Autotrophic Bacteria And Their Community In The Pearl River

Autotrophic bacteria play an important role in the processes of nitrogen, sulfur, metal,and carbon biogeochemical cycling, and keep the balance of CO2in the atmosphere. CO2fixation and assimilation are important to environment protection, resource and energyutilization. The Pearl River is the second largest river in China, whose ecosystem issignificant to the society and economics of Southern China. Ecological analysis is helpful forrevealing the community of autotrophic bacteria that mainly responsible for CO2fixation inthe Pearl River. In this paper, we collected water samples from four sites of the Pearl Riverand analyzed the concentration of NH4+、NO2-、NO3-、Fe2+、Fe3+、PO43-、SO42-andCOD of each sample. Clone libraries based on cbbL and cbbM genes which encode two formsof ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), a key enzyme in the processof CO2fixation, were constructed. The clone libraries were analyzed by the method ofRestriction Fragment Length Polymorphism (RFLP) to reveal the diversity and communitiesof autotrophic bacteria. Eight autotrophic bacteria were enriched and isolated and identifiedby their16S rRNA genes and functional cbb genes. Finally, the cbb genes and its amino acidssequences were analyzed with the method of bioinformatics.The main results are concluded as below:(1) The diversity indices, including Shannon, Simpson and Evenness values of cbbL andcbbM clone libraries indicated that the form I RubisCO gene has a higher diversity thanform II RubisCO gene in four water samples, and the diversities of form I and form IIRubisCO genes in sites SS and NS are higher than that in HXS and BXK, though theconcentrations of inorganic compounds in SS and NS are generally lower than that inBXK and HXS.(2) Sequence analysis of cbbL and cbbM clone libraries indicated that obligate lithotrophs andfacultative lithotrophs and phototrophic bacteria were found distributed in the Pearl River.Bacteria that are responsible for the CO2fixation in the Pearl River included nitrogen-fixing bacteria (NFB)-Bradyrhizobium; ammonia-oxidizing bacteria (AOB)-Nitrosomonas; sulfur-oxidizing bacteria (SOB)-Thiomonas, Sulfuricella, Beggiatoa,Ectothiorhodospira, Methylophaga, Thiothrix, Candidatus Thiodictyon, Acidithiobacillus,Thiobacillus(both in cbbL and cbbM); ferrous-oxidizing bacteria (FeOB)-Sideroxydans,Mariprofundus, Acidimicrobium, Leptothrix; magnetotactic bacteria (MTB)-Magnetovibrio, Magnetospira, Polaromonas; metal-resistant hydrogen-oxidizing bacteria-Cupriavidus metallidurans; cyanobacteria-Synechococcus, Microcystis andother bacteria-Polaromonas naphthalenivorans. The metal-resistant hydrogen-oxidizingbacteria (52.10%) and SOB (71.40%) are the predominant bacterial groups in combinedcbbL and cbbM clone libraries, respectively.(3) Eight sulfur-oxidizing bacteria strains were isolated.16S rRNA gene identificationsuggested that they are closely related to the genus Ochrobactrum, Paracoccus of-proteobacteria, the genus Acidovorax, Thiomonas of β-proteobacteria and the genusPseudomonas of γ-proteobacteria. Functional cbbL and cbbM gene identification showedthat cbbL genes could amplified from strain#B-1,#B-2,#C-1,#C-2; cbbM genes couldamplified from strain#C-1,#C-2. The cbbL amino acids sequences of strain#B-1has ahigh similarity with Thioalkalivibrio sp. ALJ17(about98%), while sequences of strain#C-1and#C-2were closely related to Thiomonas intermedia K12(about96%and98%,respectively). The cbbM amino acids sequences of strain#C-1and#C-2were related toThiomonas intermedia K12(99%and99%, respectively) as well. A strain named CMB-1was isolated, whose16S rRNA gene is closely related to Cupriavidus metallidurans(about99%); however, its cbbL and cbbM genes could not be amplified.(4) The whole cbbL and cbbS gene of strain#C-2that closely related to Thiomonasintermedia were amplified and identified. The composition of amino acids, hydrophobic,physical and chemical properties of RubisCO was analyzed and the secondary structureand three-dimensional structure of RubisCO was predicted based on the cbbL and cbbSamino acids sequence.