Promotion of bacteriophage induction and recombination by the cloned Bordetella pertussis recA gene is copy-number dependent.

Favre and coworkers (Favre et al., Biochimie 73:235-244, 1991) previously reported that the Bordetella pertussis recA gene present at high copy number could promote a low frequency of recombination, but not bacteriophage induction in Escherichia coli RecA- mutants. Reexamination of these phenotypes demonstrated that, in contrast to the previous study, when this gene is present at high copy number, it can stimulate a 2- to 4-log frequency of bacteriophage induction in the presence of mitomycin C, but no appreciable spontaneous induction. The cloned gene, whether it was present in high or low copy number, also promoted a low frequency of intrachromosomal recombination of two duplicated genes in Escherichia coli. These results suggest that a high concentration of the B. pertussis RecA protein is required to promote high-frequency mitomycin C-stimulated bacteriophage induction, but it facilitates intrachromosomal recombination at a very low frequency in E. coli RecA- mutants. The ability of the B. pertussis RecA protein to promote mitomycin C induction and its inability to appreciably stimulate spontaneous induction of bacteriophage suggest that this protein possesses a unique phenotype compared with other RecA proteins.

Construction and characterization of Bordetella pertussis RecA- mutants.

Antibiotic drug-resistance cassettes (DRCs) were used to insertionally inactivate the wild-type Bordetella pertussis recA gene cloned into a suicide vector. The mutant allele was mobilized by conjugal gene transfer from Escherichia coli strain SM10 into different genetic backgrounds of B. pertussis. Southern hybridization studies of one of these mutants showed that it contained a DRC integrated within a recA gene situated within a ClaI genomic DNA fragment. Selected mutants were assayed to quantify recombinational and DNA repair deficiencies. These mutants were shown to be highly sensitive to both chemically and physically induced DNA damage. Gene transfer studies of another RecA- mutant also indicated that it was defective in intergenic recombination. No difference in hemolytic activity or production of capsule was detected between the RecA- mutants and their corresponding wild-type strains. The results of this investigation corroborate previous studies with the cloned B. pertussis recA gene, and demonstrate that the expression of the B. pertussis recA gene in the original host promotes both DNA repair and recombination.

Correlation of teichoic acid D-alanyl esterification with the expression of methicillin resistance in Staphylococcus aureus.

Methicillin-susceptible and -resistant isolates of Staphylococcus aureus, including a teichoic acid-deficient mutant, were examined under varying physiological conditions for their degree of teichoic acid and lipoteichoic acid D-alanyl esterification. Methicillin-resistant strains grown in the presence of methicillin and NaCl possessed significantly decreased amounts of D-alanyl ester when compared with methicillin-susceptible isolates. These strains also exhibited reduced autolysis. An autoradiographic procedure was used to detect mutants, isolated by Tn917 and nitrosoguanidine mutagenesis of methicillin-susceptible strains, which were defective in D-alanyl ester formation. The quantitative uptake of D-[14C]alanine in these mutants was determined and the effect of methicillin on the growth and viability of each mutant was compared with the wild-type strain. S. aureus mutant strains, defective in the uptake and incorporation of D-alanine, were shown to exhibit slightly reduced autolysis and an enhanced expression of methicillin resistance.

Clinical evaluation of fitting presbyopic patients with contact lenses.

This clinical investigation was performed to determine the success of using a system designed as a flow chart to fit presbyopic patients with contact lenses. The value of this type of system is to provide practitioners with a simple step-by-step approach to fitting presbyopic patients and reducing the costs of several trial fitting sets. Patients were fit with monovision, simultaneous vision bifocal, alternating vision bifocal or diffractive bifocal contact lenses. The patients were fit first with monovision, and, if unsuccessful with this or any of the above lens designs, they proceeded to the next stage. Stage 2 used the simultaneous lens design, Stage 3 the alternating lens design and Stage 4 the diffractive lens design. Of the 22 patients enrolled in the study, 17 (77.3 percent) were successful through Stage 3 and 18 (81.8 percent) were successful through Stage 4. The first three stages are those proposed by Bausch & Lomb as the Presbyopic Fitting System. The Presbyopic Fitting System was found to be simple to follow and successful in fitting presbyopic patients.

Isolation and characterization of the recA gene of Bordetella pertussis.

This report describes the detection and cloning of the Bordetella pertussis recA gene. Escherichia coli clones having recombinant plasmids containing the B. pertussis recA gene were isolated by complementing an E. coli RecA- mutant's inability to survive in the presence of methylmethanesulphonate (MMS). This gene was shown to complement the deficiency of E. coli RecA- strains to tolerate the DNA-damaging effects of both a chemical agent and ultraviolet light (u.v.). Deletion mapping experiments localized the gene to a 2.5 kb StuI-EcoRI fragment, and expression of the gene in E. coli resulted in the production of a 40 kD protein. These data strongly suggest that a region of the B. pertussis chromosome that encodes RecA-like activity has been isolated and cloned.

