Conferring Rifampicin Resistance Research Articles

Insights into RNA polymerase catalysis and adaptive evolution gained from mutational analysis of a locus conferring rifampicin resistance.

S531 of Escherichia coli RNA polymerase (RNAP) β subunit is a part of RNA binding domain in transcription complex. While highly conserved, S531 is not involved in interactions within the transcription complex as suggested by X-ray analysis. To understand the basis for S531 conservation we performed systematic mutagenesis of this residue. We find that the most of the mutations significantly decreased initiation-to-elongation transition by RNAP. Surprisingly, some changes enhanced the production of full-size transcripts by suppressing abortive loss of short RNAs. S531-R increased transcript retention by establishing a salt bridge with RNA, thereby explaining the R substitution at the equivalent position in extremophilic organisms, in which short RNAs retention is likely to be an issue. Generally, the substitutions had the same effect on bacterial doubling time when measured at 20°. Raising growth temperature to 37° ablated the positive influence of some mutations on the growth rate in contrast to their in vitro action, reflecting secondary effects of cellular environment on transcription and complex involvement of 531 locus in the cell biology. The properties of generated RNAP variants revealed an RNA/protein interaction network that is crucial for transcription, thereby explaining the details of initiation-to-elongation transition on atomic level.

Molecular Epidemiology of Rifampicin Resistance in Mycobacterium tuberculosis Using the GeneXpert MTB/RIF Assay from a Rural Setting in India.

The Xpert MTB/RIF assay can detect mutations in rpoB gene that confer rifampicin resistance (RR) using five overlapping probes (A, B, C, D, and E). In this study, we described our experience with the Xpert assay in a rural setting in India. During the study period, 3250 samples were processed. The result was unsuccessful in 5.7% of cases. For extrapulmonary specimens, the risk of unsuccessful result was higher in tissue biopsy and stool samples. Among samples positive for Mycobacterium tuberculosis, rifampicin resistance was indeterminate in 1.2% of them. Our results and a review of the literature showed that the most frequent mutations conferring RR were located in the region of Probe E (63.6%; 95% confidence interval [CI] 56.26–70.94), followed by Probe B (15.02%; 95% CI 11.94–18.10), Probe D (13.35%; 95% CI 10.01–16.69), Probe A (4.73%; 95% CI 1.92–7.54), and Probe C (1.61%; 95% CI 0.67–2.54). Although the high cost of the cartridges precluded using the Xpert assay for routine diagnosis of tuberculosis, our results demonstrate that the assay can be used to diagnose RR-tuberculosis in rural areas with limited laboratory infrastructure and could be a convenient tool to investigate the molecular epidemiology of RR in resource-limited settings.

Epistatic interactions between ancestral genotype and beneficial mutations shape evolvability in Pseudomonas aeruginosa.

The idea that interactions between mutations influence adaptation by driving populations to low and high fitness peaks on adaptive landscapes is deeply ingrained in evolutionary theory. Here, we investigate the impact of epistasis on evolvability by challenging populations of two Pseudomonas aeruginosa clones bearing different initial mutations (in rpoB conferring rifampicin resistance, and the type IV pili gene network) to adaptation to a medium containing l-serine as the sole carbon source. Despite being initially indistinguishable in fitness, populations founded by the two ancestral genotypes reached different fitness following 300 generations of evolution. Genome sequencing revealed that the difference could not be explained by acquiring mutations in different targets of selection; the majority of clones from both ancestors converged on one of the following two strategies: (1) acquiring mutations in either PA2449 (gcsR, an l-serine-metabolism RpoN enhancer binding protein) or (2) protease genes. Additionally, populations from both ancestors converged on loss-of-function mutations in the type IV pili gene network, either due to ancestral or acquired mutations. No compensatory or reversion mutations were observed in RNA polymerase (RNAP) genes, in spite of the large fitness costs typically associated with mutations in rpoB. Although current theory points to sign epistasis as the dominant constraint on evolvability, these results suggest that the role of magnitude epistasis in constraining evolvability may be underappreciated. The contribution of magnitude epistasis is likely to be greatest under the biologically relevant mutation supply rates that make back mutations probabilistically unlikely.

