GENES IN AZTOBACTER VINELANDII INVOLVED IN CELLULAR RESPONSES TO FIXED NITROGEN

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: NRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 0187545
• Grant No.: 2001-35319-10013
• Proposal No.: 2000-02672
• Project Start Date: Dec 1, 2000
• Project End Date: Nov 30, 2003
• Grant Year: 2001
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF ARIZONA
888 N EUCLID AVE
TUCSON,AZ 85719-4824

Performing Department
PLANT SCIENCE

Non Technical Summary
Nitrogen fixing bacteria convert gaseous nitrogen to molecules needed by plants when the supply of fixed N in their environment is low. Inhibition of the nitrogen fixation process involves the products of several genes, only some of which are known and characterized. The new technology of analyzing microarrays, or DNA chips, will be applied to research using Azotobacter vinelandii, a free-living nitrogen fixing soil bacterium, in order to identify new genes that regulate nitrogen fixation or are regulated by nitrogenous compounds. Approximately 15,000 fragments of A. vinelandii DNA will be amplified and robotically applied to glass slides, to provide a representation of the total genome. RNA from bacterial cultures, one supplemented with fixed N, one without fixed N, or from cultures of known regulatory mutants, will be purified. DNA will be copied from each RNA sample in a reaction that will incorporate a different fluorescent label for each condition. The prepared microarrays will be treated with the resulting labeled cDNAs and the data overlaid, allowing the identification of fragments that hybridize to one fluorescent probe but not the other. New genes will be represented among these DNA fragments. The DNA chips will be made available to other laboratories wishing to identify new genes involved in responses to other dynamic environmental conditions. This technology can be usefully applied to analyze bacterial genomes for which a complete sequence is not yet available as is the case for a number of agriculturally-important microorganisms.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
206	1629	1040	10%
206	1629	1080	10%
206	1629	1100	10%
206	2499	1040	10%
206	2499	1080	10%
206	2499	1100	10%
206	4010	1040	10%
206	4010	1080	20%
206	4010	1100	10%

Knowledge Area
206 - Basic Plant Biology;

Subject Of Investigation
1629 - Perennial grasses, other; 4010 - Bacteria; 2499 - Plant research, general;

Field Of Science
1040 - Molecular biology; 1100 - Bacteriology; 1080 - Genetics;

Keywords

nitrogen fixation

gene expression

azotobacter vinelandii

glutamine synthase

molecular biology

plant microbiology

bacterial genetics

gene function

gene analysis

cell biology

proteins

nitrogen

functional analysis

dna fragments

gene regulation

gene environment interaction

reporter genes

escherichia coli

gene transfer

mutants

strains (genetics)

environmental factors

Goals / Objectives
1. Investigation of the roles of the regulatory proteins GlnK, GlnD and GlnE in NifL/NifA interactions. 2. Determination of the second vital function of the GlnK protein. 3. Microarray analysis of DNA fragments to identify genes that are regulated by N supply, by NifL/A, by NtrB/C, or by GlnK/GlnD.

Project Methods
The regulatory proteins from A. vinelandii will be transferred into an Escherichia coli strain along with a nifH-lacZ reporter fusion. Using the appropriate combinations of E. coli mutant backgrounds and A. vinelandii regulatory proteins, it will be determined whether GlnK interacts with NifL or with NifA and/or whether GlnK-UMP interacts with NifL or with NifA. The approach to Objective 2 will be mutational: a GlnE deficient strain, MV72, cannot adenylylate glutamine synthetase. While introducing glnK null mutations in wild type is lethal for two reasons, i.e. inability of GS to be deadenylylated and the other unknown, introducing glnK null mutations into MV72 and looking for stable mutants (with and without mutagenesis) should lead to second-site suppressors that block the lethal effect. These mutants will be analyzed. For Objective 3, 15,000 random DNA fragments created by nebulization will be cloned, amplified, and arrayed on glass slides. Differentially labelled cDNA prepared from mRNA in wild-type versus NifA, NtrC, GlnKY51F, and GlnD mutants will be hybridized to the microarrays. Differentially labelled DNA spots will indicate the genes that are up- or down-regulated by the regulatory protein represented by the mutant strain. mRNA from wild-type A. vinelandii grown under different growth conditions will indicate genes regulated by fixed N, by metals, by O2 and other environmental conditions.

