Biomedical subjects
Publications and source records attributed to T Edlund.
We have identified a new control or attenuator region in the chromosomal beta-lactamase operon of Escherichia coli. A single base alteration within its attenuator led to a loss in the cell's ability to coordinate its content of beta-lactamase with growth rate. We suggest a mechanism through which this mode of regulation operates.
Secondary attachment site lambda-lysogens were isolated in an Escherichia coli strain carrying multiple tandem 9.8 kb repeats. The repeat carried the structural gene for chromosomal beta-lactamase, ampC. One lysogen produced lysates with amp-transducing activity. Three types of phages with different densities were obtained from this lysogen. The one with the lowest density was found to be a helper lambda cI857S7 phage. The other two phage showed identical restriction endonuclease fragmentation patterns. The difference in density was due to the presence or absence of phage tail. In lambda damp the right cohesive end segment was deleted in a random fashion with the majority ending between 81.0% and 82.4% of lambda. The chromosomal segment of lambda damp was most likely located at the lambda attachment site. The lambda damp DNA was compared to that of ColE1 hybrid carrying the chromosomal amp segment and a ColE1 hybrid carrying the same 9.8 kb amp repeat as the lysogen from which lambda damp was isolated. It was found that the chromosomal part of lambda damp constituted 9.8 kb, i.e. the size of one repeat. Moreover, the novel joint between adjacent repeats was present. In a lambda attB-deleted E. coli K-12 strain, lysogenic for lambda damp, highly ampicillin-resistant mutants occurred at an exceedingly high frequency. They were found to contain in the chromosome an amplified 9.8 kb repeat. This suggested that integration of the novel joint for lambda damp into the amp region gives rise to an amplifiable duplication. In E. coli lysogenized for lambda damp at lambda attB highly ampicillin-resistant clones were also found at a high frequency. These clones carried multiple tandem repeats of lambda damp DNA, each with an intact right end segment.
Hybrid plasmids carrying the ampC gene of Escherichia coli K-12 that codes for the chromosomal beta-lactamase were physically studied. The ampC gene was mapped to a deoxyribonucleic acid segment encompassing 1,370 base pairs. The mapping was facilitated by the isolation of a plasmid carrying an insertion of the transposable element gamma delta (gamma delta) close to ampC. The ampA1 mutation, which increases the expression of ampC by a factor of about 20, was localized to a 370-base pair segment of the 1,370-base pair deoxyribonucleic acid segment that contains the ampC gene. Using a minicell protein labeling system, it was seen that plasmids carrying either ampA+, ampC, or ampA1 and ampC coded for a 36,000-dalton protein which comigrated with purified chromosomal beta-lactamase. In cells carrying plasmids that bore the ampA1 allele, the production of this protein was greater. In addition, a protein with a slightly higher molecular weight (38,000) was expressed by both ampA+ ampC and ampA1 ampC plasmids in this protein labeling system. This protein might represent a precursor form of chromosomal beta-lactamasee. From E. coli K-12 strains carrying the ampA1 allele, second-step mutants were isolated that hyperproduced chromosomal beta-lactamase. By reciprocal recombination, plasmid derivatives were isolated that carried these mutations. Two second-step regulatory mutations mapped within the same 370-base pair region as ampA1. This piece of deoxyribonucleic acid therefore contains ampA, a control sequence region for ampC.
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A ColEl hybrid plasmid, pNUl, carrying the amp operon coding for chromosomal beta-lactamase was isolated from the Clarke and Carbon collection and physically mapped. The physical location of ampC within this plasmid was further deduced by in vitro cloning. By reciprocal recombination between pNUl and chromosome of two unstable beta-lactamase hyperproducing E. coli K-12 mutants a large plasmid from each mutant was obtained. The respective plasmid was physically mapped and found to contain five and two repeated DNA segments. The repetitions within each plasmid were equal in size, 9,800 bp and 11,900 bp respectively and were organized in tandem. The end points of the repeats were different in the two plasmids but shared a DNA segment carrying the ampC gene. The chromosomal DNA of the beta-lactamase hyperproducing E. coli mutants were found to contain an amplified DNA segment equal in size to the repeated unit found in the respective plasmid. The data shows that up to 10 identical repeats organized in tandem can be generated by a normal mutation frequency in E. coli.
Escherichia coli K-12 ampicillin-resistant mutants hyperproducing chromosomal beta-lactamase arose spontaneously from strains carrying ampA1 ampC(+). Such mutants were found even in a recA background. Two Amp(r)-100 strains were analyzed genetically. The Amp(r)-100 resistance level of both strains could be transduced by direct selection for ampicillin resistance. Several classes of ampicillin-resistant transductants were found that differed from one another in the beta-lactamase activity and the ampicillin resistance mediated by an ampA1 ampC(+)-carrying strain. The data suggested that beta-lactamase hyperproduction was due to repetitions of the chromosomal amp genes. The size of the repeated region was calculated from cotransduction estimates, using the formula of Wu (Genetics 54:405-410, 1966), and was found to be about 1 min in one strain and 1.5 min in the other. Second-step Amp(r)-400 mutants were isolated from an Amp(r)-100 strain. The resistance of these mutants was apparently also due to repetitions, each mediating a resistance to about 10 mug/ml. Mutants of wild-type strains that were moderately resistant to ampicillin also gave rise to intermediate-resistance classes, suggesting repetitions of the wild-type amp alleles. F' factors hyperproducing chromosomal beta-lactamase by gene repetitions were constructed. They mediated levels of ampicillin resistance comparable to that of naturally occurring resistance plasmids. The expression of beta-lactamase hyperproduction was not affected by the presence of ampA and ampC alleles in trans and did not act in trans on the other alleles.
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Mutants of Bacillus thuringiensis lacking either beta-exotoxin or gamma-endotoxin were compared for their virulence using pupae of a giant silk moth. Known doses of viable log-phase bacteria were injected, and the response was followed as the number of viable bacteria in the hemolymph. The results obtained imply that, in the system used, neither the beta-exotoxin nor the gamma-endotoxin and the sporeforming ability are of importance for virulence. Results with sterile culture filtrate from B. thuringiensis have given evidence for the production of two inhibitors, A and B, which interfere with the humoral defense system in pupae of Hyalophora cecropia. Inhibitor A, which blocked the lysis of Escherichia coli,was precipitated by trichloroacetic acid and sensitive to heating. Inhibitor B, which blocked the killing of Bacillus cereus, was soluble in trichloroacetic acid and resistant to 90 degrees C for 5 min. Both inhibitors are believed to contribute to the insecticidal nature of B. thuringiensis.
DNA sequences containing the 5'-flanking regions of the insulin and chymotrypsin genes were linked to the coding sequence of the chloramphenicol acetyltransferase (CAT) gene. The insulin gene recombinant elicits preferential expression of CAT activity when introduced into cells producing insulin; similarly, the chymotrypsin gene recombinant elicits preferential expression in chymotrypsin-producing cells. Sequences located upstream of previously defined transcriptional control elements are essential for efficient expression in both cases.