Physiological conditions for nitrogen fixation in a unicellular marine cyanobacterium, Synechococcus sp. strain SF1.

A marine, unicellular, nitrogen-fixing cyanobacterium was isolated from the blades of a brown alga, Sargassum fluitans. This unicellular cyanobacterium, identified as Synechococcus sp. strain SF1, is capable of photoautotrophic growth with bicarbonate as the sole carbon source and dinitrogen as the sole nitrogen source. Among the organic carbon compounds tested, glucose and sucrose supported growth. Of the nitrogen compounds tested, with bicarbonate serving as the carbon source, both ammonia and nitrate produced the highest growth rates. Most amino acids failed to support growth when present as sole sources of nitrogen. Nitrogenase activity in Synechococcus sp. strain SF1 was induced after depletion of ammonia from the medium. This activity required the photosynthetic utilization of bicarbonate, but pyruvate and hydrogen gas were also effective sources of reductant for nitrogenase activity. Glucose, fructose, and sucrose also supported nitrogenase activity but to a lesser extent. Optimum light intensity for nitrogenase activity was found to be 70 microE/m2 per s, while the optimum oxygen concentration in the gas phase for nitrogenase activity was about 1%. A hydrogenase activity was coinduced with nitrogenase activity. It is proposed that this light- and oxygen-insensitive hydrogenase functions in recycling the hydrogen produced by nitrogenase under microaerobic conditions.     

Genome-wide expression analysis of the responses to nitrogen deprivation in the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120.

A heterocyst is a terminally differentiated cell of cyanobacteria which is specialized in dinitrogen fixation. Heterocyst differentiation in Anabaena sp. strain PCC 7120 is triggered by deprivation of combined nitrogen in the medium. Although various genes that are upregulated during heterocyst differentiation have been reported, most studies to date were limited to individual or a small number of genes. We prepared microarrays in collaboration with other members of the Anabaena Genome Project. Here we report on the genome-wide expression analysis of the responses to nitrogen deprivation in Anabaena. Many unidentified genes, as well as previously known genes, were found to be upregulated by nitrogen deprivation at various time points. Three main profiles of gene expression were found: genes expressed transiently at an early stage (1-3 hr) of nitrogen deprivation, genes expressed transiently at a later stage (8 hr), and genes expressed when heterocysts are formed (24 hr). We also noted that many of the upregulated genes were physically clustered to form 'expressed islands' on the chromosome. Namely, large, continuous genomic regions containing many genes were upregulated in a coordinated manner. This suggests a mechanism of global regulation of gene expression that involves chromosomal structure, which is reminiscent of eukaryotic chromatin remodelling. The possible implications of this global regulation are discussed.     

Identification and characterization of a gene cluster involved in nitrate transport in the cyanobacterium Synechococcus sp. PCC7942

Summary The nrtA gene, which has been proposed to be involved in nitrate transport of Synechococcus sp. PCC7942 (Anacystis nidulans R2), was mapped at 3.9 kb upstream of the nitrate reductase gene, narB. Three closely linked genes (designated nrtB, nrtC, and nrtD), which encode proteins of 279, 659, and 274 amino acids, respectively, were found between the nrtA and narB genes. NrtB is a hydrophobic protein having structural similarity to the integral membrane components of bacterial transport systems that are dependent on periplasmic substrate-binding proteins. The N-terminal portion of NrtC (amino acid residues 1–254) and NrtD are 58% identical to each other in their amino acid sequences, and resemble the ATP-binding components of binding protein-dependent transport systems. The C-terminal portion of NrtC is 30% identical to NrtA. Mutants constructed by interrupting each of nrtB and nrtC were unable to grow on nitrate, and the nrtD mutant required high concentration of nitrate for growth. The rate of nitrate-dependent O₂ evolution (photosynthetic O₂ evolution coupled to nitrate reduction) in wild-type cells measured in the presence of l-methionine d,l-sulfoximine and glycolaldehyde showed a dual-phase relationship with nitrate concentration. It followed saturation kinetics up to 10 mM nitrate (the concentration required for half-saturation = 1 M), and the reaction rate then increased above the saturation level of the first phase as the nitrate concentration increased. The high-affinity phase of nitrate-dependent O₂ evolution was absent in the nrtD mutant. The results suggest that there are two independent mechanisms of nitrate uptake and that the nrtB-nrtC-nrtD cluster encodes a high-affinity nitrate transport system.     