Occurrence and new mutations involved in rifampicin-resistant Propionibacterium acnes strains isolated from biofilm or device-related infections

We described for the first time the amino acid substitutions conferring rifampicin resistance in eight Propionibacterium acnes strains isolated from patients with biofilm or device-related infections. We identified different mutations in cluster I and one mutation, never reported, in cluster II of the rpoB gene (I480V) associated with the most frequent one in cluster I (S442L). Half of the patients previously received treatment with rifampicin.

Molecular characterization of multi drug-resistant tuberculosis isolated from Baghdad, Iraq

BackgroundTuberculosis (TB) remains a major cause of mortality and morbidity all over the world. Multi drug-resistant tuberculosis (MDR-TB) is an important cause for mortality among TB patients, and management of MDR-TB remains a major challenge for national TB programs. Early detection of MDR-TB provides better treatment outcomes and reduces the transmission of MDR. Nucleic acid amplification test (NAAT) like Line Probe Assays have been recently endorsed for use in low-income settings and can be used to screen smear-positive sputum specimens for diagnosing of resistance to rifampicin and isoniazid in 1–2days. ObjectiveOverall objective of the study was to evaluate performances of Line Probe Assay GenoType® MTBDRplus (Hain Life science, GmbH, Nehren, Germany) to move forward its introduction into routine diagnostics. Secondary objective was to determine the type of mutations in rpoB, katG and inhA genes from culture specimens isolated from Iraqi selected MDR-TB patients. Materials and methodsThe DNA was extracted by CTAB method from 51 clinical isolates which were previously characterized as MDR strains in reference laboratory/center of Chest and Respiratory Diseases obtained from patients living in Baghdad/Iraq (2010–2011). GenoType® MTBDRplus ver. 2.0 (HainLifescience, GmbH, Nehren, Germany) was performed at the Supra-national TB Reference Laboratory in Milan (Italy) as well as interpretation of mutations involved in drug resistance to rifampin and Isoniazide according to manufacturer’s instructions. Results and conclusionLine Probe Assays were performed on 51 MDR of Mycobacterium tuberculosis strains; 6 strains were excluded from the analysis due to un-interpretable results; 5 strains were identified as sensitive to both rifampicin (RIF) and isoniazid (INH) by MTBDR plus assay. The most common genetic mutation conferring RIF resistance was S531L of rpoB gene which was detected in 33 (82.5%) resistant strains. Other mutations in this gene were D516Vand H526Y which were detected in a single strain (2.5%) for each. Also, there were 5 (12.5%) RIF-resistant strains with mutations that could not be identified specifically in this assay.Isoniazid (INH) resistance due to katG mutation S315T1 was found in 17 (42.5%) strains of INH-resistant M. tuberculosis. The second most common site of mutation was in the upstream promoter sequence of inhA, which was found in 15 (37.5%) strains, 14 (35%) strains of which carried a C→T transition at the −15 position, while single strains (2.5%) carried a T→A mutation at the −8 position. The result showed 8 (20%) strains missing a band on the wild-type region of these strains, but no hybridization with mutation probes on this gene suggested resistance due to mutations other than those included in this assay.Regardless the type of mutations detected, GenoType® MTBDR plus ver. 2.0 showed an overall accuracy of 95.5% (C.I. 95% 89.9–98.1) in detecting MDR strains. Despite the relatively low number of MDR strains tested, they show very common mutations patterns being the majority mutated at the codon 531 and 315 of rpoB and katG gene. In such setting, GenoType® MTBDR plus ver. 2.0 could be a great tool to rapidly screen for MDR-TB and has the potential to substantially reduce the turnaround time of drug sensitivity test (DST) results.

