Regulation Of Nitrogen Fixation Research Articles

Continuous CO2 enrichment leads to increased nodule biomass, carbon availability to nodules and activity of carbon‐metabolising enzymes but does not enhance specific nitrogen fixation in pea

Recent research has shown that nodule nitrogen fixation is limited under a wide range of environmental constraints by lowered carbon flux within the nodule due to down‐regulation of sucrose synthase activity. The aim of this work was to elucidate whether an increase in both carbon flux and activity of enzymes of carbon metabolism in nodules may lead to an increased nitrogen fixation. We report the effects caused by a continuous exposure to atmospheric CO2 enrichment in nodulated pea plants. CO2 enrichment led to an enhanced whole‐plant growth and increased nodule biomass. Moreover, nodules of plants grown at increased CO2 showed a higher sugar content as well as enhancement of some activities related to nodule carbon metabolism, such as sucrose synthase, UDP glucose pyrophosphorylase and phosphoenolpyruvate carboxylase. Indeed, acetylene reduction activity, measured by the classical technique, was increased more than four times. However, when specific nitrogen fixation was determined as hydrogen evolution, no significant differences were detected, consistent with the lack of changes of enzymes involved in nitrogen metabolism such as glutamate synthase and aspartate aminotransferase. These results are discussed in the context of the regulation of nitrogen fixation and nodule metabolism.

Differential regulation of fixN-reiterated genes in Rhizobium etli by a novel fixL-fixK cascade.

Among the complexities in the regulation of nitrogen fixation in the Rhizobiaceae are reiteration of regulatory components as well as variant roles for each component between species. For Rhizobium etli CFN42, we reported that the symbiotic plasmid (pCFN42d) contains a key regulatory gene (fixKd) and genes for a symbiotic cytochrome oxidase (fixNOQPd). Here we discuss the occurrence of reiteration of these genes (fixKf and fixNOQPf) and the finding of an unusual fixL homolog on a plasmid previously considered cryptic (pCFN42f). The structure of the deduced FixL polypeptide is suggestive of a fusion of the receiver and transmitter modules of a two-component regulatory system as described in R. leguminosarum bv. viciae VF39. Gene fusion analysis, coupled with mutation of each regulatory element, revealed that free-living expression of FixKf was dependent fully on FixL. In contrast, synthesis of FixKd was not detected under the conditions tested. The FixKf protein is needed for microaerobic expression of both fixN reiterations, whereas the FixKd protein appears to be dispensable. Interestingly, expression of the fixN reiterations exhibits a differential dependence for FixL, where transcription of fixNf was suppressed in the absence of FixL but expression of fixNd still showed significant levels. This suggests the existence of a FixL-independent mechanism for expression of the fixNd reiteration. Surprisingly, mutations in fixL, fixKd, or fixKf (either singly or in combination) did not alter symbiotic effectiveness. A mutation in fixNd (but not in fixNf) was, however, severely affected, indicating a differential role for these reiterations in nitrogen fixation.

Signal transduction to the Azotobacter vinelandii NIFL-NIFA regulatory system is influenced directly by interaction with 2-oxoglutarate and the PII regulatory protein.

PII-like signal transduction proteins, which respond to the nitrogen status via covalent modification and signal the carbon status through the binding of 2-oxoglutarate, have been implicated in the regulation of nitrogen fixation in several diazotrophs. The NIFL-NIFA two-component regulatory system, which integrates metabolic signals to fine-tune regulation of nitrogenase synthesis in Azotobacter vinelandii, is a potential target for PII-mediated signal transduction. Here we demonstrate that the inhibitory activity of the A.vinelandii NIFL protein is stimulated by interaction with the non-uridylylated form of PII-like regulatory proteins. We also observe that the NIFL-NIFA system is directly responsive to 2-oxoglutarate. We propose that the PII protein signals the nitrogen status by interaction with the NIFL-NIFA system under conditions of nitrogen excess, and that the inhibitory activity of NIFL is relieved by elevated levels of 2-oxoglutarate when PII is uridylylated under conditions of nitrogen limitation. Our observations suggest a model for signal transduction to the NIFL-NIFA system in response to carbon and nitrogen status which is clearly distinct from that suggested from studies on other diazotrophs.

Nitrosyl adducts of FixL as probes of heme environment.

