ABSTRACT
OBJECTIVE: This study was aimed at sequence analysis and function annotation of plasmid pKKma carrying a mariner transposon. METHODS: Primers were designed based on the partial known sequence and used for directly sequencing plasmid pKKma. Transposon mutagenesis libraries were constructed to analyze the mutagenesis efficiency of plasmid pKKma. RESULTS: pKKma comprises 6879 bp with 7 open reading frames (ORFs). Among them, ORF6 encodes a mariner transposase of 348 amino acids (aa), a C9 variant of Himar1 type transposase. Two inverted terminal repeats (ITRs) are identified and of 27 bp each. ORF7 encodes gentamycin resistance gene aacC1, locating between two ITRs. Transposable sequence alignment with other mariner transposons shows that the coverage is 2.0% -47.7% and the homology is 3.2% to 99.7%. The result indicates pKKma is significantly different from the other vectors with mariner transposon. The transposition efficiency is also analyzed. It's (3.1 x 10(-4)) - (4.8 x 10(-4)) for S. marcascens and (1.3 x 10(-3)) - (1.7 x 10(-3)) for C. freundii, respectively. CONCLUSION: pKKma carries a new mariner transposon and could be used to study the role of genes by constructing transposon libraries in bacteria.
Subject(s)
Citrobacter freundii/genetics , DNA Transposable Elements , DNA-Binding Proteins/genetics , Serratia marcescens/genetics , Transposases/genetics , Open Reading Frames , Plasmids/geneticsABSTRACT
The 2,3-butanediol (2,3-BD) dehydrogenase gene budC of Serratia marcescens G12 was disrupted to construct the acetoin (AC) producing strain G12M. In shake-flask cultures, AC production was enhanced by increased concentrations of glucose or sodium acetate in G12M. In fed-batch fermentation, G12M produced 47.5 g/L AC along with 9.8 g/L 2,3-BD. The expression of the key enzymes for AC synthesis was further investigated. Alpha-acetolactate synthase gene budB decreased its expression significantly in G12M compared with G12. This probably explained the moderate AC production in G12M cultures. Additionally, overexpression of budB gene and α-acetolactate decarboxylase gene budA was conducted in G12M and no significant increase of AC was observed. The results suggested that intracellular AC accumulation might inhibit the expression of budB and budA gene and induce budC gene expression in G12M. Our analyses offered the bases for further genetic manipulations in improving AC production in microbial fermentations.
