Anaerobic Gene Expression Research Articles

Identification and molecular characterization of a transcriptional regulator from Pseudomonas aeruginosa PAO1 exhibiting structural and functional similarity to the FNR protein of Escherichia coli

A gene library of chromosomal DNA from Pseudomonas aeruginosa contained a DNA fragment which was able to restore anaerobic growth to an Escherichia coli fnr deletion mutant on glycerol/nitrate medium. The cloned gene (termed anr) was sequenced and shown to encode a protein of 244 amino acids with a calculated molecular weight of 27,129. The deduced amino acid sequence of the anr gene product showed considerable similarity to the FNR protein from E. coli. Expression of the anr gene in a T7 promoter/polymerase system identified ANR as a 31 kDa protein. Transcriptional analysis of the anr gene showed that it is monocistronic but apparently lacks the equivalent sites for negative autoregulation which have been shown to be present in the promoter region of the E. coli fnr gene. The ANR protein was shown to activate transcription of the pfl gene in E. coli in response to anaerobiosis, as well as being able to restore the activity of three anaerobically inducible enzymes. A P. aeruginosa mutant incapable of growing anaerobically with nitrate or on arginine was fully complemented by the anr gene, indicating that it probably has a function in controlling anaerobic gene expression in Pseudomonas. Further corroboration for this assumption was provided by S1 nuclease analysis of transcription of the multiple promoters of the E. coli pfl operon in P. aeruginosa. Transcription was induced by oxygen limitation and was completely ANR-dependent in both aerobic and anaerobic cells. Removal of the upstream regulatory sequence of the pfl operon, which includes the sequences required for FNR-dependent regulation in E. coli, removed ANR-dependent transcriptional control of the remaining pfl promoters, irrespective of the cellular oxygen status. These results imply that the mechanisms by which ANR and FNR regulate transcription are fundamentally similar.

Mutations in fnr that alter anaerobic regulation of electron transport-associated genes in Escherichia coli.

The fnr gene product, FNR, is a global regulator of anaerobic gene expression in Escherichia coli. When E. coli is switched from aerobic to anaerobic growth conditions, cytochrome o (cyoABCDE) and d oxidase (cydAB) genes are repressed and the anaerobic terminal reductase genes, including nitrate (narGHJI), dimethyl sulfoxide/trimethylamine (dmsABC), and fumarate (frdABCD) reductase, are induced. To determine if certain amino acid residues are essential for FNR to function in this regulatory process, site-directed mutations were introduced into the fnr gene. The resulting mutant proteins were assayed in vivo for their ability to either activate dmsA'-'lacZ and frdA'-'lacZ gene expression, or repress expression of a cyoA'-'lacZ gene fusion. The fnr mutants were grouped into four classes. Class I exhibited a severe decrease in the ability to either activate or repress fnr-dependent gene expression. Mutations in four of the five cysteine residues in the FNR protein were in this class. The sole exception was an FNR Cys16----Ser "mutant" that exhibited normal activity. Class II mutations caused a mild reduction in FNR-dependent activation or repression while Class III mutations conferred a modest increase in the ability of the FNR protein to activate gene expression under aerobic conditions (i.e. FNR*). Finally, Class IV mutations lowered the modest aerobic FNR transcriptional activation function proportionally more than the anaerobic FNR activity. These findings identify an essential role for the NH2 terminus of the FNR protein in its various activities in anaerobic gene regulation.

Induction of anaerobic gene expression in Rhodobacter capsulatus is not accompanied by a local change in chromosomal supercoiling as measured by a novel assay.

In the photosynthetic bacterium Rhodobacter capsulatus, the enzyme DNA gyrase has been implicated in the expression of genes for anaerobic metabolic processes such as nitrogen fixation and photosynthesis. To assess the involvement of supercoiling in anaerobic gene expression, we have developed an assay to detect in vivo changes in superhelicity of small regions of the bacterial chromosome. Our method is based on the preferential intercalaction of psoralen into supercoiled versus relaxed DNA, and we have demonstrated the sensitivity of the assay in vivo on chromosomal regions from 2 to 10 kilobases in size. In experiments with inhibitors of gyrase, the reactivity of individual chromosomal fragments to psoralen decreases by a factor of 1.8 compared with DNA from control cultures. We used our assay to determine whether there is a change in superhelicity near the genes coding for essential proteins for photosynthesis upon a shift from respiratory to anaerobic photosynthetic growth. For comparison, we also examined a restriction fragment containing the fbc operon, which codes for the subunits of cytochrome bc1, a membrane-bound electron transport complex utilized during both aerobic and anaerobic photosynthetic growth. During this shift in growth conditions, the puf and puh mRNAs, coding for structural polypeptides of the photosynthetic apparatus, underwent a six- to eightfold induction, while the amount of mRNA from the fbc locus remained constant. However, we detected no change in the superhelicity of either the genes for photosynthesis or those for the bc1 complex during this metabolic transition. Our data thus do not support a model in which stable changes in chromosomal superhelicity regulate anaerobic gene expression. We suggest instead that the requirement for DNA gyrase in the transcription of photosynthesis genes results from the requirement for a swivel near heavily transcribed regions of the chromosome.

