|I have worked with cyanobacteria from the time of my Ph.D. dissertation research (1967-72) on the photosynthetic properties of thermophilic unicellular cyanobacteria in the genus Synechococcus under the direction of Dick Castenholz at the University of Oregon, including a summer (1972) postdoctoral period in Yellowstone National Park with Tom Brock of the University of Wisconsin. I subsequently joined Bill (now Sir William) Stewart's laboratory at the University of Dundee, Scotland (1972-74) as a postdoctoral fellow to study nitrogen fixation, the enzymology of heterocysts and amino acid pool dynamics in Anabaena. After a one-year teaching position at the University of South Florida (1974-75), I joined Peter Wolk's laboratory at the MSU-DOE plant Research Laboratory (1975-77) as a postdoctoral fellow, where I learned from Peter the nuances of making and handling radioactive 13N (10 min half-life), while defining the routes of assimilation of exogenous and dinitrogen-derived ammonium, primarily in Anabaena. I joined the faculty of the Department of Bacteriology at UC Davis in 1977 and developed a research program that initially focused on utilizing 13N produced by the cyclotron in the Crocker Nuclear Laboratory on campus to study nitrogen fixation and metabolism in cyanobacterial symbiotic associations.
The overall goal of my research program has always been to identify and characterized the mechanisms by which cyanobacteria sense changes in their growth environment and respond to those changes through differential gene expression. We are currently monitoring the response through the appearance of two types of differentiated cells; heterocysts, terminally differentiated cells that serve as the sites of nitrogen fixation in air, and hormogonia, transiently differentiated small-celled filaments lacking heterocysts and capable of gliding and/or buoyant motility. Our experimental organism is facultatively heterotrophic Nostoc punctiforme strain ATCC 29133 (PCC 73102), whose vegetative cells have multiple developmental alternatives. Our research program can be distinguished from those of most colleagues who also study the developmental biology of filamentous cyanobacteria by the nature of one of the environmental changes. N. punctiforme and related Nostoc strains can establish a symbiotic relationship with representatives of four of the major phylogenetic groups of terrestrial plants. We utilize pure cultures of the bryophyte hornwort Anthoceros punctatus as the plant partner because it is amenable to routine experimental manipulations, including reconstitution of the symbiotic association with the wild type and mutant strains of N. punctiforme. Such plant associations are of interest because hormogonium and heterocyst differentiation of Nostoc strains are approximately 10-fold higher when in the symbiotic growth state in contrast to the free-living growth state. We have published evidence that A. punctatus produces chemical signals that both stimulate and repress hormogonium differentiation and that symbiotic heterocyst differentiation is uncoupled from the nitrogen-dependent regulatory mechanism operative in the free-living growth state.
Over the past 6 years, we have adapted and developed techniques for genetic analysis of N. punctiforme, including transposon mutagenesis, complementation in trans, gene exchange using positive selection vectors and site directed mutagenesis. Only one other heterocyst-forming cyanobacterium is currently amenable to such facile genetic analysis, the culture known as Anabaena sp. strain PCC 7120, which is an obligate photoautotroph that can only differentiate heterocysts. As a result of the initial physiological and molecular genetic analyses of N. punctiforme, we have begun to identify genes, operons and regulons that respond to the plant signals. The N. punctiforme 29133 genome is in the process of complete sequence analysis by the Joint Genome Institute sponsored by the US Department of Energy (http://www.jgi.doe.gov/). We are poised to exploit that database in functional genome analysis through proteonomics and DNA microarrays.
We are currently focused on projects in the three broad areas described below, which are supported by grants from the NSF and USDA.
1. Hormogonium differentiation. NSF IBN, Plant and Microbial Developmental Mechanisms, 8/1/00-7/31/03.
Motile hormogonium filaments function as propagules for colonization of new habitats and are the infective units of cyanobacterial symbiotic associations. Their transient formation involves cell division uncoupled from biomass increase or DNA replication. Hormogonium and heterocyst differentiation are mutually exclusive developmental processes; hormogonia also form as a consequence of global differentiation, while heterocysts reflect patterned differentiation. There have been few studies on hormogonia formation and most have been descriptive. This project has two overall objectives: (i) determine the extent of differential gene expression during hormogonium differentiation using two dimensional protein gel electrophoresis; and (ii) characterize transcription in, and the metabolic products of, the hrm regulon involved in plant controlled repression of hormogonium differentiation.
