Presence Of Novobiocin Research Articles

Plasmid stability of potential probiotic Lactobacillus plantarum strains in artificial gastric juice, at elevated temperature, and in the presence of novobiocin and acriflavine.

In this study, the presence of plasmids responsible for carbohydrate fermentation and antibiotic resistance and the stability of these plasmids in artificial gastric juice were investigated in 20 Lactobacillus plantarum strains with probiotic properties. Plasmid curing was performed with novobiocin, acriflavine and elevated incubation temperature to identify plasmids encoded with carbohydrate fermentation and antibiotic resistance genes and to compare them with artificial gastric juice. Plasmid profiling of the strains revealed that 100% of the strains were harbouring plasmids in varying sizes and numbers. The plasmid number of the potential probiotic strains ranged between 1 and 4, and the plasmid size ranged between 5.779 and 16.138kb. The potential probiotic strains could not survive in the artificial gastric juice at pH 2.0. Although the strains maintained their viability in an artificial gastric juice at pH 2.5 and 3.0, and their derivatives lost their plasmids at a high rate (100%). Similarly, high levels of cured derivatives were obtained with 8µg/mL novobiocin and 100µg/mL acriflavine applications, and 24h incubation at 43°C. All the experiments were also performed to compare with two L. plantarum-type strains containing plasmids responsible for tetracycline and tetracycline + erythromycin resistances. Artificial gastric juice and other plasmid curing treatments caused a high-frequency loss in the antibiotic resistances of type strains. Determining plasmid stability in artificial gastric juice is a novel approach. Plasmid stability in the gastrointestinal tract is important for maintaining the plasmid-encoded probiotic properties.

HU of Streptococcus pneumoniae Is Essential for the Preservation of DNA Supercoiling.

The histone-like protein HU is a conserved nucleoid-associated protein that is involved in the maintenance of the bacterial chromosome architecture. It is the only known nucleoid-associated protein in Streptococcus pneumoniae, but it has not been studied. The pneumococcal gene encoding this protein, hlp, is shown herein to be essential for cell viability. Its disruption was only possible either when it was duplicated in the chromosome and its expression induced from the PZn promoter, or when hlp was cloned into a plasmid under the control of the inducible Pmal promoter. In vitro assays indicated that pneumococcal HU shows a preference for binding to supercoiled DNA rather than to linear or nicked DNA. In vivo experiments in which the amount of HU was manipulated showed a relationship between the amount of HU and the level of DNA supercoiling. A twofold reduction in the amount of HU triggered a 21% increase in DNA relaxation in untreated cells. However, in cells treated with novobiocin, a drug that relaxes DNA by inhibiting DNA gyrase, a 35% increase in DNA relaxation was observed, instead of the expected 20% in cells with a constitutive HU amount. Conversely, a fourfold HU increase caused only 14% of DNA relaxation in the presence of novobiocin. Taken together, these results support an essential role for HU in the maintenance of DNA supercoiling in S. pneumoniae.

DNA supercoiling regulates the stress-inducible expression of genes in the cyanobacterium Synechocystis

Changes in the supercoiling of genomic DNA play an important role in the regulation of gene expression. We compared the genome-wide expression of genes in cells of the cyanobacterium Synechocystis sp. PCC 6803 when they were subjected to salt, cold, and heat stress, in the presence of novobiocin, an inhibitor of DNA gyrase, and in its absence. The analysis revealed that the expression of a large number of stress-inducible genes depends on the extent of genomic DNA supercoiling. The function of the two-component regulatory systems, which are known as sensors and transducers of salt, cold, and heat stress, depends on, and might be controlled by, the degree of supercoiling of the genomic DNA. These results suggest that stress-induced changes in superhelicity of genomic DNA provide an important permissive background for successful acclimatization of cyanobacterial cells to stress conditions.

Nuclear gyrB encodes a functional subunit of the Plasmodium falciparum gyrase that is involved in apicoplast DNA replication

The DNA replication machinery of the Plasmodium falciparum apicoplast is a validated drug target. Nuclear-encoded gyrase subunits are predicted to play a critical role in maintaining DNA topology during the D-loop/bi-directional ori replication process of the parasite. We show the presence of P. falciparum gyrase subunits in parasite lysates by using antibodies generated against recombinant gyrase A and B. The ATPase activity of PfGyrB was inhibited by novobiocin that also caused parasite death in culture. Reduction of apicoplast/nuclear DNA ratio in the presence of novobiocin indicated that the drug targets apicoplast DNA replication. Molecular modeling of gyrase A and B subunits revealed extensive fold conservation with the Escherichia coli counterparts as well as the presence of a long disordered loop adjacent to the ATPase domain of PfGyrB. Our results have implications for development of PfGyrB as a drug target against malaria.

