Cloning Chitinase Gene From Aeromonas Sp. SUWA-9 And Red Gene From Soil Bacteria

Aeromonas sp. SUWA-9 is a bacterium isolated from the Suwa Lake of Japan as a producer of several chitin-degrading enzymes. To isolating genes coding for chitin-degrading enzymes, genomic DNA was prepared from SUWA-9, partially degested with Sau 3AI, and size-fractionated by sucrose density gradient gentrifugation. The resulting 2-20 kb DNA fragments were ligated with plasmid pUC118 previously digested with Bam HI and dephosphorylated by bacterial alkaline phosphatase (BAP). After transformation into Ecoli. JM109, 8 out total 30,000 recombmant colonies were selected as producing chitinase activity on M9 selection medium cotaining substrates, methylumberyferrol (MU)-(N-acetylglucosamine)n=(1-3). By comparing restriction maps of plasmids possessed in these clones, 5 clones (clones 1, 4, 5, 6, and 8) were found to contain the same chitinase gene, while the other three clones (clones 2, 3 and 9) contain different chitinase genes one another. SDS-PAGE and activity staining analysis revealed that all of clones 2, 5 and 9 exhibited a single active band with 83-kD in size, whereas clone 3 exhibited four different bands with 64 kD, 70kD, 76kD and 83 kD After construcing a series of deleted subclones from the plasmid prepared from clone-5, a complete nucleotide sequences comprising of 3,205 bps wes determined. One open reading frame (ORF) was found in this sequence, which was comprised of 2,661 bps and could encode a polypeptide with 887 amino acid residues. A search for database in the DNA Data Bank of Japan (DDBJ) revealed that the amino acid sequence of the above ORF shows 90% identity with that of one putative chitinase from Aeromonas hydrophilu in which only a primary sequence has been reported to date.Reporter gene is very important for molecular biology. Until now more than 99% microorganisms in the nature have not been cultured. It has been wildly accepted that it is a good idea to search new genes from these uncultured microorganisms During the course of study to isolate new genes from total DNAs prepared from uncultured bacteria in soil, we obtained a 4.7-kb DNA fragment that made E. coli host cells produce a redfluorescence light under exposure of ultraviolet (UV). The minimum region of DNA fragment required for an expression of red fluorescent light could be reduced to 1.5 kb. The nucleotide sequence of this 1.5 kb DNA fragment was determined, and one ORF was found and named as red gene, which was comprised of 771 bp and coded for a polypeptide of 257 amino acid residues in size. A database search revealed that the putative sequence of the red gene shows 40-50% identity with those of uroporphyrinogen III methyltransferase (encoded by cobA gene) from various kinds of bacteria. An over-expression of the cobA gene in E. coli was reported to lead to an accumulation of trimethylated derivative of porphyrin termed trimethylpyrrocorphin and Factor II , which emit strong red fluorescence under UV. Therefore, the red gene isolated from uncultured microorganisms must be a novel cobA gene.Our interesting is to use the red gene as a reporter gene. The red gene and chitinase gene from clone-5 were ligated together with pUC118(2fcwwH I/BAP), and then transformed into Ecoli. JM109. The recombinant cells exhibited chitinase activity and shew red fluorescence under UV. So we can say that the red gene may also be used as a reporter gene.