Overexpression of the robA gene increases organic solvent tolerance and multiple antibiotic and heavy metal ion resistance in Escherichia coli.

Escherichia coli K-12 OST3410 was isolated previously as a stable cyclohexane-tolerant mutant derived from cyclohexane-sensitive strain JA300. A plasmid which provides cyclohexane tolerance to strain JA300 was isolated from the OST3410 genomic library. Subcloning and sequence analysis showed that the plasmid contained the robA gene, whose gene product was reported to bind specifically to the right border of oriC. We observed that the robA gene on the multicopy plasmid generally increased the organic solvent tolerance of several E. coli strains. We also observed an increase in the organic solvent tolerance of JA300 carrying the lac-robA fusion gene on a low-copy plasmid by isopropyl-beta-D-thiogalactopyranoside induction. Strain JA300 carrying the multicopy robA plasmid also showed an increase in resistance to a number of unrelated antibiotics and heavy metal ions, and the spectrum of resistance was significantly similar to that of the soxS-overexpressing strain.     

Organic solvent tolerance and antibiotic resistance increased by overexpression of marA in Escherichia coli.

We previously reported that overexpression of the soxS or robA gene causes in several Escherichia coli strains the acquisition of higher organic solvent tolerance and also increased resistance to a number of antibiotics (H. Nakajima, K. Kobayashi, M. Kobayashi, H. Asako, and R. Aono, Appl. Environ. Microbiol. 61:2302-2307, 1995). Most E. coli strains cannot grow in the presence of cyclohexane. We isolated the marRAB genes from a Kohara lambda phage clone and cyclohexane-tolerant mutant strain OST3408. We found a substitution of serine for arginine at position 73 in the coding region of marR of OST3408 and designated the gene marR08. Our genetic analysis revealed that marR08 is responsible for the cyclohexane-tolerant phenotype. We observed that the marA gene on high-copy-number plasmids increased the organic solvent tolerance of E. coli strains. Furthermore, exposure of E. coli cells to salicylate, which activates the mar regulon genes, also raised organic solvent tolerance. Overexpression of the marA, soxS, or robA gene increased resistance to numerous antibiotics but not to hydrophilic aminoglycosides.     

Indigo production by <Emphasis Type="Italic">Escherichia coli</Emphasis> carrying the phenol hydroxylase gene from <Emphasis Type="Italic">Acinetobacter</Emphasis> sp. strain ST-550 in a water–organic solvent two-phase system

Acinetobacter sp. strain ST-550 produces indigo from indole in the presence of a large volume of diphenylmethane and a high level of indole. Particular proteins increased remarkably in strain ST-550 grown in the two-phase culture system for indigo production. One of the proteins showed a N-terminal amino acid sequence that was identical to that of the largest subunit of phenol hydroxylase (MopN) from A. calcoaceticus NCBI8250. The indigo-producing activity was strongly induced when ST-550 was grown with phenol as a sole carbon source. Genes coding for the multicomponent phenol hydroxylase were cloned, based on the homology with mopKLMOP from A. calcoaceticus NCBI8250. Escherichia coli carrying the genes produced indigo from indole. E. coli JA300 and its cyclohexane-resistant mutant, OST3410, carrying the hydroxylase genes and the NADH regeneration system were grown in the two-phase culture system for indigo production. The OST3410 recombinant produced 52 microg indigo ml(-1) of medium in the presence of diphenylmethane. This productivity was 4.3-fold higher than that of the JA300 recombinant.     

Identification and characterization of an organic solvent tolerance gene in Helicobacter pylori