Identification and cloning of a regulatory gene for nitrogen assimilation in the cyanobacterium Synechococcus sp. strain PCC 7942.

Twenty-seven mutants that were unable to assimilate nitrate were isolated from Synechococcus sp. strain PCC 7942. In addition to mutants that lacked nitrate reductase or nitrite reductase, seven pleiotropic mutants impaired in both reductases, glutamine synthetase, and methylammonium transport were also isolated. One of the pleiotropic mutants was complemented by transformation with a cosmid gene bank from wild-type strain PCC 7942. Three complementing cosmids were isolated, and a 3.1-kilobase-pair DNA fragment that was still able to complement the mutant was identified. The regulatory gene that was cloned (ntcA) appeared to be required for full expression of proteins subject to ammonium repression in Synechococcus sp.     

一株耐盐的卓贝儿氏菌(Zobellella sp.)的分离鉴定及其硝酸盐还原能力

【背景】好氧反硝化是指在有氧条件下进行反硝化作用,使得硝化和反硝化过程能够在同一反应器中同时发生,是废水脱氮最具竞争力的技术。红树林湿地中蕴藏着丰富的微生物资源,分布着大量好氧反硝化微生物。【目的】了解耐盐微生物的脱氮机制,为含盐废水生物脱氮的工程实践提供理论依据,对一株分离于红树林湿地中的耐盐好氧细菌A63的硝酸盐异化还原能力进行分析。【方法】利用形态学特征及16S rRNA基因序列测定分析,对其种属进行了鉴定,采用单因子实验测定该菌在不同环境因子下的硝酸盐还原能力,并对其反硝化脱氮条件进行了优化。【结果】初步判定该菌株为卓贝儿氏菌(Zobellellasp.),其能在盐度0%-10%、pH5.0-10.0、温度20-40°C范围内进行反硝化脱氮和硝酸盐异化还原为氨(dissimilatorynitratereductiontoammonium,DNRA)作用。菌株A63最适生长碳源为柠檬酸钠(1.2 g/L),适宜脱氮盐度为3%、pH 7.0-7.5、温度30-35°C,且C/N为10。在最适脱氮条件下,该菌株12h内能将培养基中208.8mg/L硝态氮降至0,且仅有少量铵态氮生成...     

Temperature-independent and -dependent expression of desaturase genes in filamentous cyanobacterium Spirulina platensis strain C1 (Arthrospira sp. PCC 9438)

The alteration of the degree of unsaturated fatty acids in membrane lipids has been shown to be a key mechanism in the tolerance to temperature stress of living organisms. The step that most influences the physiology of membranes has been proposed to be the amount of di-unsaturated fatty acids in membrane lipids. In this study, we found that the desaturation of fatty acid to yield the di-unsaturated fatty acid 18:2(9,12), in Spirulina platensis strain C1, was not regulated by temperature. As shown by the fatty acid composition and gene expression patterns, the levels of 18:1(9) and 18:2(9,12) remained almost constant either when the cells were grown at 35 degrees C (normal growth temperature) or 22 and 40 degrees C. The expression of desC (Delta9) and desA (Delta12) genes, which are responsible for the introduction of first and second double bonds into fatty acids, respectively, was not affected by the temperature shift from 35 to 22 degrees C or to 40 degrees C. Only the expression and mRNA stability of the desD gene (Delta6) that is responsible for the introduction of a third double bond into fatty acids were enhanced by a temperature shift from 35 to 22 degrees C, but not the shift from 35 to 40 degrees C. The increase in the level of desD mRNA elevated the desaturation of fatty acid from 18:2(9,12) to 18:3(6,9,12) at 22 degrees C. However, the increased level of 18:3(6,9,12) was observed after 36 h of incubation at 22 degrees C, indicating a slow response to temperature of fatty acid desaturation in this cyanobacterium. These findings suggest that the desaturation of fatty acids might not be a key mechanism in the response to the temperature change of S. platensis strain C1.     

