Genome Sequencing And Comparative Genomics Of The Marine Bacterial Genus Psedoalteromonas

Marine ecosystem is one of the most important ecosystems on earth,and marine bacteria play important roles in marine ecosystem and biogeochemical cycles.Marine copiotrophs grow fast and are of high diversity.However,due to a lack of data,the molecular basis underlying the ecological differentiation for copiotrophs remains largely unknown.Pseudoalteromonas,a relatively great marine bacterial genus(currently containing 39 species),is widely distributed in global oceans and is highly abundant in many samples.Phenotypic differentiation and phylogenetic analysis demonstrated that genus Pseudoalteromonas could be divided into two large groups:pigmented and non-pigmented groups.Studies showed that strains of pigmented group exhibit stronger abilities to produce small molecular compounds with antibacterial,antifouling,algal-inhibiting and other biological activities,while non-pigmented strains have stronger abilities to produce various extracellular enzymes and possess better ecological flexibility,which indicated that these two groups may possess divergent ecological functions in marine ecosystem and marine biochemical cycle.However,the level of diversity of physiology and ecological functions as well as their genetic basis and metabolic mechanism for Pseudoalteromonas strains are not clear.In addition,studies showed that strains TAC125 and SM9913 of this genus possess two chromosomes,which is different from most bacteria.Nevertheless,few studies were discussed about structural features and replication system of Pseudoalteromonas chromosomes.In the thesis,type strains of genus Pseudoalteromonas were studied by genome sequencing and comparative genomics.1.Genome sequencing of type strains of 26 Pseudoalteromonas speciesType strains of 26 Pseudoalteromonas species(representing 2/3 of the currently recognized Pseudoalteromonas species)were collected.Fifteen non-pigmented strains are P.agarivorans,P.aliena,P.arctica,P.carrageenovora,P.espejiana,P.issachenkonii,P.lipolytica,P.marina,P.mariniglutinosa,P.nigrifaciens,P.paragorgicola,P.prydzensis,P.tetraodonis,P.translucida and P.undina.Eleven pigmented strains are P.aurantia,P.citrea,P.flavipulchra,P.luteoviolacea,P.peptidolytica,P,phenolica,P.piscicida,P.rubra,P.spongiae,P.tunicata and P.ulvae.With a combination of Solexa high-throughput sequencing and PCR gap closing,complete genome sequences of 9 type strains(non-pigmented strains P.agarivorans,P.arctica,P.espejiana,P.issachenkonii,P.nigrifaciens,P.tetraodonis,P.translucida and pigmented strains P.spongiae,P.tunicata)and high-quality draft genome sequences of 17 type strains were obtained.Genome analysis showed that genome sizes of this genus are of high diversity,strain P.undina possesses the smallest genome size(4.03 Mb)while strain P.rubra possesses the largest size(6.14 Mb).Meanwhile,pigmented strains possess larger genomes than non-pigmented ones.The genome sizes of pigmented strains are 4.75 Mb(P.ulvae)to 6.14 Mb(P.rubra),while the genome sizes of non-pigmented strains are 4.03 Mb(P.undina)to 5.21 Mb(P.prydzensis).In addition,genomic GC content of this genus are 39.0%to 47.8%.With a combination of complete genomes and systeny analysis,we discovered that all genomes of 26 Pseudoalteromonas type strains contain one large chromosome(chrI)and one small chromosome(chrII).The sizes of chrls are 3.15 Mb to 4.83 Mb,the sizes of chrIIs are 0.68 Mb to 1.60 Mb and the ratios of size of chrII to the relative genome are 15.9%(P.nigrifaciens)to 33.6%(P.spongiae).In addition,all chrIIs possess plasmid-type replication system(plasmid-originated initiator protein RepA)and plasmid-type maintenance system(plasmid-originated partition proteins ParA-ParB),but also possess essential genes(including his gene clusters responsible for histidine metabolism)and similar nucleotide composition(GC content and coding density)with the relative chrl,which indicated that Pseudoalteromonas chrIIs are plasmid-originated chromosomes.2.Discovery of significant difference of ecological adaptation strategies for the two large groupsComparative analysis showed that genomes of pigmented strains have a much larger size,higher GC content,more protein encoding genes and more tRNA genes than non-pigmented ones.Furthermore,pigmented strains have a stronger ability to produce secondary metabolites.Pigmented strains possess 5 to 19 gene clusters for secondary metabolites while non-pigmented strains possess 1 to 4 gene clusters.Therefore,non-pigmented and pigmented groups possess divergent genome constitutions.Category analysis of gene functions based on COG database showed that genomes of non-pigmented and pigmented groups are preferentially enriched in genes with different functional categories.Pigmented strains possess more genes related to defense,signal conduction,secondary metabolism.Though non-pigmented strains possess smaller genomes,they possess more genes related to the synthesis of cell wall/membrane/envelope,energy metabolism,carbohydrate transport and metabolism.Based on KEGG database,the construction of common and specific metabolic pathways showed that metabolic pathways of carbon and nitrogen sources for non-pigmented and pigmented groups are significantly different.Pigmented strains generally possess complete glycolysis pathways while many non-pigmented strains possess incomplete glycolysis pathway owing to the lost of key gene pfkA.However,these non-pigmented strains possess more copies of gene eda,which indicated that they strengthen Entner-Doudoroff pathway.Meanwhile,studies also showed that non-pigmented strains generally strengthen citrate cycle.On nitrogen metabolism,only some non-pigmented strains can use dissimilatory nitrate reduction to ammonium pathway to transform extracellular nitrate/nitrite into