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Biomedical subjects

J K Setlow

Publications and source records attributed to J K Setlow.

At least 19 recordsLinked to original sources
PubMed1992

Mutation and killing of Escherichia coli expressing a cloned Bacillus subtilis gene whose product alters DNA conformation.

Expression of the Bacillus subtilis gene coding for SspC, a small, acid-soluble protein, caused both killing and mutation in a number of Escherichia coli B and K-12 strains. SspC was previously shown to bind E. coli DNA in vivo, and in vitro this protein binds DNA and converts it into an A-like conformation. Analysis of revertants of nonsense mutations showed that SspC caused single-base changes, and a greater proportion of these were at A-T base pairs. Mutation in the recA gene abolished the induction of mutations upon synthesis of SspC, but the killing was only slightly greater than in RecA+ cells. Mutations in the umuC and umuD genes eliminated most of the mutagenic effect of SspC but not the killing, while the lexA mutation increased mutagenesis but did not appreciably affect the killing. Since there was neither killing nor mutation of E. coli after synthesis of a mutant SspC which does not bind DNA, it appears likely that the binding of wild-type SspC to DNA, with the attendant conformational change, was responsible for the killing and mutation. A strain containing the B. subtilis gene that is constitutive for the RecA protein at 42 degrees C showed a lower frequency of mutation when that temperature was used to induce the RecA protein than when the temperature was 30 degrees C, where the RecA level is low, suggesting that at the elevated temperature the high RecA level could be inhibiting binding of the B. subtilis protein to DNA.

Bacillus subtilis↗
PubMed1989

Characterization of a multiple antibiotic resistance plasmid from Haemophilus ducreyi.

Plasmid pLS88 from a clinical isolate of Haemophilus ducreyi encoded resistance determinants for sulfonamides and streptomycin related to those of RSF1010 and for kanamycin related to Tn903 but lacked the inverted repeats of the transposon. Its host range included Haemophilus influenzae, Actinobacillus pleuropneumoniae, and Escherichia coli; and it was compatible with pDM2 and RSF1010.

Autoradiography↗
PubMed1989

A plasmid carrying mucA and mucB genes from pKM101 in Haemophilus influenzae and Escherichia coli.

The plasmid pMucAMucB, constructed from the Haemophilus influenzae vector pDM2, and a similar plasmid, constructed from pBR322, increased the survival after UV irradiation of Escherichia coli AB1157 with the umu-36 mutation and also caused UV-induced mutation in the E. coli strain. In H. influenzae, pMucAMucB caused a small but reproducible increase in survival after UV irradiation in wild-type cells and in a rec-1 mutant, but there was no increase in spontaneous mutation in the wild type or in the rec-1 mutant and no UV-induced mutation.

Dose-Response Relationship, Radiation↗
PubMed1988

Characterization of the rec-1 gene of Haemophilus influenzae and behavior of the gene in Escherichia coli.

The rec-1 gene of Haemophilus influenzae was cloned into a shuttle vector that replicates in Escherichia coli as well as in H. influenzae. The plasmid, called pRec1, complemented the defects of a rec-1 mutant in repair of UV damage, transformation, and ability of prophage to be induced by UV radiation. Although UV resistance and recombination were caused by pRec1 in E. coli recA mutants, UV induction of lambda and UV mutagenesis were not. We suggest that the ability of the H. influenzae Rec-1 protein to cause cleavage of repressors but not the recombinase function differs from that of the E. coli RecA protein.

Bacterial Proteins↗
PubMed1986

Plasmid-to-plasmid recombination in Haemophilus influenzae.

No recombination between plasmids was observed after conjugal transfer of a plasmid into a cell carrying another plasmid. Two types of such recombination took place after transformation, one type being Rec+ dependent and suggesting a preferred site of recombination. The other much rarer type was at least partially Rec+ independent.

Chloramphenicol↗
PubMed1986

Plasmid-to-chromosome gene transfer in Haemophilus influenza during growth.

