Compensatory adaptation to the deleterious effect of antibiotic resistance in Salmonella typhimurium

Most chromosomal mutations that cause antibiotic resistance impose fitness costs on the bacteria. This biological cost can often be reduced by compensatory mutations. In Salmonella typhimurium, the nucleotide substitution AAA42 --> AAC in the rpsL gene confers resistance to streptomycin. The resulting amino acid substitution (K42N) in ribosomal protein S12 causes an increased rate of ribosomal proofreading and, as a result, the rate of protein synthesis, bacterial growth and virulence are decreased. Eighty-one independent lineages of the low-fitness, K42N mutant were evolved in the absence of antibiotic to ameliorate the costs. From the rate of fixation of compensated mutants and their fitness, the rate of compensatory mutations was estimated to be > or = 10-7 per cell per generation. The size of the population bottleneck during evolution affected fitness of the adapted mutants: a larger bottleneck resulted in higher average fitness. Only four of the evolved lineages contained streptomycin-sensitive revertants. The remaining 77 lineages contained mutants that were still fully streptomycin resistant, had retained the original resistance mutation and also acquired compensatory mutations. Most of the compensatory mutations, resulting in at least 35 different amino acid substitutions, were novel single-nucleotide substitutions in the rpsD, rpsE, rpsL or rplS genes encoding the ribosomal proteins S4, S5, S12 and L19 respectively. Our results show that the deleterious effects of a resistance mutation can be compensated by an unexpected variety of mutations.     

Evaluation of reasons of the MDR M. tuberculosis strains dissemination by analysis of the rifampicin and/or isoniazid resistant isolates

During the last years in Novosibirsk region of Russia the rate of TB patients infected by MDR strains of M. tuberculosis has been constantly increasing. This increase may occur as a result of the spontaneously mutated mycobacterium selection during treatment of patients or as a result of primary infection by the resistant M. tuberculosis, or also, as a result of both reasons in combination. If the main reason of MDR strain dissemination is selection of resistant bacterium during patient treatment, the equal apportionment of the dominated mutation into the mycobacterium genotypes would be observed. If the main reason is the primary infection by resistant M. tuberculosis, the unequal apportionment would be revealed. For deeper understanding of the main reasons of the fast MDR strains spreading in the region, the distribution of the main mutations over genotypes of strains in Novosibirsk (170 isolates) and Tomsk prison (51 isolates) was investigated. Mutations in rpoB gene associated with the rifampicin resistance and in katG (isoniazid resistance) were detected by biochips. M. tuberculosis genotypings were carried out by IS6110 PCR typing or MIRU typing, in the last method the twelve loci (MIRU 2, 4, 10, 16, 20, 23, 24, 26, 27, 31, 39, 40) have been used. The most frequent mutation in the rpoB gene was Ser531-->Leu (60-70% of the rifampicin resistant strains) and Ser315-->Thr in gene katG (80% of the isoniazid resistant M. tuberculosis). Both in Novosibirsk and in Tomsk prison the rates of clustered cases transmissions were high (69 and 63% respectively). Analysis of the distribution of the dominated mutations Ser531-->Leu (rpoB) and Ser315-->Thr (katG) revealed that all of them were detected in each clusters, but in Novosibirsk there were only two clusters, in which the percentage of strains, containing mutation Ser531-->Leu (rpoB) were higher (85.7% and 77.7% respectively, P < 0.05), then in others. Among the Tomsk prison's clusters it was revealed one in which the proportion of the Ser3 15-->Thr mutation in katGwas higher (96.4%, P < 0.05). The nonuniform distribution of the dominated mutations highlighted that the epidemic spread of drug-resistant strains of M. tuberculosis in region resulted from the selection of them during patient treatment and the subsequent transmission by TB patients.     

