Rif Mutants Research Articles

Unveiling the molecular basis for pleiotropy in selected rif mutants of Escherichia coli: Possible role for Tyrosine in the Rif binding pocket and fast movement of RNA polymerase

Rifampicin (RIF) is still a first line of antibiotic in the treatment of bacterial diseases, in particular the Mycobacterial infections. The antimicrobial activity of RIF is attributed to its ability to inhibit transcription by binding to the β subunit of bacterial RNA polymerase (encoded by rpoB). Continued use of this drug resulted in the emergence of RIF resistant rpoB mutations in a high frequency that compels the use of RIF almost exclusively in drug combinations. As of date, a broad array of rif mutations have been isolated and characterized by different research groups. Studies on rpoB mutations strengthen the view that the β subunit of RNA polymerase (RNAP) is very crucial in modulating transcription thereby leading to differential gene expression. Very recently we have reported the transcriptome profile of rpoB12 mutant that provides molecular evidence that presence of rpoB12 mutation modulates the transcription of about 450 genes. Here we present a maiden report that rpoB mutations that substitute Tyr at the Rif binding pocket (RBP) of β subunit of RNA polymerase are able to suppress the over-production of colanic acid capsular polysaccharide (Ces phenotype) in Δlon mutant of Escherichia coli. Further analyses of the rif mutants involving their growth pattern on LB at higher temperature (42 °C), LB media without NaCl, survival in LB media with acidic pH (pH – 3) and motility revealed that only rpoB12 (His526Tyr) and rpoB137 (Ser522Tyr) affected all the above mentioned physiological parameters in addition to the elicitation of Ces phenotype. These two rif mutations confer fast movement to RNAP and they bear Tyr as the substituted amino acid in the RBP. This is perhaps the first study that brings out the possible role of Tyr in the RBP and its participation in the global gene expression. This study also envisages the point that amino acid residues that share the properties of Tyr in the RBP can be employed as a tool to bring out differential gene expression which would certainly have basic and applied values for the mankind.

Discovery of an Antibacterial Isoindolinone-Containing Tetracyclic Polyketide by Cryptic Gene Activation and Characterization of Its Biosynthetic Gene Cluster.

The major setback in natural product screening is the decreasing hit rate of novel bioactive compounds containing new chemical skeletons. Here we report the identification and biosynthesis of isoindolinomycin (Idm), an unprecedented bioactive polyketide with a novel isoindolinone-containing tetracyclic skeleton. Idm was discovered through the screening of rifampicin-resistant ( rif) mutants that were generated from nine actinomycete strains used in this study. Of the 114 rif mutants isolated, the mutant S55-50-5 was found to overproduce Idm, which is almost undetectable in the wild-type Streptomyces sp. SoC090715LN-16. An in silico analysis coupled with gene deletion experiments revealed a biosynthetic idmB gene cluster that is responsible for the production of Idm. The biosynthetic studies of Idm primarily focused on the formation of the five-membered ring in the tetracyclic structure and the attachment of the methyl group to the core structure. In addition, a malachite green phosphate assay performed using a stand-alone adenylation domain ( idmB21) demonstrated the involvement of glycine in the formation of the isoindolinone-containing skeleton. This study contributes to an increase in the structural diversity of polyketides and paves the way toward an understanding of the complete biosynthetic pathway of a novel class of tetracyclic polyketides.

Methylbenzene-Containing Polyketides from a Streptomyces that Spontaneously Acquired Rifampicin Resistance: Structural Elucidation and Biosynthesis.

