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K Drlica

Publications and source records attributed to K Drlica.

At least 37 records · Page 2Linked to original sources
PubMed1999

Cytotoxic hammerhead ribozymes.

Small catalytic RNA molecules of the hammerhead ribozyme type were found to have cytotoxic effects unrelated to their intended activity. An expression library of ribozyme sequence variants was constructed in a recA-deficient strain of Escherichia coli such that individual library members differed in regions designed to form base pairs with human immunodeficiency virus-1 (HIV-1) tat mRNA. The parental ribozyme and many variants exhibited a bacteriostatic effect. One variant studied in detail was also bactericidal. When its expression was induced, ribozyme-dependent inhibition of bacterial growth was not observed in recA+ or recA+ lexA3 (Ind-) cells, suggesting that the recombination function of the RecA protein, not the absence of the SOS response, is sufficient to alleviate the cytotoxic effect. These data document the need for careful testing for toxic effects during intracellular studies of ribozyme action.

Base Sequence↗
PubMed1999

Gatifloxacin activity against quinolone-resistant gyrase: allele-specific enhancement of bacteriostatic and bactericidal activities by the C-8-methoxy group.

Antibacterial activities of gatifloxacin (AM1155), a new C-8-methoxy fluoroquinolone, and two structurally related compounds, AM1121 and ciprofloxacin, were studied with an isogenic set of ten quinolone-resistant, gyrA (gyrase) mutants of Escherichia coli. To compare the effect of each mutation on resistance, the mutant responses were normalized to those of wild-type cells. Alleles exhibiting the most resistance to growth inhibition mapped in alpha-helix 4, which is thought to lie on a GyrA dimer surface that interacts with DNA. The C-8-methoxy group lowered the resistance due to these mutations more than it lowered resistance arising from several gyrA alleles located outside alpha-helix 4. These data are consistent with alpha-helix 4 being a distinct portion of the quinolone-binding site of GyrA. A helix change to proline behaved more like nonhelix alleles, indicating that helix perturbation differs from the other changes at helix residues. Addition of a parC (topoisomerase IV) resistance allele revealed that the C-8-methoxy group also facilitated attack of topoisomerase IV. When lethal effects were measured at a constant multiple of the minimum inhibitory concentration for each fluoroquinolone to normalize for differences in bacteriostatic action, gatifloxacin was more potent than the C-8-H compounds, both in the presence and absence of protein synthesis (an exception was observed when alanine was substituted for aspartic acid at position 82). Collectively, these data show that the C-8-methoxy group contributes to the enhanced activity of gatifloxacin against resistant gyrase and wild-type topoisomerase IV.

Alleles↗
PubMed1999

Fluoroquinolone action against clinical isolates of Mycobacterium tuberculosis: effects of a C-8 methoxyl group on survival in liquid media and in human macrophages.

When the lethal action of a C-8 methoxyl fluoroquinolone against clinical isolates of Mycobacterium tuberculosis in liquid medium was measured, the compound was found to be three to four times more effective (as determined by measuring the 90% lethal dose) than a C-8-H control fluoroquinolone or ciprofloxacin against cells having a wild-type gyrA (gyrase) gene. Against ciprofloxacin-resistant strains, the C-8 methoxyl group enhanced lethality when alanine was replaced by valine at position 90 of the GyrA protein or when aspartic acid 94 was replaced by glycine, histidine, or tyrosine. During infection of a human macrophage model by wild-type Mycobacterium bovis BCG, the C-8 methoxyl group lowered survival 20- to 100-fold compared with the same concentration of a C-8-H fluoroquinolone. The C-8 methoxyl fluoroquinolone was also more effective than ciprofloxacin against a gyrA Asn94 mutant of M. bovis BCG. In an M. tuberculosis-macrophage system the C-8 methoxyl group improved fluoroquinolone action against both quinolone-susceptible and quinolone-resistant clinical isolates. Thus, a C-8 methoxyl group enhances the bactericidal activity of quinolones with N1-cyclopropyl substitutions; these data encourage further refinement of fluoroquinolones as antituberculosis agents.

