Production of rifamycin SV using mutant strains of Amycolatopsis mediterranei MTCC17

Production of rifamycin SV using mutant strains of Amycolatopsis mediterranei (MTCC17) has been studied. Attempts were made to increase the productivity of rifamycin SV by ultraviolet radiation and specific screening programmes for selection of superior producers. Among 20 morphologically variant mutants of A. mediterranei MTCC17 isolated, four mutants showed increased resistance to the rifamycin SV. These selected mutant isolates increased the yield of rifamycin SV from 2000 to 3250mg/l.     

MoeA,an enzyme in the molybdopterin synthesis pathway,is required for rifamycin SV production in Amycolatopsis mediterranei U32

Rifamycin SV contains one amide nitrogen atom at its C(7)N moiety. Earlier labeling studies suggested that nitrogen might be incorporated from a pathway involved in a molybdenum-dependent nitrate reductase. However, no genetic evidence is available thus far. The structural gene moeA, which is involved in molybdopterin synthesis in various organisms, has been cloned from rifamycin SV-producing Amycolatopsis mediterranei strain U32. The amino acid sequence deduced from the moeA gene showed significant similarity to members of the MoeA protein family and contains all the structural features that are highly conserved in the putative functional domains of MoeA proteins. Southern hybridization showed that there is only one moeA gene in the A. mediterranei genome. To further investigate the possible physiological function of the moeA gene, a double crossover gene replacement was achieved by inserting an aparmycin resistance gene into moeA in the A. mediterranei U32 chromosome. Phenotype analysis showed that the moeA gene is required for A. mediterranei growth in a minimal medium with nitrate as sole nitrogen source, possibly because nitrate reductase activity is diminished due to disruption of the moeA gene. Compared to the wild type strain, moeA-disrupted mutants lost 95% of their rifamycin SV production capacity in complex fermentation media. The results demonstrate that the moeA gene is necessary for rifamycin SV production in A. mediterranei, and that the nitrogen assimilation pathway involved in nitrate reductase is the major pathway for the genesis of the amide nitrogen atom in the rifamycin SV molecule.     

Stimulation of microsomal production of reactive oxygen intermediates by rifamycin SV: effect of ferric complexes and comparisons between NADPH and NADH.

Rifamycins are antibacterial antibiotics which are especially useful for the treatment of tuberculosis. Reactive oxygen intermediates are produced in the presence of rifamycin SV and metals such as copper or manganese. Experiments were carried out to evaluate the interaction of rifamycin SV with rat liver microsomes to catalyze the production of reactive oxygen species. At a concentration of 1 mM, rifamycin SV increased microsomal production of superoxide with NADPH as cofactor 3-fold, and with NADH as reductant by more than 5-fold. Rifamycin SV increased rates of H2O2 production by the microsomes twofold with NADPH, and 4- to 8-fold with NADH. In the presence of various iron complexes, microsomes generated hydroxyl radical-like (.OH) species. Rifamycin SV had no effect on NADPH-dependent microsomal .OH production, irrespective of the iron chelate. A striking stimulation of .OH production was found with NADH as the reductant, ranging from 2- to 4-fold with catalyst such as ferric-EDTA and ferric-DTPA to more than 10-fold with ferric-ATP, -citrate, or -histidine. Catalase and competitive .OH scavengers lowered rates of .OH production (chemical scavenger oxidation) and prevented the stimulation by rifamycin. Superoxide dismutase had no effect on the NADH-dependent rifamycin stimulation of .OH production with ferric-EDTA or -DTPA, but was inhibitory with the other ferric complexes. In contrast to the stimulatory effects on production of O2-., H2O2, and .OH, rifamycin SV was a potent inhibitor of microsomal lipid peroxidation. These results show that rifamycin SV stimulates microsomal production of reactive oxygen intermediates, and in contrast to results with other redox cycling agents, is especially effective with NADH as the microsomal reductant. These interactions may contribute to the hepatotoxicity associated with use of rifamycin, and, since alcohol metabolism increases NADH availability, play a role in the elevated toxic actions of rifamycin plus alcohol.     

