The signal for growth rate control and stringent sensitivity in E. coli is not restricted to a particular sequence motif within the promoter region.

Hybrid promoter constructs were used to determine the DNA sequence requirements for stringent and growth rate control within a promoter region. The promoters were obtained by fusing complementing sequence regions located upstream and downstream from the GCGC discriminator motif of the growth rate regulated rRNA P1 promoter and a non-regulated tac promoter variant. The activities and the regulatory response of the hybrid promoters were determined in vivo using a promoter test vector system with the chloramphenicol acetyltransferase (CAT) reporter gene. Measurements were made at different growth rates and after starvation for isoleucine to induce the stringent response. Neither the upstream nor the downstream sequence of P1 relative to the GCGC discriminator motif conferred comparable regulatory features when fused to the complementing sequences of the non-regulated mutant tac promoter. A minor response to amino acid deprivation or changes in the growth rate was noted for the hybrid promoter with the rrnB P1 upstream segment and the tac downstream element, pointing to a slightly different importance of the two sequence elements for regulation. The parallel effects for stringent as well as growth rate regulation of the hybrid promoters supports the view of a common mechanism for both types of control. However, none of the promoter sequence elements on its own was able to restore the complete regulatory behaviour of their 'parent' promoters.     

m-xylene-responsive Pu-PnifH hybrid sigma54 promoters that overcome physiological control in Pseudomonas putida KT2442

The sequences surrounding the -12/-24 motif of the m-xylene-responsive sigma54 promoter Pu of the Pseudomonas putida TOL plasmid pWW0 were replaced by various DNA segments of the same size recruited from PnifH sigma54 promoter variants known to have various degrees of efficacy and affinity for sigma54-RNA polymerase (RNAP). In order to have an accurate comparison of the output in vivo of each of the hybrids, the resulting promoters were recombined at the same location of the chromosome of P. putida KT2442 with a tailored vector system. The promoters included the upstream activation sequence (UAS) for the cognate regulator of the TOL system (XylR) fused to the -12/-24 region of the wild-type PnifH and its higher sigma54-RNAP affinity variants PnifH049 and PnifH319. As a control, the downstream region of the glnAp2 promoter (lacking integration host factor) was fused to the XylR UAS as well. When the induction patterns of the corresponding lacZ fusion strains were compared in vivo, we observed that promoters bearing the RNAP binding site of PnifH049 and PnifH319 were not silenced during exponential growth, as is distinctly the case for the wild-type Pu promoter or for the Pu-PnifH variant. Taken together, our results indicate that the promoter sequence(s) spanning the -12/-24 region of Pu dictates the coupling of promoter output to growth conditions.     

Genome structure of Halobacterium halobium: plasmid dynamics in gas vacuole deficient mutants

Halobacterium halobium contains two gas vacuole protein genes that are located in plasmid pHH1 (p-vac) and in the chromosomal DNA (c-vac). The mutation frequency for these genes is different: the constitutively expressed p-vac gene is mutated with a frequency of 10(-2), while the chromosomal gene expressed in the stationary phase of growth is mutated with a frequency of 10(-5). The difference in the mutation susceptibility is due to the dynamics of plasmid pHH1. p-vac gene mutations are caused (i) by the integration of an insertion element or (ii) by a deletion event encompassing the p-vac gene region. In contrast, c-vac mutants analyzed to date incurred neither insertion elements nor deletions. Deletion events within pHH1 occur at high frequencies during the development of a H. halobium culture. The investigation of the fusion regions resulting from deletion events indicates that insertion elements are involved. The analysis of pHH1 deletion variants led to a 4 kilobase pair DNA region containing the origin of replication of the pHH1 plasmid.     

Multiple promoters control the regulation of the Pseudomonas aeruginosa regA gene

Expression studies utilizing the regA promoters, fused in tandem or separately to promoterless reporter genes, indicated that regA is transcribed from two promoters (P1 and P2). Both promoters can act independently. Expression from the P1 promoter is not affected by the iron content of the medium. Expression from the P2 promoter is tightly regulated by iron.