Use of an in vivo titration method to study a global regulator: effect of varying Lrp levels on expression of gltBDF in Escherichia coli.

Most studies of global regulatory proteins are performed in vitro or involve phenotypic comparisons between wild-type and mutant strains. We report the use of strains in which the gene for the leucine-responsive regulatory protein (lrp) is transcribed from isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible promoters for the purpose of continuously varying the in vivo concentration of Lrp. To obtain a broad range of Lrp concentrations, strains were employed that contained the lrp fusion either in the chromosome (I. C. Blomfield, P. J. Calie, K. J. Eberhardt, M. S. McClain, and B. I. Eisenstein, J. Bacteriol. 175:27-36, 1993) or on a multicopy plasmid. Western blot (immunoblot) analysis with polyclonal antiserum to Lrp confirmed that Lrp levels could be varied more than 70-fold by growing the strains in glucose minimal 3-(N-morpholino)propanesulfonic acid (MOPS) medium containing different amounts of IPTG. Expression of an Lrp-regulated gltB::lacZ operon fusion was measured over this range of Lrp concentrations. beta-Galactosidase activity rose with increasing Lrp levels up to the level of Lrp found in wild-type strains, at which point expression is maximal. The presence of leucine in the medium increased the level of Lrp necessary to achieve half-maximal expression of the gltB::lacZ fusion, as predicted by earlier in vitro studies (B. R. Ernsting, J. W. Denninger, R. M. Blumenthal, and R. G. Matthews, J. Bacteriol. 175:7160-7169, 1993). Interestingly, levels of Lrp greater than those in wild-type cells interfered with activation of gltB::lacZ expression. The growth rate of cultures correlated with the intracellular Lrp concentration: levels of Lrp either lower or higher than wild-type levels resulted in significantly slower growth rates. Thus, the level of Lrp in the cell appears to be optimal for rapid growth in minimal medium, and the gltBDF control region is designed to give maximal expression at this Lrp level.     

Sequence elements determining ampC promoter strength in E. coli

A number of spontaneous up-promoter mutations have been isolated in the ampC beta-lactamase gene of Escherichia coli. The mutants were analyzed by DNA sequencing, and the level of ampC gene expression was determined. Six mutants with a 21-fold increase in promoter strength compared with the wild-type were mutated in the -35 promoter region from TTGTCA to the consensus sequence TTGACA . The -10 region sequence TACAAT was mutated to the consensus sequence TATAAT in three mutants exhibiting an ampC promoter seven times stronger than the wild-type. We have previously described a 1-bp insertion mutant ( Jaurin et al., 1981) that changes the inter-region distance to the consensus 17 bp. Thus, all the up-mutations found in the ampC promoter represent corrections of the three major discrepancies between the ampC promoter and the consensus E. coli promoter. We conclude that the three consensus elements of E. coli promoters, the -35 and -10 regions and an optimal inter-region distance of 17 bp, are the main elements determining the promoter strength.     

Synthetic promoter libraries for Corynebacterium glutamicum

The ability to modulate gene expression is an important genetic tool in systems biology and biotechnology. Here, we demonstrate that a previously published easy and fast PCR-based method for modulating gene expression in lactic acid bacteria is also applicable to Corynebacterium glutamicum. We constructed constitutive promoter libraries based on various combinations of a previously reported C. glutamicum -10 consensus sequence (gngnTA(c/t)aaTgg) and the Escherichia coli -35 consensus, either with or without an AT-rich region upstream. A promoter library based on consensus sequences frequently found in low-GC Gram-positive microorganisms was also included. The strongest promoters were found in the library with a -35 region and a C. glutamicum -10 consensus, and this library also represents the largest activity span. Using the alternative -10 consensus TATAAT, which can be found in many other prokaryotes, resulted in a weaker but still useful promoter library. The upstream AT-rich region did not appear to affect promoter strength in C. glutamicum. In addition to the constitutive promoters, a synthetic inducible promoter library, based on the E. coli lac-promoter, was constructed by randomizing the 17-bp spacer between -35 and -10 consensus sequences and the sequences surrounding these. The inducible promoter library was shown to result in β-galactosidase activities ranging from 284 to 1,665 Miller units when induced by IPTG, and the induction fold ranged from 7–59. We find that the synthetic promoter library (SPL) technology is convenient for modulating gene expression in C. glutamicum and should have many future applications, within basic research as well as for optimizing industrial production organisms.     

Manifestation of the Kanagawa phenomenon, the virulence-associated phenotype, of Vibrio parahaemolyticus depends on a particular single base change in the promoter of the thermostable direct haemolysin gene

Thermostable direct haemolysin of Vibrio parahaemolyticus has been shown to be a major virulence factor. The Kanagawa phenomenon (KP), haemolysis induced by this haemolysin on a special blood agar medium, is strongly associated with clinical strains. We have been studying the expressions of various tdh genes encoding this haemolysin to elucidate the significance of the tdh genes possessed by KP-negative strains isolated from patients. We examined the importance of the promoter sequence variation for expression level of the tdh gene in this study. Only the tdh 2 gene, one of the two tdh genes ( tdh 1 and tdh 2) present in a KP-positive strain, was previously shown to be responsible for the haemolytic activity of the KP-positive strain. The tdh 1– and tdh 2– lacZ fusions were used to determine and analyse the promoter sequence by primer extension and site-directed mutagenesis methods. Two bases (positions −24 and −34) within the determined tdh 2 promoter sequence were shown to be mostly responsible for the difference in the promoter strength between the tdh 2 and tdh 1 genes both in Escherichia coli and in V. parahaemolyticus backgrounds. Representative tdh promoters of KP-negative strains are close to the tdh 2 promoter; they differ at position −34 but have the same base at position −24 as the tdh 2 promoter. We demonstrated that base substitution of the tdh promoters of KP-negative strains only at position −34 is sufficient to increase the expression of these genes to the KP-positive level. Therefore, the tdh genes of KP-negative strains are considered to be potentially important because they can generate a KP-positive subclone by a point mutation in their promoters.     

The amino acid sequence of Lrp is highly conserved in four enteric microorganisms.

Lrp (leucine-responsive regulatory protein) is a global regulator of metabolism in Escherichia coli (J. M. Calvo and R. G. Matthews, Microbiol. Rev. 58:466-490, 1994). The lrp genes from three other enteric microorganisms, Enterobacter aerogenes, Klebsiella aerogenes, and Salmonella typhimurium, were cloned and sequenced. An analysis of these sequences and of the previously determined sequence from E. coli indicated that the vast majority of changes were synonymous rather than nonsynonymous changes. Nucleotide changes occurred at 89 of 492 positions but resulted in amino acid changes at only 2 of 164 positions. This analysis suggests that the Lrp amino acid sequence is highly adapted for function and that almost all amino acid changes lead to a protein that functions less well than the wild-type protein.     

The structural basis of the high in vivo strength of the rRNA P2 promoter of Escherichia coli

Ribosomal RNA promoters of Escherichia coli are probably the strongest promoters in vivo and they can be used on plasmid vectors to express protein-coding sequences at a high rate. In fact, the P2 promoter of the rrnB gene is stronger (in vivo) than the tac promoter, which has a perfect consensus sequence. Conversion of the rrnB P2 promoter sequence to consensus significantly increases in vivo promoter strength. The removal of four nucleotides downstream of the -10 region also increases the strength of this promoter. On the other hand, shifting of the A + T-rich region upstream of this promoter by an 11-bp insertion drastically decreases in vivo activity. It is concluded that the two functionally important hexanucleotide sequences, -35 and -10, are necessary but not sufficient factors for the optimalization of in vivo promoter strength.