A low level of recombination between homologous regions of plasmids and the host chromosome occurred during cell growth. The plasmids contained antibiotic resistance markers on the homologous regions which were only expressed when they were integrated into the chromosome. Such recombination took place in Rec+ rec-2 mutants but not in rec-1 mutants.

Chloramphenicol↗
PubMed1985

Genes from plasmid pKM101 in Haemophilus influenzae: separation of functions of mucA and mucB.

Haemophilus influenzae, normally not mutable by UV, became UV mutable with a recombinant plasmid insertion. A 7.8-kilobase-pair (kbp) fragment of the plasmid pKM101 containing the mucA and mucB genes was ligated to the shuttle vector pDM2, and a Rec- strain of H. influenzae was transformed with the ligated mixture. All of the transformants, unlike the parent Rec- strain, were resistant to UV, could carry out postreplication repair and Weigle reactivation, showed greatly increased spontaneous mutation, and contained a plasmid carrying an insert of only 1.2 rather than 7.8 kbp. This plasmid in a umuC mutant strain of Escherichia coli complemented a pKM101 derivative lacking mucA function but with an intact mucB gene, although there was no complementation with a mucA+ mucB- plasmid, suggesting that the newly constructed plasmid coded for the mucA protein; this is in accord with the restriction analysis and hybridization between the plasmid and a probe containing all of the mucA gene but only a small fraction of mucB. When one of the H. influenzae Rec- transformants lost the plasmid, the resistance to UV was retained but the high spontaneous mutation and UV mutability were not. The fact that there was hybridization between the chromosome of the "cured" strain and a probe containing both muc genes but none when almost no mucB was present suggested that at least part of the mucB gene had been integrated into the Rec- chromosome. Five different postreplication repair-proficient strains became UV mutable and had high spontaneous mutation rates caused by the putative mucA plasmid, indicating that these strains already possessed a chromosomal equivalent of the mucB gene.

Cloning, Molecular↗
PubMed1985

Differential behavior of plasmids containing chromosomal DNA insertions of various sizes during transformation and conjugation in Haemophilus influenzae.

Plasmids with chromosomal insertions were constructed by removal of a 1.1-kilobase-pair piece from the 9.8-kilobase-pair vector plasmid pDM2 by EcoRI digestion and inserting in its place various lengths of chromosomal DNA (1.7, 3.4, and 9.0 kilobase pairs) coding for resistance to novobiocin. A fourth plasmid was constructed by insertion of the largest piece of chromosomal DNA into the SmaI site of pDM2. The plasmids without inserts were taken up poorly by competent cells and thus were considered not to contain specific DNA uptake sites. The presence of even the smallest insert of chromosomal DNA caused a large increase in transformation of Rec+ and Rec- strains. The frequency of plasmid establishment in Rec+ cells by transformation increased exponentially with increasing insert size, but in Rec- cells there was less transformation by the larger plasmids. Conjugal transfer of these plasmids was carried out with the 35-kilobase-pair mobilizing plasmid pHD147. The frequency of establishment of plasmids by this method not only was not markedly affected by the presence of the insertions, but also decreased somewhat with increase in insert size and was independent of rec-1 and rec-2 genes. Recombination between plasmid and chromosome was readily detected after transformation, but could not be detected after transconjugation even when the recipient cells were Rec+ and made competent. These data suggested that there is a special processing of plasmid DNA that enters the competent cells in transformation that makes possible recombination of homologous regions of the plasmid with the chromosome and pairing with the chromosome that aids plasmid establishment.

Ampicillin↗
PubMed1985

Mutations affecting gyrase in Haemophilus influenzae.

