Raymond C. Valentine

The mechanism of ammonia assimilation in nitrogen fixing bacteria

SummaryEnzymatic and genetic evidence are presented for a new pathway of ammonia assimilation in nitrogen fixing bacteria: ammonium → glutamine → glutamate. This route to the important glutamate-glutamine family of amino acids differs from the conventional pathway, ammonium → glutamate → glutamine, in several respects. Glutamate synthetase [(glutamine amide-2-oxoglutarate aminotransferase) (oxidoreductase)], which is clearly distinct from glutamate dehydrogenase, catalyzes the reduced pyridine nucleotide dependent amination of α-ketoglutarate with glutamine as amino donor yielding two molecules of glutamate as product. The enzyme is completely inhibited by the glutamine analogue DON, whereas glutamate dehydrogenase is not affected by this inhibitor; the glutamate synthetase reaction is irreversible. Glutamate synthetase is widely distributed in bacteria; the pyridine nucleotide coenzyme specificity of the enzyme varies in many of these species.The activities of key enzymes are modulated by environmental nitrogenous sources; for example, extracts of N2-grown cells of Klebsiella pneumoniae form glutamate almost exclusively by this new route and contain only trace amounts of glutamate dehydrogenase activity whereas NH3-grown cells possess both pathways. Also, the biosynthetically active form of glutamine synthetase with a low Kmfor ammonium predominates in the N2-grown cell.Several mutant strains of K. pneumoniae have been isolated which fail to fix nitrogen or to grow in an ammonium limited environment. Extracts of these strains prepared from cells grown on higher levels of ammonium have low levels of glutamate synthetase activity and contain the biosynthetically inactive species of glutamine synthetase along with high levels of glutamate dehydrogenase. These mutants missing the new assimilatory pathway have serious defects in their metabolism of many inorganic and organic nitrogen sources; utilization of at least 20 different compounds is effected. We conclude that the new ammonia assimilatory route plays an important role in nitrogenous metabolism and is essential for nitrogen fixation.

The electron transport system in nitrogen fixation by Azotobacter. III. Requirements for NADPH-supported nitrogenase activity

Abstract Evidence has been obtained that NADPH may serve as a physiological source of reducing power for nitrogenase activity in Azotobacter vinelandii . NADH was ineffective. Electron transfer from NADPH to nitrogenase depended on four factors native to A. vinelandii cells: azotobacter ferredoxin, azotoflavin, a component replaceable by spinach ferredoxin-NADP + reductase and another soluble, heat-labile component not yet chemically characterized. The four factors probably constitute an electron transport chain between NADPH and nitrogenase.

The pathways of nitrogen fixation.

Publisher Summary Biological nitrogen fixation is the enzymic reduction of atmospheric nitrogen to ammonia. Along with the scientific interest of nitrogen fixation as a fundamental biochemical reaction, it is also of great ecological and agricultural importance because it is the most important source of the metabolizable nitrogen needed by all living organisms. Nitrogen fixation is catalyzed by nitrogenase, which requires energy in the form of ATP and a biologically strong reductant for the formation of ammonia. Moreover, the chapter also gives a general review of the field of nitrogen fixation with emphasis on recent developments. The study of the biochemical genetics of nitrogen fixation can yield important basic knowledge regarding the pathway, mechanism, and regulation of this process. Furthermore, in symbiotic nitrogen fixation, both plant and host bacterium carry genetic information relevant to this process, making the genetics of the system complex. Finally, understanding of the biochemical relationships in symbiotic nitrogen fixation might allow the extension of this process to non-leguminous agricultural crops.

Host-dependent mutants of the bacteriophage f2 II. Rescue and complementation of mutants☆

Abstract Host-dependent mutants of f2 are classed in two types on the basis of the physiological response of nonpermissive bacterial hosts. Mixed infection with helper wildtype phage rescues the mutants. Mutants of the two types will complement each other in mixed infection. Mutants of the same type do not complement each other. Two phage cistrons are thus defined.

The F-Pilus of Escherichia coli

Publisher Summary This chapter deals with F-pilus—sex hair, sex pilus, F-fimbria, and sex firnbria— of Escherichia coli . It essentially describes methods of assay and synthesis of F-pili, their functions and properties, and the mutant approach to the F-pilus problem. F-pili first observed, differed from common pili by the random adsorption of small RNA viruses along their sides. F-pili are both longer and wider than Type I or common pili. Frequent appearance of knobs or enlargements at their distal extremities is also used to distinguish F-pili in micrographs. Free F-pili from the supernatant of a culture of male cells can be treated with ultrasonic vibrations to yield smaller fragments, which are capable of adsorbing phage, largely confirm that the filaments are made of number of repeating structural units, each of which can bind the RNA phage. Knowledge of the structure of F-pili can essentially shed light on various functions, which these filaments perform in phage injection and mating. F-pili also play an important role in the mating process of E .coli and also serve the function of adsorption organelles for male phages.

