Study On The Phylogenetic Distribution And Evolution Of Polysaccharide-Degrading Enzymes In Marine Bacteria

Polysaccharides are important structural components and energy storage of organisms.In the ocean,there is a large amount of organic carbon in the form of polysaccharides.Polysaccharide degradation is an important part of the ocean carbon cycle,and bacteria play an important role in it.Polysaccharides have high molecular weights and cannot be used directly by bacteria.Before utilization,they are firstly degraded into low-molecular-weight oligosaccharides or monosaccharides by polysaccharide-degrading enzymes(PDEs).In recent years,reports of new types of PDEs derived from marine bacteria have gradually increased,but there is still a lack of systematic research on the degradation ability of various polysaccharides by marine bacteria.Analyzing the degradability of polysaccharides by various groups of marine bacteria is of great significance for elucidating the mechanism of ocean carbon cycle.This thesis systematically studied the potential of marine bacteria to produce PDEs based on the genome sequences available at public databases.In order to study the diversity of currently known marine bacterial PDEs and improve the quality of subsequent annotations of PDEs in the genome,this thesis first checked the literature and confirmed and collected 961 PDE protein sequences supported by experimental data as reference sequences,including agarases,alginate lyases,carrageenases,cellulases,chitinases,chitosanases,fucoidanases,hyaluronate lyases,hyaluronidases and ulvan lyases,10 types of PDEs.Then,BLAST and HMMER,two sequence search and comparison tools,were integrated to construct a PDE annotation pipeline.The pipeline considered the domain composition,alignment matching range and sequence identity,and adopted strict parameters to obtain high accuracy of annotation results.In order to comprehensively investigate the enzyme-production potential of marine bacteria,all genome information was downloaded from the NCBI RefSeq database.Based on the genome quality and the genome environment source information from the BioSample database,a total of 2,182 high-quality marine bacterial genomes were selected for subsequent annotation.Using the above-mentioned reference sequence and annotation pipeline,these genomes were automatically annotated,and 9 types(hyaluronidase was not found),9,335 PDE sequences were annotated from 1,370 genomes.Alginate lyases(3,756),chitinases(3,171)and cellulases(1,470)were the most abundant enzymes with the highest numbers of sequences and the widest distribution.They were distributed in 107 genera of 7 phyla,110 genera of 7 phyla and 181 genera of 12 phyla,respectively.Fucoidanases were the PDE with the smallest number of annotated sequences and the narrowest distribution and only 11 enzyme sequences were annotated in five genera from two phyla.The sequence numbers of remaining five types of PDEs were 95-470,distributed in 22-39 genera from 2-5 phyla.The analysis revealed that marine bacteria and eukaryotic groups share a few similar enzyme genes,indicating that there were gene exchanges across a few kingdoms.The phylogenetic tree was constructed for the studied marine bacteria based on the 16S rRNA gene,and the average trait depth(?d)of each type of PDE was calculated using the consenTRAIT algorithm.The ?D of different PDEs were between 0.0144-0.0281,corresponding to the 16S rRNA identities of 94.4%-97.1%,indicating that the distribution of PDE in the genus and lower taxa is conservative.The Pearson correlation coefficients and Jaccard distances were calculated to characterize whether there were correlations between the distribution of different types of PDEs.It was found that agarases and carrageenases were positively correlated(r=0.5 1,p<0.05;Jaccard distance 0.67).In twelve genera(genome number?25),a few PDEs pairs were positively correlated(r>0.4,p<0.05;Jaccard distance<0.75).It was worth noting that there were negative correlations between chitinases and the other three enzymes,cellulase,alginate lyase and agarase(r<-0.4,p<0.05;Jaccard distance>0.75),which may related to the difference in the environmental distribution between chitins and other polysaccharides in the ocean.The differences in coding abilities of PDEs within the same genus indicates that marine bacteria may have undergone multiple lifestyle differentiations within the genus,and the improvement of polysaccharide utilization capacity may be an important driving force for this differentiation.For alginate lyase,the enzyme with the largest number of sequences,three main families,PL6,PL7 and PL 17,were selected for further evolutionary study.A gene tree was constructed for each family.According to the topological structure of the tree and the source species of the sequence,the gene tree was divided into subtrees(subfamilies)which contained only single-copy genes.For each subfamily,HGT events between different orders are predicted based on the topological structure of the gene tree,the species source of the gene and the distribution of the gene in the species tree.It was found that all three families had multiple HGTs among different orders.Vibrionales and Alteromonadales from Gammaproteobacteria and Flavobacteriales from Bacteroides are the main sources of enzymes from these three families.Alginate lyases in other groups were likely to be obtained through HGTs from these groups.For PL6,PL7,and PL17 families,18,12,and 22 HGT events between orders or higher taxa were predicted,respectively.This result indicated that these HGTs,which frequently occurred among distantly related groups,had promoted the diffusion of alginate lyase genes among multiple groups,resulting in the present widespread distribution.In summary,through systematic gene annotation and evolutionary analyses,this thesis clarified the similarities and differences in the degradation potential toward various polysaccharides between different groups of marine bacteria,and revealed the conservation of degradation ability within the genus and the differences between genera.It was found that the widespread HGT events among various distant groups promoted the spread of alginate lyase genes in multiple marine bacterial groups.This thesis provided new insights in the ocean carbon cycle and the ecological differentiation of marine microorganisms.