Progress 12/01/00 to 11/30/03

Outputs
OUTPUTS: Sorry, PI passed away. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Sorry, PI passed away.

Publications

• No publications reported this period

Progress 01/01/02 to 12/31/02

Outputs
Objective 1 Examining roles of genes involved in nitrogen regulation: In several bacteria, nitrogen limitation can signal the uridylylation and expression of PII-like signal transduction proteins. These proteins influence the activity of the general Ntr regulators, NtrB and ATase (GlnE); current evidence indicates that PII-like proteins in the diazotrophs Azotobacter vinelandii and Klebsiella pneumoniae may have evolved to regulate the NifL-A two-component systems. The role of uridylylation, however, in these related bacteria might differ, essentially because A. vinelandii lacks a second PII-like protein and expression of nifLA is not controlled by the Ntr system. Furthermore, direct evidence indicates that K. pneumoniae GlnK-UMP is not required for relief of NifL inhibition. Nonetheless, glnD mutants of both organisms are Nif-, suggesting that uridylylation of A. vinelandii GlnK may be required for NifA activity. In this context, we explored the role of GlnK-UMP by constructing a glnKY51F mutation encoding an unuridylylatable form of the protein. In A. vinelandii, glnK is essential, and not unexpectedly, this allele was only viable in a glnD suppressor strain, suggesting GlnK-UMP activates GS. Moreover, mutants were severely reduced for growth in N-free media and expression of a nifH-lacZ fusion. glnKY51F nifL double mutants were Nif+. In a yeast two-hybrid assay, GlnK and GlnKY51F interacted with NifL. Together, these experiments implicate unmodified GlnK as a negative regulator of NifA whose effect is mediated through interaction with NifL. Contrary to that of K. pneumoniae, our data support a model for A. vinelandii in which relief of NifL inhibition occurs when GlnK is uridylylated in response to N-limitation. Objective 2 Microarray analysis of cloned A. vinelandii DNA fragments to identify genes that are regulated by N supply, by NifLA, by NtrBC, or by GlnK/GlnD: This objective proposed to microarray random fragments from the A. vinelandii genome because it was not expected that there would be a genome sequence available in the near future. Once this was expected (from about February of 2001 when DOE said A. vinelandii would be chosen for the 'second microbial sequencing month' at JGI), it was decided to wait for whole genome arrays available after the sequence was annotated to launch gene discovery experiments. Instead, 82 known genes were microarrayed, including several nitrogen fixation genes, to test labeling and hybridization conditions and methods for differential expression of N regulated genes. 50-mer oligonucleotides corresponding to genes in A. vinelandii were arrayed in triplicate in two subgrids on each glass slide. Cultures were grown in +N or -N conditions then mRNA was prepared, copied as cDNA, labeled with Expt A : Cy3 (-N) Cy5 (+N), Expt B: Cy5 (-N) Cy3 (+N). Genes known to be N regulated showed consistently and reproducibily higher expression in N-limited cultures than in N-sufficient conditions.

Impacts
Understanding how genes required for growth of Azotobacter vinelandii under N-deficient conditions can lead to the isolation of improved strains of agriculturally important nitrogen fixing. By analysis of mutants and recombinant plasmids, roles for regulatory proteins known as PII and NifL have been defined. Microarray technology has been developed that will lead to isolation of new genes involved in regulation of expression of genes involved in nitrogen assimilation and metabolism.

Publications

• Kennedy C (2002) Genus Beijerinckia, Genus Derxia, Genus Agromonas in Garrity et al (eds) Bergey's Manual of Systematic Bacteriology (in press)
• Rudnick P, Kunz C, Gunatilaka MK, Hines ER, Kennedy C (2002) The role of GlnK in the NifL-mediated regulation of NifA activity in Azotobacter vinelandii. J Bacteriol 184:812-820.
• Kennedy C, Rudnick P, MacDonald M, Melton T (2002) Genus Azotobacter in Garrity et al (eds) Bergey's Manual of Systematic Bacteriology (in press)
• Kennedy C, Rudnick P (2002) Genus Azomonas in Garrity et al (eds) Bergey's Manual of Systematic Bacteriology (in press)