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S Normark

Publications and source records attributed to S Normark.

At least 145 records · Page 8Linked to original sources
PubMed1983

Insertion of IS2 creates a novel ampC promoter in Escherichia coli.

A class of ampC beta-lactamase-hyperproducing mutants of Escherichia coli were shown to have the insertion element IS2 inserted into the ampC promoter. The insertion of IS2 in orientation II created a novel promoter in which the -35 region and the 17 bp long spacing sequence between the two consensus sequences are present in IS2 DNA, whereas the -10 region from the original ampC promoter is retained. In vitro transcription revealed that the transcription initiation site in the ampC::IS2 mutants was identical with that of ampC wild-type promoter. The novel promoter exhibited a 20-fold increase in promoter strength relative to the original ampC promoter, presumably due to the increase in the spacing sequence from 16 to 17 bp. The evolution of transposable elements and of control elements such as promoters are discussed on the basis of the findings described herein.

Base Sequence↗
PubMed1983

ampC beta-lactamase hyperproduction in Escherichia coli: natural ampicillin resistance generated by horizontal chromosomal DNA transfer from Shigella.

Six ampicillin-resistant clinical isolates of Escherichia coli that hyperproduced the chromosomal ampC beta-lactamase were studied. By DNA sequence analysis, we found that five of them were identical over an entire 449-base-pair sequence and carried a novel strong ampC promoter [Olsson, O., Bergström, S. & Normark, S. (1982) EMBO J. 1, 1411-1416]. Except for one base pair this sequence was identical to that of a low beta-lactamase-producing clinical isolate of Shigella sonnei. Spontaneous one-step mutants of S. sonnei that overproduced the ampC beta-lactamase by 45-fold were characterized and found to be mutated at the single base that distinguishes S. sonnei from the five E. coli hyperproducers. The most likely explanation for this result is that chromosomal DNA was transferred in vivo from Shigella to E. coli across the species barrier.

Ampicillin↗
PubMed1983

Genetics of digalactoside-binding adhesin from a uropathogenic Escherichia coli strain.

The uropathogenic strain Escherichia coli J96 mediates mannose-resistant hemagglutination owing to production of a digalactoside-binding adhesin. A cosmid clone from this strain has been isolated that, when harbored in E. coli K-12, expressed Pap pili and this adhesin (R. Hull et al., Infect. Immun. 33:933-938, 1981). By transposon mutagenesis and by the construction of a number of hybrid plasmid derivatives, we have demonstrated that about 8.5 kilobases of DNA is required to generate a mannose-resistant hemagglutination-positive phenotype in E. coli K-12 strain P678-54. The structural gene for the Pap pili monomer, papA, has been identified and mapped close to the promotor-proximal end of the Pap operon. Although strain P678-54 that harbored a Tn5 insertion within papA showed a mannose-resistant hemagglutination-positive phenotype, it was negative in a competitive enzyme-linked immunosorbent assay with anti-Pap pilus serum. This could mean that a Pap adhesin is encoded by a region on the Pap operon that is distinct from papA.

Adhesins, Escherichia coli↗
PubMed1983

Comparison of the overlapping frd and ampC operons of Escherichia coli with the corresponding DNA sequences in other gram-negative bacteria.

Specific DNA probes from Escherichia coli K-12 were used to analyze the sequence divergence of the frd and ampC operons in various species of gram-negative bacteria. These operons code for the fumarate reductase complex and the chromosomal beta-lactamase, respectively. We demonstrate that the two operons show the same general pattern of divergence, although the frd operon is considerably more conserved than is the ampC operon. The major exception is Salmonella typhimurium LT2, which shows a strong homology to the E. coli frd probe but none to the E. coli ampC probe. The operons from Citrobacter freundii and Shigella sonnei were cloned and characterized by physical mapping, Southern hybridization, and protein synthesis in minicells. In S. sonnei, as in E. coli K-12, the frd and ampC operons overlap (T. Grundström and B. Jaurin, Proc. Natl. Acad. Sci. U.S.A. 79:1111-1115, 1982). Only minor discrepancies between the two operons were found over the entire frd-ampC region. In C. freundii, the ampC and frd operons do not overlap, being separated by about 1,100 base pairs. Presumably the inducible property of the C. freundii chromosomal beta-lactamase is encoded by this 1,100-base-pair DNA segment.

