Compatible solutes in new moderately halophilic isolates

Abstract Using high performance liquid chromatography and nuclear magnetic resonance techniques, the compatible solutes of some moderately halophilic bacteria were studied. The following accepted species of moderately halophilic bacteria were included: Volcaniella eurihalina and Deleya salina among Gram-negative rods, and Salinicoccus roseus and Salinicoccus hispanicus among Gram-positive cocci. Besides these strains we have also screened other new isolates, including Marinomonas species and Gram-positive cocci and rods. The tetrahydropyrimidine carboxylic acid 'ectoine' was found to be the main compatible solute in the Gram-negative strains tested when these were grown in glucose-mineral medium. In addition, betaine was accumulated from complex media containing yeast extract. Among the Gram-positive strains investigated, the solutes proline (bacillus 30, Salinicoccus ) and hydroxyectoine (coccus 28) also played an important role, while alanine, glucose, glutamate, glutamine and trehalose occurred as minor components. We also detected two recently described compatible solutes: Nδ -acetylornithine and a homologous compound, Nε -acetyllysine. Representatives of distinct phenotypic groups of Gram-positive cocci and rods were clearly distinguished by their solute pattern.     

以四氢嘧啶为主要相容性溶质的中度嗜盐菌I15的分离和特性研究

从草地土壤中分离到一株中度嗜盐菌I15,经过16S rDNA(GenBank登录号为DQ010162)序列分析、形态学和生理生化特征分析,该菌株初步鉴定为Virgibacillus marismortui。I15能在0%～25%NaCl的培养基中生长,最适生长NaCl浓度为10%,最适生长温度为30℃,最适pH为7.5～8.0。在高盐条件下,I15细胞内主要的相容性溶质为四氢嘧啶,在15%NaCl培养基中其含量达到1.608mmol/(g\5cdw),占到相容性溶质总摩尔含量的89.6%。渗透冲击试验表明I15细胞内四氢嘧啶在低渗冲击时能够快速分泌到细胞外,在高渗冲击冲剂时能够较快地重新合成。     

Biodegradation of N-Methylpyrrolidone by Paracoccus sp. NMD-4 and its degradation pathway

N-Methylpyrrolidone (NMP), a kind of nitrogen-containing heterocyclic pollutant, is widely used in chemical industry. Microbial degradation is an important environmental fate process in soil and water, however, the microbial metabolic mechanism is still unknown. Strain NMD-4, capable of utilizing NMP as the sole source of carbon and nitrogen, was isolated from the activated sludge of a pesticide plant in Jiangsu, China, and identified as Paracoccus sp. based on its physiological–biochemical properties, as well as 16S rRNA gene sequence analysis. The degradation characteristic of NMP by strain NMD-4 was studied in a liquid culture, and the metabolic pathway of NMP by the strain was investigated. Two metabolites, 1-methyl-2,5-pyrrolidinedione and succinic acid, were detected and identified by liquid chromatography-mass spectrometry analysis, and a plausible microbial degradation pathway of NMP was proposed by the first time.     

蜡状芽孢杆菌菌株Jp-A的分离鉴定及其降解苯酚特性

从某钢铁厂处理废水的活性污泥中驯化分离一株能高效降解苯酚的细菌（Jp-A）.通过形态观察、生理生化实验和16srRNA序列分析,初步鉴定Jp A为蜡状芽孢杆菌（Bacillus cereus）.在实验条件下,该菌在16、24和32 h内能将浓度分别为5、10和15 mmol·L^-1的苯酚完全降解,而30 mmol·L^-1的苯酚则完全抑制该菌的生长.该菌也能以甲苯、氯酚类和硝基酚类等芳香烃类物质作为唯一碳源和能源生长.双加氧酶检测表明,其通过间位途径开环裂解苯酚,该途径的关键酶邻苯二酚2,3 双加氧酶主要定位在细胞膜上,为诱导酶,补加葡萄糖能抑制该酶的产生.     

Involvement of the plasmid pPGH1 in the phenol degradation of Pseudomonas putida strain H

Pseudomonas putida strain H (wildtype) was shown to harbour two plasmids with a molecular mass of about 50 kb and 200–220 kb, respectively. Evidence is presented that the larger one, pPGH1, is involved in the phenol degradation via the meta-cleavage pathway.     

