Characterization Of Mixotrophic And Environmental Adaptation Mechanism Of Marinobacter Spp.

Marinobacter are widely distributed in various marine environments and are highly adaptable to their environment.They have been isolated from surface seawater,sediments,pollutants,salt field crystals and marine biological samples,and are important players in biogeochemical cycles and play an important role in marine ecosystems.Marinobacter is generally regarded as a typical heterotrophic bacterium,but a few studies have shown that some Marinobacter have autotrophic ability,suggesting that bacteria of the genus Marinobacter may have mixed trophic ability,but related metabolic identification,characterization and genetic analysis studies are less reported.In this study,we analyzed the mixotrophic metabolic ability（organic heterotrophic and inorganic chemoautotrophic）of 27 Marinobacter type species and 5Marinobacter of hydrothermal origin by physiological and biochemical experiments,including the detection of autotrophic/heterotrophic sulfur oxidation and iron oxidation ability,and preliminarily resolved the genomic characteristics of 59 Marinobacter strains at the genomic level,predicted iron sulfur oxidation,carbon fixation phase The genomic characteristics of 59 strains of Marinobacter were preliminarily resolved at the genomic level,and genes related to iron-sulfur oxidation,carbon fixation phase and marine environment adaptation were predicted to reveal their mixotrophic metabolic potential and environmental adaptability in marine environment.The results of the study are as follows.1.Validation of autotrophic/heterotrophic mixotrophic metabolic capacity of Marinobacter.From 27 type strains and 5 newly isolated deep-sea hydrothermal-zone sourced Marinobacter,5 Marinobacter type strains and 1 hydrothermal-zone sourced Marinobacter were found to have mixotrophic metabolic ability.In an autotrophic sulfur oxidation medium with HCO₃^-as the sole carbon source and Na₂S₂O₃ as the sole electron donor,the model strains Marinobacter guineae M3B^T,Marinobacter aromaticivorans D15-8P^T,Marinobacter vulgaris F01^T,Marinobacter profundi PWS21^T and Marinobacter denitrificans JB02H27^T and the hydrothermal origin of Marinobacter sp.ST-1M（99.93%similarity to Marinobacter salsuginis SD-14B^T）could be grown continuously by transfer.the cell volume（number of cells）of the five type species of Marinobacter sp.increased from 10⁵ cells/ml to between 8.88×10⁷cells/m L and 9.72×10⁷ cells/m L,with thiosulfate consumption ranging from 3.59 m M to 9.64 m M and sulfate production ranging from 0.10 m M～0.96 m M.In addition,incubation experiments in autotrophic medium iron oxidation gradient tubes revealed that 14 type strains of Marinobacter formed growth strips and produced brown stripes of iron-rich oxides,presumably with autotrophic iron oxidation ability.2.Genomic analysis of inorganic chemoautotrophic potential:Genome-wide annotation and analysis revealed the presence of a thiosulfate oxidizing Sox multi-enzyme complex（Sox B-Sox A-Sox Z-Sox Y-Sox X-Sox C）in six of the 53Marinobactertype species.Gene phylogenetic evolutionary analysis showed that the Marinobacter sox B genes were independently clustered and were closest to Thiomicrorhabdus arctica and Aequoribacter fuscus of the Gammaproteobacteria.The analysis of carbon fixation pathway-related genes revealed that the CBB carbon fixation pathway and r TCA carbon fixation pathway of the five autotrophic Marinobacter type species were incomplete,but all of them had key enzymes of the CBB pathway,while some key enzymes of the r TCA pathway were lacking,implying that their autotrophic carbon fixation pathways had unknown functional genes.It was also tentatively demonstrated that 14 strains of Marinobacter had autotrophic iron oxidation-related genes cyc1,fox Y,fox Z.3.Analysis of the marine environment adaptation mechanism of Marinobacter.The genome analysis of 59 strains of Marinobacter revealed that their GC content（53.7～63.2 mol%）was high compared with other Gammaproteobacteria,and they had an open pan-genome.Among them,27 strains had CRISPR/Cas system;comparing the genetic differences between marine origin（100m～9600m seawater and sediment）and other sources of Marinobacter,the marine origin Marinobacter core genes were enriched to more genes related to lipid metabolism,energy metabolism and heterologous substance metabolism,and the accessory genes were enriched to more genes related to cell motility,energy metabolism,membrane transport and signal transduction.The specific genes were enriched to more genes related to carbohydrate metabolism,lipid metabolism,other amino acid metabolism and heterologous substance metabolism.Among the core genes of marine origin,glutamate synthase and glutamate dehydrogenase encoding compatible solute glutamate,ABC transporter proteins related to iron（III）,lipopolysaccharide,zinc and urea transport,chemotactic protein signal transduction histidine kinase che A related to cell motility,flagellin flh G,two-component system Pho R-Pho P related to phosphate regulation,chlorotropic protein related to heterotrimeric substance metabolism,and the heterotrimeric substance metabolism.These characteristics are favorable for marine origin Marinobacter with physiological properties such as maintenance of genetic traits,cell membrane stability,resistance to phage infestation,and better adaptation to changing environments.In summary,representative species of the genus Marinobacter were analyzed for their mixotrophic metabolic potential based on pure culture strains with genomic information.It was found that a high proportion of the traditionally considered heterotrophic ascomycetes had autotrophic sulfur oxidation（about 19%）and iron oxidation autotrophic ability（about 52%）;their metabolic diversity and open pangenome may provide a possibility for their marine environmental adaptation and wide distribution.The results of the study provide a reference for further understanding of the environmental adaptation mechanisms of Marinobacter.