Streptococcus pneumoniae is a facultative anaerobe lacking catalase enzyme and requires exogenous catalase supplemented to culture media for aerobic growth. We introduced a catalase gene (kat) of Listeria seeligeri into S. pneumoniae and tried to see if this listerial kat gene was expressed within the pneumococcal host. To clone the listerial kat gene in the pneumococcal chromosome, a non-replicating plasmid pAHA-LSt3, along with its original promoter region was used for integration the chromosome via homologous recombination. One of three resulting transformants was confirmed to contain the kat gene and designated as EHS2. In addition, the kat gene was subcloned in Escherichia coli in frame to the lac promoter of a shuttle vector to generate pDL-Kat, which was subsequently used for pneumococcal transformation. Four identical recombinants were identified to contain the plasmid with the kat gene. By performing RT-PCR, it was observed that the listerial kat gene was indeed transcribed within pneumococcal recombinants from its original promoter in the chromosome of EHS2 and from the lac promoter in the plasmid pDL-Kat. In contrast to the E. coli kat+ recombinants, however, the pneumococcal kat+ recombinants failed to reveal any catalase activities detectable by ferricyanide staining on non-denaturing PAGE. When the pDL-Kat plasmid DNA purified from pneumococci was allowed to transform E. coli again, many kat+ recombinants were obtained, ruling out the possibility of the defective kat E. coli transformants gene within pneumococci. The observation that the listerial kat gene in pneumococci was unable to produce the functional catalase enzyme, which requires a heme group at its active site and a cofactor NADPH, suggests pneumococcal defect in heme production.
Catalase* ; Catalytic Domain ; Clone Cells ; Culture Media ; DNA ; Escherichia coli ; Genetic Vectors ; Heme ; Homologous Recombination ; Listeria* ; NADP ; Native Polyacrylamide Gel Electrophoresis ; Plasmids ; Pneumonia ; Promoter Regions, Genetic ; Streptococcus pneumoniae* ; Streptococcus*

An in vitro detection method of the gastrointestinal absorption of Pilose Antler protein was established for mixed protein activity. Five bands of protein with molecular weight of 17.8-160 kD derived from the Pilose Antler were extracted and sufficiently labeled with FITC (FITC-PE). The stability and variation of FITC-PE in gastrointestinal circumstances were detected by native polyacrylamide gel electrophoresis and confocal laser scanning microscope. Results showed that the main component of FITC-PE kept invariant after being reacted with artificial gastric fluid and artificial intestinal fluid. The fluorescence signal was detected 20 min after administration in the valgus intestinal purse experiment, and three kinds of protein, with molecular weight of 45, 25, and 17.8 kD, were detected in the mixture of absorbent protein. The research laid the foundation for the further in vivo study of Pilose Antler protein. Meanwhile, it would be an in vitro screening method for the absorption, distribution and metabolism of mixed protein from traditional Chinese medicine.
Animals ; Antlers ; chemistry ; Deer ; Fluorescein-5-isothiocyanate ; Intestinal Absorption ; Intestinal Mucosa ; metabolism ; Male ; Materia Medica ; chemistry ; isolation & purification ; pharmacokinetics ; Microscopy, Confocal ; Molecular Weight ; Native Polyacrylamide Gel Electrophoresis ; Proteins ; chemistry ; isolation & purification ; pharmacokinetics ; Rats ; Rats, Wistar ; Stomach ; metabolism