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Biomedical subjects

I Chopra

Publications and source records attributed to I Chopra.

At least 73 records · Page 4Linked to original sources
PubMed1991

Origin of tetracycline efflux proteins: conclusions from nucleotide sequence analysis.

The sequences of six tetracycline efflux proteins and three transport proteins which have some resemblance to them were compared. The tetracycline efflux proteins fall into three families: (i) those encoded by pBR322, RP1, and Tn10 (Escherichia coli); (ii) pT181 (Staphylococcus aureus) and pTHT15 (Bacillus subtilis); and (iii) tet347 (Streptomyces rimosus). There is global sequence homology within each of the first two families, but there is none between the families. The pT181/pTHT15 family shares close homology with the N-terminal half of the methylenomycin A efflux protein (Streptomyces coelicor), while tet347 resembles the C-terminal half. Portions of the N-terminal half of the Tn10-encoded protein show significant resemblance to portions in the N-terminal half of the pT181/pTHT15 family, but this sometimes occurs among transport proteins which do not have a common substrate. Tetracycline efflux proteins, therefore, appear to have arisen on at least two, or possibly three, separate occasions, probably from other transport proteins.

Amino Acid Sequence↗
PubMed1991

Molecular basis of tetracycline action: identification of analogs whose primary target is not the bacterial ribosome.

Tetracycline analogs fell into two classes on the basis of their mode of action. Tetracycline, chlortetracycline, minocycline, doxycycline, and 6-demethyl-6-deoxytetracycline inhibited cell-free translation directed by either Escherichia coli or Bacillus subtilis extracts. A second class of analogs tested, including chelocardin, anhydrotetracycline, 6-thiatetracycline, anhydrochlortetracycline, and 4-epi-anhydrochlortetracycline, failed to inhibit protein synthesis in vitro or were very poor inhibitors. Tetracyclines of the second class, however, rapidly inhibited the in vivo incorporation of precursors into DNA and RNA as well as protein. The class 2 compounds therefore have a mode of action that is entirely distinct from the class 1 compounds, such as tetracycline that are used clinically. Although tetracyclines of the second class entered the cytoplasm, the ability of these analogs to inhibit macromolecular synthesis suggests that the cytoplasmic membrane is their primary site of action. The interaction of class 1 and class 2 tetracyclines with ribosomes was studied by examining their effects on the chemical reactivity of bases in 16S rRNA to dimethyl sulfate. Class 1 analogs affected the reactivity of bases to dimethyl sulfate. The response with class 2 tetracyclines varied, with some analogs affecting reactivity and others (chelocardin and 4-epi-anhydrotetracycline) not.

Bacteria↗
PubMed1991

Lack of evidence for a saturable tetracycline transport system in Staphylococcus aureus.

Previous studies on tetracycline transport into Staphylococcus aureus identified a high-affinity, saturable uptake system for the antibiotic (Km, 4.76 microM) (B.L. Hutchings, Biochim. Biophys. Acta 174:734-738, 1969). However, the earlier results could not be confirmed using conditions that permitted energy-dependent, concentrative uptake of tetracycline. Kinetic artifacts introduced by inappropriate washing procedures may explain the previous results.

Biological Transport, Active↗
PubMed1990

Effect of short-chain organic acids on macromolecular synthesis in Escherichia coli.

Incubating cultures of Escherichia coli with propionic acid (5 mmol/l) or formic acid (10 mmol/l) at pH 5.0 produced bacteriostasis lasting 30 and 120 min respectively. During this time rates of RNA, DNA, protein, lipid and cell wall synthesis were reduced. Growth resumed after continued incubation in the presence of acid, but cells from acid-treated cultures were larger than controls. DNA synthesis was particularly sensitive to the presence of the propionic or formic acid.

Bacterial Proteins↗
PubMed1990

Sensitive biological detection method for tetracyclines using a tetA-lacZ fusion system.

A sensitive microbiological detection system for tetracyclines, utilizing an Escherichia coli strain containing a cloned tetA-lacZ gene fusion, is described. Expression of beta-galactosidase by the fusion plasmid pUB3610 remained subject to regulatory control by the TetR repressor protein, with the presence of tetracyclines in the growth medium leading to a 12-fold induction of beta-galactosidase synthesis. Because synthesis of beta-galactosidase was influenced to a small extent by the carbon source and the addition of cyclic AMP to the medium, cells were grown in the presence of cyclic AMP to enhance the sensitivity of the assay. All commonly marketed tetracyclines and some derivatives at concentrations as low as 0.1 ng/ml could be detected in the growth medium. A plate assay utilizing the fusion plasmid that detects 1 ng of tetracycline has also been developed.

