Generation Of Complementation Strain In Cbba Mutant Derivative Of Neisseria Meningitidis: A Molecular Approach Based At Allelic Exchange System

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.