Dietmar Meletzus

Characterization of a Major Cluster of nif,fix, and Associated Genes in a Sugarcane Endophyte,Acetobacter diazotrophicus

A major 30.5-kb cluster of nif and associated genes of Acetobacter diazotrophicus (syn. Gluconacetobacter diazotrophicus), a nitrogen-fixing endophyte of sugarcane, was sequenced and analyzed. This cluster represents the largest assembly of contiguous nif-fix and associated genes so far characterized in any diazotrophic bacterial species. Northern blots and promoter sequence analysis indicated that the genes are organized into eight transcriptional units. The overall arrangement of genes is most like that of the nif-fix cluster in Azospirillum brasilense, while the individual gene products are more similar to those in species of Rhizobiaceae or in Rhodobacter capsulatus.

Regulation of nitrogen fixation by ammonium in diazotrophic species of proteobacteria

Abstract 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.

Analysis of nif and regulatory genes in Acetobacter diazotrophicus

Abstract The focus of our collaborative research program is the identification and characterization of nif and related regulatory genes of Acetobacter diazotrophicus in order to understand what factors influence nitrogen fixation in this unique diazotroph. To date, the following genes have been isolated from genomic libraries and are being analyzed: nifHDK, nifA, nifB, nifV, nifE and ntrBC. In addition, Nif− mutants have been constructed by insertional mutagenesis. These mutants are currently being used in inoculation experiments of sterile sugarcane plants to determine whether nitrogen fixed by A. diazotrophicus is significant for plant nutrition.

Identification of Three Genes Encoding PII-Like Proteins in Gluconacetobacter diazotrophicus: Studies of Their Role(s) in the Control of Nitrogen Fixation

In our studies on the regulation of nitrogen metabolism in Gluconacetobacter diazotrophicus, an endophytic diazotroph of sugarcane, three glnB-like genes were identified and their role(s) in the control of nitrogen fixation was studied. Sequence analysis revealed that one P(II) protein-encoding gene, glnB, was adjacent to a glnA gene (encoding glutamine synthetase) and that two other P(II) protein-encoding genes, identified as glnK1 and glnK2, were located upstream of amtB1 and amtB2, respectively, genes which in other organisms encode ammonium (or methylammonium) transporters. Single and double mutants and a triple mutant with respect to the three P(II) protein-encoding genes were constructed, and the effects of the mutations on nitrogenase expression and activity in the presence of either ammonium starvation or ammonium sufficiency were studied. Based on the results presented here, it is suggested that none of the three P(II) homologs is required for nif gene expression, that the GlnK2 protein acts primarily as an inhibitor of nif gene expression, and that GlnB and GlnK1 control the expression of nif genes in response to ammonium availability, both directly and by relieving the inhibition by GlnK2. This model includes novel regulatory features of P(II) proteins.

Ammonium sensing in nitrogen fixing bacteria: Functions of the glnB and glnD gene products

A plentiful supply of fixed nitrogen as ammonium (or other compounds such as nitrate or amino acids) inhibits nitrogen fixation in free-living bacteria by preventing nitrogenase synthesis and/or activity. Ammonium and nitrate have variable effects on the ability ofRhizobiaceae (Rhizobium, Bradyrhizobium andAzorhizobium) species to nodulate legume hosts and on nitrogen fixation capacity in bacteroid cells contained in nodules or in plant-free bacterial cultures. In addition to effects on nitrogen fixation, excess ammonium can inhibit activity or expression of other pathways for utilization of nitrogenous compounds such as nitrate (through nitrate and nitrite reductase), or glutamine synthetase (GS) for assimilation of ammonium. This paper describes the roles of two key genesglnB andglnD, whose gene products sense levels of fixed nitrogen and initiate a cascade of reactions in response to nitrogen status. While work onEscherichia coli and other enteric bacteria provides the model system,glnB and, to a lesser extent,glnD have been studied in several nitrogen fixing bacteria. Such reports will be reviewed here. Recent results on the identity and function of theglnB andglnD gene products inAzotobacter vinelandii (a free-living soil diazotroph) and inRhizobium leguminosarum biovarviciae, hereinafter designatedR.l. viciae will be presented. New data suggests thatAzotobacter vinelandii probably contains aglnB-like gene and this organism may have twoglnD-like genes (one of which was recently identified and namednfrX). In addition, evidence for uridylylation of theglnB gene product (the PII protein) ofR. l. viciae in response to fixed nitrogen deficiency is presented. Also, aglnB mutant ofR. l. viciae has been isolated; its characteristics with respect to expression of nitrogen regulated genes is described.

Characterization of Genes Involved in Regulation of Nitrogen Fixation and Ammonium Sensing in Acetobacter diazotrophicus, an Endophyte of Sugarcane

A. diazotrophicus is the major nitrogen fixing bacterium isolated from the leaves, stems and roots of sugarcane collected in various sites of Brazil, Australia, Mexico and Cuba and is considered to be a true endophyte based on its initial isolation from surface sterilized tissues and more recently on ultra-microscopic examinations of infected tissues. Other unique characteristics of this alpha-group proteobacteria diazotroph include the ability to grow and fix nitrogen at low pH, production of acetic acid from sucrose or glucose, and growth at high sucrose concentrations. Our objective was to isolate regulatory and ammonium assimilatory genes in order to understand what factors influence nitrogen fixation in this unique habitat.

Analysis of nitrogen fixation and regulatory genes in the sugar cane endophyte Acetobacter diazotrophicus

The specific endophytic association between Acetobacter diazotrophicus and sugarcane may represent a beneficial symbiosis with the bacterial partner providing fixed N and/or plant growth promoting factors such as auxin. Our goals are to identify and characterize the genes of A. diazotrophicus necessary for nitrogen fixation and effective symbiosis. In summary, we have isolated a gene cluster including nifAB HDK ENXQ USVW, fixABCX in one region of the A. diazotrophicus genome. Also, two other genes, modD (molybdenum transporter protein) and mcpA (methyl-accepting chemotaxis protein) were located downstream of fixABCX. The mcpA gene product is involved in responses to extracellular chemotactic signals, which may play an important role in plant-microbe interactions. A mcpA mutant of A. diazotrophicus lost chemotaxis toward a wide range of attractant stimuli and will be used to determine whether mcpA + bacteria have a competitive advantage with respect to cell survival and colonization of sugarcane.

Identification and Characterization of Genes Involved in the Ammonium Sensing Mechanism in Acetobacter diazotrophicus

The PII protein encoded by the glnB gene and the uridylyltransferase, product of the glnD gene, play a major role in the ammonia sensing mechanism of bacteria.