BACKGROUND: Pre-cleaning and soaking in glutaraldehyde is the necessary procedure to disinfect endoscopes. However, some chemical-solvent-tolerant bacteria may survive after incomplete endoscopic disinfection. The goal of this study was to identify glutaraldehyde resistance-related genes in Helicobacter pylori. MATERIALS AND METHODS: Lambda-Zap phagemid expression library of H. pylori strain NTUH-C1 was selected with 0.1% glutaraldehyde. The minimal inhibitory concentration (MIC) of glutaraldehyde-resistant DNA fragments of H. pylori NTUH-C1 strain were determined. Imp/OstA recombinant protein was expressed, purified, and used to generate anti-Imp/OstA polyclonal antibody. Imp/ostA knockout, deletion, and complementation strains were constructed. The function of Imp/OstA was monitored by organic solvent tolerance assay, antibiotics susceptibility test, and N-phenylnapthylamine assay. RESULTS: Using Imp/ostA polyclonal antibody against cell lysate of wild-type and imp/ostA mutant showed that it is not essential in H. pylori. Organic solvent tolerance assay demonstrated the role of Imp/ostA in n-hexane tolerance. MIC test showed that the mutation of imp/ostA was susceptible to hydrophobic and beta-lactam antibiotics. NPN assay demonstrated that the level of outer membrane permeability was increased by 50% in mutant strain comparing to wild-type strain (p < .001). CONCLUSIONS: We have identified an Imp/OstA protein that was associated with glutaraldehyde resistance in our clinical strain H. pylori NTUH-C1 by screening of lambda-Zap expression library. Disruption of this protein results in altering membrane permeability, sensitivity to organic solvent, and susceptibility to antibiotics.     

arcA基因提高大肠杆菌对有机溶剂的耐受性

【目的】将来源于恶臭假单胞菌(Pseudomonas putida JUCT1)的基因arc A(编码精氨酸脱亚胺酶)整合到Escherichia coli JM109(DE3)基因组中,以提高该菌对有机溶剂的耐受性。【方法】以P.putida JUCT1的基因组为模板扩增基因arc A,并与p ET-20b(+)连接后导入E.coli JM109(DE3)中,验证该基因提高E.coli JM109(DE3)对有机溶剂的耐受性。利用Red同源重组的方法将arc A整合到E.coli JM109(DE3)基因组中。【结果】E.coli JM109(DE3)/p ET-20b(+)-arc A在添加了2.0%(体积比)环己烷、0.1%(体积比)甲苯、4.0%(体积比)萘烷和0.1%(体积比)丁醇的培养基中培养8 h后,其OD660由初始的0.2分别上升到0.8、0.9、1.8和1.3。将arc A成功整合到E.coli JM109(DE3)基因组中,获得了具有较好遗传稳定性的溶剂耐受E.coli JM109(DE3)宿主菌株。【结论】外源基因arc A能提高大肠杆菌菌株的有机溶剂耐受性,为工业化应用中耐溶剂微生物菌株的构建提供了实验依据和理论基础。     

Discovery of glpC, an organic solvent tolerance-related gene in Escherichia coli, using gene expression profiles from DNA microarrays

Gene expression profiles were collected from Escherichia coli strains (OST3410, TK33, and TK31) before and after exposure to organic solvents, and the six genes that showed higher gene expression were selected. Among these genes, glpC encoding the anaerobic glycerol-3-phosphate dehydrogenase subunit C remarkably increased the organic solvent tolerance.     

Discovery of glpC, an Organic Solvent Tolerance-Related Gene in Escherichia coli, Using Gene Expression Profiles from DNA Microarrays

Gene expression profiles were collected from Escherichia coli strains (OST3410, TK33, and TK31) before and after exposure to organic solvents, and the six genes that showed higher gene expression were selected. Among these genes, glpC encoding the anaerobic glycerol-3-phosphate dehydrogenase subunit C remarkably increased the organic solvent tolerance.     

Improvement in organic solvent tolerance by double disruptions of proV and marR genes in Escherichia coli

Aims: To investigate the involvement of osmoprotectant transporters in organic solvent tolerance in Escherichia coli and to construct an E. coli strain with high organic solvent tolerance. Methods and Results: The organic solvent tolerance of ΔbetT, ΔproV, ΔproP or ΔputP single‐gene knockout mutants of E. coli K‐12 strain was examined. Among these mutants, the organic solvent tolerance of the ΔproV mutant remarkably increased compared with that of the parent strain. It has been known that a marR mutation confers tolerance on E. coli to organic solvents. A ΔproV and ΔmarR double‐gene mutant was more tolerant to organic solvents than the ΔproV or ΔmarR single‐gene mutant. The n‐hexane amount accumulated in E. coli cells was examined after incubation in an n‐hexane‐aqueous medium two‐phase system. The intracellular n‐hexane level in the ΔproV and ΔmarR double‐gene mutant was kept lower than those of the parent strain, ΔproV mutant and ΔmarR mutant. Conclusions: The organic solvent tolerance level in E. coli highly increased by dual disruption of proV and marR. Significance and Impact of the Study: This study suggests a new strategy for increasing the organic solvent tolerance level in E. coli to improve the usability of the whole‐cell biocatalysts in two‐phase systems employing organic solvents.     