Antimicrobial drug resistance in Salmonella: problems and perspectives in food- and water-borne infections

Strains of Salmonella spp. with resistance to antimicrobial drugs are now widespread in both developed and developing countries. In developed countries it is now increasingly accepted that for the most part such strains are zoonotic in origin and acquire their resistance in the food-animal host before onward transmission to humans through the food chain. Of particular importance since the early 1990s has been a multiresistant strain of Salmonella typhimurium definitive phage type (DT) 104, displaying resistance to up to six commonly used antimicrobials, with about 15% of isolates also exhibiting decreased susceptibility to ciprofloxacin. Mutations in the gyrA gene in such isolates have been characterised by a PCR LightCycler-based gyrA mutation assay, and at least four different mutations have been identified. Multiple resistance (to four or more antimicrobials) is also common in the poultry-associated pathogens Salmonella virchow and Salmonella hadar, with an increasing number of strains of these serotypes exhibiting decreased susceptibility to ciprofloxacin. Multiple resistance is also being found in other serotypes in several other European countries, and has been associated with treatment failures. For Salmonella typhi, multiple drug resistance is now the norm in strains originating in the Indian subcontinent and south-east Asia. Such multiresistant strains have been responsible for several epidemics and some of these have been associated with contaminated water supplies. Furthermore, an increasing number of multiresistant strains of S. typhi are now exhibiting decreased susceptibility to ciprofloxacin, with concomitant treatment failures. In developed countries antimicrobial resistance in zoonotic salmonellas has been attributed to the injudicious use of antimicrobials in food-producing animals. It is hoped that the application of Codes of Practice for the use of such agents, which have been prepared by the pharmaceutical industry in response to widespread international concern about the development of drug resistance in bacterial pathogens, will now result in a widespread reduction in the incidence of drug-resistant salmonellas in food production animals and humans on an international scale.     

Genetic characteristics and protective properties of neamin-resistant mutant Salmonella typhimurium and S. dublin of the Nea(r) Str(s) and Nea(r) Str(r) 500 classes

The genetic analysis of attenuated mutants, class Nea(r) Str(s), with the use of bacteriophage P 22 has shown that mutation rendering the mutants resistant to neamine is localized in gene nea A. In experiments with the intraperitoneal infection of mice, the appearance of this mutation in S. typhimurium and S. dublin virulent strains has been found to lead to the decrease of virulence in 100% of clones. On the basis of the data obtained in this investigation, region str-spc in S. typhimurium and S. dublin has been mapped. In contrast to mutation spc A, mutations nea A and str A have been shown to inhibit the action of amber suppressor. The investigation has confirmed the regularity, previously established for Shigella flexneri, concerning the relationship between the influence of mutations, occurring in the genes which determine resistance to neamine and streptomycin and control the synthesis of ribosomal proteins S4, S5, S12 and S17, on the virulence of S. typhimurium and S. dublin and the effect of these mutations on the accuracy of the translation of genetic information in the biosynthesis of protein: mutation spc A has been found to produce no changes in the virulence of salmonellae, while mutations nea A and str A cause its loss. Salmonella strains carrying mutations nea A and nea B have shown pronounced protective properties in experiments on mice.     

Role of an acrR mutation in multidrug resistance of in vitro-selected fluoroquinolone-resistant mutants of Salmonella enterica serovar Typhimurium

Quinolone resistance in Salmonella spp. is usually attributed to both active efflux and mutations leading to modification of the target enzymes DNA gyrase and topoisomerase IV. Here, we investigated the presence of mutations in the efflux regulatory genes of fluoroquinolone- and multidrug-resistant mutants of Salmonella enterica serovar Typhimurium (S. Typhimurium) selected in vitro with enrofloxacin that both carried a mutation in the target gene gyrA and overproduced the AcrAB efflux pump. No mutations were detected in the global regulatory loci marRAB and soxRS for the four strains studied. A mutation in acrR, the local repressor of acrAB, was found for two ciprofloxacin-resistant selected-mutants, leading to duplication of amino acids Ile75 and Glu76. Complementation experiments with wild-type acrR showed that the mutation identified in acrR partially contributed to the increase in resistance levels to several unrelated antibiotics. The acrR mutation also contributed to acrAB overexpression as shown by RT-PCR. Thus, this study underlines the role of an acrR mutation, in addition to the mutation in gyrA, in the fluoroquinolone and multidrug resistance phenotype of S. Typhimurium mutants, through overexpression of acrAB.     