Effect of metal ion catalyzed oxidation of rifamycin SV on cell viability and metabolic performance of isolated rat hepatocytes

The effect of rifamycin SV on metabolic performance and cell viability was studied using isolated hepatocytes from fed, starved and glutathione (GSH) depleted rats. The relationships between GSH depletion, nutritional status of the cells, glucose metabolism, lactate dehydrogenase (LDH) leakage and malondialdehyde (MDA) production in the presence of rifamycin SV and transition metal ions was investigated. Glucose metabolism was impaired in isolated hepatocytes from both fed and starved animals, the effect is dependent on the rifamycin SV concentration and is enhanced by copper (II). Oxygen consumption by isolated hepatocytes from starved rats was also increased by copper (II) and a partial inhibition due to catalase was observed. Cellular GSH levels which decrease with increasing the rifamycin SV concentration were almost depleted in the presence of copper (II). A correlation between GSH depletion and LDH leakage was observed in fed and starved cells. Catalase induced a slight inhibition of the impairment of gluconeogenesis, GSH depletion and LDH leakage in starved hepatocytes incubated with rifamycin SV, iron (II) and copper (II) salts. Lipid peroxidation measured as MDA production by isolated hepatocytes was also augmented by rifamycin SV and copper (II), especially in hepatic cells isolated from starved and GSH depleted rats. Higher cytotoxicity was observed in isolated hepatocytes from fasted animals when compared with fed or GSH depleted animals. It seems likely that in addition to GSH level, there are other factors which may have an influence on the susceptibility of hepatic cells towards xenobiotic induced cytotoxicity.     

Effect of uracil on rifamycin SV production by Amycolatopsis mediterranei MV35R

The effect of different organic nitrogen compounds on the production of rifamycin SV by Amycolatopsis mediterranei MV35R and their optimum concentrations have been described. Results obtained indicate that rifamycin SV production increased from 4020 mg l-1 to 4575 mg l-1 when organic nitrogen compound uracil was added at 0.2% (w/v) concentration to the fermentation medium by A. mediterranei MV35R. The rifamycin SV yield was enhanced by 505 mg l-1 using uracil (2 g l-1) when compared with barbital.     

Genetic analysis of Staphylococcus aureus RNA polymerase mutants.

Spontaneous mutants of Staphylococcus aureus resistant to rifampin, rifamycin SV, streptovaricin, or streptolydigin were isolated and shown to be resistant due to chromosomal rather than plasmid mutations. Based on data concerning spontaneous mutation rates, genetic cotransduction rates, and in vitro sensitivity studies, four major antibiotic cross-resistance patterns were found. The genetic markers responsible for these cross-resistance patterns were shown to be separable by transduction. Nonpurified RNA polymerase activity in lysates of mutants showed the same sensitivity to these antibiotics as shown by the mutants on solid media. A model is proposed explaining possible structure-function relationships involved in the binding of these antibiotics to the RNA polymerase molecule and the mutations resulting in resistance to these antibiotics. This model includes generally overlapping but different-sized binding sites on the RNA polymerase protein coded for by similarly arranged mutable sites on the DNA.     

