James P. Shapleigh, Ph. D.

Associate Professor
Department of Microbiology

Cornell University
257A Wing Hall
Ithaca, NY 14853-8101

Phone: (607) 255-8535
Fax: (607) 255-3904
E-Mail: jps2@cornell.edu

 

The focus of the research in the Shapleigh laboratory is to increase our understanding of the regulation and physiological role of denitrification at the cellular level. We are currently undertaking genetic, biochemical and genomic-based approaches to identify and characterize genes and gene products involved in denitrification and nitric oxide metabolism.

Reactions of the nitrogen cycle transform molecules of nitrogen gas (N2) into fixed forms and then back into gaseous forms (Figure 1). The transformation of fixed forms of nitrogen to gaseous forms is termed denitrification (Figure 1). Denitrification is a beneficial process in some situations, for example, during waste treatment. In contrast, the production of gaseous intermediates during denitrification is unfavorable from an agricultural perspective since fixed nitrogen is frequently a growth-limiting nutrient. A detailed understanding of the factors controlling denitrification will increase our understanding of the fate of fixed nitrogen in the environment. While some of the fundamental regulators of denitrification are known (oxygen, carbon and nitrogen oxide availability) the interplay between these various factors is not fully understood at the field or cellular level.

Denitrification is carried out by a variety of prokaryotes including archaea such as Pyrobaculum aerophilum and Haloferax denitrificans, gram-positives such as Bacillus azotoformans and Streptomyces species and a variety of gram-negative bacteria. The majority of currently characterized denitrifiers belong to the group of gram negatives referred to as proteobacteria. The most heavily studied denitrifiers are found in the a and g subdivisions of this group. Our primary model organism is the photosynthetic bacterium Rhodobacter sphaeroides, which is in the a subdivision. Of the many strains of R. sphaeroides that have been characterized, only a few are complete denitrifiers. The type strain of R. sphaeroides, strain 2.4.1 whose genome has recently been sequenced, is, in fact, a partial denitrifier. It has been found to encode and express the genes for nitrate and nitric oxide reductase but to lack nitrite reductase (Figure 1). The strain we study, 2.4.3, encodes a copper-containing nitrite reductase as well as the other nitrogen oxide reductases, thus making it a complete denitrifier.

Our research efforts have focused on studying the production and reduction of nitric oxide (NO). While the physiological function and regulation of NO metabolism in R. sphaeroides 2.4.3 shares many mechanistic similarities with similar reactions in other denitrifiers, strain 2.4.3 has many unique features which make it a very useful organism for studying NO metabolism. For example, changes in pigmentation during denitrification make it easy to determine if cells are actively denitrifying (Figure 2). Also, 2.4.3 is one of the few R. sphaeroides strains that shows nitrogen oxide dependent taxis (Figure 3). The taxis response is useful in detecting subtle changes in the ability of mutants to detect and metabolize nitrogen oxides. One of the most unique features in 2.4.3 is the complex regulation of the gene that encodes for nitrite reductase (Nir), which is the key enzyme in microbial denitrification. Nir expression appears to be repressed under dark, anaerobic conditions (Figure 4). Very recently we have found that NO production in 2.4.3 has some unique features that distinguish it from most other denitrifiers (unpublished).

Current research projects are focused on identifying additional components of the electron transfer chain required for denitrification in 2.4.3, studying the promoter structure of the genes encoding Nir and nitric oxide reductase, better quantifying NO production and reduction, and identifying additional genes required for denitrification. A project studying structural and mechanistic details of nitrite reductase is being undertaken in collaboration with Prof. Charles P. Scholes of SUNY –Albany. Another area of interest is to take advantage of what we have learned about the capacity of denitrifiers like 2.4.3 to sense and metabolize NO to develop useful reporters. For example, we have developed a GFP fusion reporter system for detecting NO utilizing a gene from the NnrR regulon of R. sphaeroides 2.4.3 (Figure 5) (manuscript submitted). An ongoing collaboration with Prof. Hector Abruña here at Cornell has also resulted in the development and application of useful nitrogen oxide sensing electrodes. 

Publications.

Hartsock, A. and J.P. Shapleigh. 2010. Identification, functional studies, and genomic comparisons of new members of the NnrR regulon in Rhodobacter sphaeroides. J. Bacteriol. 192:903-911.

Lee, B. O.M. Usov, V.M. Grigoryants, W.K. Myers, J.P. Shapleigh and C.P. Scholes. 2009. The role of arginine-127 at the proximal NO-binding site in determining hte electronic structure and function of 5-coordinate NO-heme in cytochrome c" of Rhodobacter sphaeroides. Biochemistry 48:8985-8993.