Mutants separately resistant to novobiocin, coumermycin, nalidixic acid, and oxolinic acid contained gyrase activity as measured in vitro that was resistant to the antibiotics, indicating that the mutations represented structural alterations of the enzyme. One Novr mutant contained an altered B subunit of the enzyme, as judged by the ability of a plasmid, pNov1, containing the mutation to complement a temperature-sensitive gyrase B mutation in Escherichia coli and to cause novobiocin resistance in that strain. Three other Novr mutations did not confer antibiotic resistance to the gyrase but appeared to increase the amount of active enzyme in the cell. One of these, novB1, could only act in cis, whereas a new mutation, novC, could act in trans. An RNA polymerase mutation partially substituted for the novB1 mutation, suggesting that novB1 may be a mutation in a promoter region for the B subunit gene. Growth responses of strains containing various combinations of mutations on plasmids or on the chromosome indicated that low-level resistance to novobiocin or coumermycin may have resulted from multiple copies of wild-type genes coding for the gyrase B subunit, whereas high-level resistance required a structural change in the gyrase B gene and was also dependent on alteration in a regulatory region. When there was mismatch at the novB locus, with the novB1 mutation either on a plasmid or the chromosome, and the corresponding wild-type gene present in trans, chromosome to plasmid recombination during transformation was much higher than when the genes matched, probably because plasmid to chromosome recombination, eliminating the plasmid, was inhibited by the mismatch.

Aminocoumarins↗
PubMed1985

Gyrase activity and number of copies of the gyrase B subunit gene in Haemophilus influenzae.

Gyrase activities in extracts of various strains of Haemophilus influenzae can differ by more than an order of magnitude (J. K. Setlow, E. Cabrera-Juárez, W. L. Albritton, D. Spikes, and A. Mutschler, J. Bacteriol. 164:525-534, 1985). Measurements of in vitro activity and copy number indicated that most of these differences arose from variations in the number of copies of the gene for the gyrase B subunit, with some strains containing multicopy plasmids coding for that subunit. The quantitative relationship between gyrase and copy number depended on the mutations in the plasmids and in the host. The gyrase and copy number were considerably lower in plasmid-bearing strains carrying the prophage HP1c1. Two mutations affecting gyrase that are apparently regulatory caused an increase in gyrase without a concomitant increase in copy number. The possibility that the in vivo gyrase activity did not reflect the in vitro data was explored by measurement of alkaline phosphatase and ATPase activity in the extracts. Alkaline phosphatase activity increased with increasing gyrase activity measured in vitro, but ATPase activity did not. We conclude that extra supercoiling enhanced transcription of the alkaline phosphatase gene but not the ATPase gene and that it is unlikely that there is much discrepancy between gyrase activity assayed in vitro and the activity in the cell.

Adenosine Triphosphatases↗
PubMed1984

Heterospecific transformation in the genus Haemophilus.

The relationship between nine Haemophilus species and Haemophilus influenzae was studied by DNA-DNA hybridization, by transformation of H. influenzae to streptomycin resistance with heterospecific DNA, by competition of heterospecific DNA for transformation by homospecific DNA and by the lethal effect of heterospecific DNA on competent H. influenzae. H. parainfluenzae, H. parasuis, and H. aegyptius DNA transformed at more than 10% efficiency when compared to homologous transformation, but only H. aegyptius demonstrated, by hybridization, a relative binding ratio of more than 80%. H. aphrophilus and H. paraphrophilus DNA demonstrated a relative binding ratio of less than 30% and transformed H. influenzae at only 10(-5) the efficiency of homologous DNA, but they competed for H. influenzae transformation as well as or better than homospecific DNA. The data indicated that in some of the species sharing the common ecological habitat of the mammalian respiratory tract, sequences necessary for competition and efficient uptake into H. influenzae are present in large numbers in their DNAs, which nevertheless have little overall homology with H. influenzae DNA.

DNA, Bacterial↗
PubMed1984

Prophage induction in Haemophilus influenzae and its relationship to mutation by chemical and physical agents.