Conventional screening for novel bioactive compounds in actinomycetes often results in the rediscovery of known compounds. In contrast, recent genome sequencing revealed that most of the predicted gene clusters for secondary metabolisms are not expressed under standard cultivation conditions. To explore the potential metabolites produced by these gene clusters, we implemented a cryptic gene activation strategy by screening mutants that acquire resistance to rifampicin. The induction of rifampicin resistance in 11 actinomycete strains generated 164 rifampicin-resistant mutants (rif mutants). The comparison of the metabolic profiles between the rif mutants and their wild-type strains indicated that one mutant (TW-R50-13) overproduced an unidentified metabolite (1). During the isolation and structural elucidation of metabolite 1, an additional metabolite was found; both are unprecedented compounds featuring a C5N unit and a methylbenzene moiety. Of these partial structures, the biosynthesis of the latter has not been reported. A feeding experiment using (13)C-labeled precursors demonstrated that the methylbenzene moiety is most likely synthesized by the action of polyketide synthase. The gene deletion experiments revealed that the genes for the methylbenzene moiety are located at a different locus than the genes for the C5N unit.

Mutational Improvement of <i>Lactobacillus acidophilus</i> GH 201 Intended for Ground Beef Preservation in Refrigerator Storage

Lactic acid bacteria widely used in food preservation at refrigerator temperatures due to their ability produce high amount of hydrogen peroxide and/or other antibacterial substances at refrigerator temperatures to inhibit food-borne pathogens and psychrophilic spoilage microorganisms. In order to improve of bio-preservation efficacy of Lactobacillus acidophilus GH 201 mutations causing resistance to rifampicin (rif) were used. Among UV-mutagenized population of L. acidophilus five Rif mutants producing high amounts of H2O2 were selected. Rif mutants produced significant amounts of hydrogen peroxide 50-55 μg/ml in sodium phosphate buffer (0.2 M, pH 6.5) and in beef broth (BB) at 5°C for 5 days submerged cultivation without of growth. The mutants possess higher impact against food-borne pathogen Escherichia coli O157: H7 at refrigeration temperatures and for 3 days reduces the pathogen total amount practically undetectable level. Rif mutants L. acidophilus reduced initial amount 2x10<sup5 of E. coli O157: H7 in ground beef up to 3 log for 3 days of solid-state cocultivation when the wild strain reduced only 2 log. The application of L. acidophilus GH 201 mutants did not cause any changes in sensory characteristics of ground beef, moreover promotes expanding of shelf-life due to inhibition of psychrophilic spoilage microorganisms.

Plasmid pUPI126-encoded pyrrolnitrin production by Acinetobacter haemolyticus A19 isolated from the rhizosphere of wheat

An Acinetobacter species identified as A. haemolyticus A19 produces an antibiotic and the enzyme chitinase. The antibiotic produced by A. haemolyticus A19 was extracellular and inducible by co-cultivation with Klebsiella pneumoniae in the optimum ratio 2:1, respectively. pH 7, temperature 28 °C, and addition of 2% (w/v) NaCl are the most suitable environmental conditions for production and activity of the antibiotic. The antibiotic was produced in the early stationary growth phase (48 h) of A. haemolyticus A19. It has a very broad spectrum of antimicrobial activity against plant and human pathogenic bacteria and fungi. The antibiotic was extracted with ethyl acetate and purified by column chromatography with further purification by preparative thin-layer chromatography. Yield of the antibiotic was 15 mg/l. The antibiotic was active at very low concentrations, for example 50 μg/ml, and was water-soluble. It was stable at room temperature for up to 7 days. (1)H NMR analysis revealed the antibiotic was a pyrrolnitrin. It was found that pyrrolnitrin production by A. haemolyticus A19 was encoded by plasmid pUPI126 of molecular weight 25.7 kb. Plasmid pUPI126 was transferred to E. coli HB101 at a frequency of 5 × 10(-5) per μg DNA. It was also conjugally transformed to E. coli HB101 rif (r) mutants at a frequency of 5.9 × 10(-8) per recipient cell. Plasmid pUPI126 was 100% stable in Acinetobacter and 95% stable in E. coli HB101. Transconjugants and transformants both produced the antibiotic. This is the first report of plasmid-mediated pyrrolnitrin production by A. haemolyticus A19 isolated from wheat rhizosphere.

