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

Molecular Biotechnology of Microbial Systems
Plant Growth-Promoting Bacteria
Nitrogenase

Interest in diazotrophs as biofertilizers was revived following The Development of Gene Isolation and modification techniques, which stimulated research into the biochemical and molecular biological MECHANISMS OF NITROGEN fixation. Scientists hoped that this research would lead to the creation of more efficient nitrogen-fixing microorganisms to boost crop yields, and some researchers even envisioned introducing bacterial Nitrogen Fixation genes directly into plants so they could fix nitrogen on their own. Although these overly ambitious plans have not yet been realized, the nitrogen fixation process has been studied in such detail that the Introduction/32.html">Genetic Engineering OF certain diazotrophs is now a much more realistic prospect.

Components

All known nitrogenases contain two oxygen-sensitive components: I and II. Component I is a complex of two α-subunits (each weighing approximately 50,000 Da), two β-subunits (each approximately 60,000 Da), 24 iron atoms, two molybdenum atoms, and an iron-molybdenum cofactor designated as FeMoCo (Fig. 14.1). Component II consists of two α-subunits (each approximately 32,000 Da) and an unknown number of iron atoms, and its α-subunits are not homologous to those in component I. Nitrogen fixation requires both components, a magnesium-ATP complex, and a source of reducing equivalents:

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Fig. 14.1. Proposed Structure OF THE iron-molybdenum cofactor bound to a nitrogen molecule (N₂).

In addition to nitrogen fixation, nitrogenase also catalyzes the reduction of acetylene gas to Ethylene:

Nitrogenase activity can be estimated by measuring The amount of synthesized ethylene using gas Chromatography. Assays can be performed on whole Cells in solution (Fig. 14.2), on Bacteria associated with plant roots, on crude Cell extracts, or on highly purified enzyme preparations. Component I catalyzes the actual reduction of N₂, while component II serves as an electron donor. Both are extremely sensitive to oxygen and are rapidly and irreversibly inactivated at high oxygen concentrations. The functioning of nitrogenase also depends on 15–20 accessory Proteins. Some of these play a role in transferring electrons to component II, as well as in The Biosynthesis of the iron-molybdenum cofactor.

Fig. 14.2. Determination of nitrogenase activity by the reduction of acetylene to ethylene. A. Nitrogenase-producing bacteria (either in culture or associated with plant roots) or a purified enzyme preparation (not shown) are placed in a sealed container under an acetylene atmosphere. B. Samples are periodically drawn from the container, and the amounts of acetylene and ethylene are measured by gas chromatography. Nitrogenase activity is proportional to the amount of ethylene formed.

Genetic Engineering of the Nitrogenase Gene Cluster

Nitrogen fixation is a highly complex process requiring the coordinated action of many different proteins. Therefore, it was unlikely that all the Genetic information required for nitrogen fixation would be contained within a single DNA fragment, or that this fragment could be isolated from The Genome of a diazotrophic microorganism and transferred into a non-diazotrophic Organism. Furthermore, the physiological conditions within the recipient organism must be suitable for the functioning of active nitrogenase. A more feasible approach to isolating nitrogen fixation genes (nif genes) was to identify and characterize clones from a wild-type DNA library that restore the nitrogen-fixing ability of various mutants of the organism. This method is known as genetic complementation.

The first nif genes identified by complementation were isolated from a clone library of the diazotrophic bacterium Klebsiella pneumoniae. This is a well-studied enterobacterium found in soil and Water, as well as in the human gut. The isolation scheme is as follows (Fig. 14.3).

1. K. pneumoniae cells are treated with a mutagen dose that results in approximately 0.1–1.0% survival. Some of the mutant cells, which are able to grow on minimal medium containing a source of bound nitrogen such as NH₄Cl, but not in the absence of bound nitrogen, likely carry a mutation in a nif gene; they are designated as Nif^-.

2. Using broad-host-range expression Plasmid Vectors, a clone library of wild-type (Nif⁺) K. pneumoniae chromosomal DNA is constructed and maintained in E. coli.

3. Conjugation is performed between Nif^- K. pneumoniae cells and E. coli cells harboring the clone library in plasmid vectors.

4. Transformed K. pneumoniae cells that have acquired the Nif⁺ phenotype are selected by plating them on minimal medium lacking a bound nitrogen source. Under these conditions, only Nif^- K. pneumoniae cells containing a plasmid that encodes the protein missing or non-functional in the Nif^- mutant will grow.

The plasmid DNA fragment that complements the chromosomal Nif^- mutation contains a nif gene, which can be characterized in detail and used to isolate other nif genes.