阿特拉津降解菌Acinetobacter sp. DNS32对无机氮源的响应

【目的】研究Acinetobacter sp.DNS32的生长、阿特拉津降解能力和降解基因转录水平的表达对无机氮素的响应关系,为菌株的工程应用提供指导与理论基础。【方法】以Acinetobactersp.DNS32为对象,采用摇瓶法研究菌株在阿特拉津培养基中菌株生长情况及降解能力对外加硝态氮与铵态氮的响应关系,利用荧光定量PCR技术检测DNS32降解基因表达量对外加无机氮源的响应关系。【结果】外加无机氮源可以促进DNS32菌株的生长,提高阿特拉津降解能力,无机氮源对DNS32菌株的trzN、atzB和atzC 3种降解基因表达均有促进作用,加入无机氮源的试验处理中DNS32菌株trzN基因的表达量最高可达对照的11.252±2.408倍,推断DNS32菌株的这3种降解基因所编码的酶是稳定表达的组成酶。【结论】DNS32降解阿特拉津不受"氮饥饿"诱导机制调控,且无机氮源的存在对菌株的生长与降解有促进作用,因此菌株在土壤修复实践中具有广阔的应用前景。     

Heterocyst-specific expression of patB,a gene required for nitrogen fixation in Anabaena sp. strain PCC 7120

The patB gene product is required for growth and survival of the filamentous cyanobacterium Anabaena sp. strain PCC 7120 in the absence of combined nitrogen. A patB::gfp fusion demonstrated that this gene is expressed exclusively in heterocysts. patB mutants have a normal initial pattern of heterocyst spacing along the filament but differentiate excess heterocysts after several days in the absence of combined nitrogen. Expression of hetR and patS, two critical regulators of the heterocyst development cascade, are normal for patB mutants, indicating that patB acts downstream of them in the differentiation pathway. A patB deletion mutant suffers an almost complete cessation of growth and nitrogen fixation within 24 h of combined nitrogen removal. In contrast, a new PatB mutant that is defective in its N-terminal ferredoxin domain, or a previously described mutant that has a frameshift removing its C-terminal helix-turn-helix domain, grows very slowly and differentiates multiple contiguous heterocysts under nitrogen-deficient conditions.     

Concerted changes in gene expression and cell physiology of the cyanobacterium Synechocystis sp. strain PCC 6803 during transitions between nitrogen and light-limited growth

Physiological adaptation and genome-wide expression profiles of the cyanobacterium Synechocystis sp. strain PCC 6803 in response to gradual transitions between nitrogen-limited and light-limited growth conditions were measured in continuous cultures. Transitions induced changes in pigment composition, light absorption coefficient, photosynthetic electron transport, and specific growth rate. Physiological changes were accompanied by reproducible changes in the expression of several hundred open reading frames, genes with functions in photosynthesis and respiration, carbon and nitrogen assimilation, protein synthesis, phosphorus metabolism, and overall regulation of cell function and proliferation. Cluster analysis of the nearly 1,600 regulated open reading frames identified eight clusters, each showing a different temporal response during the transitions. Two large clusters mirrored each other. One cluster included genes involved in photosynthesis, which were up-regulated during light-limited growth but down-regulated during nitrogen-limited growth. Conversely, genes in the other cluster were down-regulated during light-limited growth but up-regulated during nitrogen-limited growth; this cluster included several genes involved in nitrogen uptake and assimilation. These results demonstrate complementary regulation of gene expression for two major metabolic activities of cyanobacteria. Comparison with batch-culture experiments revealed interesting differences in gene expression between batch and continuous culture and illustrates that continuous-culture experiments can pick up subtle changes in cell physiology and gene expression.     

