Phenol degradation by halophilic bacteria isolated from hypersaline environments

Phenol is a toxic aromatic compound used or produced in many industries and as a result a common component of industrial wastewaters. Phenol containing waste streams are frequently hypersaline and therefore require halophilic microorganisms for efficient biotreatment without dilution. In this study three halophilic bacteria isolated from different saline environments and identified as Halomonas organivorans, Arhodomonas aquaeolei and Modicisalibacter tunisiensis were shown to be able to grow on phenol in hypersaline media containing 100 g/L of total salts at a concentration of 3 mM (280 mg/L), well above the concentration found in most waste streams. Genes encoding the aromatic dioxygenase enzymes catechol 1,2 dioxygenase and protocatechuate 3,4-dioxygenase were present in all strains as determined by PCR amplification using primers specific for highly conserved regions of the genes. The gene for protocatechuate 3,4-dioxygenase was cloned from the isolated H. organivorans and the translated protein was evaluated by comparative protein sequence analysis with protocatechuate 3,4-dioxygenase proteins from other microorganisms. Although the analysis revealed a wide range of sequence divergence among the protocatechuate 3,4-dioxygenase family, all of the conserved domain amino acid structures identified for this enzyme family are identical or conservatively substituted in the H. organivorans enzyme.     

Growth and nitrogen removal characteristics of Halomonas sp. B01 under high salinity

At present, the nitrogen (N) removal efficiency of the microbial treatment in the high-salinity nitrogenous wastewaters is relatively low. Study on the N removal behavior and properties of moderately halophilic bacteria Halomonas under high salinity is of great significance for the microbial treatment of high-salinity nitrogenous wastewater. The response mechanism of Halomonas sp. B01 to high osmotic pressure stress was investigated by measuring the compatible solute ectoine concentration and superoxide dismutase (SOD) activity. The salt tolerance during growth and N removal of the strain was evaluated by measuring the activities of growth-related and N removal–related enzymes and the mRNA expression abundance of ammonia monooxygenase-encoding gene (amoA). The process of simultaneous heterotrophic nitrification and aerobic denitrification (SND) under high salinity was described by measuring the concentration of inorganic N. Halomonas sp. B01 synthesized ectoine under NaCl stress, and the intracellular ectoine concentration increased with increased NaCl concentration in the growth medium. When the NaCl concentration of the medium reached 120 g L−1, the malondialdehyde concentration and SOD activity were significantly increased to 576.1 μg mg−1 and 1.7 U mg−1, respectively. The growth-related and N removal–related enzymes of the strain were active or most active in medium with 30–60 g L−1 NaCl. The amoA of the strain cultured in medium with 60 g L−1 NaCl had the highest mRNA expression abundance. In the N removal medium containing 60 g L−1 NaCl and 2121 mg L−1 NH4+-N, SND by Halomonas sp. B01 was performed over 96 h and the N removal rate reached 98.8%. In addition to the protective mechanism of synthetic compatible solutes, Halomonas sp. B01 had the repair mechanism of SOD for lipid peroxidation. The growth-related and N removal–related enzymes of the strain were most active at a certain salt concentration; amoA also had the highest mRNA expression abundance under high salinity. Halomonas sp. B01 could efficiently perform N removal by SND under high salinity.     

Synchronous effects of temperature, hydrostatic pressure, and salinity on growth, phospholipid profiles, and protein patterns of four Halomonas species isolated from deep-sea hydrothermal-vent and sea surface environments

Four strains of euryhaline bacteria belonging to the genus Halomonas were tested for their response to a range of temperatures (2, 13, and 30 degrees C), hydrostatic pressures (0.1, 7.5, 15, 25, 35, 45, and 55 MPa), and salinities (4, 11, and 17% total salts). The isolates were psychrotolerant, halophilic to moderately halophilic, and piezotolerant, growing fastest at 30 degrees C, 0.1 MPa, and 4% total salts. Little or no growth occurred at the highest hydrostatic pressures tested, an effect that was more pronounced with decreasing temperatures. Growth curves suggested that the Halomonas strains tested would grow well in cool to warm hydrothermal-vent and associated subseafloor habitats, but poorly or not at all under cold deep-sea conditions. The intermediate salinity tested enhanced growth under certain high-hydrostatic-pressure and low-temperature conditions, highlighting a synergistic effect on growth for these combined stresses. Phospholipid profiles obtained at 30 degrees C indicated that hydrostatic pressure exerted the dominant control on the degree of lipid saturation, although elevated salinity slightly mitigated the increased degree of lipid unsaturation caused by increased hydrostatic pressure. Profiles of cytosolic and membrane proteins of Halomonas axialensis and H. hydrothermalis performed at 30 degrees C under various salinities and hydrostatic pressure conditions indicated several hydrostatic pressure and salinity effects, including proteins whose expression was induced by either an elevated salinity or hydrostatic pressure, but not by a combination of the two. The interplay between salinity and hydrostatic pressure on microbial growth and physiology suggests that adaptations to hydrostatic pressure and possibly other stresses may partially explain the euryhaline phenotype of members of the genus Halomonas living in deep-sea environments.     