Two approaches were used to isolate other genes involved in nitrogen fixation. First, a K. pneumoniae clone library was used to Complement independently arising Nif^- mutants, thereby increasing the likelihood of isolating a different nif gene in each case. Second, the isolated nif genes were used as Hybridization probes to screen a K. pneumoniae chromosomal DNA library containing large inserts (7 to 10 kb), based on the premise that prokaryotic genes of a single biosynthetic pathway are typically clustered. As a result of comprehensive studies, the entire set of K. pneumoniae nif genes was identified and characterized. These genes are organized into a single cluster approximately 24 kb in length (Fig. 14.4), which contains seven separate operons encoding a total of 20 different proteins (Table 14.2). For active nitrogenase to be formed, all nif genes must be transcribed and translated simultaneously (under the regulatory control of the nifA and nifL genes). The NifA protein is a transcriptional activator of all nif operons except its own. It binds to a specific DNA sequence (5'-TGT-N₁₀-ACA-3') located in the promoter of each nif Operon. The NifA binding site is located approximately 80–150 NUCLEOTIDES upstream of each METABOLISM/31.html">Transcription initiation site. Prior to the initiation of transcription from the nif promoter, the DNA-bound NifA protein interacts with the specific transcription initiation factor σ⁵⁴. The NifL protein is a repressor. In the presence of either oxygen or bound nitrogen, it acts as a NifA antagonist, thereby inhibiting the transcription of all other nif genes.

Table 14.2. K. pneumoniae genes involved in Nitrogen Fixation and the proteins (or Functions) they encode

Fig. 14.3. Isolation of nif genes by genetic complementation. A DNA clone library from Nif⁺ cells is used to complement a Nif^- strain of K. pneumoniae. Transformed cells are selected based on their ability to grow on minimal medium lacking bound nitrogen.

Fig. 14.4. Organization of nif genes within the cluster and some of their encoded functions. Genes are designated by capital letters: the red arrow under each group of letters indicates a specific nif operon and its direction of transcription. Arrows extending from the gene designations show the involvement of some of these gene products in nitrogen fixation. F — flavodoxin, FO — pyruvate:flavodoxin oxidoreductase.

The Role of K. pneumoniae in the overall biological process of nitrogen fixation is not major. Therefore, to modify the nitrogen fixation process in soil bacteria, which are of great interest for promoting plant growth, nif genes from other sources have been cloned and characterized. In doing so, the nif genes of K. pneumoniae were used as hybridization probes to isolate the corresponding genes from clone libraries of other diazotrophic microorganisms. Most diazotrophs share a similar set of genes encoding the nitrogen-fixing apparatus, and the DNA sequences of these genes differ very little among various organisms.

Based on the results of molecular genetic studies, it is likely possible to increase the level of Nitrogen fixation by diazotrophic bacteria by modifying the nifA and nifL genes. Following the genetic engineering introduction of extra copies of the nifA gene into a Rhizobium meliloti strain, alfalfa plants inoculated with this recombinant strain grew larger and produced more biomass than plants treated with the untransformed strain. Apparently, a similar approach could be applied to the nifL gene, making the NifL protein (a negative regulatory factor) less sensitive to the presence of fixed nitrogen. Under such deregulated conditions, the microorganism would supply more nitrogen to its symbiotic partner. However, current evidence indicates that not all nitrogen-fixing organisms synthesize the NifL protein (in some, essential regions of NifL may be an integral part of NifA), so this approach is not universal. Furthermore, increasing the amount of nitrogen a microorganism can fix demands more energy (typically in the form of fixed carbon) to support its metabolism. Consequently, the recombinant microorganism might fail to stimulate plant growth simply because its own growth is slowed.

Given The complexity of microbial nitrogen fixation, it can be concluded that simply introducing one or two nif genes into a non-diazotrophic recipient cell is insufficient for it to acquire nitrogen-fixing ability. Moreover, even introducing a complete 24-kb nif gene cluster into the plant genome will not yield the desired effect, because nitrogenase is inactivated at the oxygen concentrations typical of plant cells. If the oxygen concentration is lowered, The plant cell will most likely die. First and foremost, however, attempts to engineer nitrogen-fixing plant cells require solving fundamental problems of transcription, Translation, and regulation. For example, it is difficult to envision how fixation would be regulated, since plants lack promoters that can bind the NifA protein. Consequently, Transcription of the nif genes would not be initiated in such a transgenic plant. Furthermore, to respond to the level of fixed nitrogen in The Cell, all nif genes would need to be under the control of individual promoters, as plant cells are unable to process multigenic transcripts. Considering all of the above, it must be concluded that creating nitrogen-fixing plants is highly unlikely to be feasible.