Optimal microbial adaptation routes for the rapid degradation of high concentration of phenol

Pseudomonas fluorescence KNU417 was able to degrade up to 700 mg/L of phenol in 65 h but could not degrade 1,000 mg/L of phenol. Phenol degradation rate was noticeably enhanced by pre-adaptation. In addition, the cell was able to degrade up to 1,300 mg/L of phenol by pre-adapting to 700 mg/L of phenol. Repeated adaptations to the same concentration of phenol showed negligible increase in degradation rate. Also, relatively low concentration of phenol (100–700 mg/L) required only one pre-adaptation while high concentration (1,000 mg/L) did two consecutive stepwise pre-adaptations for rapid degradation. Optimal adaptation routes were suggested for the fast phenol degradation. For example, 1,000 mg/L of phenol was degraded as fast as in 48 h when the cell was pre-adapted to 100 and 300 mg/L of phenol sequentially. The mechanism of adaptation was explained in terms of catechol 1,2-dioxygenase induction, related to aromatic ring cleavage.     

Metabolic profiling analysis of the degradation of phenol and 4-chlorophenol by Pseudomonas sp. cbp1-3

To investigate the enhancement of phenol on the biodegradation of 4-chlorophenol (4-cp), metabolic profiling approach was performed for the first time to analyze metabolite changes of Pseudomonas sp. cbp1-3 using single substrate (succinate, phenol, and 4-cp) and dual substrate (mixtures of phenol and 4-cp). Phosphoric acid, γ-aminobutyric acid, 4-cp, 4-chlorocatechol, and catechol were shown to change significantly. Results indicated that phenols, especially 4-cp, depressed cell growth by inhibiting its primary metabolic pathway. In addition, the addition of phenol into the 4-cp-containing medium had a global influence on cells including the accumulation of amino acids, amines, saturated fatty acids, and monoacylglycerols as well as the concentration changes of metabolite participating in phenols biodegradation, thus enhancing the degradation of 4-cp. This study provided novel insights into the biodegradation of mixed phenolic compounds and the method could be used to investigate the biodegradation of complicated multi-pollutants.     

Elementary mode analysis: a useful metabolic pathway analysis tool for characterizing cellular metabolism

Elementary mode analysis is a useful metabolic pathway analysis tool to identify the structure of a metabolic network that links the cellular phenotype to the corresponding genotype. The analysis can decompose the intricate metabolic network comprised of highly interconnected reactions into uniquely organized pathways. These pathways consisting of a minimal set of enzymes that can support steady state operation of cellular metabolism represent independent cellular physiological states. Such pathway definition provides a rigorous basis to systematically characterize cellular phenotypes, metabolic network regulation, robustness, and fragility that facilitate understanding of cell physiology and implementation of metabolic engineering strategies. This mini-review aims to overview the development and application of elementary mode analysis as a metabolic pathway analysis tool in studying cell physiology and as a basis of metabolic engineering.     

Hydrocarbon degradation and enzyme activities of cold-adapted bacteria and yeasts

The potential of 89 culturable cold-adapted isolates from uncontaminated habitats, including 61 bacterial and 28 yeast strains, to utilize representative fractions of petroleum hydrocarbons (n-alkanes, monoaromatic and polycyclic aromatic hydrocarbons) for growth and to produce various enzymes at 10°C was investigated. The efficiency of bacterial and yeast strains was compared. The growth temperature range of the yeast strains was significantly smaller than that of the bacterial strains. Sixty percent of the yeasts but only 8% of the bacteria could be classified as true psychrophiles, showing no growth above 20°C. A high percentage (89%) of the yeast strains showed lipase activity. More than one-third of the 61 bacterial strains produced amylase, -lactamase, -galactosidase or lipase; more than two-thirds were protease producers. Only 6% of the bacterial strains but 79% of the yeast strains utilized n-hexadecane for growth; 13% of the bacterial strains and 21–32% of the yeast strains utilized phenol, phenanthrene or anthracene for growth. Only four yeast strains but none of the bacterial strains could grow with all hydrocarbons tested. The biodegradation of phenol was investigated in fed-batch cultures at 10°C. Three yeast strains degraded phenol concentrations as high as 10 mm (one strain) or 12.5 mm (two strains). Of eight bacterial strains, two strains degraded up to 10 mm phenol. The optimum temperature for phenol degradation was 20°C for all eight bacterial strains and for two yeast strains. Biodegradation by five yeast strains was optimal at 10°C and faster at 1°C than at 20°C. All phenol-degrading strains produced catechol 1,2 dioxygenase activity.Communicated by K. Horikoshi     

