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Biomedical subjects

S Normark

Publications and source records attributed to S Normark.

At least 127 records · Page 7Linked to original sources
PubMed1986

Induction of chromosomal beta-lactamase expression in enterobacteria.

Enterobacteria and Pseudomonas express evolutionarily related chromosomally encoded beta-lactamases. These enzymes have a high affinity for modern cephalosporins, and beta-lactamase overproduction is the most important factor in the development of resistance against such drugs. In some species the beta-lactamase is produced constitutively at a low level, and mutations to high expression occur only infrequently. In other species beta-lactamase synthesis can be induced by beta-lactams, and mutation to constitutive overexpression of the enzyme is a frequent event. We discuss the current knowledge concerning the genetic basis of the two different modes of beta-lactamase regulation, as well as the mechanisms through which a high level of beta-lactamase synthesis is reached.

Base Sequence↗
PubMed1986

Use of genetically marked minicells as a probe in measurement of predation on bacteria in aquatic environments.

Minicells produced by Escherichia coli M2141 were used as probes to measure predation on pelagic bacteria in situ. The minicells, labeled with [S]methionine in one specific protein, were shown to disappear in the presence of a microflagellate (Ochromonas sp.), as seen by a decrease in the amount of labeled marker protein with time. Incubation in filtered (pore size, 0.2 mum) and autoclaved seawater did not affect the amount of labeled marker protein in the minicell. The generation time of flagellates feeding on minicells was determined to be similar to that found for flagellates grown on seawater bacteria or living E. coli NC3. Data indicate that minicells are seen as true food particles by the flagellates. The minicell probe was used in recapture experiments, in which predation in situ on pelagic bacteria was demonstrated. The rate of bacterial production showed a clear covariation with the rate of predation, both in different sea areas and in depth profiles. The obtained results (11 field experiments) showed that the rate of predation, on average, accounts for the consumption of 62% of the bacteria produced.

Journal Article↗
PubMed1986

Intragenic variation by site-specific recombination in the cryptic plasmid of Neisseria gonorrhoeae.

Cryptic plasmid DNA of Neisseria gonorrhoeae was found integrated into the gonococcal chromosome in both plasmid-bearing strains and plasmid-free strains. At several chromosomal locations only segments of the plasmid were found. However, in at least two strains an intact copy of the plasmid seemed to be present with the joints between the plasmid and the chromosomal DNA being located within the cppB gene of the cryptic plasmid. The cppB gene was shown to undergo a sequence-specific intragenic deletion. The deletion removed 54 base pairs, representing 18 amino acids, and did not affect the reading frame. It is proposed that the cryptic plasmid integrates into the chromosome and other gonococcal plasmids within this site-specific deletion region. Models for the site-specific recombination are presented.

Amino Acid Sequence↗
PubMed1986

Chromosomal beta-lactam resistance in enterobacteria.

Most enterobacterial species carry a chromosomal ampC beta-lactamase gene. In Escherichia coli and Shigella, expression from ampC is non-inducible and the beta-lactamase is synthesized at low levels. Mutations leading to increased beta-lactamase synthesis occur rather infrequently, making resistance to modern cephalosporins a rare event in these species. In other enterobacteria and Pseudomonas, ampC beta-lactamase synthesis is induced by beta-lactams. In Enterobacter cloacae, Citrobacter freundii and probably also in other species with inducible beta-lactamase expression, ampC is regulated by at least two genes, ampR and ampD. Mutations affecting ampR abolish beta-lactamase inducibility, and mutants devoid of ampR, produce ampC beta-lactamase at low constitutive levels. Mutations in ampD lead to constitutive overproduction of inducible beta-lactamase if an intact ampR protein is present in the cell. The latter type of mutations occur at a high frequency and result in clinical resistance to several third-generation cephalosporins.

Anti-Bacterial Agents↗
PubMed1985

Transcriptional activation of a pap pilus virulence operon from uropathogenic Escherichia coli.

A gene cluster mediating production of pili in uropathogenic Escherichia coli was analysed with respect to regulation of pili synthesis. Two cistrons, papB and papI, were localized upstream of the major pilus subunit gene, papA. The papI-papB-papA region was characterized by nucleotide sequencing and by transcriptional analysis. The papA gene was primarily represented by an 800 nucleotide long transcript but was also co-transcribed with papB as a less abundant 1300 nucleotide long mRNA. Both transcripts presumably terminated at the same site downstream of the papA coding sequence. The weakly expressed papI gene was transcribed in the opposite direction to that of papB and papA. Studies with lacZ operon fusions showed that the papB gene encoded a trans-active effector required for papA transcription. Similarly, the papI gene stimulated papB transcription in trans. Furthermore, full expression of papA was cis dependent upon the papI-papB region. Transcription of the papB gene was shown to be dependent upon cAMP and its receptor protein. A binding site for the cAMP-CRP complex was postulated in the DNA sequence upstream of the papB promoter.

Amino Acid Sequence↗
PubMed1985

Initiation of translation makes attenuation of ampC in E. coli dependent on growth rate.

