Analysis Of Algal Polysaccharide Utilization Enzymes And The Characteristics Of Laminaria Japonica Degradation By Tamlana Sp.S12,an Alginate Degrading Bacterium

In coastal marine ecosystems,large quantities of algae serve as primary producers and act as a carbon sink,making them a valuable source of renewable biomass.The complex polysaccharides found in algal cell walls provide suitable nutritional substrates for bacterial survival.Marine flavobacteria play a crucial role in the degradation and utilization of these polysaccharides.These bacteria possess carbon-active enzymes(CAZymes)that rely on specific genomic regions known as polysaccharide utilization loci(PULs)to break down polysaccharides.By studying marine flavobacteria with fast algae-dissolving abilities and understanding their characteristics,we can enhance our understanding of the role of algal polysaccharide-utilizing bacteria in marine carbon cycling and explore the potential for utilizing algal biomass.In this study,we investigated a high-efficiency alginate-degrading strain,Tamlana sp.S12,which was isolated from the gut of a sea cucumber.Our objective was to gain insight into the characteristics of this strain in utilizing and obtained a series of findings.Our results demonstrated that strain S12 possesses multiple PULs that play a role in the degradation of algae polysaccharides.Furthermore,it was observed that this strain can rapidly degrade Laminaria japonica,thereby occupying a unique ecological niche in the carbon cycle of marine algae.These findings have made a significant contribution to our understanding of carbohydrate-active enzymes(CAZymes).The research progress and achievements obtained in this study are summarized as follows:1.We conducted a comparative genomics analysis after performing whole-genome sequencing of Tamlana sp.S12 and conducted a comparative genomics analysis.This analysis revealed the adaptability of Tamlana sp.S12 to the ecological niche of algal polysaccharides.The complete genome of Tamlana sp.S12 was sequenced in its entirety,and its potential to degrade complex algal polysaccharides was examined.This was accomplished by identifying different PULs associated with the utilization of alginate,carrageenan,agar,and pectin.A comparative analysis was then conducted on 47 strains of Tamlana,Algibacter,Maribacter,and Zobellia.This analysis uncovered shared CAZyme genes that seemed to be conserved within strains and potentially had homology.Furthermore,we identified additional features of strain S12 that are related to the usage of algal polysaccharides and its adaptation to the gut environment of sea cucumbers.These features include the presence of gene clusters associated with the synthesis of extracellular polysaccharides(EPS).These clusters may have various physiological and biochemical functions,facilitating the absorption of polysaccharides in the gut of sea cucumbers by acting as decomposers of algal polysaccharides.Overall,this study demonstrates the adaptability of marine flavobacteria to algal polysaccharide environments and extends our understanding of CAZymes.2.The study analyzed the degradation process of L.japonica by Tamlana sp.S12 and proposed the characteristics of secondary release of alginates during the process,as well as the secondary growth of bacterial cells.When L.japonica was used as the sole nutrient source,strain S12 was found to rapidly degraded it.Under L.japonica induction,strain S12 exhibited secondary growth after entering the platform period until 42 h later.Further observations using electron microscopy revealed that strain S12 caused two disruptions to the cell wall of L.japonica during the degradation process.Experimental results indicate that strain S12 initially breaks down the cell wall of L.japonica to release and utilize some of the Laminaria polysaccharides for bacterial growth.During the secondary growth phase,strain S12 further dismantles the cell wall of L.japonica to release the remaining polysaccharides for secondary growth.Analysis of the sugar components in L.japonica fermentation liquid at different times showed that the major sugar components in the broth were soluble polysaccharides and unknown oligosaccharides.Further investigation demonstrated that the undigested polysaccharides remaining in the fermentation broth after complete degradation of L.japonica were primarily composed of fucoidan.3.Enrichment analysis was performed on the differentially expressed genes associated with the degradation of L.japonica by Tamlana sp.S12.Furthermorce,a protein-protein interaction(PPI)network was employed to examine the interactions among these differentially expressed