Modification of Rifamycin Polyketide Backbone Leads to Improved Drug Activity against Rifampicin-resistant Mycobacterium tuberculosis

Rifamycin B, a product of Amycolatopsis mediterranei S699, is the precursor of clinically used antibiotics that are effective against tuberculosis, leprosy, and AIDS-related mycobacterial infections. However, prolonged usage of these antibiotics has resulted in the emergence of rifamycin-resistant strains of Mycobacterium tuberculosis. As part of our effort to generate better analogs of rifamycin, we substituted the acyltransferase domain of module 6 of rifamycin polyketide synthase with that of module 2 of rapamycin polyketide synthase. The resulting mutants (rifAT6::rapAT2) of A. mediterranei S699 produced new rifamycin analogs, 24-desmethylrifamycin B and 24-desmethylrifamycin SV, which contained modification in the polyketide backbone. 24-Desmethylrifamycin B was then converted to 24-desmethylrifamycin S, whose structure was confirmed by MS, NMR, and X-ray crystallography. Subsequently, 24-desmethylrifamycin S was converted to 24-desmethylrifampicin, which showed excellent antibacterial activity against several rifampicin-resistant M. tuberculosis strains.     

Relationship between beta-lactamase activity and resistance to beta-lactam antibiotics in Mycobacterium smegmatis

Penicillin-susceptible mutants and beta-lactamase-negative mutants were isolated from Mycobacterium smegmatis after nitrosoguanidine mutagenesis. Both the mutants were found to be susceptible to low levels of penicillin and cephalosporins by twofold dilution testing. Clavulanic acid reduced the minimal inhibitory concentrations of beta-lactamase-labile beta-lactams for the penicillin-susceptible mutants and the parent strain, but had no effect on the susceptibility of the beta-lactamase-negative mutants. Comparison of the beta-lactamase activities found in these mutants and the parent strain indicated that there was a rough correlation between the beta-lactamase level in these organisms and their susceptibility to beta-lactams.     

Diffusion of beta-lactam antibiotics through oligomeric or monomeric porin channels of some gram-negative bacteria

The penetration of anionic beta-lactam antibiotics through porins was evaluated as a mechanism of drug resistance. The major proteins with porin activity were purified from the outer membranes of six bacteria. Three of the six porins were oligomeric porins. The molecular weights of their monomers were 37 kDa from Photobacterium damsela, 42 kDa from Serratia liquefaciens, and 36 kDa from E. coli B. The other three porins were heat-modifiable monomeric porins with molecular weights of 43 kDa from Porphyromonas asaccharolytica and Acinetobacter baumannii, and 37 kDa from Escherichia coli K12.Comparison of the six porin proteins revealed that, independent of their aggregation state, their amino acid content is similar but not identical. All have double the amount of negatively charged amino acids compared with positively charged amino acids. They have a similar polarity and polarity index. Two of the six tested bacteria do not produce beta-lactamase. These two bacteria were sensitive to the different beta-lactams tested. The other four bacteria were resistant to all or to several beta-lactams.A modified liposome swelling method was used for determining the rate of penetration of charged beta-lactam antibiotics. Zwitterionic beta-lactams were found to penetrate into liposomes at a rate that more or less fits their molecular weight, whether the porins are monomeric or oligomeric. The penetration rates of negatively charged beta-lactams are different for oligomeric and monomeric porins. Negatively charged beta-lactams penetrate through oligomeric porins better than estimated by their molecular weight, whereas monomeric porins are less penetrable to negatively charged beta-lactams than estimated by their molecular weight. The contribution of all types of porins to the susceptibility of bacteria to beta-lactam antibiotics (zwitterionic or negatively charged) is apparently doubtful. The porins may decrease or increase bacterial penetration rates to beta-lactams, and only the existence of a potential beta-lactamase that can destroy the penetrating drug will cause resistance.     

Deacylation kinetics analysis of Streptococcus pneumoniae penicillin-binding protein 2x mutants resistant to beta-lactam antibiotics using electrospray ionization- mass spectrometry

Penicillin-binding proteins (PBPs) catalyze the transpeptidase reaction involved in peptidoglycan synthesis and are covalently inhibited by the beta-lactam antibiotics. In a previous work we have focused on acylation efficiency measurements of various Streptococcus pneumoniae PBP2x* mutants to study the molecular determinants of resistance to beta-lactams. In the present paper we have developed a method to improve an accurate determination of the deacylation rate constant using electrospray ionization-mass spectrometry. This method is adaptable to the analysis of deacylation of any beta-lactam. Compared to the fluorographic technique, the ESI-MS method is insensitive to variations in the concentration of functional proteins and is therefore more reliable. We have established that the resistance of PBPs to beta-lactams is mostly due to a decrease of the acylation efficiency with only marginal effects on the deacylation rates.     

