[Cloning, expression and elementary characterization of phosphofructokinase from Bacillus sphaericus C3-41].
OBJECTIVE
Bacillus sphaericus is unable to use hexose and pentoses as the sole carbon source, due to the lack of key enzymes in Embden-Meyerhof-Parnas pathway (EMP), Hexose Monophophate Pathway (HMP) and Entner-Doudoroff (ED) pathway, such as phosphofructokinase (PFK). Based on the genome sequence annotation results of B. sphaericus C3-41, the phosphofructokinase gene pfk was verified with a single copy on chromosome, the aim of this research is to analysis the EMP pathway in B. sphaericus further, and confirm the function of phosphofructokinase.


METHODS
The methods of southern-blot of pfk gene among different B. sphaericus strains, pfk ORF cloning from C3-41 and expressing in Escherichia coli, the corresponding sequence analysis and anlignment were used.


RESULTS
The pfk ORF of B. sphaericus was composed of 960 bp nucleitides encoding a protein about 42 kDa, and the PFK sequence analysis showed it had the conservative amino acids-binding sites and an ATP-binding domain. The expression of pfk in recombinant E. coli strain could complement the PFK activity of a pfk mutated E. coli strain DF1020.


CONCLUSIONS
The expressed PFK had the conventional phosphofructokinase activity, and settled the foundation for the further research of catabolism of B. sphaericus.
Han
Bei
B
Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China. tydyhan@gmail.com
Cai
Yajun
Y
Hu
Xiaomin
X
Yuan
Zhiming
Z
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D003001:Cloning, Molecular; D019295:Computational Biology; D017124:Conserved Sequence; D004926:Escherichia coli / Q000235:genetics; D018628:Gene Dosage; D015870:Gene Expression; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D025221:Phosphofructokinases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D016415:Sequence Alignment
D000255:Adenosine Triphosphate; D025221:Phosphofructokinases