有机溶剂胁迫处理对菌株Bacillus subtilis OST23a产胞外多糖的影响

【目的】研究有机溶剂胁迫处理对菌株分泌胞外多糖的影响并确定最佳条件。【方法】利用分泌抗氧化活性胞外多糖海洋细菌Bacillus subtilis OST23a及其突变菌株UD292为出发菌株,在考察菌株有机溶剂耐受性的基础上,测定不同浓度正己烷胁迫处理不同时间后该菌株抗氧化胞外多糖产量。【结果】结果表明最佳胁迫处理浓度和时间分别为3%和6 h,此时Bacillus subtilis OST23a和菌株UD292胞外多糖分泌量分别从9.02 mg/L和43.92 mg/L显著提高到52.97 mg/L和201.81 mg/L,且胞外多糖的抗氧化性能无显著变化。Bacillus subtilis OST23a和菌株UD292连续传代试验结果表明菌株遗传性状较稳定。【结论】有机溶剂胁迫可以提高细菌分泌胞外多糖的能力,在微生物育种方面有潜在的应用。     

基于蛋白质组学的假单胞菌耐溶剂机制研究

【目的】有机溶剂对微生物有强烈的毒害作用致使绝大多数微生物不能在较高的有机溶剂浓度下生长。为了探究微生物的耐溶剂性机制,由野生型假单胞菌Pseudomonas putida JUCS驯化获得一株能够在60%(V/V)的环己烷中生长的菌株P.putidaJUCT1。【方法】采用蛋白质二维电泳对P.putida JUCT1在不同溶剂条件下的蛋白组分表达量的差异进行分析比对。【结果】从总共22个表达量差异均超过50%的蛋白质中,选取了3个高丰度蛋白质,通过MALDI-TOF/TOF鉴定为:3-羟基异丁酸水解酶、蛋白质延伸因子EF-Ts、异分支酸水解酶超家族(编码基因分别为mmsB、tsf、PSEEN0851)。将这3个基因在大肠杆菌中重组表达,3个蛋白均能不同程度地提高E.coli JM109的耐溶剂性,其中3-羟基异丁酸脱氢酶(编码基因mmsB)对菌株的溶剂耐受性影响最为显著。【结论】证明了运用蛋白组学的方法研究微生物的耐溶剂性的可行性,并为构建适用于工业化应用的溶剂耐受性整体细胞生物催化剂提供理论依据。     

YLR099C (ICT1) encodes a soluble Acyl-CoA-dependent lysophosphatidic acid acyltransferase responsible for enhanced phospholipid synthesis on organic solvent stress in Saccharomyces cerevisiae

One of the major determinants of organic solvent tolerance is the increase in membrane phospholipids. Here we report for the first time that an increase in the synthesis of phosphatidic acid is responsible for enhanced phospholipid synthesis that confers tolerance to the organic solvent in Saccharomyces cerevisiae. This increase in phosphatidic acid formation is because of the induction of Ict1p, a soluble oleoyl-CoA:lysophosphatidic acid acyltransferase. YLR099C (ICT1) was reported to be maximally expressed during solvent tolerance (Miura, S., Zou, W., Ueda, M., and Tanaka, A. (2000) Appl. Environ. Microbiol. 66, 4883-4889); however, its physiological significance was not understood. In silico analysis revealed the absence of any transmembrane domain in Ict1p. Domain analysis showed that it has a hydrolase/acyltransferase domain with a distinct lipid-binding motif and a lysophospholipase domain. Analysis of ict1Delta strain showed a drastic reduction in phosphatidic acid suggesting the role of Ict1p in phosphatidic acid biosynthesis. Overexpression of Ict1p in S. cerevisiae showed an increase in phosphatidic acid and other phospholipids on organic solvent exposure. To understand the biochemical function of Ict1p, the gene was cloned and expressed in Escherichia coli. The purified recombinant enzyme was found to specifically acylate lysophosphatidic acid. Specific activity of Ict1p was found to be higher for oleoyl-CoA as compared with palmitoyl- and stearoyl-CoAs. This study provides a mechanism for organic solvent tolerance from the point of membrane dynamics in S. cerevisiae.     