高盐盐湖可分离嗜盐耐盐菌的种群多样性及四氢嘧啶产量评价

为了解柴达木盆地茶卡盐湖、柯柯盐湖和小柴旦盐湖等三大硫酸镁亚型高盐盐湖可分离嗜盐耐盐菌的种群多样性,采用RM中、高盐培养基筛选分离可培养的嗜盐菌和耐盐菌,扩增16S rRNA基因序列进行种属鉴定和环境因子典范对应分析(CCA),选取优势菌属构建系统发育树,并采用高效液相色谱法(HPLC)检测次级代谢产物四氢嘧啶(Ect...     

Biodegradation of organic pollutants by halophilic bacteria and archaea

Hypersaline environments are important for both surface extension and ecological significance. As all other ecosystems, they are impacted by pollution. However, little information is available on the biodegradation of organic pollutants by halophilic microorganisms in such environments. In addition, it is estimated that 5% of industrial effluents are saline and hypersaline. Conventional nonextremophilic microorganisms are unable to efficiently perform the removal of organic pollutants at high salt concentrations. Halophilic microorganisms are metabolically different and are adapted to extreme salinity; these microorganisms are good candidates for the bioremediation of hypersaline environments and treatment of saline effluents. This literature survey indicates that both the moderately halophilic bacteria and the extremely halophilic archaea have a broader catabolic versatility and capability than previously thought. A diversity of contaminating compounds is susceptible to be degraded by halotolerant and halophile bacteria. Nevertheless, significant research efforts are still necessary in order to estimate the true potential of these microorganisms to be applied in environmental processes and in the remediation of contaminated hypersaline ecosystems. This effort should be also focused on basic research to understand the overall degradation mechanism, to identify the enzymes involved in the degradation process and the metabolism regulation.     

Characterization of beta-ketoadipate pathway from multi-drug resistance bacterium, Acinetobacter baumannii DU202 by proteomic approach

In this study, the biodegradative activities of monocyclic aromatic compounds were determined from the multi-drug resistant (MDR) Acinetobacter baumannii, which were studied in the form of clinical isolates from a hospital in Korea. These bacteria were capable of biodegrading monocyclic aromatic compounds, such as benzoate and p-hydroxybenzoate. In order to determine which pathways are available for biodegradation in these stains, we conducted proteome analyses of benzoate and p-hydroxybenzoate-cultured A. baumannii DU202, using 2-DE/MS analysis. As genome DB of A. baumannii was not yet available, MS/MS analysis or de novo sequencing methods were employed in the identification of induced proteins. Benzoate branch enzymes [catechol 1,2-dioxygenase (CatA) and benzoate dioxygenase alpha subunit (BenA)] of the beta-ketoadipate pathway were identified under benzoate culture condition and p-hydroxybenzoate branch enzymes [protocatechuate 3,4-dioxygenase alpha subunit (PcaG) and 3-carboxy-cis,cis-muconate cycloisomerase (PcaB)] of the beta-ketoadipate pathway were identified under p-hydroxybenzoate culture condition, respectively, thereby suggesting that strain DU202 utilized the beta-ketoadipate pathway for the biodegradation of monocyclic aromatic compounds. The sequence analysis of two purified dioxygenases (CatA and PcaGH) indicated that CatA is closely associated with the CatA of Acinetobacter radiresistance, but PcaGH is only moderately associated with the PcaGH of Acinetobacter sp. ADP1. Interestingly, the fused form of PcaD and PcaC, carboxymuconolactone decarboxylase (PcaCD), was detected on benzoate-cultured A. baumannii DU202. These results indicate that A. baumannii DU202 exploits a different beta-ketoadipate pathway from other Acinetobacter species.     

