Source: UNIVERSITY OF CALIFORNIA, DAVIS submitted to
CONTROL OF CELLULAR DIFFERENTIATION IN SYMBIOTIC NITROGEN-FIXING NOSTOC PUNCTIFORME
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0074508
Grant No.
(N/A)
Project No.
CA-D-MIC-3620-H
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2007
Project End Date
Dec 31, 2011
Grant Year
(N/A)
Project Director
Meeks, J. C.
Recipient Organization
UNIVERSITY OF CALIFORNIA, DAVIS
410 MRAK HALL
DAVIS,CA 95616-8671
Performing Department
MICROBIOLOGY
Non Technical Summary
This project addresses biological nitrogen fixation and supply of nitrogen for growth of crop plants. The experimental system is a filamentous nitrogen fixing cyanobacterium NOSTOC PUNCTIFORME. This organisms fixes nitrogen as a free-living entity in soils and when in symbiotic association with a variety of non-crop plants. We propose to apply the new technology of global transcription assays to identify genes and regulatory pathways essential for nitrogen fixation and symbiotic association. The results will be used to engineer new nitrogen-fixing symbioses with crop plants. This project is relevant to biological supply of fixed nitrogen in sustainable agricultural systems, especially paddy rice. The cyanobacterium NOSTOC PUNCTIFORME is unusual in its ability to converted nitrogen gas to ammonium in both the free-living growth state and in symbiotic association with non-crop plants. We seek to understand the regulatory mechanisms for the differentiation of motile filaments called hormogonia, which function as the infective units for symbiosis, and of heterocysts, cells specialized for nitrogen fixation. We especially want to know haw plants have exploited those mechanisms in establishment of a stable nitrogen fixing association. This knowledge will be used in attempts to engineer nitrogen-fixing associations with crop plants.
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
20615301100100%
Knowledge Area
206 - Basic Plant Biology;

Subject Of Investigation
1530 - Rice;

Field Of Science
1100 - Bacteriology;
Goals / Objectives
Overall objective 1 is to determine whether a branched hierarchal signal transduction pathway operates for the differentiation of both nitrogen-fixing heterocysts and motile hormogonium filaments of the symbiotically competent cyanobacterium NOSTOC PUNCTIFORME. Overall objective 2 is to conduct a genetic and biochemical characterization of a locus of genes encoding chemotaxis-like proteins that hypothetically regulate hormogonium differentiation.
Project Methods
A DNA microarray of the NOSTOC PUNCTIFORME genome that consists of 200 to 700 bp fragments of 6,893 genes has been constructed. The microarray will be used to determine all of the genes transcribed during cellular differentiation by the wild-type and specific mutants blocked in various stages of heterocyst and hormogonium formation. The mutants are in hand for these experiments to identify components of the signal transduction pathway. Transcription profiles will also be defined for N. PUNCTIFORME in symbiosis with ANTHOCEROS PUNCTATUS and of hormogonia during exit from the hormogonium cycle. Genetic and biochemical approaches will be used to characterize components of chemotaxis-like locus 2 in N. PUNCTIFORME that may operate as part of the signal transduction pathway of hormogonium differentiation. Insertion mutants will be generated using standard protocols. Transcriptional start points will be determined by 5 prime RACE. Phosphotransfer from CheA to CheY proteins using radioactive phosphate will be examined at two time points, 10 s and 60 min, to distinguish specific and non-specific interaction, respectively. Identification of the composition of signal transduction complexes will be done by immumoprecipitation and mass spectrometric protein analysis, as well as by bacterial two hybrid assays. Cellular localization of signal transduction complexes will be determined by fluorescence microscopy using a receptor protein-green fluorescent protein fusion.

Progress 10/01/07 to 12/31/11

Outputs
OUTPUTS: This project was initiated in 1978. The overall goal was to understand and manipulate the differentiation of nitrogen-fixing heterocysts and motile filaments called hormogonia of filamentous cyanobacteria in the genus NOSTOC. These cyanobacteria are unique in fixing nitrogen in both free-living and plant-associated symbiotic growth states. A model symbiotic association was established between pure cultures of NOSTOC species and gametophytes of the bryophyte hornwort ANTHOCEROS PUNCTATUS. This has been recognized internationally as the most robust symbiotic cyanobacterial experimental system and provided unequivocal evidence that hormogonia are the infective units of cyanobacteria-plant associations. Biochemical and radiotracer experiments using nitrogen-13 (10 min half-life), generated at the Crocker Nuclear Laboratory on the UC Davis campus, were employed to establish that dinitrogen-derived ammonium is made available to the plant partner. We also identified plant sources reductant as carbohydrates for symbiotically associated NOSTOC. We then developed protocols for genetic analysis of NOSTOC PUNCTIFORME which has lead to the identification of genes involved in both heterocyst and hormogonium differentiation and to delineation of genes/gene products essential for symbiotic infection and function. We continue to use the ANTHOCEROS system to analyze symbiotic phenotype of NOSTOC mutants and as a source of a hormogonium-inducing factor. We identified a genetic locus (HRM) that is involved in plant-dependent repression of hormogonium differentiation. Genetic analysis of the genes, PATN, HETF and HETR have established that the initiation of heterocyst differentiation involves two of the broad strategies of pattern differentiation employed by plants and animals of biased inheritance of cell fate determinants (PatN) and self-enhancing activator (HetR and HetF)-diffusible inhibitor (PatS-5) interactions for competitive resolution of developmentally unstable cells. In 2001, the complete genome sequence of N. PUNCTIFORME was determined with the support of the USA Department of Energy. The genome sequence opened the way for systems level genomic studies. We defined a 1750 member proteome of an ammonium grown N. PUNCTIFORME culture, and are currently working on dinitrogen-grown and symbiotically grown proteomes. We developed a DNA microarray of the N. PUNCTIFORME genome and have determined transcriptomes of ammonium and dinitrogen grown steady state cultures, as well as time courses during the differentiation of heterocysts and hormogonia. The results of these studies have defined the numerical limits of genes differentially transcribed during cellular development, verified transcriptional patterns of known genes and identified candidate genes for in depth genetic analysis. Substantial progress was made in understanding cellular differentiation in these cyanobacteria. Ultimately, we would hope to engineer new nitrogen-fixing cyanobacterial-plant associations with crop species such as rice and corn. The immediate approach should be a systems level focus on the plant partner ANTHOCEROS to identify genes and metabolites essential for symbiotic association. PARTICIPANTS: PI: John C. Meeks. Provided overall direction for the project, interpretation of data and publication of the results. SRA: Elsie L. Campbell. Maintained the laboratory, trained undergraduate and graduate students in basic protocols, as well as visitors to the laboratory. Actively involved in generation of experimental data, pioneered the DNA microarray experiments. Provided rough drafts of manuscript in which she is the senior author. Postdoctoral fellow: Miriam Martin. Involved in development of the original DNA microarray. Postdoctoral fellow: Doug Risser. Characterization of the PATN mutant of heterocvyst differentiation and of chemotaxis locus 2 that is involved in regulation of motility in hormogonia. Postdoctoral fellow: Daniella Ferreira. Characterization of chemotaxis locus 4 that is required for phototaxis of hormogonia and of the cyanobacteriochrome Cph1. Graduate Student: Harry Christman. Characterization of the role of HetF in heterocyst differentiation and transctriptomic analyses of wild type and HETF and HETR mutants during induction of heterocyst differentiation. The laboratory has recently hosted a high school student (Mira Loma HS in Sacramento) for 6 months of training. We have also hosted five graduate students from other laboratory (4 international and 1 national) for training in genetics and genomics of Nostoc. TARGET AUDIENCES: Microbial cellular differentiation. Plant-microbe interactions. Sustainable agriculture. Genomic studies. Nitrogen fixation. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
This project has made major contributions to understanding both cyanobacterial development and symbiotic association. The contributions are reflected in the invited review articles produced over the years. We have been the major international force in the genetics and genomics of hormogonium differentiation, the physiology and genetics of cyanobacteria symbioses, and a major contributor to understanding heterocyst differentiation and function. Hormogonia are biologically interesting filaments in that they reflect a transient non-growth state whose physiological role is in short-distance dispersal. We determined that approximately 40% (2,750 genes) of the 9.06 Mbp genome is differentially transcribed during their differentiation. The question then becomes, what are the transcriptional regulatory mechanisms involved in such an extensive and global change, and what are the signals that allow for a return to the vegetative growth state. The genetic regulatory pathway of heterocyst differentiation is slowly being defined; our transcriptome studies have determined that only between 270 and 500 genes are differentially transcribed, with 75 per-cent up-regulated. Nitrogen fixation is basically an alternative route of acquisition of ammonium, relative to nitrate, nitrite, urea or ammonium in the environment. Nitrogen fixation in NOSTOC differs from nitrate, nitrite or urea not only by the number of catalytic proteins involved in generation of ammonium, but primarily in the proteins required for the development of the terminally differentiated heterocysts. Our genomic data support the prior statement, while our genetic analyses have contributed to the identification of regulatory factors essential of the differentiation of heterocyst in their specific spacing pattern in the filaments. Our most unique contribution has been in the development of the NOSTOC-ANTHOCEROS experimental system. This association grows rapidly in submerged liquid culture, it can be reconstituted in pure culture with various NOSTOC isolates and mutant strains and it can be uniformly treated with chemicals, all for comparative studies. We have supplied researchers, nationally and internationally, with pure cultures of the ANTHOCEROS gametophytes, but, unfortunately, few have been successful in maintaining pure stock cultures.