Antibiotic Resistance Acquired through a DNA Damage-Inducible Response in Acinetobacter baumannii

Acinetobacter baumannii is an emerging nosocomial, opportunistic pathogen that survives desiccation and quickly acquires resistance to multiple antibiotics. Escherichia coli gains antibiotic resistances by expressing genes involved in a global response to DNA damage. Therefore, we asked whether A. baumannii does the same through a yet undetermined DNA damage response akin to the E. coli paradigm. We found that recA and all of the multiple error-prone DNA polymerase V (Pol V) genes, those organized as umuDC operons and unlinked, are induced upon DNA damage in a RecA-mediated fashion. Consequently, we found that the frequency of rifampin-resistant (Rif(r)) mutants is dramatically increased upon UV treatment, alkylation damage, and desiccation, also in a RecA-mediated manner. However, in the recA insertion knockout strain, in which we could measure the recA transcript, we found that recA was induced by DNA damage, while uvrA and one of the unlinked umuC genes were somewhat derepressed in the absence of DNA damage. Thus, the mechanism regulating the A. baumannii DNA damage response is likely different from that in E. coli. Notably, it appears that the number of DNA Pol V genes may directly contribute to desiccation-induced mutagenesis. Sequences of the rpoB gene from desiccation-induced Rif(r) mutants showed a signature that was consistent with E. coli DNA polymerase V-generated base-pair substitutions and that matched that of sequenced A. baumannii clinical Rif(r) isolates. These data strongly support an A. baumannii DNA damage-inducible response that directly contributes to antibiotic resistance acquisition, particularly in hospitals where A. baumannii desiccates and tenaciously survives on equipment and surfaces.

Study of the Rifampin Monoresistance Mechanism in Mycobacterium tuberculosis

Rifampin (RIF) susceptibility is a key factor in determining the treatment effectiveness of the standardized treatment regimens. In Mycobacterium tuberculosis, both target gene mutation and the efflux pump play major roles in the resistance to antituberculosis drugs. By eliminating RIF-resistant strains with rpoB mutation, the choice of RIF-monoresistant strains may allow us to identify the RIF-specific efflux pump genes. This study explored the RIF monoresistance mechanism in M. tuberculosis. Data from DNA sequencing and MIC measurements revealed that specific mutations, including Ser531Leu and His526Asp in RpoB, show high-level drug resistance. Three-dimensional structure modeling provided further evidence that the affinity between RIF and RpoB mutants was in accordance with the drug resistance level of the corresponding isolates. Furthermore, transcription-level analysis among the nonmutated isolates indicated that three efflux pumps (Rv0783, Rv2936, and Rv0933) might be involved in exporting RIF from the cell. Compared to 8 μg/ml for wild-type Escherichia coli, the MICs for the transgenic E. coli strains with either Rv0783 or Rv2936 were 32 and 16 μg/ml, respectively. In conclusion, our study indicated that several RpoB mutant types, including Ser531Leu and His526Asp, show high-level RIF resistance attributed to low affinity between RpoB mutant proteins and RIF. In addition, this work demonstrates that Rv2936 and Rv0783 may be responsible for low-level resistance to RIF by exporting RIF from cells. The predicted structure of RpoB and the newly identified efflux pumps in this study will provide a novel approach to design new drugs and develop novel diagnosis technologies.

Role of altered rpoB alleles in Bacillus subtilis sporulation and spore resistance to heat, hydrogen peroxide, formaldehyde, and glutaraldehyde

Mutations in the RNA polymerase β-subunit gene rpoB causing resistance to rifampicin (Rif(R)) in Bacillus subtilis were previously shown to lead to alterations in the expression of a number of global phenotypes known to be under transcriptional control. To better understand the influence of rpoB mutations on sporulation and spore resistance to heat and chemicals, cells and spores of the wild-type and twelve distinct congenic Rif(R) mutant strains of B. subtilis were tested. Different levels of glucose catabolite repression during sporulation and spore resistance to heat and chemicals were observed in the Rif(R) mutants, indicating the important role played by the RNA polymerase β-subunit, not only in the catalytic aspect of transcription, but also in the initiation of sporulation and in the spore resistance properties of B. subtilis.