Base Sequence↗
PubMed1983

Sequence-specific DNA modification in Neisseria gonorrhoeae.

Neisseria gonorrhoeae 82409/55(pJD1) is postulated to possess six DNA sequence-specific cytosine methyltransferases and one DNA sequence-specific N6-adenine methyltransferase. From the DNA sequencing of the plasmid pJD1 (manuscript in preparation) by a modification of the Maxam and Gilbert chemical cleavage procedure, the cytosine methylation specificities were demonstrated. Five of these methylating enzymes and their respective specificities are M . NgoI (formula; see text) does not methylate the cytosine of its recognition sequence, in agreement with a detected adenine modification. A biological implication of these different DNA methylating activities is discussed.

Base Sequence↗
PubMed1983

Overlapping genes.

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Base Sequence↗
PubMed1982

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.

Ampicillin↗
PubMed1982

Identification of a novel ampC beta-lactamase promoter in a clinical isolate of Escherichia coli.

A clinical strain of Escherichia coli, C16, that overproduces the ampC beta-lactamase was isolated. A 203-bp DNA segment from this strain, including the promoter and attenuator region of the ampC structural gene, was sequenced. A comparison with the corresponding sequence of E. coli K12 revealed four base pair differences between the ampC segments from these strains. DNA sequence data and in vitro transcription indicated that the ampC promoter in the clinical isolate was displaced 5 bp upstream of the promotor defined in the E. coli K12 strain. Like the ampC gene of E. coli K12, the ampC gene from the clinical isolate was metabolically regulated. However, the increase in the specific amount of beta-lactamase relative to the increase in specific growth rate was much higher in the clinical isolate. These data imply that the growth rate-dependent anti-termination acting on the ampC attenuator in vivo is more pronounced in the clinical E. coli isolate than in E. coli K12. A possible molecular mechanism for this is discussed.

Base Sequence↗
PubMed1982

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.

Base Sequence↗
PubMed1981

Recombination between short DNA homologies causes tandem duplication.

The ampC gene of Escherichia coli K-12 codes for a beta-lactamase which can hydrolyse the beta-lactam ring of ampicillin. Ampicillin resistance is strictly related to ampC gene copy number thus we have been able to isolate ampicillin-resistant mutants carrying multiple ampC repeats. We have isolated on a plasmid a segment of chromosomal DNA carrying multiple ampC repeats, and compared the nucleotide sequence of the region joining repeat units to the sequence of the DNA segments that fused to create the joint. The fusion had occurred within a 12-base pair (bp) sequence of perfect homology. We suggest that recombination between randomly occurring short homologies (12-13-bp long), could be a general mechanism to generate tandem duplications in the size range of 10 kilobases (kb).

Base Sequence↗
PubMed1981

The E. coli beta-lactamase attenuator mediates growth rate-dependent regulation.

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.

Base Sequence↗
PubMed1981

A mutant of Salmonella typhimurium with an abnormal septation pattern associated with an inhibition of RNA synthesis.

