Comparative Genomics Study And Endogenous Plasmid Analysis Of Pseudomonas Berry A22 And B25

Cold-adapted microorganisms are widespread among subzero region,they are important biological groups among low temperature environments such as polar region,deep sea and refrigerated storages.Their cold tolerance render them high efficient of nutrition absorption and conversion,even biomass productions at low temperature.They play a major role in energy and substrate circles in cold ecosystems,and have important applications in biotechnology.Cold-adapted microorganisms store large amount of cellular energetic macromolecules to survive from cold environments,studies on metabolism provides us theoretical basis about discovery of novel biomass energy and better understandings about cold adaption.This study focuses on two Pseudomonas fragi strains A22 and B25.A22 produce and accumulate igloo-like structures among cell-wall,named A22B,and secrete high-level lipopolysaccharide during lifetime.Previous studies of A22B suggested that A22B belong to polyester,and extensive studies were followed to discover the biosynthesis pathway and structure of A22B.B25 strain was applied in this study as a type strain of P.fragi,they don't produce A22B-like structures and have low yield of lipopolysaccharide.Hence,for providing us better comprehension of A22B synthesis and cold adaptation,it naturally leads us to the sequencing and comparative genomics studies of these two strains.1.Sequencing of A22 and B25 were performed by next generation sequencing technology,whole genome sequences polishing were performed manually.They are the first two whole genome sequences of P.fragi ever been reported A22 genome is 5,070,897 bp in length,bioinformatics analysis predicted 3818 functional genes,while the genome of B25 is 5,020,398 bp,and consisting 3856 functional genes.Structural analysis revealed several genomic elements,and the clustering of both genes and proteins were performed for further comparative analysis.2.Genomic structural comparison analysis suggested a highly constitutional similarity between A22 and B25 genomes,genomic insertion or missing is rarely observed,as well as rearrangement.In A22 genome,two genomic island harboring lipopolysaccharides biosynthesis gene clusters were discovered,which are missing in B25's.A22 also contains much more tandem repeats sequences,insertion sequences and genomic mobile elements than B25,suggested that those elements provide A22 genomic diversities in adaption of cold environments.Comparative analysis between A22 and B25 suggested that A22 is more alternative in carbon and nitrogen resources selection,thus they have more survival rate in its native infertile environments.On the other hand,genomic comparison among five Pseudomonas strains,P.putida KT2440,P.syringae B728a,and P.protegens Pf-5,showed P.fragi have larger ratio of genes associated with metabolism and lipid transport in its genome.Which may help them coping with cold environments.On the level of gene clustering and orthologous comparison,both strains(A22 and B25)showed unique gene products associated with cold adaptation,those genes functioned as cold-inducible proteins or regulator factors.In summary,A22 is more flexible in many metabolism pathways,which makes A22 more competitive and adaptive in natural environment.3.Four cryptic plasmids from P.fragi were first time reported,A22 harboring plasmids pPF01-03,while only one plasmid was isolated from B25 strain.Those plasmids were extracted and sequenced.Three replication proteins were predicted by sequence alignments,their function and flanked sequence were analyzed by ORF deletion or sequence deletion,results suggested that those predicted Rep protein all participate in plasmid replication.Interestingly,pPF02 and pB25 shares high identity in nucleotide sequence and shows little genomic rearrangement in their sequence,which suggested that they were more native plasmid that others in P.fragi,and during the evolution of genome or plasmid,A22 has more opportunity to absorb genomic transfer elements from others to adapt cold and starving environment,Which causes the genomic sequence and phage sequence insertion in other two plasmids.Besides Rep protein,there is an orf5-parE operon in pPF02,sub-cloning and plasmid maintenance analysis suggested the parE and its adjacent orf5 were responsible for plasmids maintenance.Three shorten shuttle vectors were constructed,with stable maintenance and transformation efficiency.During plasmid curing,a mutant strain B25M was screened,in which the pB25 was cured.These were excellent genetic tools for study unknown genes in A22 genome.