Clonal expansion may account for high levels of quinolone resistance in Salmonella enterica serovar enteritidis

We have observed a high incidence of isolated nalidixic acid resistance in Salmonella enterica serovar Enteritidis isolates in Ireland, particularly isolates of phage type 1 (PT1). A group of nalidixic acid-resistant (n = 22) and nalidixic acid-susceptible (n = 28) isolates of serovar Enteritidis from multiple sites in Ireland were selected. Isolates were typed by pulsed-field gel electrophoresis (PFGE) with XbaI, and the MICs for nalidixic acid and ciprofloxacin were determined. Mutations associated with nalidixic acid resistance in clinical isolates and laboratory mutants of serovar Enteritidis and 32 nalidixic acid-resistant isolates of 15 other salmonella serovars were identified. PFGE had limited discriminatory power. A specific point mutation (G246T) associated with amino acid substitution Asp87Tyr in the quinolone resistance determining region of the gyrA gene accounted for 95% of all mutations in serovar Enteritidis and for all mutations in PT1 isolates. Greater diversity of mutations was observed among all non-Enteritidis salmonella serovars studied. Rates of nalidixic acid resistance in serovar Enteritidis may predominantly reflect clonal expansion after infrequent mutation or selection events.     

Clonal Expansion May Account for High Levels of Quinolone Resistance in Salmonella enterica Serovar Enteritidis

We have observed a high incidence of isolated nalidixic acid resistance in Salmonella enterica serovar Enteritidis isolates in Ireland, particularly isolates of phage type 1 (PT1). A group of nalidixic acid-resistant (n = 22) and nalidixic acid-susceptible (n = 28) isolates of serovar Enteritidis from multiple sites in Ireland were selected. Isolates were typed by pulsed-field gel electrophoresis (PFGE) with XbaI, and the MICs for nalidixic acid and ciprofloxacin were determined. Mutations associated with nalidixic acid resistance in clinical isolates and laboratory mutants of serovar Enteritidis and 32 nalidixic acid-resistant isolates of 15 other salmonella serovars were identified. PFGE had limited discriminatory power. A specific point mutation (G246T) associated with amino acid substitution Asp87Tyr in the quinolone resistance determining region of the gyrA gene accounted for 95% of all mutations in serovar Enteritidis and for all mutations in PT1 isolates. Greater diversity of mutations was observed among all non-Enteritidis salmonella serovars studied. Rates of nalidixic acid resistance in serovar Enteritidis may predominantly reflect clonal expansion after infrequent mutation or selection events.     

Gain-of-function mutations associated with hereditary pancreatitis enhance autoactivation of human cationic trypsinogen

Hereditary pancreatitis (HP), an autosomal dominant disorder, has been associated with mutations in the cationic trypsinogen gene. Here we demonstrate that the two most frequent HP mutations, Arg117 --> His and Asn21 --> Ile, significantly enhance autoactivation of human cationic trypsinogen in vitro, in a manner that correlates with the severity of clinical symptoms in HP. In addition, mutation Arg117 --> His inhibits autocatalytic inactivation of trypsin, while mutation Asn21 --> Ile has no such effect. The findings strongly argue that increased trypsinogen activation in the pancreas is the common initiating step in both forms of HP, whereas trypsin stabilization might also contribute to HP associated with the Arg117 --> His mutation.     

