ABSTRACT
A third generation promoter probe shuttle vector pKG was constructed, using the green fluorescent protein as a reporter, for in situ evaluation of Deinococcal promoter activity in Escherichia coli or Deinococcus radiodurans. The construct yielded zero background fluorescence in both the organisms, in the absence of promoter sequences. Fifteen Deinococcal promoters, either harbouring Radiation and Desiccation Response Motif (RDRM) or not, were cloned in vector pKG. Only the RDRM-promoter constructs displayed (i) gamma radiation inducible GFP expression in D. radiodurans, following gamma irradiation, (ii) DdrO-mediated repression of GFP expression in heterologous E. coli, or (iii) abolition in GFP induction following gamma irradiation, in pprI mutant of D. radiodurans. Utility of pKG vector for real-time in situ assessment of Deinococcal promoter function was, thus, successfully demonstrated.
ABSTRACT
K+, the dominant intracellular cation, is required for various physiological processes like turgor homeostasis, pH regulation etc. Bacterial cells have evolved many diverse K+ transporters to maintain the desired concentration of internal K+. In E.coli, the KdpATPase (comprising of the KdpFABC complex), encoded by the kdpFABC operon, is an inducible high-affinity K+ transporter that is synthesised under conditions of severe K+ limitation or osmotic upshift. The E.coli kdp expression is transcriptionally regulated by the KdpD and KdpE proteins, which together constitute a typical bacterial two-component signal transduction system. The Kdp system is widely dispersed among the different classes of bacteria including the cyanobacteria. The ordering of the kdpA, kdpB and kdpC is relatively fixed but the kdpD/E genes show different arrangements in distantly related bacteria. Our studies have shown that the cyanobacterium Anabaena sp. strain L-31 possesses two kdp operons, kdp1 and kdp2, of which, the later is expressed under K+ deficiency and desiccation. Among the regulatory genes,the kdpD ORF of Anabaena L-31 is truncated when compared to the kdpD of other bacteria, while a kdpE -like gene is absent. The extremely radio-resistant bacterium, Deinococcus radiodurans strain R1, also shows the presence of a naturally short kdpD ORF similar to Anabaena in its kdp operon. The review elaborates the expression of bacterial kdp operons in response to various environmental stress conditions, with special emphasis on Anabaena. The possible mechanism(s)of regulation of the unique kdp operons from Anabaena and Deinococcus are also discussed.