Identification Of Three Purple Nonsulfur Bacteria Species And Their Response Of Photopigment Accumulation To Environmental Factors

Anoxygenic photosynthetic bacteria (APB) represent an assemblage of predominantly aquatic bacteria that are able to grow under anoxic conditions by photosynthesis, without oxygen production. They are widely distributed in nature and have been found in freshwater, marine and hypersaline environments that are exposed to light. Purple nonsulfur bacteria (PNSB) are a physiologically versatile group of APB that can grow well either phototrophically or in darkness. Discovering different unique habitats of microbial resources has been a topic of research for several years.Few studies have been reported on PNSB strains isolated from unique habitats in China. Therefore, it is significant to explore PNSB resource from special habitats in China and investigate the effect of the environmental factors on the composition and content of photosynthetic pigments.We isolated and gained three kinds of PNSB, including halotolerant Rhodobacter capsulatus XJ-1 (from Halosol soil in Xinjiang), Rhodobacter sphaeroides TS (from Tianchi lake in Xinjiang) and Rhodopseudomonas palustris 98144 (from acidic coal mine in Datong). The three strains were identified and characterized on the basis of cell morphology observation, physiological and biochemical characteristics investigation, as well as phylogenetic analysis of 16S rRNA sequence. This study evaluated the tolerance to NaCl as well as arsenic, and mainly examined the composition and relative content of accumulated photosynthetic pigments regulated by light, oxygen, salinity, arsenic, acetate and pH. The experiments were performed with UV-Vis、high-performance liquid chromatography (HPLC), liquid chromatography/mass spectrometry (LC/MS), thin-layer chromatography (TLC) and grayscale intensity image analysis using Gel-Pro analyzer software. This research provides an important reference for gaining insight into the environmental adaptive mechanism of PNSB from special habitats and their role in the ecosystem, lays the foundation for carrying out the further study of molecular regulation mechanism of photosynthetic pigment in APB adaptability to environment, and demonstrates the potential of testing environment arsenic as well as developing pigment producion.The main results were as follows:1. Strain XJ-1 was isolated from the Halosol soil sample which was collected from Xinjiang Autonomous Region, China. The strain was capable of growing well at salt concentrations in the range of 0-0.4 mol/L NaCl. Under anaerobic-light conditions, the median effective concentrations (EC50) for arsenite [As (III)] and arsenate [As (V)] to strain XJ-1 were calculated to be 0.61 mM and 2.03 mM, respectively. Strain XJ-1 could reduce As(V) to As(III), but As(III) could not be transformed back to As(V) or other organic As compounds. Strain XJ-1 was similar to Rhodobacter capsulatus in cell morphology, characteristic zigzag chain formation, the ability of utilization most carbon and nitrogen sources. The sequence similarities for strain XJ-1 were found with respect to the type strain of Rhodobacter capsulatus 99%, which revealed that strain XJ-1 was most closely related to Rhodobacter capsulatus. However, strain XJ-1 was different from reported Rhodobacter capsulatus strains in habitat, psychrotrophic nature, halotolerance, requirement of no vitamins, utilization of glycerol, nitrate and thiosulfate, tolerance to arsenic. Strain XJ-1 was identified as a novel isolate of Rhodobacter capsulatus and deposited in Nucleotide Sequence Databases under accession number HM370064.2. Regulation of light and oxygen on bacteriochlorophyll (BChl) and carotenoids biosynthesis in Rhodobacter capsulatus XJ-1 was investigated. Strain XJ-1 accumulated almost only one kind of BChl a anaerobically in the light, aerobically in the light and dark, and the relative contents of the BChl a were 44.61%,74.89% and 77.53% of the total pigments, respectively. A new purple pigment appeared only in aerobic-light grown cells, exhibited absorption maxima at 355,389,520,621 and 755 nm, whereas vanished in anaerobic-light and in aerobic-dark culture. Spheroidene and OH-spheroidene predominated in anaerobic phototrophic cultures. Spheroidenone was the sole carotenoid when exposed to both light and oxygen. The second keto-carotenoids, OH-spheroidenone, presented only in aerobic-dark culture in addition to spheroidenone. Strain XJ-1 would be a good model organism for the further illustration of the regulation of bacteriochlorophyll biosynthesis gene expression in response to unique habitat.The effect of NaCl on cells growth and photosynthetic pigment biosynthesis in halotolerant Rhodobacter capsulatus XJ-1 under anaerobic light conditions was investigated. The results showed that bacteria growth rate, the final biomass and the contents of BChl and carotenoids increased with the increasing concentrations of NaCl within the range of 0-0.2 mol/L However, the growth rate, the final biomass and the accumulation yields of BChls and carotenoids gradually decreased with further increasing NaCl concentrations. Rhodobacter capsulatus XJ-1 could accumulate BChl a, bacteriopheophytin, OH-spheroidene, spheroidenone and spheroidene when grown in the absence of NaCl. However, in the presence of 0.2-0.4 mol/L NaCl, strain XJ-1 also accumulated an additional bacteriopheophytin in addition to the mentioned above pigments. With the increasing concentrations of NaCl, the relative contents of BChls and spheroidene decreased, whereas two bacteriophaeophytins increased, the relative contents of OH-spheroidene and spheroidenone firstly