Physical mapping and expression of hybrid plasmids carrying chromosomal beta-lactamase genes of Escherichia coli K-12.

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.     

Escherichia coli K-12 mutants hyperproducing chromosomal beta-lactamase by gene repetitions.

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.     

Inactivation of the ampD gene causes semiconstitutive overproduction of the inducible Citrobacter freundii beta-lactamase.

In Citrobacter freundii and Enterobacter cloacae, synthesis of AmpC beta-lactamase is inducible by the addition of beta-lactams to the growth medium. Spontaneous mutants that constitutively overproduce the enzyme occur at a high frequency. When the C. freundii ampC beta-lactamase gene is cloned into Escherichia coli together with the regulatory gene ampR, beta-lactamase expression from the clone is inducible. Spontaneous cefotaxime-resistant mutants were selected from an E. coli strain carrying the cloned C. freundii ampC and ampR genes on a plasmid. Virtually all isolates had chromosomal mutations leading to semiconstitutive overproduction of beta-lactamase. The mutation ampD2 in one such mutant was caused by an IS1 insertion into the hitherto unknown ampD gene, located between nadC and aroP at minute 2.4 on the E. coli chromosome. The wild-type ampD allele cloned on a plasmid could fully trans-complement beta-lactamase-overproducing mutants of both E. coli and C. freundii, restoring the wild-type phenotype of highly inducible enzyme synthesis. This indicates that these E. coli and C. freundii mutants have their lesions in ampD. We hypothesize that induction of beta-lactamase synthesis is caused by blocking of the AmpD function by the beta-lactam inducer and that this leads directly or indirectly to an AmpR-mediated stimulation of ampC expression.     

Common evolutionary origin of chromosomal beta-lactamase genes in enterobacteria

A 32P-labeled fragment of DNA, encoding the major part of the chromosomal ampC beta-lactamase gene of Escherichia coli K-12, was used as a hybridization probe for homologous DNA sequences in colonies of Neisseria gonorrhoeae, Pseudomonas aeruginosa, and different enterobacterial species. The ampC probe detected the presence of homologous DNA sequences in clinical isolates of E. coli, Shigella flexneri, Shigella sonnei, Klebsiella pneumoniae, Salmonella typhimurium, Serratia marcescens, and P. aeruginosa. No hybridization was found with N. gonorrhoeae colonies. In Southern blotting experiments the ampC probe hybridized to chromosomal DNA fragments of the same size in all enterobacterial species tested. However, the degree of hybridization differed with DNA from different species. DNA from the Shigella species strongly hybridized to the ampC probe. Furthermore, antibodies raised against purified E. coli K-12 ampC beta-lactamase precipitated beta-lactamases from the Shigella species, suggesting extensive sequence similarities between the ampC genes of these genera. The production of chromosomal beta-lactamase in S. sonnei increased with increasing growth rate similar to E. coli K-12. This growth rate response was abolished in two beta-lactamase-hyperproducing S. sonnei mutants, which thus seem similar to E. coli K-12 attenuator mutants. We propose that both the structure and regulation of the chromosomal beta-lactamase genes are very similar in E. coli and in S. sonnei.     

Isolation and characterization of extragenic suppressor mutants of the tolA-876 periplasmic-leaky allele in Escherichia coli K-12

tolA mutants of Escherichia coli K-12 release periplasmic proteins into the extracellular medium; they are sensitive to growth inhibitors such as cholic acid and tolerant to group A colicins and filamentous bacteriophage. Suppressor mutants of the tolA-876 allele were isolated by selecting for cholic acid resistant clones that did not release periplasmic ribonuclease I. One class of tolA suppressor strains carried mutations in the staA gene (for suppressor of tolA) located a 41 min. tolA-876 staA strains partially recovered a wild-type phenotype: they exported alkaline phosphatase and beta-lactamase into the periplasm and only released very low amounts of periplasmic proteins; moreover, they were sensitive to E1 and A colicins and more resistant than tolA-876 staA+ strains to various growth inhibitors. Furthermore, tolA-876 staA-2 and tolA+staA-2 mutants were 10- to 2700-times more resistant than staA+ strains to bacteriophages TuIa, TuIb and T4, and TuII whose receptors are major outer membrane proteins OmpF, OmpC and OmpA, respectively. SDS-PAGE analysis suggested that cell envelopes of staA or staA+ strains contained similar amounts of these proteins but characterization of strains carrying ompF (or C or A)-phoA gene fusions showed that mutation stA-2 reduced ompF gene expression by a factor of two. Analysis of double mutants strains carrying mutation staA-2 and a tolA, tolB, excC or excD periplasmic-leaky mutation showed that staA suppression was allele specific which suggested that proteins TolA and StaA might directly interact.     

