| Since the autoradiography report of Escherichia coli DNA first proved that its genome is composed of a single circular chromosome,people have always believed that all bacterial genomes are composed of a single circular chromosome.With the development of sequencing technology,the explosive growth of bacterial genome sequencing results showed that about 10%of bacterial genomes also possess a second chromosome.The second chromosome carries the core genes for bacterial growth and also has a plasmid-like replication system.It has been found that the genomes of many important bacteria contain second chromosomes,such as plant symbiotic bacteria and zoonotic bacteria.However,the formation mechanism and significance of the existence of the second chromosome are unclear.Flammeovirga sp.bacteria are typical polysaccharide degrading bacteria with high efficiency.Flammeovirga sp.MY04 is a strain isolated from coastal sediments by the research team,which is rich in polysaccharide degrading enzyme resources.A series of genes for glycosyl hydrolase such as agarase,mannose hydrolase and polysaccharide lyase such as algin lyase and xanthan lyase have been cloned and identified from strain MY04.However,there is only one report on the draft genome of Flammeovirga sp.bacteria,and no official report on the complete genome.The lack of information on the composition,structure and function of the genome of the genus strain has led to a further in-depth understanding of its ecological functions,characteristics and genetic basis is difficult.Therefore,we combined the second-and third-generation sequencing technologies to sequence,assemble and annotate the entire genome of the MY04 strain,and to name its replicons.Through COG function annotation,CAZy function annotation,drug resistance gene prediction,prephage prediction,CRISPR Cas system and restriction endonuclease analysis and prediction,we have compared and analyzed the functions of different chromosomes of the MY04 strain.Through BLAST,the duplication genes and horizontal transfer genes of different chromosomes of the MY04 strain were identified and functionally annotated.Through comparison with other genomes in this genus,homology analysis,phylogenetic tree construction and selection pressure calculation were carried out.Through the sequencing,assembly and annotation of the genomes of other standard strains of this genus,evolution and functional analysis were carried out.The transcriptome was determined by setting different culture conditions of sodium chloride concentration,and the genes expression of different replicons were compared.The main research results of this article are as follows:1.Sequencing,assembly and annotation of the complete genome of strain MY04 showed that it contained three replicons:chromosome 1(5.06 Mb,4063 genes);chromosome 2(2.19 Mb,1478 genes);plasmid(0.08 Mb,71 genes).2.The functional analysis of different replicons of strain MY04 showed that COG G and COG P were significantly enriched on chromosome 2.COG J,COG E,COG I,etc.are enriched on chromosome 1.Up to 11.26%of the protein in chromosome 2 is annotated as Glycoside Hydrolases(GH),while only 2.34%in chromosome 1.GH2,GH3,GH16,GH29,GH74,GH82,GH86,GH92,GH117 and GH136 are obviously enriched in chromosome 2.There are 14 predicted resistance-related genes on chromosome 1,and 7 predicted resistance genes on chromosome 2.There is a prophage region on chromosome 1 and the plasmid,but not on chromosome 2.There is a complete type I R-M system on chromosome 1,three type ? R-M systems(two complete),and three complete type I R-M systems on chromosome 2;there is no R-M system on the plasmid.There are three CRISPR system regions on chromosome 1,which are degenerate CRISPR sequences,and there is no Cas protein.There is no CRISPR Cas system on chromosome 2;there is a larger CRISPR region and 6 Cas proteins on the plasmid.3.The analysis of duplication genes and horizontal transfer genes of different chromosomes of strain MY04 showed that there are 26.83%of the duplication genes on chromosome 1,43.17%of the duplication genes on chromosome 2,and 39.44%of the duplication genes on the plasmid.There are 1.6%HTG on chromosome 1,and 6.6%HTG on chromosome 2.There is 0.49%transposase on chromosome 1,1.62%transposase on chromosome 2,and 5.63%transposase on the plasmid.The functions of chromosome 1 duplication genes are concentrated in the transport and metabolism of carbohydrates and inorganic ions(COG G/P),and the functions of chromosome 2 duplication genes are concentrated in signal transduction and cell membrane biogenesis(COG T/M).The phylogenetic tree construction of duplication genes shows that the phylogenetic tree of duplication genes between chromosomes is type B,and the phylogenetic tree of duplication genes within chromosomes is type A.4.Comparative genomics analysis results:Phylogenetic analysis based on the complete genome sequence showed that the most similar strains to strain MY04 are F.yaeyamensis sp.NBRC 100898 and F.kamogawensis strain DSM 26273.The homology analysis of the two chromosomes of strain MY04 with other strains of the same genus showed that chromosome 1 has higher homology.Evolutionary pressure analysis showed that the median Ka/Ks value of chromosome 1 gene is 0.117,chromosome 2 gene is 0.143,plasmid gene is 0.198,chromosome 2 has low evolutionary selection pressure.Complete genome sequencing of standard strains of the genus Flammeovirga sp.showed that their genomes all contained second chromosome.Collinearity analysis of MY04 strain and these genomes showed that chromosome 1 was evolutionarily conserved and chromosome 2 was plastic.Therefore,the functional preference of the proteins encoded by the two chromosomes in these genomes was analyzed,and the results showed that similar to the MY04 strain,the proteins encoded by their chromosome 1 are also responsible for core cell functions,and chromosome 2 is responsible for the transport and metabolism of carbohydrates and inorganic ions with preference.5.Preliminary analysis of the transcriptome of the MY04 strain showed that the differentially expressed genes of the MY04 strain at 0.9%sodium chloride concentration were significantly enriched in ribosome assembly,partial amino acid metabolism,nitrogen metabolism,carbon metabolism and other pathways.Differentially expressed genes at a concentration of 2%sodium chloride were significantly enriched in the carbon metabolism pathway,and differentially expressed genes at a concentration of 4%sodium chloride were significantly enriched in fatty acid metabolism,signal transduction,and carbon metabolism pathways.Under the group sodium chloride concentration,the differentially expressed genes annotated as COGP were counted.With the increase of sodium chloride concentration,the differential expression of inorganic ion transport and metabolism-related genes showed an increase in the proportion of up-regulated genes and a decrease in the proportion of down-regulated genes.At 0.9%and 2%sodium chloride concentrations,the abundance of COGP differentially expressed genes on chromosome 2 is greater than that of chromosome 1,while at 4%,the abundance of the two is the same.In summary,this study has completed the complete map sequencing of the Flammeovirga sp.MY04 strain genome for the first time,and compared and analyzed the differences of its different replicons from two aspects of function and evolution.It was discovered that its two chromosomes played different roles in the survival of the strain.Chromosome 1 maintained the basic survival of the strain,and chromosome 2 promoted the strain to better adapt to the environment.This supports the view that multiple replicons have the advantage of adapting to new ecological niches.The evolutionary difference betwe.en the two chromosomes is reflected in the evolutionary conservation of chromosome 1 and the stronger plasticity of chromosome 2,and the characteristics of plasmids on chromosome 2,which supports the hypothesis that multiple replicons are evolved from plasmids to a certain extent.This basically answers the possible reason and characteristic function of the formation of the second chromosome of this strain,and provides a reference for the study of the multiple replicator system.In particular,the enrichment of polysaccharide degrading enzymes on the second chromosome will provide theoretical support for the development and utilization of tool-type carbohydrases from a genomic perspective.At the same time,the genome sequencing and functional analysis of the standard strains of the genus indicated that having multiple chromosomes may be a common feature of the genus.And these extra chromosomes are of great significance in promoting the adaptation of strains of this genus to the environment,and they play an important role in the ocean carbon cycle. |
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