Glucose repression of anaerobic genes of Escherichia coli is independent of cyclic AMP

Several anaerobically regulated gene fusions were examined for the effects of catabolite repression. Glucose repressed the expression of most of the genes represented in our collection of anaerobically induced fusions. However, addition of cyclic AMP did not reverse the effects of glucose. Furthermore, introduction of cya and crp mutations into selected anaerobically induced fusion strains did not reduce anaerobic gene expression as expected from the known mechanism of aerobic catabolite repression. In fact, in different fusion strains, cya or crp mutations caused from 2 to 20-fold increases in gene expression. Although glucose repression occurs anaerobically its mechanism would appear to be quite different from that under aerobic conditions.

Glucose repression of anaerobic genes ofEscherichia coliis independent of cyclic AMP

Several anaerobically regulated gene fusions were examined for the effects of catabolite repression. Glucose repressed the expression of most of the genes represented in our collection of anaerobically induced fusions. However, addition of cyclic AMP did not reverse the effects of glucose. Furthermore, introduction of cya and crp mutations into selected anaerobically induced fusion strains did not reduce anaerobic gene expression as expected from the known mechanism of aerobic catabolite repression. In fact, in different fusion strains, cya or crp mutations caused from 2 to 20-fold increases in gene expression. Although glucose repression occurs anaerobically its mechanism would appear to be quite different from that under aerobic conditions.

Novel regulatory loci controlling oxygen- and pH-regulated gene expression in Salmonella typhimurium.

Three new loci were discovered, each of which participates in the regulation of anaerobic gene expression. The regulatory gene earA negatively regulates the expression of the anaerobiosis-inducible gene aniG as well as that of at least three other genes, as determined by two-dimensional polyacrylamide gel electrophoresis. The earA locus maps at 86 min. The expression of aniG was also shown to be controlled by changes in external pH under aerobic and anaerobic conditions. Maximal expression was observed under anaerobic conditions at an external pH of 6.0. Significant transcriptional activity was also observed under aerobic conditions at pH 6.0. This was in contrast to hyd, whose expression was dependent upon anaerobiosis and varied with external pH. The pH dependence disappeared under fully aerobic conditions. Mutations in earA had no effect upon hyd expression. The two other regulators identified were oxrF, which controls aniH, and oxrG, which, in concert with oxrA and oxrB, controls aniC and aniI. The oxrG locus was mapped to 88 min and appears to code for a positive regulator. Various oxr mutants were subjected to two-dimensional polyacrylamide electrophoretic analysis of anaerobiosis-inducible proteins. Several pathways of anaerobic control were observed by means of these techniques.

Two genetically distinct pathways for transcriptional regulation of anaerobic gene expression in Salmonella typhimurium.

Expression of the tripeptide permease gene tppB is anaerobically induced. This induction is independent of the fnr (oxrA) gene product, which is known to be required for the anaerobic induction of several respiratory enzymes. We isolated, characterized, and mapped mutations in two genes, oxrC and tppR, which prevent the anaerobic induction of tppB expression. Mutations in oxrC were highly pleiotropic, preventing the anaerobic expression of the formate dehydrogenase component of formate hydrogen lyase (fhl), a tripeptidase (pepT), and two of the three known hydrogenase isoenzymes (hydrogenases 1 and 3). On the other hand, expression of nitrate reductase, fumarate reductase, and a number of other fnr (oxrA)-dependent enzymes was not affected by mutations in oxrC. Thus, there appeared to be at least two distinct classes of anaerobically induced genes, those which required fnr for their expression and those which required oxrC. It seems that fnr-dependent enzymes perform primarily respiratory functions, whereas oxrC-dependent enzymes served fermentative or biosynthetic roles. We found the primary defect of oxrC mutants to be a deficiency in phosphoglucose isomerase activity, implying that a product of glycolysis functions as an anaerobic regulatory signal. Mutations in tppR were specific for tppB and did not affect expression of other oxrC-dependent genes. However, tppR did exhibit phenotypes other than the regulation of tppB. Both oxrC and tppR mutants were hypersensitive to the toxic NAD analog 6-aminonicotinic acid. This suggests that oxrC and tppR may play a role in the regulation of NAD biosynthesis or, alternatively, that NAD or a related nucleotide serves as the anaerobic signal for oxrC-dependent enzymes.

Cyclic AMP and anaerobic gene expression in E. coli

The expression of the fumarate reductase system of Escherichia coli is completely dependent on the presence of adenylate cyclase or cyclic AMP, but the cyclic AMP receptor protein (CRP) is not required. This suggests that cyclic AMP may function as an effector for a second gene activator protein, possibly Fnr, and thus form part of a redox-sensitive regulatory mechanism controlling the expression of anaerobic respiratory functions.