Diversity of moderately halophilic bacteria producing extracellular hydrolytic enzymes

AIMS: The aim of this study was to determine the diversity of moderately halophilic bacteria with hydrolase activities. METHODS AND RESULTS: Screening bacteria from different hypersaline environments in South Spain led to the isolation of a total of 122 moderately halophilic bacteria able to produce different hydrolases (amylases, DNases, lipases, proteases and pullulanases). These bacteria are able to grow optimally in media with 5-15% salts and in most cases up to 20-25% salts. In contrast to strains belonging to previously described species, that showed very little hydrolase activities, environmental isolates produced a great variety of hydrolases. These strains were identified as members of the genera: Salinivibrio (55 strains), Halomonas (25 strains), Chromohalobacter (two strains), Bacillus-Salibacillus (29 strains), Salinicoccus (two strains) and Marinococcus (one strain), as well as eight non-identified isolates. CONCLUSIONS: Moderately halophilic bacteria are a source of hydrolytic enzymes such as amylases, DNases, lipases, proteases and pullulanases. SIGNIFICANCE AND IMPACT OF THE STUDY: Although most culture collection strains are not able to produce hydrolases, it has been shown that environmental isolates can produce these potentially biotechnological important enzymes.     

Isolation and characterization of Halomonas sp. strain IMPC, a p-coumaric acid-metabolizing bacterium that decarboxylates other cinnamic acids under hypersaline conditions

A moderately halophilic, mesophilic, Gram-negative, motile, nonsporulating bacterium, designated strain IMPC, was isolated from a table-olive fermentation rich in aromatic compounds, after enrichment on p-coumaric acid under halophilic conditions. Strain IMPC was able to degrade p-coumaric acid. p-hydroxybenzaldehyde and p-hydroxybenzoic acid were detected as breakdown products from p-coumaric acid. Protocatechuic acid was identified as the final aromatic product of p-coumaric acid catabolism before ring fission. Strain IMPC transformed various cinnamic acids with substituent H, OH, CH(3) or OCH(3) in the para- and/or meta-position of the aromatic ring to the corresponding benzoic acids, indicating a specific selection. A beta-oxidation pathway was proposed for these transformations. Phylogenetic analysis of the 16S rRNA gene revealed that this isolate was a member of the genus Halomonas. Strain IMPC was closely related to Halomonas elongata ATCC 33173(T)and Halomonas eurihalina ATCC 49336(T).     

Halomonas and Marinobacter ecotypes from hydrothermal vent, subseafloor and deep-sea environments

Moderately halophilic and euryhaline bacteria are routinely found in cool to warm hydrothermal vent and nearby cold, deep-sea environments. To elucidate the diversity of these microorganisms - with the goal of determining which among them constitute ecotypes specifically associated with hydrothermal vent and subseafloor habitats - PCR primers were designed to detect natural populations of euryhaline Gammaproteobacteria belonging to the cosmopolitan genera Halomonas and Marinobacter. The distribution patterns of 16S rRNA gene sequence data revealed that Halomonas group 2A comprised a subseafloor population at Axial Seamount on the Juan de Fuca Ridge. Complementary biogeographic and physiological data suggested that other Halomonas clades include members that are cold adapted (Halomonas group 2B) or associated with massive sulfide deposits (Halomonas group 2C). Similarly, a monophyletic Marinobacter clade may represent Fe(2+) -oxidizing facultative chemoautotrophs based on the phylogenetic data presented here and previously reported phenotypic characterizations. The biogeographic distributions of Halomonas and Marinobacter isolates and clones reveal that these are cosmopolitan genera, commonly found in the deep sea and in hydrothermal vent settings. As such, they are good candidates for further laboratory investigations into the biogeochemical processes in these environments.     

