Identification and characterization of the nitrate assimilation genes in the isolate of Streptomyces griseorubens JSD-1

Microbial Cell Factories, Dec 2014

Background Streptomyces griseorubens JSD-1 is a novel actinomycete isolated from soil that can utilize nitrate as its sole nitrogen source for growth and these nitrate assimilation genes active in this biotransformation are expected to be crucial. However, little is known about its genomic or genetic background related to nitrogen metabolism in this isolate. Thus, this study concentrates on identification and characterization of genes involved in nitrate assimilation. Results To investigate the molecular mechanism of nitrate metabolism, genome sequencing was performed by Illumina Miseq platform. Then the draft genome of a single linear chromosome with 8,463,223 bp and an average G+C content of 72.42% was obtained, which has been deposited at GenBank under the accession number JJMG00000000. Sequences of nitrate assimilation proteins such as nitrate reductase (EC 1.7.99.4), nitrite reductase (EC 1.7.1.4), glutamine synthetase (EC 6.3.1.2), glutamate synthase (EC 1.4.1.13) and glutamate dehydrogenase (EC 1.4.1.2) were acquired. All proteins were predicted to be intracellular enzymes and their sequences were highly identical to those from their similar species owing to the conservative character. Putative 3D structures of these proteins were also modeled based on the templates with the most identities in the PDB database. Through KEGG annotated map, these proteins proved to be located on the key positions of nitrogen metabolic signaling pathway. Finally, quantitative RT-PCR indicated that expression responses of all genes were up-regulated generally and significantly when stimulated with nitrate. Conclusion In this manuscript, we describe the genome features of an isolate of S. griseorubens JSD-1 following with identification and characterization of these nitrate assimilation proteins such as nitrate reductase, nitrite reductase, glutamine synthetase, glutamate synthase and glutamate dehydrogenase accounts for the ability to utilize nitrate as its sole nitrogen source for growth through cellular localization, multiple sequence alignment, putative 3D modeling and quantitative RT-PCR. In summary, our findings provide the genomic and genetic background of utilizing nitrate of this strain.

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Identification and characterization of the nitrate assimilation genes in the isolate of Streptomyces griseorubens JSD-1

Microbial Cell Factories Identification and characterization of the nitrate assimilation genes in the isolate of Streptomyces griseorubens JSD-1 Haiwei Feng 0 1 Yujing Sun 1 Yuee Zhi 0 1 Xing Wei 0 1 Yanqing Luo 0 1 Liang Mao 0 1 Pei Zhou 0 1 0 Key Laboratory of Urban Agriculture (South), Ministry of Agriculture, Shanghai Jiao Tong University , Shanghai 200240 , China 1 School of Agriculture and Biology, Shanghai Jiao Tong University , Shanghai 200240 , China Background: Streptomyces griseorubens JSD-1 is a novel actinomycete isolated from soil that can utilize nitrate as its sole nitrogen source for growth and these nitrate assimilation genes active in this biotransformation are expected to be crucial. However, little is known about its genomic or genetic background related to nitrogen metabolism in this isolate. Thus, this study concentrates on identification and characterization of genes involved in nitrate assimilation. Results: To investigate the molecular mechanism of nitrate metabolism, genome sequencing was performed by Illumina Miseq platform. Then the draft genome of a single linear chromosome with 8,463,223 bp and an average G+C content of 72.42% was obtained, which has been deposited at GenBank under the accession number JJMG00000000. Sequences of nitrate assimilation proteins such as nitrate reductase (EC 1.7.99.4), nitrite reductase (EC 1.7.1.4), glutamine synthetase (EC 6.3.1.2), glutamate synthase (EC 1.4.1.13) and glutamate dehydrogenase (EC 1.4.1.2) were acquired. All proteins were predicted to be intracellular enzymes and their sequences were highly identical to those from their similar species owing to the conservative character. Putative 3D structures of these proteins were also modeled based on the templates with the most identities in the PDB database. Through KEGG annotated map, these proteins proved to be located on the key positions of nitrogen metabolic signaling pathway. Finally, quantitative RT-PCR indicated that expression responses of all genes were up-regulated generally and significantly when stimulated with nitrate. Conclusion: In this manuscript, we describe the genome features of an isolate of S. griseorubens JSD-1 following with identification and characterization of these nitrate assimilation proteins such as nitrate reductase, nitrite reductase, glutamine synthetase, glutamate synthase and glutamate dehydrogenase accounts for the ability to utilize nitrate as its sole nitrogen source for growth through cellular localization, multiple sequence alignment, putative 3D modeling and quantitative RT-PCR. In summary, our findings provide the genomic and genetic background of utilizing nitrate of this strain. Streptomyces griseorubens; Genome sequencing; Nitrogen metabolism; Nitrate reductase; Nitrite reductase; Glutamine synthetase; Glutamate synthase; Glutamate dehydrogenase - Background Nutritionally, physically and biologically, soil is a particularly complex and variable environment, of which the indispensable component is inorganic salts. Among these numerous elements, nitrogen is crucial as it supplies essential nutrient for plants and microorganisms. Nitrogen in soil is existed in the organic or inorganic forms. Generally, the inorganic nitrogen mainly includes nitrate and ammonium. Nitrate is preferentially found in temperate climates, while ammonium is dominant in a number of tropical soil types. Nitrate is metabolized through various reduction processes in organisms. Nitrate reduction, the most important stages of nitrogen recycle in nature, has various functions as follows: (1) as a source of nitrogen by utilization of NO3 (nitrate assimilation); (2) as terminal acceptor of electrons by producing metabolic energy during NO3 utilization (nitrate respiration); (3) maintain oxidation reduction balance by dissipating excessed energy (nitrate dissimilation). Totally, three different nitrate reducing systems (Nas, Nar and Nap) have been described in microorganisms [1-4]. Assimilatory nitrate reductases (Nas) are usually cytoplasmic enzymes stimulated by nitrate or nitrite and repressed by ammonium with the character of using either NAD(P)H or ferredoxin as physiological electron donor. Membrane-bounded nitrate reductases (Nar) are mainly involved in anaerobic nitrate respiration and denitrification. Finally, periplasmic dissimilatory reductases (Nap) contribute to redox balancing and aerobic or anaerobic denitrification. Nitrate assimilation is a key process of nitrogen recycling carried out by higher plants [5,6], algae [7], yeasts [8,9], and bacteria [10,11]. The assimilatory process starts when nitrate is transported into the cell by an active transport system. Nitrate is converted to nitrite with the function of nitrate reductase following with the reduction of nitrite to ammonia and then the conversion of ammonia to glutamine through nitrite reductase and glutamine synthetase. Finally, glutamine is transformed into glutamate by glutamate synthase [12]. Both (...truncated)


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Haiwei Feng, Yujing Sun, Yuee Zhi, Xing Wei, Yanqing Luo, Liang Mao, Pei Zhou. Identification and characterization of the nitrate assimilation genes in the isolate of Streptomyces griseorubens JSD-1, Microbial Cell Factories, 2014, pp. 174, 13, DOI: 10.1186/s12934-014-0174-4