Cloning and DNA sequence of a mycoplasmal recA gene.

Mycoplasmas are wall-less prokaryotes phylogenetically related to gram-positive bacteria. In order to investigate DNA recombination in these organisms, we have cloned the recA gene from the mycoplasma Acholeplasma laidlawii. DNA sequence data indicate extensive homology between the A. laidlawii recA gene and recA genes from other bacteria, particularly Bacillus subtilis. The recA sequences from three A. laidlawii strains (strains JA1, K2, and 8195) were compared, and surprisingly, the gene from A. laidlawii 8195 was found to contain a nonsense mutation that results in truncation of 36 amino acids from the carboxyl terminus of the RecA protein. By using sensitivity to UV irradiation as a measure of DNA repair, strain 8195 had an apparent RecA- phenotype. When carried on a multicopy plasmid, the wild-type A. laidlawii recA gene was detrimental to growth of Escherichia coli, perhaps because of improper regulation of the RecA protein.     

Organization of Ureaplasma urealyticum urease gene cluster and expression in a suppressor strain of Escherichia coli.

Ureaplasma urealyticum is a pathogenic ureolytic mollicute which colonizes the urogenital tracts of humans. A genetic polymorphism between the two biotypes of U. urealyticum at the level of the urease genes was found. The urease gene cluster from a biotype 1 representative of U. urealyticum (serotype I) was cloned and sequenced. Seven genes were found, with ureA, ureB, and ureC encoding the structural subunits and ureE, ureF, ureG, and a truncated ureI) gene encoding accessory proteins. Urease expression was not obtained when the plasmid containing these genes was incorporated into an opal suppressor strain of Escherichia coli, although this enzymatic activity was found in the same E. coli strain transformed with pC6b, a plasmid with previously cloned urease genes from the U. urealyticum T960 strain of biotype 2 (serotype 8). Although there are 12 TGA triplets encoding tryptophan within urease genes, the level of expression obtained was comparable to the levels reported for other bacterial genes expressed in E. coli. Nested deletion experiments allowed us to demonstrate that ureD is necessary for urease activity whereas another open reading frame located downstream is not. The promoter for ureA and possibly other urease genes was identified for both serotypes.     

Screening of genes involved in isooctane tolerance in Saccharomyces cerevisiae by using mRNA differential display

A Saccharomyces cerevisiae strain, KK-211, isolated by the long-term bioprocess of stereoselective reduction in isooctane, showed extremely high tolerance to the solvent, which is toxic to yeast cells, but, in comparison with its wild-type parent, DY-1, showed low tolerance to hydrophilic organic solvents, such as dimethyl sulfoxide and ethanol. In order to detect the isooctane tolerance-associated genes, mRNA differential display (DD) was employed using mRNAs isolated from strains DY-1 and KK-211 cultivated without isooctane, and from strain KK-211 cultivated with isooctane. Thirty genes were identified as being differentially expressed in these three types of cells and were classified into three groups according to their expression patterns. These patterns were further confirmed and quantified by Northern blot analysis. On the DD fingerprints, the expression of 14 genes, including MUQ1, PRY2, HAC1, AGT1, GAC1, and ICT1 (YLR099c) was induced, while the expression of the remaining 16 genes, including JEN1, PRY1, PRY3, and KRE1, was decreased, in strain KK-211 cultivated with isooctane. The genes represented by HAC1, PRY1, and ICT1 have been reported to be associated with cell stress, and AGT1 and GAC1 have been reported to be involved in the uptake of trehalose and the production of glycogen, respectively. MUQ1 and KRE1, encoding proteins associated with cell surface maintenance, were also detected. Based on these results, we concluded that alteration of expression levels of multiple genes, not of a single gene, might be the critical determinant for isooctane tolerance in strain KK-211.     

Molecular cloning with a pMEA300-derived shuttle vector and characterization of the Amycolatopsis methanolica prephenate dehydratase gene.

An efficient restriction barrier for methylated DNA in the actinomycete Amycolatopsis methanolica could be avoided by using a nonmethylating Escherichia coli strain for DNA isolations. The A. methanolica prephenate dehydratase gene was cloned from a gene bank in a pMEA300-derived shuttle vector in E. coli and characterized.