| Biological nitrogen fixation is the dominant natural process by which ecosystems obtain nitrogen. It is mediated by diazotrophic bacteria that capable of catalysing the reduction of atmospheric N2 gas to biologically available ammonium with the nitrogenase. The Mo nitrogenase consists of Fe protein and MoFe protein is the longest-known and best-studied nitrogenase. Due to the highly conserved through evolution and the phylogenetic tree largely resembles the 16S rRNA phylogenetic tree, the nifH gene encoding Fe protein, has become an ideal and common molecular tool to measure the potential of a system to fix dinitrogen and diversity of diazotrophic organisms in a culture-independent manner.In desert ecosystems, nitrogen deficiency could be caused by the few fertilizing, the low nitrogen contents of plant litter and the loss of nitrogen through mineralization. The nitrogen nutrient for plant growth mostly acquired via biological dinitrogen fixation and groundwater. Several studies suggest that the available nitrogen for shrub’s growth is mainly gotten from active symbiotic nitrogen fixation, and biological crust is a dominant source of fixed nitrogen in desert soils, mature crusts input approximately 9kg of N ha-1 year-1.The TaklaMakan Desert located in the temperate desert region of southern Xinjiang is the largest desert in China. It is characterized by dryness, extreme temperatures, high soil salinity, low nutrient levels and high summer UV radiation levels. In order to monitor the microbial potential for nitrogen fixation in desert soil of TaklaMakan Desert, we present a survey based on the nifH gene in the barren desert soils and P. euphratica forest soils by clone libraries and RFLP analysis in this study. We also used cultured-based and molecular phylogeny approaches for evaluating nitrogen-fixing bacteria in arid P. euphratica forest soils. This work represents the first study of the diversity and the community composition of the diazotrophic bacteria in desert soils of TaklaMakan Desert. The main contributions of this dissertation are as follows.(1) A total of 38 bacteria were isolated from three P. euphratica forest soils. Phylogenetic analysis based on 16S rRNA sequences of the 16 representative type strains and their closely related taxa revealed that 16 isolates were distributed into 16 ribotypes of 4 phylum: Actinobacteriaa, Firmicutes, Protobacteria and Bacteroidetes, and Actinobacteria and Firmicutes were the dominant phyla. Compared with other two P. euphratica forest soils, the microbial community is more diversity in the soil collected from TaklaMakan Desert.(2) A total of 16 culturable diazotrophs that can grow very well in a nitrogen-free medium were isolated from three P. euphratica forest soils. Their nitrogenase gene nifH were amplified and sequenced. Acetylene reduction assay showed that one strain possesses nitrogen-fixing activity, suggesting that free-living diazotrophs actually inhabit in desert ecosystems.(3) Three nifH libraries from P. euphratica forest soil sample and three nifH libraries from barren desert soil sample were constructed successfully. The values of six libraries coverage ranged from 74.1 to 91.9%. A total of 197 nifH sequences that were grouped into 84 operational taxonomic units (OTUs) by using MOTHUR at a sequence identity threshold of 0.03 at the DNA level were sequenced from these libraries.65,14 and 5 OTUs fell into the three known nifH clusters, referred to as cluster Ⅰ, Ⅱ and Ⅲ, respectively. It is noteworthiness that the nifH sequences within two clades were distantly related to any known cultured bacteria. They all derived from uncultured bacterium and showed<83% homologous to nifH sequences previously isolated from various environment.(4) All nifH sequences of culturable diazotrophic isolates and the overwhelming majority of nifH sequences from six libraries fall into α-proteobacteria. These results suggest that α-proteobacteria nifH sequences were common in desert soils, and the horizontal gene transfer of nifH gene may plays a major role in the genome evolution of diazotrophs inhabiting in TaklaMakan Desert.(5) The analysis of relative percentage abundance of nifH phylogenetic clusters between the libraries revealed that the libraries retrieved from P. euphratica forest soil exhibited a higher diversity and abundance of nifH sequence than those from barren desert soil. These result indicated there is a direct correlation between N2-fixing potential and soil nitrogenous compounds and the biological N2-fixing in desert ecosystems should closely connected with the vegetable or biological crust.(6) Five potential new taxa were found in the TaklaMakan Desert, two strains, H4XT and H9XT were subjected to a taxonomic analysis using a polyphasic approach to confirm their position in taxonomy in this study.Strain H4XT is a Gram-negative, rod-shaped bacterium that forms red colonies. Phylogenetic and biochemical analysis indicated that the isolate is a new species of the genus Pontibacter. The 16S rRNA gene of H4X displays 94.2-96.8% sequence similarities to those of other strains in Pontibacter. The major respiratory quinone is menaquinone-7 (MK-7). The major cellular fatty acids are iso-C15:0, C16:1ω5c, summed feature 3 (containing C16:1ω6c and/or C16:1ω7c) and summed feature 4 (comprising anteiso-C17:1B and/or iso-C17:1I). The major polar lipids are phosphatidylethanolamine (PE), one aminophospholipid (APL) and some unknown phospholipids (PLs). The DNA G+C content is 46.6 mol%. It is interesting to see that this bacterium can grow very well in a nitrogen-free medium. PCR amplification suggested that the bacterium possesses at least one type of nitrogenase gene. Acetylene reduction assay showed that the strain H4XT was able to convert acetylene into ethylene at the rate of 7.13± 1.2 nmol per hour per 108 cells. Therefore, it can be concluded that H4X is a new diazotroph. We thus referred it to as Pontibacter diazotrophicus sp. nov. The type strain is H4XT (=CCTCC AB 2013049T=NRRL B-599741).Strain H9XT is a Gram-staining-negative, rod-shaped, and pink bacterium.16S rRNA gene sequence homology search indicated that the isolate is most closely related to the family Cytophagacea. The 16S rRNA gene of strain H9XT displays 94.2-96.3% sequence identities to those of other type strains of the genus Pontibacter. It only possesses menaquinone-7. The major cellular fatty acids of the novel isolate are iso-C15:0, C16:1ω5c summed feature 3 (containing C16:1ω6c and/or C16:1ω7c) and summed feature 4 (comprising anteiso-C17:1 B and/or iso-C17:1 I). The major polar lipids are phosphatidylethanolamine (PE), one unknown aminophospholipid (APL), one glycophospholipids (GPL) and several unknown phospholipids (PLs). The G+C content of this bacterium is 55.2 mol%. Based on the phenotypic and genotypic data presented, it can be concluded that this isolate represents a novel species of the genus Pontibacter, for which the name Pontibacter yuliensis sp. nov. is proposed. The type strain is H9XT (=CCTCC AB 2013047T=KCTC 32396T).These results indicate that the potential and special diazotrophs is widely distributed in TaklaMakan Desert. Obviously, it is significant to explore these microbial resources and utilize their nitrogenase genes. Our results provide a scientific basis to understand the desert biological nitrogen fixation. It is helpful to reveal the processes and mechanisms of microbial community in nitrogen cycle in the extreme drought and oligotrophic desert ecosystems. |
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