Publications

  • Campbell. E.L., Christman, H. and Meeks, J.C.. 2008. DNA microarray comparisons of plant factor and nitrogen deprivation, stress induced hormogonia reveal decision making transcriptional regulation patterns in Nostoc punctiforme. J. Bacteriol. 190:7382-7391.
  • Meeks, J.C. 2009. Physiological adaptations in nitrogen-fixing Nostoc-plant symbiotic associations. In: Prokaryotic endosymbionts in plants, (K. Pawlowski, ed.), pp 181-205. Springer-Verlag, Berlin.
  • Christman, H.D., Campbell, E.L. and Meeks. J.C. 2011. Global transcription profiles of the nitrogen stress response resulting in heterocyst or hormogonium development in Nostoc punctiforme. J. Bacteriol. 193:6874-6886.
  • Meeks, J.C. 2011. Perspective: Closing the circle. Science 334:1508-1509.
  • Risser, D.D., Wong, F.Y.C. and Meeks, J.C.. 2012. Biased inheritance of PatN frees vegetative cells to initiate patterned heterocyst differentiation in cyanobacteria (pending).
  • Ferreira, P.D.P., Rockwell N.C., Chen, R., Hagen, K., Risser, D.D. Martin, S.S., Lagarias, J.C. and Meeks, J.C. 2012. How Nostoc punctiforme sees and responds to light: characterization of the taxis photosensor in a filamentous cyanobacterium (pending).


Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: NOSTOC PUNCTIFORME has an extraordinary wide range of physiological properties, vegetative cell developmental alternatives and ecological niches. Two of the most interesting traits are the three developmental alternatives of its vegetative cells (nitrogen fixing heterocysts, spore-like akinetes and motile hormogonium filaments) and its ability to fix nitrogen in free-living and plant associated symbiotic growth states. N PUNCTIFORME has broad symbiotic competence with three major phylogenetic groups of terrestrial plants (bryophyte hornworts, gymnosperm cycads and the angiosperm family Gunneraceae). We are continuing genetic and genomic analyses of plant-induced hormogonium and heteorcyst differentiation. The three major projects are supported by the NSF and DOE. We are examining the role of chemotaxis-like proteins encoded by a seven gene operon in the differentiation of hormogonia (NSF support). Transcriptomics and proteomics are being applied to define the physiological differences between the free-living and symbiotic growth states wherein heterocyst differentiation and rates of nitrogen fixation are symbiotically enhanced three to five fold. The proteomics are being done in collaboration with Brett Phiney in the UC Davis genome center; in these instances we are defining not only protein abundance, but also modification state. We have switched from an in-house constructed PCR gene fragment based two-color hydridization platform to one of Nimblegen generated, synthesized on the slide, oligomer array platform and one color hybridization. The Nimble system allows for greater reproducibility and sensivity. All genes previously identified by the two-color array as differentially transcribed during heterocyst differentiation are also detected in the Nimblegen format, in addition to 150 genes not previously observed (the systems level studies are supported by the DOE). We are collaborating with J. Clark Lagarius in The Department of Molecular and Cell Biology, David Britt and Delmar Larson in the Department of Chemistry, and Thomas Huser in the Department of Internal Medicine on Photoreceptor regulation and optimization of energy harvesting. In particular, we are examining energy transfer in the receptor of a phototaxis system and two different bacteriochromes. We have defined the transcriptional units of the multiprotein phototaxis complex and the action spectrum of hormogonium phototaxis (supported by a collaborative DOE grant). PARTICIPANTS: PI John C. Meeks, who is responsible for direction of the project, reporting and dissemination of the results. SRA Elsie Campbell, who is involved in training of undergraduate and graduate students, and visitors to the laboratory, as well as her own research projects. Graduate Student Harry Christman, whose dissertation project is to apply transcriptominc, genetic and biochemical approaches to identify the role of the HETF gene product in heterocyst differentiation. Postdoctoral Fellow Doug Risser, who is examining the role of a chemotaxis-like system in hormogonium differentiation and characterizing a mutant strain of N PUNCTIFORME that differentiates 30 percent of its vegetative cells into heterocysts in free-living cultured, similar to the symbiotic growth state. TARGET AUDIENCES: This project has applications to sustainable agriculture in biological nitrogen fixation. The target crop is most likely paddy rice, but could be adapted to corn. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The results are helping us to understand the parameters involved in plant-induced differentiation of hormogonia. We propose that understanding the mechanisms of hormogonium and heterocyst differentiation can lead to the engineering of new cyanobacterial-crop plant symbiotic nitrogen fixing associations. The initial step in establishment of such associations is control of the infection stage involving the differentiation and behavior of hormogonia.

Publications

  • Christman, H., Campbell, E.L. and Meeks, J.C. 2009. Heterocyst specific genes are expressed in Nostoc punctiforme destined to become hormogonia. Proceeding of the 13th International Symposium on Phototrophic Prokaryotes. Montreal, Canada. pp. 52-53.
  • Meeks, J.C., Campbell, E. and Chen, Rui. 2009. Chemotaxis-like signal transduction systems in development and behavior of hormogonia of Nostoc punctiforme. Proceeding of the 13th International Symposium on Phototrophic Prokaryotes. Montreal, Canada. p. 72.
  • Christman, H., Campbell, E., Risser, D., Phinney, B., Chiu, W-L. and Meeks, J.C. 2009. Systems level approaches to understanding and manipulating heterocyst differentiation in Nostoc punctiforme. Genomic Science 2010, Awardee Workshop VII and Knowledgebase Workshop. W@ashington DC. P. 85.


Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: NOSTOC PUNCTIFORME has an extraordinary wide range of physiological properties, vegetative cell developmental alternatives and ecological niches. Two of the most interesting traits are the three developmental alternatives of its vegetative cells (nitrogen fixing heterocysts, spore-like akinetes and motile hormogonium filaments) and its ability to fix nitrogen in free-living and plant associated symbiotic growth states. N PUNCTIFORME has broad symbiotic competence with three major phylogenetic groups of terrestrial plants (bryophyte hornworts, gymnosperm cycads and the angiosperm family Gunneraceae). The physiological characteristics of symbiotic NOSTOC were reviewed in the context of the evolution of cyanobacterial-plant symbioses for a book chapter. Symbiotic association involves plant dependent manipulation of two of the NOSTOC developmental alternatives: (1) the differentiation and behavior of hormogonium filaments, which function as infective units; and (2) the differentiation and behavior of heterocysts whose frequency and rates of nitrogen fixation increase more than 3-fold, while ammonium assimilation is repressed, resulting in release of dinitrogen-derived ammonium). We are applying global gene transcription assays to determine the extent of differential gene expression using DNA microarray methodology during hormogonium and heterocyst differentiation in free-living and symbiotic growth sates. Hormogonium differentiation can be induced by abrupt combined nitrogen differentiation or exposure to a plant exudate containing a hormogonium inducing factor (HIF). Approximately 40% of the N PUNCTIFORME genome (6,978 open reading frames) is differentially transcribed during hormogonium differentiation, approximately half of the genes are up-regulated and half down-regulated. The down-regulated genes are primarily involved in core metabolic activity of growth and division. The up-regulated genes are enriched in those encoding sensory transduction proteins, including proteins involved in photo- and chemo-taxis. Clustering analysis of time course data revealed both common and distinct transcription patterns for the two modes of induction. A transcription check point was observed at 12 h following induction by nitrogen starvation that was not seen in HIF hormogonia. These results indicated that hormogonium differentiation is considerably more complex than heterocyst differentiation, that a common cluster of 1,328 differentially express hormogonium genes can be defined and the HIF is a stronger inducing signal than nitrogen starvation. We are continuing this analysis by examination of mutations blocked in steps of hormogonium differentiation and extending the analyses to heterocyst differentiation. We have completed a time course of differential gene expression during heterocyst differentiation. The pattern is less complex than of hormogonium differentiation. Many of the up-regulated genes are ones that were predicted from prior genetic and physiological experiments. However, about 40% of the up-regulated genes fall into the category of unassigned function, which need to be characterized with respect to phenotype and biochemical activity. PARTICIPANTS: SRA Elsie Campbell, who is involved in training of undergraduate and graduate students, and visitors to the laboratory, as well as her own research projects. Graduate Student Harry Christman, whose dissertation project is to apply transcriptomic, genetic and biochemical approaches to identify the role of the HETF gene product in heterocyst differentiation. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The results are helping us to understand the parameters involved in plant-induced differentiation of hormogonia. We propose that understanding the mechanisms of hormogonium and heterocyst differentiation can lead to the engineering of new cyanobacterial-crop plant symbiotic nitrogen fixing associations. The initial step in establishment of such associations is control of the infection stage involving the differentiation and behavior of hormogonia.

