In order to investigate the above hypothesis, a series of nif promoters from Klebsiella pneumoniae (i.e. nifB, nifE, nijF, nifH, nif J and nifU promoters) were cloned and fused in frame with the laeZ reporter gene on plasmid pGD926. When these constructs were introduced into Escherichia coli cya crp mutants respectively, their activities were measured. The results show that: first, these promoters can all be activated by their cognate activator NifA, expressed by a constitutive promoter; secondly, their activity can all be repressed by the CRP protein in a cAMP dependent fashion, ranging from 5- to 48-fold. Sequence analysis of the above promoters indicates that some CRP-binding sites may exist on the upstream control sequences, and several of them overlap with previously identified NifA-binding sites. However, little relationship has been found between the location and the conservation of these putative CRP-binding sites and the fold of repression.
The crp genes from K. oxytoca and K. pneumoniae have been isolated and sequenced. At the nucleotide sequence level, they are about 85% homologous to E. coli crp gene. At the deduced amino acid sequence level, they are virtually identical to that of E. coli. The only difference is a serine at position 118 of K. oxytoca and K. pneumoniae rather than the alanine in E. coli. This difference has also been observed in Salmonella typhimurium and Klebsiella aerogenes CRP proteins. Previous studies demonstrated that it did not cause significant differences in function.
Taken together, these results indicate that although our experiments were conducted in a heterogeneous genetic background, the same repression phenomenon could also exist in homogeneous strain. It suggests that CRP-cAMP-mediated repression on nif promoters has physiological significance.
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