Biomedical subjects
Publications and source records attributed to W D Lawton.
An F-lac plasmid from Escherichia coli was transferred to virulent Yersinia pestis, resulting in the repression of virulence. The Y. pestis F-lac clones retained all of the known virulence traits but were avirulent and calcium independent. Every lac segregant derived from the F-lac clones was fully virulent and calcium dependent.
The Gono Gen (Micro-Media Systems, Inc., Potomac, Md.) coagglutination test was compared with the sugar utilization test and with a direct fluorescent antibody test for confirmation of Neisseria gonorrhoeae. Of 110 gonococcal clinical isolates, 109 were positive by the Gono Gen test. Of 57 nongonococcal gram-negative diplococci, all were negative by the Gono Gen test. We conclude that the Gono Gen test is sensitive and highly specific and provides a rapid method for the confirmation of N. gonorrhoeae.
In 100 clinical specimens containing Neisseria gonorrhoeae, commercially prepared New York City medium in JEMBEC plates was superior to Martin-Lewis medium for recovery of gonococci in 49 specimens, equal to Martin-Lewis medium in 43 specimens, and less effective in 8 specimens.
Seventeen apparently unrelated isolates of Neisseria gonorrhoeae out of 2,123 tested produced a diffusible growth-inhibitory substance against other gonococci. The inhibitor was destroyed by trypsin, not blocked by bovine serum albumin, and not soluble in chloroform-methanol; each isolate was resistant to the inhibitor it produced. Thus, the substance differs from previously described gonococcal inhibitors, and since it fits the description of a bacteriocin we designated it gonocin. The use of gonocin for typing was complicated by the observation that susceptibility to gonocin appears to depend on the gonococcal colony type.
A study of 27 clinical specimens from which Neisseria gonorrhoeae and Candida albicans were isolated simultaneously indicated that 44% of the gonococcal isolates were resistant to inhibition by the C. albicans with which they were found and an additional 33% were totally resistant to inhibition by all C. albicans tested. All 27 C. albicans showed inhibitory activity against standard indicator strains of N. gonorrhoeae.
Of 171,089 mailed specimens examined for Neisseria gonorrhoeae, 88% were submitted by family planning clinics, physicians in private practice, and other non-VD clinics. To process a large volume of specimen bottles, we developed a swab-oxidase test and demonstrated it to be reliable for the initial screening of all specimens. Our protocol permitted the isolation and confirmation by sugar fermentation of 95% of all presumed N. gonorrhoeae cultures within 1 or 2 days after growth was observed. The results obtained over a 2-year period indicate that the positivity rate is directly related to the lag period between specimen collection and examination. There was no adverse effect of cold weather on the positivity rate, but, irrespective of the season, 3 to 9% of the positive specimens required incubation for 3 days before gonococcal growth appeared.
When Candida albicans is present on Transgrow specimens, Neisseria gonorrhoeae is detected less frequently or else can be seen in Gram stains but cannot be readily cultured. When C. albicans and N. gonorrhoeae are grown together on Transgrow, the gonococcal cells die off much more readily than N. gonorrhoeae grown on Transgrow alone. By use of a cross-streaking technique on agar plates, it has been demonstrated that C. albicans produces a soluble substance inhibitory to N. gonorrhoeae, although not to other microorganisms tested. Preliminary results indicate that this inhibitory factor can be extracted by the use of tertiary butanol. Since approximately one-third of the Transgrow specimens with growth contains yeasts, of which C. albicans is by far the most frequent, this factor presents an important complication in the diagnosis of gonorrhea in women.
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A strain of Pasteurella pestis, harboring the F'Cm plasmid from Escherichia coli, was able to donate its chromosome to auxotrophic recipient strains of P. pestis. The frequency of gene transfer in P. pestis was approximately 10(-6) per donor cell, 100 times less efficient than gene transfer in Pasteurella pseudotuberculosis, but efficient enough to determine entry times for the markers histidine, threonine, and tryptophan and to show linkage to the markers arginine and pigmentation. An attempt to extend the conjugation system to different serotypes of P. pseudotuberculosis and to Yersinia enterocolitica did not succeed.
Bacteriophage P1 CM can convert Pasteurella pestis or P. pseudotuberculosis to chloramphenicol resistance and phage restriction, but no viable phage was induced from converted Pasteurella strains.
