GENERATION OF COMPLEMENTATION STRAIN IN CBBA MUTANT DERIVATIVE OF NEISSERIA MENINGITIDIS: A MOLECULAR APPROACH BASED AT ALLELIC EXCHANGE SYSTEM

S. A. TUNIO, N.J. OLDFIELD, K.G. WOOLDRIDGE, D.P.J. TURNER

Abstract


N. meningitidis remains an important cause of septicemia and meningitis and is associated with high morbidity and mortality. The bacterium is a naturally competent in uptake of foreign DNA and this unique feature has been exploited extensively  for  genetic  manipulation  in  the  laboratory. In  this  study,  we present a molecular  method  for  construction  of gene complementation strain of N. meningitidis in cbbA-isogenic mutant strain. A complemented strain was created in N. meningitidis cbbA- isogenicmutant using PCR based molecular approaches and allelic exchange methodology. A  wild-copy  of  the  cbbA  gene together  with  its  predicted  promoter  sequences  were  amplified  by PCR and  cloned  between  two meningococcal genes: NMB0102 and NMB0103 in complementation vector pSAT-11. The pSAT-11 was then used to reintroduce the single copy of wild-type cbbA at ectopic site in isogenic mutant strain. Complemented strain was shown to restore the expression of cbbA to the wild-type level at ectopic site. In growth profiling experiments both strains were shown to grow at the same rate suggesting that the gene has been returned back properly. In  conclusion, the  cbbA  mutation  in  N.  meningitidis MC58 was  successfully  complemented  by  using  allelic  exchange  method  and  the newly  constructed complemented mutant strain was demonstrated to restore the expression of the gene to the wildtype level by using western blot analysis.


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