Proteome analysis of Pseudomonas sp. K82 biodegradation pathways

Pseudomonas sp. K82 is a soil bacterium that can degrade and use monocyclic aromatic compounds including aniline, 3-methylaniline, 4-methylaniline, benzoate and p-hydroxybenzoate as its sole carbon and energy sources. In order to understand the impact of these aromatic compounds on metabolic pathways in Pseudomonas sp. K82, proteomes obtained from cultures exposed to different substrates were displayed by two-dimensional gel electrophoresis and were compared to search for differentially induced metabolic enzymes. Column separations of active fractions were performed to identify major biodegradation enzymes. More than thirty proteins involved in biodegradation and other types of metabolism were identified by electrospray ionization-quadrupole time of flight mass spectrometry. The proteome analysis suggested that Pseudomonas sp. K82 has three main metabolic pathways to degrade these aromatic compounds and induces specific metabolic pathways for each compound. The catechol 2,3-dioxygenase (CD2,3) pathway was the major pathway and the catechol 1,2-dioxygenase (beta-ketoadipate) pathway was the secondary pathway induced by aniline (aniline analogues) exposure. On the other hand, the catechol 1,2-dioxygenase pathway was the major pathway induced by benzoate exposure. For the degradation of p-hydroxybenzoate, the protocatechuate 4,5-dioxygenase pathway was the major degradation pathway induced. The nuclear magnetic resonance analysis of substrates demonstrated that Pseudomonas sp. K82 metabolizes some aromatic compounds more rapidly than others (benzoate > p-hydroxybenzoate > aniline) and that when combined, p-hydroxybenzoate metabolism is repressed by the presence of benzoate or aniline. These results suggest that proteome analysis can be useful in the high throughput study of bacterial metabolic pathways, including that of biodegradation, and that inter-relationships exist with respect to the metabolic pathways of aromatic compounds in Pseudomonas sp. K82.     

Key enzymes of the protocatechuate branch of the β-ketoadipate pathway for aromatic degradation in Corynebacterium glutamicum

Although the protocatechuate branch of the β-ketoadipate pathway in Gram- bacteria has been well studied, this branch is less understood in Gram⁺ bacteria. In this study, Corynebacterium glutamicum was cultivated with protocatechuate, p-cresol, vanillate and 4-hydroxybenzoate as sole carbon and energy sources for growth. Enzymatic assays indicated that growing cells on these aromatic compounds exhibited protocatechuate 3,4-dioxygenase activities. Data-mining of the genome of this bacterium revealed that the genetic locus ncg12314-ncg12315 encoded a putative protocatechuate 3,4-dioxygenase. The genes, ncg12314 and ncg12315, were amplified by PCR technique and were cloned into plasmid (pET21aP34D). Recombinant Escherichia coli strain harboring this plasmid actively expressed protocatechuate 3,4-dioxygenase activity. Further, when this locus was disrupted in C. glutamicum, the ability to degrade and assimilate protocatechuate, p-cresol, vanillate or 4-hydroxybenzoate was lost and protocatechuate 3,4-dioxygenase activity was disappeared. The ability to grow with these aromatic compounds and protocatechuate 3,4-dioxygenase activity of C. glutamicum mutant could be restored by gene complementation. Thus, it is clear that the key enzyme for ring-cleavage, protocatechuate 3,4-dioxygenase, was encoded by ncg12314 and ncg12315. The additional genes involved in the protocatechuate branch of the β-ketoadipate pathway were identified by mining the genome data publically available in the GenBank. The functional identification of genes and their unique organization in C. glutamicum provided new insight into the genetic diversity of aromatic compound degradation.     

Salinity Influences the Response of Halomonas hydrothermalis to Artificial Fossilization by Evaporative Silicification

Evaporative silicification can drive microbial fossilization within diverse natural habitats. Research into this process is pivotal to understanding the terrestrial fossil record and the preservation of biomarkers within extreme environments. We employed laboratory experiments to silicify the polyextremotolerant bacterium Halomonas hydrothermalis cultured at low, intermediate and high salinities (1, 3.5 or 11.8% w/v of NaCl) under iron-rich or iron-deprived conditions. Silicification was achieved by adding sodium silicate solution (30 or 150 ppm of Si) onto cultures, followed by evaporation. Scanning electron microscopy demonstrated the presence of mineralized bacteria with intact morphology across all culture conditions. However, multivariate analysis of the attenuated total reflectance Fourier-transform infrared (ATR-FT-IR) spectra of silicified cultures showed significant differences between the examined salinities, most notably between cultures silicified after incubation at high salinity and those at lower salinities. Although the spectra of mineralized low- and intermediate-salinity cultures appeared distinct from their nonsilicified counterparts, these differences were less pronounced at high salinity. By showing that differences in salinity can influence microbial responses to mineralization at the molecular level, these data indicate that the potential for evaporative silicification to contribute to microbial fossilization may differ between freshwater and hypersaline environments.     