It is known that UV, X-rays, MMC and MMS are not mutagenic for H. influenzae, whereas HZ, EMS and MNNG are potent mutagens for this bacterium. All of these agents, however, are known to be both mutagenic and able to induce prophage in E. coli. We report here that all the agents except HZ induce prophage in H. influenzae, and EMS even induces in the recombination-defective recl mutant, which is non-inducible by UV, MMC, MNNG and MMS. MMS did not cause single-strand breaks or gaps in DNA synthesized after treatment of H. influenzae, but EMS and MNNG produced them. EMS caused more breaks in DNA synthesized before treatment than in that synthesized after treatment. On the other hand we did observe such breaks or gaps induced in E. coli in DNA synthesized posttreatment by EMS as well as by MMS and MNNG, at comparable survival levels.

Bacteriophages↗
PubMed1984

Plasmid containing a DNA ligase gene from Haemophilus influenzae.

A ligase gene from Haemophilus influenzae was cloned into the shuttle vector pDM2 . Although the plasmid did not affect X-ray sensitivity, it caused an increase in UV sensitivity of the wild-type but not excision-defective H. influenzae and a decrease in UV sensitivity of the rec-1 mutant.

Cloning, Molecular↗
PubMed1984

Loss of plasmids containing cloned inserts coding for novobiocin resistance or novobiocin sensitivity in Haemophilus influenzae.

Plasmids pNov1 and pNov1s , coding for resistance and sensitivity to novobiocin, respectively, were readily lost from wild-type Haemophilus influenzae but retained in a strain lacking an inducible defective prophage. The plasmid loss could be partly or wholly eliminated by a low-copy-number mutation in the plasmid or by the presence of certain antibiotic resistance markers in the host chromosome. Release of both phage HP1c1 , measured by plaque assay, and defective phage, measured by electron microscopy, was increased when the plasmids were present. The frequency of recombination between pNov1 and the chromosome, causing the plasmid to be converted to pNov1s , could under some circumstances be decreased from the normal 60 to 70% to below 10% by the presence of a kanamycin resistance marker in the chromosome. This suggested that a gene product coded for by the plasmid, the expression of which was affected by the kanamycin resistance marker, was responsible for the high recombination frequency. Evidence was obtained from in vitro experiments that the gene product was a gyrase.

Anaerobiosis↗
PubMed1984

Mechanism of acquisition of chromosomal markers by plasmids in Haemophilus influenzae.

The hybrid plasmid pNov1 readily acquired genetic information from the chromosome of wild-type, but not rec-2, cells. Most of the recombination had taken place 1 h after entrance of the plasmid into the cell, as judged by transformation of rec-2 by lysates made from wild-type cells exposed to pNov1. Measurement of physical transfer from radioactively labeled cellular DNA to plasmids recombining in wild-type cells failed, since there was little more radioactivity in plasmids from such cells than from labeled rec-2 recipients, in which no recombination took place. EcoRI digestion of pNov1 divided the DNA into a 1.7-kilobase-pair fragment containing the novobiocin resistance marker and a 13-kilobase-pair fragment containing all of the original vector and considerable portions homologous to the chromosome. Transformation by the large fragment alone resulted in a plasmid the size of the original pNov1. Our hypothesis to explain the data is that genetic transfer from chromosome to plasmid took place by a copy choice mechanism.

Base Sequence↗
PubMed1982

Novobiocin resistance marker in Haemophilus influenzae that is not expressed on a plasmid.

The plasmid pNov2, carrying a cloned chromosomal marker conferring resistance to at least 2.5 micrograms of novobiocin per ml, was constructed with a new Haemophilus influenzae cloning vehicle, pDM2. The novobiocin marker of pNov2 was not normally expressed, but in Rec+ cells approximately one in 10(4) cells in a culture of a transformant became novobiocin resistant, a frequency about four orders of magnitude higher than the spontaneous mutation frequency. Variants of such cells that had lost the plasmid were also novobiocin resistant. Since Rec- cultures bearing pNov2 showed novobiocin resistance only at the normal mutation frequency, we concluded that the Rec+ novobiocin-resistant transformants arose because of a rare recombination between plasmid and chromosome in which the chromosome acquired the novobiocin marker from the plasmid. Evidence is presented that novobiocin sensitivity is dominant over this particular novobiocin resistance marker.

Chloramphenicol↗