Publications

  • Campbell. E.L., H. Christman and J.C. Meeks 2008. DNA microarray comparisons of plant factor and nitrogen deprivation, stress induced hormogonia reveal decision making transcriptional regulation patterns in Nostoc punctiforme. J. Bacteriol. 190:7382-7391.
  • Meeks, J.C. 2009. Physiological adaptations in nitrogen-fixing Nostoc-plant symbiotic associations. In: Prokaryotic endosymbionts in plants (K. Pawlowski, ed.), in press, published on line September 2007. Springer-Verlag, Berlin.


Progress 01/01/07 to 12/31/07

Outputs
We are continuing systems level approaches to understanding and manipulation of two developmental states of the symbiotic cyanobacterium NOSTOC PUNCTIFORME; nitrogen-fixing heterocysts and motile hormogonium filaments. Hormogonia are the infective units of cyanobacteria-plant symbioses. We have constructed a DNA microarray consisting of 6,893 internal gene fragments. The array has been challenged by cDNA derived from total RNA of cultures grown in the steady state with ammonia and dinitrogen as nitrogen sources and at single time points during the differentiation of spore-like akinetes and hormogonia. The ammonia-grown culture yielded a transcriptome of 2,935 genes, the majority of which are associated with core metabolic functions. The dinitrogen grown cultures showed the differential transcription of 373 and 122 up- and down-regulated genes, respectively. We are most interested in the 26 up-regulated genes encoding signal transduction and transcriptional regulatory proteins. The akinete differentiating culture showed differential transcription of 255 and 242 up- and down-regulated genes. The down regulated genes were enriched in core metabolic function and there was minimal overlap between the heterocyst and akinete up-regulated genes, implying little evolutionary linkage. Surprisingly, hormogonia showed differential expression of 944 and 883 up- and down-regulated genes, respectively. Since hormogonia are non-growing, the down-regulation of core metabolic genes was predicted, although not at the extent observed. The up-regulated genes are highly enriched in sensory functions. We also collaborated with colleagues at Arizona State University to identify essential genes for the production of UV-light absorbing compounds by N PUNCTIFORME and at the University of Sevilla in reviewing gene transfer in cyanobacteria.

Impacts
We propose that understanding the mechanisms of heterocyst differentiation in N PUNCTIFORME can lead to production of dinitrogen-derived ammonia in aquatic agricultural systems and in engineering of new plant-NOSTOC nitrogen-fixing symbiotic associations, as well as possible bioreactor production of hydrogen gas.

Publications

  • Soule, T, V Stout, JC Meeks and F Garcia-Pichel. 2007. Molecular genetics and genomic analysis of scytonemin biosynthesis in NOSTOC PUNCTIFORME ATCC 29133. J Bacteriology 189:4465-4472.
  • Campbell, EL, ML Summers, H Christman, ME Martin and JC Meeks. 2007. Global gene expression patterns of NOSTOC PUNCTIFORME in steady state dinitrogen-grown heterocyst-containing cultures and at single time points during the differentiation of akinetes and hormogonia. J Bacteriology 189:5247-5256.
  • Flores, E, AM Muro-Pastor and JC Meeks. 2008. Gene transfer to cyanobacteria in the laboratory and in nature. In: The cyanobacteria: molecular biology, genomics and evolution, (A. Herrero and E. Flores, eds.), pp. 45-57. Horizon Sci Press, Norwich.


Progress 01/01/06 to 12/31/06

Outputs
This project involves identification of the chemical interactions in the model symbiotic association between the nitrogen-fixing cyanobacterium NOSTOC PUNCTIFORME and the hornwort ANTHOCEROS PUNCTATUS. Two cyanobacterial developmental processes are essential in establishment of a symbiotic association: formation and behavior of motile hormogonia, the infective units, and of heterocysts, sites of nitrogen fixation. Based on the sequenced genome of N. PUNCTIFORME we are now utilizing global, systems level approaches to identifying developmental and symbiotic genes in this cyanobacterium. We have completed a proteomics analysis of an ammonium grown culture; this is the most permissive growth state upon which to define the basic proteome for future comparisons. Multiple liquid chromatography steps were used to fractionate proteins and then peptides prior to entry into a tandem mass spectrometer for analysis. First pass analysis identified 1,575 proteins, approximately 51% of which can be assigned to core metabolic and transport functions, 10% to adaptive metabolism and 38% to unassigned functions. Near complete precursor, monomer, pigment, lipid, nucleic acid and protein biosynthetic pathways can be constructed from the proteomic data, which suggests broad coverage. Sixty-one percent of the adaptive proteins (99/162), including 44 sensor histidine kinases and 7 protein kinases, are involved in signal transduction, indicating N. PUNCTIFORME has a substantial capacity to sense and respond to environmental signals. We are specifically interested in genetic and biochemical analyses of these genes/gene products. The proteome results were published.

Impacts
We propose that N. PUNCTIFORME, or related cyanobacteria can be used in aquatic agricultural applications, either as free-living entities, overproducing and excreting ammonium, or in a program to engineer new nitrogen-fixing associations with, for example, rice. Basic research provides a foundation to pursue the applied goals.

Publications

  • Anderson, DC, EL Campbell and JC Meeks. 2006. A soluble 3D LC/MS/MS proteome of the filamentous cyanobacterium NOSTOC PUNCTIFORME. J Proteome Research 5:3096-3104.


Progress 01/01/05 to 12/31/05

Outputs
This project involves identification of the chemical interactions in the symbiotic association between the nitrogen-fixing cyanobacterium NOSTOC PUNCTIFORME and the hornwort ANTHOCEROS PUNCTATUS. We have completed construction of a DNA microarray of 6,890 genes in NOSTOC genome and are challenging the array with cDNA from a variety of developmental states and transitions between states. We are specifically focusing on the differentiation of nitrogen-fixing heterocysts and of motile filaments called hormogonia. Hormogonia appear to respond to chemoattractants produced by ANTHOCEROS in establishment of the association. Five loci of genes encoding chemotaxis-like proteins are annotated in the NOSTOC genome. Northern hybridization analyses established that genes encoding the methylaccepting chemotaxis-like proteins (MCP) from three of the loci are expressed predominately, or exclusively, in the hormogonium developmental state. We hypothesize that the three loci have distinct physiological roles; one locus may be involved in synthesis of the motility motor, one may be involved in phototaxis and the third in chemotaxis. We are testing the hypothesis by in vitro construction of insertion mutations in the MCP and gene replacement in the NOSTOC chromosome. We predict mutation in the MCP associated with phototaxis will infect ANTHOCEROS at wild type frequencies, but mutations in genes of loci involved in chemotaxis or synthesis of the motility motor(s) will be deficient in infection. A review was published on the molecular mechanisms involved in the NOSTOC-bryophyte symbiosis.

Impacts
We hypothesize that the low specificity of NOSTOC in establishment of associations with plants that span the phylogenetic spectrum can ultimately be exploited in a program to engineer new nitrogen fixing associations with crop plants, such as cereal grains, especially rice.

Publications

  • Meeks, JC. 2005. Molecular mechanisms in the nitrogen-fixing NOSTOC-Bryophyte symbiosis. In: J. Overmann (ed.), Molecular Basis of Symbiosis, pp. 165-196. Springer-Verlag, Berlin.