Investigation of catechol 2,3-dioxygenase and 16S rRNA gene diversity in hypoxic, petroleum hydrocarbon contaminated groundwater

Detection of catechol 2,3-dioxygenase genes in aromatic hydrocarbon contaminated environments gives the opportunity to measure the diversity of bacteria involved in the degradation of the contaminants under aerobic conditions. In this study, we investigated the diversity and distribution of Comamonadaceae family (Betaproteobacteria) related catechol 2,3-dioxygenase genes, which belong to the I.2.C subfamily of extradiol dioxygenase genes. These catabolic genes encode enzymes supposed to function under hypoxic conditions as well, and may play a notable role in BTEX degradation in oxygen limited environments. Therefore, their diversity was analyzed in oxygen limited, petroleum hydrocarbon contaminated groundwater by terminal restriction fragment length polymorphism and cloning. Subfamily I.2.C related catechol 2,3-dioxygenase genes were detected in every investigated groundwater sample and a dynamic change was observed in the case of the structure of C23O gene possessing bacterial communities. To link the metabolic capability to the microbial structure, 16S rRNA gene-based clone libraries were generated and it was concluded that Betaproteobacteria were abundant in the bacterial communities of the contaminated samples. These results support the opinion that Betaproteobacteria may play a significant role in BTEX degradation under hypoxic conditions.     

Development of catechol 2,3-dioxygenase-specific primers for monitoring bioremediation by competitive quantitative PCR

Benzene, toluene, xylenes, phenol, naphthalene, and biphenyl are among a group of compounds that have at least one reported pathway for biodegradation involving catechol 2,3-dioxygenase enzymes. Thus, detection of the corresponding catechol 2,3-dioxygenase genes can serve as a basis for identifying and quantifying bacteria that have these catabolic abilities. Primers that can successfully amplify a 238-bp catechol 2,3-dioxygenase gene fragment from eight different bacteria are described. The identities of the amplicons were confirmed by hybridization with a 238-bp catechol 2,3-dioxygenase probe. The detection limit was 10(2) to 10(3) gene copies, which was lowered to 10(0) to 10(1) gene copies by hybridization. Using the dioxygenase-specific primers, an increase in catechol 2, 3-dioxygenase genes was detected in petroleum-amended soils. The dioxygenase genes were enumerated by competitive quantitative PCR with a 163-bp competitor that was amplified using the same primers. Target and competitor sequences had identical amplification kinetics. Potential PCR inhibitors that could coextract with DNA, nonamplifying DNA, soil factors (humics), and soil pollutants (toluene) did not impact enumeration. Therefore, this technique can be used to accurately and reproducibly quantify catechol 2, 3-dioxygenase genes in complex environments such as petroleum-contaminated soil. Direct, non-cultivation-based molecular techniques for detecting and enumerating microbial pollutant-biodegrading genes in environmental samples are powerful tools for monitoring bioremediation and developing field evidence in support of natural attenuation.     

Characteristics of bacteria showing high denitrification activity in saline wastewater

AIMS: Denitrification efficiency at 10% salinity was compared with that at 2% salinity. The characteristics of bacterial strains isolated from the denitrification system, where an improvement of denitrification efficiency was observed at a high salinity were investigated. METHODS AND RESULTS: Two continuous feeding denitrification systems for saline solutions of 2% and 10% salinity, were operated. Denitrification efficiency at 10% salinity was higher than that at 2% salinity. The bacterial strains were isolated using the trypticase soy agar (TSA) medium at 30 degrees C. The phylogenetic analysis of 16S rRNA gene sequences of isolates indicated that halophilic species were predominant at 10% salinity. CONCLUSIONS: The improvement of denitrification efficiency at a high salinity was demonstrated. The strains isolated from the denitrifying system with 10% salinity were halophilic bacteria, Halomonas sp. and Marinobacter sp., suggesting that these bacteria show a high denitrifying activity at 10% salinity. SIGNIFICANCE AND IMPACT OF THE STUDY: The long-term acclimated sludge used in this study resulted in high denitrification performance at a high salinity, indicating that the design of a high-performance denitrification system for saline wastewater will be possible.     