Rapid molecular TB diagnosis: evidence, policy making and global implementation of Xpert MTB/RIF

If tuberculosis (TB) is to be eliminated as a global health problem in the foreseeable future, improved detection of patients, earlier diagnosis and timely identification of rifampicin resistance will be critical. New diagnostics released in recent years have improved this perspective but they require investments in laboratory infrastructure, biosafety and staff specialisation beyond the means of many resource-constrained settings where most patients live. Xpert MTB/RIF, a new assay employing automated nucleic acid amplification to detect Mycobacterium tuberculosis, as well as mutations that confer rifampicin resistance, holds the promise to largely overcome these operational challenges. In this article we position Xpert MTB/RIF in today's TB diagnostic landscape and describe its additional potential as an adjunct to surveillance and surveys, taking into account considerations of pricing and ethics. In what could serve as a model for the future formulation of new policy on diagnostics, we trace the unique process by which the World Health Organization consulted international expertise and systematically assessed published evidence and freshly emerging experience from the field ahead of its endorsement of the Xpert MTB/RIF technology in 2010, summarise subsequent research findings and guidance on who to test and how, and provide perspectives on scaling up the new technology.

Epidemiological and molecular aspects of rifampicin-resistant Staphylococcus aureus isolated from wounds, blood and respiratory samples

To study the incidence of rifampicin-resistant Staphylococcus aureus in Gipuzkoa, Northern Spain, and to characterize representative resistant isolates and mutations associated with resistance. For rifampicin-resistant isolates, the rpoB gene fragment that includes the most frequent mutations conferring rifampicin resistance in S. aureus was amplified and sequenced. The role of new mutations responsible for rifampicin resistance was confirmed by cloning and complementation in trans. Resistant isolates were characterized by multilocus sequence typing and PFGE. Between 1999 and 2008, 0.59% (96/16 348) of S. aureus clinical isolates studied showed rifampicin resistance. Rifampicin resistance was higher in methicillin-resistant S. aureus (MRSA) than in methicillin-susceptible S. aureus (MSSA) (3.26% versus 0.26%; P < 0.001). Twenty-two randomly selected rifampicin-resistant isolates were studied in depth, 11 showing low-level and 11 showing high-level rifampicin resistance (rifampicin MICs of 2-4 mg/L and ≥8 mg/L, respectively). Overall, 12 different mutations in the rpoB gene were detected, including a newly described N474K mutation followed by the insertion of a glycine residue at position 475. Among the eight different sequence types (STs) found, the most frequent were ST8 and ST863, the latter being associated with respiratory infections. Ten of the 11 low-level rifampicin-resistant isolates were MRSA ST8 and had the same H481N mutation, while the 11 high-level rifampicin-resistant isolates, 6 MSSA and 5 MRSA, belonged to eight different STs and had distinct rpoB mutations. Low-level rifampicin-resistant isolates were mainly clonal while high-level resistant isolates showed a high genetic diversity. Most mutations observed coincided with those found in other studies, but a new mutation conferring rifampicin resistance was detected.

Rifampicin Resistance Phenotyping of Brucella melitensis by rpoB Gene Analysis in Clinical Isolates

Rifampicin resistance of Brucella melitensis by rpoB gene analysis has not yet been performed in Turkey, where brucellosis is endemic. In this study, we investigated the efficacy of E-test and single nucleotide polymorphism (SNP) analysis of the B. melitensis rpoB gene, for the detection of mutations conferring rifampicin resistance, by sequencing 21 human B. melitensis strains from the Southeast and Marmara regions of Turkey. On CLSI slow-growing bacteria standards, all isolates were sensitive to rifampicin except for 6 which showed intermediate resistance to rifampicin. MIC50 and MIC90 values were 1 μg/ml and 1.5 μg/ml respectively (range 0.50 -1.5 μg/ml). The rifampicin-resistant phenotype was investigated at Cd 154 (GTT/TTT), Cd 526 (GAC/TAC, GAC/AAC, GAC/GGC), Cd 536 (CAC/CTC, CAC/TAC), Cd 539 (CGC/AGC), Cd 541 (TCG/TTG) and Cd 574 (CCG/CTG) of the rpoB gene in B. melitensis 16M and B115 strains, and in clinical isolates. No missense mutations were found in any of the B. melitensis isolates, which indicates that all isolates were rifampicin-susceptible. In conclusion, SNP analysis was useful as a molecular tool for rifampin resistance testing. Although resistance to rifampicin was not detected in our strains of B. melitensis; the presence of strains with intermediate resistance to rifampicin indicates that susceptibility testing should be performed periodically.

