Brown - Nitrogen Fixation

• N₂ cannot be used directly by plants or animals to synthesize amino acids and nucleotides

• Must be fixed into NH₃

• Requires large amount of energy because of triple bond  of N₂ (N       N) is very stable

• Energy obtained from hydrolysis of ATP

• Chemical conversion of N₂ to NH₃ requires high temp and pressure.

• 100 million tons of fixed N₂ required annually to sustain global food production

• Chemical fertilizers account for half of this N₂ supply

• Diazotrophic (nitrogen fixing) bacteria  supplies the remainder

• Examples of diazotrophic bacteria include:

  • Rhizobium

  • Frankia

  • Azospirillum

  • Azotobacter and cyanobacteria

• Prohibitive cost of chemical fertilizers acts as an incentive for the development of biofertilizers

• Most important organisms being used in agriculture belong to two related genera:

  • Rhizobium

  • Bradyrhizobium

LIFE CYCLE OF RHIZOBIAL BACTERIA

• As part of their lifecycle, rhizobial bacteria invade plant root cells and initiate developmental changes that lead to the formation of root nodules

Inside the nodule nitrogenase is protected from the toxic effects of O₂ in two ways:

• O₂ does not readily diffused into a nodule

• The nitrogenase content within a nodule is regulated by leghemoglobin

The heme moiety is synthesized by the bacterium

The globin portion is encoded by a plant gene

• The plant provides the bacterium with photosynthetically fixed carbon (needed for bacterial growth)

• Plant receives fixed nitrogen from bacterium

TEXTBOOK

Nitrogen Fixation

Nitrogen gas (N2 ), which makes up approximately 80% (by volume) of the air that we breathe, cannot be used directly by plants or animals to synthe size essential nitrogen-containing biomolecules, such as amino acids and nucleotides. Rather, it must first be converted (fixed) into ammonia. This conversion requires a high input of energy because the triple bond of N2 (N≡N) is extremely stable. The energy for the biological fixation of nitrogen comes from the hydrolysis of large amounts of ATP. Similarly, the chemical (industrial) conversion of N2 to ammonia uses a considerable amount of energy in the form of high temperature and pressure.

More than 100 million tons of fixed nitrogen is needed annually to sustain global food production. Synthetic (chemically produced) fertilizers account for about half of this nitrogen supply, and most of the remainder is derived from diazotrophic bacteria. No eukaryote is known to fix nitrogen. Chemical fertilizers have helped considerably in increasing crop yields, but their continual use has led to pollution problems as a result of runoff and to depletion of the nutrient reserves in the soil. Moreover, their cost has been rising steadily. These factors have provided an incentive for devel oping alternative sources of fixed nitrogen, including the development of diazotrophic microorganisms as “bacterial fertilizers.”

A wide range of bacteria can fix nitrogen, and a number of them have potential as crop fertilizers. However, until a bacterial fertilizer has been shown conclusively to be as effective as a chemical formulation, there will be reluctance to change current practices, especially in those countries where the cost of chemical fertilizer is not significant relative to the value of the crop.

For example, soybeans, which constitute the second largest crop in the United States in terms of both cash value and total acres planted, form a beneficial symbiotic relationship with the bacterium Bradyrhizobium japonicum. In this symbiosis, the bacteria provide the plant with fixed nitrogen and, in turn, receive photosynthetically fixed carbon from the plant. When plants are inoculated with specific strains of B. japonicum, the final yield of plant material can be increased by 25 to 50%, and the inocu lated plants no longer require the addition of chemically fixed nitrogen.

Although approximately 40% of the world’s soybean crop is produced in just a few locales in the United States and agricultural practices tend to be similar throughout these locales, at present, only a small fraction of this crop is treated with B. japonicum. Most of these farmers continue to depend on the naturally occurring strains of B. japonicum in the soil and chemical fertilizers.

The most important microorganisms that are currently used agricultur ally to improve the nitrogen content of plants include a range of rhizobial genera and species (Table 15.7). These bacteria are gram negative, flagel lated, and rod shaped, and they form symbiotic relationships with legumes. Generally, each rhizobial species is specific for a limited number of plants and will not interact with plants other than its natural hosts (Table 15.7).

As part of their life cycle, rhizobial bacteria invade plant root cells and initiate a complex series of developmental changes that lead to the formation of a root nodule. Inside the root nodule, the bacteria proliferate and persist in a form that has no cell wall. The bacteria within the nodules fix atmospheric nitrogen by means of the enzyme nitrogenase. The structural and biochemical interactions between a symbiotic rhizobacterium and a host plant are quite intricate and mutually beneficial. Inside a nodule, nitrogenase is protected from the toxic effects of atmospheric oxygen in two ways. First, oxygen does not readily diffuse into a nodule. Second, the oxygen content within a nodule is regulated by the protein leghemoglobin.

The heme moiety of this oxygen-binding protein is synthesized by the bacterium, and the globin portion of the molecule is encoded by a plant gene. The plant also provides the bacterium with photosynthetically fixed carbon, which the bacterium requires for growth. For its part, the plant benefits from this symbiotic relationship by receiving fixed nitrogen from the bacterium.

NITROGENASE

All known nitrogenases have two oxygen sensitive components.

• Component I consists of :

  • Two β- protein subunits  (60,000 daltons each)

  • Two α- protein subunits (50,000 daltons each)

  • 22 molecules of Fe

  • 2 molecules of molybdenum

  • Iron-molybdenum cofactor called FeMoCo

• Component II consists of

  • Two α – protein subunit (32,000 daltons each)

  • Associated iron-molybdenum  molecules

The catalysis of nitrogen to ammonia requires the combination of components I and II, a complex of magnesium and ATP, and a source of reducing equivalents

N₂ + 8H+ + 8e- + 16MgATP        2NH₃ + H₂ + 16MgADP + 16 Pi

• Nitrogenase can reduce the gas acetylene to ethylene

• The production of ethylene can be used as a measure of nitrogenase activity

• Components I and II are extremely sensitive to O₂

• Functional nitrogenase is dependent on 15 to 20 additional accessory proteins

• These accessory proteins have been known to transfer electrons to component II and the biosynthesis of the iron-molybdenum that is a part of component I

TEXTBOOK

NITROGENASE

The renewed interest in diazotrophs as biological fertilizers overlapped the development of techniques for gene isolation and manipulation and pro vided the impetus for studying the biochemical and molecular biological aspects of nitrogen fixation. Initially, scientists believed that these studies would lead to the development of improved nitrogen-fixing organisms that would enhance crop yields. Some researchers even went so far as to sug gest that bacterial genes for nitrogen fixation might be introduced directly into plants to enable them to fix their own nitrogen. Although this overly optimistic prediction has not materialized, a detailed understanding of the process of nitrogen fixation has emerged. And with this understanding, the possibility of improving the nitrogen-fixing activity of some diazotrophs by genetic manipulation is a little closer to becoming a reality.

COMPONENTS OF NITROGENASE

All known nitrogenases have two oxygen-sensitive components. Component I is a complex of two identical α-protein subunits (approximately 50,000 daltons each), two identical β-protein subunits (approximately 60,000 dal tons each), 24 molecules of iron, 2 molecules of molybdenum, and an iron molybdenum cofactor, often called FeMoCo (Fig. 15.17). Component II has two α-protein subunits (approximately 32,000 daltons each), which are not the same as the α-protein subunits of component I, and a number of associ ated iron molecules. The catalysis of nitrogen to ammonia requires the combination of components I and II, a complex of magnesium and ATP, and a source of reducing equivalents (reaction 1; the upward-pointing arrow indicates a gas and Pi is inorganic phosphate).

N 2 + 8H+ + 8e− + 16MgATP → 2NH3 + H2 ↑ + 16MgADP + 16Pi (1)

In addition to fixing nitrogen, the nitrogenase can reduce the gas acetylene to ethylene (reaction 2).

H—C≡C—H + 2H+ → H2 =C=C=H2 (2)

The measurement by gas chromatography of ethylene production as a func tion of time provides a convenient assay for nitrogenase activity. This assay can be performed with intact cells in solution (Fig. 15.18), bacteria associated with plant roots, crude cell extracts, or highly purified enzyme preparations.

Component I catalyzes the actual reduction of N2 , and component II donates electrons to component I. Both components are extremely sensitive to oxygen and can be rapidly and irreversibly inactivated when the oxygen concentration is too high. In addition to components I and II, the activity of a complete, functional nitrogenase depends on 15 to 20 additional accessory proteins. The roles of most of the accessory proteins have been delineated and include the transfer of electrons to component II and the biosynthesis of the iron–molybdenum cofactor that is a part of component I.

GENETIC ENGINEERING OF THE NITROGENASE GENE CLUSTER

• Nitrogen fixation is a very complex process

• Most direct way to isolate the genes involved in nitrogen fixation (nif genes) is to identify and characterize clones of a wild-type library that restore nitrogen fixation to various mutants of the original organism

• This process is called genetic complementation

• 1^st nif genes identified by complementation were isolated from clone banks of Klebsiella pneumoniae.