The chromosomal beta-lactamase gene of E. coli, ampC, shows increased expression with increased growth rate of the bacteria. We have previously shown that transcription of ampC is attenuated, and that a mutation in the terminator stem of this attenuator abolishes the growth rate-dependency of ampC expression. We now present studies using mutations, made in vitro, located such that the 5'-end of ampC mRNA carries a possible recognition sequence for initiation of translation close to the attenuator stem. Alteration of the supposed initiation codon AUG to UUG resulted in a reduced and growth rate-independent expression of ampC beta-lactamase. AmpC mRNA starts with the sequence AUC, which might be a non-typical ribosome binding site, situated four bases before the AUG. Deletion of the C in this sequence caused a partial reduction of ampC expression and also a partial loss of the growth rate-dependent regulation. The phenotypes of these mutants support a model in which formation of a ribosome initiation complex at a level increasing with the growth rate inhibits termination of transcription at the ampC attenuator.

Bacterial Proteins↗
PubMed1985

Regulatory components in Citrobacter freundii ampC beta-lactamase induction.

Citrobacter freundii encodes an inducible chromosomal beta-lactamase similar to the constitutively expressed ampC beta-lactamase of Escherichia coli. In the latter species the ampC gene is located next to the fumarate reductase (frd) operon, whereas in C. freundii the ampC gene is known to be separated from frd by 1100 base pairs. This intervening DNA segment carries a gene, ampR, coding for a 31-kilodalton polypeptide. The cloned C. freundii OS60 ampC gene is inducible by beta-lactam antibiotics in E. coli, but only in the presence of an intact ampR gene. In the absence of inducer the AmpR protein represses C. freundii ampC synthesis 2.5-fold. Addition of beta-lactams induced expression from the cloned ampC beta-lactamase gene 11-fold. Thus, the AmpR protein has a positive effect on ampC expression in the presence of inducing beta-lactams. Two spontaneous mutants of C. freundii were isolated that constitutively overproduce the ampC beta-lactamase. The mutations in both these strains occurred outside the frd-amp region, suggesting that there is at least one additional component in the regulatory system. With the cloned C. freundii ampC gene in E. coli, mutants with the same phenotype could be obtained. These mutations were located on the E. coli chromosome. The constitutive beta-lactamase overproduction in these mutants requires the presence of an intact ampR gene.

Ampicillin↗
PubMed1985

Adhesion to human cells by Escherichia coli lacking the major subunit of a digalactoside-specific pilus-adhesin.

Pathogenic bacteria frequently possess pili with specific binding properties that allow them to attach to epithelial tissue. In Escherichia coli, the pili associated with pyelonephritis (Pap pili) bind to digalactoside-containing glycolipids on the uroepithelium. Transposon-insertion mutants and deletion mutants of the cloned genetic determinant encoding synthesis of such digalactoside-binding Pap pili have been studied in E. coli K-12. Mutants that completely lack synthesis of the major Pap pili subunit protein, the papA gene product, and thereby no longer produce pili were shown to retain the binding specificity of intact Pap pili. Reduced expression of some of the remaining pap genes, presumably due to polarity effects from papA::Tn5 insertions, was circumvented by the use of a copy-number mutant plasmid vector. Derivatives carrying the papA-D genes produced Pap pili but did not bind to human cells. The products of the genes papE-G are essential for digalactoside-specific hemagglutination and for attachment to urinary bladder cells. The papC and papD genes presumably aid in surface localization and/or polymerization of the pili-adhesin subunits and are required for expression of pili as well as of the binding properties. Serological evidence is presented that suggests that a minor pilus component(s), presumably produced by the papE, -F, or -G gene, is the actual binding moiety in the digalactoside-specific interaction of Pap pilus-adhesin.

Adhesins, Escherichia coli↗
PubMed1985

Type III 5-methylcytosine modification of DNA in Neisseria gonorrhoeae.

We present here the first report of a type III methyltransferase that modifies a cytosine. Neisseria gonorrhoeae 82409/55 (pJD1) modifies the first cytosine on only one strand from the 5' end of the nonpalindromic sequence: (Formula; see text). We have called this modifying activity M X NgoVIII.

5-Methylcytosine↗
PubMed1985

Globoside-specific adhesins of uropathogenic Escherichia coli are encoded by similar trans-complementable gene clusters.

Uropathogenic Escherichia coli frequently express globoside-specific adhesins, shown to mediate binding to uroepithelial cells. For one gene cluster pap, it recently has been demonstrated that globoside binding is not dependent on expression of the pilus subunit gene papA. Instead, two other pap genes papF and papG are specifically required for globoside binding (F. P. Lindberg et al., EMBO J. 3:1167-1173, 1984). By restriction enzyme mapping, DNA hybridization, DNA sequencing, and protein expression in minicells, we show that three gene clusters encoding globoside binding have a very similar structure and gene organization, although they were cloned from different E. coli isolates. Major differences between the adhesin clones were restricted to the central part of the pilin gene (papA) and to one of the two adhesin gene (papG). The three functional units required for biogenesis of globoside-binding pili, i.e., pilin synthesis, pilin export, and pilin assembly, as well as expression of adhesion function, were all trans complementable among the gene clusters.

