Identification of a point mutation in the promoter region of cps operon responsible for capsular polysaccharide deficiency in Streptococcus pneumniae SPY1

Abstract:

Abstract: ［Objective］ To determine if the point mutation of nt313713 T → C in the promoter region of capsular polysaccharide biosynthesis (cps) operon is responsible for the deficiency of capsular polysaccharide in S．pneumoniae SPY1 strain.［Methods］Western blot was used to compare the amounts of capsular polysaccharide between the wild-type strain and SPY1 strain． Ｒeal-time quantitative PCR was used to determine transcription levels of the first four genes of cps operon，cps2A，cps2B，cps2C and cps2D． The lacZ gene was used as a reporter gene to report the strength of the promoters on cps transcription．The cps promoter was amplified by PCＲ from the wild-type strain or SPY1 strain．The amplified fragments were cloned into shuttle vector pEVP3，transformed into S．pneumoniae D39 or SPY1 strain．The transcription activities of the promoters on capsular polysaccharide biosynthesis were determined by using β-galactosidase as the reporter．Transmission electron microscopy and the Neufeld test were used to reveal the changes in capsule．［Results］Compared to that in the wild-type strain，mＲNA levels of the cps genes were significantly decreased in SPY1 strain．The amount of CPS was also decreased in SPY1 strain． β-galactosidase activities in SPY1-pEVP3-cps promoterSPY1 and D39-pEVP3-cps promoterSPY1 were decreased by about 79% and 76%，respectively，compared to that of the control． Transmission electron microscopy showed that the amount of the capsular polysaccharide of SPY1-pEVP3-cps promoterD39 strain was restored to the wild-type level. In addition，capsular polysaccharide was absent in the D39-pEVP3-cps promoterSPY1 (NC_008533.1 313713 T→C) strain as determined by Neufeld test.［Conclusion］The point mutation of nt313713 T→C in the cps promoter region results in a significantly reduced transcription of the cps genes，which is responsible for the significant reduction or even absence of the biosynthesis of capsular polysaccharide in SPY1 strain.