Distribution of rpoB mutations among multidrug-resistant Mycobacterium tuberculosis (MDRTB) strains from Thailand and development of a rapid method for mutation detection

Since rifampicin resistance is a surrogate marker for multidrug-resistant Mycobacterium tuberculosis (MDRTB), the present study aimed to investigate rpoB mutations conferring rifampicin resistance in M. tuberculosis strains from Thailand, and to develop a rapid, inexpensive and simple PCR-based method for rapid detection of MDRTB. Overall, 267 M. tuberculosis isolates, including 143 MDRTB isolates, were investigated. Isolates of the Beijing strain predominated among the MDRTB isolates (79.1%), but accounted for only 45.5% of the susceptible isolates. Mutations in the rpoB gene were found most commonly at codons 531, 526 and 516 (58%, 25.2% and 9.1%, respectively). A multiplex allele-specific PCR was developed and tested with 216 clinical isolates. In comparison with the proportion method, the method showed 94.2% sensitivity and 100% specificity, and had a 100% positive predictive value and a 95% negative predictive value, which suggested that this method could be useful for screening for MDRTB, particularly in resource-limited countries.

Oxygen induces mutation in a strict anaerobe, Prevotella melaninogenica

Strict anaerobes are highly sensitive to oxygen, but the mutagenicity of oxygen in strict anaerobes has not been well understood. Prevotella melaninogenica, a strict anaerobe, is susceptible to oxygen and shows an increase in oxidative DNA damage upon exposure to oxygen. In this study, we have investigated the mutagenicity of oxygen and the types of mutations induced by oxygen. Exposure to oxygen decreased cell survival and increased the levels of 8-oxo-deoxyguanosine (8-oxodG). The frequency of rifampicin-resistant mutants was markedly increased after exposure to oxygen. After sequencing a 254-bp fragment of the rpoB gene, which encodes the β subunit of bacterial RNA polymerase, a target molecule of rifampicin, we found that most mutants induced by oxygen had GC to TA transversions, a signature of 8-oxodG. In addition, all detected single-nucleotide changes would lead to amino acid changes that confer rifampicin resistance. These results indicate that oxygen is mutagenic in a strict anaerobe, P. melaninogenica, and its mutagenic characteristics could be analyzed with this experimental system.

Development and evaluation of a novel multiple-primer PCR amplification refractory mutation system for the rapid detection of mutations conferring rifampicin resistance in codon 425 of the rpoB gene of Mycobacterium leprae

Rifampicin-resistant Mycobacterium leprae is regularly reported and drug resistance is a major threat for the elimination of leprosy. There is an urgent need for a simple method that can detect rifampicin resistance in clinical isolates. This study developed a multiple-primer PCR amplification refractory mutation system, a simple, reliable and economical method for clinical specimens that allowed the rapid detection of mutations in the nucleotides of the codon for Ser425 of the M. leprae rpoB gene, mutation of which to Leu, Met or Phe is associated with rifampicin resistance. The approach involved a multiple-primer PCR in which both mutant-specific and normal sets of primers were included in the reaction. The mutant-specific primer was complementary to the corresponding sequence of the wild-type gene except for one additional deliberate mismatch at the fourth nucleotide from the 3'-OH terminus. A single mismatch has little influence on the yield of PCR products, but if there are two mismatches as a result of mutation at the position being tested, the mutant-specific primer will not function in PCR under appropriate conditions, leading to no yield of PCR product from the mutant allele. The assay was evaluated successfully using a panel of plasmids and M. leprae reference strains carrying the wild-type or known rpoB mutations. The assay was subsequently applied to M. leprae DNA extracts from skin biopsies taken from patients. In all biopsy samples, the wild-type allele was detected for Ser425. The PCR results correlated with rifampicin susceptibility, as also measured by the traditional in vivo mouse footpad technique.