Predicting the clinical lethality of osteogenesis imperfecta from collagen glycine mutations

Osteogenesis imperfecta (OI), or brittle bone disease, often results from missense mutation of one of the conserved glycine residues present in the repeating Gly-X-Y sequence characterizing the triple-helical region of type I collagen. A composite model was developed for predicting the clinical lethality resulting from glycine mutations in the alpha1 chain of type I collagen. The lethality of mutations in which bulky amino acids are substituted for glycine is predicted by their position relative to the N-terminal end of the triple helix. The effect of a Gly --> Ser mutation is modeled by the relative thermostability of the Gly-X-Y triplet on the carboxy side of the triplet containing the substitution. This model also predicts the lethality of Gly --> Ser and Gly --> Cys mutations in the alpha2 chain of type I collagen. The model was validated with an independent test set of six novel Gly --> Ser mutations. The hypothesis derived from the model of an asymmetric interaction between a Gly --> Ser mutation and its neighboring residues was tested experimentally using collagen-like peptides. Consistent with the prediction, a significant decrease in stability, calorimetric enthalpy, and folding time was observed for a peptide with a low-stability triplet C-terminal to the mutation compared to a similar peptide with the low-stability triplet on the N-terminal side. The computational and experimental results together relate the position-specific effects of Gly --> Ser mutations to the local structural stability of collagen and lend insight into the etiology of OI.     

Crystal structure of PBP2x from a highly penicillin-resistant Streptococcus pneumoniae clinical isolate: a mosaic framework containing 83 mutations.

Penicillin-binding proteins (PBPs) are the main targets for beta-lactam antibiotics, such as penicillins and cephalosporins, in a wide range of bacterial species. In some Gram-positive strains, the surge of resistance to treatment with beta-lactams is primarily the result of the proliferation of mosaic PBP-encoding genes, which encode novel proteins by recombination. PBP2x is a primary resistance determinant in Streptococcus pneumoniae, and its modification is an essential step in the development of high level beta-lactam resistance. To understand such a resistance mechanism at an atomic level, we have solved the x-ray crystal structure of PBP2x from a highly penicillin-resistant clinical isolate of S. pneumoniae, Sp328, which harbors 83 mutations in the soluble region. In the proximity of the Sp328 PBP2x* active site, the Thr(338) --> Ala mutation weakens the local hydrogen bonding network, thus abrogating the stabilization of a crucial buried water molecule. In addition, the Ser(389) --> Leu and Asn(514) --> His mutations produce a destabilizing effect that generates an "open" active site. It has been suggested that peptidoglycan substrates for beta-lactam-resistant PBPs contain a large amount of abnormal, branched peptides, whereas sensitive strains tend to catalyze cross-linking of linear forms. Thus, in vivo, an "open" active site could facilitate the recognition of distinct, branched physiological substrates.     

Absence of insertions among spontaneous mutants of Salmonella typhimurium

Summary While insertion sequences (IS) in Escherichia coli transpose frequently to generate spontaneous insertion mutants, such mutations are rare in Salmonella typhimurium: the only documented insertion mutation is a hisD mutation caused by the Salmonella-specific IS element IS200. To obtain more examples of IS200 insertion mutations and to seek additional types of IS elements in Salmonella, we selected and characterized 422 independent, spontaneous His^– mutants and some 2100 additional mutants that are not necessarily independent. None of the mutants showed the absolute polar effect characteristic of insertion mutations or the reversion properties characteristic of insertions (low spontaneous reversion frequency and no reversion induction by chemical mutagens). A few mutants, showing a high spontaneous reversion frequency, were screened physically. No insertion mutations were found. Thus insertion mutations appear to be rare in S. typhimurium, in strong contrast to E. coli and despite the possession in Salmonella of at least one type of insertion element (IS200). These results suggest that in Salmonella transposition of the endogenous elements has been controlled. The transposition ability of the elements may have been reduced or favored target sites removed from the host genome.     