Sequence elements determining ampC promoter strength in E. coli

A number of spontaneous up-promoter mutations have been isolated in the ampC beta-lactamase gene of Escherichia coli. The mutants were analyzed by DNA sequencing, and the level of ampC gene expression was determined. Six mutants with a 21-fold increase in promoter strength compared with the wild-type were mutated in the -35 promoter region from TTGTCA to the consensus sequence TTGACA . The -10 region sequence TACAAT was mutated to the consensus sequence TATAAT in three mutants exhibiting an ampC promoter seven times stronger than the wild-type. We have previously described a 1-bp insertion mutant ( Jaurin et al., 1981) that changes the inter-region distance to the consensus 17 bp. Thus, all the up-mutations found in the ampC promoter represent corrections of the three major discrepancies between the ampC promoter and the consensus E. coli promoter. We conclude that the three consensus elements of E. coli promoters, the -35 and -10 regions and an optimal inter-region distance of 17 bp, are the main elements determining the promoter strength.     

Resistance of Escherichia coli to Penicillins I. Genetic Study of Some Ampicillin-Resistant Mutants.

Eriksson-Grennberg, Kerstin G. (University of Uppsala, Uppsala, Sweden), Hans G. Boman, J. A. Torbj?rn Jansson, and Sone Thorén. Resistance of Escherichia coli to penicillins. I. Genetic study of some ampicillin-resistant mutants. J. Bacteriol. 90:54-62. 1965.-A number of ampicillin-resistant mutants have been isolated and characterized. Of these strains, two groups have been genetically investigated: members of one group, which are moderately resistant to ampicillin (Amp(r) (10)) carry mutations in a locus which we have designated ampA; another strain, which is resistant to high levels (50 mug/ml) of ampicillin, is a multistep mutant for which a genotype cannot yet be written. The phenotype of this strain has been designated Amp(r) (50). The location of ampA was studied by the interrupted-conjugation technique, with argF, metB, mtl, and serA as reference markers. The phenotypic expression was the same for ampA and argF. These experiments, as well as recombination without selection for ampicillin resistance, indicate that ampA is located between argF and pyrB. "Broken slopes" on the recombinant curves and failure to demonstrate cotransduction make a more accurate mapping difficult. Phage P1bt transduced ampA from resistant donors to sensitive recipient strains with a frequency of 5 x 10(-7). The ampA locus segregated in conjugation and transduction experiments with an Amp(r) (50) donor strain, but neither method gave a genetic transfer of high resistance to ampicillin. Penicillinase activity was demonstrated in two independent mutants carrying the ampA locus.     

Genetic analysis of Escherichia coli K-12 mutants defective for the structural and regulatory genes for second purine nucleoside phosphorylase

Two types of mutants lacking the second purine nucleoside phosphorylase (PNPase 2) activity were isolated using the Escherichia coli K-12 pndR strains with constitutive or inosine-inducible synthesis of the PNPase 2. The mutations of the first type are recessive to the pndR+ allele on the F' episome. They are closely linked to the original pndR+ mutations and therefore affect the pndR gene encoding the activator protein. The mutations of the second type affect the PNPase 2 structural gene (pndA) and are recessive to the pndA+ allele on the F' episome. The nupC-pndR-pndA-ptsH-cysA gene order was established by means of four- and five-factorial transductional crosses.     

Analysis of antibiotic resistance gene expression in Pseudomonas aeruginosa by quantitative real-time-PCR

In Pseudomonas aeruginosa many of the clinically relevant resistance mechanisms result from changes in gene expression as exemplified by the Mex drug efflux pumps, the AmpC beta-lactamase and the carbapenem-specific porin OprD. We used quantitative real-time-PCR to analyze the expression of these genes in susceptible and antibiotic-resistant laboratory and clinical strains. In nalB mutants, which overexpress OprM, we observed a four- to eightfold increase in the expression of mexA, mexB, and oprM genes. MexX and mexY genes were induced eight to 12 times in the presence of 2 mg L(-1) tetracycline. The mexC/oprJ and mexE/oprN gene expression levels were increased 30- to 250-fold and 100- to 760-fold in nfxB and nfxC mutants, respectively. We further found that in defined laboratory strains expression levels of ampC and oprD genes paralleled beta-lactamase activity and OprD protein levels, respectively. Our data support the use of quantitative real-time-PCR chain reaction for the analysis of the antimicrobial resistance gene expression in P. aeruginosa.     