Modeling and predicting the simultaneous growth of Escherichia coli O157:H7 and ground beef background microflora for various enrichment protocols.

The simultaneous growth of Escherichia coli O157:H7 (O157) and the ground beef background microflora (BM) was described in order to characterize the effects of enrichment factors on the growth of these organisms. The different enrichment factors studied were basal medium (Trypticase soy broth and E. coli broth), the presence of novobiocin in the broth, and the incubation temperature (37 degrees C or 40 degrees C). BM and O157 kinetics were simultaneously fitted by using a competitive growth model. The simple competition between the two microfloras implied that O157 growth stopped as soon as the maximal bacterial density in the BM was reached. The present study shows that the enrichment protocol factors had little impact on the simultaneous growth of BM and O157. The selective factors (i.e., bile salts and novobiocin) and the higher incubation temperature (40 degrees C) did not inhibit BM growth, and incubation at 40 degrees C only slightly improved O157 growth. The results also emphasize that when the level of O157 contamination in ground beef is low, the 6-h enrichment step recommended in the immunomagnetic separation protocol (ISO EN 16654) is not sufficient to detect O157 by screening methods. In this case, prior enrichment for approximately 10 h appears to be the optimal duration for enrichment. However, more experiments must be carried out with ground beef packaged in different ways in order to confirm the results obtained in the present study for non-vacuum- and non-modified-atmosphere-packed ground beef.

Characterizing lipopolysaccharide and core lipid A mutant O1 and O139 Vibrio cholerae strains for adherence properties on mucus-producing cell line HT29-Rev MTX and virulence in mice

Components of lipopolysaccharide (LPS), i.e. capsule, O antigen, core oligosaccharide, as well as the toxin-coregulated pili are among the factors which significantly contribute to intestinal colonization by Vibrio cholerae O1 and O139. To further address the contribution of LPS to V. cholerae virulence, we performed in vivo colonization experiments and mucus layer attachment studies with defined LPS and capsule mutants of O1 and O139. We investigated the interaction of V. cholerae strains with the differentiated human intestinal cell line HT29-Rev MTX, and found 3–5-fold reduced efficiencies for attachment by defined LPS and capsule mutants of O1 and O139 in comparison with the wild-type strains. In addition, two O1/O139-specific core oligosaccharide biosynthetic gene products, WavJ and WavD, were characterized and tested for colonization. We demonstrate that single and double knockout mutants in wavJ and wavD have an effect on core oligosaccharide biosynthesis, and that these mutants show an attenuated growth in the presence of novobiocin. Curiously, in the mouse intestinal colonization model, only the O139 wavJ,D mutants demonstrated reduced colonization.

Silencing of Glycopeptide Resistance in Enterococcus faecalis BM4405 by Novobiocin

Enterococcus faecalis BM4405-1, a susceptible derivative of the VanE-type vancomycin-resistant E. faecalis strain BM4405, was obtained after growth in the presence of novobiocin, an inhibitor of the GyrB subunit of DNA gyrase. In contrast to findings for BM4405, UDP-MurNAc-L-Ala-gamma-D-Glu-L-Lys-D-Ala-D-Ala (pentapeptide[D-Ala]) was the only peptidoglycan precursor found in BM4405-1, and no VanXY(E) D,D-peptidase or VanT serine racemase activities were detected in that strain, even after induction by subinhibitory concentrations of vancomycin. Sequencing of the vanE operon of BM4405-1 revealed two mutations leading to substitutions in VanE (D200N) and in the C-terminal amino acid of VanR(E) (Y225F). Cloning of the vanE, vanXY(E), and vanT(E) genes of BM4405-1 into the susceptible E. faecalis strain JH2-2 conferred resistance to vancomycin, indicating that the mutation in vanE was not responsible for susceptibility. Transcriptional analysis of the vanE operon in BM4405 by quantitative reverse transcription-PCR indicated that novobiocin did not affect the expression level of the vanE operon. Sequencing of the gyrB gene of BM4405-1 revealed a mutation responsible for substitution of a residue (K337Y) required for ATPase activity and thus implicated in DNA supercoiling. Cloning of the gyrB gene of BM4405 restored vancomycin resistance to BM4405-1. Taken together, these data suggest that alteration of DNA supercoiling following a mutation in GyrB was responsible for lack of expression of the vanE operon and thus for vancomycin susceptibility in BM4405-1.

