII-digested chromosomal DNAs confirmed the diversity of chromosomal sites involved and demonstrated that the inserts were Tn916 insertions rather than integrations of all or part of pAD1::Tn916. Both pAD1::Tn916 and pPD5::Tn916 were transferred to S. aureus by membrane-filter matings. These plasmids remained intact and expressed tetracycline resistance in S. aureus. S. aureus strains carrying pAD1::Tn916, but not a chromosomal insert of Tn916, and any one of several conjugal gentamicinresistance plasmids lost their ability to serve as conjugal donors of the gentamicin-resistance plasmids.