Selection of resistant bacteria at very low antibiotic concentrations

The widespread use of antibiotics is selecting for a variety of resistance mechanisms that seriously challenge our ability to treat bacterial infections. Resistant bacteria can be selected at the high concentrations of antibiotics used therapeutically, but what role the much lower antibiotic concentrations present in many environments plays in selection remains largely unclear. Here we show using highly sensitive competition experiments that selection of resistant bacteria occurs at extremely low antibiotic concentrations. Thus, for three clinically important antibiotics, drug concentrations up to several hundred-fold below the minimal inhibitory concentration of susceptible bacteria could enrich for resistant bacteria, even when present at a very low initial fraction. We also show that de novo mutants can be selected at sub-MIC concentrations of antibiotics, and we provide a mathematical model predicting how rapidly such mutants would take over in a susceptible population. These results add another dimension to the evolution of resistance and suggest that the low antibiotic concentrations found in many natural environments are important for enrichment and maintenance of resistance in bacterial populations.     

Oxygen Enhancement of bactericidal activity of rifamycin SV on Escherichia coli and aerobic oxidation of rifamycin SV to rifamycin S catalyzed by manganous ions: the role of superoxide

Oxygen enhanced the bactericidal activity of rifamycin SV to Escherichia coli K12. Anaerobically grown cells, which had a low level of superoxide dismutase, were more susceptible to the bactericidal activity than aerobically grown cells, which contained a high level of superoxide dismutase. Oxygen also enhanced the inhibition of RNA polymerase activity of rifamycin SV, when Mn2+ was used as a cofactor. Rifamycin S was reduced to rifamycin SV by NADPH catalyzed by cell-free extracts of Escherichia coli K12. These results indicate that the inhibition of bacterial growth by rifamycin SV is due to the production of active species of oxygen resulting from the oxidation-reduction cycle of rifamycin SV in the cells. The aerobic oxidation of rifamycin SV to rifamycin S was induced by metal ions, such as Mn2+, Cu2+, and Co2+. The most effective metal ion was Mn2+. In the presence of Mn2+, accompanying the consumption of 1 mol of oxygen and the oxidation of 1 mol of rifamycin SV, 1 mol of hydrogen peroxide and 1 mol of rifamycin S were formed. Superoxide was generated during the autoxidation of rifamycin SV. Superoxide dismutase inhibited the formation of rifamycin S, but scavengers for hydrogen peroxide and the hydroxyl radical did not affect the oxidation. A mechanism of Mn2+-catalyzed oxidation of rifamycin SV is proposed and its relation to bactericidal activity is discussed.     

The increased sensitivity of rifamycin-resistant mutants of Methylobacterium AM1 to a variety of antimicrobial agents

Rifamycin-resistant strains of Methylobacterium AM1 are more sensitive to β-lactam antibiotics, chloramphenicol, tetracycline, spectinomycin, puromycin, nalidixic acid, benzalkonium chloride and p -fluorophenylalanine than are rifamycin-sensitive strains. This suggests that mutation to rifamycin resistance is associated with a general increase in membrane permeability and may have a bearing on the use of rifamycin-resistant mutants in genetic and biochemical studies of this organism.     

Increase of the deacylation rate of PBP2x from Streptococcus pneumoniae by single point mutations mimicking the class A beta-lactamases.