Fitness-compensatory mutations in rifampicin-resistant RNA polymerase

Mutations in rpoB (RNA polymerase β-subunit) can cause high-level resistance to rifampicin, an important first-line drug against tuberculosis. Most rifampicin-resistant (Rif(R)) mutants selected in vitro have reduced fitness, and resistant clinical isolates of M. tuberculosis frequently carry multiple mutations in RNA polymerase genes. This supports a role for compensatory evolution in global epidemics of drug-resistant tuberculosis but the significance of secondary mutations outside rpoB has not been demonstrated or quantified. Using Salmonella as a model organism, and a previously characterized Rif(R) mutation (rpoB R529C) as a starting point, independent lineages were evolved with selection for improved growth in the presence and absence of rifampicin. Compensatory mutations were identified in every lineage and were distributed between rpoA, rpoB and rpoC. Resistance was maintained in all strains showing that increased fitness by compensatory mutation was more likely than reversion. Genetic reconstructions demonstrated that the secondary mutations were responsible for increasing growth rate. Many of the compensatory mutations in rpoA and rpoC individually caused small but significant reductions in susceptibility to rifampicin, and some compensatory mutations in rpoB individually caused high-level resistance. These findings show that mutations in different components of RNA polymerase are responsible for fitness compensation of a Rif(R) mutant.     

Determination of acaricide resistance allele frequencies in field populations of Tetranychus urticae using quantitative sequencing

Resistances to monocrotophos, fenpropathrin and abamectin in Tetranychus urticae are primarily conferred by reduced sensitivities of respective target sites [i.e., acetylcholinesterase (TuAChE), voltage-sensitive sodium channel (TuVSSC) and glutamate-gated chloride channel (TuGluCl)], which are due to point mutations (G228S and F439W in TuAChE; L1022V in TuVSSC; G323D in TuGluCl). As a population-based genotyping technique, a quantitative sequencing (QS) protocol was developed for the determination of the resistance-associated mutation frequencies in T. urticae. Standard prediction equations revealed high correlation coefficients (r² = 0.993–0.999), demonstrating that the resistant nucleotide signal ratio is highly proportional to the resistance allele frequencies. The lower and higher detection limits for the four resistance mutations were 3.7–13.1% (7.8 ± 3.3%) and 89.4–97.3% (93.3 ± 3.2%), respectively, suggesting that QS can be employed as a preliminary monitoring tool for the detection of resistance allele frequencies, which ranged approximately 7.8–93.3% at the 95% confidence level. The QS was successfully employed for the determination of resistance allele frequencies in 26 T. urticae populations. The two TuAChE mutations responsible for monocrotophos resistance were almost saturated in most field populations. The TuVSSC L1022V mutation tentatively associated with fenpropathrin resistance was also found in 9 field populations. However, the TuGluCl G323D mutation conferring abamectin resistance was found only in one field population, suggesting that abamectin resistance is not yet widespread. The QS protocol, as an alternative to traditional bioassays, will greatly facilitate resistance monitoring of T. urticae.     

Genetic Characterization of the SufJ Frameshift Suppressor in SALMONELLA TYPHIMURIUM

A new suppressor of +1 frameshift mutations has been isolated in Salmonella typhimurium. This suppressor, sufJ, maps at minute 89 on the Salmonella genetic map between the argH and rpo(rif) loci, closely linked to the gene for the ochre suppressor tyrU(supM). The suppressor mutation is dominant to its wild-type allele, consistent with the suppressor phenotype being caused by an altered tRNA species. The sufJ map position coincides with that of a threonine tRNA(ACC/U) gene; the suppressor has been shown to read the related fourbase codons ACCU, ACCC, ACCA.--The ability of sufJ to correct one particular mutation depends on the presence of a hisT mutation which causes a defect in tRNA modification. This requirement is allele specific, since other frameshift mutations can be corrected by sufJ regardless of the state of the hisT locus.--Strains carrying both a sufJ and a hisT mutation are acutely sensitive to growth inhibition by uracil; the inhibition is reversed by arginine. This behavior is characteristic of strains with mutations affecting the arginine-uracil biosynthetic enzyme carbamyl phosphate synthetase. The combination of two mutations affecting tRNA structure may reduce expression of the structural gene for this enzyme (pyrA).     