This report presents a spectroscopic investigation of the nitrosyl adducts of FixL, the sensor in the signal transduction system responsible for regulating nitrogen fixation in Rhizobium meliloti. Variable-temperature resonance Raman (RR), electron spin resonance (ESR), and variable-temperature UV-visible absorption data are presented for the ferrous NO adducts of two FixL deletion derivatives, FixLN (the heme-containing domain) and FixL* (a functional heme-kinase). A temperature-dependent equilibrium is observed between the five-coordinate (5-c) and six-coordinate (6-c) ferrous nitrosyl adducts, with lower temperatures favoring formation of the 6-c nitrosyl adduct. This equilibrium is perturbed as the solution freezes, and the amount of 5-c FixL-NO increases sharply until a nearly constant ratio of 6-c to 5-c adducts is obtained. Complexation between the heme domain of FixL and its response regulator, FixJ, is revealed through specfic FixJ-induced increase in the energy separation between 5-c and 6-c FixL-NO. Ferric nitrosyl adducts of FixL* and FixLN autoreduce to their corresponding ferrous nitrosyl adducts. The kinetic behavior of this reduction is monophasic for FixL*-NO, while the reaction for ferric FixLN-NO is biphasic. These results suggest conformational inhomogeneity in the heme pocket of FixLN and conformational homogeneity in that of FixL*. Hence the kinase domain plays a role in distal pocket conformational stability. Implications for the signal transduction mechanism are discussed.

Symbiotic induction of pyruvate dehydrogenase genes from Sinorhizobium meliloti.

Genes coding for components of the pyruvate dehydrogenase (PDH) multienzyme complex (PDHc) from Sinorhizobium meliloti, the alfalfa symbiont, have been isolated on the basis of their high expression in symbiotic bacteria. The Elp component, PDH, is encoded by two genes, pdhAalpha (1,047 bp) and pdhAbeta (1,383 bp), a situation encountered in the alpha-proteobacteria Rickettsia prowazekii and Zymomonas mobilis as well as in some gram-positive bacteria and in mitochondria. pdhAalpha and pdhAbeta precede pdhB (1,344 bp), which encodes the E2p component, dihydrolipoamide acetyltransferase, of the PDHc. No gene encoding the E3 component, lipoamide dehydrogenase, was found in the immediate vicinity of pdhA and pdhB genes. pdhAalpha, pdhAbeta and pdhB likely constitute an operon. Here, we provide evidence that pdhA expression is induced in the symbiotic stage, compared with free-living conditions. We demonstrate that symbiotic expression of pdhA genes does not depend on the fix LJ regulatory cascade that regulates nitrogen fixation and respiration gene expression in symbiotic S. meliloti cells. Induction of pdhA expression could be obtained under free-living conditions upon the addition of pyruvate to the culture medium. Induction by pyruvate and symbiotic activation of pdh gene expression take place at the same promoter.

Mutagenesis and functional characterization of the glnB, glnA, and nifA genes from the photosynthetic bacterium Rhodospirillum rubrum.

Nitrogen fixation is tightly regulated in Rhodospirillum rubrum at two different levels: transcriptional regulation of nif expression and posttranslational regulation of dinitrogenase reductase by reversible ADP-ribosylation catalyzed by the DRAT-DRAG (dinitrogenase reductase ADP-ribosyltransferase-dinitrogenase reductase-activating glycohydrolase) system. We report here the characterization of glnB, glnA, and nifA mutants and studies of their relationship to the regulation of nitrogen fixation. Two mutants which affect glnB (structural gene for P(II)) were constructed. While P(II)-Y51F showed a lower nitrogenase activity than that of wild type, a P(II) deletion mutant showed very little nif expression. This effect of P(II) on nif expression is apparently the result of a requirement of P(II) for NifA activation, whose activity is regulated by NH(4)(+) in R. rubrum. The modification of glutamine synthetase (GS) in these glnB mutants appears to be similar to that seen in wild type, suggesting that a paralog of P(II) might exist in R. rubrum and regulate the modification of GS. P(II) also appears to be involved in the regulation of DRAT activity, since an altered response to NH(4)(+) was found in a mutant expressing P(II)-Y51F. The adenylylation of GS plays no significant role in nif expression or the ADP-ribosylation of dinitrogenase reductase, since a mutant expressing GS-Y398F showed normal nitrogenase activity and normal modification of dinitrogenase reductase in response to NH(4)(+) and darkness treatments.