Progress 01/01/04 to 12/31/04

Outputs
This project involves characterization of the regulatory systems governing cellular differentiation in the nitrogen-fixing cyanobacterium NOSTOC PUNCTIFORME in the context of its symbiotic interaction with the hornwort ANTHOCEROS PUNCTATUS. The principle effort in the past year has been to synthesize an internal fragment of all 7,353 genes known to be present in the sequenced genome of NOSTOC by high throughput PCR amplification. This gene fragment library will be utilized in two subsequent experimental approaches. First, they will be arrayed onto glass slides and function as hybridization targets for cDNA isolated from cells cultured under a variety of conditions; these experiments will define the transcriptome. Second, they will be cloned into a plasmid that does not replicate in NOSTOC and used to generate antibiotic resistant mutants following homologous recombination of the internal fragments in NOSTOC; the survivors will represent mutations in all genes nonessential for growth under the permissive culture conditions. We have thus far been unable to detect transfer of DNA from AGROBACTERIUM into ANTHOCEROS by selection for antibiotic resistance; the major problem has been effectiveness of antibiotic killing of ANTHOCEROS, in contrast to plant tissue cultures. A review was published on the information present in the genome of NOSTOC PUNCTIFORME that is relevant to its free-living and symbiotic nitrogen fixation.

Impacts
We hypothesize that the low specificity of NOSTOC in establishment of associations with plants that span the phylogenetic spectrum can ultimately be exploited in a program to engineer new nitrogen fixing associations with crop plants, such as cereal grains.

Publications

  • Meeks, JC. 2005. The genome of the filamentous cyanobacterium NOSTOC PUNCTIFORME. What can we learn from it about free-living and symbiotic nitrogen fixation? In W.E. Newton (Ed.), Nitrogen Fixation: 1888 - 2001. Vol. VI: Genomes and Genomics of Nitrogen-Fixing Organisms, pp. 27-70. Kluwer Academic Publishers.


Progress 01/01/03 to 12/31/03

Outputs
We have continued to define the cellular developmental regulatory circuits and symbiotic interactions of the nitrogen fixing cyanobacterium NOSTOC PUNCTIFORME and its association with the bryophyte ANTHOCEROS PUNCTATUS. We are now taking two broad experimental approaches. First, utilizing the completed genome sequence of N. PUNCTIFORME, we have initiated a comprehensive systems approach involving global transcriptional and proteome analyses. Second, we are continuing our physiological genetic and biochemical analyses of specific processes, genes and gene products. The specific process include induction and repression of hormogonium formation, and induction of heterocyst differentiation and nitrogen fixation. We have also initiated preliminary studies on genetic analysis of A. PUNCTATUS. Based on our progress, we have developed and published a model of the symbiotic interactions as akin to the domestication of N. PUNCTIFORME by A. PUNCTATUS in the establishment of ammonium factory. The model is quite distinct from those proposed in dissection of the rhizobia-legume associations.

Impacts
We propose that understanding, and ultimately manipulating the regulation of symbiotic and free-living nitrogen fixation by N. PUNCTIFORME will lead to engineering of new nitrogen-fixing symbiotic associations with plants and to provision of fixed nitrogen in aquatic agricultural systems.

Publications

  • Meeks, J.C. 2003. Symbiotic interactions between NOSTOC PUNCTIFORME, a multicellular cyanobacterium, and the hornwort ANTHOCEROS PUNCTATUS. Symbiosis 34: 55-71.


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

Outputs
We have continued to define the cellular developmental regulatory circuits and symbiotic interactions of the nitrogen-fixing cyanobacterium NOSTOC PUNCTIFORME and its association with the bryophyte ANTHOCEROS PUNCTATUS. We compiled a solicited comprehensive review of the regulation of cellular differentiation in cyanobacteria in symbiotic and free-living growth states and a more focused review on the breadth of developmental alternatives of N. PUNCTIFORME. We have made significant progress in defining chemical signaling between partners of the symbiotic association by establishing the role of a DNA binding protein, HrmR, which we discovered by genome sequence analysis. HrmR functions as a transcriptional repressor in the regulation of hormogonium differentiation in N. PUNCTIFORME. HrmR binding to operator regions of its own structural gene and an adjacent gene called HRME is alleviated following exposure of cells to an aqueous extract of its symbiotic plant partner. The extract either contains or stimulates the synthesis of a sugar related to galacturonate in the N. PUNCTIFORME cells that then binds to HrmR, thereby allowing transcription of HRME and subsequent repression of hormogonium differentiation. We are now testing the physiological role of HrmE. We have made unpublished progress in proteome analysis of N. PUNCTIFORME and anticipate initiation of global transcription global transcription analyses in a pending proposal to the USDA NRI. We will also initiate experiments on feasibility of genetic manipulation of A. PUNCTATUS using polyethylene glycol induced DNA uptake and homologous recombination.

Impacts
We propose that understanding and manipulating the regulation of symbiotic and free-living nitrogen fixation by N. PUNCTIFORME will lead to engineering of new nitrogen fixing symbiotic associations with crop plants, such as rice.

Publications

  • Meeks, J.C and J. Elhai. 2002. Regulation of cellular differentiation in filamentous cyanobacteria in free-living and plant-associated symbiotic growth states. Microbiol. Mol. Biol. Rev. 66: 94-121.
  • Meeks, J.C., E.L. Campbell, M.L. Summers and F.C. Wong. 2002. Cellular differentiation in the cyanobacterium NOSTOC PUNCTIFORME. Arch. Microbiol. 178: 395-403.
  • Campbell, E.L., F.C.Y. Wong and J.C. Meeks. 2003. DNA binding properties of the HrmR protein of NOSTOC PUNCTIFORME responsible for transcriptional regulation of genes involved in the differentiation of hormogonia. Mol. Microbiol. 47:573-582.


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

Outputs
We have continued to dissect the interactions between the nitrogen fixing cyanobacterium NOSTOC PUNCTIFORME and the bryophyte ANTHOCEROS PUNCTATUS that result in establishment of the diazotrophic symbiotic association and of genes differentially expressed in heterocysts and hormogonia in free-living and symbiotic growth states. Activity of glucose-6-phosphate dehydrogenase (G6PD) is essential for nitrogen fixation in heterocysts. G6PD activity is regulated by a protein (OpcA) whose structural gene is cotranscribed with G6PD. We established that OpcA is an allosteric activator of G6PD. This work defines a new catalytic regulatory mechanism. We authored a collaborative overview of the sequenced genome of NOSTOC PUNCTIFORME, which at 9.5 Mb is amongst the largest bacterial genomes and contains genetic information that essentially defines all of the cyanobacteria. Most relevant for this project is that genes involved in cellular differentiation or symbiotic interaction cannot be identified by sequence analysis alone, therefore functional genome analyses will be necessary. We determined that HetR, is not required for cyanobacterial akinete formation, in contrast to another published report implying both heterocyst and akinete differentiation have a common developmental pathway.

Impacts
The potential to increase available biologically fixed nitrogen in aquatic agricultural systems and to engineer new nitrogen fixing symbiotic associations with crop plants such as rice.

Publications

  • Hagen, K. and Meeks, J.C. 2001. The unique cyanobacterial protein OpcA is an allosteric effector of glucose-6-phosphate dehydrogenase in NOSTOC PUNCTIFORME ATCC 29133. J. Biol. Chem. 276: 11477-11486.
  • Meeks, J.C., J. Elhai, T. Thiel, M. Potts, F. Larimer, J. Lamerdin, P. Predki and R. Atlas. 2001. An overview of the genome of Nostoc punctiforme, a multicellular, symbiotic cyanobacterium. Photosyn. Res. 70:85-106.
  • Wong. F.C. and J.C. Meeks. 2002. Establishment of a functional symbiosis between the cyanobacterium Nostoc punctiforme and the bryophyte hornwort Anthoceros punctatus requires genes involved in nitrogen control and initiation of heterocyst differentiation. Microbiology 148:315-323.