Functional interactions within 23S rRNA involving the peptidyltransferase center.

A molecular genetic approach has been employed to investigate functional interactions within 23S rRNA. Each of the three base substitutions at guanine 2032 has been made. The 2032A mutation confers resistance to the antibiotics chloramphenicol and clindamycin, which interact with the 23S rRNA peptidyltransferase loop. All three base substitutions at position 2032 produce an erythromycin-hypersensitive phenotype. The 2032 substitutions were compared with and combined with a 12-bp deletion mutation in domain II and point mutations at positions 2057 and 2058 in the peptidyltransferase region of domain V that also confer antibiotic resistance. Both the domain II deletion and the 2057A mutation relieve the hypersensitive effect of the 2032A mutation, producing an erythromycin-resistant phenotype; in addition, the combination of the 2032A and 2057A mutations confers a higher level of chloramphenicol resistance than either mutation alone. 23S rRNAs containing mutations at position 2058 that confer clindamycin and erythromycin resistance become deleterious to cell growth when combined with the 2032A mutation and, additionally, confer hypersensitivity to erythromycin and sensitivity to clindamycin and chloramphenicol. Introduction of the domain II deletion into these double-mutation constructs gives rise to erythromycin resistance. The results are interpreted as indicating that position 2032 interacts with the peptidyltransferase loop and that there is a functional connection between domains II and V.     

Carbon starvation of Salmonella typhimurium does not cause a general increase of mutation rates.

Mutation rates in bacteria can vary depending on the genetic target studied and the specific growth conditions of the cells. Here, two different methods were used to determine how rates of mutation to antibiotic resistance, auxotrophy, and prototrophy were influenced by carbon starvation on agar plates. The rate of mutation to rifampin resistance was increased by starvation as measured by fluctuation tests, similar to what has been reported previously for Escherichia coli. In contrast, the rates of mutation to various types of auxotrophy were unaffected or decreased as measured by both fluctuation tests and a repeated-streaking procedure. Similarly, the rates of reversion to prototrophy of his and lac nonsense and missense mutations were unaffected by starvation. Thus, mutation rates of different genetic targets can be affected differently by starvation and we conclude that carbon starvation is not generally mutagenic in Salmonella typhimurium.     

5-fluorouracil forward mutation assay in Salmonella: determination of mutational target and spontaneous mutational spectra

A forward mutation assay in Salmonella typhimurium that selects for 5-fluoruracil (FU) resistance has been developed. The two genes possibly involved in FU resistance, the uracil phosphoribosyl transferase gene (upp) and the uracil transport protein (uraA), have been cloned from S. typhimurium and sequenced. One hundred percent of FU-resistant clones display sequence changes in the upp gene, indicating that its loss is the major mechanism involved in FU resistance. The spontaneous mutational spectra at the upp locus were then determined in two S. typhimurium strains, FU100 and FU1535, that differ only in the presence of pKM101 plasmid. The pKM101 plasmid provides error-prone replicative bypass of DNA lesions and renders FU100 more susceptible to induced mutagenesis. Fluctuation analysis of FU-resistant clones demonstrated a 10-fold higher spontaneous mutation rate at the upp locus in FU100 relative to FU1535. Over 300 independent FU-resistant clones were then used to generate the spectra at the upp locus in both the strains. Approximately 40% of all the mutations were base substitutions, present at the same relative percentage in both the strains. Frameshift mutations also accounted for approximately 40% of the total; however, their incidence was slightly elevated in FU100. The remaining mutations were larger insertions and deletions, which were both slightly elevated in FU1535. pKM101 significantly elevated the rate of all classes of mutations at the upp locus, with profound effects on A:T to T:A transversions and -2-base frameshift mutations. These initial mutational spectra at the upp locus reveal 147 mutable sites, or 23% of the total 627-base coding sequence and suggest that the target can detect a diverse spectrum of mutagenic events.     