A mutant strain of S. typhimurium that is disturbed in the regulation of cell division and macromolecular synthesis is described. The life cycle of the mutant can be divided into two discrete stages. When growing in rich medium at a low cell density, cell division is inhibited and the cells filament at the same time as the relative amount of RNA shows a continuous increase. However, at a certain stage, RNA synthesis stops and the filaments start to septate resulting in chain-formation. These chains can thereafter segregate into individual cells of unit cell length. The accumulation of RNA is rather due to a regulatory defect in th synthesis of ths stable RNA species than to a unusual stability of messenger RNA (mRNA) as the half life of mRNA was estimated to 2.3 minutes during the period of RNA accumulation. Latter inhibition of RNA synthesis affects only stable species of RNA. The ppGpp pools of the strain did not fluctuate during growth, showing that inhibition of RNA. The ppGpp pools of the strain did not fluctuate during growth, showing that inhibition of RNA synthesis is not correlated to changes in the level of ppGpp. Different treatments that reduce the level of transcription such as sublethal concentrations of rifampicin, shift-down or high concentrations of nalidixic acid, all induced cell division of filamentous cells, suggesting that there exists an intimate relationship between macromolecular synthesis and cell division. The behavior of this mutant its best with the proposed hypothesis that the biomass to volume ratio is of importance in the regulation of cell division in bacteria.

Cell Division↗
PubMed1981

Deoxyribonucleic acid modifications and restriction endonuclease production in Neisseria gonorrhoeae.

Modification of gonococcal deoxyribonucleic acid (DNA) was investigated, and the relationship with endonuclease production was explored. Both chromosomal and plasmid DNA from different gonococcal strains, irrespective of their plasmid content, was poorly cleaved by the restriction endonucleases HaeII, HaeIII, SacII, and BamHI. The fragment pattern of the Tn3 segment present on the 7.2-kilobase gonococcal resistance plasmid, when compared to its known DNA sequence, allowed us to conclude that the HaeIII and BamHI resistance was due to modification of these sites. A comparison of the fragment pattern of the resistance plasmid, when isolated from Escherichia coli or Neisseria gonorrhoeae, revealed that the resistance of HaeII must also be due to modification of its recognition sequence. Isoschizomers of HaeII and HaeIII can be found in isolates of N. gonorrhoeae (NgoI and NgoII, respectively). A new restriction endonuclease in gonococci, NgoIII, with a specificity similar to SacII, is reported here. High-pressure liquid chromatography of gonococcal DNA showed the presence of 5-methylcytosine. It is suggested that the methylation of cytosine residues in the HaeII (NgoI), HaeIII (NgoII), and SacII (NgoIII) recognition sites is the basis for the resistance of gonococcal DNA to cleavage by these enzymes. This methylation may be part of a host restriction modification system. In two out of five gonococcal strains the sequence -GATC- was modified. One strain unable to modify this sequence was a spontaneous mutant of a strain carrying such a modifying function.

Base Sequence↗
PubMed1980

A relationship between plasmid structure, structural lability, and sensitivity to site-specific endonucleases in Neisseria gonorrhoeae.

Nearly all gonococcal strains carry a small "phenotypically cryptic" plasmid of approximately 4,200 basepairs. A detailed physical map of this plasmid has been constructed, revealing the presence of numerous putative inverted repeats. These studies also revealed the presence on the plasmid of recognition sequences for several site-specific endonucleases (particularly HpaII, MspI and AluI) that are particularly resistant to cleavage, and confirmed previous reports of structural lability. Both the sites that are resistant to cleavage, and the observed structural variation are associated with the inverted repetitive sequences.

Base Composition↗
PubMed1980

Tandem duplication induced by an unusual ampA1-, ampC-transducing lambda phage: a probe to initiate gene amplification.

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.

Ampicillin↗
PubMed1980

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.

Cloning, Molecular↗
PubMed1980

Overproduction of outer membrane protein suppresses envA-induced hyperpermeability.

A quantitative study on outer membrane components was performed in a number of envelope mutants of Escherichia coli K-12 exhibition different permeability properties for antimicrobial agents. The envA1 allele causing an increased influx for both hydrophobic and hydrophilic drugs was found to be associated with a deficiency in the amount of lipopolysaccharides. The sefA1 envA1 double mutant was found to have a higher outer membrane buoyant density, apparently due to an increase in protein content. This double mutant was still low in lipopolysaccharide content.

Alleles↗