Phenotypes and gene expression profiles of Saccharopolyspora erythraea rifampicin-resistant (rif) mutants affected in erythromycin production

BackgroundThere is evidence from previous works that bacterial secondary metabolism may be stimulated by genetic manipulation of RNA polymerase (RNAP). In this study we have used rifampicin selection as a strategy to genetically improve the erythromycin producer Saccharopolyspora erythraea.ResultsSpontaneous rifampicin-resistant (rif) mutants were isolated from the parental strain NRRL2338 and two rif mutations mapping within rpoB, S444F and Q426R, were characterized. With respect to the parental strain, S444F mutants exhibited higher respiratory performance and up to four-fold higher final erythromycin yields; in contrast, Q426R mutants were slow-growing, developmental-defective and severely impaired in erythromycin production. DNA microarray analysis demonstrated that these rif mutations deeply changed the transcriptional profile of S. erythraea. The expression of genes coding for key enzymes of carbon (and energy) and nitrogen central metabolism was dramatically altered in turn affecting the flux of metabolites through erythromycin feeder pathways. In particular, the valine catabolic pathway that supplies propionyl-CoA for biosynthesis of the erythromycin precursor 6-deoxyerythronolide B was strongly up-regulated in the S444F mutants, while the expression of the biosynthetic gene cluster of erythromycin (ery) was not significantly affected. In contrast, the ery cluster was down-regulated (<2-fold) in the Q426R mutants. These strains also exhibited an impressive stimulation of the nitrogen regulon, which may contribute to lower erythromycin yields as erythromycin production was strongly inhibited by ammonium.ConclusionRifampicin selection is a simple and reliable tool to investigate novel links between primary and secondary metabolism and morphological differentiation in S. erythraea and to improve erythromycin production. At the same time genome-wide analysis of expression profiles using DNA microarrays allowed information to be gained about the mechanisms underlying the stimulatory/inhibitory effects of the rif mutations on erythromycin production.

Genetic and physiological characterization of rpoB mutations that activate antibiotic production in Streptomyces lividans.

Antibiotic production in Streptomyces lividans can be activated by introducing certain mutations (rif) into the rpoB gene that confer resistance to rifampicin. Working with the most typical (rif-17) mutant strain, KO-417, the rif-17 mutation was characterized. The rif-17 mutation was shown to be responsible for activating antibiotic production and for reducing the growth rate of strain KO-417, as demonstrated by gene-replacement experiments. Gene-expression analysis revealed that introduction of rif into S. lividans elevates expression of the pathway-specific regulatory gene actII-ORF4 to nearly the same level seen in Streptomyces coelicolor. The rif effect on antibiotic production was still evident in the genetic background of relC, indicating that the rif mutation can provoke its effect without depending on ppGpp. Accompanying the restoration of antibiotic production, rif mutants also exhibited a lower rate of RNA synthesis compared to the parental strain when grown in a nutritionally rich medium, suggesting that the mutant RNA polymerases may behave like 'stringent' RNA polymerases. These results indicate that the rif mutation can alter the gene-expression pattern independent of ppGpp. The impaired growth of strain KO-417 (rif-17) was largely restored by introducing the second rif mutation (rif-18) just adjacent to the rif-17 position. Proteome analysis using two-dimensional PAGE revealed that the rif mutant strain KO-418 (rif-17 rif-18) displayed a temporal burst of expression especially of two enzymes, glutamine synthetase (type II) and oxidoreductase, during the late growth phase.

Streptomycin-resistant (rpsL) or rifampicin-resistant (rpoB) mutation in Pseudomonas putida KH146-2 confers enhanced tolerance to organic chemicals.