Progress 01/01/00 to 12/31/00

Outputs
We have continued to dissect the interactions between the nitrogen fixing cyanobacterium NOSTOC PUNCTIFORME and the bryophyte ANTHOCEROS PUNCTATUS that result in establishment of the diazotrophic symbiotic association and of genes differentially expressed in heterocysts and hormogonia in free-living and symbiotic growth states. We have initiated a proteome approach to analyze the differentiation of hormogonia, the infective units of cyanobacterial symbioses. Activity of glucose-6-phosphate dehydrogenase (G6PD) is essential for nitrogen fixation in heterocysts. G6PD activity is regulated by a protein (OpcA) whose structural gene is cotranscribed with G6PD. By application of immunoblots to define oligomer size and structure, and detailed kinetic analysis of substrate dependent G6PD activity in the presence and absence of OpcA, we have established that OpcA is an allosteric activator of G6PD. This is the first known protein allosteric activator; all know activators are small molecules or metabolites. A major advance in our studies of cellular differentiation and symbiotic association occurred this year when NOSTOC PUNCTIFORME was selected for complete genome sequencing by the DOE. We have supplied DNA and biological expertise to the sequencing effort. The shotgun sequencing phase was completed by September, 2000; finishing may take an additional 18 months. The NOSTOC PUNCTIFORME genome is amongst the largest bacterial genomes sequenced and contains genetic information that essentially defines all of the cyanobacteria. This database will define much of our future experimental approach and will be instrumental in our ability to understand and manipulate nitrogen-fixing cyanobacterial associations with plants.

Impacts
The potential to increase available biologically fixed nitrogen in aquatic agricultural systems and to engineer new nitrogen fixing symbiotic associations with crop plants such as rice.

Publications

  • Meeks, J., Campbell, E. Hagen, K, Wong, F., Atlas, R., Elhai, J., Potts, M., Thiel, T. and Lamerdin. 2000. Preliminary results from the genome sequencing project of NOSTOC PUNCTIFORME and the phenotypic characteristics that enhance genome analysis of a complex cyanobacterium. Abstracts, 10th International Symposium on Phototrophic Prokaryotes, pp. 63.


Progress 01/01/99 to 12/31/99

Outputs
We have continued to dissect the interactions between the nitrogen fixing cyanobacterium NOSTOC PUNCTIFORME and the bryophyte ANTHOCEROS PUNCTATUS that result in establishment of a diazotrophic symbiotic association. Establishment of the association is dependent first on the differentiation of motile filaments called hormogonia, which function as infective units and then on inhibition of their differentiation once infection has occurred. We have identified a regulon involved in inhibition of hormogonium differentiation. The regulon encodes proteins that have high similarity to the enzymatic pathway of hexuronic acid catabolism in heterotrophic bacteria. We are currently analyzing transcriptional regulation in the regulon. We previously determined that developmental of a mature nitrogen-fixing heterocysts is dependent on the activity of a truncated response regulator protein termed DevR. Deletion and site-directed mutagenesis of DEVR verified that the protein acts in a phosphorelay signaling pathway, most likely in the synthesis of the polysaccharide layer of the heterocyst wall.

Impacts
Potential to increase available biologically fixed nitrogen in aquatic agricultural systems and understanding of plant-microbe interactions leading to a symbiotic relationship.

Publications

  • Meeks, J.C., Campbell, E. Hagen, K., Hanson, T. Hitzman, N., Wong, F. 1999. Developmental alternatives of symbiotic NOSTOC PUNCTIFORME in response to its plant partner ANTHOCEROS PUNCTATUS. In: The Phototrophic Prokaryotes, G.A. Peschek, W. Loffelhardt and G. Schmetterer eds. Kluwer Academic, New York, pp.665-678.
  • Hagen, K., Meeks, J.C. 1999. Biochemical and genetic evidence for participation of DevR in a phosphorelay signal transduction pathway essential for heterocyst maturation in NOSTOC PUNCTIFORME ATCC 29133. Journal of Bacteriology 181, 4430-4434.


Progress 01/01/98 to 12/01/98

Outputs
Interactions between the cyanobacterium NOSTOC PUNCTIFORME and bryophyte ANTHOCEROS PUNCTATUS during the formation of a nitrogen fixing symbiosis results in an enhancement in two of the developmental pathways of NOSTOC; differentiation of motile filaments called hormogonia and of heterocysts, cells specialized for nitrogen fixation. The properties of this symbiotic experimental system were reviewed in 1998 and the methods for transposon mutageneis of NOSTOC that we have developed over the past few years were complied for a volume of Methods in Enzymology. The GLNB gene product PII, which signals nitrogen starvation in enteric bacteria was characterized in NOSTOC. Genetic analysis indicated that GLNB is essential for growth of NOSTOC, while biochemical assays established that PII can be modified by phosphorylation. However, it is not clear whether PII has an essential role in initiation of heterocyst diffentiation in either the free-living or symbiotic growth states. We established that mutation of an alternative sigma factor (SigH) of RNA polymerase in NOSTOC results in an increased frequency of infection of ANTHOCEROS tissue by NOSTOC. SigH appears to direct transcription of a gene, operon or regulon that controls the behavior of hormogonia filaments.

Impacts
(N/A)

Publications

  • Meeks, J.C. 1998. Symbiotic associations between nitrogen fixing cyanobacteria and plants. BioScience 48, 266-276.
  • Hanson, T.E., Forchhammer, K., Tandeau De Marsac, N., Meeks, J.C. 1998. Characterization of the GLNB gene product of NOSTOC PUNCTIFORME strain ATCC 29133; GLNB or the PII protein may be essential.
  • Cohen, M.F., Meeks, J.C., Cai, Y.A., Wolk, C.P. 1998. Transposon mutagenesis of heterocyst-forming filamentous cyanbacteria. Methods
  • Campbell, E.L., Brahamsha, B., Meeks, J.C. 1998. Mutation of an alternative sigma factor in the cyanobacterium NOSTOC PUNCTIFORME results in increased infection of its symbiotic partner ANTHOCEROS PUNCTATUS. Journal of Bacteriology 180, 493


Progress 01/01/97 to 12/01/97

Outputs
Transient hormogonium filaments of NOSTOC ssp. are the intective units of their symbiosis with various plants, while terminally differentiated heterocysts are the sites of nitrogen fixation. A transposon mutant of NOSTOC PUNCTIFORME was isolated that showed a 10-fold higher frequency of initial infection of its symbiotic plant partner ANTHOCEROS PUNCTATUS. The mutant differentiated more hormogonia in the presence of the plant and it appeared to immediately reenter the hormogonium state, thereby extending the infection window. Two genes of operon were sequenced; HRMA has no significant similarities in the data bases, while HRMU has similarity to a family of oxidoreductases. The operon is induced by an extract of the plant, but not by the factor released by the plant that induces hormogonium differentiation. The operon may encode enzymes involved in metabolism of an autogenic repressor of hormogonium differentiation. A second transposon-induced mutant of N. PUNCTIFORME was isolated that failed to make the characteristic heterocyst glycolipids and thus could not fix nitrogen in the presence of air (oxygen-sensitive). Sequence analysis indicated that the transposon had inserted into a gene encoding a large multidomain protein characteristic of genes involved in polyketide synthesis. Transcriptional analysis indicated the gene is expressed during the early steps of heterocyst maturation.

Impacts
(N/A)

Publications

  • Cohen, M.F., Meeks, J.C. 1997. A hormogonium regulating locus, HRMUA, of the cyanobacterium NOSTOC PUNCTIFORME strain ATCC 29133 and its response to an extract of a symbiotic plant partner ANTHOCEROS PUNCTATUS. Molecular Plant-Microbe Inter
  • Campbell, E.L., Cohen, M.L., Meeks, J.C. 1997. A polyketide synthase-like gene is involved in synthesis of heterocyst glycolipids in NOSTOC PUNCTIFORME strain ATCC 29133. Archives of Microbiology


Progress 01/01/96 to 12/30/96

Outputs
A transposon mutant of NOSTOC PUNCTIFORME was isolated that is unable to fix nitrogen in air, but does fix nitrogen when in symbiosis with ANTHOCEROS PUNCTATUS. The interrupted gene encodes a receiver-domain only response regulator protein (DevR) characteristic of two component regulatory systems. This result indicates operation of a signaling pathway for heterocyst maturation in the free-living growth state that is not required in symbiosis. Previous work established that glucose-6-phosphate dehydrogenase (G6PD) is essential in supplying reductant for nitrogen fixation in heterocysts. Five transcript start points (TSP) for differential G6PD expression were identified by primer extension. Three of the TSP were specifically activated upon nitrogen starvation, one TSP was enhanced under these conditions and one TSP was activated by the presence of fructose. Sequence analysis of the promoter regions revealed two regions of the nitrogen regulated promoters that could be involved in binding of regulatory proteins. One region is present at -16 bp from the TSP in all five promoters, but most highly conserved 5' of the P5 promoter; it may bind a repressor protein. The second site consists of a pair of purine rich boxes separated by 12-14 bases starting at -46 bp from the TSP of the P1 promoter and may reflect the binding site of an transcriptional activator protein.