A. Initiation of differentiation. A multitude of environmental changes, acting in an additive manner, will induce hormogonium differentiation, including an extracellular hormogonium-inducing factor (HIF) from the symbiotic plant partners. The targets of HIF are unknown. SRA Elsie Campbell determined that a gene encoding an alternative sigma factor (sigH) of RNA polymerase is transiently transcribed between 1.5 and 12 h after exposure to HIF. The sigH mutant differentiates hormogonia at the same frequency as the wild type, but the hormogonia are more effective in infection of A. punctatus. Thus, SigH appears to be involved in transcription of genes that influence hormogonium behavior and not in initiation of differentiation. We attempted to establish a comprehensive cDNA library of genes induced during the initiation of hormogonium differentiation by three different subtractive approaches. Seven of the nine HIF responsive genes identified by this approach are transcribed at a low level in vegetative cells; thus, an efficient subtraction of those genes by vegetative cell RNA, even when their transcription is enhanced following HIF induction, removes a significant fraction of any clonable or hybridizable sequence. We will now apply 2D isoelectric focusing and PAGE protein analysis to determine the extent of differential gene expression during hormogonium differentiation. The corresponding genes will be identified from derived protein sequences in the N. punctiforme database.
B. Repression of differentiation. Hormogonium differentiation is mutually exclusive to heterocyst differentiation and plants appear to restrict hormogonium differentiation in the mature symbiotic association. We have identified a regulon that appears to repress hormogonium differentiation. The regulon was identified following characterization of a transposon mutant with a high infection phenotype. Interruption of genes in the regulon results in continued entry into the hormogonium cycle when in the presence of HIF. The five genes identified in the regulon have high similarity to genes encoding enzymes of glucuronic and gluconic acid metabolism in Escherichia coli and Bacillus species. We propose that the end product of the metabolic pathway may function as an inhibitor of hormogonium differentiation. Transciption in the regulon is induced by a plant product termed a hormogonium repressing factor (HRF). We are especially interested in the role and mechanisms of action of a putative transcriptional repressor (hrmR) that may regulate expression in the regulon and are currently involved in a detailed characterization of HrmR binding sites and cellular factors activated by HRF that disrupt in vitro binding.
2. Heterocyst differentiation. USDA NRICGP, Nitrogen Fixation/Nitrogen Metabolism, 12/15/98 -12/31/01.
This cellular differentiation process is the most highly studied in cyanobacteria and understanding heterocyst differentiation and function has biotechnological implications in the supply of fixed nitrogen in agricultural systems. We have previously identified and characterized three genes involved in the maturation and function of heterocysts (hglE, devR and zwf) in N. punctiforme. However, our primary interest is identifying the symbiotic signaling system that enhances heterocyst differentiation and regulates heterocyst spacing. This project has two overall objectives: (i) isolate a growing collection of Tn5-1063 induced mutants of N. punctiforme, screen the mutants for a defect in the initiation of heterocyst differentiation in the free-living growth state and analyze these mutants for a symbiotic phenotype; and (ii) characterize two recently isolated mutants whose phenotypes reflect the activity of a positive (hetF) and a negative (patN) regulator of heterocyst differentiation.
A. Initiation of differentiation. Our working hypothesis is that a plant-derived signal overrides the normal free-living nitrogen control system and initiates heterocyst differentiation in the symbiotic growth state. Three genes have been identified as essential positive regulators of heterocyst differentiation in Anabaena 7120 and they operate in the following epistatic order: ntcA (encoding a global nitrogen regulator in the CRP family of regulatory proteins that is not restricted to heterocyst differentiation), hanA (encoding a DNA binding protein analogous to HU; the mutant has an extremely pleiomorphic/pleiotrophic phenotype) and hetR (encoding a putative serine protease and is temporally the first protein identified to specifically accumulate in heterocysts). Mutations in ntcA and hetR of N. punctiforme yield phenotypes that are similar to those of Anabaena 7120 in the free-living growth state, but express contrasting symbiotic phenotypes. The hetR mutant infects Anthoceros tissue, but cannot support N2-dependent growth, indicating that hetR is essential for symbiotic heterocyst differentiation. The ntcA mutant differentiates hormogonia, but fails to infect Anthoceros tissue, therefore the requirement for a functional ntcA gene in symbiotic heterocyst differentiation remains unknown. We suggest additional regulatory genes have been overlooked due to screening for a nitrogen-responsive (bleaching) phenotype and propose rather to screen for a petite colony phenotype.