Chromosomes, Bacterial↗
PubMed1990

Induction of the Citrobacter freundii group I beta-lactamase in Escherichia coli is not dependent on entry of beta-lactam into the cytoplasm.

Induction of the cloned ampC Citrobacter freundii beta-lactamase in Escherichia coli by 6-aminopenicillanic acid was prevented by periplasmic TEM beta-lactamase in the same cell. In contrast, cytoplasmic TEM beta-lactamase did not prevent induction. This result implies that entry of the beta-lactam into the cytoplasm is not necessary for induction of ampC.

Anti-Bacterial Agents↗
PubMed1989

Effects of tetracyclines on the production of extracellular proteins by members of the propionibacteriaceae.

The effect of tetracyclines on the synthesis of proteins in Propionibacterium avidum and P. acnes was examined. Synthesis of an extracellular lipase by P. avidum was slightly more sensitive to inhibition by tetracycline than total (cellular and extracellular) protein synthesis. The effect of tetracycline and other analogues on the synthesis of secreted proteins was also examined in P. avidum and P. acnes by protein radiolabelling experiments. In all cases the synthesis of secreted proteins was only about 2-fold more sensitive to inhibition by tetracyclines than total protein synthesis. These results contrast with previously published findings in Escherichia coli which show that synthesis of secreted proteins is highly susceptible to inhibition by tetracycline. The implications of these findings in relation to inhibition of membrane bound ribosomes by tetracyclines are discussed.

Bacterial Proteins↗
PubMed1989

Penicillin-binding protein 2 is required for induction of the Citrobacter freundii class I chromosomal beta-lactamase in Escherichia coli.

Involvement of Escherichia coli penicillin-binding proteins (PBPs) in induction of the cloned ampC Citrobacter freundii beta-lactamase by 6-aminopenicillanic acid was investigated. The enzyme was not inducible at 42 degrees C in a mutant thermosensitive for expression of PBP 2. The results imply that PBP 2 is involved in the process leading to induction of ampC.

Acyltransferases↗
PubMed1988

Prediction of signal sequence-dependent protein translocation in bacteria: assessment of the Escherichia coli minicell system.

The use of phenethyl alcohol (PEA) as a probe for signal sequence-dependent protein translocation in minicells was examined. Processing of beta-lactamases and tonA was inhibited by PEA at concentrations which did not affect production of the alpha and gamma forms of penicillin binding protein (PBP) lb. The PBPlbs are believed to lack leader sequences whereas the other proteins contain them. Processing of a beta-lactamase which shares the murein-lipoprotein export pathway was relatively resistant to PEA, consistent with previous findings in whole bacteria. The results reported here suggest that PEA is a suitable probe for leader sequences in the minicell system. By using PEA we predict that PBP4 does not require a leader sequence for membrane insertion.

Bacterial Proteins↗
PubMed1988

Molecular mechanisms involved in the transport of antibiotics into bacteria.

Many clinically useful antibacterial drugs have intracellular target sites. Therefore, in order to reach their targets, these compounds must be able to cross bacterial outer and cytoplasmic membranes. Considerable information is available on the mechanisms by which antibiotics cross bacterial membranes and, in many cases, it is now possible to define the molecular basis of their uptake. Passage of drugs across the outer membrane of Gram-negative bacteria can occur by diffusion through porin channels (e.g. beta-lactams and tetracyclines), by facilitated diffusion using specific carriers (e.g. albomycin), or by self-promoted uptake (e.g. aminoglycosides and polymyxins). Transfer of antibiotics across the bacterial cytoplasmic membrane is usually mediated by active, carrier-mediated, transport systems normally operating to transport essential solutes into the cell. For example, the antibiotic streptozotocin bears sufficient structural resemblance to N-acetyl-D-glucosamine to be transported by the phosphoenolpyruvate:phosphotransferase system, and D-cycloserine is recognized by the D-alanine, proton motive force dependent transport system. However, in some cases (e.g. tetracycline) although carrier-mediated transport is implied by the observation that drug uptake is energy dependent, the nature of the membrane carrier(s) responsible is unknown. Knowledge acquired from studies on bacterial peptide transport has been successfully used to deliver (or smuggle) amino acid mimetics disguised as peptides into the bacterial cell. These amino acid mimetics, although often poorly transported in their own right, are frequently potent inhibitors of bacterial peptidoglycan or lipopolysaccharide synthesis once they have gained access to the interior of the cell.