Rhizobium etli Genetically Engineered for the Heterologous Expression of Vitreoscilla sp. Hemoglobin: Effects on Free-Living and Symbiosis

Oxygen concentration is an environmental signal that regulates nitrogen fixation in the Rhizobium-legume symbiosis. We investigated the effect of the heterologous expression of Vitreoscilla sp. hemoglobin (VHb), which is an oxygen-binding protein, in Rhizobium etli. The vhb gene and its native promoter were subcloned in the plasmid pMR4 and transformed into the R. etli strain CE3. Free-living cultures of engineered R. etli CE3 expressed the vhb gene, as shown by the CO-difference spectral and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses of cell extracts. The expression of vhb in free-living R. etli grown under most limiting oxygen concentrations resulted in an increase in respiratory activity, chemical energy content, and expression of the nitrogen-fixation gene nifHc. Bacteroids isolated from nodules of bean plants inoculated with the engineered R. etli CE3 expressed the vhb gene, as shown by RNA slot-blot analysis. Bean plants inoculated with the engineered strain exhibited higher nitrogenase activity and total nitrogen content (68% and 14 to 53%, respectively) than bean plants inoculated with the R. etli wild type. These results suggest that the synthesis of VHb in engineered R. etli stimulated the respiratory efficiency of free-living rhizobia, and also probably of symbiotic bacteroids, thus leading to higher levels of symbiotic nitrogen fixation.

Interaction of Azospirillum lipoferum with wheat germ agglutinin stimulates nitrogen fixation.

In vitro, the nitrogen fixation capability of A. lipoferum is efficiently increased in the presence of wheat germ agglutinin (WGA). A putative WGA-binding receptor, a 32-kDa protein, was detected in the cell capsule. The stimulatory effect required N-acetyl-D-glucosamine dimer (GlcNAcdi) terminated sugar side chains of the receptor and was dependent on the number of GlcNAcdi links involved in receptor-WGA interface. Binding to the primary sugar binding sites on WGA had a larger stimulatory effect than binding to the secondary sites. The WGA-receptor complex generated stimulus led to elevated transcription of the nifH and nifA genes and of the glnBA gene cluster but not of the glnA gene from its own promoter. There may well be a signalling cascade contributing to the regulation of nitrogen fixation.

Structural transitions in the FixJ receiver domain.

Two-component signal transduction pathways are sophisticated phosphorelay cascades widespread in prokaryotes and also found in fungi, molds and plants. FixL/FixJ is a prototypical system responsible for the regulation of nitrogen fixation in the symbiotic bacterium Sinorhizobium meliloti. In microaerobic conditions the membrane-bound kinase FixL uses ATP to transphosphorylate a histidine residue, and the response regulator FixJ transfers the phosphoryl group from the phosphohistidine to one of its own aspartate residues in a Mg(2+)-dependent mechanism. Seven X-ray structures of the unphosphorylated N-terminal receiver domain of FixJ (FixJN) have been solved from two crystal forms soaked in different conditions. Three conformations of the protein were found. In the first case, the protein fold impairs metal binding in the active site and the structure reveals a receiver domain that is self-inhibited for catalysis. In the second conformation, the canonical geometry of the active site is attained, and subsequent metal binding to the protein induces minimal conformational changes. The third conformation illustrates a non-catalytic form of the protein where unwinding of the N terminus of helix alpha 1 has occurred. Interconversion of the canonical and self-inhibited conformations requires a large conformational change of the beta 3-alpha 3 loop region. These unphosphorylated structures of FixJN stress the importance of flexible peptide segments that delineate the active site. Their movements may act as molecular switches that define the functional status of the protein. Such observations are in line with structural and biochemical results obtained on other response regulator proteins and may illustrate general features that account for the specificity of protein-protein interactions.

Heme speciation in alkaline ferric FixL and possible tyrosine involvement in the signal transduction pathway for regulation of nitrogen fixation.