Phage phi29 proteins p1 and p17 are required for efficient binding of architectural protein p6 to viral DNA in vivo.

Bacteriophage phi29 protein p6 is a viral architectural protein, which binds along the whole linear phi29 DNA in vivo and is involved in initiation of DNA replication and transcription control. Protein p1 is a membrane-associated viral protein, proposed to attach the viral genome to the cell membrane. Protein p17 is involved in pulling phi29 DNA into the cell during the injection process. We have used chromatin immunoprecipitation and real-time PCR to analyze in vivo p6 binding to DNA in cells infected with phi29 sus1 or sus17 mutants; in both cases p6 binding is significantly decreased all along phi29 DNA. phi29 DNA is topologically constrained in vivo, and p6 binding is highly increased in the presence of novobiocin, a gyrase inhibitor that produces a loss of DNA negative superhelicity. Here we show that, in cells infected with phi29 sus1 or sus17 mutants, the increase of p6 binding by novobiocin is even higher than in cells containing p1 and p17, alleviating the p6 binding deficiency. Therefore, proteins p1 and p17 could be required to restrain the proper topology of phi29 DNA, which would explain the impaired DNA replication observed in cells infected with sus1 or sus17 mutants.

Protein A gene expression is regulated by DNA supercoiling which is modified by the ArlS–ArlR two-component system of Staphylococcus aureus

Bacterial pathogens such as Staphylococcus aureus undergo major physiological changes when they infect their hosts, requiring the coordinated regulation of gene expression in response to the stresses encountered. Several environmental factors modify the expression of S. aureus virulence genes. This report shows that the expression of spa (virulence gene encoding the cell-wall-associated protein A) is down-regulated by high osmolarity (1 M NaCl, 1 M KCl or 1 M sucrose) in the wild-type strain and upregulated by novobiocin (a DNA gyrase inhibitor that relaxes DNA). A gyrB142 allele corresponding to a double mutation in the B subunit of DNA gyrase relaxed DNA and consequently induced spa expression, confirming that spa expression is regulated by DNA topology. Furthermore, in the presence of novobiocin plus 1 M NaCl, a good correlation was observed between DNA supercoiling and spa expression. The ArlS-ArlR two-component system is involved in the expression of virulence genes such as spa. Presence of an arlRS deletion decreased the effect of DNA supercoiling modulators on spa expression, suggesting that active Arl proteins are necessary for the full effect of DNA gyrase inhibitors and high osmolarity on spa expression. Indeed, evidence is provided for a relationship between the arlRS deletion and topological changes in plasmid DNA.

Novobiocin induces a distinct conformation of Hsp90 and alters Hsp90-cochaperone-client interactions.

Hsp90 functions to facilitate the folding of newly synthesized and denatured proteins. Hsp90 function is modulated through its interactions with cochaperones and the binding and hydrolysis of ATP. Recently, novobiocin has been shown to bind to a second nucleotide binding site located within the C-terminal domain of Hsp90. In this report, we have examined the effect of novobiocin on Hsp90 function in reticulocyte lysate. Novobiocin specifically inhibited the maturation of the heme-regulated eIF2alpha kinase (HRI) in a concentration-dependent manner. Novobiocin induced the dissociation of Hsp90 and Cdc37 from immature HRI, while the Hsp90 cochaperones p23, FKBP52, and protein phosphatase 5 remained associated with immature HRI. Proteolytic fingerprinting of Hsp90 indicated that novobiocin had a distinct effect on the conformation of Hsp90, and molybdate lowered the concentration of novobiocin required to alter Hsp90's conformation by 10-fold. The recombinant C-terminal domain of Hsp90 adopted a proteolytic resistant conformation in the presence of novobiocin, indicating that alteration of Hsp90/cochaperone interactions was not the cause of the novobiocin-induced protease resistance within Hsp90's C-terminal domain. The concentration dependence of this novobiocin-induced conformation change correlated with the dissociation of Hsp90 and Cdc37 from immature HRI and novobiocin-induced inhibition of Hsp90/Cdc37-dependent activation of HRI's autokinase activity. The data suggest that binding of novobiocin to the C-terminal nucleotide binding site of Hsp90 induces a change in Hsp90's conformation leading to the dissociation of bound kinase. The unique structure and properties of novobocin-bound Hsp90 suggest that it may represent the "client-release" conformation of the Hsp90 machine.