Anti-Bacterial Agents↗
PubMed1988

Multiple isoelectric forms of bacterial penicillin-binding proteins: artefacts or genuine cellular components?

Some bacterial penicillin-binding proteins (PBPs) are reported to exist as multiple isoelectric forms. Other PBPs were examined by two-dimensional electrophoresis to establish whether multiple isoelectric forms are widespread amongst PBPs. Bacillus subtilis PBPs 3 and 5 and Escherichia coli PBPs 1b alpha and 1b gamma focussed as discrete spots with no evidence of multiple isoforms. However, E. coli PBPs 1b beta and 4 displayed isoelectric variants. The results are discussed in relation to current models of bacterial peptidoglycan structure.

Bacillus subtilis↗
PubMed1987

Inhibition of Providencia stuartii cell envelope enzymes by chlorhexidine.

The possibility that chlorhexidine is a specific inhibitor of membrane bound bacterial adenosine triphosphatase (ATPase) was addressed. The in-vitro susceptibilities of several Providencia stuartii cell envelope enzymes, including ATPase, to chlorhexidine were compared. The following concentrations of chlorhexidine were required to cause 50% inhibition of enzyme activity in preparations from chlorhexidine-sensitive strains (MIC 50 mg chlorhexidine/l): ATPase (160 mg/l), succinic dehydrogenase (greater than 300 mg/l), penicillin binding protein 7 (300 mg/l) and beta-lactamase (45 mg/l). Fifty per cent inhibition of the ATPase from a chlorhexidine-resistant strain (MIC 1600 mg/l) was achieved at an in-vitro concentration of 225 mg chlorhexidine/l. Our observations do not support the suggestion that bacterial membrane-bound ATPases are specific targets for chlorhexidine.

Adenosine Triphosphatases↗
PubMed1987

Mutations affecting penicillin-binding proteins 2a, 2b and 3 in Bacillus subtilis alter cell shape and peptidoglycan metabolism.

Bacillus subtilis mutants with altered penicillin-binding proteins (PBPs), or altered expression of PBPs, were isolated by screening for changes in susceptibility to beta-lactam antibiotics. Mutations affecting only PBPs 2a, 2b and 3 were isolated. Cell shape and peptidoglycan metabolism were examined in representative mutants. Cells of a PBP 2a mutant (UB8521) were usually twisted whereas PBP 2b (UB8524) and 3 (UB8525) mutants produced helices, particularly after growth at 41 degrees C. The PBP 2a mutant (UB8521) had a higher peptidoglycan synthetic activity than its parent strain whereas the opposite applied to the PBP 2b mutant UB8524. The PBP 3 mutant (UB8525) had a similar peptidoglycan synthetic activity to that of the parent strain when grown at 37 degrees C, but 40% higher activity after growth at 41 degrees C. The PBP 2a mutant (UB8521) exhibited the same wall thickening activity as the parent, but the PBP 2b and 3 mutants (UB8524 and UB8525) were partially defective in this respect. The changes in the susceptibility of PBP 2a, 2b and 3 mutants to beta-lactam antibiotics imply that these PBPs are killing targets, consistent with the fact that these PBPs are also important for shape determination and peptidoglycan synthesis.

Acyltransferases↗
PubMed1987

Fractionation of the Providencia stuartii cell envelope.

Cell envelopes (i.e. unfractionated inner and outer membranes) were obtained from Providencia stuartii by following procedures previously applied to the isolation of envelopes from Escherichia coli. The P. stuartii envelopes contained known inner membrane enzymes that included a variety of dehydrogenases and ATPase. The catalytic activity of the ATPase depended upon the concentration of magnesium ions, the substrate (ATP) level and the ratio of magnesium ions to ATP. Cell envelopes from P. stuartii were further fractionated to recover inner and outer membrane polypeptides by treatment with the detergent Sarkosyl. Proteins from the periplasmic region were recovered by a simple osmotic shock procedure also previously applied to E. coli. The purity of the various P. stuartii cell envelope fractions was assessed by a combination of techniques that included one- and two-dimensional gel electrophoresis of proteins, enzyme assays and detection of penicillin-binding proteins.

Bacterial Outer Membrane Proteins↗