The pH-dependent behavior of the ferric forms of two soluble truncations of Rhizobium meliloti FixL, FixL (heme and kinase domains, functional), and FixLN (heme domain) are examined by UV-visible, resonance Raman, and electron paramagnetic resonance spectroscopy. Global analysis of UV-visible data indicates that the pKa for hydroxide binding is slightly higher in FixL than in FixLN. Spectroscopic data show that high-spin and low-spin hydroxide adducts of FixLN and FixL exist in a thermal spin-state equilibrium with a significant fraction of the heme in the high spin form at room temperature. FixLN and FixL differ from myoglobin and hemoglobin in that their hemes are not fully ligated by hydroxide ion under strongly alkaline conditions. In addition to the binding of hydroxide ion, both FixLN and FixL undergo additional alkaline transitions that involve the deprotonation of tyrosine residues. FixLN contains four tyrosine residues. One has a pKa of 9.6, which is indistinguishable from that for hydroxide binding to the heme. The other three tyrosines have pKas greater than 11. At pH 11, the alkaline species react with cyanide to yield the familiar low-spin cyanide adduct. Upon reduction of the heme iron, the alkaline forms of the FixL deletion derivatives are converted to their deoxy forms. Resonance Raman spectra reveal that the Fe-His stretching vibrations of deoxyFixLN and deoxyFixL are not measurably shifted from those of their neutral counterparts. Treatment of the alkaline deoxyFixLs with O2 yields the respective oxy forms. Spectroscopic evidence indicates that the loss of activity at elevated pH cannot be attributed solely to generation of a low-spin heme hydroxide. Involvement of one or more tyrosines in signal transmission between the heme and kinase domains of FixL is proposed.

Regulation of nitrogen fixation by different nitrogen sources in the filamentous non-heterocystous cyanobacterium Microcoleus sp.

The pattern of N 2 fixation, the synthesis and activity of nitrogenase under different nitrogen sources was studied in the filamentous, non-heterocystous cyanobacterium Microcoleus sp. grown under defined culture conditions. Cells grown under a 10 h light/14 h dark (10L/14D) cycle with N 2 as an inorganic nitrogen source showed highest nitrogenase activity (acetylene reduction) at the end of the light phase and then a decrease after entering the dark phase. Nitrogenase synthesis was neither suppressed after 7 days of growth with 2 mM NaNO 3 or 0.2 mM (NH 4) 2SO 4 or 0.3 mM urea nor with 20 mM NaNO 3 or 3 mM (NH 4) 2SO 4 or 4 mM urea under the 10L/14D cycle. Western immunoblots tested with polyclonal antisera against the Fe-protein revealed the following: (1) the Fe-protein was synthesized in cells grown with N 2 as well as in cells grown with NaNO 3 or (NH 4) 2SO 4 under the 10L/14D cycle; (2) the Fe-protein was found in cells grown with urea under the 10L/14D cycle, but not in the darkness; (3) only one protein band, corresponding to the Fe-protein, was found in cells harvested during the light phase of the 10L/14D cycle under the tested conditions. No nitrogenase activity was observed when chloramphenicol was added to the cultures 4 h before the onset of the light period. This observation suggest de novo synthesis of nitrogenase in Microcoleus sp.

Regulation of nitrogen fixation by ammonium in diazotrophic species of proteobacteria

Nitrogenase enzyme is not synthesized in diazotrophs growing with a sufficient or excess supply of fixed nitrogen in their environment. In addition, in some bacteria the nitrogenase enzyme is inactivated by ammonium. The mechanisms by which cells respond to ammonium have been studied in several members of the Proteobacteria, including species from three of the four subgroups of this major taxon. In all of them, NifA is a transcriptional activator of expression of all other nif genes, the products of which are required for synthesis of active nitrogenase. NifA is either inactivated or not synthesized in ammonium-grown cultures of the Proteobacterial diazotrophs. The mechanisms by which NifA synthesis and activity are controlled by fixed nitrogen vary widely, as does the function of the PII protein, a major component of the nitrogen regulatory cascade, with respect to nitrogen fixation.

Regulation of nif gene expression and nitrogen metabolism in Azospirillum

The Azospirillum genus comprises nitrogen-fixing bacteria which have been isolated from the roots of numerous grasses, including cereals, sugar cane and forage grasses. Among the five species known, Azospirillum brasilense is the best documented in terms of genetics and regulation of nitrogen fixation and ammonia assimilation. Several key regulatory and structural genes have been characterized, including nifA, ntrBC, glnA, glnB and rpoN. Regulation of their expression has been studied at the free-living state and in association with wheat. A model of regulation is reported and the challenge of engineering nitrogen-fixing associations is also discussed.