genes,with the objective of identifying potential core proteins involved in the transcriptional process..Based on transcriptome analysis of strain S12 during the degradation of L.japonica,we observed that the oxidation-reduction and carbohydrate metabolism pathways were induced and upregulated after 12 h of growth.This suggests that strain S12 utilized the polysaccharide components in L.japonica,which were released and metabolized during this stage.After 24 h,strain S12 entered a plateau phase in the L.japonica culture medium.During this phase,there was an induction of transcription and translation processes,sugar-bound transport and metabolic pathways,protein synthesis pathways,and a large number of ribosomal proteins.Previous studies on the degradation of L.japonica by strain S12 support our findings,indicating that after 24 h,strain S12 mainly relies on the transportation and utilization of Laminaria polysaccharide degradation products and related reducing sugars in the environment to maintain community survival.However,its ability to degrade and bind alginate in the environment tends to stagnate.Additionally,glutamate dehydrogenase participates in multiple redox processes,while membrane insertion enzymes play a crucial role in the assembly of transmembrane proteins.These two proteins may occupy central positions in the transcriptional metabolism of strain S12 during L.japonica degradation at 12 and 24 h,respectively.4.This study aims to clarify the molecular transcription characteristics involved in the degradation of L.japonica by Tamlana sp.S12.Additionally,it investigates the regulatory role of the lactose operon LacI in the utilization of the PUL associated with alginates.During the initial stage of degradation,strain S12 showed significant expression of PULs associated with the degradation of cellulose,xylan,starch,and pectin.This expression led to the disruption of the cell wall of L.japonica and the subsequent release of Laminaria polysaccharides.As a result,PULs involved in the utilization of Laminaria polysaccharides were subsequently induced for expression.Upon entering the first plateau phase,strain S12 transported the oligosaccharides from the environment into its interior,which provided the necessary energy for bacterial growth.As the sugar substances in the environment became depleted,strain S12 was stimulated to produce enzymes that further degraded the cell wall of L.japonica.This resulted in a secondary release of Laminaria polysaccharides,triggering the re-expression of genes associated with the utilization of Laminaria polysaccharides and initiating the second growth phase of strain S12.Eventually,the Laminaria polysaccharides were completely released and consumed.This study revealed a noteworthy inhibitory effect of lactose on the expression of alginate lyase,which is potentially regulated by LacI.These findings offer new insights into the regulation of alginate degradation and utilization by strain S12.5.A thorough analysis was conducted to classify and identify a new strain of Bacteroidetes,named N1E240T.The analysis revealed that this strain shares complementary characteristics with strain S12 in terms of its ability to utilize algal polysaccharides.This finding underscores the potential synergistic effects between these strains in efficiently utilizing algal polysaccharides..A novel Bacteroidetes species,designated as strain N1E240T,was successfully isolated from the sediment of sea cucumber aquaculture ponds,the same environment where strain S12 was previously isolated.Through multiple classification analyses,strain N1E240T was identified as a new species within the genus Marinifilum and named Marinifilum sediminis.Genomic analysis revealed the presence of numerous secondary metabolic gene clusters and a wide array of genes associated with carbohydrate metabolism in strain N1E240T,indicating its adaptability to the utilization of polysaccharides.The investigation of CAZymes and physiological and biochemical analyses demonstrated that strain S12 exhibits a preference for and possesses the capability to degrade mannans and laminarin,highlighting its adaptation to the polysaccharide-rich environment found in the sediment of aquaculture ponds.By comparing strain S12 with strain N1E240T,significant differences were observed in their respective CAZyme repertoires and polysaccharide utilization capacities,which likely contribute to their distinct roles in marine polysaccharide utilization.This discovery significantly enhances our understanding of the synergistic utilization of polysaccharides and the ecological interactions among microorganisms in sea cucumber aquaculture ponds.