• This organism is found in soil and water and in the human intestine

ISOLATION PROTOCOL

1. K. pneumonia cells are treated with a  dose of mutagentic agent that allows approximately 0.1 to 1% of the cells to survive.   

• Some of the mutant cells may grow on a minimal   medium containing NH₄Cl but do not grow in the   absence of fixed nitrogen.

• These cells are likely to a have a mutation in the   nif gene and are designated Nif-

2. A clone bank that consists of a chromosomal DNA from wild type (Nif+) K. pneumoniae is constructed  into a broad host range plasmid expression vector and maintained in  E. coli.

3. The Nif- K. pneumoniae cells are conjugated with the E.coli cells that carry the clone bank on a plasmid shuttle vector.

4. The transformed K. pneumoniae cells are selected for the acquisition of the Nif+ phenotype by plating onto a minimal medium that does not contain a source of fixed nitrogen

The only cells expected to grow under these conditions will be the Nif- K. pneumoniae cells containing a plasmid encoding the protein that is either missing or nonfunctional in the Nif- mutant.

OTHER PROTOCOLS

1^st  — The K. pneumoniae clone bank has been used to complement a series of independently derived Nif- mutants, increasing the likelyhood that in each case a different nif gene will be isolated.

2^{nd —} Isolated nif genes have been used as DNA hybridization probes which have been used to screen  a K. pneumoniae chromosomal DNA clone bank that carries large inserts (7-10 kb)

• This is based on the premise that in prokaryotes the genes involved in a pathway are clustured on the chromosomal DNA and are arranged in operons

DNA hybridization then allows for the identification of clones containing additional nif genes that are adjacent to the sequence initially isolated

• The entire set of nif genes in K. pneumoniae has been isolated and characterized

• They occupy a single cluster that occupies 24 kb of the bacterial genome

• Cluster contains 7 operons that encode 20 distinct proteins

• All of the nif genes must be  under the regulatory control of nifA and nifL  genes in order to produce a functional nitrogenase

• NifA protein is a positive regulatory factor

• It turns on the transcription factor of all the other nif genes except its own

• It does this by binding to a specific DNA sequence

• (5’-TGT-N10-ACA-3') that is part of each promoter of each operon

• NifA protein binds to σ⁵⁴ before transcription from the nif promoter is initiated

• The nifL protein is a negative regulatory factor

• In the presence of O₂ or fixed N it acts as an antagonist to nifA protein

• Turns off the transcription of all other nif genes

• To genetically engineer nitrogen fixation in important plant growing bacteria, other nif genes  have been cloned and characterized

• The nif genes from K. pneumoniae have been used as hybridization probes to isolate nif genes from clone banks of other diazotrophic micro-organisms

• Most diazotrophic microrganisms have a similar set of genes encoding their nitrogen fixation apparatus

• The sequences of the genes do not vary much

• It may be possible to increase the amount of nitrogen fixed by diazotrophic microorganisms by manipulating nifA and nifL genes

• NifL genes could be manipulated so that they are less sensitive to the presence of fixed nitrogen.

• This will allow the micro-organism to fix more nitrogen

• Increasing the amount of fixed nitrogen may inturn increase the amount energy (Carbon) that is needed  to support its metabolism.

• Could lose its effectiveness as a nitrogen fixing agent

CAVEATS OF THE PROCESS

• The addition of nif genes to a non-diazotrophic microorganisms may not confer on it the ability to fix nitrogen

• Insertion of the nif gene cluster may not be effective as O₂ in the host cell will inactivate nitrogenase

• Transcriptional, translational and regulatory problems

• There are no plant promoters that respond to the nifA protein

• Therefore nif genes will not be turned on in transgenic plants

• There will also be the need for separate promoters for each of the nif genes in order for it to respond to the level of fixed nitrogen in the cell

• This is because plant cells do not process multigene transcripts

• The introduction of functional nitrogen fixation capabilities in plants is therefore extremely unlikely

TEXTBOOK

Genetic Engineering of the Nitrogenase Gene Cluster

Nitrogen fixation is a very complicated process requiring the concerted actions of a large number of different proteins. Therefore, it was not real istic to expect either that an intact single DNA fragment containing all the genetic information for nitrogen fixation could be readily cloned from a diazotrophic microorganism and transferred into a nondiazotrophic organism or that a recipient organism could maintain the physiological conditions needed for nitrogenase activity.

Consequently, the most direct way to isolate the genes involved in nitrogen fixation (nif genes) was to identify and characterize those clones of a wild-type library that restore nitrogen fixation to various mutants of the original organism. This process is called genetic complementation.

The first nif genes identified by complementation were isolated from clone banks of the diazotroph Klebsiella pneumoniae. This well-studied organism is found in soil and water, as well as in the human intestine. The isolation protocol comprises the following steps (Fig. 15.19)

1. K. pneumoniae cells are treated with a dose of a mutagenic agent that allows approximately 0.1 to 1.0% of the cells to survive. Some of the mutagenized cells are able to grow on a minimal medium containing a source of fixed nitrogen, such as NH4 Cl, but do not grow in the absence of fixed nitrogen. These cells are likely to have a mutation in a nif gene and are designated Nif−.

2. A clone bank that consists of chromosomal DNA from wild-type (Nif+) K. pneumoniae cells is constructed in a broad-host-range plasmid expression vector and maintained in Escherichia coli.

3. The Nif− K. pneumoniae cells are conjugated with the E. coli cells that carry the clone bank on a plasmid shuttle vector

4. The transformed K. pneumoniae cells are selected for the acquisition of the Nif+ phenotype by plating them onto a minimal medium that does not contain a source of fixed nitrogen. The only cells that grow under these conditions are Nif− K. pneumoniae cells containing a plasmid encoding and expressing the protein that is either missing or nonfunctional in the Nif− mutant.

Fig 15.18. Assay for nitrogenase activity based on the conversion of acetylene to ethylene.

(A) The source of the nitrogenase enzyme, for example, bacteria growing in culture, bacteria associated with plant roots, or a purified enzyme preparation (not shown), is placed in a sealed container under an atmosphere of acetylene.

(B) Samples are periodically withdrawn from the sealed container, and the levels of acetylene and ethylene are measured by gas chromatography. The extent of nitrogenase activity is proportional to the amount of ethylene produced

Fig 15.19. Procedure for isolating nif genes by genetic complementation. A clone bank that was constructed with Nif+ K. pneumoniae DNA is used to complement a Nif− K. pneumoniae strain. Transformants are selected for growth on minimal medium that does not contain fixed nitrogen.

Fig 15.20. Partial restriction endonuclease digestion of the region of the chromo somal DNA encoding nif genes. The nif gene that was isolated by genetic comple mentation is used as a DNA hybridization probe (red). The probe and the DNA fragments from the partial digest are ordered as they would be in the chromosomal DNA. Colony hybridization of a clone bank consisting of the illustrated fragments, using the probe shown, would be expected to yield clones containing plasmids with fragments C and D. Using fragments C and D separately as probes to screen the same clone bank would be expected to yield clones containing plasmids with frag ments B and E, respectively. Eventually, in this way, a large contiguous section of the host chromosome is isolated as a set of overlapping clones.

The DNA fragment in the plasmid that complements the Nif− chromosomal mutation contains a nif gene that can be characterized more thoroughly and used to isolate other nif genes.

Two approaches have been used to isolate other genes that are involved in the nitrogen fixation process.

First, the K. pneumoniae clone bank has been used to complement a series of independently derived Nif− mutants, increasing the likelihood that in each case a different nif gene will be isolated.

Second, isolated nif genes have been used as DNA hybridization probes, which have then been used to screen a K. pneumoniae chromosomal DNA clone bank that carries large (7- to 10-kb) inserts (Fig. 15.20). The premise behind the latter scheme is based on the observation that in prokaryotic organisms many of the genes involved in one pathway are clus tered on the chromosomal DNA and are often arranged in operons. Thus, DNA hybridization enables investigators to identify clones containing addi tional nif genes that are adjacent to the sequence initially isolated.

As a result of a considerable amount of research, the entire set of nif genes from K. pneumoniae has been isolated and characterized. These genes are arranged in a single cluster that occupies approximately 24 kb of the bacterial genome (Fig. 15.21). The cluster contains seven separate operons that together encode 20 distinct proteins (Table 15.8). All of the nif genes must be transcribed and translated in a concerted fashion, under the regu latory control of the nifA and nifL genes, to produce a functional nitroge nase. The NifA protein is a positive regulatory factor. It turns on the transcription of all of the nif operons except its own by binding to a specific 2nd Proof 3rd Proof 1st Proof Final 4rd Proof DNA sequence (5′-TGT-N10-ACA-3′) that is part of each promoter of each nif operon. There is a site on the DNA approximately 80 to 150 nucleotides upstream of each transcriptional start site where the NifA protein binds.

