Mark R. O’Brian, Ph.D. Professor Department of Biochemistry Director of the Witebsky Center for Microbial Pathogenesis & Immunology Phone: (716) 829-3200 Fax: (716) 829-2725 Email: mrobrian@buffalo.edu

Regulation of heme biosynthesis in Rhizobium
Rhizobia, the collective term for the genera Rhizobium, Bradyrhizobium and Azorhizobium, belong to the alpha proteobacteria, and numerous organisms within this taxon form intracellular associations with higher eukaryotes. 16S rRNA analysis of mitochondrial genomes places the organelle within this group as well, and agrees with an endosymbiotic origin hypothesis. Whereas Brucella and Rickettsia are mammalian pathogens, Rhizobium is symbiotic with leguminous plants, and the differentiated bacterium converts atmospheric N2 to ammonia which is subsequently assimilated by the host to fulfill its nutritional nitrogen requirement. Numerous virulence genes have been identified that contain homologs in Rhizobium that are essential for symbiosis. Although the outcomes of successful infection and colonization by a bacterial symbiont are very different from a pathogen from the host perspective, it is clear that they are variations of similar molecular and cellular processes. My lab is interested in metabolic regulation in Bradyrhizobium japonicum, and in understanding how the microbe redirects host processes for symbiotic development.

Symbiosis is energy-intensive, and requires changes in heme protein content for oxidative metabolism. We have shown that heme biosynthesis is highly regulated in B. japonicum, and are focused on control by iron and oxygen. Heme is an iron-protoporphyrin, and we identified a novel protein called the iron response regulator (Irr) that mediates iron control of the heme biosynthesis. An irr mutant accumulates the immediate heme precursor protoporphyrin under iron limited conditions, and we suggest that Irr prevents protoporphyrin accumulation from exceeding iron availability. The irr gene is regulated by iron, and Irr is only detectable under iron limitation. Iron exerts weak control of irr transcription, but strongly regulates Irr protein turnover. We have shown that iron-regulated protein stability is mediated by a heme group, which binds to Irr directly to facilitate its degradation. Furthermore, Irr interacts directly with the heme synthesis enzyme ferrochelatase, and its activity is affected by the ferrochelatase substrates iron and protoporphyrin. Our current model argues that Irr responds to heme at the site of synthesis, and does not rely on free “pools” of effector molecules.

We have proposed that B. japonicum heme found in soybean root nodules can be synthesized from the tetrapyrrole precursor d-aminolevulinic acid (ALA) derived from the plant host, thus segments of a single metabolic pathway are spatially separated between two organisms. We found that ALA uptake by B. japonicum is controlled by the ptsP gene. Interestingly, a homolog was identified in the pathogen Pseudomonas aeruginosa, and is essential for its infection of an animal host C. elegans. Finally, our data show that induction of ALA synthesis by the host in response to bacterial colonization involves an altered spatial expression of genes that are otherwise induced strongly only in photosynthetic tissues of uninfected plants. We have identified a host protein induced in nodules (the symbiotic organ) that may allow this altered expression pattern.

Professional Activities

Member, Gordon Research Conference Council, 2004-2006.
Editorial Board, Applied and Environmental Microbiology , 2001-2006.
Monitoring Editor, Plant Physiology , 2005-2010.
Recipient, University at Buffalo Sustained Achievement Award, 2006

SELECTED PUBLICATIONS
Sangwan, I., and M.R. O'Brian. 1999. Expression of a soybean gene encoding the tetrapyrrole synthesis enzyme glutamyl-tRNA reductase in symbiotic root nodules. Plant Physiol. 119: 593-598.

Hamza, I., R. Hassett and M.R. O'Brian. 1999. Identification of a functional fur gene in Bradyrhizobium japonicum. J. Bacteriol. 181: 5843-5846.

Qi, Z., I. Hamza and M.R. O'Brian. 1999. Heme is an effector molecule for iron-dependent degradation of the bacterial iron response regulator (Irr) protein. Proc. Natl. Acad. Sci. U.S.A. 96: 13056-13061.

Hamza, I., Z. Qi, N.D. King and M.R. O’Brian. 2000. Fur-independent regulation of iron metabolism by Irr in Bradyrhizobium japonicum. Microbiol. 146: 669-676.

King, N.D., D. Hojnacki and M.R. O'Brian. 2000. The Bradyrhizobium japonicum proline biosynthesis gene proC is essential for symbiosis. Appl. Env. Microbiol. 66: 5469-5471.

King, N.D. and M.R. O'Brian. 2001. Evidence for direct interaction between Enzyme INtr and aspartokinase to regulate bacterial oligopeptide transport. J. Biol. Chem. 276: 21311-21316.

Qi, Z. and M.R. O’Brian. 2002. Interaction between the bacterial iron response regulator (Irr) and ferrochelatase mediates genetic control of heme biosynthesis. Mol. Cell, 9:155-162.

Sangwan, I. And M.R. O’Brian. 2002. Identification of a soybean protein that interacts with GAGA element dinucleotide repeat DNA. Plant Physiol. 129: 1788-1794.

Panek, H. and M.R. O’Brian. 2002. A genomic view of prokaryotic heme biosynthesis. Microbiol. 148: 2273-2282.

