A new mechanism of antibiotic resistance in Enterobacteriaceae induced by a structural modification of the major porin

In Enterobacter aerogenes, multidrug resistance involves a decrease in outer membrane permeability associated with changes in an as yet uncharacterized porin. We purified the major porin from the wild-type strain and a resistant strain. We characterized this porin, which was found to be an OmpC/OmpF-like protein and analysed its pore-forming properties in lipid bilayers. The porin from the resistant strain was compared with the wild-type protein and we observed (i) that its single-channel conductance was 70% lower than that of the wild type; (ii) that it was three times more selective for cations; (iii) a lack of voltage sensitivity. These results indicate that the clinical strain is able to synthesize a modified porin that decreases the permeability of the outer membrane. Mass spectrometry experiments identified a G to D mutation in the putative loop 3 of the porin. Given the known importance of this loop in determining the pore properties of porins, we suggest that this mutation is responsible for the novel resistance mechanism developed by this clinical strain, with changes in porin channel function acting as a new bacterial strategy for controlling beta-lactam diffusion via porins.     

New major outer membrane proteins found in an Escherichia coli tolF mutant resistant to bacteriophage TuIb.

Cell envelopes prepared from an Escherichia coli tolF strain selected as resistant to phage TuIb contained a new major outer membrane protein related to outer membrane proteins Ia and Ib. The strain that produces this protein is a tolF par double mutant but contains an additional mutation leading to the production of the new major outer membrane protein. Antibiotic sensitivity lost as a result of the tolF mutation is regained in strains that contain the new major outer membrane protein. This indicates that this protein functions to restore the selective permeability of the outer membrane to low-molecular-weight hydrophilic molecules.     

Genetic and physiological analysis of an envB spherelike mutant of Escherichia coli K-12 and characterization of its transductants.

The envB1 mutation mediating a distorted cell morphology of Escherichia coliK-12 was cotransducible with strA, aroE, aspB, and argG. The mapping data is consistent with a gene location for envB around 62.5 min. In partial diploids envB1 was recessive to its wild-type allele. The original envB mutant contained a second mutation in a locus denoted sloB close to strA. The following gene order is suggested: sloB-strA-aroE-envB-aspB-argG. The sloB1 mutation caused a marked reduction in the growth rate of both envB and envB+ strains. Moreover, this mutation in the presence of envB1 appears to increase the ratio between deoxyribonucleic acid and protein in cells growing in rich medium. The phenotypic properties of envB1, sloB+ and envB+ transductants were characterized. Cells with envB1, sloB+ genotype were hypersensitive to several penicillins including the beta-lactam compound, amidino penicillin. Penicillin hypersensitivity could not be explained by increased outer membrane penetrability. The original envB mutant (envB1,SLOB1), as well as envB1, sloB1 or envB+, SLOB1 transductants were resistant to amidino penicillin. Resistance was explained by the slow growth rate mediated by the sloB1 mutation. The similarity between envB cells and wild-type cells treated with sublethal concentrations of amidino penicillin was emphasized.     

Characterisation of atovaquone resistance in Leishmania infantum promastigotes

Atovaquone, an antiparasitic agent, could possibly represent an alternative therapy after relapse following classical treatment for visceral leishmaniasis. Atovaquone-resistant strains were selected in vitro by stepwise drug pressure to study the mechanism of resistance in Leishmania. Characteristics of a promastigote strain resistant to 250 microg/ml of atovaquone were compared with those of the wild type (WT) strain. Resistant strains were shown to have a high level of resistance (45 times). They were stable in drug-free medium for 6 months, and showed no cross-resistance with other antileishmanial drugs. Rhodamine uptake and efflux were studied. They were not modified in the resistant strain, indicating the absence of P-glycoprotein overexpession. The effect of atovaquone on membrane lipidic composition was determined in both WT and atovaquone-resistant promastigotes. Analysis of lipid composition of the atovaquone-resistant strain showed that sterol biosynthesis was decreased in atovaquone-resistant parasites. Cholesterol was found to be the major membrane sterol as opposed to the WT strain. Cholesterol, due to its ordering effect, could decrease membrane fluidity and subsequently block the passage of atovaquone through the membrane. Increased membrane cholesterol content and altered drug membrane fluidity resulted from possible decrease of ergosterol biosynthesis by atovaquone, incorporation of cholesterol by promastigotes in the culture medium, solubilisation of atovaquone by cholesterol and co-passage of the two compounds or influence of dimethylsulfoxide. These results indicate that different cellular alterations may participate in the resistant phenotype, by altering drug membrane permeability.     