Control of bacterial motility by environmental factors in polarly flagellated and peritrichous bacteria isolated from Lake Baikal.

Despite numerous studies on bacterial motility, little is known about the regulation of this process by environmental factors in natural isolates. In this study we investigated the control of bacterial motility in response to environmental parameters in two strains isolated from the natural habitat of Lake Baikal. Morphological characterization, carbon source utilization, fermentation analysis, and sequence comparison of 16S rRNA genes showed that these strains belong to two distinct genera, i.e., Enterobacter and Pseudomonas; they were named strains 22 and Y1000, respectively. Both strains swarmed at 25 degrees C and remained motile at low temperatures (4 degrees C), especially the Pseudomonas strain, which further supports the psychrotrophic characteristics of this strain. In contrast, a strong inhibition of motility was observed at above 30 degrees C and with a high NaCl concentration. The existence of flagellar regulatory proteins FlhDC and FleQ was demonstrated in Enterobacter strain 22 and Pseudomonas strain Y1000, respectively, and environmental conditions reduced the expression of the structural genes potentially located at the first level in the flagellar cascade in both organisms. Finally, as in Enterobacter strain 22, a strong reduction in the transcription of the master regulatory gene fleQ was observed in Pseudomonas strain Y1000 in the presence of novobiocin, a DNA gyrase inhibitor, suggesting a link between DNA supercoiling and motility control by environmental factors. Thus, striking similarities observed in the two organisms suggest that these processes have evolved toward a similar regulatory mechanism in polarly flagellated and laterally flagellated (peritrichous) bacteria.

Chips with everything?

Researchers from Hoffman-La Roche have been using ‘bacteria chips’ to study the action of two currently popular antibiotics and their results suggest new directions for developing antibiotic drugs. Hans Gmuender and colleagues have taken advantage of so-called ‘gene chips’, tiny devices that monitor gene activity in a specific organism. Using a chip for Haemophilus influenzae, they monitored gene activity in the presence of novobiocin and ciprofloxacin. The two antibiotics produced clearly different gene ‘signatures’ on the bacteria chip, indicating that each affects a specific set of genes, which are distinguishable using genome technology. This technology will help in classifying and developing novel antibiotics to combat increasingly resistant bacterial strains. CKhttp://www.eurekalert.org

Novobiocin Inhibits Vaccinia Virus Replication by Blocking Virus Assembly

Novobiocin inhibits the replication of vaccinia virus in cultured BSC40 cells. All classes of viral proteins were synthesized during synchronous infection in the presence of drug. The onset of DNA replication was delayed slightly, yet the extent of DNA replication in the presence of novobiocin was comparable to that of a control infection. A delay in the temporal transition to late viral protein synthesis was in keeping with the effects on DNA replication. Although the precursor forms of the major viral structural proteins were synthesized normally at late times, the proteolytic processing of these polypeptides was inhibited, which suggested an impediment to virus assembly. Electron microscopy revealed that novobiocin blocked virus morphogenesis at an early stage. Conversion of the concatameric DNA replication intermediates into hairpin telomeres occurred in the presence of novobiocin, confirming that telomere resolution was not coupled to virus assembly. Novobiocin is the latest addition to a class of antipoxviral agents, which includes rifampin and IMCBH, that arrest morphogenesis.