Fig 15.21. Arrangement of the genes in the K. pneumoniae nif gene cluster and some of the functions that they encode. The nif genes are represented by italic uppercase letters; each red arrow below sets of these letters denotes a specific nif operon and the direction of its transcription. The arrows pointing up and away from the gene designations show how some of the various gene products partici pate in nitrogen fixation. F, flavodoxin molecule; FO, pyruvate–flavodoxin oxidoreductase; FeMoCo, the iron–molybdenum cofactor; CoA, coenzyme A.

The DNA-bound NifA protein then interacts with a specific transcription initiation protein called sigma-54 (σ54) before transcription from the nif promoter is initiated. The NifL protein is a negative regulatory factor. In the presence of either oxygen or high levels of fixed nitrogen, it acts as an antagonist of the NifA protein and, as a result, turns off the transcription of all other nif genes.

The bacterium K. pneumoniae does not make a major contribution to the overall global biological fixation of nitrogen. Therefore, to genetically engi neer nitrogen fixation in soil bacteria that are more important in promoting plant growth, other nif genes have been cloned and characterized. To do this, the nif genes from K. pneumoniae have been used as DNA hybridization probes to isolate nif genes from clone banks of other diazotrophic microor ganisms. Most diazotrophic organisms have a similar array of genes encoding their nitrogen-fixing apparatus, and the DNA sequences of these genes do not vary much from one organism to another.

It may be possible to increase the amount of nitrogen fixed by diazotrophic organisms by manipulating the nifA and nifL genes. After researchers genetically engineered extra copies of the nifA gene into a Sinorhizobium meliloti strain, alfalfa plants that were inoculated with this transformant grew larger and produced more biomass than plants that were treated with the non-transformed strain. Similarly, it may be possible to engineer the nifL gene so that the NifL protein, the negative regulator, is less sensitive to the presence of fixed nitrogen. With this kind of deregulation, an organism would fix more nitrogen for its plant partner.

However, not all nitrogen-fixing organisms have a NifL protein, so this sort of manipulation may be limited to only certain bacterial strains. In some organisms, the essential regions of NifL are part of NifA. Moreover, increasing the amount of nitrogen that an organism can fix also increases the amount of energy, usually in the form of fixed carbon, that is needed to power its metabolism. Consequently, an engineered microorganism that can fix a higher than normal level of nitrogen may lose its effective ness as a plant growth-promoting agent because of a diminished growth rate.

Because of the complexity of nitrogen fixation by microorganisms, the simple addition of one or two nif genes will not confer on a nondiazotrophic recipient cell the ability to fix nitrogen.

Moreover, genetic modification of plants with the entire 24-kb nif gene cluster would not be effective because the normal level of oxygen in the host cell would inactivate nitrogenase, and if this level were reduced, the host plant cell would probably die. In addition, the engineering of nitrogen fixation in plant cells requires resolving major, if not insurmountable, transcriptional, translational, and regulatory problems. For example, it is difficult to con ceive how the regulation of nitrogen fixation could be achieved, since there are no plant promoters that respond to the NifA protein.

Consequently, nif genes would not be turned on in such a transgenic plant. Each of the nif genes would also have to be under the control of separate promoters because plant cells cannot process multigene transcripts. The introduction of a functional nitrogen fixation capability into plants is therefore extremely unlikely.

NOTES

GLYCOGEN SYNTHASE MUTANTS

• Rhizobial respiration plays an important role in N fixation

  • Oxidative phosphorylation provides the energy needed for N fixation

  • Respiration removes O₂

  • Prevents inactivation of nitrogenase

• It would be more advantageous if the C reserves were driven towards ATP production instead of glycogen synthesis

[TEXTBOOK]

Fig 15.22. Schematic representation of the partitioning of rhizobial intracellular glucose between glycogen synthesis and respiration. A mutation in the gene for glycogen synthase prevents glycogen from being synthesized, so that all of the glucose enters the tricarboxylic acid (TCA) cycle. In this cycle, the acetyl group of acetyl coenzyme A (CoA) is enzymatically degraded to form carbon dioxide and hydrogen. The hydrogen (or the corresponding electrons) is fed into the electron transport chain, and a large portion of the energy released is conserved by the phos phorylation of ADP to ATP. The ATP is then available to “power” a large number of energy-requiring metabolic processes, including nitrogen fixation.

[BACK TO NOTES]

• A strain of Rhyzobium tropici was constructed with a deletion mutant for glycogen synthase

• This strain was unable to produce glycogen and was able to increase both the  nodule number and the dry weight of treated plants

• Deletion mutants cannot revert to the wild type

• Good for agriculture

• Inability to synthesize glycogen can have deleterious effects hence these microbes will not persist for a long time.

• Reinnoculation field necessary

ENGINEERING OXYGEN LEVELS

• Oxygen is inhibitory to nitrogenase and is a negative regulator of nif gene expression

• Oxygen is required for bacteriod respiration. How can we solve the problem?

  • Introduction of leghemoglobin that which binds free oxygen tightly

  • This will ensure that the nif genes and functioning of nitrogenase are not impaired

• It is possible to engineer rhizobial strains with genes encoding the bacterial equivalent of the leghemoglobin.

• The hemoglobin containing strain can grow to a greater extent under conditions where oxygen is limited.

• This expression of bacterial hemoglobin genes maybe advantageous to Rhyzobium both when they are free living and when they are bacteriods.

TEXTBOOK

The concentration of oxygen is a critical factor in determining the amount of nitrogen that is fixed by a rhizobial strain. On one hand, oxygen is inhibitory to nitrogenase and is a negative regulator of nif gene expression. On the other hand, oxygen is required for bacteroid respiration. This conundrum can be resolved by the introduction of leghemoglobin, which binds free oxygen tightly (Fig. 15.23) so that both the transcription of nif genes and the functioning of nitrogenase can proceed unimpaired. In fact, the addition of exogenous leghemoglobin to isolated bacteroids results in a dramatic increase in nitrogenase activity. Thus, it is possible to engineer more efficient strains of Rhizobium by overproducing leghemoglobin.

Alternatively, since the globin portion of leghemoglobin is produced by the plant, it may be more efficient to transform rhizobial strains with genes encoding a bacterial equivalent of leghemoglobin.

Following the transformation of a strain of Rhizobium etli with a broad host-range plasmid carrying the Vitreoscilla sp. (a gram-negative aerobic or microaerophilic bacterium) hemoglobin gene at low levels of dissolved oxygen (0.25 to 1.0%) in the growth medium, the rhizobial cells had a two- to threefold-higher respiratory rate than the nontransformed strain. These data suggest that free-living R. etli with a Vitreoscilla sp. hemoglobin gene may have a competitive advantage over nontransformed rhizobial strains in soil (which usually has a low level of oxygen). As has been observed in the laboratory for numerous other free-living bacteria, the hemoglobin containing strain can grow to a greater extent because it is able to sequester oxygen and provide it to the reactions, where it is necessary for bacterial metabolism (nitrogenase activity is at its peak at this time).

In greenhouse experiments, when bean plants were inoculated with either nontransformed or hemoglobin-containing R. etli, the plants inocu lated with the hemoglobin-containing strain had approximately 68% more nitrogenase activity. This difference in nitrogenase activity leads to a 25 to 30% increase in leaf nitrogen content at 40 to 50 days after infection and a 16% increase in the nitrogen content of the seeds that are produced. Thus, the expression of a bacterial hemoglobin gene may be advantageous to Rhizobium bacteria both when they are free-living and when they are in bacteroids as part of a symbiotic relationship with their host plant.

HYDROGENASE [NOTES]

• Undesirable side effect of nitrogen fixation is the conversion of H⁺ to H₂ gas by nitrogenase

• This utilizes a lot of energy and H₂ is lost to the atmosphere

• As a result only 40-60% of the electrons flux through the nitrogenase system is transferred to N₂

• Efficiency of nitrogen fixation is reduced

• Theoretically if H₂ could be recycled back to H+ the energy loss would be less and the nitrogen fixing process more efficient

• The enzyme hydrogenase is able to take up H₂ from the atmosphere and convert it to H+

• Some strains of B. japonicum have the hydrogenase enzyme and they are able to utilize this for growth

• Soybean plants inocculated with strains that produced Hydrogenase (Hup+) had more biomass and nitrogen than plants that were innoculated with non-hydrogenase producing (Hup-) strains

• This was observed despite that fact that the Hup- strains had higher nitrogenase activity

• From this experiment it was concluded that the presence of a hydrogen uptake system in diazotrophs eg B. japonicum improved its ability to stimulate plant growth

• The majority of the naturally occurring Rhyzobium and Bradyrhizobium strains are Hup-

GENETIC ENGINEERING OF HYDROGENASE

• The structure and function of hydrogenases remain to be understood

• Many organism consists of more than one hydrogenase

• Many hydrogenases have more than one polypeptide chain

• Some hydrogenases are active only in the uptake of H₂ from the atmosphere

• Others can synthesize H₂ depending on the conditions

• Conversion of Hup- strains to Hup+ strains may not be readily achieved by just introducing one hydrogenase gene

• The introduced gene must encode all the enzymes subunit

• The introduced gene must also be able to interact with the appropriate electron transport molecule within the host organism

• The most common strategy for isolating hytrogenase genes is through genetic complementation

• The first hydrogenase gene to be isolated was from E.coli

• This was accomplished by complementation of E. coli mutants that did not express this activity

• Conversion of Hup- strains to Hup+ strains may not be readily achieved by just introducing one hydrogenase gene

• The introduced gene must encode all the enzymes subunit

• The introduced gene must also be able to interact with the appropriate electron transport molecule within the host organism

• The most common strategy for isolating hytrogenase genes is through genetic complementation

• The first hydrogenase gene to be isolated was from E.coli

• This was accomplished by complementation of E. coli mutants that did not express this activity.