Increased loss of duplicated genes in streptomycin-resistant (strA) mutants of Escherichia coli k-12.

The recombination-dependent loss of a duplicated portion of the Escherichia coli chromosome is five- to tenfold greater in strains containing streptomycin resistance (strA) mutations than in the strA+ parental strain. Streptomycin (500 mug/ml) partially reverses the increase. These results suggest an interaction between strA mutations and recombination.     

Self-cloning in Streptomyces griseus of an str gene cluster for streptomycin biosynthesis and streptomycin resistance.

An str gene cluster containing at least four genes (strR, strA, strB, and strC) involved in streptomycin biosynthesis or streptomycin resistance or both was self-cloned in Streptomyces griseus by using plasmid pOA154. The strA gene was verified to encode streptomycin 6-phosphotransferase, a streptomycin resistance factor in S. griseus, by examining the gene product expressed in Escherichia coli. The other three genes were determined by complementation tests with streptomycin-nonproducing mutants whose biochemical lesions were clearly identified. strR complemented streptomycin-sensitive mutant SM196 which exhibited impaired activity of both streptomycin 6-phosphotransferase and amidinotransferase (one of the streptomycin biosynthetic enzymes) due to a regulatory mutation; strB complemented strain SD141, which was specifically deficient in amidinotransferase; and strC complemented strain SD245, which was deficient in linkage between streptidine 6-phosphate and dihydrostreptose. By deletion analysis of plasmids with appropriate restriction endonucleases, the order of the four genes was determined to be strR-strA-strB-strC. Transformation of S. griseus with plasmids carrying both strR and strB genes enhanced amidinotransferase activity in the transformed cells. Based on the gene dosage effect and the biological characteristics of the mutants complemented by strR and strB, it was concluded that strB encodes amidinotransferase and strR encodes a positive effector required for the full expression of strA and strB genes. Furthermore, it was found that amplification of a specific 0.7-kilobase region of the cloned DNA on a plasmid inhibited streptomycin biosynthesis of the transformants. This DNA region might contain a regulatory apparatus that participates in the control of streptomycin biosynthesis.     

An A666G mutation in transmembrane helix 5 of the yeast multidrug transporter Pdr5 increases drug efflux by enhancing cooperativity between transport sites

Resistance to antimicrobial and chemotherapeutic agents is a significant clinical problem. Overexpression of multidrug efflux pumps often creates broad‐spectrum resistance in cancers and pathogens. We describe a mutation, A666G, in the yeast ABC transporter Pdr5 that shows greater resistance to most of the tested compounds than does an isogenic wild‐type strain. This mutant exhibited enhanced resistance without increasing either the amount of protein in the plasma membrane or the ATPase activity. In fluorescence quenching transport assays with rhodamine 6G in purified plasma membrane vesicles, the initial rates of rhodamine 6G fluorescence quenching of both the wild type and mutant showed a strong dependence on the ATP concentration, but were about twice as high in the latter. Plots of the initial rate of fluorescence quenching versus ATP concentration exhibited strong cooperativity that was further enhanced in the A666G mutant. Resistance to imazalil sulfate was about 3–4x as great in the A666G mutant strain as in the wild type. When this transport substrate was used to inhibit the rhodamine 6G transport, the A666G mutant inhibition curves also showed greater cooperativity than the wild‐type strain. Our results suggest a novel and important mechanism: under selection, Pdr5 mutants can increase drug resistance by improving cooperative interactions between drug transport sites.     

