Construction of chimaeric promoter regions by exchange of the upstream regulatory sequences from fdhF and nif genes

Hybrid 5' regulatory regions were constructed in which the upstream activator sequence (UAS) and promoter of various nif genes were exchanged with the upstream regulatory sequence (URS) of the fdhF gene from Escherichia coli. They were analysed for their regulatory response under different growth conditions with the aid of fdhF'-'lacZ or nif'-'lacZ fusions. Placement of the UAS from the Bradyrhizobium japonicum nifH gene in front of the spacer (DNA region between URS and promoter) plus promoter from fdhF renders fdhF expression activatable by the Klebsiella pneumoniae NIFA protein, both under aerobic and anaerobic conditions. This excludes the possibility that the spacer of the fdhF5' flanking region contains a site recognized by a putative oxygen- or nitrate-responsive repressor. There was also considerable activation by NIFA of fdhF expression in a construct lacking the nifH UAS but containing the fdhF spacer plus promoter. Further experimental evidence suggests that this reflects a direct interaction between NIFA and RNA polymerase at the ntrA-dependent promoter. A second set of hybrid constructs in which the URS from fdhF (E. coli) was placed in front of the nifD spacer plus promoter from B. japonicum or in front of the K. pneumoniae nifH, nifU, nifB spacers and promoters, delivered inactive constructs in the case of the nifD, nifU and nifB genes. However, a nifH'-'lacZ fusion preceded by its own spacer and promoter plus the foreign fdhF URS displayed all the regulatory characteristics of fdhF expression, i.e. anaerobic induction with formate and repression by oxygen and nitrate. Although it is not known why only one out of the four nif promoters could be activated by the fdhF URS, this result nevertheless demonstrates that the various regulatory stimuli affecting expression of fdhF in E. coli have their target at the upstream regulatory sequence.     

prrA, a putative response regulator involved in oxygen regulation of photosynthesis gene expression in Rhodobacter sphaeroides.

A new locus, prrA, involved in the regulation of photosynthesis gene expression in response to oxygen, has been identified in Rhodobacter sphaeroides. Inactivation of prrA results in the absence of photosynthetic spectral complexes. The prrA gene product has strong homology to response regulators associated with signal transduction in other prokaryotes. When prrA is present in multiple copies, cells produce light-harvesting complexes under aerobic growth conditions, suggesting that prrA affects photosynthesis gene expression positively in response to oxygen deprivation. Analysis of the expression of puc::lacZ fusions in wild-type and PrrA- cells revealed a substantial decrease in LacZ expression in the absence of prrA under all conditions of growth, especially when cells were grown anaerobically in the dark in the presence of dimethyl sulfoxide. Northern (RNA) and slot blot hybridizations confirmed the beta-galactoside results for puc and revealed additional positive regulation of puf, puhA, and cycA by PrrA. The effect of truncated PrrA on photosynthesis gene expression in the presence of low oxygen levels can be explained by assuming that PrrA may be effective as a multimer. PrrA was found to act on the downstream regulatory sequences (J. K. Lee and S. Kaplan, J. Bacteriol. 174:1146-1157, 1992) of the puc operon regulatory region. Finally, two spontaneous prrA mutations that abolish prrA function by changing amino acids in the amino-terminal domain of the protein were isolated.     

Identification of an upstream repressor site controlling the expression of an anaerobic gene (ANB1) in Saccharomyces cerevisiae

The Saccharomyces cerevisiae anaerobic gene (ANB1) is negatively regulated both by oxygen and heme. A 299-base pair-long fragment from the 5'-flanking region of the ANB1 gene was found to confer oxygen-mediated negative regulation to an heterologous CYC1-LacZ hybrid gene. Studies with deletions of predefined length in this fragment demonstrated the presence of separate elements that comprise an upstream promoter that is active in the absence or presence of oxygen, and an upstream repressor site (URS) which confers strong repression upon the promoter element when oxygen is present. The promoter element is located 5' to the URS in the ANB1 gene. Mixed oligonucleotide-directed mutagenesis was used to obtain nucleotide substitutions in the URS which partially or completely inactivated this sequence without affecting the promoter activity. The URS region has three short direct repeats which seem to be important for function, as nucleotide substitutions within the repeats and not outside them, inactivated URS function. A model to explain the negative regulation of the ANB1 gene by oxygen and heme is proposed.     

Molecular genetic analysis suggesting interactions between AppA and PpsR in regulation of photosynthesis gene expression in Rhodobacter sphaeroides 2.4.1.