Null mutations in the essential gene yrfF (mucM) are not lethal in rcsB,yojN or rcsC strains of Salmonella enterica serovar Typhimurium

Insertion of factor MudJ in the intergenic region between divergent genes yrfF and yrfE, at centisome 76 in the genome of Salmonella enterica serovar Typhimurium LT2, confers the characteristics recently described for mucM mutants, i.e. mucoidy and resistance to mecillinam. Cloning of the intergenic region plus either the yrfF or the yrfE gene in a multicopy plasmid showed that only the plasmid carrying the yrfF gene complemented mucM mutants, thus suggesting that mucM mutations are in fact yrfF mutations. A null yrfF mutation obtained by insertion of a kanamycin cassette into the yrfF open reading frame (yrfF28::Kan) produced abortive colonies when transduced to a wild-type strain but was normally accepted by rcsB, rcsC or yojN strains. Neither mutations preventing synthesis of the capsular exopolysaccharide colanic acid (cps, galE) nor rcsA mutations, which reduce expression of cps genes, conferred tolerance to the lethal yrfF28::Kan mutation. Spontaneous suppressor mutations arose very frequently in abortive yrfF28::Kan colonies, and all of them affected either rcsC, yojN, or rcsB genes. Thus, the lethal effect caused by inactivation of gene yrfF appears to be mediated by a function that is dependent on the rcsC-yojN-rcsB phosphorelay system but does not involve synthesis of colanic acid.     

The mechanism of ciprofloxacin resistance in dihydrogen peroxide-induced mutants of Salmonella enterica subsp. enterica serovar typhimurium consists mainly in mutations in gyrA gene and less in mutations affecting ciprofloxacin uptake

The effect of H(2)O(2) on the induction of ciprofloxacin (CFL) resistant mutants of Salmonella enterica subsp. enterica serovar Typhimurium was evaluated and determinants of CFL resistance in the mutants were analyzed. Factors associated with CFL resistance in H(2)O(2)-induced mutants included (i) mutations in gyrA gene, predominantly (63 %) Asp(87)-->Asn and less (37 %) Ser(83)-->Phe substitutions, (ii) mutations in the regulatory genes of MarRAB or SoxRS or in the individual structural genes of these operons. Such mutations are induced by H(2)O(2) in a much lower extent. Reduced OmpF expression simultaneously with enhanced efflux was detected only in one mutant strain and 20 % of mutant strains had increased CFL efflux from the cells.     

Modeling the Leigh syndrome nt8993 T-->C mutation in Escherichia coli F1F0 ATP synthase.

The mutations in human mitochondrial DNA at nt8993 are associated with a range of neuromuscular disorders. One mutation encodes a proline in place of a leucine conserved in all animal mitochondrial ATPase-6 subunits and bacterial a subunits of F1F0 ATP synthases. This conserved site is leu-156 and leu-207 in humans and Escherichia coli, respectively. An aleu-207-->pro substitution mutation has been constructed in the E. coli F1F0 ATP synthase in order to model the biochemical basis of the human disease mutation. The phenotype of the aleu-207-->pro substitution has been compared to that of the previously studied aleu-207-->arg substitution (Hartzog and Cain, 1993, Journal of Biological Chemistry 268, 12250-12252). The leu-207-->pro mutation resulted in approximately a 35% decrease in the number of intact enzyme complexes as determined by N, N'-dicyclohexylcarbodiimide-sensitive membrane associated ATP hydrolysis activity and western analysis using an anti-a subunit antibody. A 75% reduction in the efficiency of proton translocation through F1F0 ATP synthase was observed in ATP-driven proton pumping assays. Interestingly, the loss in F1F0 ATP synthase activity resulting from the leu-207-->pro substitution was markedly less dramatic than had been observed for the leu-207-->arg mutation studied earlier. By analogy, the human enzyme may also be affected by the leu-156-->pro substitution to a lesser extent than the leu-156-->arg substitution, and this would account for the milder clinical manifestations of the human leu-156-->pro disease mutations.