Anti-Infective Agents↗
PubMed1999

Effect of fluoroquinolone concentration on selection of resistant mutants of Mycobacterium bovis BCG and Staphylococcus aureus.

When Mycobacterium bovis BCG and Staphylococcus aureus were plated on agar containing increasing concentrations of fluoroquinolone, colony numbers exhibited a sharp drop, followed by a plateau and a second sharp drop. The plateau region correlated with the presence of first-step resistant mutants. Mutants were not recovered at concentrations above those required for the second sharp drop, thereby defining a mutant prevention concentration (MPC). A C-8-methoxy group lowered the MPC for an N-1-cyclopropyl fluoroquinolone.

Anti-Infective Agents↗
PubMed1998

Topoisomerase I of Helicobacter pylori: juxtaposition with a flagellin gene (flaB) and functional requirement of a fourth zinc finger motif.

Cloning and nucleotide sequence analysis showed that in Helicobacter pylori the gene encoding topoisomerase I (topA) lies about 170 nucleotides upstream from flaB, a gene encoding one of the two flagellin proteins that is required for virulence. The topA and flaB genes are divergently transcribed. The orientation and spatial relationship between flaB and topA are remarkably conserved among strains of a bacterium in which genomic rearrangements are common. The deduced amino acid sequence of topoisomerase I revealed four zinc finger motifs, one more than has been reported previously for the Escherichia coli homologue. The additional motif, which is near the C-terminus of the protein, appears to be essential for function since mutations in that region are lethal. These data show that TopA proteins can be divided into several classes on the basis of zinc finger motifs and raise the interesting possibility that the H. pylori enzyme has local topological effects focussed on a flagellin gene.

Bacterial Proteins↗
PubMed1998

Degradation of hammerhead ribozymes by human ribonucleases.

Hammerhead ribozymes were used as substrates to examine endoribonucleolytic activities in cell extracts and cultured human cells. Primer-extension analyses showed that ribozymes directed against tumor necrosis factor-alpha mRNA and human immunodeficiency virus type 1 tat mRNA were cleaved at UA and CA dinucleotides by extracts. Preferred cleavage sites were similar to those observed following digestion with RNase A, and cleavage was blocked by RNasin, an inhibitor of pyrimidine-specific ribonucleases. Removal of UA and CA dinucleotides rendered ribozymes more stable when incubated in cell extracts that were not significantly contaminated by extracellular nucleases. Placement of UA dinucleotides adjacent to a ribozyme in mRNA led to excision of the ribozyme from long transcripts during incubation in extracts. UA dinucleotides also made mRNA more labile than a control RNA when expressed from an endogenous plasmid gene in the human myeloid cell line U937. Similarly, UA and CA dinucleotides caused ribozymes to have a shorter half-life when delivered to U937 cells by lipofectin-mediated transformation. Taken together, these data indicate that one or more members of the pyrimidine-specific ribonuclease family is involved in the intracellular degradation of RNA, and they explain the paucity of UA dinucleotides in eukaryotic mRNA. Judicious manipulation of preferred target sequences of pyrimidine-specific ribonucleases may be useful in designing effective hammerhead ribozymes.

Base Sequence↗
PubMed1998

Fluoroquinolone action against mycobacteria: effects of C-8 substituents on growth, survival, and resistance.