The AppA protein plays an essential regulatory role in development of the photosynthetic apparatus in the anoxygenic phototrophic bacterium Rhodobacter sphaeroides 2.4.1 (M. Gomelsky and S. Kaplan, J. Bacteriol. 177:4609-4618, 1995). To gain additional insight into both the role and site of action of AppA in the regulatory network governing photosynthesis gene expression, we investigated the relationships between AppA and other known regulators of photosynthesis gene expression. We determined that AppA is dispensable for development of the photosynthetic apparatus in a ppsR null background, where PpsR is an aerobic repressor of genes involved in photopigment biosynthesis and puc operon expression. Moreover, all suppressors of an appA null mutation thus far isolated, showing improved photosynthetic growth, were found to contain mutations in the ppsR gene. Because ppsR gene expression in R. sphaeroides 2.4.1 appears to be largely independent of growth conditions, we suggest that regulation of repressor activity occurs predominately at the protein level. We have also found that PpsR functions as a repressor not only under aerobic but under anaerobic photosynthetic conditions and thereby is involved in regulating the abundance of the light harvesting complex II, depending on light intensity. It seems likely therefore, that PpsR responds to an integral signal (e.g., changes in redox potential) produced either by changes in oxygen tension or light intensity. The profile of the isolated suppressor mutations in PpsR is in accord with this proposition. We propose that AppA may be involved in a redox-dependent modulation of PpsR repressor activity.     

Three classes of Escherichia coli mutants selected for aerobic expression of fumarate reductase

Fumarate reductase (encoded by frd) and succinate dehydrogenase (encoded by sdh) of Escherichia coli are both known to catalyze the interconversion of fumarate and succinate. Fumarate reductase, however, is not inducible aerobically and therefore cannot participate in the dehydrogenation of succinate. Three classes of suppressor mutants, classified as frd oxygen-resistant [frd(Oxr)], constitutive [frd(Con)], and gene amplification [frd(Amp)] mutants, were selected from an sdh strain as pseudorevertants that regained the partial ability to grow aerobically on succinate. All contained increased aerobic levels of fumarate reductase activity. In frd(Oxr) mutants expression of the operon showed increased resistance to aerobic repression. Under anaerobic conditions expression of the operon became less dependent on the fnr+ gene product, a pleiotropic activator protein for genes encoding anaerobic respiratory enzymes. Exogenous fumarate, however, was still required for full induction, and repression by nitrate was undiminished. Thus, aerobic repression and anaerobic nitrate repression appear to involve separate mechanisms. In frd(Con) mutants expression of the operon became highly resistant to aerobic repression. Under anaerobic conditions expression of the operon no longer required the fnr+ gene product or exogenous fumarate and became immune to nitrate repression. In partial diploids bearing an frd(Oxr) or an frd(Con) allele and phi(frd+-lac) there was no mutual regulatory influence between the two genetic loci. Thus, the frd mutations act in cis and hence are probably in the promoter region. In frd(Amp) mutants the frd locus was amplified without significant alteration in the pattern of regulation.     

Identification and sequence analysis of the gene encoding the transcriptional activator of the formate hydrogenlyase system of Escherichia coli

Through complementation of a trans-acting regulatory mutation a gene has been cloned whose product is required for the formate induction of the anaerobic expression of the formate hydrogenlyase structural genes. By restriction analysis, and from the size of the encoded protein, the gene could be identified as being equivalent to fhlA described by Sankar et al. (1988). The nucleotide sequence of the fhlA gene was determined and it was shown to code for a protein with a calculated Mr of 78,467. Analysis of the derived amino acid sequence showed that the carboxy-terminal domain of FHLA shares considerable sequence similarity with NIFA and NTRC, which are the 'regulators' of two-component regulatory systems. Carboxy-terminal truncation of, and introduction of amino-terminal deletions in, the fhlA gene delivered inactive gene products. When overexpressed, FHLA mediates activation of expression of the formate dehydrogenase and hydrogenase structural genes in the presence of formate also under aerobic growth conditions. FHLA appears to bind to the upstream regulatory sequence (URS) in the 5' flanking region of the fdhF gene since activation of fdhF expression was dependent on the presence of the URS.     

Involvement of ArcA and Fnr in expression of Escherichia coli thiol peroxidase gene

To explore the oxygen response regulators involved in thiol peroxidase gene (tpx) expression in Escherichia coli, we constructed a single-copy tpx-lacZ operon fusion and monitored tpx-lacZ expression in various genetic backgrounds. Expression of the tpx-lacZ fusion was increased 4-fold by aerobic growth. Anaerobic expression of tpx-lacZ in either (delta)arcA or delta(fnr) strains was 2.5-fold depressed compared with that of the wild-type strain. The results of immunoblotting experiments also demonstrated that ArcA and Fnr regulatory proteins repressed thiol peroxidase gene expression during anaerobic growth. Inspection of the tpx promoter region revealed putative binding sites for ArcA and Fnr. It thus appears that ArcA and Fnr function as repressors by blocking the binding of RNA polymerase to the tpx promoter in E. coli under anaerobic growth conditions.