Chloridazon-catechol dioxygenases, a distinct group of meta-cleaving enzymes

We previously described a new meta-cleaving enzyme, termed chloridazon-catechol dioxygenase. The present paper describes the comparison of this enzyme with the meta-cleaving enzymes of eighteen strains of soil bacteria isolated with various aromatic compounds. Four of these strains were isolated with the herbicide chloridazon, six with the analgeticum aminopyrine and one with the analgeticum antipyrine as sole carbon source. These strains all belonged to a new type of bacteria, called Phenylobacteria. The seven other strains were isolated with aromatic compounds such as toluene, 3-phenylpropionate, benzoate, papaverine and 4-chlorobenzoate, and belonged to various species including Pseudomonas, Acinetobacter and Nocardia. In double diffusion experiments with antibodies, prepared against chloridazon-catechol dioxygenase, extracts from the eleven strains of Phenylobacteria gave a cross reaction, whereas the extracts of the seven other strains showed no reaction. The enzymes of the eleven positive strains showed the same characteristic kinetic behaviour as the previously described enzyme. In contrast to catechol 2, 3-dioxygenase they needed the addition of exogenous Fe2+ ions for activity. On ion-exchange chromatography they emerged at the same buffer concentration as chloridazon-catechol dioxygenase. In polyacrylamide electrophoresis they migrated identically. The linkage map derived from the activities of the various enzymes with 10 different substrates revealed an identity of more than 80% for these eleven enzymes. So the meta-cleaving enzymes of the Phenylobacteria seem to form a distinct group among the non-heme iron-containing dioxygenases.     

Degradation of naphthalene by thermophilic bacteria <Emphasis Type="Italic">via</Emphasis> a pathway,through protocatechuic acid

A number of thermophilic bacteria capable of utilizing naphthalene as a sole source of carbon were isolated from a high-temperature oilfield in Lithuania. These isolates were able to utilize several other aromatic compounds, such as anthracene, benzene, phenol, benzene-1, 3-diol, protocatechuic acid as well. Thermophilic isolate G27 ascribed to Geobacillus genus was found to have a high aromatic compound degrading capacity. Spectrophotometric determination of enzyme activities in cell-free extracts revealed that the last aromatic ring fission enzyme in naphthalene biotransformation by Geobacillus sp. G27 was inducible via protocatechuate 3, 4-dioxygenase; no protocatechuate 4, 5-dioxygenase, protocatechuate 2, 3-dioxygenase activities were detected. Intermediates such as o-phthalic and protocatechuic acids detected in culture supernatant confirmed that the metabolism of naphthalene by Geobacillus sp. G27 can proceed through protocatechuic acid via ortho-cleavage pathway and thus differs from the pathways known for mesophilic bacteria.     

Development of Catechol 2,3-Dioxygenase-Specific Primers for Monitoring Bioremediation by Competitive Quantitative PCR

Benzene, toluene, xylenes, phenol, naphthalene, and biphenyl are among a group of compounds that have at least one reported pathway for biodegradation involving catechol 2,3-dioxygenase enzymes. Thus, detection of the corresponding catechol 2,3-dioxygenase genes can serve as a basis for identifying and quantifying bacteria that have these catabolic abilities. Primers that can successfully amplify a 238-bp catechol 2,3-dioxygenase gene fragment from eight different bacteria are described. The identities of the amplicons were confirmed by hybridization with a 238-bp catechol 2,3-dioxygenase probe. The detection limit was 10² to 10³ gene copies, which was lowered to 10⁰ to 10¹ gene copies by hybridization. Using the dioxygenase-specific primers, an increase in catechol 2,3-dioxygenase genes was detected in petroleum-amended soils. The dioxygenase genes were enumerated by competitive quantitative PCR with a 163-bp competitor that was amplified using the same primers. Target and competitor sequences had identical amplification kinetics. Potential PCR inhibitors that could coextract with DNA, nonamplifying DNA, soil factors (humics), and soil pollutants (toluene) did not impact enumeration. Therefore, this technique can be used to accurately and reproducibly quantify catechol 2,3-dioxygenase genes in complex environments such as petroleum-contaminated soil. Direct, non-cultivation-based molecular techniques for detecting and enumerating microbial pollutant-biodegrading genes in environmental samples are powerful tools for monitoring bioremediation and developing field evidence in support of natural attenuation.     

盐单胞菌属BYS1四氢嘧啶合成基因ectABC克隆及其盐激表达

利用SEFA-PCR技术从中度嗜盐菌Halomonassp.BYS-1总DNA中克隆了四氢嘧啶合成基因ectABC及其上游序列(GenBank accession number DQ017757);OMIGA软件分析结果显示ectA、ectB、ectC位于同一个操纵子上,大小分别为573bp1、251bp和387bp,预测编码的DAT(L-二氨基丁酸转氨酶)、DAA(L-二氨基丁酸乙酰转移酶)和ES(四氢嘧啶合酶)大小分别为21.1kDa(191 amino acid)、45.7kDa(417 amino acid)和14.5kDa(129 amino acid);将包含ectABC基因及其上游1000bp序列的片段克隆到pUC19中并转化E.coliDH5α,转化子E.coli(pUC19ECT)能够在盐激条件下合成四氢嘧啶,但其耐盐能力没有得到显著改善。