B. Characterization of heterocyst regulatory genes. Graduate student Francis Wong has isolated a fourth positive regulatory gene essential for heterocyst differentiation, which we have termed hetF. hetF is constitutively expressed and displays neither distinct motifs nor similarity to database sequences. Insertion mutants and expression of hetF in trans from a multicopy plasmid yield phenotypes identical to hetR. hetF is also required for symbiotic heterocyst differentiation. Based on mRNA analyses and expression of a PhetR-GFP fusion, hetF is not directly involved in hetR transcription. HetF appears to modulate the activity or stability of HetR in some manner.
Francis is also charactering a gene, patN, that influences the pattern of heterocyst spacing in the free-living filaments. Mutations in patN result in a three-fold higher heterocyst frequency following nitrogen deprivation; the heterocysts appear singly in the filaments with a decrease in the number of vegetative cells between adjacent heterocysts. Again, the sequence of patN has no significant similarity in the databases. However, the frequency and pattern of heterocyst spacing in this mutant is similar to that observed in the symbiotic growth state. We are designing experiments to test the hypothesis that patN modulates the activity or translocation of PatS, the dominant negative regulator of heterocyst differentiation in Anabaena 7120.
3. Glucose-6-phosphate dehydrogenase (G6PD). NSF, MCB Metabolic Biochemistry, 6/1/97 - 5/31/01.
Mike Summers, while in our laboratory, unequivocally established that G6PD is essential for dark and dinitrogen-dependent growth of N. punctiforme. Mike also determined that its transcription is complex within the four-gene opc operon and dependent on the nitrogen and carbon source for growth. The requirement for G6PD in nitrogen fixation implies that zwf (the gene encoding G6PD) may be differentially expressed in vegetative cells and heterocysts. In addition, G6PD catalytic activity is dependent on a second gene product, OpcA, whose structural gene is 3' from zwf and the two genes are cotranscribed in N. punctiforme. This project has two overall objectives: (i) characterize transcriptional regulation of zwf; and (ii) determine the role of OpcA in activation of G6PD.
A. Transcriptional regulation of zwf. Based on a sequence alignment of other nitrogen regulated genes in Anabaena 7120, two conserved sequences can be observed in two of the nitrogen regulated putative promoter regions of the opc operon of N. punctiforme. One region is upstream of the -10 region (-21 to -30) in the 5' sequence of the P5 putative promoter, which we hypothesize is a repressor binding site. The second conserved sequence is upstream of the -35 region (-46 to -80) of the putative P1 promoter, which we hypothesize is an activator binding site. Because mutations that block transcription from the P5 region do not yield a N2 or dark-dependent growth phenotype, we are focusing our efforts on the P1 region. We have proposed in vivo and in vitro assays to verify the putative promoter activity in the 5' region and deletion/substitution analysis to support the regulatory role for the conserved sequence. We have PCR amplified the putative P1 promoter region, cloned it into a luciferase expression vector and detected promoter activity in vivo from a multicopy plasmid in trans. The promoter activity was unregulated implying limited concentrations of a regulatory element. While on sabbatical leave, I developed a purification protocol for RNA polymerase from N. punctiforme that involves affinity precipitation, affinity chromatography and size exclusion chromatography that is prelude to in vitro transcription analyses.
B. OpcA activation of G6PD. Postdoctoral fellow Kari Hagen has completed a kinetic analysis (varying G6P and NADP) of the effect of His-tagged OpcA on His-tagged G6PD, both purified from E. coli, and with reciprocal mixed cell extracts of zwf and opcA mutants. It is clear that when synthesized in the presence or absence of OpcA, G6PD aggregates into a tetrameric form. However, the G6PD synthesized in the absence of OpcA has a high Km (65 mM) for G6P. Addition of purified OpcA lowers the Km for G6P to about 0.6 mM, but has no effect on the affinity of NADP. Maximal activation of G6PD using the native proteins occurs in at 1:1 stoichiometry. Both G6PD and OpcA have conserved cysteine residues that could be subject to redox regulation. The purified OpcA can be reversibly reduced by (DTT-reduced) thioredoxin and oxidized by tetrathionate; the oxidized form of OpcA activates G6PD whereas the reduced form is unable to do so. These results are in contrast to suggestions by others that the redox state of G6PD is regulated by thioredoxin and OpcA activates by promoting aggregation of tetramers to a higher ordered state. The situation is somewhat complicated by the observation that opcA or OpcA also influences the accumulation of G6PD in N. punctiforme by stabilization of the mRNA, the polypeptide, or the mature protein. These possibilities are now being examined.