Evaluation of a rapid screening test for rifampicin resistance in re-treatment tuberculosis patients in the Eastern Cape.

Patients with multidrug-resistant (MDR) tuberculosis (TB) are at high risk of treatment failure. It is anticipated that early identification of MDR-TB and appropriate treatment will improve patient outcome and disease control. We evaluated the rapid detection of rifampicin resistance in previously treated TB patients, directly from acidfast bacilli (AFB)-positive sputum using a phage-based test, FASTPlaque-Response (Biotec Laboratories Ltd, Ipswich, UK). The ability of rifampicin resistance to predict MDR-TB was also determined. A prospective study was done comparing performance of the rapid phage test with conventional culture and drug susceptibility testing (DST) in AFB-positive TB patients. Five primary health clinics and one TB referral centre in the Port Elizabeth Metropolitan area, Eastern Cape. Sensitivity, specificity and overall accuracy of the phage test were determined compared with gold standard culture and DST. Discrepant results were resolved by molecular detection of mutations conferring rifampicin resistance. The proportion of rifampicin-resistant strains that were MDR was also determined. Previously treated patients were at a high risk of MDRTB (35.7%). Sensitivity, specificity and overall accuracy of FASTPlaque-Response for rifampicin resistance determination were 95.4% (95% confidence interval (CI): 91.0 - 99.8%), 97.2% (95% CI: 94.5 - 99.9%) and 96.5% (95% CI: 94.1 - 98.9%) respectively compared with conventional DST (unresolved), calculated for specimens that had both FASTPlaque-Response and conventional DST results available. FASTPlaque-Response results were available in 2 days instead of 28 - 85 days with conventional DST. However, only 70.6% of FASTPlaque-Response results were interpretable compared with 86.3% of conventional DST results. The majority (95.5%) of rifampicinresistant strains were MDR-TB. Rapid detection of rifampicin resistance using FASTPlaque-Response could contribute to improved management of patients at risk of MDR-TB, such as previously treated patients. However, improvement in control of specimen-related contamination is needed to ensure that a higher proportion of FASTPlaque-Response results are interpretable. Where indicated, early modification of therapy could improve patient prognosis and reduce disease transmission.

Role of a MutY DNA glycosylase in combating oxidative DNA damage in Helicobacter pylori

MutY is an adenine glycosylase that has the ability to efficiently remove adenines from adenine/7,8-dihydro-8-oxoguanine (8-oxo-G) or adenine/guanine mismatches, and plays an important role in oxidative DNA damage repair. The human gastric pathogen Helicobacter pylori has a homolog of the MutY enzyme. To investigate the physiological roles of MutY in H. pylori, we constructed and characterized a mutY mutant. H. pylori mutY mutants incubated at 5% O 2 have a 325-fold higher spontaneous mutation rate than its parent. The mutation rate is further increased by exposing the mutant to atmospheric levels of oxygen, an effect that is not seen in an E. coli mutY mutant. Most of the mutations that occurred in H. pylori mutY mutants, as examined by rpoB sequence changes that confer rifampicin resistance, are GC to TA transversions. The H. pylori enzyme has the ability to complement an E. coli mutY mutant, restoring its mutation frequency to the wild-type level. Pure H. pylori MutY has the ability to remove adenines from A/8-oxo-G mismatches, but strikingly no ability to cleave A/G mismatches. This is surprising because E. coli MutY can more rapidly turnover A/G than A/8-oxo-G. Thus, H. pylori MutY is an adenine glycosylase involved in the repair of oxidative DNA damage with a specificity for detecting 8-oxo-G. In addition, H. pylori mutY mutants are only 30% as efficient as wild-type in colonizing the stomach of mice, indicating that H. pylori MutY plays a significant role in oxidative DNA damage repair in vivo.

