Molecular Biotechnology: Principles and Applications - Glick B., Pasternak J. 2002

Molecular Biotechnology of Microbial Systems
Plant Growth-Promoting Bacteria
Hydrogenase

An undesirable side reaction of Nitrogen Fixation is the reduction of H⁺ to H₂ (hydrogen gas) by Nitrogenase, during which energy (in the form of ATP) is consumed to produce hydrogen, which ultimately simply escapes. As a result, only 40 to 60% of the total electron flow passing through the nitrogenase complex is transferred to N₂, which significantly reduces the efficiency of the nitrogen fixation process. In principle, if H₂ could be converted back to H⁺, energy losses would be lower, and the nitrogen fixation process would become more efficient. However, it is impossible to eliminate this side reaction directly because it is determined by the specific Chemical Structure of the nitrogenase Active Site; any attempt to block it by altering the enzyme's structure would inevitably lead to a decrease in nitrogenase activity.

Hydrogen METABOLISM

In the mid-1970s, it was shown that certain strains of Bradyrhizobium japonicum can grow under microaerophilic conditions (at low oxygen concentrations) using hydrogen as an energy source. To do this, they synthesize the enzyme hydrogenase, which is capable of converting atmospheric H₂ to H⁺ (Fig. 14.5). To test whether these strains could influence soybean growth, plants were inoculated with hydrogenase-synthesizing (Hup⁺) B. japonicum. The plants produced greater biomass and assimilated more nitrogen than those inoculated with Hup^- strains, despite the higher level of nitrogenase activity in the latter (Table 14.3). Based on the results of this and similar experiments, it was concluded that the presence of a hydrogen uptake system in symbiotic diazotrophs such as B. japonicum enhances their ability to stimulate plant growth, apparently as a result of capturing and recycling hydrogen gas produced in the nodules by nitrogenase (Fig. 14.5).

Class="center">

Fig. 14.5. Recycling of hydrogen gas, a side product of nitrogen fixation. Nitrogenase catalyzes The formation of hydrogen using the energy of ATP Hydrolysis, while hydrogenase catalyzes its utilization.

Despite the benefits that a plant derives from Symbiosis with a diazotrophic microorganism possessing a hydrogen recycling system, such a system involving Rhizobium strains is rare under natural conditions. According to the test results presented in Table 14.4, most of the surveyed natural strains of Rhizobium and Bradyrhizobium exhibit the Hup^- phenotype. Several strains of each of these species were tested, and for B. japonicum, there were more than 1400. Clearly, as soon as the genetic Nature of the hydrogenase system is studied in sufficient detail and the corresponding genes are identified, commercial Hup^- Rhizobium strains will be the primary candidates for conversion into strains with the Hup⁺ phenotype.

Modification of hydrogenase genes

Much effort has been devoted over the last 20 years to studying the hydrogenases of both diazotrophic and non-diazotrophic microorganisms, yet the STRUCTURE AND Functions of these Enzymes are still not fully understood. Many microorganisms synthesize more than one hydrogenase, and they often consist of more than one polypeptide chain. Some hydrogenases only uptake atmospheric hydrogen, while others can also synthesize it under appropriate conditions. It follows from all this that simply introducing a Gene for one of the hydrogenases into The Genome of a Hup^- Rhizobium strain is unlikely to be sufficient to convert it to Hup⁺. The introduced gene(s) must encode all subunits of the enzyme, which must be compatible with The electron transport System of the host Organism.

Table 14.3. Relative activities of nitrogenase and hydrogenase and the ability of B. japonicum Hup⁺ (SR) and three Hup^- mutants (SR1, SR2, and SR3) to stimulate plant growth^{1) 2)}

¹⁾ From Albrecht et al. Science 203: 1255—1257, 1979.

²⁾ Nitrogenase activity was assayed by the time-dependent reduction of acetylene to Ethylene; hydrogenase activity was determined using a hydrogen electrode. Plant dry weight includes the weight of shoots and roots. Nitrogen content was calculated as the fraction of dry weight attributable to nitrogen. All values are normalized relative to those of the parental strain.

Table 14.4. Proportion of natural Rhizobium and Bradyrhizobium strains possessing a hydrogen uptake system (Hup⁺)¹⁾

¹⁾ From Evans et al. Annu. Rev. Microbiol. 41: 335—361, 1987

The most common strategy for isolating hydrogenase genes is genetic complementation. The first of these genes, the gene for the membrane-bound hydrogenase of E. coli, was identified by complementation in a mutant E. coli unable to synthesize active hydrogenase, using a wild-type E. coli DNA clone bank constructed with plasmid pBR322. The mutant containing the defective membrane-bound hydrogenase failed to grow on minimal medium in the presence of formate, while The activity of the soluble hydrogenase remained unaffected. Transformed Cells capable of growing on this medium were screened for the presence of active hydrogenase. A transformant in which hydrogenase activity was restored to the wild-type level contained a plasmid encoding a protein with a Molecular Weight of approximately 60,000 Da, which corresponds to the molecular

weight of one of the subunits of the membrane-bound hydrogenase of E. coli. Further studies showed that the hydrogenase system of E. coli comprises multiple genes.

Subsequently, the hydrogenase (hup) genes of B. japonicum were identified using a wild-type DNA clone bank constructed with the broad-host-range cosmid vector pLAFR1, and Hup^- mutants of B. japonicum. The presence of uptake hydrogenase in the transformed Hup^- mutant cells was determined by the ability of the active enzyme to reduce methylene blue under a hydrogen atmosphere. A more detailed study showed that the B. japonicum hup genes form at least two, and possibly three, operons spanning approximately 15 kb, with the hup genes of Rhizobium leguminosarum being similar to those of B. japonicum in both nucleotide sequence and Introduction/29.html">Gene Organization. Thus, the identified B. japonicum hup genes can be used as Hybridization probes to screen for homologous genes from an R. leguminosarum clone bank.

Following the identification of the R. leguminosarum hup genes, despite The complexity of the hydrogenase system, it was successfully "transferred" from a Hup⁺ strain of R. leguminosarum into a Hup^- strain (Table 14.5). Pea plants nodulated by the recombinant Hup⁺ strain of R. leguminosarum grew faster and contained more nitrogen than plants inoculated with the Hup^- strain (Table 14.5).

Although research on hydrogenase genes has not generated as much interest as studies on nif genes, it has nevertheless convincingly demonstrated the feasibility of using Genetic Engineering techniques to enhance the plant growth-promoting ability of diazotrophic microorganisms. It remains to be tested whether the introduction of hup genes into the genomes of other diazotrophic microorganisms (both free-living and symbiotic) will produce a similar effect.

The hydrogenase system has Applications beyond increasing the efficiency of nitrogen fixation. For instance, purified hydrogenase can be used for solar energy conversion and storage; regeneration of Cofactors involved in industrial enzymatic processes; Synthesis of specific chemical compounds requiring H₂ as a reducing agent; removal of tritium from Water used to cool nuclear reactors; synthesis of H₂ from organic waste; and The production of hydrogen-oxygen fuel cells. However, although more than a dozen hydrogenase genes have been identified and characterized, none have yet been used for the large-scale synthesis of these enzymes.

Table 14.5. Plant Growth and nitrogen assimilation after introduction of hup genes into a Hup^- strain of R. leguminosarum¹⁾

¹⁾ From Brewin, Johnston, U.S. patent 4,567,146, January 1986.