• Hydrogenase hup genes from B. japonicum were isolated from a clone bank of wild-type DNA constructed in  abroad host range cosmid vector pLAFR1

• This was done by complementation of B. japonicum Hup- mutants

  The hup genes from B. japonicum are organized into two or 3 transcriptional units covering 20 kb of the genome  and encoding 18 separate genes

• Study of the hup gene of Rhyzobium leguminosarum has indicated that the genes are similar in both DNA sequence and gene organization to the hup genes in B. japonicum

• Therefore hup genes isolated from B. japonicum can now be used as a hybridization probe to select homologous genes from R. leguminosarum

• Plants treated with the R. leguminosarum that had been transformed to Hup+ grew larger and contained more nitrogen than the plants inoculated by the Hup- parental strain

TEXTBOOK

HYDROGENASE

An undesirable side reaction of nitrogen fixation is the reduction of H+ to H2 (hydrogen gas) by nitrogenase. Energy in the form of ATP is wasted on the production of hydrogen, which is eventually lost to the atmosphere. Because of this side reaction, only 40 to 60% of the electron flux through the nitrogenase system is transferred to N2 , thereby significantly lowering the overall efficiency of the nitrogen-fixing process. Theoretically, if H2 could be recycled to H+, the extent of energy loss could be diminished and the nitrogen-fixing process would become more efficient. It is probably impossible to prevent this side reaction directly, because it is a consequence of the chemistry of the active site of the nitrogenase; hence blocking the side reaction by altering nitrogenase would concomitantly inhibit nitrogenase activity

HYDROGEN METABOLISM

In the mid-1970s, it was discovered that some strains of B. japonicum could use hydrogen as an energy source for growth under microaerophilic (low oxygen-concentration) conditions. These strains have an enzyme called hydrogenase that is able to take up H2 from the atmosphere and convert it into H+ (Fig. 15.25). Experiments were undertaken to test whether the pres ence of hydrogenase in B. japonicum had an impact on the growth of soy bean plants. Plants inoculated with strains that produce hydrogenase (Hup+) had more biomass and nitrogen than plants that were treated with non-hydrogenase-producing (Hup−) strains, despite higher levels of nitrogenase activity in the Hup− strains (Table 15.10).

From this and similar experiments, it was concluded that the presence of a hydrogen uptake system in a symbiotic diazotroph, such as B. japonicum, improves its ability to stimulate plant growth, presumably by binding and then recycling the hydrogen gas that is formed inside the nodule by the action of nitrogenase (Fig. 15.25). Within the nodule, the contribution of atmospheric hydrogen is negligible.

Although it is clearly beneficial to the plant to obtain its nitrogen from a symbiotic diazotroph that has a hydrogen uptake system, this trait is not common in naturally occurring rhizobial strains. In one study, it was found that the majority of naturally occurring Rhizobium and Bradyrhizobium strains examined were Hup− (Table 15.11). In that study, the data were based on a small number of strains for each species except B. japonicum, for which over 1,400 strains were assayed. The conclusion that can be drawn from this work is that commercial Hup− rhizobial strains are prime candidates for transformation to a Hup+ phenotype.

GENETIC ENGINEERING OF HYDROGENASE GENES

Although a considerable amount of effort has been directed over the past 30 years or so toward studying hydrogenases from both diazotrophic and nondiazotrophic microorganisms, an in-depth understanding of the struc tures and functions of these enzymes remains elusive. Many organisms have more than one hydrogenase, and many hydrogenases consist of more than a single polypeptide chain. Some hydrogenases are active only in the uptake of hydrogen from the atmosphere, whereas others, depending on the conditions, can also synthesize hydrogen. One result of this complexity is that the conversion of a Hup− strain of Rhizobium into a Hup+ strain may not be readily achieved by the introduction of just any hydrogenase gene. Rather, the introduced gene(s) must encode all of the enzyme’s subunits and must be able to interact with the appropriate electron transport molecule within the host organism.

The most common strategy for isolating hydrogenase genes has been genetic complementation. The first hydrogenase gene to be isolated was the gene for an E. coli membrane-bound hydrogenase, and it was selected by complementation of an E. coli mutant that did not express this activity.

After the work with E. coli, hydrogenase (hup) genes from B. japonicum were isolated from a clone bank of wild-type DNA constructed in the broad-host-range cosmid vector pLAFR1 by complementation of B. japonicum Hup− mutants. The presence of a hydrogenase that takes up hydrogen from the atmosphere in the complemented Hup− mutant strains was indicated by the ability of the active hydrogenase to reduce the dye methylene blue in a hydrogen atmosphere. More detailed studies of the B. japonicum hup genes showed that they were organized into at least two, and possibly three, transcriptional units covering approximately 20 kb of the genome and including 18 separate genes. Subsequent work on the hup genes from Rhizobium leguminosarum has indicated that these genes are similar in both DNA sequence and gene organization to the hup genes from B. japonicum. Thus, the isolated hup genes from B. japonicum may be used as DNA hybridization probes to select homologous genes from a clone bank of R. leguminosarum.

Following the isolation of R. leguminosarum hup genes, and despite the complexity of this system, it has been possible to use cosmid vectors to transfer a complete set of uptake hydrogenase genes from a Hup+ strain of R. leguminosarum to a Hup− strain (Table 15.12). Plants treated with R. legu minosarum that had been transformed to Hup+ grew larger and contained more nitrogen than the plants inoculated with the Hup− parental strain (Table 15.12). Although hydrogenase genes have not received as much attention as nif genes, this simple gene transfer experiment is a convincing demonstration of the use of genetic manipulation to improve the ability of a diazotroph to stimulate plant growth.

More recently, one group of scientists modified the hup gene promoter in R. leguminosarum and in the process engineered a more efficient rhizobial strain. In R. leguminosarum, 18 genes are associated with hydrogenase activity. There are 11 hup genes (Fig. 15.26) responsible for the structural components of the hydrogenase, the processing of the enzyme, and elec tron transport. There are also seven hyp (hydrogenase pleitropic) genes, which are involved in processing the nickel that is part of the active center of the enzyme. The hup promoter is dependent on the NifA protein (which is also required to activate the synthesis of nif genes), so that hup genes are expressed only within bacteroids. On the other hand, the hyp genes are transcriptionally regulated by an FnrN-dependent promoter, which is turned on by low levels of oxygen.

Thus, the hyp genes are expressed both in bacteroids and microaerobically. By modifying the chromosomal DNA of R. leguminosarum and exchanging the hup promoter for an FnrN-dependent promoter (Fig. 15.26), a derivative of the original bacterium with an increased level of hydrogenase was created (Table 15.13). The engineered R. leguminosarum strain displayed a twofold increase in hydrogenase activity compared to the wild type, and no discernible amount of hydrogen gas was produced as a by-product of nitrogen fixation.

This is expected to make this strain of R. leguminosarum much more effective at promoting plant growth and increasing plant nitrogen content. Moreover, regardless of whether nickel was added to the system, the amount of hydrogen evolved from nitrogen-fixing nodules was extremely low, indicating that virtually all of the hydrogen produced by nitrogenase was recycled. The reason that nickel was added to this system is that in many soils, the avail ability of nickel limits hydrogenase activity. In some soils, the level of nickel is so low that even if a naturally occurring strain contains hydrogenase genes, the hydrogenase activity may be so low as to be ineffectual.

On the other hand, when the engineered R. leguminosarum was tested with various field soils, hydrogenase overproduction invariably overcame the limitation of low nickel levels, with the net result that the amount of fixed nitrogen, and hence plant productivity, was greater.