Friedman, Y.E. and M.R. O’Brian. 2003. A novel DNA-binding site for the ferric uptake regulator (Fur) protein from Bradyrhizobium japonicum. J. Biol. Chem. 278: 38395-38401.

Friedman, Y.E. and M.R. O’Brian. 2004. The ferric uptake regulator (Fur)
protein from Bradyrhizobium japonicum is an iron-responsive transcriptional repressor in vitro. J. Biol. Chem. 279: 32100-32105.

Platero, R., L. Peixoto, M.R. O’Brian and E. Fabiano. 2004. Fur is involved manganese-dependent regulation of mntA (sitA) expression in Sinorhizobium meliloti. Appl. Environ. Microbiol. 70: 4349-4355.

Panek, H.R. and M.R. O’Brian. 2004. KatG is the primary detoxifier of hydrogen peroxide produced by aerobic metabolism in Bradyrhizobium japonicum. J. Bacteriol. 186: 7874-7880.

Yang, J., K. Ishimori and M.R. O’Brian. 2005. Two heme binding sites are involved in the regulated degradation of the bacterial iron response regulator (Irr) protein. J. Biol. Chem. 280: 7671-7676.

Gao, T. and M.R. O’Brian. 2005. Iron-dependent cytochrome c1 expression is mediated by the status of heme in Bradyrhizobium japonicum. J. Bacteriol. 187: 5084-5089.

Yang J. Sangwan I. O'Brian MR. The Bradyrhizobium japonicum Fur protein is an iron-responsive regulator in vivo. Molecular Genetics & Genomics: MGG. 276(6):555-64, 2006 Dec.

Puri S. O'Brian MR. The hmuQ and hmuD genes from Bradyrhizobium japonicum encode heme-degrading enzymes. Journal of Bacteriology. 188(18):6476-82, 2006 Sept.

Yang J. Sangwan I. Lindemann A. Hauser F. Hennecke H. Fischer HM. O'Brian MR. Bradyrhizobium japonicum senses iron through the status of haem to regulate iron homeostasis and metabolism. Molecular Microbiology. 60(2):427-37, 2006 Apr.

Gao T. O'Brian MR. Control of DegP-dependent degradation of c-type cytochromes by heme and the cytochrome c maturation system in Escherichia coli. Journal of Bacteriology. 189(17):6253-9, 2007 Sep.

O'Brian MR. Vance CP. Legume biology: sequence to seeds. Plant Physiology. 144(2):537, 2007 Jun.

BOOK CHAPTERS
Chauhan, S. and M.R. O’Brian. 1997. Regulation of Bradyrhizobium japonicum hemB, a heme synthesis gene. In “Biological Fixation of Nitrogen for Ecology and Sustainable Agriculture” (A. Legocki, H. Bothe, and A. Puhler, eds.), Springer Verlag, New York, pp. 95-98.

O'Brian, M.R. 1999. Regulation of bacterial heme biosynthesis by iron. In "Iron Metabolism: Inorganic Biochemistry and Regulatory Mechanisms " (G.C. Ferreira, J.G.G. Moura, R. Franco, , eds.), Wiley-VCH, New York, pp. 1-13.

O'Brian, M.R. 2000. Heme biosynthesis and function in the Rhizobium-legume symbiosis. In "Prokaryotic Nitrogen Fixation: A Model System for the Analysis of a Biological Process". (E.W. Triplett, ed.), Horizon Scientific Press, pp. 509-528.

O'Brian, M.R.and L. Thony-Meyer. Biochemistry, regulation and genomics of heme Biosynthesis in Prokaryotes. In Adv. Microbial. Physiol. (R.K. Poole, ed), Academic Press, London, pp. 257-318.

Book Edited: Nitrogen Fixation: Global Persepectives. 2002. T.M. Finan, M.R. O'Brian, D.B. Layzell, J.K., Vessey and W. Newton (eds), CABI Publishing Wallingford, UK, 553 p.

Gao, T. and M.R. O'Brian. 2005. Iron-dependent cytochrome c 1 expression is mediated by the status of heme in Bradyrhizobium japonicum . J. Bacteriol. 187: 5084-5089.

Yang, J., H.R.. Panek, and M.R. O'Brian. 2006 Oxidative stress promotes degradation of the Irr protein to regulate heme biosynthesis in Bradyrhizobium japonicum . Mol. Microbiol. 60: 209-218.

Yang, J., I. Sangwan, A. Lindemann, F. Hauser, H. Hennecke, H.-M. Fischer, and M.R. O'Brian. 2006. Bradyrhizobium japonicum senses iron through the status of heme to regulate iron homeostasis and metabolism. Mol. Microbiol. 60: 427-437.

Yang, J., Y. Friedman and M.R. O'Brian. 2005. Regulation of heme and iron metabolism in Bradyrhizobium japonicum . In "Biological Nitrogen Fixation, Sustainable Agriculture and the Environment" (Y.-P. Yang, M. Lin, Z.-X. Tian, C. Elmerich, W.E. Newton, eds), Springer, Dordrecht , pp. 303-306.

Gao T. O'Brian MR. Control of DegP-dependent degradation of c-type cytochromes by heme and the cytochrome c maturation system in Escherichia coli. Journal of Bacteriology. 189(17):6253-9, 2007 Sep.

O'Brian MR. Vance CP. Legume biology: sequence to seeds. Plant Physiology. 144(2):537, 2007 Jun.