Number of mutations required to evolve a new lactase function in Escherichia coli.

The frequency of mutation of the ebgAo allele to ebgA+ was compared with the frequency of mutation of strA+ to strA-. The observation that both spontaneous and ethyl methane sulfonate-induced mutations to ebgA+ occurred more frequently than mutations to strA- suggests that ebgA+ mutants arise as the result of single-point mutations.     

DNA damage and prophage induction and toxicity of nitrofurantoin in Escherichia coli and Vibrio cholerae cells

Repair-deficient and repair-proficient strains of E. coli K12 were sensitive to nitrofurantoin treatment to varying degrees with the double mutant strain (uvrA 6, recA 13) being most sensitive. Ultraviolet absorption data and thermal chromatography through a hydroxyapatite column revealed that nitrofurantoin treatment of V. cholerae strain OGAWA 154 produced a maximal amount of 55% reversibly bihelical DNA at a nitrofurantoin dose of 120 micrograms/ml/h, which indicated the formation of inter-strand cross-links in DNA. Nitrofurantoin also produced prophage-lambda induction in E. coli K12 strain GY 5027: envA, uvrB, ampA 1, strA (lambda), in a dose-dependent manner, the maximum induction being highly significant (P less than 0.001). Previously published mutation data coupled with the prophage induction data presented here suggest that the genotoxic properties of nitrofurantoin are mediated through the SOS pathway.     

Morphologic mutant of Escherichia coli K12 having disorders in morphology, division and cell wall biosynthesis]

A study was made of the properties of a spherical mutant obtained from the E. coli K12 HfrC strain under the effect of N-nitroso-N-methyl-urea. The growth of the mutant of full value media was characterized by a marked reduction of the cell division at the rest phase, but exponential growth phase failed to differ from the growth of the parental strain. Electron microscopic study of surface structures of the mutant cells which grew under physiological conditions permitted to distinguish two types: the first type had a typical structure of the cell wall characteristic of Gram negative microbes; the second type was framed by a bicontour membrane without any distinct structure. The presence of these two types of cells was also confirmed by their different sensitivity to the ionic detergents. On the basis of chemical analysis of peptidoglycan of the cell wall (which was markedly decreased in amount in the mutant cells), and also of the unsually high accumlation of the UDP-precursors of peptidoglycan under conditions of penicillin action it is supposed that normal regulation of metabolism of the cell walls was deranged. Mutation designated by 11rA symbol was plotted by phase PI transduction alongside of strA gene.     

The biosynthetic pathway of new polyamines in Caldariella acidophila

A spontaneous mutant of Escherichia coli (strain AB2847), selected for resistance to the aminoglycoside antibiotic neamine, shows severe restriction of amber suppressors in vivo. Ribosomes isolated from the mutant exhibit only low misreading in vitro in the presence of the antibiotic. Genetic and biochemical analyses indicate that the neamine-resistant phenotype is the result of two distinct mutations. The first, res3128, appears to affect the gene (strA) coding for the ribosomal protein S12. Although it leads to a restrictive phenotype it does not, however, confer resistance to streptomycin. The second mutation, X3128, is located between the sirA and AROB loci and is lethal when segregated from the res3128 mutation. It may affect the ribosome at the level of a post-translational modification.     

