Genomic Insights Into Polysaccharides Degradation And Metabolic Pathways Of Different Species In Pseudoalteromonas Genus

Bacteria are important participants in the biogeochemical cycle and play key roles in the degradation of particulate organic matters in the ocean,but the mechanism underlying the degradation of particulate organic matters by marine bacteria remains unclear.Pseudoalteromonas is a genus of Gammaproteobacteria ubiquitous in global oceans.Forty-one species have been identified in Pseudoalteromonas,which implies the high diversity in this genus.So far,a variety of extracellular enzymes have been found in this genus,indicating that it may play important roles in the degradation of high molecular organic matters in the ocean.However,there is still a lack of systematic understanding of the role of this genus in the degradation of marine particulate organic matters.Alginate is the main component of the cell wall of brown algae,while chitin is a typical component of the exoskeleton of shellfish.It has been reported that Pseudoalteromonas strains can produce alginate lyases and chitinases,implying that they have the ability to degrade alginate and/or chitin,respectively.Whole genome sequencing of multiple species in Pseudoalteromonas have been carried out in our group.In this thesis,we performed a research on the utilization of alginate and chitin by different species in this genus by using comparative genomic analyses and experimental verification.1.Degradation and metabolic pathways for chitin and alginateBased on genome analysis,chitin metabolic pathway was found in Pseudoalteromonas rubra(P.rubra),Pseudoalteromonas flavipulchra(P.flavipulchra)and Pseudoalteromonas tetraodonis(P.tetraodonis),and related genes were distributed in several gene clusters in their genomes.A total of four chitinase gene clusters were found in P.rubra geome.One cluster containing three chitinase genes(PRUBb1435,PRUBbl437,and PRUBbl438)is on the small chromosome,and three clusters(PRUBa0113,PRUBa0115,and PRUBa0117;PRUBa4419,PRUBa4420,PRUBa4421,and PRUBa4423;PRUBa1269 and PRUBal270)are on the large chromosome.These chitinases are responsible for converting chitin to oligosaccharides and monosaccharides.We also found a conserved gene cluster(containing 4 genes)for converting the lysis product(N-acetylglucosamine,NAG)of chitin to fiructose 6-phosphate.Three genes are responsible for the phosphorylation(PRUBa4423),deacetylation(PRUBa4421)and deamination(PRUBa4419)in the utilization of NAG,respectively.Similar to P.rubra,four chitinase gene clusters are distributed on the two chromosomes in P.flavipulchra.Three genes(PFLAb0431,PFLAb0432,and PFLAb0434)encoding chitinases form a cluster on the small chromosome;two genes(PFLAb0710 and PFLAb0719)encoding hexosaminidases,together with two genes encoding signaling protein and transcriptional regulator form another cluster.These genes on small chromosome are mainly responsible for converting chitin to NAG.Two clusters are distributed on the big chromosome,four genes(PFLAa0064,nagA:PFLAa0067,nagB;PFLAa0064,GspK;PFLAa0066)form one cluster;three genes(PFLAa3543,agaA;PFLAa3544,nagK;PFLAa3548,agaR)form another cluster,and these genes are mainly responsible for the further utilization of NAGUnlike P.rubra and P.flavipulchra,the clusters in P.tetraodonis are mainly distributed on the small chromosome,one of which contains 2 chitinase genes(PTETb0328 and PTETb0329).While another cluster containing 6 genes.One gene(PTETh0171,nagZ)encodes a hexosaminidase,responsible for the degradation of chitin oligosaccharide.Two genes(PTETb0172 and PTETbo174)encode ATPase and deacetylase,respectively,converting NAG to N-acetylglucosamine-6-phosphate.The fourth gene(PTETb0173)encodes an aminotransferase,converting fructose-6-phosphate to glucosamine-6-phosphate.Furthermore,this cluster also contains an MFS tansporter(PTEThO175),which i5 responsible for the uptake of the products of chitin degradation.The gene(PTETb0169)encoding a lacI family transcription factor may be responsible for the transcriptional regulation of this gene clusterAlginate degradation and metabolic pathway was found in P.agarivorans and related genes are distributed in one cluster.Three genes(PAGAa1382,PA GAal383,and PAGAal393)encode alginate lyases,converting the environment al alginate into 4,5-unsaturated monouronates.KdgF(PAGAal384)and dehydro genase(PAGAal381)are responsible for the conversion of 4,5-unsaturated mon ouronates to 2-keto-3-deoxy-D-gluconate(KDG).A 2-dehydro-3-deoxygluconoki nase(PAGAal386)is responsible for converting 2-keto-3-deoxy-D-gluconate to 2-keto-3-deoxy-6-phosphogluconate(KDPG),a metabolite in Entner-Doudoroff(ED)pathway.Furthermore,a gene(eda,PAGAa1388)encoding 2-keto-3-deoxy-6-phosphogluconate aldolase in ED pathway,a gene(PAGAal390,fbaA)enco ding fructose-bisphosphate aldolase in the glycolysis and gluconeogenesis and a