Pasteurella pseudotuberculosis, containing the Escherichia coli plasmid F'lac, transferred its chromosome in an oriented manner to each of five multiply auxotrophic strains of P. pseudotuberculosis. In a mating system containing gelatin, glucose, and phosphate buffer, a maximum of 0.02% of the donor cells transferred lead markers. The donor population was counterselected with nalidixic acid. We established the entry time of seven markers as follows: proline (11 min); arginine (14 min); histidine (14 min); threonine (25 min); lysine (50 min); tyrosine (67 min); and tryptophan (77 min). However, an analysis of the inheritance of unselected markers did not support the simplest assumption that the chromosome was transferred as Origin... pro... arg his... thr... lys... tyr... trp.... The markers common to all five recipients, arg and his, were closely linked, but of the five other markers, each unique to a different recipient strain, only trp was linked to arg and his. Our data suggest that the Pasteurella chromosome is transferred in more than one linkage group.
When either the F' lac or the F'Cm plasmid was transferred from Escherichia coli into Pasteurella pseudotuberculosis, the P. pseudotuberculosis (F') strains isolated formed plaques with both ribonucleic acid (RNA)-containing and deoxyribonucleic acid-containing male-specific phages. In contrast, strains of P. pestis harboring E. coli (F') plasmids did not form plaques with male-specific phages, although such strains permitted limited multiplication of phage MS2. The adsorption and burst size of MS2 were approximately the same in both species of Pasteurella, but the per cent of adsorbed MS2 that produced infective centers was much lower in P. pestis than it was in P. pseudotuberculosis. By use of a sib-selection technique of P. pestis (F') cells, we isolated a single clone that could form MS2 plaques. (32)P-labeled MS2 adsorbed equally to and its RNA penetrated equally into both the typical MS2-nonpermissive P. pestis cells and the MS2-permissive P. pestis cells. No host modification occurred after growth of MS2 in Pasteurella. Our data suggest that typical strains of P. pestis inhibit the intracellular development of phage MS2.
Several Bacillus subtilis W-23 auxotrophs were unable to produce wild-type pigment normally on minimal agars supplemented sufficiently for growth. This offers a reliable means for scoring genotypes.
When donor and recipient strains of Escherichia coli were added to columns containing Cellex-P (a cation-exchange cellulose), more than 80% of the female cells passed through the column but only 11% or less of the male cells were eluted. However, when donor strains were blended before their addition to the column, the majority of these cells were eluted. These results indicated that the filamentous appendages termed F pili (which are removed by blending) were the structures responsible for the adherence of donor cells to the cellulose.
The requirements and characteristics of Pasteurella novicida transformations in liquid suspensions were studied. Transformation frequencies of 0.1 to 0.3% were routinely obtained when recipient cells were harvested from 16-hr agar plates and higher than 1% when logarithmic-phase broth-grown cells were used. Calcium ions were essential for transformations. The deoxyribonucleic acid dose response curve, kinetics of transformation, and pH optimum for transformations were similar to those of other bacterial transformation systems. The genetic relatedness of P. novicida and P. tularensis was established by transforming P. novicida mutants with deoxyribonucleic acid extracted from P. tularensis.
Bacteriophage SP-15, a large generalized transducing phage of Bacillus, was compared with phages PBS-1 and SP-10 for the ability to cotransduce pairs of genetic markers exhibiting different degrees of linkage. When auxotrophs of B. subtilis W-23 were used as recipients, SP-15 and PBS-1 effected a much higher frequency of cotransduction than did SP-10 with markers that were not closely linked. With more closely linked loci, the differences were not as great. SP-15 cotransduced linked markers at a higher mean frequency than PBS-1, suggesting that SP-15 is able to transfer a larger fragment of the Bacillus genome than any phage heretofore described. The frequency of the joint transfer of genetic markers in B. licheniformis was lower via transforming deoxyribonucleic acid than by transduction with phage SP-10. The availability of three procedures for genetic exchange-transduction by SP-15 and SP-10 as well as transformation-each of which reveals a different degree of linkage, makes B. licheniformis 9945A especially amenable to genetic analysis.
Deoxyribonucleic acid from a streptomycin-resistant mutant of Pasteurella novicida transformed portions of P. novicida streptomycin-sensitive populations to streptomycin-resistant. Similarly, mutants auxotrophic for tryptophan or purine biosynthesis were also transformed to nutritional independence.
Phage H, thought to be specific for Pasteurella pestis, was shown to plate efficiently on F(-) strains of Escherichia coli but not on F(+), F', or Hfr strains. The phage was adsorbed rapidly to F(-) strains but was not adsorbed to strains carrying F. Comparison with seven other reported female-specific phages showed that, although phage H was similar to the other phages in some characteristics, the exceptionally low efficiency of plating (<10(-9)) on F-containing cells makes phage H a particularly useful female-specific phage.