Impacts
(N/A)

Publications

  • CAMPBELL, E.L., HAGEN, K.D., COHEN, M.F., SUMMERS, M. L., MEEKS, J.C. 1996. The DEVR gene product is characteristic of receivers of two-component regulatory systems and is essential for heterocyst development in the filamentous cyanabacteriu SUMMERS, M.
  • L., MEEKS, J.C. 1996. Transcriptional regulation of ZWF, encoding glucose-6-phosphate dehydrogenase, from the cyanobacterium NOSTOC PUNCTIFORME strain ATCC 29133. Molecular Microbiology 22, 473-480.
  • COHEN, M.F. 1996. Transposon mutagenesis and characterization of a hormogonium regulating locus, HRMUA, of the cyanobacterium NOSTOC PUNCTIFORME strain ATCC 29133. Ph.D. Thesis, University of California, Davis.


Progress 01/01/95 to 12/30/95

Outputs
Biochemical studies have not resolved whether the oxidative pentose phosphate (OPP) cycle or the glycolytic pathway, or both, is the primary route of carbon catabolism and reductant supply to nitrogenase in heterocysts. From NOSTOC sp. strain ATCC, we have cloned and sequenced the gene (ZWF) encoding glucose-6-phosphate dehydrogenase (G6PD), the initial enzyme of the OPP cycle, and a cotranscribed gene (OPCA) encoding a protein of unknown function. Insertion mutations in either ZWF or OPCA resulted in cultures unable to grow with dinitrogen as the sole nitrogen source, or to grow heterotophically in the dark. The results unequivocally establish that: (1) the OPP cycle is essential in providing reductant in heterocysts for nitrogenase catalytic activity and for respiratory oxygen consumption in partial maintenance of an anoxic environment for the synthesis of nitrogenase; (2) the glycolytic pathway, pyruvate-ferredoxin oxidoreductase and isocitrate dehydrogenase are not important reactions in heterocyst reductant supply; (3) the OPP cycle is the major route of carbon catabolism in supply of metabolic precursors for dark heterotrophic growth; (4) OpcA has an essential role in catalytic activation of G6PD. Preliminary studies indicate differential expression of genes in the OPC operon in response to either carbon or nitrogen sources for growth; detailed studies are in progress.

Impacts
(N/A)

Publications


    Progress 01/01/94 to 12/30/94

    Outputs
    The interrupted genes of three transposon (Tn5-1063)-induced mutants of diazotrophic, symbiotically competent NOSTOC have been isolated and sequenced. Two of the mutants are unable to fix nitrogen in air in the free-living state and the third mutant has increased symbiotic infection. One mutation, yielding a defective heterocyst but symbiotically effective phenotype, is in a gene with similarity to the receiving domain of eubacterial response-regulator proteins. This regulatory gene may function to integrate multiple environmental signals controlling heterocyst maturation. A mutation blocking synthesis of the unique heterocyst glycolipids and having a symbiotic negative phenotype is in a gene encoding a protein with multiple active site domains that are typically involved in fatty acid synthesis and portions are analogous to a rhizobial gene involved in nodulation. The interrupted gene of the mutant with an increased symbiotic infection phenotype has no identity in data bases; it is preceded by a gene with similarity to dehydrogenases functioning in polyketide synthesis. A mutant NOSTOC strain lacking glucose-6-phosphate dehydrogenase (G6PD) activity was constructed by gene replacement; the mutant cannot grow with dinitrogen as the sole nitrogen source, nor in the dark with any carbon or nitrogen source. These results are genetic evidence for the essential activity of G6PD in supplying reductant to nitrogenase in heterocysts and in catabolism of carbon reserves for dark growth.

    Impacts
    (N/A)

    Publications


      Progress 01/01/93 to 12/30/93

      Outputs
      This program emphasizes a genetic characterization of heterocyst differentiationin symbiotically-competent diazotrophic cyanobacteria of the genus NOSTOC. Two projects continue on characterization of transposon-induced mutants of NOSTOC 29133 with altered symbiotic (Sym) phenotypes. The interrupted gene of a superinfective mutant has been cloned and sequenced; its sequence has no significant homology in data banks. The mutation has been reconstructed as an interposon. Rescue of the transposon and characterization of the flanking genomic sequences continue in the noninfective mutants. Portions of the rescued flanking genomic DNA from two Fox- Sym-and one Fox-Sym+ mutants have been sequenced; again with no detectable homology to known genes. Work continues to finish the nucleotide sequences and begin transcript analysis. The glucose-6-phosphate gene (ZWF) and three additional genes that constitute an operon have been sequenced. Long range S1 nuclease analysis indicates ten transcripts cover ZWF, five different ones being major transcripts under ammonia, ammonia + glucose, or dinitrogen growth conditions. The GLNB gene (PII protein) was cloned and sequenced from NOSTOC 29133 by the PI on sabbatical leave. Experiments continue to identify cellular nitrogen status-dependent modification of PII in NOSTOC.

      Impacts
      (N/A)

      Publications


        Progress 01/01/92 to 12/30/92

        Outputs
        Physiological and genetic experiments continued on identifying mechanisms regulating heterocyst differentiation and expression of nitrogenase in symbiotically competent NOSTOC spp. A primary physiological approach in this project was use of the radionuclide 13N to analyze the products of symbiotic N2 fixation in situ. The methodology developed for the synthesis of 13N, generation of various 13N labeled inorganic nitrogen molecules and analysis of organic products of 13N assimilation was published. The experimental approach has turned to molecular and physiological genetic analysis. Mutants of NOSTOC 29133 defective in heterocyst differentiation, nitrogen fixation and symbiotic interaction were isolated by transposition; 18 Fix-, 1 Het- and 19 Sym- mutants have been phenotypically characterized. All Sym- mutants appear to be in genes regulating infection of host tissue. Methods were developed for rescue of the transposon and flanking NOSTOC genomic sequences. A random shear library of NOSTOC genomic DNA was established from which to isolate and subclone interrupted genes. Molecular characterization of the DNA sequences is in progress.

        Impacts
        (N/A)

        Publications


          Progress 01/01/91 to 12/30/91

          Outputs
          We have continued physiological and genetic experiments on the regulation of heterocyst differentiation and nitrogenase expression in NOSTOC spp., grown in the free-living state or in symbiotic association with the hornwort ANTHOCEROS or water fern AZOLLA. Mutants of NOSTOC sp. UCD 7801, resistant to the herbicide DCMU, were used in situ to verify the photosynthetic potential of symbiotically associated NOSTOC and to establish that the reductant for symbiotic nitrogenase activity is provided as photosynthate from both NOSTOC and ANTHOCEROS. Mutants of NOSTOC sp. ATCC 29133 unable to assimilate nitrate were used to prove that the repression of nitrogenase activity in symbiotic NOSTOC in response to the presence of combined nitrogen is mediated by ANTHOCEROS. The structural gene for glucose-6-phosphate dehydrogenase (G6PD) was isolated from a genomic library of NOSTOC 29133 and its nucleotide sequence determined. The activity of G6PD is 70-fold higher in heterocysts than in vegetative cells. Transcript analysis of the gene is now underway to determine if its expression is under developmental regulation. Attempts continue to complement, by electroporation or conjugation of cloned parental NOSTOC DNA, a growing collection of NOSTOC 29133 mutants unable to differentiate heterocysts, fix N2 or form a functional symbiotic association.

          Impacts
          (N/A)

          Publications


            Progress 01/01/90 to 12/30/90

            Outputs
            We have continued physiology and genetic experiments on the regulation of heterocyst differentiation and nitrogenase expression in cyanobacteria grown free-living or in symbiotic association with the water fern AZOLLA or bryophyte ANTHOCEROS. We have discovered cross talk between inducers of Rhizobial NOD gene transcription produced by PHASEOLUS sp. that induce hormogonia formation in NOSTOC and hormogonia inducing factors from ANTHOCEROS that induce NOD gene transcription in R. LEGUMINOSARIUM. Purification and identification of the factors continue. Use of aerobic nitrogen fixation minus mutants and oxygen microelectrodes have established that symbiotic NOSTOC colonies in ANTHOCEROS tissue are in a microaerobic environment. Characterization and genetic complementation of the mutants continue. Barriers exist for conjugational transfer of cosmid cloned DNA from E. COLI into symbiotic NOSTOC strains. We are continuing to define the parameters for conjugation by using donor E. COLI strains with mutations in various DNA restriction/modification systems, and are examining methods of electroporation. The routes of nitrogen assimilation in cyanobacteria were reviewed in Meeks (1990).