Alteration of N nutrition in Myrica gale induces changes in nodule growth, nodule activity and amino acid composition

Changes in nodule growth and activity and in the concentrations of soluble N compounds in nodules, leaves and xylem sap under conditions of altered N nutrition in the actinorhizal plant Myrica gale L. are reported. Altering the N nutrition of symbiotic plants may alter the internal regulation of combined N which in turn may regulate nodule growth and activity. Flushing nodules daily with 100% O2 caused a decline in amide concentration and an increase in nodule growth although plants had recovered some nitrogenase activity within 4 h of exposure to O2. Samples of nodules, leaves and xylem sap were derivatized and amino acids identified and quantified using either reverse phase high performance liquid chromatography or gas chromatography‐mass spectrometry in single ion monitoring mode. The ratio of asparagine in the nodules to that in the xylem was much higher in plants fed N (6.7 for NH+4‐fed and 8.3 for NO−3‐fed plants) than for N2‐fixing plants (2.5). Significant amounts of 15N added as 15NH+4 or 15NO−3 accumulated in nodules following accumulation in the shoot which is consistent with the translocation of N to the nodules via the phloem. The uptake of 15NH+4 led to the synthesis and subsequent translocation of glutamine in the xylem sap. These results are discussed in terms of the feedback mechanisms that may regulate nitrogen fixation in Myrica root nodules.

Rapid assimilation of recently fixed N2 in root nodules of Myrica gale

In Myrica gale L. plants the assimilation of ammonia released by symbiotic Frankia was observed by 15N2 labelling and subsequent analysis of the isotopic enrichment of nodule amino acids over time by single ion monitoring gas chromatography‐mass spectrometry. In detached nodules of Myrica, glutamine was the first amino acid labelled at 30 s and subsequently the amino acids glutamate, aspartate, alanine and γ‐amino butyric acid (GABA) became labelled. This pattern of labelling is consistent with the incorporation of ammonium via glutamine synthetase [GS; EC 6.3.1.2]. No evidence for the ammonium assimilation via glutamate dehydrogenase [GDH; EC 1.4.1.2] was observed as glutamate became labelled only after glutamine. Using attached nodules and pulse‐chase labelling, we observed synthesis of glutamine, glutamate, aspartate, alanine, GABA and asparagine, and followed the transport of fixed nitrogen in the xylem largely as glutamine and asparagine. Estimation of the cost of nitrogen fixation and asparagine synthesis in Myrica nodules suggests a minimum of one sucrose required per asparagine produced. Rapid translocation of recently fixed nitrogen was observed in Myrica gale nodules as 80% of the nitrogen fixed during a 1‐h period was translocated out of the nodules within 9 h. The large pool of asparagine that is present in nodules may buffer the transport of nitrogen and thus act to regulate nitrogen fixation via a feedback mechanism.

Regulation of nitrogen fixation in infected cells of leguminous root nodules in relation to O 2 supply

Respiration and nitrogen fixation in legume root nodules is considered to be limited by the rate at which O2 from the atmosphere can enter nodules. A thin diffusion barrier in the inner cortex, restricts access to the central tissue where there is a high demand for and low concentration of O2. Observed variations in rates of nodule activities in response to imposed stresses, are often attributed to variations in the diffusion resistance of the barrier. In the present work, alternative or supplementary metabolic mechanisms are considered. Aspects of nodule structure and of metabolism underlying nodule activities are reviewed in terms of components of the symbiotic system, the nature of steady states and in relation to homeostasis of low concentration of O2 within the bacteroid-filled host cells. It is suggested that variations in O2-demand of both mitochondria and bacteroids, serve to preserve nitrogenase activity by poising O2 concentration within ‘safe’ limits. Further, data from isolated soybean bacteroids suggest that nitrogenase is converted to a less active but more robust form, in the presence of O2 in excess of about 70 nM, thus protecting nitrogenase from irreversible inactivation by excess O2. This regulation is rapidly-reversible when O2 concentration falls below about 0.1 µM. Respiration by large numbers of host mitochondria in the periphery of infected nodule cells, adjacent to gas-filled intercellular spaces, is considered to play an important part in maintaining a steep gradient of O2 concentration in this zone. Also, it is possible that variations in nodule O2 demand may be involved in the apparent variations in resistance of the diffusion barrier. It is concluded that there are many biochemical components which should be considered, along with possible changes to the diffusion barrier, when the effects of imposed stresses on nodule activities are being analysed.