Genetic interaction between the Escherichia coli AcpT phosphopantetheinyl transferase and the YejM inner membrane protein

Strain LH530, a mutant of Escherichia coli K-12, was reported by others to show increased outer membrane permeability, temperature-sensitive growth, and reduced synthesis of lipid A. The unmapped mutant gene was found to be suppressed by high-copy-number plasmids carrying the wild-type acpT gene, which encodes a protein that catalyzes a post-translational protein modification, the attachment of 4'-phosphopantetheine. We mapped the strain LH530 mutation to a gene of unknown function, yejM, known to encode an inner membrane protein. The mutation is a yejM nonsense mutation that produces a truncated protein lacking the predicted periplasmic domain. Reconstruction of the mutation gave a strain having the same phenotypes as LH530. In contrast to the nonsense mutants, deletion of the entire yejM gene was lethal. Suppression by AcpT overexpression of the yejM nonsense mutants encoding the truncated proteins was specific to AcpT. Moreover, AcpT overexpression also suppressed the lethality due to deletion of the entire yejM gene and this suppression also did not require that AcpT be enzymatically active. The mechanism whereby overexpression of a specific cytosolic protein bypasses the essentiality of an inner membrane protein is unknown.     

Genetic determinant of pyocin R2 in Pseudomonas aeruginosa PAO. I. Localization of the pyocin R2 gene cluster between the trpCD and trpE genes

Thirty-seven mutants defective in pyocin R2 production in the P. aeruginosa PAO strain were subjected to fine mapping of pyocin R2 genes by transduction with phage F116L. Sixteen complementation groups (designated prtA through prtP) involved in pyocin R2 production were tentatively identified by complementation tests using phage F116L. Their linkages to trpC and trpE markers and fine mapping by three point crosses demonstrated that most of the mutations (prtA through prtN) were located in between trpC and trpE, and that the prtP mutation was localized outside this major prt cluster but in the proximity of the rifA and strA region.     

Energy transduction in Escherichia coli: physiological and biochemical effects of mutation in the uncB locus.

The transduction of energy through biological membranes was investigated in Escherichia coli strains defective in the ATP synthetase complex. Everted vesicles prepared from strains containing an uncA or uncB mutation were compared with those of the parental strain for their ability to couple energy derived from the oxidation of substrates by the electron transport chain or from the hydrolysis of ATP by the Mg2+-adenosine triphosphatase, as measured by the energy-dependent quenching of quinacrine fluorescence or the active transport of 45Ca2+. Removal of the Mg2+-adenosine triphosphatase from membranes derived from the parental or an uncA strain caused a loss of energy-linked functions and a concomitant increase in the permeability of the membrane for protons. Proton impermeability was restored by treatment with N,N'-dicyclohexylcarbodiimide. When membranes of the uncB strain were treated in a similar manner, there was no loss of respiratory-driven functions, nor was there a change in proton permeability. These observations suggest that the uncB mutation specifically results in alteration of an intrinsic membrane protein channel necessary for the generation of utilzation of the electrochemical gradient of protons by that complex. Loss of the function of the proton channel is believed to prevent the transduction of energy through the ATP synthetase complex.     

Suppression of spectinomycin resistance in a mutant of Escherichia coli K-12.

A mutant of Escherichia coli has been isolated which exhibits partial suppression of spectinomycin resistance. The site of mutation is in the streptomycin (strA) region and is closely linked to the spcA gene. However, this gene, which we propose to call mod, is phenotypically distinguishable from both the neomycin-kanamycin (nek) and the ribosomal ambiguity gene (ram). The relative gene order is mod spcA strA. In a cell-free protein synthesizing system, altered ribosomes appear to be responsible for the suppression of spectinomycin resistance caused by mod.     