Fluoroquinolones trap gyrase on DNA as bacteriostatic complexes from which lethal DNA breaks are released. Substituents at the C-8 position increase activities of N-1-cyclopropyl fluoroquinolones against several bacterial species. In the present study, a C-8-methoxyl group improved bacteriostatic action against gyrA (gyrase-resistant) strains of Mycobacterium tuberculosis and M. bovis BCG. It also enhanced lethal action against gyrase mutants of M. bovis BCG. When cultures of M. smegmatis, M. bovis BCG, and M. tuberculosis were challenged with a C-8-methoxyl fluoroquinolone, no resistant mutant was recovered under conditions in which more than 1, 000 mutants were obtained with a C-8-H control. A C-8-bromo substituent also increased bacteriostatic and lethal activities against a gyrA mutant of M. bovis BCG. When lethal activity was normalized to bacteriostatic activity, the C-8-methoxyl compound was more bactericidal than its C-8-H control, while the C-8-bromo fluoroquinolone was not. The C-8-methoxyl compound was also found to be more effective than the C-8-bromo fluoroquinolone at reducing selection of resistant mutants when each was compared to a C-8-H control over a broad concentration range. These data indicate that a C-8-methoxyl substituent, which facilitates attack of first-step gyrase mutants, may help make fluoroquinolones effective antituberculosis agents.

Anti-Infective Agents↗
PubMed1998

Role of superoxide in catalase-peroxidase-mediated isoniazid action against mycobacteria.

Isoniazid (INH) activation in vitro is associated with reduction of the mycobacterial ferric KatG catalase-peroxidase by hydrazine and reaction with O2 to form an oxyferrous enzyme complex. Since this complex could also form directly via reaction of ferric KatG with superoxide, intracellular activation might be responsive to superoxide concentration. When Mycobacterium smegmatis carrying the M. bovis katG gene was treated with nontoxic levels of plumbagin, a generator of superoxide, the bacteriostatic activity of INH increased unless a plasmid-borne superoxide dismutase gene was also present. Thus, endogenous superoxide probably contributes to intracellular activation of INH.

Antitubercular Agents↗
PubMed1998

Killing of Staphylococcus aureus by C-8-methoxy fluoroquinolones.

C-8-methoxy fluoroquinolones were more lethal than C-8-bromine, C-8-ethoxy, and C-8-H derivatives for Staphylococcus aureus, especially when topoisomerase IV was resistant. The methoxy group also increased lethality against wild-type cells when protein synthesis was inhibited. These properties encourage refinement of C-8-methoxy fluoroquinolones to kill staphylococci.

Anti-Bacterial Agents↗
PubMed1997

DNA topoisomerase targets of the fluoroquinolones: a strategy for avoiding bacterial resistance.

Fluoroquinolones are antibacterial agents that attack DNA gyrase and topoisomerase IV on chromosomal DNA. The existence of two fluoroquinolone targets and stepwise accumulation of resistance suggested that new quinolones could be found that would require cells to obtain two topoisomerase mutations to display resistance. For wild-type cells to become resistant, the two mutations must be acquired concomitantly. That is expected to occur infrequently. To identify such compounds, fluoroquinolones were tested for the ability to kill a moderately resistant gyrase mutant. Compounds containing a C8-methoxyl group were particularly lethal, and incubation of wild-type cultures on agar containing C8-methoxyl fluoroquinolones produced no resistant mutant, whereas thousands arose during comparable treatment with control compounds lacking the C8 substituent. When the test strain contained a preexisting topoisomerase IV mutation, which by itself conferred no resistance, equally high numbers of resistant mutants were obtained for C8-methoxyl and control compounds. Thus C8-methoxyl fluoroquinolones required two mutations for expression of resistance. Although highly lethal, C8-methoxyl fluoroquinolones were not more effective than C8-H controls at blocking bacterial growth. Consequently, quinolone action involves two events, which we envision as formation of drug-enzyme-DNA complexes followed by release of lethal double-strand DNA breaks. Release of DNA breaks, which must occur less frequently than complex formation, is probably the process stimulated by the C8-methoxyl group. Understanding this stimulation should provide insight into intracellular quinolone action and contribute to development of fluoroquinolones that prevent selection of resistant bacteria.

Anti-Infective Agents↗
PubMed1997

Monovalent cations differ in their effects on transcription initiation from a sigma-70 promoter of Escherichia coli.