Physiological responses of the halophilic archaeon Halobacterium sp. strain NRC1 to desiccation and gamma irradiation

We report that the halophilic archaeon Halobacterium sp. strain NRC-1 is highly resistant to desiccation, high vacuum and ⁶⁰Co gamma irradiation. Halobacterium sp. was able to repair extensive double strand DNA breaks (DSBs) in its genomic DNA, produced both by desiccation and by gamma irradiation, within hours of damage induction. We propose that resistance to high vacuum and ⁶⁰Co gamma irradiation is a consequence of its adaptation to desiccating conditions. Gamma resistance in Halobacterium sp. was dependent on growth stage with cultures in earlier stages exhibiting higher resistance. Membrane pigments, specifically bacterioruberin, offered protection against cellular damages induced by high doses (5 kGy) of gamma irradiation. High-salt conditions were found to create a protective environment against gamma irradiation in vivo by comparing the amount of DSBs induced by ionizing radiation in the chromosomal DNA of Halobacterium sp. to that of the more radiation-sensitive Escherichia coli that grows in lower-salt conditions. No inducible response was observed after exposing Halobacterium sp. to a nonlethal dose (0.5 kGy) of gamma ray and subsequently exposing the cells to either a high dose (5 kGy) of gamma ray or desiccating conditions. We find that the hypersaline environment in which Halobacterium sp. flourishes is a fundamental factor for its resistance to desiccation, damaging radiation and high vacuum.     

pbpB, a gene coding for a putative penicillin-binding protein, is required for aerobic nitrogen fixation in the cyanobacterium Anabaena sp. strain PCC7120

Transposon mutagenesis of Anabaena sp. strain PCC7120 led to the isolation of a mutant strain, SNa1, which is unable to fix nitrogen aerobically but is perfectly able to grow with combined nitrogen (i.e., nitrate). Reconstruction of the transposon mutation of SNa1 in the wild-type strain reproduced the phenotype of the original mutant. The transposon had inserted within an open reading frame whose translation product shows significant homology with a family of proteins known as high-molecular-weight penicillin-binding proteins (PBPs), which are involved in the synthesis of the peptidoglycan layer of the cell wall. A sequence similarity search allowed us to identify at least 12 putative PBPs in the recently sequenced Anabaena sp. strain PCC7120 genome, which we have named and organized according to predicted molecular size and the Escherichia coli nomenclature for PBPs; based on this nomenclature, we have denoted the gene interrupted in SNal as pbpB and its product as PBP2. The wild-type form of pbpB on a shuttle vector successfully complemented the mutation in SNa1. In vivo expression studies indicated that PBP2 is probably present when both sources of nitrogen, nitrate and N₂, are used. When nitrate is used, the function of PBP2 either is dispensable or may be substituted by other PBPs; however, under nitrogen deprivation, where the differentiation of the heterocyst takes place, the role of PBP2 in the formation and/or maintenance of the peptidoglycan layer is essential.     

一株假单胞菌(Pseudomonas sp.)L-1菌株γ-丁基甜菜碱羟化酶基因bbh 的克隆、表达及酶学性质

【目的】γ-丁基甜菜碱羟化酶是生物体内合成L-肉碱的关键酶。从假单胞菌(Pseudomonas sp.)L-1中克隆γ-丁基甜菜碱羟化酶基因,实现其在大肠杆菌(Escherichia coli)中的高效表达,并对表达产物进行酶学性质分析,为生物转化生产L-肉碱奠定基础。【方法】通过PCR克隆γ-丁基甜菜碱羟化酶基因,并将其开放阅读框(ORF)克隆至融合表达载体pET-15b;表达产物经His.Bind Resin纯化后对BBH进行酶学性质及三维空间结构分析;并以静止细胞进行L-肉碱的转化。【结果】成功地克隆了一个γ-丁基甜菜碱羟化酶基因bbh(GenBank:JQ250036),并实现了其在E.coli中的高效表达。融合蛋白以同源二聚体的形式存在,单个亚基的分子量约46.5 kDa,最适反应温度为30℃,最适反应pH为7.5。该酶在45℃以下稳定。在pH6.0时该酶有最高的pH稳定性。以表达bbh基因的重组大肠杆菌静止细胞转化L-肉碱,L-肉碱产量可达12.7mmol/L。【结论】Pseudomonas sp.L-1γ-丁基甜菜碱羟化酶与现有报道的bbh基因有较大的差异。由该基因表达的γ-丁基甜菜碱羟化酶能有效地转化γ-丁基甜菜碱生成L-肉碱。本研究不仅丰富了γ-丁基甜菜碱羟化酶基因资源,而且为L-肉碱的生物转化提供了一种新的转化方案。