The class A beta-lactamases and the transpeptidase domain of the penicillin-binding proteins (PBPs) share the same topology and conserved active-site residues. They both react with beta-lactams to form acylenzymes. The stability of the PBP acylenzymes results in the inhibition of the transpeptidase function and the antibiotic activity of the beta-lactams. In contrast, the deacylation of the beta-lactamases is extremely fast, resulting in a high turnover of beta-lactam hydrolysis, which confers resistance to these antibiotics. In TEM-1 beta-lactamase from Escherichia coli, Glu166 is required for the fast deacylation and occupies the same spatial location as Phe450 in PBP2x from Streptococcus pneumoniae. To gain insight into the deacylation mechanism of both enzymes, Phe450 of PBP2x was replaced by various residues. The introduction of ionizable side chains increased the deacylation rate, in a pH-dependent manner, for the acidic residues. The aspartic acid-containing variant had a 110-fold faster deacylation at pH 8. The magnitude of this effect is similar to that observed in a naturally occurring variant of PBP2x, which confers increased resistance to cephalosporins.     

Determination of antibiotic hypersensitivity among 4,000 single-gene-knockout mutants of Escherichia coli

We have tested the entire Keio collection of close to 4,000 single-gene knockouts in Escherichia coli for increased susceptibility to one of seven different antibiotics (ciprofloxacin, rifampin, vancomycin, ampicillin, sulfamethoxazole, gentamicin, or metronidazole). We used high-throughput screening of several subinhibitory concentrations of each antibiotic and reduced more than 65,000 data points to a set of 140 strains that display significantly increased sensitivities to at least one of the antibiotics, determining the MIC in each case. These data provide targets for the design of “codrugs” that can potentiate existing antibiotics. We have made a number of double mutants with greatly increased sensitivity to ciprofloxacin, and these overcome the resistance generated by certain gyrA mutations. Many of the gene knockouts in E. coli are hypersensitive to more than one antibiotic. Together, all of these data allow us to outline the cell's “intrinsic resistome,” which provides innate resistance to antibiotics.     

Production of rifamycin B and SV by free and immobilized cells of Amycolatopsis mediterranei

The effect of growth conditions (incubation time, inoculum size, initial pH value) and some nutrient concentrations on the growth and rifamycin B and SV production by free and immobilized cells of Amycolatopsis mediterranei CBS 42 575 was studied. In alginate beads, the immobilized cells behaved like the free cells, but a pronounced difference was observed in antibiotic production and cell growth. The rifamycin production by the immobilized cells was higher than that obtained by the free cells. The immobilized cells were also reused repeatedly for six batch cultivations with a fresh medium charged into flasks at the beginning of each batch. It was found that the immobilized cells were stable, and the rifamycin yield was almost constant during the first three batches and then decreased.     

等离子体作用结合氧限制模型选育利福霉素SV高产菌株

利福霉素SV毒性低、疗效高、抗菌谱广,主要由地中海拟无枝酸菌发酵生产,其发酵过程属于耗氧发酵,供氧直接影响产物形成.为减少发酵过程氧限制影响,进一步提高利福霉素发酵产量,通过构建定向氧限制模型,将常温常压等离子体诱变和无水亚硫酸钠氧限制筛选模型相结合,建立了利福霉素生产菌株24孔板快速培养的高通量筛选方法,高效选育出能...     

Role of the PhoP-PhoQ system in the virulence of Erwinia chrysanthemi strain 3937: involvement in sensitivity to plant antimicrobial peptides, survival at acid Hh, and regulation of pectolytic enzymes