Crystallization of Inhibitor Complexes of an N-Terminal 24 kDa Fragment of the DNA Gyrase B Protein

A 24 kDa N-terminal fragment of the Escherichia coli DNA gyrase B protein has been crystallized in the presence of novobiocin. One crystal form has been obtained that is orthorhombic, P 212121, with unit cell dimensions a = 40·3 Å, b = 47·7 Å, c = 111·9 Å. The asymmetric unit of this crystal form contains one molecule (Vm=2·24 Å3/Da). Complete native data have been collected to 2·5 Å resolution. This same protein fragment has also been crystallized in the presence of GR122222X, an inhibitor that is structurally related to cyclothialidine. These crystals also exhibit P212121 symmetry but have unit cell dimensions of a = 68·8 Å, b = 68·6 Å, c = 48·6 Å. The Vm value of this crystal form is 2·39 Å3/Da, assuming one molecule in the asymmetric unit, and native data have been collected to 2·0 Å resolution. Molecular replacement studies of both complexes are underway.

The effect of topoisomerase inhibitors on the expression of differentiation markers and cell cycle progression in human K-562 leukemia cells

Treatment of human K-562-J leukemia cells for 1 h with the topoisomerase II-reactive drugs VP-16, VM-26, or mAMSA resulted in a dose-dependent inhibition of proliferation and in an increase in the percentage of cells staining positive for hemoglobin, a marker of erythroid differentiation. Staining for hemoglobin of up to about 60% of the cells was observed at 20 μ M VP-16, 1 μ M VM-26, and 8 μ M mAMSA. Such treatment also caused a G2 M arrest in the cell cycle. Incubation of the cells with radiolabeled VP-16 indicated that the induced erythroid differentiation was not due to continuous cell exposure to a residual amount of the drug. VP-16-induced erythroid differentiation was also not affected by DNA, RNA, or protein synthesis inhibitors. Differentiation induction and the G2 M arrest evoked by VP-16, VM-26, and mAMSA were, however, reduced in the presence of novobiocin. Our results indicate that topo-reactive drugs that cause G2 M arrest in the K-562-J cell cycle can induce in these cells erythroid differentiation after a short and irreversible interaction with their target molecule(s).

Novobiocin Treatment Reverses Radiation-Induced Alterations in Higher-Order DNA Structure in L5178Y Nucleoids

We have studied the effect of novobiocin treatment on radiation-induced damage and its repair in higher-order DNA structure in two mouse leukemia cell lines differing in their radiosensitivity, L5178Y-R (LY-R) and L5178Y-S (LY-S). We used the fluorescent halo technique to measure alterations in the superhelical density and the topological constraints of DNA in LY-R and LY-S nucleoids. The results for untreated cells show that both cell lines reached maximal DNA unwinding at the same concentration of propidium iodide (PI), whereas LY-S nucleoids were less efficient in their ability to rewind their DNA. The loop size did not differ significantly between the cell lines. Incubation of LY-R and LY-S cells with novobiocin at a concentration which does not influence survival (0.1 mM for 45 min), but inhibits DNA synthesis in LY-R cells (by 28%) to a greater extent than in LY-S cells (by 10%), also causes more DNA unwinding in LY-R nucleoids than in LY-S nucleoids. However, a decreased superhelical density was observed in nucleoids from both cell lines. Novobiocin applied before, and present during, irradiation prevents radiation-induced alterations in DNA supercoiling more efficiently in LY-R than in LY-S cells. The presence of novobiocin during the repair period increased DNA rewinding to levels not significantly different from control values in nucleoids from both cell lines.

Molecular analysis of the Bacillus subtilis recF function

recF resides between the dnaN and gyrB genes of Bacillus subtilis. The recF15 mutation results in replacement of a glutamate residue in the wild type with a lysine residue in the mutant RecF protein. We investigated the in vivo regulation of recF using a transcriptional fusion to the xylE gene and assaying mRNA production. We found that novobiocin leads to a four-fold induction in recF gene expression, but this is not observed in a gyrB mutant strain. Enhancement of expression of the recF gene in the presence of novobiocin is unrelated to the SOS response. The RecF protein, which has a predicted molecular mass of 42.2 kDa, does not seem to be involved in its own regulation.