Automated system for gene annotation and metabolic pathway reconstruction using general sequence databases

Despite the growing number of genomes published or currently being sequenced, there is a relative paucity of software for functional classification of newly discovered genes and their assignment to metabolic pathways. Available software for such analyses has a very steep learning curve and requires the installation, configuration, and maintenance of large amounts of complex infrastructure, including complementary software and databases. Many such tools are restricted to one or a few data sources and classification schemes. In this work, we report an automated system for gene annotation and metabolic pathway reconstruction (ASGARD), which was designed to be powerful and generalizable, yet simple for the biologist to install and run on centralized, commonly available computers. It avoids the requirement for complex resources such as relational databases and web servers, as well as the need for administrator access to the operating system. Our methodology contributes to a more rapid investigation of the potential biochemical capabilities of genes and genomes by the biological researcher, and is useful in biochemical as well as comparative and evolutionary studies of pathways and networks.     

Halomonas alkaliantarctica sp. nov., isolated from saline lake Cape Russell in Antarctica, an alkalophilic moderately halophilic, exopolysaccharide-producing bacterium

The taxomony of strain CRSS (DSM 15686(T)=ATCC BAA-848(T)) isolated from Cape Russell in Antarctica (Ross Sea, 74 52.35 S 163 53.03 E) was investigated in a polyphasic approach. The morphological, physiological and genetic characteristics were compared with that of related species of the genus Halomonas. The isolate grew optimally at pH 9.0, 10% NaCl at 30 degrees C. The cells were Gram-negative aerobic rods able to produce exopolysaccharide. They accumulated glycine-betaine, as a major osmolyte, with minor components ectoine and glutamate. The strain CRSS biosynthetised alpha-glucosidase. The polar lipid profile consisted of phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol as major components. Ubiquinone with nine repetitive unities (Q9) was the only quinone found and the fatty acid composition was dominated by C18:1 (53%). The G+C content of DNA was 55.0mol% and its phylogenetic position was established by 16S rRNA gene sequencing as a member of the genus Halomonas. For physiological, chemotaxonomic and genetic features (DNA-DNA hybridisation) it is proposed to classify the isolate as a new species for which we propose the name Halomonas alkaliantarctica sp. nov.     

Extracellular degradation of phenol by the cyanobacterium Synechococcus PCC 7002

Investigations of the unicellular marine cyanobacterium Synechococcus PCC 7002 revealed its ability to metabolize phenol under non-photosynthetic conditions up to 100 mg L^–1. Under continuous light, photoautotrophic growth was reduced only slightly in the presence of this phenol concentration, but no transformation was observed. However neither under photoautotrophic nor heterotrophic conditions were the cells able to use phenol for growth. During the degradation of phenol in the dark cis,cis-muconic acid was produced as the major product, which was identified by gas chromatography/mass spectrometry. This result was confirmed by an identical absorption spectrum and an identical retention time in high performance liquid chromatographic analysis with authentic muconic acid as standard. This provides the first record for an ortho-fission of a phenolic substance by cyanobacteria. Further investigations of the breakdown mechanism of phenol have shown that the transformation is an extracellular process inhibited by heat, proteases and metal ions and is probably catalyzed by a protein.     

Feasibility study of exponential feeding strategy in fed-batch cultures for phenol degradation using Cupriavidus taiwanensis

An indigenous phenol-degrading bacterial isolate Cupriavidus taiwanensis R186 was used to degrade phenol from an aqueous solution under fed-batch operation. An exponential feeding strategy combined with dissolved oxygen control was applied based on kinetic characteristics of cell growth and phenol degradation to meet sufficient metabolic needs for cellular growth and achieve the best phenol removal efficiency. Without the stress of phenol inhibition, the optimal set point of specific growth rate of exponential feeding for fed-batch phenol degradation was found to be 0.50–0.55μ_max (μ_max denotes the maximum specific growth rate from Monod model). Meanwhile, the sufficient set point of dissolved oxygen for maximal phenol degradation efficiency was approximately at 10–55% air saturation. With the optimal operation conditions, the best phenol degradation rate was 0.213 g phenol h⁻¹, while a shortest treatment time of 15 h was achieved for complete degradation of 11.35 mM (ca. 3.20 g) of phenol.     

