| 2-keto-D-gluconic acid(2KGA) is one of the organic that is produced in a large-scale during industrialized bacterial fermentation.This product is mostly used as D-isoascorbic acid, which is a kind of food antioxidant,used in the sodium salt synthesis.Pseudomonas fluorescens A46,which was a mutant strain by ultraviolet ray(UV) inducing from P. fluorescens K1005,was a bacteriophage used in domestic 2KGA fermentation,because its high conversion ratio and short fermentation period.In recently years,there were new phages infecting A46 resulted in abnormal fermentation broth and the factory serious damage.The species of bacteriophages are remarkably numerous in the different environments. So,finding pollution of bacteriophage as early as we can and taking remediation measure in time can reduce economic expense.Deep-looking insight into the genetic background of phage and its host is very important for no matter investigating the interaction of bacteria and phage,and revealing the biological diversity mechanism resulting from gene horizontal transfer caused by phage,or engineering remodeling of bacteriophage and looking for new phage-resistant bacteria strains.It is possible to sequence and remodel the genome of bacteriophage easily,which just has thousands or ten thousands bases or base pairs.If it is once successful,there will be a great value of science and application.The bacteriophage KS461-2,which was first separated from the abnormal fermentation broth of P.fluorescens A46,infection on A46 and the corresponding control were researched. The genome sequencing of KS461-2 has been completed,and the gene function of this genome has been initiated.The main results were as follows:1.The changes of cell morphology of P.fluorescens A46,a strain producing 2-keto-D-gluconic acid,were observed by transmission electron microscope after infected by the bacteriophage KS461-2.The current results showed that the morphology changed significantly as infection time went by.2.The mutation frequency of P.fluorescens A46 resistant to the bacteriophage was determined to be 2.5×10-6.3.The bacteriophage was seen easily adhere the bacteria at 7.5～8.0 pH value.4.Ammonium oxalate could remarkably restrain the multiplication of the bacteriophage, but had no effect on the growth of the strain A46.The optimal concentration of addition ammonium oxalate was 0.7%.5.Analysis of 85229bp with EditSeq software in DNAStar showed the 4 base constituent: %A=30.51,%G=21.07,%T=29.45,%C=18.97;%A+T=59.96,%C+G=40.04.6.163 candidate genes have been predicated from the genome using the software of ORF Finer,Glimmer and Gene Finding in Viral Genomes of the web softberry.Each ORF was queried with BLASTx(basic local alignment search tool).The functions of 44 ORFs were defined with homology sequences present in the public data bases.7.Two tRNAs were predicted using the software of the web Pasteur in the genome,and the secondary structures of two tRNAs were simulated by DNA Master.8.Analyzing the genome using the software CpG islands of the web EMBL-EBI and CpGFinder of the web softberry.The result showed that 7 GC islands,which were more than 200bp,were found,among which two ORFs,ORF99 and ORF100,having the highest G+C content,may be acquired genes by horizontal transfer.9.101 promoters were found using BPRM(promoter finding in bacteria) of the web softberry.10.Analyzing the genome using the software PALINDROME of the web Pasteur,15 inverted repeats were found.No tandem repeats was found using the software EQUICKTANDEM.11.24 possible terminators are found in the intergenic regions by analyzing the genome using the software FindTerm of the web softberry.12.Choosing 9 function genes,which have more homology proteins,multiple sequence alignment was made to its homologous proteins with software MegAlign in DNAStar,and then the phylogenetic trees were drown.13.The transmembrane regions of lysozyme and holin were predicted.The result showed that lysozyme has one transmembrane region,while holin has seven.
|
PDF Full Text Request