Robert Haselkorn, Ph.D.

Organization and Regulation of Genes Involved in Nitrogen Fixation and Photosynthesis in Cyanobacteria and Photosynthetic Bacteria; Acetyl CaA Carboxylase in Plants

Research Summary

We study the molecular genetics of nitrogen fixation and photosynthesis in cyanobacteria and purple bacteria. We also study genes encoding the enzyme acetyl-CoA carboxylase in plants.

The cyanobacterium Anabaena grows in filaments of 100 cells or more. When starved for nitrogen, specialized cells called heterocysts differentiate from the photosynthetic vegetative cells at regular intervals along each filament. Heterocysts are anaerobic factories for nitrogen fixation; in them, the nitrogenase enzyme complex synthesized and the components of the oxygen-evolving photosystem II are turned off. More than 1000 genes are believed to be differentially expressed during the (irreversible) development of a heterocyst from a vegetative cell. We have cloned and sequenced genes for nitrogen fixation (nif) and others encoding RuBP carboxylase, glutamine synthetase, the D1, CP-47 and water-oxidizing proteins of photosystem II, all the components of phycobilisome rods, and the sigma and core sub-units of RNA polymerase. Many mutants unable to fix nitrogen aerobically have been isolated. Among these are some that have altered heterocyst morphology or an altered pattern. Four of these have been studied in detail, using a complementation system to isolate the wild-type gene defective in the mutants. One mutant fails to deposit the necessary glycolipid layer that forms part of the heterocyst envelope. A second mutant fails to make any heterocysts at all. A third makes them only at the ends of filaments. A fourth makes them too late and too frequently! In these cases, the sequences of the complementing genes are highly informative, corresponding to proteins that participate in environment-sensing regulatory cascades. The relationships among these regulatory proteins are being worked out by using the Green Fluorescent Protein from the jellyfish as a cell-specific reporter of gene expression.

The purple bacterium Rhodobacter capsulatus carries out photosynthesis and nitrogen fixation at the same time. Its chromosome is a circle containing 3.7 Mb of DNA. We have constructed a fine-structure physical map of the chromosome based on a set of overlapping cosmids that cover it completely. Nearly all of the known genes of Rhodobacter have been located on the physical map. We have almost determined the complete sequence of the
chromosomal DNA, but four gaps remain. We are attempting to close the gaps by sequencing lambda clones that bridge the gaps.

Fatty acid synthesis, in plants as well as in cyanobacteria, begins with the reaction catalyzed by acetyl-CoA carboxylase (ACC). ACC in bacteria, including cyanobacteria, is comprised of four subunits: biotin carboxyl carrier protein (BCCP), biotin carboxylase (BC), and two subunits of carboxyltransferase. In chicken, rat, yeast and plants all of these domains reside in a single polypeptide. We have cloned and sequenced genes encoding the two isozymes of ACC from wheat, one of which is cytoplasmic while the other is located in the chloroplast and mitochondria. The corresponding cDNAs have been cloned in yeast strains that lack their own ACC.

Yeast using the wheat enzyme are sensitive to herbicides that target the wheat enzyme, allowing a full study of structure/function relationships for this important enzyme. The wheat/yeast system will also be useful for production of crystallizable amounts of protein for structure determinations. It turns out that apicomplexan parasites such as Toxoplasma and Plasmodium contain ACC enzymes that are sensitive to the same compounds that inhibit the wheat chloroplast ACC. We have constructed yeast recombinant strains dependent for growth on the parasite ACC and are using these strains to screen for new drugs to treat the parasitic diseases.

Selected Papers

Vlcek C, Paces V, Maltsev N, Paces J, Haselkorn R and Fonstein M. (1997). Sequence of a 189-kb segment of the chromosome of Rhodobacter capsulatus SB1003. Proc. Natl. Acad. Sci. USA 94, 9384-9388.

Joachimiak M, Tevzadze G, Podkowinski J, Haselkorn R and Gornicki P. (1997). Wheat cytosolic acetylCoA carboxylase complements an ACC1 null mutation in yeast. Proc. Nat. Acad. Sci. USA 94, 9990-9995.

Gornicki P, Faris J, King I, Podkowinski J, Gill B and Haselkorn R. (1997). Plastid-localized acetylCoA carboxylase of bread wheat is encoded by a single gene on each of the three ancestral chromosome sets. Proc. Natl. Acad. Sci. USA 94, 14179-14284.

Buikema WJ and Haselkorn R. (2001). Expression of the Anabaena hetR gene from a copper-regulated promoter leads to heterocyst differentiation under repressing conditions. Proc. Natl. Acad. Sci. USA 98: 2729-2734.

Jelenska J, Sirikhachornkit A, Haselkorn R and Gornicki P. (2002). The carboxyltransferase activity of the apicoplast ACCase of Toxoplasma gondii is the target of aryloxyphenoxypropionate inhibitors. J. Biol. Chem. 277: 23208-23215.

Podkowinski J, Jelenska J, Sirikhachornkit A, Zuther E, Haselkorn R and Gornicki P. (2003). Expression of cytosolic and plastid acetyl-CoA carboxylase genes in young wheat plants. Plant Physiol. 131: 763-772.