Initiation of transcription from the sigma-70 rep promoter of plasmid pBR322 was measured by abortive transcription assays at various concentrations of potassium, rubidium, and sodium acetate. When linear and negatively supercoiled templates were compared, each salt generated a characteristic response. Increasing the salt concentration decreased transcription from a linear template but produced an increase (potassium) or a bell-shaped response (rubidium) with a supercoiled template. In the case of sodium ions, increasing concentration inhibited transcription initiation from both linear and supercoiled templates. These results are discussed with respect to effects of monovalent cations on DNA twist.

Base Sequence↗
PubMed1997

DNA gyrase, topoisomerase IV, and the 4-quinolones.

For many years, DNA gyrase was thought to be responsible both for unlinking replicated daughter chromosomes and for controlling negative superhelical tension in bacterial DNA. However, in 1990 a homolog of gyrase, topoisomerase IV, that had a potent decatenating activity was discovered. It is now clear that topoisomerase IV, rather than gyrase, is responsible for decatenation of interlinked chromosomes. Moreover, topoisomerase IV is a target of the 4-quinolones, antibacterial agents that had previously been thought to target only gyrase. The key event in quinolone action is reversible trapping of gyrase-DNA and topoisomerase IV-DNA complexes. Complex formation with gyrase is followed by a rapid, reversible inhibition of DNA synthesis, cessation of growth, and induction of the SOS response. At higher drug concentrations, cell death occurs as double-strand DNA breaks are released from trapped gyrase and/or topoisomerase IV complexes. Repair of quinolone-induced DNA damage occurs largely via recombination pathways. In many gram-negative bacteria, resistance to moderate levels of quinolone arises from mutation of the gyrase A protein and resistance to high levels of quinolone arises from mutation of a second gyrase and/or topoisomerase IV site. For some gram-positive bacteria, the situation is reversed: primary resistance occurs through changes in topoisomerase IV while gyrase changes give additional resistance. Gyrase is also trapped on DNA by lethal gene products of certain large, low-copy-number plasmids. Thus, quinolone-topoisomerase biology is providing a model for understanding aspects of host-parasite interactions and providing ways to investigate manipulation of the bacterial chromosome by topoisomerases.

Bacteria↗
PubMed1996

Hammerhead ribozyme structure probed by cell extracts.

To examine hammerhead ribozyme structure and vulnerability to cellular nucleases, ribozymes were incubated with soluble extracts from Escherichia coli, and cleavage sites were identified by primer extension analysis. Mapping of endonuclease-sensitive sites revealed that the most sensitive were in the 3'-substrate-binding region of the ribozyme. The catalytic domain was much less susceptible, although some cleavage preference was seen at two positions known to be twisted out of parallel stacking in a ribozyme-substrate analogue complex. Changes in substrate-binding domain nucleotide sequence had no effect on cleavage patterns of catalytic domains. Hammerhead ribozymes, in solution and free from substrate, appear to have structurally independent, asymmetrically arranged domains.

Base Sequence↗
PubMed1996

DNA gyrase and topoisomerase IV on the bacterial chromosome: quinolone-induced DNA cleavage.

DNA gyrase, the bacterial enzyme that supercoils DNA, is trapped on chromosomal DNA by the 4-quinolone compounds, as drug-gyrase complexes that contain DNA breaks. Examination of chromosomal DNA extracted from Escherichia coli indicated that bacteriostatic concentrations of oxolinic acid trap gyrase and block DNA synthesis without releasing broken DNA from gyrase-DNA complexes. Release, detected as free rotation of DNA in the presence of an intercalating dye, occurred only at high, bactericidal oxolinic acid concentrations. Release of DNA breaks and cell death were both blocked by chloramphenicol, an inhibitor of protein synthesis, suggesting that synthesis of additional protein activity is required to free the DNA ends. Ciprofloxacin, a more potent quinolone, released DNA breaks and killed cells even in the presence of chloramphenicol. It is proposed that this second, chloramphenicol-insensitive mode for release of DNA breaks and cell killing arises from dissociation of gyrase subunits. Ciprofloxacin also killed a gyrase (gyrA) mutant resistant to the prototype of quinolone, nalidixic acid, and created complexes on DNA detected by DNA fragmentation. This lethal effect of ciprofloxacin was eliminated by additional mutations mapping in parC, one of the two genes encoding topoisomerase IV. Thus, the fluoroquinolone compounds have two intracellular targets. In the absence of the gyrA mutation, the parC (CipR) allele did not by itself confer resistance to ciprofloxacin, indicating that gyrase is the major quinolone target in E. coli. These findings provide a molecular explanation for quinolone action in bacteria and a new way to study topoisomerase IV-chromosome interactions.