Broad-host-range plasmid vector for the in vitro construction of transcriptional/translational lac fusions.

A broad-host-range plasmid was constructed that allows the in vitro formation of beta-galactosidase fusions. DNA from the photosynthetic bacterium Rhodopseudomonas sphaeroides was cloned into this plasmid and a number of R. sphaeroides isolates were recovered that had varying levels of beta-galactosidase activity. beta-galactosidase antigenic activity from the fusion strains could be localized immunologically in polypeptides with an Mr of 120 000 or greater. Expression of beta-galactosidase activity under control of fusion derivatives was either very low or nonexistent in Escherichia coli relative to R. sphaeroides, indicating that R. sphaeroides promoters or translational start signals function poorly in E. coli.

Plasmidless, photosynthetically incompetent mutants of Rhodospirillum rubrum.

Ethyl methanesulfonate rendered a high percentage of Rhodospirillum rubrum cells plasmidless and photosynthetically incompetent (Kuhl et al., J. Bacteriol. 156:737-742, 1983). By probing restriction endonuclease-digested chromosomal DNA from these plasmidless strains with 32P-labeled R. rubrum plasmid DNA, we showed that no homology exists between the plasmid and the chromosomal DNA of the mutant. Loss of the plasmid in all the nonphotosynthetic isolates was accompanied by the synthesis of spirilloxanthin under aerobic growth conditions, resistance to cycloserine and HgCl2, and loss of ability to grow fermentatively on fructose. Changes in both the protein and lipid composition of the membranes and the impaired uptake of 203HgCl2 in the plasmidless strains (compared with the wild type) suggest either that membrane modification occurs as a result of plasmid loss, accounting for several of the acquired phenotype characteristics of the cured strains, or that both membrane modification and plasmid loss are part of the same pleiotropic mutation.

Characterization of a Rhodospirillum rubrum plasmid: loss of photosynthetic growth in plasmidless strains.

A single plasmid of 55 kilobases was found in crude cell lysates of each of nine strains of Rhodospirillum rubrum. Restriction endonuclease analysis showed identical fragment patterns with a given nuclease for all plasmids except one, for which an additional EcoRI site was observed. Elimination of the plasmid required that the cells be passaged several times in 25 mM calcium-containing medium, followed by at least two passages under photosynthetic growth conditions in low-calcium medium before treatment with ethyl methanesulfonate. The resulting plasmidless mutants only grew aerobically and were all incapable of pigment formation and photosynthetic growth, suggesting that plasmid DNA is required for photosynthetic competence in R. rubrum.

Construction of a genetic map for Caulobacter crescentus.

RP4-mediated conjugation has been used to transfer large fragments of chromosomal material in Caulobacter crescentus. In this system, conjugation proceeds from multiple origins, and haploid recombinants are recovered at frequencies of 10(-6) and 10(-7) per donor cell. The data from five-factor crosses were subjected to computer-assisted crossover analyses as a rapid method to determine marker order. Using this information and data from additional two- and three-factor crosses mediated by RP4 or the generalized transducing bacteriophage phi Cr30, we constructed the first genetic map for C. crescentus.

Plasmid Profiles of Lactose-Negative and Proteinase-Deficient Mutants of Streptococcus lactis C10, ML(3), and M18.

Lactose- and proteinase-negative (Lac Prt) mutants of Streptococcus lactis C10, ML3, and M18 were isolated after treatment with ethidium bromide. The Lac Prt mutants of C10 were missing a 40-megadalton plasmid. A 33-megadalton plasmid was absent in the ML3 mutants, and the M18 variants lacked a 45-megadalton plasmid. The results suggest a linkage of these metabolic traits to the respective plasmids. The possible complexity of the interrelationship between lactose metabolism and proteinase activity is presented.

Chromosomal map location of the methicillin resistance determinant in Staphylococcus aureus.

Three-factor genetic crosses performed by transformation have shown that the methicillin resistance determinant of Staphylococcus aureus strain DU4916 (the mec-4916 marker) is linked to a novobiocin resistance (Novr) marker (nov-142) and mutational sites affecting pyrimidine (pyr-141), purine (pur-102), and histidine (hisG15) biosynthesis in S. aureus strain 8325. The linkage group thus defined is pyr-141-hisG15-nov-142-pur-102-mec-4916. Phage 80alpha previously propagated on a novobiocin-resistant, methicillin-sensitive (Mecs) 8325 strain was used to infect 21 novobiocin-sensitive, methicillin-resistant clinical isolates (including strain DU4916). Among the novobiocin-resistant transductants so obtained from each recipient, between 1 and 5% were methicillin sensitive (reflecting cotransduction of Novr and Mecs). These results are consistent with the genetic determinant of methicillin resistance having a single chromosomal locus in most, if not all, strains of S. aureus.