Association of rpoB mutations with rifampicin resistance in Mycobacterium avium

The susceptibility of clinical isolates of Mycobacterium avium to rifampicin (RIF) was examined. All 32 clinical isolates tested, including 18 from Japan, 13 from Poland and 1 from Thailand, were resistant to RIF (minimum inhibitory concentrations (MICs) ≥32 μg/mL for 17 isolates and 2–16 μg/mL for 15 isolates), whereas the type strain of M. avium ATCC 25291 was susceptible to RIF (MIC ≤ 0.03 μg/mL). Mutations in nucleotides 1276–1356 of the rpoB gene, termed the 81 bp core region, are associated with RIF resistance in Mycobacterium tuberculosis. No mutations were found in this region in any of the M. avium clinical isolates tested. However, mutation of G → A to give a Gly544 → Asp substitution was identified within the rpoB gene downstream of the 81 bp region in all clinical isolates. A RIF-resistant strain (ATCC 25291 Rif r; MIC ≥ 32 μg/mL) obtained by culturing the type strain in RIF-containing broth possessed a mutation C → T to give a His445 → Tyr substitution within the 81 bp region. When the rpoB gene of the ATCC 25291 Rif r strain and of a clinical isolate were inserted into Mycobacterium smegmatis, organisms with the ATCC 25291 Rif r sequence, but not those with the clinical isolate sequence, showed resistance to RIF. These results suggest that mutations of the 81 bp region of rpoB, as well as factors other than rpoB mutation, confer RIF resistance in M. avium.

Use of a high-density DNA probe array for detecting mutations involved in rifampicin resistance in Mycobacterium tuberculosis

A Mycobacterium high-density DNA probe array designed to detect rpoB mutations conferring rifampicin resistance in Mycobacterium tuberculosis was evaluated. The rpoB hybridisation patterns produced by 41 susceptible (RifS) and 59 rifampicin-resistant (RifR) clinical isolates of M. tuberculosis were compared with the results of conventional dideoxynucleotide sequencing of the rpoB gene. For all the RifR isolates, the rpoB hybridisation patterns correlated with the rpoB sequencing results. Among the 59 isolates, 11 distinct amino-acid changes were detected by the DNA probe array. Of these, 36 (61%) corresponded to replacement of the serine residue found in position 531 (S531L in 34 isolates and S531W in two isolates), 16 (27%) affected histidine 526 (five H526D, five H526Y, four H526L, one H526N and one H526R), four (6.8%) replaced aspartate 516 with a valine, and one (1.7%) replaced glutamine 513 with a leucine. Deletion of the asparagine residue at position 519 was detected in one isolate susceptible to rifampicin, but yielding c. 0.1% resistant colonies on rifampicin-containing medium. No mutation was detected in the rpoB region from one isolate yielding c. 5% of resistant colonies on rifampicin-containing medium. Finally, a D516Y substitution was detected in association with an unexpected mutation, G523W, not tiled on the DNA probe array, but which could be detected by analysing the hybridisation pattern obtained with the wild-type probes covering codon 523. In conclusion, the Mycobacterium probe array is a promising approach to rapid detection of mutations involved in rifampicin resistance in M. tuberculosis.

A Mutant RNA Polymerase Reveals a Kinetic Mechanism for the Switch between Nonproductive Stuttering Synthesis and Productive Initiation during Promoter Clearance

A Mutant RNA Polymerase Reveals a Kinetic Mechanism for the Switch between Nonproductive Stuttering Synthesis and Productive Initiation during Promoter Clearance

Genetic and Transcriptional Organization of the Region Encoding the β Subunit of Bacillus subtilis RNA Polymerase

The gene encoding the beta subunit of Bacillus subtilis RNA polymerase was isolated from a lambda gt11 expression library using an antibody probe. Gene identity was confirmed by the similarity of its predicted product to the Escherichia coli beta subunit and by mapping an alteration conferring rifampicin resistance within the conserved rif coding region. Including the rif region, four colinear blocks of sequence similarity were shared between the B. subtilis and E. coli beta subunits. In E. coli, these conserved blocks are separated by three regions that either were not conserved or were entirely absent from the B. subtilis protein. The B. subtilis beta gene was part of a cluster with the order rplL (encoding ribosomal protein L7/L12), orf23 (encoding a 22,513-dalton protein that is apparently essential for growth), rpoB (beta), and rpoC (beta'). This organization differs from the corresponding region in E. coli by the inclusion of orf23. Experiments using promoter probe vectors and site-directed mutagenesis located a major rpoB promoter overlapping the 3'-coding region of orf23, 250 nucleotides upstream from the beta initiation codon. Thus, the B. subtilis rpoB region differs from its E. coli counterpart in both genetic and transcriptional organization.