ammonia,which subsequently participates in the biosynthesis of amino acids.Meanwhile,non-pigmented strains strengthen the transformation between acetyl-CoA and acetoacetyl-CoA,which results in the strengthened communications among glucose,ketone body,fatty acid and amino acid metabolisms.The strengthened communications offers more advantages on the transformation and storage of carbon sources and energy metabolism.In addition,metabolic pathway analysis and API tests demonstrated that non-pigmented strains generally possess complete catabolism pathways and degradation activities of various carbohydrates(including cellulose,sucrose and maltose).In contrast,pigmented strains can utilize fewer kinds of carbohydrates.Noteworthily,pigmented strains generally strengthen the catabolism pathway of chitin,which indicated that pigmented strains can utilize chitin(shell of shrimps and crabs)as carbon sources and energy.The difference on gene functions and metabolic pathway indicated that non-pigmented and pigmented groups of this genus possess divergent physiology and ecological functions.By strengthening the interplay with other biological groups,pigmented groups probably possess more competitive advantages in symbiotic or adnascent environments with marine animal or algae,where nutrients are relatively abundant but space is limited.However,by strengthening the acquisition of nutrients,the production of energy and the synthesis of cellular structures,non-pigmented strains possess more competitive advantages in nutrients and possess better ecological flexibility in nutrient-limited environments.3.Discovery of the diversity of replication orientation for Pseudoalteromonas chrllsReplication system analysis showed that Pseudoalteromonas chrIs possess a similar set of replication system with chrI of Vibrio cholerae(model strain for studies of bacteria with multiple chromosomes),they share high similarity in replication origin site(oriI),gene arrangement surrounding oril site and genes participating in replisome(replication initiator gene dnaA,DNA polymerase subunit P dnaN,DNA gyrase subunit B gyrB and DNA replication and repair protein gene recF).In addition,Pseudoalteromonas chrIs possess some other replication-related systems,including chromosome dimer resolution system(FtsK-XerCD complexes),chromosome structure maintenance system(SMC-ScpAB complexes)and chromosome segregation system(ParA1-ParB1).Studies also showed that Pseudoalteromonas chrIIs possess a divergent set of replication system from chrII of V.cholerae,but share some similarity with the replication system of plasmid R1.Pseudoalteromonas chrIIs and plasmid R1 both initiate replication by RepA,terminate replication by Tus and segregate replicated chromosomes by ParA2-ParB2.In addition,Pseudoalteromonas chrIIs encode some unique replication-related proteins,including MinCDE complexes participating in cell division,DNA recombination protein RmuC and DNA helicase YwqA.GC skew analysis of 66 completely sequenced bacterial species with multiple chromosomes(belonging to 24 genera of 5 phyla except Pseudoalteromonas genus)showed that all main chromosomes and additional chromosomes of these species replicate bidirectionally.However,GC skew analysis and the gradients of sequencing coverages analysis demonstrated that most Pseudoalteromonas chrIIs replicate unidirectionally,which indicated that the unidirectional mode of replication is unique in bacteria kingdom.In addition,among studied Pseudoalteromonas chrIIs,only chrII of species P.spongiae replicates bidirectionally,which showed the diversity of replication orientation for Pseudoalteromonas chrIIs,this is the first time to discover such a case in bacteria.Homology analysis showed that chrII of P.spongiae shares a common ancestor with other chrIIs.Because Pseudoalteromonas chrIIs were originated from a plasmid R1-like ancestor and plasmid R1 replicates unidirectionally,then chr? of P.spongiae probably converted replication from unidirectionally to bidirectionally.Chromosome structure analysis showed that during the conversion of replication orientations,insertions of exogenous segments probably played key roles.4.The genetic mechanism of strain Neptunomonas untarctica S3-22T to adapt to marine sediment in AntarcticaMarine bacterial genus Neptunomonas attracts a broad attention because of its potential in dealing with marine oil pollution.A new species of this genus,Neptunomonas antarctica S3-22T,was isolated from Antarctic marine sediment and its genome was de novo sequenced.Genome analysis revealed some genetic mechanisms of strain S3-22T to adapt to Antarctic marine sediments,including the adaptation to cold environments by cold/heat shock proteins and dioxygenase,the adaptation to sediment environments by flagellar motility and resistance to heavy-metal ions and toxic drug compounds.Genome analysis also revealed that strain S3-22T can utilize extracellular nitrate/nitrite as nitrogen sources and utilize numerous toxic aromatic compounds as carbon sources.In conclusion,the thesis de novo sequenced and comparatively analyzed 26 Pseudoalteromonas species,constructed metabolic pathways of this genus and revealed the genetic basis of physiological characteristics and ecological differentiation for the two large groups of this genus.These studies not only provide abundant genomic data for the study of this genus and other bacterial groups,but also provide further insights into the study of ecological differentiation for marine bacteria especially for marine copiotrophs.In addition,the thesis studied the structural features of Pseudoalteromonas chromosomes,and discovered the diversity of replication orientation of bacterial chromosomes for the first time.These studies provide a better knowledge of replication mode for bacterial chromosomes and are valuable for further studies.