Cotransduction of strA and Ribosomal Protein Cistrons in Escherichia coli-Salmonella typhimurium Hybrids

Genetic mapping of ribosomal protein cistrons of Salmonella typhimurium and Escherichia coli was performed by phage P1 mediated, generalized transduction. From an E. coli hybrid strain which carried a S. typhimuirum F' factor, an E. coli strain was constructed which had integrated S. typhimurium genetic material including the region of the strA locus. Salmonella genetic material from this hybrid was transduced into E. coli recipients. The ribosomal protein electrophoretic patterns of these hybrid transductants were correlated with the presence of markers contributed by each parent.The results of these studies indicate that cistrons for at least three characteristic S. typhimurium and two E. coli 30S ribosomal proteins are closely linked to the strA locus on the genetic maps of both organisms. At least one cistron coding for a 50S ribosomal protein is also closely linked to this locus on both maps. These findings support the concept that cistrons coding for the ribosomal proteins are clustered in one area of the genome. Mutations to spectinomycin and streptomycin resistance are closely linked in S. typhimurium and are located at strA.     

A design-constraint trade-off underpins the diversity in ecologically important traits in species Escherichia coli

Bacterial species are internally diverse in genomic and multi-locus gene comparisons. The ecological causes of phenotypic and genotypic diversity within species are far less well understood. Here, we focus on the competitive fitness for growth on nutrients within Escherichia coli, an internally rich species. Competition experiments in nutrient-limited chemostats revealed that members of the ECOR collection exhibited a wide continuum of competitive abilities, with some fitter and some less fit than the lab strain MG1655. We observed an inverse relationship between competitiveness and the resistance of strains to detergent and antibiotic, consistent with the notion that membrane permeability and competitive fitness are linked by a trade-off between self-preservation and nutritional competence (SPANC); high permeability has a postulated cost in antibacterial sensitivity whereas a low permeability has a cost in nutrient affinity. Isolates moved along the markedly nonlinear trade-off curve by mutational adaptation; an ECOR strain sensitive to antibacterials and a good competitor was easily converted by mutation into a mutant with higher resistance but poorer competition in the presence of low antibiotic concentrations. Conversely, a resistant ECOR strain changed into a better competitor after a short period of selection under nutrient limitation. In both directions, mutations can affect porin proteins and outer membrane permeability, as indicated by protein analysis, gene sequencing and an independent assay of outer membrane permeability. The extensive, species-wide diversity of E. coli in ecologically important traits can thus be explained as an evolutionary consequence of a SPANC trade-off driven by antagonistic pleiotropy.     

Mutation in Escherichia coli K-12 Mediating Spherelike Envelopes and Changed Tolerance to Ultraviolet Irradiation and Some Antibiotics

In a mutation experiment with a rough, ampicillin-resistant strain of Escherichia coli K-12, two smooth ampicillin-sensitive mutants were isolated. One of the mutants (with the envA gene) was recently described. The second mutant (strain D23) with the envB gene which has been mapped to a position close to streptomycin resistance (strA) at 64 min is described. The envB gene gives rise to spherelike cells. Electron microscopy revealed an abnormal septum formation and a circular distribution of the nuclear material. Strain D23 (with envB) showed a changed resistance to several antibiotics as well as an increased tolerance to ultraviolet irradiation. The envB gene decreased the ampicillin resistance mediated by the ampA gene (at 82 min), but no effect was found on episomally mediated penicillin resistance.     

30S subunit mutations relieving restriction of ribosomal misreading caused by L6 mutations

Summary Mutants were analyzed biochemically and genetically in which restriction of translational misreading by ribosomes containing an altered L6 protein is relieved. Amongst 100 such strains eight possessed an altered S4 and two a mutant S5 protein. The protein-chemical type of L6 mutation seems to influence the kind of S4 mutant form selected. Also, only a few types of S4 ram mutations are obtained and they are different from those usually found amongst suppressors of streptomycin-dependent (Sm^D) strains. The S4 mutations selected are able to reduce the level of streptomycin-resistance of strA1 or strA40 strains and they confer extreme hypersensitivity to aminoglycosides when present alone. On the other hand, S4 mutations from Sm^D suppressor strains only weakly reverse L6 restriction. The results imply that control of translational fidelity is an intersubunit function and that protein L6 (an interface protein) cooperates with 30S proteins by directly or indirectly determining parameters involved in aminoacyl-tRNA recognition.