Evidence for the endogenous GTP‐dependent ADP‐ribosylation of the α‐subunit of the stimulatory guanyl‐nucleotide‐binding protein concomitant with an increase in basal adenylyl cyclase activity in chicken spleen cell membrane

We investigated the endogenous GTP-dependent ADP-ribosylation of the alpha-subunit of the stimulatory guanyl-nucleotide-binding protein (Gs alpha) concomitant with an increase of basal adenylyl cyclase activity in chicken spleen cell membranes. When these membranes were incubated with [adenylate-32P]NAD, there was significant incorporation of [32P]ADP-ribose into a 45-kDa acceptor protein in the membranes. This reaction was inhibited when 20 mM arginine was present during the incubation. When the membranes were incubated with unlabelled NAD, subsequent ADP ribosylation by cholera toxin was diminished significantly. Thus, chicken spleen cell membranes have the potential to endogenously ADP-ribosylate the arginine residue of Gs alpha. The endogenous ADP-ribosylation Gs alpha was enhanced by the addition of 0.1 mM GTP or 0.1 mM guanosine 5'-[gamma-thio]triphosphate (GTP[S]), but not 0.1 mM GDP, 0.1 mM ATP or 0.1 mM ADP. The endogenous GTP-dependent ADP-ribosylation of Gs alpha stimulated basal adenylyl cyclase activity. Furthermore, NAD-induced stimulation of basal adenylyl cyclase activity was suppressed, when the membranes were incubated with NAD in the presence of novobiocin, an inhibitor of arginine-specific ADP-ribosyltransferase. These data represent the first demonstration that a eukaryotic cell membrane contains an ADP-ribosyltransferase which can catalyze the endogenous GTP-dependent ADP-ribosylation of the arginine residue of Gs alpha and that this modification enhances basal adenylyl cyclase activity in the membrane. In light of this evidence, the possible control of basal adenylyl cyclase activity via endogenous GTP-dependent ADP-ribosylation in eukaryotic cells warrants further attention.

Production and release of human proinsulin by recombinant Escherichia coli immobilized in agarose microbeads

Escherichia coli cells, releasing human proinsulin, were entrapped in 2% agarose microbeads. In order to optimize production of proinsulin in shake flask cultures, a number of parameters were evaluated, including temperature, pH, initial cell loading, bead size, agarose concentration, and bead/volume ratios. Maximum production was about 2 mg l −1 day −1. Production decreased by over 70% per day. Storage stability, t 0.5, was 6 and 10 days at 21 and 4°C, respectively, with regard to proinsulin production. Extensive protein and cell release was observed after induction. Cell leakage decreased as the agarose concentration, bead size, and initial cell loading increased. In addition, cell leakage (free cell growth) could be reduced by decreasing the temperature or the shaking rate. In the latter case, proinsulin production increased. Cell leakage (growth) was also decreased by adding novobiocin, nalidixic acid, or immobilized polymyxin. In the presence of novobiocin, proinsulin production increased.

Suppressive effect of novobiocin on the frequency of chromosome-type aberrations induced by ara C in the G1 phase of human lymphocytes

1-beta-D-Arabinofuranosylcytosine (ara C) induces chromosome-type aberrations in mammalian cells by inhibiting repair replication in the G1 phase. The effect of novobiocin, an inhibitor of prokaryotic gyrases, on G1 repair in human cells was studied cytogenetically using this characteristic of ara C. The experiment was based on the assumption that if novobiocin inhibits the relaxation of chromatin required prior to repair replication, it would reduce the frequency of chromosome-type aberrations in cells treated with a mutagen followed by posttreatment with ara C. It has also been shown that in lymphocytes ara C induces chromosome-type aberrations which were not caused by any induced DNA lesion, and that the frequency of these aberrations changes with the age of the blood donor. The effect of novobiocin on the frequency of chromosome-type aberrations induced by ara C in lymphocytes without mutagen pretreatment was also investigated for blood samples from donors of different ages. Human peripheral blood lymphocytes, which were either untreated of treated with 4-nitroquinoline-N-oxide (4NQO) or methyl methanesulfonate (MMS), were posttreated in their early G1 phase with ara C only or ara C and novobiocin. The resulting chromosome-type aberrations were observed in cells in their first mitoses, and a comparison was made between the frequency of aberrations occurring in the presence of novobiocin and in its absence. The results showed that novobiocin reduced the frequency of chromosome-type aberrations induced by ara C in both mutagen-pretreated and -non-pretreated cells, and that lymphocytes from younger donors were less sensitive to novobiocin. The present study demonstrated cytogenetically the existence of a novobiocin-sensitive process to induce chromosome recombination in G1 lymphocytes.