The Replication Cycle of RNA Bacteriophages

Publisher Summary The RNA phages indeed deserve to be considered among the “living world.” They possess well-developed powers of genetic regulation, morphogenesis, chromosomal replication, and even adaptation. In the broadest sense the virus must use all of its genetic and chemical potential, while making maximal use of existing host reactions. In other words, by prudent use of its chemical potential these tiny phages have achieved the “living” state with far fewer genes than higher organisms. The viral proteins themselves are highly versatile in function, and it seems clear that studies directed toward understanding the functional nature of a given viral protein will lead to the discovery of multiple “active sites” on the protein. The tiny RNA chromosome, itself a functional partner in many of these reactions, must also be graced with a multitude of “active sites.” These sites behave as special genetic elements and function, for example, as adsorption points for viral polymerase or capsid as repressor. These sites give the chromosome a type of communication system with the cytoplasm. In one case, a chromosome-cytoplasm circuit of communication is achieved when the capsid protein locks on the chromosome, and switches off the polymerase at a certain time in the infective cycle. This feedback circuit using capsid, itself a product of the infection, endows the virus with powers of self-regulation. The initiation of the RNA replication cycle may require an even more sophisticated protein-chromosome interaction, one where the polymerase is forced to seek out a single parental molecule to use as template among a variety of different host RNA species. When this union is achieved, the infection switches from a purely translational state to the transcriptional cycle where many more copies of RNA are made. The chromosome then appears to monitor the work of the infection going on around it through this form of communication.

An endogenous electron carrier for the nitrogenase system of Rhizobium bacteroids.

Abstract A partly purified electron carrier isolated from an extract of Rhizobium bacteroids (from soybean root nodules) mediated the transfer of reducing power generated by illuminated spinach chloroplasts to the nitrogenase of Rhizobium bacteroids or of Azotobacter vinelandii . The electron carrier is reducible by dithionite and, according to preliminary evidence, is similar to the recently reported Azotobacter type of ferredoxin.

PHOTOBIOLOGY OF RNA BACTERIOPHAGES—I. ULTRAVIOLET INACTIVATION AND PHOTOREACTIVATION STUDIES*

Abstract— Biologically active f2‐RNA, Obtained from bacteriophage f2, was inactivated by ultraviolet (u.v) light (2537 Å) with a quantum yield of 3.3 ± 0.3 times 10‐3 when assayed in the dark with protoplasts of an F‐ strain of E. coli k12. Assay under “black light” gave a quantum yield of 2.7 ± 0.5 times 10‐3 which was just enough lower to suggest that 17 per cent photorecovery of the u.v. lesions has taken place.

PHOTOBIOLOGY OF RNA BACTERIOPHAGES–II U.V.–IRRADIATION OF f2: EFFECTS ON EXTRACELLULAR STAGES OF INFECTION AND ON EARLY REPLICATION*

Abstract— The effect of u.v. irradiation (2537 Å) on the RNA bacteriophage f2 has been studied with respect to the adsorption of f2 to E. coli K12 (male strain), the penetration of f2‐RN A into the host cell and the conversion of the phage nucleic acid to the double‐stranded replicative intermediate. The biological parameter most sensitive to u.v. was the plaque‐forming ability of the phage. Its loss could be attributed to several factors. (1). A binding of capsid protein to phage nucleic acid interfering with host penetration by the f2‐RNA. (2). Desorption of some irradiated phage at 37° from their attachment sites on the host. (3). Molecular alterations in the RNA preventing formation of the replicative intermediate within the host.

High-Energy Electrons in Bacteria

Publisher Summary High-energy electrons are of fundamental importance to the bacterial cell. They play important roles in many of the basic processes of bacterial metabolism, both in aerobic and anaerobic cells. High-energy electrons produced in one biochemical reaction are linked via specific electron carriers to reductive cellular processes. In the simplest instances, ferredoxin works with a dehydrogenase and a reductase linking these two enzymes together. In some cases, the electron-transport chain of such pathways is more complex, as in Azobacter, where additional carriers are involved. Electrons that feed into electron-transport chains are supplied from donors such as a-keto acids, formate, molecular hydrogen, hypoxanthine, acetaldehyde, “excited” chlorophyll, and reduced nicotinamide nucleotides. A variety of compounds function as acceptors of high-energy electrons such as nicotinamide nucleotides (NAD and NADP), carbon dioxide, and molecular nitrogen. One of the reactions, the reduced nicotinamide nucleotide to ferredoxin couple, acts as an important source of high-energy electrons mid and may be found to play a key role in several organisms. This nicotinamide nucleotide-Fd reaction also appears to be vital for the nitrogen-fixation pathway of Azotobacter and photosynthetic bacteria. One of the most interesting aspects of the low-redox chains discussed is the high degree of specificity of the redox reactions. It seems clear that the high-energy carriers must possess a complex series of active sites on their surfaces to allow the selective coupling to other carriers. The specificity of high-energy carriers means the individual electrons themselves must be tucked away—or perhaps buried—in the interior of the protein body, thus protecting them from seizure by surrounding electron-deficient compounds.