NODULATION [NOTES]

• Many of the commercial inoculant strains are great nitrogen fixers but are not very effective at establishing nodules

• Conversely there are microorganisms that are great at establishing nodules but are poor nitrogen fixers

• Therefore to make use of the commercial strains  either the nodulation capability must be enhanced or indigenous rhizobial strains must be inhibited

GENETIC ENGINEERING OF NODULATION GENES

• DNA from wild type S. meliloti is cloned in broad range cosmid pLAFRI

• It is packaged into bacteriophage λ and introduced into E. coli

• The clone bank is then transferred from E.coli to a

• Nod^-- S. meliloti by conjugation

• Alfalfa plants are inoculated with transformed Nod^- S. meliloti.

• Those plants that develop root nodules have been infected with the Nod+ S. meliloti cells that were carrying complementing nodulation genes inserted in cosmid vector

• Those plants that develop root nodules have been infected with the Nod+ S. meliloti cells that were carrying complementing nodulation genes inserted in cosmid vector

• The transformed Nod⁺ S. meliloti cells can be isolated directly from root nodules

• The complete set of nodulation genes of R. meliloti has been characterized

• Nodulation is a very complex process

• Some nodulation genes are highly conserved among nodulation organisms while some are species specific.

• Nod genes are grouped into three classes:

  • Common genes

  • Host specific genes

  • Regulatory nodD genes

• The nodABC genes are common in all Rhizobium species and are found on a single operon

EVENTS DURING NODULATION

• The nodD gene product recognizes and binds flavonoids

• Each rhizobial species recognizes only a limited number of flavonoid structures

• Each plant species produces its own specific set of flavonoid molecules

• The binding of flavonoid molecules activates the NodD gene products and enables the flavonoid-NodD complex to attach to a nodulation promoter element (nod box)

• The nod box is located upstream from all nodulation genes except NodD gene and turns on transcription of these genes

• The nodABC genes encode proteins that cause the plant root hair tips to curl

• The bacteria synthesize a Nod factor that is probably modified by NodH gene product

• This factor elicits in plants a host-specific response that includes root hair curling and deformation and is important for Rhizobium to form nodules

• After the initial change in root morphology the bacteria attaches to the root hair.

• The bacterial cells penetrate the plant cell through an infection thread

• Depending on the the Rhizobium or Bradyrhizobium strain ~ 20 additional gene products  are synthesized

• These proteins along with plant encoded protein contribute to the nodule

• More research is needed to elucidate the role of each of the nod genes

• To date despite the fact that nod genes from several different rhizobial strains have been isolated  and characterized

• No simple genetic means has been devised  for using nod genes to enable inoculated strains of Rhizobium to outcompete indigenous strains

NODULATION AND ETHYLENE

• Ethylene is produced by plants during the initial stages of infection (eventually leading to nodule formation by rhizobia)

• Small rise in ethylene can inhibit and limit rhizobial infection

• Some species increase nodule formation by limiting the rise of ethylene

• Some produce rhizobitoxin that inhibits ACC synthase

• Some also produce ACC deaminase that removes ACC before it can be converted to ethylene

TEXTBOOK

NODULATION

Competition among Nodulating Organisms

A major goal of agricultural biotechnology research is the development, by genetic manipulation, of Rhizobium strains that can increase plant produc tivity more effectively than naturally occurring strains. Many commercial inoculant strains that have been developed by mutation and selection to be superior nitrogen fixers are not very effective at establishing nodules on host plant roots when placed in competition with Rhizobium strains that are already present in the soil.

Conversely, although many of the strains that are indigenous to the soil are highly successful in establishing nodules in competitive situations, they are not especially efficient at nitrogen fixation. Therefore, to make use of the commercial inoculant strains, either the nodulation capability of these strains must be enhanced or indigenous rhizobial strains must be inhibited.

Studies were undertaken to determine the genetic basis of this “competitiveness” with the aim of adding these particular genes to the strains that are used as inoculants. The nature of the competitive advantage of soil rhizobial species is not known, but it was reasoned that the indigenous bacteria might be more efficient at nodulation and, as a consequence, might prevent an inoculated strain from becoming established and forming its own nodules

GENETIC ENGINEERING OF NODULATION GENES

When scientists first attempted to isolate nodulation (nod) genes, the absence of any specific information about the biochemical or genetic basis of nodulation meant that a strategy had to be devised for the identification of the genes. Therefore, once again, genetic complementation was used. Nodulation-defective (Nod−) mutants of S. meliloti were transformed with a clone bank of wild-type chromosomal DNA from S. meliloti, and those colonies that had acquired the ability to nodulate alfalfa roots were isolated (Fig. 15.27). More specifically, the steps of the procedure were as follows.

1. A clone bank of wild-type (Nod+) S. meliloti was constructed by partial digestion of R. meliloti DNA with EcoRI and insertion into the unique EcoRI site of the broad-host-range cosmid pLAFR1, which can carry up to 40 kb of cloned DNA.

2. The clone bank was packaged into bacteriophage λ, introduced into E. coli, and subsequently transferred to Nod− mutants of S. meliloti by conjugation. The vector carries a tetracycline resistance gene that can be used as a selectable marker in both E. coli and S. meliloti.

3. After conjugation, suspensions of 200 to 300 transformed S. meliloti cells were tested for the ability to nodulate sterile alfalfa plants, with the expectation that only those transformants that carried and expressed a gene that complemented the nodulation defect in the S. meliloti host would produce nodules.

4. The bacteria that formed nodules on the test plants were recovered from within the nodules. These bacteria were then grown in cul ture and used to retrieve the vector carrying the complementing gene. The specific portion of the large insert DNA that carried the complementing gene was then subcloned onto another plasmid vector and analyzed further.

5. Once a single nodulation gene was identified, it was used as a DNA hybridization probe to identify adjacent regions of S. meliloti chromosomal DNA in a genomic library (Fig. 15.20).

The complete repertoire of nodulation genes from S. meliloti has been characterized. Detailed biochemical and genetic studies have revealed that nodulation and its regulation are complex processes that require the func tioning of a large number of genes (Table 15.14). Some of the nodulation genes are highly conserved (common) among nodulating microorganisms, and others are species specific. The nod genes are grouped into three sepa rate classes: common genes, host-specific genes, and the regulatory nodD gene. Thus, for example, the nodABC genes are common to all Rhizobium species and are structurally interchangeable. In most species, the nodABC genes are found on a single operon.

A number of events are now known to occur during nodulation. First, the nodD gene product, which is constitutively expressed, recognizes and binds to a flavonoid molecule, which is excreted by the roots of the poten tial host plants. Flavonoids are a class of plant phenolic molecules with a basic structure that consists of 15 carbons arranged as two aromatic rings connected by a 3-carbon bridge. They perform a number of different func tions for the plant, such as pigmentation and defense against fungi or insects.

The binding of flavonoids to the NodD gene product is one of the major determinants of rhizobial host specificity, because each rhizobial spe cies recognizes only a limited number of flavonoid structures and each plant species produces its own specific set of flavonoid molecules (Table 15.15). In a limited number of instances, other small organic molecules, such as aldonic acids and betaines, that are exuded by plant roots or germi nating seeds and are present in large amounts can interact with the NodD protein. Some strains, such as R. leguminosarum biovar (bv.) trifolii, have a very narrow host range, responding to only a few kinds of flavonoids, while others, such as Rhizobium sp. strain NGR234, have a very broad host range and respond to a much larger number of different flavonoids.

The binding of a flavonoid molecule activates the NodD gene product, presumably causing it to undergo a conformational change, and enables the flavonoid–NodD complex to attach to a nodulation promoter element called a nod box. This promoter element is located upstream from all the nodulation genes except the nodD gene, and it activates the transcription of these genes. The nodABC genes encode proteins that cause the plant root hair tips to swell and curl, an effect that is recognized as the initial step in the infection of the plant root by the bacterium. The bacteria synthesize an oligosaccharide Nod factor that is modified by the NodH gene product and perhaps also by the NodQ and NodP gene products.

This factor (Fig. 15.28) elicits in the plant a host-specific response that includes root hair curling and deformation and is essential for Rhizobium to induce nodules. After the initial change in the root morphology, the bacterium attaches to the root hair. Next, the bacterial cell penetrates the plant cell through an infection thread. Finally, depending on the bacterial strain, up to approximately 20 additional nod gene products are synthesized. These proteins, together with some plant-encoded proteins, contribute to the formation of the nodule.

DNA sequencing and computer analysis revealed that in a slow growing variant of a Bradyrhizobium sp., the region of the DNA between the nodD and nodABC genes contained an open reading frame that the fast growing form lacked. This open reading frame was designated nodK. When plants were inoculated with a Bradyrhizobium sp. that had a mutagenized nodK gene (NodK−) and were compared with those treated with the wild type strain (NodK+), the onset of nodulation in the NodK− strain-treated plants was 5 days earlier, the nodulation number was doubled, and there was a 120% enhancement of plant yield.