We found that certain Str-, Gen- or Rif- mutants derived from Pseudomonas putida KH146-2, which are resistant to streptomycin, gentamicin or rifampicin, respectively, are tolerant to the aromatic compound 4-hydroxybenzoate (4HBA). The minimum inhibitory concentration (MIC) of 4HBA as the sole carbon source for the wild-type strain was 1%, whereas the MIC for the mutants was 1.7%. Frequency of 4HBA-tolerant mutants among spontaneous Str-, Gen- and Rif- mutants was 5-15%, 3-5%, and 3% respectively. These 4HBA-tolerant mutants also tolerated to a variety of organic chemicals such as 3-hydroxybenzoate, aliphatic and heterocyclic compounds, chlorobenzoates, as well as organic solvents toluene and m-xylene. The Str mutants had a point mutation in the rpsL gene, which produces the ribosomal protein S12. The Rif mutants were found to have a point mutation in the rpoB gene, which encodes the RNA polymerase beta-subunit. Mutation points in Gen mutants still remain unknown. Str-, Gen- and Rif-phenotypes occurred in spontaneous 4HBA-tolerant mutants which had been selected by successively increasing concentrations (from 0.8% to 5%) of 4HBA. Complementation experiments with one of the Str mutants demonstrated a causal relationship between a rpsL mutation (str-1) and 4HBA tolerance. Uptake experiments using [14C]-4HBA revealed that apparent ability of 4HBA to be taken up by the membrane transport system was reduced two to threefold in the mutants compared to the wild-type strain, accounting at least partly for the enhanced tolerance to 4HBA. Our approaches thus could be effective in improvement of tolerance to aromatic compounds of bacteria applicable for bioremediation.

A rifampicin resistance mutation in the rpoB gene confers ppGpp-independent antibiotic production in Streptomyces coelicolor A3(2).

In Streptomyces coelicolor A3(2), deletion of relA or a specific mutation in rplK ( relC) results in an inability to synthesize ppGpp (guanosine 5'-diphosphate 3'-diphosphate) and impairs production of actinorhodin. We have found that certain rifampicin-resistant ( rif) mutants isolated from either relA or relC strains regain the ability to produce actinorhodin at the same level as the wild-type strain, although their capacity to synthesize ppGpp is unchanged. These rif mutants were found to have a missense mutation in the rpoB gene that encodes the RNA polymerase beta-subunit. This rpoB mutation was shown to be responsible for the observed changes in phenotype, as demonstrated by gene replacement experiments. Gene expression analysis revealed that the restoration of actinorhodin production in both relA and relC strains is accompanied by increased expression of the pathway-specific regulator gene actII-ORF4, which is normally decreased in the rel mutants. In addition to the restoration of antibiotic production, the rif mutants also exhibited a lower rate of RNA synthesis compared to the parental strain when grown in a rich medium, suggesting that these mutant RNA polymerases behave like "stringent" RNA polymerases. These results indicate that rif mutations can alter gene expression patterns independently of ppGpp. We propose that RNA polymerases carrying particular rif mutations in the beta-subunit can functionally mimic the modification induced by binding of ppGpp.

Mutations in the ss subunit of the Bacillus subtilis RNA polymerase that confer both rifampicin resistance and hypersensitivity to NusG.

Mutations conferring resistance to the antibiotic rifampicin (Rif(r)) occur at specific sites within the ss subunit of the prokaryotic RNA polymerase. Rif(r) mutants of Escherichia coli are frequently altered in the elongation and termination of transcription. Rif(r) rpoB mutations were isolated in Bacillus subtilis and their effects on transcription elongation factor NusG and Rho-dependent termination were investigated. RNase protection assay, Northern analysis and the expression of nusG-lacZ fusions in cells with an inducible NusG suggested the B. subtilis nusG gene was autoregulated at the level of transcription. Rif(r) mutations that changed residue Q469 to a basic residue (Q469K and Q469R) enhanced autoregulation of nusG. A mutant expressing a truncated form of NusG, due to a nonsense mutation within the nusG gene, was isolated on the basis of the loss of autoregulation. The mechanism of autoregulation was found to be independent both of transcription termination factor Rho and of the promoter transcribing nusG. Autoregulation required sequences within the 5' coding sequence of the nusG gene or immediately upstream. This is the first evidence from any bacterium that Rif(r) RNA polymerases can display altered transcription regulation by NusG.

Repair and plasmid R46 mediated mutation requires inducible functions in Proteus mirabilis.