fructose-1,6-biophosphatase gene(PAGAal391,fbp)in gluconeogenesis are als o in this cluster.2.Comparative analysis of chitin and alginate utilization in genus PseudoalteromonasTo gain further insights into the utilization of chitin and alginate by the whole genus,the distribution of the gene clusters found above in 18 species from different lineages were studied.The gene clusters for the utilization of chitin and alginate are remarkably different.Almost all species in lineages L-?,L-?,L-?,L-?.l,L-?.2 and L-?.3 only contain gene clusters for the chitin degradation and metabolic pathway;in contrast,all species in lineage L-?.4 only contain gene clusters for the alginate degradation and metabolic pathway.This result suggests that the lineages L-I,L-?,L-?,L-?.1,L-?.2 and L-?.3 can utilize chitin,while the lineage L-?.4 can utilize alginate.The ability of these 18 species to use chitin,NAG or alginate as the sole carbon source was tested.While only a part of chitin utilization cluster-containing strains could grow with chitin as the sole carbon source,most of the strains could grow with NAG as the sole carbon source.Most alginate utilization cluster-containing strains could grow with alginate as the sole carbon source.This result is consistent with our results based on genomic analyses,and reveals the differences in the degradation and metabolic pathways of the two polysaccharides in different species and lineages in Pseudoalteromonas.3.Transcription levels of genes related to the degradation and metabolisms of chitin and alginatePrevious studies in our laboratory have shown that P.tetraodonis(lineage L-?.3)and P.agarivorans(lineage L-IV.4)are widely distributed in the marine environments.Furthermore,the experimental analysis showed that P.flavipulchra(Lineage L-?)had stronger ability to degrade chitin.In this thesis,transcriptome sequencing was performed to analyze the transcription levels of genes related to the degradation and metabolism of chitin and alginate.For P.tetraodonis,compared with those in Marine Broth 2216 or in minimal medium containing glucose as the sole carbon source,genes related to NAG utilization were up-regulated in minimal medium containing NAG as the sole carbon source,indicating that NAG may induce the transcription of these genes.On the other hand,we noted that,compared to those with glucose as the sole carbon source,genes in glycolysis and gluconeogenesis were up-regulated and genes in ED pathway were down-regulated in minimal medium containing NAG as the sole carbon source,which indicated that glycolysis may play important roles in NAG utilization of P.tetraodonis.For P.flavipulchra,compared with those in Marine Broth 2216 or in minimal medium containing glucose as the sole carbon source,the genes encoding chitinases,deacetylase and deaninase were up-regulated in minimal medium containing NAG or chitin as the sole carbon source,respectively,indicating that NAG and chitin may induce the transcription of these genes.For P.agarivorans,compared with those in Marine Broth 2216 or in minimal medium containing glucose as the sole carbon source,genes in the alginate utilization cluster were up-regulated in minimal medium containing alginate as the sole carbon source,indicating that alginate may induce the transcription of these genes.Also,we noted that genes in glycolysis and gluconeogenesis were down-regulated,while genes in ED pathway were up-regulated,which indicated that Entner-Doudoroff pathway may play important roles in alginate utilization of P.agarivorans.4.Gene knockout of phosphofructokinase and preliminary analysis of its functionComparative genomic analysis showed that most strains that have the degradation and metabolic pathway for chitin have phosphofructokinase(pfkA),but strains that have the degradation metabolic pathway for alginate do not have this enzyme.As the key enzyme in glycolysis,phosphofructokinase is responsible for the conversion of fructose-6-phosphate,the product of chitin degradation,to fructose-1,6-bisphosphate.Therefore,this enzyme may play an important role in the degradation metabolism of chitin.In order to address this,we disrupted the gene pfkA with gene knockout technique,and got the mutant ?pfkA.However,there was no significant differences in the growth curves of P.tetraodonis and ?pfkA when they were cultured in minimal medium containing glucose or NAG as the sole carbon source.This result indicates that the ED pathway may play a compensatory role for glycolysis,but further study is needed.In summary,by using a combination of comparative genomics,transcriptomics and experiments,this thesis revealed differences in chitin and alginate utilization by the ubiquitous genus Pseudoalteromonas and its genomic basis.These results provide important insights into the ecological differentiation of marine microorganisms and diversified roles in the biogeochemical cycle of organic matters in the ocean.