Isolation, Identification And Fluorescent Marking Of A Heierotrophic Mitrifier And Clone Of The Key Enzyme Invovled In Nitrification

Nitrogen pollution is one of the major problems that caused waterpollution and eutrophication. Generally, biological nitrogen removal wascomposed of two-step processes, that is, nitrification and denitrification.Autotrophic nitrifying bacteria have occupied a predominant position inthe nitrification. However, recent researches have shown that someheterotrophic bacteria such as Paracoccus denitrificans andPseudomonas putida are also capable of carrying out nitrification.One heterotrophic nitrifier was isolated. According to morphological,physiological,biochemical properties and 16S rDNA homology analysis,the strain was identified as Pseudomonas stutzeri and named LYS-86.The result showed that the optimal temperature,pH and salt concentrationfor nitrite oxidation were 8.0¹0.0 , 30℃and 1g/L respectively. Inaddition, the strain showed the maximum nitrification activity when theinitial concentration of NO2-N was 0.5g/l and its nitrification activity wasgreatly inhibited by increasing the initial concentrations of NO2-N.In order to monitor and investigate the formation and change ofbiofilm, recombinant suicidal vector-pUT/mini-Tn5-Km2-GFP was firstlyconstructed. GFP was then chromosomally integrated into the genomic DNA of the strain LYS-86, leading to the stable fluorescent behavior.PUT/mini-Tn5-Km2-GFP has been successfully constructed and GFPgene has also been expressed in the E.coli S17-λpir which wassuccessfully tagged with GFP. In addition, several positive conjugantswere isolated from the selective medium used to culture nitrifiers withaddition of appropriate concentration kanamycin.Two positive conjugantsconfirmed by PCR were investigated if they were tagged with GFPsuccessfully with fluorescence microscope. Unfortunately, GFP wasn'texpressed in these conjugants. It was speculated that the lac promotercould not work in the Pseudomonas stutzeri LYS-86, which leaded tofailure of GFP expression in it.A pair of degenerate primers was designed to amplify the partialfragment of amoA from Pseudomonas putida xyz according to the twomost conservative regions of amoA. A DNA band of 817bp was obtained.The DNA fragment had 94% identity with the amoA from Pseudomonasputida F1. Furthermore, a pair of specific primers was designed based onamoA from Pseudomonas putida F1 to amplify amoA from Pseudomonasputida xyz. A DNA fragment of 1056bp was also acquired. The DNAfragment could encode 315aa and had 97%, 93%, 75% and 74% identitywith amoA from Pseudomonas putida F1,Pseudomonas entomophilaL48,Pseudomonas fluorescens Pf-5 and Pseudomonas syringae pv.oryzae str. 1₆ respectively. Based on these results, it was confirmed thatamoA gene was successfully cloned and isolated from the Pseudomonasputida xyz.