Antimicrobial cationic surfactant, cetyltrimethylammonium bromide, induces superoxide stress in Escherichia coli cells

Aims: To clarify whether an antibacterial surfactant, cetyltrimethylammonium bromide (CTAB), induces superoxide stress in bacteria, we investigated the generation of superoxide and hydrogen peroxide and expression of soxR, soxS and soxRS regulon genes in Escherichia coli cells with the treatment of CTAB. Methods and Results: In situ oxidative stress analyses with BES fluorescent probes revealed that generation of both superoxide and hydrogen peroxide were significantly increased with the CTAB treatment at a sublethal concentration in wild‐type strain OW6, compared with the CTAB‐resistant strain OW66. The activity of manganese–superoxide dismutase (Mn–SOD), a member of the soxRS regulon proteins, was decreased by the CTAB treatment only in strain OW6. Furthermore, quantitative real‐time PCR analyses revealed that expression of the soxRS regulon genes was not upregulated, although soxS was upregulated by the CTAB treatment in strain OW6. Conclusions: Cetyltrimethylammonium bromide treatment led E. coli cells to a generation state of superoxide and hydrogen peroxide. It was also suggested that superoxide generation was caused by inhibiting SoxS function and decreasing Mn–SOD activity. Significance and Impact of the Study: It was revealed that excess superoxide generation in bacterial cells play a key action of antibacterial surfactants.     

Repression of Invasion Genes and Decreased Invasion in a High-Level Fluoroquinolone-Resistant Salmonella Typhimurium Mutant

Background

Nalidixic acid resistance among Salmonella Typhimurium clinical isolates has steadily increased, whereas the level of ciprofloxacin resistance remains low. The main objective of this study was to characterize the fluoroquinolone resistance mechanisms acquired in a S. Typhimurium mutant selected with ciprofloxacin from a susceptible isolate and to investigate its invasion ability.

Methodology/Principal Findings

Three different amino acid substitutions were detected in the quinolone target proteins of the resistant mutant (MIC of ciprofloxacin, 64 µg/ml): D87G and G81C in GyrA, and a novel mutation, E470K, in ParE. A protein analysis revealed an increased expression of AcrAB/TolC and decreased expression of OmpC. Sequencing of the marRAB, soxRS, ramR and acrR operons did not show any mutation and neither did their expression levels in a microarray analysis. A decreased percentage of invasion ability was detected when compared with the susceptible clinical isolate in a gentamicin protection assay. The microarray results revealed a decreased expression of genes which play a role during the invasion process, such as hilA, invF and the flhDC operon. Of note was the impaired growth detected in the resistant strain. A strain with a reverted phenotype (mainly concerning the resistance phenotype) was obtained from the resistant mutant.

Conclusions/Significance

In conclusion, a possible link between fluoroquinolone resistance and decreased cell invasion ability may exist explaining the low prevalence of fluoroquinolone-resistant S. Typhimurium clinical isolates. The impaired growth may appear as a consequence of fluoroquinolone resistance acquisition and down-regulate the expression of the invasion genes.     

All genomic mutations in the antimicrobial surfactant-resistant mutant, Escherichia coli OW66, are involved in cell resistance to surfactant

The spontaneous antimicrobial surfactant-resistant mutant, Escherichia coli OW66, has been isolated, and its physiological properties have been characterized in our previous paper (Ishikawa et al., J Appl Microbiol 92:261–268, 2002b). This report revealed that strain OW66 had seven mutations in their chromosomal DNA by comparative genomic hybridization microarray, and that their alternative functions were involved in cell resistance to antimicrobial surfactants. These mutations were located in oppB, ydcR, IVR(vacJ-yfdC), rpoN, rpoB, rpoC, and soxR. Furthermore, seven of the single-mutated isogenic strains and seven of the six-mutated isogenic strains were constructed from strains OW6 (NBRC106482) and OW66, respectively, through homologous recombination, and their resistances to an antimicrobial surfactant were measured using the minimum inhibitory concentration method. These results revealed that all six-mutated strains were more sensitive than strain OW66, and that the soxR66 mutation was independently involved in antimicrobial surfactant resistance of E. coli cells. Expression of soxR66 and soxS was increased in both strains OW66 and OW6-soxR66 without the surfactant treatment by the quantitative real time-polymerase chain reaction analysis, compared with strain OW6. Two-dimensional polyacrylamide gel electrophoresis analysis also revealed that some proteins in the soxRS regulon, including Mn-SOD, were overexpressed in both strains OW66 and OW6-soxR66. These results indicate that the soxR66 mutation leads to the constitutive expression of the soxRS regulon, resulting in the acquired resistance of E. coli cells to an antimicrobial surfactant.     