To date, despite the fact that nod genes from several different rhizobial strains have been isolated and characterized, no simple genetic means has been devised for using nod genes to enable inoculated strains of Rhizobium to outcompete indigenous strains. Nevertheless, host specificity can be altered by the transfer of a nodD gene from a broad-specificity rhizobial strain to one with narrow specificity.

It has become clear that the process of nodulation is quite complicated. Thus, considerable additional effort will be required before it is possible to further enhance the competitiveness of rhizobial strains by genetic engineering.

NODULATION AND ETHYLENE

Ethylene is often produced by plants following the initial stages of infec tion (eventually leading to nodule formation) by rhizobia. This small rise in the plant ethylene level is generally localized to a portion of the root and can inhibit, and therefore limit, subsequent rhizobial infection and nodula tion. One way in which some strains of Rhizobium naturally increase the number of nodules that they can form on the roots of a host legume is to limit the rise in ethylene that occurs following the initial infection. Different Rhizobium species decrease ethylene levels either by synthesizing a small molecule called rhizobitoxin that chemically inhibits ACC synthase, one of the ethylene biosynthetic enzymes, or by producing ACC deaminase and removing some of the ACC before it can be converted to ethylene. The result of lowering the local level of ethylene is that both the number of nodules and the biomass of the plant are increased by 25 to 40%. Assays of isolated rhizobia indicate that in the field approximately 1 to 10% of rhizobial strains possess ACC deaminase.

It should therefore be possible to increase the nodulation efficiency of Rhizobium strains that lack ACC deaminase by genetically engineering these strains with isolated Rhizobium ACC deaminase genes (and their regulatory regions). In fact, insertion of a single copy of an ACC deaminase gene from R. leguminosarum bv. viciae into the chromosomal DNA of a strain of S. meliloti that lacked this enzyme dramatically increased both the nodule numbers and biomass of host alfalfa plants (Fig. 15.29). While genetically engineered strains of Rhizobium may not be acceptable for use in the field in all jurisdictions at this time, as a result of this work, several commercial inoculant producers are already screening their more recently isolated Rhizobium strains for active ACC deaminase.

TUTORIAL 5

What would you expect the effect of mutagenizing either nifA or nifL to be on the amount of nitrogen that an organism can fix?

Here’s what you’d predict from mutations in each of those two key regulators, based squarely on your textbook description:

1. Mutation of nifA (positive regulator)

• Normal role: NifA binds the conserved 5′-TGT-N₁₀-ACA-3′ sequence in each nif operon’s promoter, recruits σ⁵⁴ (the alternative σ‐factor), and drives transcription of all nif genes except its own.

• If nifA is inactivated or knocked out:

  • Transcription of virtually every nif operon will plummet. Without NifA, σ⁵⁴ can’t be properly engaged at the nif promoters, so the mRNAs encoding both nitrogenase components (plus all the accessory proteins) aren’t made.

  • Outcome: Near‐complete loss of nitrogenase activity—cells are unable to convert N₂ to NH₃, and “nitrogen fixation” assays (e.g. acetylene → ethylene) will show essentially zero activity.

In short: a nifA mutant behaves like a classic Nif⁻ mutant—no positive “on” signal, no nif gene expression, no nitrogen fixation.

2. Mutation of nifL (negative regulator)

• Normal role: NifL senses oxygen and fixed‐nitrogen levels, and in those conditions binds and antagonizes NifA—turning nif transcription off when fixing N₂ would be wasteful or impossible.

• If nifL is inactivated or rendered non‐functional:

  • Constitutive or hyperactive transcription of the nif operons—even in the presence of NH₄⁺ or O₂—because there’s no NifL “brake” to shut NifA down.

  • Outcome: Elevated levels of nitrogenase components and thus increased nitrogen‐fixation capacity, particularly under conditions where a wild‐type strain would normally repress nif expression.

  • Caveat: Unchecked nitrogenase synthesis is energetically expensive (16 MgATP per N₂ reduced), so you may see slower overall growth or competitive disadvantage unless the extra fixed nitrogen is worth the extra ATP cost.

In practice: researchers have indeed engineered nifL mutants (or NifL proteins less sensitive to NH₄⁺) and observed higher-than-normal N₂-fixation rates—and in symbiotic bacteria, improved plant growth—though only up to the point where ATP supply or oxygen sensitivity become limiting.

Summary Table

By tinkering with nifA, you shut the system off; by disabling nifL, you let it run full-blast (for better or worse).

Discuss whether it is possible to genetically engineer plants to fix their own nitrogen

Genetically engineering plants to fix their own nitrogen—a “holy grail” of agricultural biotechnology—remains, at present, an extraordinarily daunting challenge. Below is an in-depth discussion of why this is so, drawing directly on the detailed biology and engineering hurdles you provided.

1. Enormous Genetic Complexity

• Twenty distinct proteins in one 24 kb cluster. In Klebsiella pneumoniae, the nif genes form seven operons spanning ~24 kb and encoding twenty different proteins: the two oxygen-sensitive nitrogenase subunits (components I and II), plus 15–20 accessory factors for electron transfer and FeMoCo cofactor biosynthesis. Each of these proteins must be expressed at the right stoichiometry for a functional enzyme.

• Operon versus eukaryotic gene structure. Bacterial nif genes are organized into operons—single polycistronic transcripts under the control of common promoters and regulators (NifA/NifL, σ⁵⁴). Plants, however, cannot process polycistronic messages: each nif ORF would need its own promoter, terminator, and untranslated regions to ensure proper transcription and mRNA processing.

2. Regulatory Incompatibilities

• Lack of plant promoters responsive to NifA. In bacteria, the NifA protein binds a conserved 5′–TGT-N₁₀-ACA–3′ motif upstream of each operon and, in concert with σ⁵⁴, directly activates nif transcription. No known plant transcription factor or promoter element responds to NifA, so simply transferring nifA won’t trigger expression in plants.

• Need for separate regulatory circuits. Even if you replaced the bacterial promoters with plant-compatible, tissue- or oxygen-responsive promoters, you’d have to engineer twenty distinct transcriptional units and coordinate their induction precisely under nitrogen-deficient conditions.

3. Oxygen Sensitivity and Cellular Environment

• Nitrogenase is irreversibly inactivated by O₂. Both nitrogenase components (I and II) are exquisitely oxygen sensitive. In rhizobia, root nodules—thanks to leghemoglobin and low‐O₂ diffusion—provide a microaerobic environment. In plant cells, oxygen levels are orders of magnitude higher; maintaining an O₂-free compartment would require novel organellar engineering or synthetic micro-compartments.

• Energy supply constraints. Biological nitrogen fixation consumes at least 16 ATP per N₂ reduced. Diazotrophic bacteria channel large amounts of cellular ATP (and reducing power) into nitrogenase. A plant cell would need to divert significant photosynthetic energy to fuel an endogenous nitrogenase—potentially compromising growth unless tightly regulated.

4. Physiological and Metabolic Trade-Offs

• Metabolic burden on the host. Even in bacteria, mutations that derepress nif expression (e.g. nifL knockouts) yield hyperactive nitrogen fixation at the cost of slower growth because of ATP depletion. In plants, unregulated nitrogenase expression could starve other vital processes or trigger stress responses.

• Subcellular targeting. To incorporate nitrogenase into a plant, one must decide whether to target it to chloroplasts (to tap into photosynthetic ATP), mitochondria, or the cytosol—each presenting unique targeting‐signal and redox‐balance challenges.

5. Practical Engineering and Synthetic Biology Hurdles

• Multi-gene assembly at large scale. Delivering a 24 kb cluster plus customized plant promoters and terminators for twenty genes exceeds the capacity of most plant transformation vectors. Modular cloning methods (e.g. Golden Gate) help, but ensuring stable integration and expression of all components remains a major bottleneck.

• Chromatin and epigenetic silencing. Introduced transgenes often become methylated or silenced over generations. Twenty separate expression cassettes amplify this risk, jeopardizing long-term trait stability.

Are There Paths Forward?

While a fully self-reliant nitrogen-fixing plant remains out of reach, several complementary strategies are under exploration:

1. Engineering bacterial symbionts. Instead of embedding nif genes in the plant genome, optimize naturally symbiotic rhizobia—e.g. by tweaking nifL regulation—to boost their nitrogen output in nodules.

2. Synthetic micro-compartments. Recent work in non-plant systems uses protein scaffolds or encapsulins to shield oxygen-sensitive enzymes. Adapting these to plants could, in principle, sequester nitrogenase away from O₂.

3. Horizontal gene transfer into chloroplasts. Chloroplasts share a prokaryotic heritage and may better accommodate multi-gene operons; engineering them as “nitrogenase factories” is an active area of synthetic biology.

4. Signaling-based induction. Creating plant regulatory circuits—using synthetic promoters responsive to internal nitrogen levels—might allow conditional nif expression only when needed, mitigating energy and oxygen challenges.