Enhancing GDP-fucose production in recombinant Escherichia coli by metabolic pathway engineering

Guanosine 5′-diphosphate (GDP)-fucose is the indispensible donor substrate for fucosyltransferase-catalyzed synthesis of fucose-containing biomolecules, which have been found involving in various biological functions. In this work, the salvage pathway for GDP-fucose biosynthesis from Bacterioides fragilis was introduced into Escherichia coli. Besides, the biosynthesis of guanosine 5′-triphosphate (GTP), an essential substrate for GDP-fucose biosynthesis, was enhanced via overexpression of enzymes involved in the salvage pathway of GTP biosynthesis. The production capacities of metabolically engineered strains bearing different combinations of recombinant enzymes were compared. The shake flask fermentation of the strain expressing Fkp, Gpt, Gmk and Ndk obtained the maximum GDP-fucose content of 4.6 ± 0.22 μmol/g (dry cell mass), which is 4.2 fold that of the strain only expressing Fkp. Through fed-batch fermentation, the GDP-fucose content further rose to 6.6 ± 0.14 μmol/g (dry cell mass). In addition to a better productivity than previous fermentation processes based on the de novo pathway for GDP-fucose biosynthesis, the established schemes in this work also have the advantage to be a potential avenue to GDP-fucose analogs encompassing chemical modification on the fucose residue.     

Quorum sensing-mediated protein degradation for dynamic metabolic pathway control in Saccharomyces cerevisiae

Dynamic regulation has been widely applied to optimize metabolic flux distribution. However, compared with prokaryotes, quorum sensing-mediated pathway control is still very limited in Saccharomyces cerevisiae. In this study, we designed quorum sensing-regulated protein degradation circuits for dynamic metabolic pathway control in S. cerevisiae. The synthetic quorum sensing circuits were developed by integration of a plant hormone cytokinin system with the endogenous yeast Ypd1-Skn7 signal transduction pathway and the positive feedback circuits were optimized by promoter engineering. We then constructed an auxin-inducible protein degradation system and used quorum sensing circuits to regulate auxin synthesis to achieve dynamic control of protein degradation. As a demonstration, the circuits were applied to control Erg9 degradation to produce α-farnesene and the titer of α-farnesene increased by 80%. The population-regulated protein degradation system developed here extends dynamic regulation to the protein level in S. cerevisiae and is a promising approach for metabolic pathway control.     

Synergy between methylerythritol phosphate pathway and mevalonate pathway for isoprene production in Escherichia coli

Isoprene, a key building block of synthetic rubber, is currently produced entirely from petrochemical sources. In this work, we engineered both the methylerythritol phosphate (MEP) pathway and the mevalonate (MVA) pathway for isoprene production in E. coli. The synergy between the MEP pathway and the MVA pathway was demonstrated by the production experiment, in which overexpression of both pathways improved the isoprene yield about 20-fold and 3-fold, respectively, compared to overexpression of the MEP pathway or the MVA pathway alone. The ¹³C metabolic flux analysis revealed that simultaneous utilization of the two pathways resulted in a 4.8-fold increase in the MEP pathway flux and a 1.5-fold increase in the MVA pathway flux. The synergy of the dual pathway was further verified by quantifying intracellular flux responses of the MEP pathway and the MVA pathway to fosmidomycin treatment and mevalonate supplementation. Our results strongly suggest that coupling of the complementary reducing equivalent demand and ATP requirement plays an important role in the synergy of the dual pathway. Fed-batch cultivation of the engineered strain overexpressing the dual pathway resulted in production of 24.0 g/L isoprene with a yield of 0.267 g/g of glucose. The synergy of the MEP pathway and the MVA pathway also successfully increased the lycopene productivity in E. coli, which demonstrates that it can be used to improve the production of a broad range of terpenoids in microorganisms.     