            Impacts
            (N/A)

            Publications


              Progress 01/01/89 to 12/30/89

              Outputs
              Diazotrophic cyanobacteria in symbiotic associations with plants differentiate ahigh frequency of heterocysts, fix N(2) and provide fixed nitrogen for growth of the associated tissues. Our work in defining the basic biology of symbiotic cyanobacteria was reviewed for the associations with the water fern Azolla (2) and the bryophyte Anthoceros (4). The association with Anthoceros can be experimentally reconstituted, whereas such manipulations with Azolla are not yet possible. The initial stage of symbiotic association in Anthoceros is dependent on plant production of a extracellular metabolite that stimulates formation of Nostoc infective units (Hormogonia). Physiological characteristics of metabolite production and of hormogonia were described (1). The catalytic activity of the major carbon dioxide assimilating enzyme, ribulose bisphosphate carboxylase (rubisco), of the Nostoc is regulated by a posttranslational mechanism when in association with Anthoceros (3). Any covalent modification, however, does not appear to involve the ribulose bisphosphate binding site (3). Physiological and molecular gentics studies continue in attempts to identify a symbiotic specific regulatory system of heterocyst differentiation and nitrogenase expression in Nostoc.

              Impacts
              (N/A)

              Publications


                Progress 01/01/88 to 12/30/88

                Outputs
                We have continued to use symbiotic associations with the bryophyte Anthoceros and the fern Azolla as a growth state of N(subscript 2)-fixing Nostoc strains in comparison to free-living growth to study regulation of enzyme activity, gene expression and heterocyst differentiation. DNA restriction fragment length polymorphism analysis of the cyanobacteria associated with Azolla caroliniana indicate that A. caroliniana contains a dominant symbiont and a minor symbiont (1). The minor symbiont can be cultured in the free-living state, but it is doubtful that the dominant symbiont of this or any other Azolla sp. has ever been cultured. The dominant symbiont is characterized by a contiguous nifHDK operon, while the nifD gene in vegetative cells of the minor symbiont and all Nostoc Spp. associated with Anthoceros is interrupted by a segment of DNA of unknown length (1). The major ammonium assimilating enzyme, glutamine synthetase (GS), in the dominant symbiotic cyanobacteria associated with Azolla is synthesized at a lower rate than free-living cyanobacteria (2). This is in contrast to posttranslational control of GS in Nostoc spp. associated with Anthoceros and neither synthesis nor catalytic control of the enzyme in Nostoc spp. when in association with cycads (2). Anthoceros produces a currently unidentified extracellular metabolite (s) that stimulates hormogonia formation in symbiotic Nostoc spp. (3).

                Impacts
                (N/A)

                Publications


                  Progress 01/01/87 to 12/30/87

                  Outputs
                  We have continued to use symbiotic associations with Anthoceros and Azolla as a growth state of the N(2)-fixing Nostoc strains in comparison to free-living growth to study regulation of gene expression and to characterize the phenotype of mutant strains. Using N generated at Crocker Nuclear Laboratory and direct in vivo assays, we showed that the eucaryotic partner in the Azolla association assimilated N(2)-derived NH(4) by the same glutamine synthetase-glutamate synthase pathway as do the symbiotic cyanobacteria and Anthoceros tissue. The activity of the initial enzyme of the pathway, glutamine synthetase, in Nostoc strains associated with Anthoceros is apparently controlled by a posttranslational mechanisms, rather than at the level of transcription. Biochemical experiments are continuing in attempts to identify both the modification mechanism and how it is activated in symbiosis. In a collaborative effort, the N methodology was also applied in an in vivo study to show the glutamine synthetase-glutamate synthase pathway functional under both nitrogen-limited and excess growth conditions in the associated N(2)-fixing Azospirillum. An increasing portion of our effort is now being directed toward genetic analysis of symbiotic and free-living heterocyst differentiation and nitrogenase expression. This effort includes mutagenesis and on-going phenotypic characterization and complementation.

                  Impacts
                  (N/A)

                  Publications


                    Progress 01/01/86 to 12/30/86

                    Outputs
                    We have continued to study the interactions between partners of the Anthoceros-Nostoc symbiotic association. Our major effort has been physiological characterization of carbon and nitrogen metabolism by symbiotic Nostoc are catalytically-inhibited, rather than transcriptionally-repressed (manuscripts in preparation). Nevertheless, using herbicide-resistant mutants we have also determined that symbiotic Nostoc does directly supply a portion of the photosynthate for reduction of molecular nitrogen. Work has continued in purification and identification of the extracellular compound produced by Anthoceros that induces formation of infective units of Nostoc. More effort has been directed toward genetic analysis of symbiotic Nostoc. We have shown that Nostoc DNA can be manipulated in vitro, but that barriers exist in expression of cloned DNA in Escherichia coli. Therefore shuttle vectors are now being modified for cosmid cloning and mutants of symbiotically competent and noncompetent strains of Nostoc. Using Southern hybridization, we determined that major rearrangement of the DNA do not occur in the transition between free-living and symbiotic growth of Nostoc. Similar analysis of a number of Nostoc strains indicated that the presence of an excisable element between the nifD and nifK genes is variable, & related neither to symbiotic potential nor the regulations of heterocyst differentiation.

                    Impacts
                    (N/A)

                    Publications


                      Progress 01/01/85 to 12/30/85

                      Outputs
                      We have continued to study nitrogen metabolism, and its control, in partners of cyanobacterial symbiotic association, with efforts in two major categories: physiological and biochemical characteristics of the parners; and preliminary molecular genetic analysis. We have determined that nitrogen limited Anthoceros produced a heat stable extracellular metabolite that induces all symbiotically competent and some non-competent Nostoc strains to form infective units called homogonia. The induction process and the biochemical characteristics of hormogonia are under investigation. Symbiotically-associated Nostoc assumes a growth rate lower than free-living cultures that appears to be constitutively imposed by Anthoceros metabolism. We have determined that this growth state is accompanied by an inhibition of the activity of the primary CO(2) and NH(4) assimilation enzymes and not by transcriptional control of their synthesis. Efforts are continuing to identify the post-translational control mechanism(s) and to characterize the photochemical properties of symbiotic Nostoc. We have demonstrated that symbiotic Nostoc DNA can be manipulated in vitro by construction of a genomic library using a cosmid vector. Preliminary analysis of this library indicated infrequent cleavage of Nostoc DNA by common restriction endonucleases, that only a few Nostoc genes are expressed in E. coli, and that some segments of Nostoc DNA are not stably replicated in E. coli.

                      Impacts
                      (N/A)

                      Publications


                        Progress 01/01/84 to 12/30/84

                        Outputs
                        We have continued to study nitrogen metabolism, and its control, principally in partners of cyanobacterial symbiotic associations, with efforts in two broad categories: (1) physiology and biochemistry, and (2) preliminary molecular biology experiments. In collaboration with S. Kustu, we showed that in Salmonella the adenylylation of GS is important in protection of the metabolic pool of glutamate. We speculate that most, if not all, prototrophic organisms must maintain a substantial pool of glutamate for optimal, competitive growth. Using PGDTN and in-vivo experimentation we determined that symbiotic Anabaena isolated from Azolla releases about 40% of its fixed N(2) (primarily as NH4RG) to host tissue and that Anabaena assimilates the remaining N(2)-derived NH(4)RG by the GS-GOGAT pathway with little or no contribution by GDH. These data indicate that the lower expression of GS in symbiotic cyanobacteria has potential regulatory significance as well as physiological significance in depressing the rates of glutamate metabolism. We recently observed that Anthoceros produces an extracellular metabolite that induces symbiotically-competent Nostoc to differentiate normal N(2)-fixing filaments into hormogonia, small celled gliding filaments important in symbiotic infection, at a frequency of essentially 100%. We are now examining the chemical interaction important in hormogonia induction and their physiological characteristics.