Conclusion

Introducing a functional, autonomous nitrogen-fixation pathway into plants is, with today’s technology, extremely unlikely. The sheer complexity of the nif gene cluster, its operon-based regulation, the oxygen sensitivity of nitrogenase, and the energy demands together pose multiple, interlocking hurdles. However, by focusing on engineering improved bacterial fertilizers, designing protective micro-compartments, and exploiting synthetic biology advances, researchers continue to chip away at this grand challenge—keeping alive the vision of crops that fix their own nitrogen without costly fertilizers.

ANOTHER RESPONSE

Genetically endowing a crop plant with the ability to fix atmospheric N₂ into ammonia—thereby eliminating the need for synthetic fertilizers—remains one of the most alluring yet formidable goals in biotechnology. In principle, the core chemistry is known and the genes identified; in practice, a convergence of molecular, physiological, and ecological hurdles makes a fully self-sufficient “nitrogen-fixing plant” essentially out of reach with current technology. Below is an in-depth discussion of why this is so, and what (if any) paths forward might exist.

1. The Sheer Genetic Complexity of Nitrogenase

• Twenty proteins in seven bacterial operons
  In Klebsiella pneumoniae, the complete nif cluster spans ~24 kb, organized into seven separate operons that collectively encode:

  • The two nitrogenase components (I and II), each multi-subunit and oxygen-sensitive.

  • Fifteen to twenty accessory factors for FeMoCo biosynthesis and electron transfer.

• Poly-cistronic operons vs. monocistronic eukaryotic transcription
  Bacteria co-transcribe these genes as single mRNAs under the control of common promoters and regulators (NifA/σ⁵⁴). Plant nuclei lack the machinery to process such polycistronic messages—each nif ORF would require its own promoter, terminator, and mRNA-processing signals.

Implication: Simply transferring the entire nif cluster into a plant genome would create a tangle of twenty independent expression cassettes—an enormous cloning and regulatory challenge.

2. Regulatory Incompatibilities

• NifA/σ⁵⁴ activation
  Bacterial NifA binds a conserved 5′-TGT–N₁₀–ACA-3′ motif to recruit σ⁵⁴ and trigger nif transcription. No known plant transcription factors or promoter elements respond to NifA, so the simplest “copy-and-paste” approach fails at step one.

• Environmental sensing
  Bacteria tightly repress nif under high O₂ or fixed-N; plants have their own nitrogen-responsive networks (e.g., glutamine-sensing) that do not intersect with the bacterial circuitry.

Implication: You would need to redesign twenty promoters to respond to plant-friendly signals—such as nitrate deprivation or root-specific cues—and ensure correct timing and stoichiometry.

3. Oxygen Sensitivity of Nitrogenase

• Irreversible inactivation by O₂
  Nitrogenase components are exquisitely oxygen-labile. In root nodules, leghemoglobin and low diffusional O₂ combine to create a microaerobic niche. A free-living plant cell or chloroplast sits in an O₂-rich environment.

• Synthetic micro-compartments needed
  To protect nitrogenase, one would have to engineer novel, O₂-impermeable compartments—perhaps proteinaceous microbodies or modified plastids—while still supplying the enzyme with ATP and reducing equivalents.

Implication: Without a “nodule-like” structure inside plant cells, nitrogenase would be swiftly inactivated.

4. Energetic and Metabolic Trade-Offs

• High ATP demand
  Each N₂ → 2 NH₃ conversion costs 16 MgATP and requires a steady stream of reduced ferredoxin. Aerial plant tissues must divert enormous amounts of photosynthate to fuel this process, at the risk of starving growth and yield.

• Carbon “sink” vs. photosynthesis
  Unlike rhizobia, which can tap soil carbon, plants would must balance their own sugar needs against an ATP-hungry nitrogenase.

Implication: Even if nif expression and O₂ protection were solved, the metabolic drain could cripple the plant unless tightly regulated to match developmental or environmental demands.

5. Practical and Technical Barriers

• Vector capacity and stability
  Delivering ~24 kb of coding sequence plus engineered promoters in one or multiple T-DNA constructs strains current plant‐transformation methods.

• Transgene silencing
  Twenty separate expression cassettes amplify the risk of methylation and small-RNA–mediated silencing over generations.

• Field performance and biosafety
  Uncontrolled or leaky nif expression could cause unanticipated ecological interactions or fitness penalties.

6. Is It Totally Impossible?—Emerging Strategies

While a wholesale transfer of the bacterial nif system into plants is currently impractical, several alternative or intermediate approaches are under active research:

1. Symbiont Engineering
   – Super-fixing rhizobia: Enhance nifL deregulation, hydrogenase uptake, or leghemoglobin expression to boost bacterial fixation in nodules.

2. Chloroplast Transformation
   – Operon‐like expression in plastids: Plastids retain prokaryotic gene‐expression features (polycistronic RNAs, bacterial ribosomes), making them a more natural home for nif operons.

3. Synthetic Nodule Formation
   – Engineering micro-organs: Induce nodule‐like structures in non-legume roots by co-expressing nodulation genes, then introduce symbiotic bacteria.

4. Protein Micro-Compartments
   – Designer organelles: Use self-assembling protein shells (encapsulins) to create O₂-impermeable nanocompartments loaded with nitrogenase and ATP-regenerating enzymes.

5. Signal‐Triggered Expression
   – Nitrogen‐deprivation–responsive promoters: Identify or create plant promoters that switch on under low soil-nitrate conditions to limit ATP drain to times when the benefit outweighs the cost.

Conclusion

A fully autonomous, nitrogen-fixing crop plant—one that expresses and regulates a complete bacterial nif system, protects it from oxygen, and meets its enormous ATP needs—remains beyond our current capabilities. However, hybrid strategies—improving symbiont performance, exploiting plastid genetics, and building synthetic compartments—offer incremental but tangible steps toward reducing fertilizer dependence. So, while a “magic bullet” transgenic plant isn’t possible today, the toolbox of synthetic biology continues to grow—and with it, the promise of one day achieving biologically fixed nitrogen in the plant kingdom.

Explain how the hydrogenase enzyme makes the nitrogen fixation process of diazotrophic microbes more efficient.

Hydrogenase improves the overall efficiency of biological nitrogen fixation by recapturing energy and electrons that would otherwise be wasted as H₂ gas. Here’s exactly how it works, step by step:

1. The Nitrogenase Side Reaction Squanders Energy

• Primary reaction:
  N₂ + 8H⁺ + 8e⁻ + 16 MgATP → 2 NH₃ + H₂ ↑ + 16 MgADP + 16 Pi

• Energetic cost: For every molecule of N₂ reduced, nitrogenase hydrolyzes 16 ATP.

• Side reaction: A substantial fraction (40–60%) of the electron flux through nitrogenase ends up reducing protons to H₂ rather than N₂. That H₂ simply diffuses away, representing both lost reducing equivalents (electrons) and wasted ATP.

2. Hydrogenase Recovers Lost Reducing Power

• Reaction catalyzed by uptake hydrogenase:
  H₂ + acceptor → 2 H⁺ + 2 e⁻

• Mechanism: Specialized membrane-oriented hydrogenases (Hup) bind H₂ released by nitrogenase and oxidize it back to protons, feeding the liberated electrons into the cell’s electron‐transport chain.

• Energetic payoff: Those electrons regenerate the reduced carriers (e.g. ferredoxin, flavodoxin) that drive nitrogenase and produce a proton gradient that can be used to synthesize ATP. In effect, ATP spent to make H₂ is partially recovered when hydrogenase funnels H₂ back through the respiratory chain.

3. Shifting the Nitrogenase Equilibrium

• Le Châtelier’s principle: By continually removing H₂ from the vicinity of nitrogenase, hydrogenase helps drive the reaction toward even more N₂ reduction (less product inhibition by H₂).

• Low local H₂ concentration: Prevents buildup of inhibitory H₂ partial pressure inside the nodule or bacterial cell, allowing nitrogenase to operate closer to its maximal rate.

4. Improved Plant Growth and Nitrogen Yield

• Empirical observations:
  – Soybean plants inoculated with Hup⁺ Bradyrhizobium japonicum strains accumulated more biomass and fixed nitrogen than those inoculated with Hup⁻ strains—despite slightly lower raw nitrogenase activity in the Hup⁺ bacteria.
  – Engineered Hup⁺ Rhizobium leguminosarum (with overproduced hydrogenase) showed a two‐fold increase in H₂ uptake and virtually eliminated H₂ evolution from nodules, resulting in higher plant nitrogen content and growth across nickel‐poor soils.

• Conclusion: The ability to recycle H₂ back into the bacterial energy metabolism makes the whole nitrogen‐fixation process more ATP‐efficient, translating into more net fixed nitrogen delivered to the plant partner.

5. Genetic Basis and Engineering Implications

• hup gene clusters: In B. japonicum and R. leguminosarum, uptake hydrogenase genes (hup) span ~20 kb and include multiple subunits plus accessory hyp genes for nickel‐cofactor assembly.