increased and then decreased. This work indicated that the accumulation of photosynthetic pigment was related with the growth of bacteria, and the composition and relative content of accumulated pigments varied in the change of salinity in Rhodobacter capsulatus XJ-1. The results provide reference for the further study of salinity adaptability mechanism of photosynthetic pigment in anoxygenic phototrophic bacteria.The effect of arsenic (As) on cells growth and photosynthetic pigment biosynthesis in strain XJ-1 under anaerobic light conditions was investigated. The total yields of BChls and carotenoids were significantly enhanced at low concentrations of As(Ⅲ)(0.2 mM), but were only slightly enhanced under low concentrations of As(V). When strain XJ-1 was exposed to different concentrations of As(III) and As(V), the pigment OH-spheroidene was not present. The pigment bacteriophaeophytin(BPhe) was not present when the cells were treated with 0.5 mM of As(V) and 0.2 mM of As(III). The results indicate that photosynthetic pigments accumulated in strain XJ-1 were sensitive to As(V) and As(III), and that the concentrations of As significantly influenced the biosynthesis of photosynthetic pigments. When exposed to As, the ratios of Tetrahydrogeranylgeranyl BChl a(T1, BCh1 aTHGG) to phytylated BChl a, and spheroidenone to spheroidene varied. The relative content of accumulated spheroidenone decreased from 32% to 28% of the total photosynthetic pigments with increasing concentrations of As(V), Whereas the relative content of accumulated spheroidenone significantly increased from 25% to 35% with increasing concentrations of As. Therefore, strain XJ-1 could be a promising candidate for quantifying higher As concentrations in the environment. Strain XJ-1 could be useful for As measurement in real-world.The effect of acetate on cells growth, compositions and relative contents of accumulated photopigments in Rhodobacter capsulatus XJ-1 under anaerobic light conditions was investigated. Photosynthetic pigment accumulation in Rhodobacter capsulatus strain XJ-1 varied in the presence of 2-24 g/L acetate. The total yields of BChls as well as carotenoids were maximum in the presence of 6 g/L acetate. The synthesis of bacteriopheophytin was inhibited by high concentrations of acetate. Cells accumulated one or two kinds of bacteriopheophytin in the presence of low concentrations of acetate. When strain XJ-1 was grown with 6 g/L�'�8 g/L acetate, the pigment OH-spheroidenone was present on the TLC. Furthermore, only a kind of BCh1 a was accumulated with different concentrations of acetate as carbon source. If further study is carried out, the purified BChl a should be gained when the organic wastewater was treated.3. Strain TS was isolated from a Tianchi lake sample from Xinjiang Autonomous Region with modified Ormerod medium HP83. Strain TS was similar to Rhodobacter sphaeroides in cell morphology, slime production, the ability of utilization most carbon and nitrogen sources. The sequence similarities for strain TS were found with respect to the type strain of Rhodobacter sphaeroides 99%. Phylogenetic analysis of 16S rRNA gene sequence revealed that strain TS was most closely related to Rhodobacter sphaeroides. However, strain TS was different from reported Rhodobacter sphaeroides strains in habitat, the in vivo absorption spectrum of the living cells, requirement of vitamins, optimal pH and temperature. Thus, strain TS was identified as a novel isolate of Rhodobacter sphaeroides and deposited in Nucleotide Sequence Databases under accession number EU882155.The effect of pH on cells growth, compositions and relative contents of accumulated BChls as well as carotenoids in Rhodobacter sphaeroides TS under anaerobic light conditions was investigated. The wavelengths of maximum absorption that occurred at around 376,423,453,480,508,592, 802,854 nm and that humped at 874-877 nm indicated the presence of BChl a and the carotenoids from the spheroidene series. Different carbon sources, nitrogen sources, pH and different concentrations of NaCl affected the characteristic absorption peaks of the living cells, especially the change of characteristic absorption peak at 423 nm.The final biomass of cells and the total yields of BChl as well as carotenoids were slightly enhanced with pH increasing. The relative contents of BChl a accumulated in cells grown with pH 6.5 and 8.0 were 55.46% and 55.43%of the total pigments, respectively. The relative contents of of BPhe and carotenoids were obviously affected by pH. Strain TS did not accumulate spheroidenone. The results provide reference for the further study of pH mechanism of photosynthetic pigment in anoxygenic phototrophic bacteria.4. Strain 98144 was isolated from acidic environment with modified medium Pfennig 1988. Strain 98144 was able to tolerate elemental sulfur, thiosulfate and sulfite. Growth was observed in cells growing with 0-6 mmol/L Na2S. Phylogenetic analysis of 16S rRNA gene sequence revealed that strain 98144 was most closely related to Rhodopseudomonas palusteris ATCC 17001. However, they were distinguishable in vivo absorption spectrum of the living cells, some carbon and sulfur sources. Hence, strain 98144 was identified as a novel isolate of Rhodopseudomonas palusteris and deposited in Nucleotide Sequence Databases under accession number EU882156.The results of thin layer chromatography and image gray intensity analysis showed that Rhodopseudomonas palusteris 98144 accumulated four kinds of carotenoids, one kind of bacteriophaeophytin and BChl a, respectively. The relative content of BCh1 a was 65.66%. This isolate would be a promising candidate for obtain BChl a production.