Control of MarRAB Operon in Escherichia coli via Autoactivation and?Autorepression

Choice of network topology for gene regulation has been a question of interest for a long time. How do simple and more complex topologies arise? In this work, we analyze the topology of the marRAB operon in Escherichia coli, which is associated with control of expression of genes associated with conferring resistance to low-level antibiotics to the bacterium. Among the 2102 promoters in E. coli, the marRAB promoter is the only one that encodes for an autoactivator and an autorepressor. What advantages does this topology confer to the bacterium? In this work, we demonstrate that, compared to control by a single regulator, the marRAB regulatory arrangement has the least control cost associated with modulating gene expression in response to environmental stimuli. In addition, the presence of dual regulators allows the regulon to exhibit a diverse range of dynamics, a feature that is not observed in genes controlled by a single regulator.     

Molecular characterization of a novel Na+/H+ antiporter cDNA from Eucalyptus globulus

Environmental stress factors such as salt, drought and heat are known to affect plant productivity. However, high salinity is spreading throughout the world, currently affecting more than 45 million ha. One of the mechanisms that allow plants to withstand salt stress consists on vacuolar sequestration of Na⁺, through a Na⁺/H⁺ antiporter. We isolated a new vacuolar Na⁺/H⁺ antiporter from Eucalyptus globulus from a cDNA library. The cDNA had a 1626 bp open reading frame encoding a predicted protein of 542 amino acids with a deduced molecular weight of 59.1 KDa. Phylogenetic and bioinformatic analyses indicated that EgNHX1 localized in the vacuole. To assess its role in Na⁺ exchange, we performed complementation studies using the Na⁺ sensitive yeast mutant strain Δnhx1. The results showed that EgNHX1 partially restored the salt sensitive phenotype of the yeast Δnhx1 strain. However, its overexpression in transgenic Arabidopsis confers tolerance in the presence of increasing NaCl concentrations while the wild type plants exhibited growth retardation. Expression profiles of Eucalyptus seedlings subjected to salt, drought, heat and ABA treatment were established. The results revealed that Egnhx1 was induced significantly only by drought. Together, these results suggest that the product of Egnhx1 from E. globulus is a functional vacuolar Na⁺/H⁺ antiporter.     

RecBCD- RecFOR-independent pathway of homologous recombination in Escherichia coli

The RecA protein is a key bacterial recombination enzyme that catalyzes pairing and strand exchange between homologous DNA duplexes. In Escherichia coli, RecA protein assembly on DNA is mediated either by the RecBCD or RecFOR protein complexes. Correspondingly, two recombination pathways, RecBCD and RecF (or RecFOR), are distinguished in E. coli. Inactivation of both pathways in recB(CD) recF(OR) mutants results in severe recombination deficiency. Here we describe a novel, RecBCD- RecFOR-independent (RecBFI) recombination pathway that is active in ΔrecBCD sbcB15 sbcC(D) ΔrecF(OR) mutants of E. coli. In transductional crosses, these mutants show only four-fold decrease of recombination frequency relative to the wild-type strain. At the same time they recombine 40- to 90-fold better than their sbcB⁺ sbcC⁺ and ΔsbcB sbcC counterparts. The RecBFI pathway strongly depends on recA, recJ and recQ gene functions, and moderately depends on recG and lexA functions. Inactivation of dinI, helD, recX, recN, radA, ruvABC and uvrD genes has a slight effect on RecBFI recombination. After exposure to UV and gamma irradiation, the ΔrecBCD sbcB15 sbcC ΔrecF mutants show moderately increased DNA repair proficiency relative to their sbcB⁺ sbcC⁺ and ΔsbcB sbcC counterparts. However, introduction of recA730 allele (encoding RecA protein with enhanced DNA binding properties) completely restores repair proficiency to ΔrecBCD sbcB15 sbcC ΔrecF mutants, but not to their sbcB⁺ sbcC⁺ and ΔsbcB sbcC derivatives. Fluorescence microscopy with UV-irradiated recA-gfp fusion mutants suggests that the kinetics of RecA filament formation might be slowed down in the RecBFI pathway. Inactivation of 3′-5′ exonucleases ExoVII, ExoIX and ExoX cannot activate the RecBFI pathway in ΔrecBCD ΔsbcB sbcC ΔrecF mutants. Taken together, our results show that the product of the sbcB15 allele is crucial for RecBFI pathway. Besides protecting 3′ overhangs, SbcB15 protein might play an additional, more active role in formation of the RecA filament.     