• Engineering strategy:

  1. Genetic complementation of Hup⁻ mutants with hup-containing cosmids restores hydrogen uptake.

  2. Promoter swapping (e.g. replacing the NifA‐dependent hup promoter with an FnrN‐dependent promoter) decouples hydrogenase expression from the stringent nif regulatory circuit, boosting Hup levels under low‐O₂ in nodules.

• Outcome: Enhanced hydrogenase expression converts more of the wasted H₂ back into cellular energy, further optimizing nitrogen fixation efficiency.

In summary, hydrogenase acts as an internal recycler, capturing the H₂ “leak” from nitrogenase, reconverting it into protons and electrons that feed back into ATP production and electron carriers—thereby increasing the net yield of ammonia per ATP invested and delivering more fixed nitrogen to the plant.

How does glycogen synthesis affect the ability of a strain of Rhizobium to fix nitrogen?

Glycogen synthesis and nitrogen fixation in rhizobia are in direct competition for the same intracellular carbon—and thus energy—resources. Because biological N₂-fixation by nitrogenase is one of the most ATP-hungry processes in nature, anything that diverts carbon away from energy production toward storage (glycogen) will limit the capacity of the cell to fix nitrogen. Here’s how it works in mechanistic detail:

1. The Energetic Demands of Nitrogen Fixation

• Nitrogenase reaction:

  N2​+8H++8e−+16 MgATP⟶2NH3​+H2​+16 MgADP+16 Pi​

  Fixing one molecule of N₂ consumes 16 molecules of ATP plus a continuous supply of reduced electron carriers (ferredoxin or flavodoxin).

• Respiration’s dual role:

  1. Energy generation—oxidative phosphorylation in the TCA cycle/ETC regenerates the ATP pool.

  2. O₂ scavenging—high respiratory rates keep intracellular O₂ low, protecting oxygen-sensitive nitrogenase.

2. Carbon Partitioning: Glycogen vs. Respiration

Rhizobia break down glucose via glycolysis to pyruvate, which enters the TCA cycle as acetyl-CoA. At each turn of the cycle, electrons are fed into the electron-transport chain (ETC), driving ADP → ATP.

• Glycogen synthesis:

  • Glucose-6-P is diverted into the glycogen pathway by glycogen synthase, producing insoluble glycogen granules.

  • Glycogen is a carbon reserve, but its synthesis withdraws glucose from glycolysis/TCA and thus reduces ATP production and O₂ consumption.

• Respiratory flux:

  • When all glucose flows into glycolysis/TCA, maximum NADH/FADH₂ is produced, fueling the ETC and ATP synthase.

  • High respiration both powers nitrogenase and maintains a low-O₂ environment in nodules or free-living cells.

3. Glycogen Synthase Mutants: Shifting the Balance

In the classic Rhizobium tropici glycogen synthase (glgA) deletion mutant:

1. No glycogen formation: all glucose-6-P must proceed through glycolysis and the TCA cycle.

2. Elevated respiration: increased flux of electrons into the ETC boosts ATP yield per unit glucose.

3. Enhanced O₂ removal: faster oxygen consumption protects nitrogenase from inactivation.

4. Improved nitrogen fixation:

   • Plants inoculated with the ΔglgA strain developed more nodules and showed higher dry weight compared to wild-type inoculations.

   • The extra ATP and better microaerobic conditions translate into higher rates of ammonia production per bacterial cell.

4. Agricultural Implications and Trade-Offs

• Benefits:

  • ΔglgA strains act like “super-fixers,” channeling every available carbon toward N₂ reduction rather than storage.

  • These strains can give immediate boosts in plant biomass and nitrogen content, especially in early growth stages.

• Drawbacks:

  1. Lack of carbon reserve: without glycogen, bacteria cannot survive transient starvation or environmental stress as well—they cannot revert to wild-type and may fail to persist in the field.

  2. Re-inoculation needed: the ΔglgA population will decline over time, requiring periodic reapplication of inoculant.

  3. Potential metabolic imbalance: in fluctuating environments, inability to buffer carbon fluxes might sensitize cells to sudden nutrient shortages.

5. Conclusion

By knocking out glycogen synthase, rhizobia are forced to drive all their glucose into energy-producing pathways. This increases both ATP availability for the 16-ATP‐per‐N₂ nitrogenase reaction and the rate of O₂ consumption to protect nitrogenase. The net result is a strain with higher effective nitrogen‐fixation capacity, at the cost of cellular resilience and long-term persistence in the soil.

What strategies can be employed to increase the effectiveness of biocontrol strains?

1. Give it better tools

   • Stronger “weapon” genes: Swap in promoters (the on-switches) that make more of its antibiotic or lytic-enzyme proteins so it can better attack pathogens.

   • Stress-proofing: Add genes that help it survive heat, UV or dryness (e.g. by making protective sugars), so more cells stay alive on seeds or leaves.

2. Partner it up

   • Microbe teams: Instead of one strain, use a small mix of complementary bugs—one makes antibiotics, another primes the plant’s immune system, a third helps gather nutrients—so you cover more bases.

   • Friendly food: Add simple sugars or other “prebiotics” to the soil or seed coating that your biocontrol strain loves, giving it a head-start over unwanted microbes.

3. Smarter delivery

   • Coatings & carriers: Encapsulate cells in beads or mix them into peat, biochar or alginate so they survive storage and release slowly when planted.

   • Seed coatings: Coat seeds directly so the bug jumps right into the young root’s neighborhood where it’s needed.

4. Wake up the plant

   • Immune-priming signals: Choose or tweak strains that naturally release small molecules (like flagellin fragments) that tell the plant, “Hey, gear up your defenses!”

5. Keep the bad guys out

   • Quorum-quenchers: Arm your strain with enzymes that block the chemical “chat” (quorum signals) pathogens use to coordinate an attack.

   • Phage backup: In some cases, adding bacteriophages (viruses that attack specific bacteria) alongside can thin out tough bacterial pathogens without harming your good strain.

Key takeaway:

• Boost its survival (stress-proof, better food)

• Boost its weapons (more antibiotics/enzymes)

• Deliver it smartly (seed coats, carriers)

• Use teamwork (microbial cocktails, phages)

• Help the plant too (immune-primers)

What would you expect the effect of mutagenizing either nifA  or nifL to be on the amount of nitrogen that an organism can fix?

1. Mutating nifA (the positive activator)

Normal role of NifA

• Binds a specific 5'-TGT-N₁₀-ACA-3' motif in the promoter of every nif operon

• Recruits σ⁵⁴ (the alternative sigma factor) and the RNA polymerase II-like machinery in bacteria

• Turns on transcription of all nif genes (except its own), so that nitrogenase components I & II plus the 15–20 accessory proteins are made in the right amounts

What happens when you knock out or inactivate nifA

1. No “go” signal: σ⁵⁴ can’t be stably recruited to any nif promoter.

2. Transcription collapse: mRNAs for both nitrogenase subunits and all accessory factors essentially drop to zero.

3. No enzyme → no activity: Without the proteins in place, the N₂→NH₃ reaction cannot occur.

4. Net effect: The mutant becomes a classic Nif⁻ strain—virtually zero nitrogen‐fixing ability under any condition.

2. Mutating nifL (the negative regulator)

Normal role of NifL

• Senses both oxygen and fixed-nitrogen (e.g. NH₄⁺) levels

• Binds and antagonizes NifA under non-ideal conditions (high O₂ or plenty of NH₄⁺)

• Turns off nif transcription when fixing nitrogen would be wasteful or when oxygen would irreversibly damage nitrogenase

What happens when you knock out or inactivate nifL

1. Brake removed: NifA is free to activate its target promoters regardless of ambient O₂ or NH₄⁺.

2. Constitutive nif expression: The operons encoding nitrogenase I & II and all accessory proteins are transcribed continuously, even when the cell would normally shut them down.

3. Higher enzyme levels: More nitrogenase complexes assemble, ready to reduce N₂ whenever ATP and electrons are available.

4. Increased N₂‐fixation capacity: Under many conditions, a nifL⁻ mutant will fix more nitrogen than wild type—especially when external NH₄⁺ or fluctuating O₂ would normally inhibit the process.

5. Physiological trade-off: Because nitrogenase consumes 16 ATP per N₂, the strain may grow more slowly or require extra carbon/energy to support its hyperactive nitrogenase.

Summary Table

In practical terms:

• A nifA mutant cannot fix nitrogen at all. Such strains serve as genetic “null” controls in complementation studies.

• A nifL mutant or a strain carrying “deregulated” NifA (e.g. NifL less sensitive to NH₄⁺) often shows enhanced plant growth promotion in symbiosis—so long as there’s enough photosynthate to pay the ATP bill.

By choosing which regulator to tweak, you can either knock out nitrogen fixation (nifA mutation) or turbo-charge it (nifL mutation)—each with predictable, opposite effects on the amount of ammonia an organism can produce.