一株苯酚降解菌的鉴定及其降解特性

【目的】鉴定从某化工厂附近土样中分离到的一株耐高浓度苯酚的菌株T10,通过优化菌株的培养条件提高菌株对苯酚的降解率。【方法】根据菌株的形态、生理生化鉴定及16S rDNA测序分析确定其种属,以液体摇瓶培养菌株T10对苯酚的降解率为指标,对菌株的生长条件进行优化。【结果】菌株T10属恶臭假单胞菌(Pseudomonas putida)。添加葡萄糖、蛋白胨能有效缩短T10菌的生长周期,并使苯酚的降解率提高1.7倍。在菌体初始接种浓度为10%、温度为30°C、转速为180 r/min条件下,对初始苯酚浓度、pH和装液量的响应面优化结果如下:初始苯酚浓度3 000 mg/L、pH 7.5和装液量80 mL/250 mL,苯酚去除率最高可达到87.56%。【结论】T10菌能够耐受较高浓度的含酚废水,并且对苯酚有较强的降解能力,为下一步利用生物法处理含酚废水提供科学依据。     

Comparison of the downstream pathways for degradation of nitrobenzene by Pseudomonaspseudoalcaligenes JS45 (2-aminophenol pathway) and by Comamonas sp. JS765 (catechol pathway)

Nitrobenzene is degraded by Pseudomonas pseudoalcaligenes JS45 via 2-aminophenol to 2-aminomuconic semialdehyde, which is further degraded to pyruvate and acetaldehyde. Comamonas sp. JS765 degrades nitrobenzene via catechol to 2-hydroxymuconic semialdehyde. In this study we examined and compared the late steps of degradation of nitrobenzene by these two microorganisms in order to reveal the biochemical relationships of the two pathways and to provide insight for further investigation of their evolutionary history. Experiments showed that 2-hydroxymuconate, the product of the dehydrogenation of 2-hydroxymuconic semialdehyde, was degraded to pyruvate and acetaldehyde by crude extracts of Comamonas sp. JS765, which indicated the operation of a classical catechol meta-cleavage pathway. The semialdehyde dehydrogenases from Comamonas sp. JS765 and P. pseudoalcaligenes JS45 were able to metabolize both 2-amino- and 2-hydroxymuconic semialdehyde, with strong preference for the physiological substrate. 2-Aminomuconate was not a substrate for 4-oxalocrotonate decarboxylase from either bacterial strain. The close biochemical relationships among the classical catechol meta-cleavage pathway in Comamonas sp. JS765, 2-aminophenol meta-cleavage pathways in P. pseudoalcaligenes JS45, and an alternative 2-aminophenol meta-cleavage pathway in Pseudomonas sp. AP-3 suggest a common evolutionary origin. Received: 23 November 1998 / Accepted: 3 February 1999     

Malathion biodegradation by a psychrotolerant bacteria Ochrobactrum sp. M1D and metabolic pathway analysis

An organophosphorus pesticide malathion biodegradation was investigated by using the bacteria Ochrobactrum sp. M1D isolated from a soil sample of peach orchards in Palampur, District Kangra, Himachal Pradesh (India). The bacterium was able to utilize malathion as the sole source of carbon and energy. The isolated bacterium was found psychrotolerant and could degrade 100% of 100 mg l⁻¹ malathion in minimal salt medium at 20°C, pH 7·0 within 12 days with no major significant metabolites left at the end of the study. Through GCMS analysis, methyl phosphate, diethyl maleate, and diethyl 2-mercaptosuccinate were detected and identified as the major pathway metabolites. Based on the GCMS profile, three probable degradation pathways were interpreted. The present study is the first report of malathion biodegradation at both the psychrophilic and mesophilic conditions by any psychrotolerant strain and also through multiple degradation pathways. In the future, the strain can be explored to bio-remediate the malathion contaminated soil in the cold climatic region and to utilize the enzymatic systems for advanced biotechnology applications.     

Model-based design of different fedbatch strategies for phenol degradation in acclimatized activated sludge cultures

Microbial degradation of phenol was studied using batch and fedbatch cultures of acclimatized activated sludge under a wide range of phenol (0-793 mg l⁻¹) and biomass (0.74-6.7 g l⁻¹) initial concentrations. As cell growth continued after total phenol removal, the production and later consumption of a main metabolic intermediate was considered the step governing the biodegradation kinetics. A model that takes explicitly into account the kinetics of the intermediate was developed by introducing a specific growth rate model associated with its consumption and the incorporation of a dual-substrate inhibitory effect on phenol degradation. Biomass growth and phenol removal were adequately predicted in all the cultures. Moreover, the model-based design of the fedbatch feeding strategies allowed driving separately the phenol degradation under substrate-limitation and substrate-inhibition modes. A sensitivity analysis was also performed in order to establish the importance of the parameters in the accuracy of model predictions.