Erwinia chrysanthemi is a phytopathogenic bacterium that causes soft-rot diseases in a broad number of crops. The PhoP-PhoQ system is a key factor in pathogenicity of several bacteria and is involved in the bacterial resistance to different factors, including acid stress. Since E. chrysanthemi is confronted by acid pH during pathogenesis, we have studied the role of this system in the virulence of this bacterium. In this work, we have isolated and characterized the phoP and phoQ mutants of E. chrysanthemi strain 3937. It was found that: (i) they were not altered in their growth at acid pH; (ii) the phoQ mutant showed diminished ability to survive at acid pH; (iii) susceptibility to the antimicrobial peptide thionin was increased; (iv) the virulence of the phoQ mutant was diminished at low and high magnesium concentrations, whereas the virulence of the phoP was diminished only at low magnesium concentrations; (v) in planta Pel activity of both mutant strains was drastically reduced; and (vi) both mutants lagged behind the wild type in their capacity to change the apoplastic pH. These results suggest that the PhoP-PhoQ system plays a role in the virulence of this bacterium in plant tissues, although it does not contribute to bacterial growth at acid pH.     

State of penicillin-binding proteins and requirements for their bactericidal interaction with beta-lactam antibiotics in Serratia marcescens highly resistant to extended-spectrum beta-lactams

The quantities of penicillin-binding proteins (PBPs), and sensitivity to extended-spectrum beta-lactams, were measured in isogenic strains of Serratia marcescens with high (HR) and low (LR) resistance to extended-spectrum beta-lactam antibiotics and with constitutively overproduced chromosomal beta-lactamase in the periplasm. The binding of structurally different beta-lactams to PBPs in growing resistant bacteria was determined quantitatively. In S. marcescens HR, the amounts of PBPs 3 and 6 were, respectively, 1.5 and 2 times those in strain LR and in sensitive reference strains. Sensitivities of the essential PBPs in S. marcescens LR and HR to the tested beta-lactams were identical. Only a single target, PBP 3, was highly sensitive to cefotaxime, ceftazidime and aztreonam. In contrast, three PBPs (2, 1A and 3) were highly sensitive to imipenem. In growing S. marcescens HR and LR, all antibiotics, even at fractions of their minimal growth inhibitory concentrations (MICs), bound extensively to those PBPs which were highly sensitive to them. Thus, overproduced beta-lactamase did not prevent PBP-beta-lactam interaction. Only at or above their (high) MICs did cefotaxime, ceftazidime and aztreonam bind to multiple targets. Growth inhibition of the otherwise highly resistant S. marcescens HR at the lower MIC of imipenem was correlated with the binding of this antibiotic to multiple, highly sensitive targets in the bacteria. Killing of the bacteria by inactivation of multiple targets was suggested. This assumption was supported by the synergistic killing of HR bacteria by combinations of the PBP-2-specific mecillinam with PBP-3-specific beta-lactams.     

Characterization of novel Mycobacterium tuberculosis and Mycobacterium smegmatis mutants hypersusceptible to beta-lactam antibiotics

Our laboratory previously constructed mutants of Mycobacterium tuberculosis and Mycobacterium smegmatis with deletions in the genes for their major beta-lactamases, BlaC and BlaS, respectively, and showed that the mutants have increased susceptibilities to most beta-lactam antibiotics, particularly the penicillins. However, there is still a basal level of resistance in the mutants to certain penicillins, and the susceptibilities of the mutants to some cephalosporin-based beta-lactams are essentially the same as those of the wild types. We hypothesized that characterizing additional mutants (derived from beta-lactamase deletion mutants) that are hypersusceptible to beta-lactam antibiotics might reveal novel genes involved with other mechanisms of beta-lactam resistance, peptidoglycan assembly, and cell envelope physiology. We report here the isolation and characterization of nine beta-lactam antibiotic-hypersusceptible transposon mutants, two of which have insertions in genes known to be involved with peptidoglycan biosynthesis (ponA2 and dapB); the other seven mutants have insertions which affect novel genes. These genes can be classified into three groups: those involved with peptidoglycan biosynthesis, cell division, and other cell envelope processes. Two of the peptidoglycan-biosynthetic genes (ponA2 and pbpX) may encode beta-lactam antibiotic-resistant enzymes proposed to be involved with the synthesis of the unusual diaminopimelyl linkages within the mycobacterial peptidoglycan.