Anti-Infective Agents↗
PubMed1996

Cross-talk between topoisomerase I and HU in Escherichia coli.

In Escherichia coli about one half of the negative supercoiling of DNA is constrained by proteins, in contrast to the situation in eukaryotic cells where most of the DNA is constrained by histones. The level of supercoiling in the unrestrained portion is controlled by a balance between the supercoiling activity of gyrase and the relaxing activity of DNA topoisomerase I. In the present work we show, by disrupting one or both genes encoding the heterodimeric protein HU, that an interplay exists in bacteria between HU and topoisomerase I activity: a decrease in the intracellular concentration of HU was accompanied by an increase in relaxing activity as measured in cell extracts. Conversely, a topA10 mutant of topoisomerase I, which has low levels of relaxing activity, was unable to accept an HU deficiency introduced by transduction. Thus it appears that the ability to increase relaxing activity, or to decrease an excess of supercoiling, is important for cells to survive in the absence of HU. These data can be explained in terms of HU constraining supercoiling in vivo as it does in vitro: the absence of HU would generate more unconstrained supercoiling, which in turn would require an increase in relaxing activity to maintain physiological levels.

Bacterial Proteins↗
PubMed1996

Histone-like protein HU and bacterial DNA topology: suppression of an HU deficiency by gyrase mutations.

The abundant bacterial protein called HU has the ability to wrap and bend DNA in vitro, and thus it has long been thought to play a role in DNA supercoiling. In the absence of HU, Escherichia coli formed tiny colonies on agar, rapidly accumulated suppressor mutations, and was hypersensitive to novobiocin. Three types of evidence implicated gyrase in the suppression of an HU deficiency. First, spontaneous suppressors that restored normal growth and reduced sensitivity to novobiocin mapped in gyrB, one of the genes encoding DNA gyrase. Second, a pair of known gyrB mutations (gyrB-203 Ts gyrB-221 NovR) allowed normal growth at permissive (30 degrees C) but not at intermediate (37 degrees C) conditions. Third, introduction of a gyrB-expressing plasmid restored normal colony size. DNA supercoiling comparisons showed that chromosomal supercoiling decreased in the absence of HU and increased toward wild-type levels in the presence of a spontaneous gyrB suppressor. Taken together, these data establish that HU has a physiological role in chromosomal DNA topology, probably by facilitating the action of gyrase.

Alleles↗
PubMed1996

Fluoroquinolone resistance associated with specific gyrase mutations in clinical isolates of multidrug-resistant Mycobacterium tuberculosis.

Fluoroquinolones are potent antibacterial agents being used clinically against multidrug-resistant tuberculosis. Treatment failure is thought to arise from acquisition of fluoroquinolone resistance by Mycobacterium tuberculosis. A collection of 13 resistant clinical isolates of M. tuberculosis was examined for ciprofloxacin sensitivity relative to controls exhibiting the same IS6110 DNA type. Specific alleles were associated with distinct levels of drug susceptibility for 11 isolates that contained nucleotide changes expected to alter the amino acid sequence of the A subunit of DNA gyrase. Five different gyrA (ciprofloxacin resistance) alleles were present among 7 isolates having the W DNA subtype. These isolates, which are representative of an outbreak strain, constitute a panel of organisms that can be used to evaluate contributions of gyrase and DNA topoisomerase IV to resistance.

Anti-Infective Agents↗