Mutations in ORF D13L and Other Genetic Loci Alter the Rifampicin Phenotype of Vaccinia Virus

Phenotypic analysis of vaccinia virus temperature-sensitive mutants identified several virus isolates that were either resistant or hypersensitive to rifampicin. Temperature-sensitive mutants 1085, 7743, 1085R, and 7743R were found to be rifampicin resistant (Rif R). Each virus contained at least one mutation in ORF D13L which resulted in a change of amino acid sequence. Analysis of each point mutation by marker rescue demonstrated that mutations gly to asp at residue 80, pro to phe at 458, lys to asn at 484, and ile to val at 485 conferred the resistance phenotype, while the mutation changing ser to leu at 176 did not affect the rifampicin phenotype. Temperature-sensitive mutants C6, C17, and C43 were found to be rifampicin hypersensitive. Only C43, however, was found to contain a lesion in ORF D13L. This mutation, ser to asn at 169 was confirmed by marker rescue to confer the hypersensitive phenotype. The results presented here along with those from previous reports show that known mutations conferring rifampicin resistance cluster at the C or N-terminus of D 13L while a mutation mapping at residue 169 confers rifampicin hypersensitivity. Further, the finding that the hypersensitive mutants C6 and C17 contain a wild-type D 13L ORF suggests a possible role of other vital functions in the interaction of rifampicin with vaccinia virus.

Amino acid changes in conserved regions of the beta-subunit of Escherichia coli RNA polymerase alter transcription pausing and termination.

Control of transcription at pause and termination sites is common in bacteria. Many transcriptional pause and termination events are thought to occur in response to formation of an RNA hairpin in the nascent transcript. Some mutations in the beta-subunit of Escherichia coli RNA polymerase that confer resistance to the transcription inhibitor rifampicin also alter the response to transcriptional pause and termination signals. Here, we report isolation of termination-altering mutations that do not confer rifampicin resistance and show that such mutations occur predominantly in limited regions of the beta-subunit polypeptide. One region is between amino acid residues 500 and 575, which encompasses the locations of almost all known rifampicin-resistance mutations. Many termination-altering mutations also occur in two other regions: between amino acid residues 740 and 840 and near the carboxyl terminus of the beta-subunit (amino acid residues 1225-1342). Amino acid sequences in these three regions of the beta-subunit are conserved between prokaryotic and eukaryotic beta-subunit homologs. Several mutations that alter transcription termination in vitro affect amino acid residues that are identical in prokaryotic and eukaryotic RNA polymerase beta-subunit homologs, suggesting that they alter an important function common to multisubunit RNA polymerases. We propose that these three regions of the beta-subunit may contact the nascent RNA transcript, the RNA-DNA heteroduplex, or the DNA template in the transcription complex and that mutations in these regions alter transcription pausing and termination by affecting these contacts.

Rifampicin prevents virosome localization of L65, an essential vaccinia virus polypeptide

In contrast to its irreversible effect on the Escherichia coli RNA polymerase β-subunit, the antibiotic rifampicin reversibly inhibits vaccinia virus morphogenesis at a step during the formation of immature viral particles. The protein affected by the presence of rifampicin is L65, a major late vaccinia polypeptide to which mutations that confer rifampicin resistance have been mapped. We now provide evidence using a monospecific anti-L65 serum in concert with immunofluorescence and sucrose gradient analysis that the mechanism of action of rifampicin on vaccinia virus replication involves the inhibition of localization of L65 to the viral factories (virosomes) thereby blocking further development. Studies on the expression and distribution of L65 during the infection cycle reveal that L65 is a stable, nonglycosylated late protein associated with virions. These results are discussed in relationship to the possible in vivo functions of the L65 protein.