In Proteus mirabilis nalidixic acid or a predose of UV induce Rec protein formation, a portion of post-UV replication repair and "post-UV replication enhancement." These inducible functions are not significantly affected by the plasmid R46, which renders P. mirabilis efficiently UV-mutable. The R46-mediated UV induction of rif mutations requires additional inducible functions, as existing after nalidixic acid treatment in rec+ strains. After a nalidixic acid pretreatment UV efficient induction of rif mutations occurs without an otherwise obligatory period of post-UV incubation prior to plating on rifampicin agar. THe inducible character of this "qualification" of plasmid R46-mediated UV mutagenesis in P. mirabilis is evident from the inhibitory effects of chloramphenicol and starvation. Constitutive high-level synthesis of Rec protein in cells harboring the recombinant (multi-copy) rec+ plasmid pPM1 reduced plasmid R46-mediated UV mutagenesis, probably by preventing (inducible?) functions required by the plasmid R46 repair-mutator.

Genetic evidence for possible interaction between a ribonucleic acid polymerase subunit and the spo0C gene product of Bacillus subtilis.

Spontaneous rifampin-resistant mutants (9V Rifr) were isolated from a mutant strain of Bacillus subtilis, 9V, which has a spo0C mutation. Whereas 90% of the 9V Rifr double mutants maintained the Spo0C phenotype (Spo- Abs +/-), the remaining 10% had the Spo0A phenotype (Spo- Abs-). The latter mutants, termed 9V Rifr Spo- Abs-, were revealed to have other Spo0A characters, such as reduced transformability, higher sensitivity to phage phi 2, and reduced frequency of lysogenization by phage phi 105. The rif mutation of these 9V Rifr Spo- Abs- strains was mapped near the cysA locus. The phenotype of the Rifr transformants of strain 9V by deoxyribonucleic acid derived from these 9V Rifr Spo- Abs- strains was Spo0A, and that of the Rifr transformants of strain 168 was Spo+ Abs+. The ribonucleic acid polymerase of the 9V Rifr Spo- Abs- strains was shown to be resistant to rifampin.

Increased levels of dihydrofolate reductase in rifampin-resistant mutants of Bacillus subtilis.

Several independent, spontaneous rifampin-resistant mutants of Bacillus subtilis were isolated and found to have an increased resistance to trimethoprim, an inhibitor of dihydrofolate reductase. This increased resistance in the rif mutants was the result of a specific threefold increase in the activity of dihydrofolate reductase, since six other enzymes examined remained unchanged. This increased level of dihydrofolate reductase and the trimethoprim resistance were cotransformed (100%) with the rif marker. These results suggest that the RNA polymerase is altered in its recognition of the gene that specifies dihydrofolate reductase.

Interaction between mutations of ribosomes and RNA polymerase: a pair of strA and rif mutants individually temperature-insensitive but temperature-sensitive in combination.

A temperature-sensitive lethal mutant of Escherichia coli has been constructed by combining two temperature-insensitive mutations: a rif180 mutation that modifies RNA polymerase (RNA nucleotidyltransferase; nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) and a strA24 mutation that modifies the ribosomal protein S12. The temperature sensitivity is a property of the combination of these two particular alleles; replacement of either of the alleles relieves the temperature sensitivity. An isogenic strain containing a different strA mutation (i.e., rif180 strA11) is not temperature sensitive. Evidently ribosomes modified by the particular strA24 polymerase altered by the rif180 mutation, which suggests that in vivo there may exist some interaction between structures of ribosomes and the RNA polymerase.

A link between streptomycin and rifampicin mutation.

Introduction of str A mutations frequently make "male" strains of Escherichia coli permissive to bacteriophage T7; certain rif mutations reverse the permissive effect of strA mutation. Permissiveness of the strA mutation is accompanied by enhanced transcription of bacteriophage T7 genome. Introduction of the nonpermissive rif allele to the permissive strA strain reduces or abolishes the transcription of T7 genome. Thus, a link is implied in the functioning of the ribosome and the RNA polymerase (RNA nucleotidyltransferase, EC 2.7.7.6).