                        Impacts
                        (N/A)

                        Publications


                          Progress 01/01/83 to 12/30/83

                          Outputs
                          We have continued our studies of nitrogen metabolism in cyanobacterial symbioticassociations. In collaboration with Dr. G. A. Peters, C.F. Kettering Research Foundation, we have applied our PGDTN tracer methodology to the fixation and transfer of fixed nitrogen in the Azolla-Anabaena association. Time course and inhibitor experiments indicate that glutamate is formed from dinitrogen-derived ammonium through the glutamine synthetase-glutamate synthase pathway by isolated Anabaena and by the intact Azolla-Anabaena association. [PGDTN]N(2) pulse with PG,N(2) chase incubations followed by microdissection of Zolla main stem axes show that nitrogen fixed by Anabaena in mature fronds is translocated to the apical meristem regions. Additional chase periods are necessary before the translocated compound(s) can be unequivocaly identified. We have previously established that symbiotic Nostoc, then rapidly isolated from Anthoceros of when examined in situ, assimlated less than 20% of its N(2)-derived NH(4)RG. However, in vitro assays show that symbiotic Nostoc has 88% of the glutamine synthetase specific activity of free-living Nostoc (mean of 14 data points, 6 separate experiments). Thus, regulation of nitrogen assimilation in symbiotic Nostoc does not now appear to involve repression of of the synthesis of glutamine synthetase.

                          Impacts
                          (N/A)

                          Publications


                            Progress 01/01/82 to 12/30/82

                            Outputs
                            The major goal of the past year was to identify the nitrogenous compound(s) translocated from symbiont to host tissue in pure cultures of the intact Anthoceros-Nostoc association. Both free-living Nostoc and Anthoceros tissue grown under nitrogen-limited conditions assimilated exogenous -13NH(4) plus by the glutamine synthetase-glutamate synthase pathway. Consistent with this pathway, total -13NH(4) plus assimilation was inhibited over 95% in the presence of methionine sulfoximine (MSX). Strains of Nostoc resistant to MSX were selected for reconstitution in the association. [13N]-N(2) assimilation into organic metabolites was inhibited by MSX in Anthoceros association reconstituted with MSX-resistant or wild-type Nostoc strains, although there was about 15% less inhibition in the association with MSX-resistant Nostoc. These results indicate that ammonium is the nitrogenous compound translocated in vivo from symbiont to host tissue. Rapidly isolated symbiotic Nostoc had 5% of the glutamine synthetase specific activity and 20% of the exogenous 13-NH(4) plus assimilatory capacity of free-living Nostoc. These results indicate that -N(2) -derived -NH(4) plus was released by symbiotic Nostoc because the initial enzyme of the primary assimilatory pathway is controlled at the level of either gene expression or activity of preformed enzyme. To differentiate between these two possibilities, we are now purifying glutamine synthetase from Nostoc.

                            Impacts
                            (N/A)

                            Publications


                              Progress 01/01/81 to 12/30/81

                              Outputs
                              To examine competition between Nostoc species during pure culture reconstitutionof the Anthoceros/Nostoc symbiotic association, to determine if a symbiotic Nostoc colony arises from one or multiple infections, and ultimately to inhibit specific Anthoceros metabolic reactions, we have selected and are characterizing analogue resistant strains of three Nostoc species: nalidixic acid-resistant Nostoc g 7901 (originally from Gunnera); fluorocytosine-resistant Nostoc 27895 (originally a free-living species); and methionine sulfoximine (MSX)-resistant Nostoc ac 7801 (the original isolate from Anthoceros). We have verified by time course, pulse-chase, and amide-nitrogen distillation experiments that Anthoceros gameophyte tissue assimilated 1 3NF(4)+ by th glutamine synthetase/glutamate synthase pathway. In the presence of 50 MuM MSX, 1 3NH(4) + assimilation into total organic products, including glutamate, is inhibited more than 99%, indicating little or no activity of glutamic acid dehydrogenase. After a 10 min incubation period, the products of ( 1 3N)N(2) assimilation by Anthoceros/Nostoc are glutamine, glutamate, and alanine, with less than 1.5% of the total fixed 1 3N appearing as free ammonium. Following pretreatment with digitonin to inhibit total metabolism by Anthoceros issue, the products of ( 1 3N)N(2) assimilation are also glutamaine, glutamate, and alanine.

                              Impacts
                              (N/A)

                              Publications


                                Progress 01/01/80 to 12/30/80

                                Outputs
                                We are continuing our research on nitrogen control of heterocyst differentiationand nitrogenase expression in symbiotic cyanobacteria. Our current emphasis is on the association between the hornwort Anthoceros and Nostoc sp. We have obtained rapid growth of pure Anthoceros tissue in submerged liquid culture. We have reconstituted the symbiotic association between Anthoceros and the original purified Nostoc isolate, as well as Nostoc isolates from a variety of other symbiotic associations and one normally free-living Nostoc strain. This is a major accomplishment as we now have pure liquid cultures of symbiotic material for physiological and biochemical analyses. All of the Nostoc strains in association show a high heterocyst frequency (25 to 45% of the total cells) and a diminished photosynthetic capacity (ca. 10% of the rate of O(2) evolution by comparable free-living cultures). We have determined that Anthocerpos tissue assimilates 1 3NH(4) + primarily by the outline synthetase/glutamate synthase pathway whether grown symbiont-free with NH(4)NO(3) or reconstituted and dependent on fixed nitrogen from symbiotic Nostoc. We have found that glutamine synthetase also catalyzes the first step in the assimilation of 1 3NH(4) + by symbiotic Nostoc colonies. In preliminary experiments, the in vitro transferase activity of glutamine synthetase in symbiotic Nostoc appears nearly equal to that of free-living cultures, however, the rate of in vivo 1 3NH(4) assimilation appears to be about 10-fold lower by symbiotic colonies.

                                Impacts
                                (N/A)

                                Publications


                                  Progress 01/01/79 to 12/30/79

                                  Outputs
                                  We are continuing our research on nitrogen control of heterocyst differentiationin free-living and symbiotic cyanobacteria. Procedures have been devised for reconstitution of the symbiotic association between pure cultures of the N(2)-fixing cyanobacterium Nostoc sp. a.c. 7801 and gametophytes of the hornwort Anthoceros crispulus. Reconstituted associations can be maintained on nitrogen-free medium for 2-3 weeks. After 2-3 weeks, some of the symbiotic Nostoc filaments tend to glide out of the underside cavities of the Anthoceros tissue and form free-living colonies on the agar and gametophyte surfaces. To obtain experimental material, growth of the free-living Nostoc is controlled during the 2-3 week period by inclusion of 200 units/ml of penicillin in the medium. Nitrogen metabolism by Nostoc-free gametophytes and by the reconstituted symbiotic Nostoc is currently being investigated, using 1 3N as a pribe. Procedures for the synthesis of 1 3NO from target 1 3NO(3) were developed and published. The effect of nitrate on parameters such as growth, nitrogenase activity (acetylene reduction) and assimilation of 1 3NO(2) were determined in two free-living Anabaena species that respond differently, in terms of heterocyst formation, to nitrate in the growth medium. The accumulated data indicate that assimilation of nitrate in excess of that required for maximal growth results in repression of heterocyst formation.

                                  Impacts
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                                  Publications


                                    Progress 01/01/78 to 12/30/78

                                    Outputs
                                    Nostoc spp from the hornwort Anthoceros crispulus and the lichen Peltigera aphthosa have been isolated in pure culture. Spores of A. crispulus have been surface sterilized and germinated on defined minimal medium. Pure cultures of gametophyte tissue are being accumulated for biochemical analysis. Experiments to reconstitute the association are underway using A. crispulus gametophytes and the two isolated symbiotic Nostoc species, as well as the Nostoc symbiont from the angiosperm Gunnera. We intend to study nitrogen metabolism by the reconstituted intact association, rapidly isolated and separately cultured symbiotic Nostoc, and separately cultured A. crispulus gametophytes by using 1 3N (as ( 1 3N)N(2) and 1 3NH(4) +) as a tracer. The experimental procedures for 1 3N (t(1/2) equal to 10 min) generation, synthesis of pure 1 3NO(3) - and 1 3NH(4) +, assimilation by cyanobacteria, and extraction and analysis of the formed metabolites have now been developed for use in the CNL, Davis cyclotron. During this period of experimental development, 1 3NO(3) - assimilation by NO(3) --, NH(4) +- and N(2)-grown cyanobacteria is being studied. In Anabaena cylindrica, 1 3NO(3) - uptake is by diffusion only. The initial organic products of assimilation by cells grown with all three nitrogen sources are similar to those observed when ( 1 3N)N(2) or 1 3NH(4) + are assimilated by N(2)- grown cells (Meeks et al., 1978. J. Bacteriol. 134:125-130).

                                    Impacts
                                    (N/A)

                                    Publications