Ectopic overexpression of a mungbean vacuolar Na+/H+ antiporter gene (VrNHX1) leads to increased salinity stress tolerance in transgenic Vigna unguiculata L. Walp

Salinity is a major threat to sustainable agriculture worldwide. Plant NHX exchangers play an important role in conferring salt tolerance under salinity stress. In this study, a vacuolar Na⁺/H⁺ antiporter gene VrNHX1 (Genbank Accession No. JN656211.1) from mungbean (Vigna radiata) was introduced into cowpea (Vigna unguiculata) by the Agrobacterium tumefaciens-mediated transformation method. Polymerase chain reaction and Southern blot hybridization confirmed the stable integration of VrNHX1 into the cowpea genome. Comparative expression analysis by semi-quantitative RT-PCR revealed higher expression of VrNHX1 in transgenic cowpea plants than wild-type. Under salt stress conditions, T2 transgenic 35S:VrNHX1 cowpea lines exhibited higher tolerance to 200 mM NaCl treatment than wild-type. Furthermore, T2 transgenic 35S:VrNHX1 lines maintained a higher K⁺/Na⁺ ratio in the aerial parts under salt stress and accumulated higher [Na⁺] in roots than wild-type. Physiological analysis revealed lower levels of lipid peroxidation, hydrogen peroxide and oxygen radical production but higher levels of relative water content and proline, ascorbate and chlorophyll contents in T2 transgenic 35S:VrNHX1 lines.     

The genome sequence of Salmonella enterica serovar Choleraesuis, a highly invasive and resistant zoonotic pathogen

Salmonella enterica serovar Choleraesuis (S.Choleraesuis), a highly invasive serovar among non-typhoidal Salmonella, usually causes sepsis or extra-intestinal focal infections in humans. S.Choleraesuis infections have now become particularly difficult to treat because of the emergence of resistance to multiple antimicrobial agents. The 4.7 Mb genome sequence of a multidrug-resistant S.Choleraesuis strain SC-B67 was determined. Genome wide comparison of three sequenced Salmonella genomes revealed that more deletion events occurred in S.Choleraesuis SC-B67 and S.Typhi CT18 relative to S.Typhimurium LT2. S.Choleraesuis has 151 pseudogenes, which, among the three Salmonella genomes, include the highest percentage of pseudogenes arising from the genes involved in bacterial chemotaxis signal-transduction pathways. Mutations in these genes may increase smooth swimming of the bacteria, potentially allowing more effective interactions with and invasion of host cells to occur. A key regulatory gene of TetR/AcrR family, acrR, was inactivated through the introduction of an internal stop codon resulting in overexpression of AcrAB that appears to be associated with ciprofloxacin resistance. While lateral gene transfer providing basic functions to allow niche expansion in the host and environment is maintained during the evolution of different serovars of Salmonella, genes providing little overall selective benefit may be lost rapidly. Our findings suggest that the formation of pseudogenes may provide a simple evolutionary pathway that complements gene acquisition to enhance virulence and antimicrobial resistance in S.Choleraesuis.     

Dss1 associating with the proteasome functions in selective nuclear mRNA export in yeast

Dss1p is an evolutionarily conserved small protein that interacts with BRCA2, a tumor suppressor protein, in humans. The Schizosaccharomyces pombe strain lacking the dss1⁺ gene (Δdss1) shows a temperature-sensitive growth defect and accumulation of bulk poly(A)⁺ RNA in the nucleus at a nonpermissive temperature. In situ hybridization using probes for several specific mRNAs, however, revealed that the analyzed mRNAs were exported normally to the cytoplasm in Δdss1, suggesting that Dss1p is required for export of some subsets of mRNAs. We identified the pad1⁺ gene, which encodes a component of the 26S proteasome, as a suppressor for the ts⁻ phenotype of Δdss1. Unexpectedly, overexpression of Pad1p could suppress neither the defect in nuclear mRNA export nor a defect in proteasome function. In addition, loss of proteasome functions does not cause defective nuclear mRNA export. Dss1p seems to be a multifunctional protein involved in nuclear export of specific sets of mRNAs and the ubiquitin-proteasome pathway in fission yeast.     

The Contribution of Superoxide Radical to Cadmium Toxicity in E. coli

Numerous reports suggest the involvement of oxidative stress in cadmium toxicity, but the nature of the reactive species and the mechanism of Cd-induced oxidative damage are not clear. In this study, E. coli mutants were used to investigate mechanisms of Cd toxicity. Effects of Cd on metabolic activity, production of superoxide radical by the respiratory chain, and induction of enzymes controlled by the soxRS regulon were investigated. In E. coli, the soxRS regulon controls defense against O₂·⁻and univalent oxidants. Suppression of metabolic activity, inability of E. coli to adapt to new environment, and slow cell division were among the manifestations of Cd toxicity. Cd increased production of O₂·⁻ by the electron transport chain and prevented the induction of soxRS-controlled protective enzymes, even when the regulon was induced by the redox-cycling agent, paraquat. The effect was not limited to soxRS-dependent proteins and can be attributed to previously reported suppression of protein synthesis by Cd. Increased production of superoxide, combined with inability to express protective enzymes and to replace damaged proteins by de novo protein synthesis, seems to be the main reason for growth stasis and cell death in Cd poisoning.