Closed system O2uptake: a simple technique of nodule oxygen permeability estimation

Regulation of nitrogen fixation in response to various environmental conditions often involves an adjustment in nodule permeability and, because of the importance of nodule permeability in nitrogen fixation, several methods to estimate it have been developed. In the present study, these methods are reviewed and their limitations are highlighted. A simple, rapid and inexpensive technique that can be used to estimate permeability of nodules and respiration of other plant tissues is described. The technique was evaluated by comparing it to the lag-phase technique as an independent and reliable method for estimating nodule permeability. Over a wide range of nodule permeability estimates, the closed system O 2 uptake technique was linearly related to lag-phase permeability estimates. The technique was tested further by studying the response to sub- and supra-ambient pO 2 in the root environment, and the responses agreed well with published reports on the effect of O 2 on nodule permeability. The technique was found to be very satisfactory in estimating nodule permeability and may be used to measure the ability of other plant tissues to take up O 2 .

GROWTH AND NITROGEN FIXATION OF THE DIAZOTROPHIC FILAMENTOUS NONHETEROCYSTOUS CYANOBACTERIUM TRICHODESMIUM SP. IMS 101 IN DEFINED MEDIA: EVIDENCE FOR A CIRCADIAN RHYTHM1

ABSTRACT Trichodesmium sp. IMS 101, originally isolated from coastal western Atlantic waters by Prufert‐Bebout and colleagues and maintained in seawater‐based media, was successfully cultivated in two artificial media. Its characteristics of growth, nitrogen fixation, and regulation of nitrogen fixation were compared to those of natural populations and Trichodesmium sp. NIBB 1067. Results indicate that the culture grown in artificial media had nitrogen fixation characteristics similar to those when the culture is grown in seawater‐based medium and to those of Trichodesmium sp. in the natural habitat. The study provides practical artificial media to facilitate the physiological studies of these important diazotrophic cyanobacteria, as well as the cultivation of other Trichodesmium species in future studies. Manipulations of the light/dark cycle were performed to determine whether or not the daily cycle of nitrogen fixation is a circadian rhythm. Cultures grown under continuous light maintained the cycle for up to 6 days. We demonstrated that the daily cycle of nitrogen fixation in Trichodesmium sp. IMS 101 was at least partially under the control of a circardian rhythm.

Seasonal variation in rates of heterotrophic nitrogen fixation (acetylene reduction) in Zostera noltii meadows and uncolonised sediments of the Bassin d'Arcachon, south-west France

Nitrogen fixation (acetylene reduction) rates were measured over an annual cycle in meadows of the seagrass Z. noltii and uncolonised sediments of the Bassin d'Arcachon, south-west France, using both slurry and whole core techniques. Measured rates using the slurry technique in Z. noltii colonised sediments were consistently higher than those determined in isolated cores. This was probably due to the release of labile organic carbon sources during preparation of the slurries. Thus, in colonised sediments the whole core technique may provide a more accurate estimate of in situ activity. Acetylene reduction rates measured by the whole core technique in colonised sediments were 1.8 to 4-fold greater, dependent upon the season, in the light compared with those measured in the dark, indicating that organic carbon released by the plant roots during photosynthesis was an important factor regulating nitrogen fixation. In contrast acetylene reduction rates in uncolonised sediments were independent of light.

Coexistence of two structurally similar but functionally different PII proteins in Azospirillum brasilense

The coexistence of two different PII, proteins in Azospirillum brasilense was established by comparing proteins synthesized by the wild-type strain and two null mutants of the characterized glnB gene (encoding PII) adjacent to glnA. Strains were grown under conditions of nitrogen limitation or nitrogen excess. The proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or isoelectric focusing gel electrophoresis and revealed either by [32P]phosphate or [3H]uracil labeling or by cross-reaction with an anti-A. brasilense PII-antiserum. After SDS-PAGE, a single band of 12.5 kDa revealed by the antiserum in all conditions tested was resolved by isoelectric focusing electrophoresis into two bands in the wild-type strain, one of which was absent in the glnB null mutant strains. The second PII protein, named Pz, was uridylylated under conditions of nitrogen limitation. The amino acid sequence deduced from the nucleotide sequence of the corresponding structural gene, called glnZ, is very similar to that of PII. Null mutants in glnB were impaired in regulation of nitrogen fixation and in their swarming properties but not in glutamine synthetase adenylylation. No glnZ mutant is yet available, but it is clear that PII and Pz are not functionally equivalent, since glnB null mutant strains exhibit phenotypic characters. The two proteins are probably involved in different regulatory steps of the nitrogen metabolism in A. brasilense.