Common cis-acting region responsible for transcriptional regulation of Bradyrhizobium japonicum hydrogenase by nickel, oxygen, and hydrogen

Abstract

Bradyrhizobiumjaponicum expresses hydrogenase in microaerophilic free-living conditions in the presence of nickel. Plasmid-borne hup-lacZ transcriptional fusion constructs were used to study the regulation of the hydrogenase gene. The hydrogenase gene was transcriptionally induced under microaerobic conditions (0.1 to 3.0% partial pressure 02). The hydrogenase gene was not transcribed or was poorly transcribed in strictly anaerobic conditions or conditions above 3.0% 02. Hydrogen gas at levels as low as 0.1% partial pressure induced hydrogenase transcription, and a high level of transcription was maintained up to at least 10% H2 concentration. No transcription was observed in the absence of H2. Hydrogenase was regulated by H2, 02, and Ni when the 5'-upstream sequence was pared down to include base number -168. However, when the upstream sequence was pared down to base number -118, the regulatory response to 02, H2, and Ni levels was negated. Thus, a common cis-acting regulatory region localized within 50 bp is critical for the regulation of hydrogenase by hydrogen, oxygen, and nickel. As a control, the B. japonicum hemA gene which codes for 8-aminolevulinic acid synthase was also fused to the promoterless lacZ gene, and its regulation was tested in the presence of various concentrations of 02 and H2. hemA-lacZ transcription was not dependent on levels of Ni, 02, or H2. Two different hup-lacZ fusions were tested in a Hup-background, strain JH47; these hup-lacZ constructs in JH47 demonstrated dependency on nickel, 02, and H2, indicating that the hydrogenase protein itself is not a sensor for regulation by 02, H2, or nickel.In soybean root nodules, the Bradyrhizobium japonicum bacterium undergoes a morphological transformation into N2-fixing bacteroids. Within the nodule, conditions are microaerobic because of an 02 diffusion barrier and 02-binding leghemoglobins; because of this, the 02 concentration at the bacteroid surface is estimated to be 11 nM (1). Many new proteins are expressed in the 02-limited nodule environment. In addition to those encoded by the nitrogen fixation genes, a membrane-bound H2-oxidizing hydrogenase enzyme that utilizes the H2 evolved by nitrogenase is expressed. The Ni-containing hydrogenase (3,32,33) activates H2 and sends the electrons through an energy-generating respiratory electron transport chain (reviewed in reference 27). Therefore, it is thought to be a component responsible for maintaining efficient N2 fixation in the nodules.Studying the regulation of bacteroid hydrogenase in response to environmental stimuli supplied to the root nodules is difficult if not physically impossible. Conditions have been worked out so that B. japonicum hydrogenase activity can be induced in the free-living form in an H2-and C02-containing atmosphere (16)(17)(18). It is within these controlled circumstances that most hydrogenase regulation studies have been done (16-19, 22, 26, 35, 36). (30) and 20 bases downstream of it. The transcription initiation site has previously been mapped (30). pBJ3-1 is ...

HoxA is a transcriptional regulator for expression of the hup structural genes in free‐living Bradyrhizobium japonicum

“…In free-living B. japonicum, hydrogenase activity is induced by hydrogen, low oxygen concentrations and trace amounts of nickel (Maier et al, 1978;Stults et al, 1986;van Berkum, 1987). The induced activity is caused by an increased expression of the hydrogenase structural genes (Kim et al, 1991). Our results demonstrate that HoxA is necessary to obtain increased hupSL gene expression upon induction in a microaerobic, hydrogen-containing atmosphere.…”

“…Thus the hoxA gene product must be involved in positive regulation of the expression of the hydrogenase structural genes in free-living conditions. A region upstream of the hup structural genes is necessary for transcriptional regulation of hupSL gene expression by hydrogen, nickel and oxygen (Kim et al, 1991). As the predicted amino acid sequence of HoxA is characteristic for DNA-binding transcriptional regulators (Van Soom et al, 1993b), protein binding at this region was studied by means of gel retardation.…”

“…This offers the advantage that the factors regulating the expression of the hup genes can be studied ex planta. To obtain hydrogenase activity in free-living B. japonicum cultures, the cells must be incubated in a microaerobic atmosphere in the presence of hydrogen and trace amounts of nickel (Maier et al, 1978;Stults et al, 1986;van Berkum, 1987;Kim et al, 1991). A region of 50 bp in the promoter region of the hupSL structural genes is necessary for transcriptional regulation of hupSL expression by oxygen, hydrogen and nickel (Kim et al, 1991;1993).…”

“…To obtain hydrogenase activity in free-living B. japonicum cultures, the cells must be incubated in a microaerobic atmosphere in the presence of hydrogen and trace amounts of nickel (Maier et al, 1978;Stults et al, 1986;van Berkum, 1987;Kim et al, 1991). A region of 50 bp in the promoter region of the hupSL structural genes is necessary for transcriptional regulation of hupSL expression by oxygen, hydrogen and nickel (Kim et al, 1991;1993). Besides these environmental prerequisites, expression of the hupSL genes is dependent on RpoN and stimulated by integration host factor (IHF) binding (Black and Maier, 1995).…”

The hupTUV operon is involved in negative control of hydrogenase synthesis in Rhodobacter capsulatus

“…In contrast, we have shown that in R. capsulatus, the HupV Ϫ or Hup(UV) Ϫ mutant expressed hydrogenase at a high level, especially in the absence of H 2 . Since B. japonicum requires microaerophilic conditions to synthesize hydrogenase (19), there may be additional superimposed controls in response to oxygen, either in R. capsulatus or in B. japonicum or in both, which may explain the differences observed in the two bacteria.…”