Escherichia coli strain with a deletion of the chromosomalampC gene marked with TcR, suitable for production of penicillin G acylase

Escherichia coli strain which contains a marker of tetracycline resistance gene (TcR) placed by P1 transduction beside the chromosomal deletion of ampC gene (delta ampC) coding for beta-lactamase was constructed. Such introduction of TcR marker permits a fast and simple selection for the transfer of delta ampC by P1 transduction into industrial E. coli strains. This approach was used for constructing an E. coli strain suitable for penicillin acylase production.     

Evidence for a unique first position codon-anticodon mismatch in vivo

The Ser68(AGC) codon of the beta-lactamase gene was changed to the glycine codons GGA and GGC. With glycine at position 68, beta-lactamase is inactive because it does not have a nucleophilic side-chain to function in the reaction mechanism. The mutant SG68(GGA) allele had no detectable beta-lactamase activity; however, the mutant SG68(GGC) did produce a small amount of activity. Both mutant alleles produce comparable amounts of beta-lactamase protein in a maxi-cell system. To identify why these two "same-sense" beta-lactamase mutants differ phenotypically, we introduced the alleles into Escherichia coli strains with mutations that affect translational fidelity. The rpsD mutation, which decreases fidelity, significantly increased activity with the SG68(GGC) allele, while the rpsL mutation, which increases translational fidelity, had little effect on the beta-lactamase activity. The rpsD and rpsL alleles had no effect on the SG68(GGA) allele. From the allele specificity of the activity produced by the bla mutants, and from the differential effect of translational fidelity on the activity of the SG68(GGC) allele, we infer that tRNA(GCU)Ser, the AGU/C reading tRNA(Ser), mistranslates SG68(GGC) at a frequency of about 0.1%, and subsequently produces active beta-lactamase. This is the first observation of an A/G wobble with a wild-type tRNA at the first position of the codon-anticodon interaction.     

mut-25, a mutation to mutator linked to purA in Escherichia coli.

The mutation mut-25 that results in a mutator phenotype is closely linked to purA on the chromosome of Escherichia coli. The gene order in this region is ampA mut-25 purA. purA mut-25 double mutants retained mutator activity indicating that mut-25 is not a mutation in the purA gene. The repair mutations uvrA6, recA56, and exrA1 had no effect on mutation frequencies in mut-25 strains, and mut-25 strains were normally resistant to ultraviolet irradiation. Frequencies of host range mutations were not increased in phages T1, T2, and T7 grown on mut-25 strains. mut-25 could act trans, reverting the trpA46 mutation either on the chromosome or on an F episome. The transitions AT yields GC (adenine-thymine yields guanine-cytosine) and GC yields AT were induced by mut-25.     

Resistance of Escherichia coli to penicillins. VI. Purification and characterization of the chromosomally mediated penicillinase present in ampA-containing strains

The chromosomally mediated penicillinase present in three strains of Escherichia coli K-12 has been purified and characterized. Two of the strains carried the ampA gene and the third the wild-type allele. The purification involves release of the enzyme by spheroplast formation, dialysis, chromatography on sulfoethyl cellulose, and chromatography on hydroxylapatite. Enzyme from the two mutants appeared homogeneous in polyacrylamide gel electrophoresis. Enzyme from the wild-type strain gave two bands. Immunologically, the enzymes from all three strains were identical. Ultracentrifugation gave a homogeneous peak with a sedimentation coefficient of 3.4S. Gel filtration gave an estimated molecular weight of 29,000. The N-terminal amino acid residue was found to be alanine. Complete amino acid analysis showed a lack of cysteine. Ultraviolet spectra were recorded at three different pH values. The extinction coefficient at 280 nm is 21.0 for a 1% solution at pH 6.8. The optimal pH is 7.3. With enzyme from one of the resistant mutants, the following K(m) and turnover number values were obtained: for penicillin G, 12 mum and 2,080; for d-ampicillin, 6 mum and 83; for cephalosporin C, 217 mum and 18,400. The effect of different salts on the enzyme activity was tested. Under many conditions the enzyme was found to be unstable.