Adhesiveness↗
PubMed1985

Cryptic plasmid of Neisseria gonorrhoeae: complete nucleotide sequence and genetic organization.

The naturally occurring cryptic plasmid pJD1 of Neisseria gonorrhoeae is 4,207 base pairs long and is found in about 96% of gonococcal strains. The total probable coding capacity of pJD1 was determined from the complete nucleotide sequence by using computational probes to identify open reading frames with similar codon usage and by screening for the presence of ribosomal binding sites before the start codons. Candidates for promoters and terminators were also found in the sequence. Based on these findings, we propose a model for the genetic organization of the plasmid. The model predicts two transcriptional units, each composed of five compactly spaced genes. A promoter of one of the transcripts was shown to function in Escherichia coli, and the products of three of the five genes in this operon were identified in minicell expression experiments. Of these, the cppA gene encoded a 9-kilodalton protein, and the cppB and cppC genes both coded for 24-kilodalton proteins. No expression of the other transcriptional unit was detected, but two genes in this operon were expressed in minicells when transcribed from an E. coli promoter. The experimental data were consistent with the model.

Amino Acid Sequence↗
PubMed1984

Mutations in E coli cistrons affecting adhesion to human cells do not abolish Pap pili fiber formation.

A chromosomal DNA fragment which mediates Pap (pili associated with pyelonephritis) pili formation, mannose-resistant hemagglutination ( MRHA ) and binding to uroepithelial cells has been isolated from the uropathogenic Escherichia coli clinical isolate J96 , and genetically studied. Analysis of polypeptides expressed by the Pap DNA led to detection of a number of polypeptides ranging in mol. wt. from 13 000 to 81 000 daltons. The gene order and transcriptional orientation for four of the corresponding cistrons was: 13 000 ( papB ) 19 500 ( papA , structural gene for the Pap pilus subunit), 81 000 ( papC ) and 28 500 ( papD ). Analyses of a lacZ- papA gene fusion located a promoter upstream from papA within the cloned DNA. Transposon Tn5 insertions in any of these four cistrons decreased or eliminated Pap pili formation. A number of transposon Tn5 mutations were identified in a region distal to papD that expressed normal levels of the papA protein on the cell surface in the form of recognizable pili structures but did not agglutinate human erythrocytes or adhere to uroepithelial cells. This region expressed polypeptides of 15 000, 24 000, 26 000 and 35 000 daltons. This finding shows that Pap pili formation and binding properties can be genetically dissociated.

Antigens, Bacterial↗
PubMed1984

Genes of pyelonephritogenic E. coli required for digalactoside-specific agglutination of human cells.

Most pyelonephritic Escherichia coli strains bind to digalactoside-containing glycolipids on uroepithelial cells. Purified Pap pili (pili associated with pyelonephritis) show the same binding specificity. A non-polar mutation early in the papA pilin gene abolishes formation of Pap pili but does not affect the degree of digalactoside-specific hemagglutination. Three novel pap genes, papE , papF and papG are defined in this report. The papF and papG gene products are both required for digalactoside-specific agglutination by whole bacteria cells as well as for agglutination by pilus preparations. Pili prepared from a papE mutant have lost their binding ability although whole cells from this mutant retain it, implying an adhesin anchoring role for the papE gene product. A mutant with lesions both in the papA and the papE genes does not mediate digalactoside-specific agglutination. The implications of this finding for pilus biogenesis are discussed.

DNA Restriction Enzymes↗
PubMed1984

Nucleotide sequence of the papA gene encoding the Pap pilus subunit of human uropathogenic Escherichia coli.

The papA gene of the uropathogenic strain Escherichia coli J96, coding for the Pap pili subunit, was subjected to DNA sequencing, and found to code for an 185-amino acid-long polypeptide with a 22-amino acid-long signal peptide. Here we present the primary sequence, the hydrophilicity profile, and the predicted polypeptide secondary structure of the Pap pili subunit.

Amino Acid Sequence↗
PubMed1983

Mannose-sensitive and Gal-Gal binding Escherichia coli pili from recombinant strains. Chemical, functional, and serological properties.

Chromosomal genes encoding the MS and Gal-Gal binding properties have been cloned into separate recombinants and their respective pili characterized. Hapten inhibition of hemagglutination with synthetic carbohydrate receptor analogues and carbohydrate-adsorbed latex agglutination studies indicate that Gal-Gal and MS pili collectively exhibit the binding properties of the parent strain. MS pili migrated in SDS-PAGE with an Mr of 19 kdaltons and 17 kdaltons; the Mr of Gal-Gal pili was 17.5 kdaltons. The pili are chemically similar by amino acid composition and when the N-terminal cysteines are aligned, 8 of the 13 residues between positions 9 and 22 are homologous. Further, carboxy-terminal sequence homology was inferred from the carboxypeptidase digestion of a MS pili and the sequence of a carboxy-terminal tryptic peptide from Gal-Gal pili.

Agglutination Tests↗