Bergaust, L., J. P. Shapleigh, Å. Frostegård, and L. R. Bakken. in press. Transcription and activities of NOx reductases in Agrobacterium tumefaciens: the influence of nitrate, nitrite and oxygen availability. Env. Microbiol.

Baek, S. H., A. Hartsock, and J. P. Shapleigh. 2008. Agrobacterium tumefaciens C58 uses ActR and FnrN to control nirK and nor expression. J Bacteriol 190:78-86.

Shapleigh, J. P. 2007. The denitrifying prokaryotes, p. 769-792. In M. Dworkin (ed.), The Prokaryotes:A Handbook on the Biology of Bacteria: Volume 2: Ecophysiology and Biochemistry. Springer-Verlag New York, NY.

Basaglia, M., A. Toffanin, E. Baldan, M. Bottegal, J. P. Shapleigh, and S. Casella. 2007. Selenite-reducing capacity of the copper-containing nitrite reductase of Rhizobium sullae. FEMS Microbiol Lett 269:124-130.

Laratta, W. P., M. J. Nanaszko, and J. P. Shapleigh. 2006. Electron transfer to nitrite reductase of Rhodobacter sphaeroides 2.4.3: examination of cytochromes c₂ and c_Y. Microbiology 152:1479-1488.

Casella, S., J. P. Shapleigh, A. Toffanin, and M. Basaglia. 2006. Investigation into the role of the truncated denitrification chain in Rhizobium sullae strain HCNT1. Biochem Soc Trans

Choi, P. S., Z. Naal, C. Moore, E. Casado-Rivera, H. D. Abruna, J. D. Helmann, and J. P. Shapleigh. 2006. Assessing the impact of denitrifier-produced nitric oxide on other bacteria. Appl Environ Microbiol 72:2200-2205.

Usov, O. M., P. S. Choi, J. P. Shapleigh, and C. P. Scholes.2006. ENDOR of NO-ligated cytochrome c'. J Am Chem Soc 128:5021-5032.

Usov, O. M., Y. Sun, V. M. Grigoryants, J. P. Shapleigh, and C. P. Scholes. 2006. EPR-ENDOR of the Cu(I)NO complex of nitrite reductase. J Am Chem Soc 128:13102-13111.

Baek, S. H., and J. P. Shapleigh. 2005. Expression of nitrite and nitric oxide reductases in free-living and plant-associated Agrobacterium tumefaciens C58 cells. Appl Environ Microbiol 71:4427-4436.<

Choi, P. S., V. M. Grigoryants, H. D. Abruna, C. P. Scholes, and J. P. Shapleigh. 2005. Regulation and function of cytochrome c' in Rhodobacter sphaeroides 2.4.3. J Bacteriol 187:4077-4085.

Usov, O. M., P. S. Choi, J. P. Shapleigh, and C. P. Scholes. 2005. ENDOR investigation of the liganding environment of mixed-spin ferric cytochrome c'. J Am Chem Soc 127:9485-9494.

Baek, S. H., G. Rajashekara, G. A. Splitter, and J. P. Shapleigh. 2004. Denitrification genes regulate Brucella virulence in mice. J Bacteriol 186:6025-6031.

Basumallick, L., R. K. Szilagyi, Y. Zhao, J. P. Shapleigh, C. P. Scholes, and E. I. Solomon. 2003. Spectroscopic studies of the Met182Thr mutant of nitrite reductase: role of the axial ligand in the geometric and electronic structure of blue and green copper sites. J Am Chem Soc. 125:14784-14792.

Laratta, W. P., and J. P. Shapleigh.2003. Site-directed mutagenesis of NnrR: a transcriptional regulator of nitrite and nitric oxide reductase in Rhodobacter sphaeroides. FEMS Microbiol. Lett. 229:173-178.

Yin, S., M. Fuangthong, W. P. Laratta, and J. P. Shapleigh. 2003. Use of a green fluorescent protein-based reporter fusion for detection of nitric oxide produced by denitrifiers. Appl Environ Microbiol 69:3938-3944.

Lee, D. Y., A. Ramos, L. Macomber, and J. P. Shapleigh. 2002. The taxis response of various denitrifying bacteria to nitrate and nitrite. Appl. Environ. Microbiol. 68: 2140-2147.

Laratta, W. P., P. S. Choi, I. E. Tosques, and J. P. Shapleigh. 2002. Involvement of the PrrB/PrrA two-component system in nitrite respiration in Rhodobacter sphaeroides 2.4.3: evidence for transcriptional regulation. J. Bacteriol. 184:3251-3259.

Takada, K., Z. Naal, J.-H. Park, J. P. Shapleigh, S. Bernhard, C. A. Batt, and H. D. Abruña. 2002. Study of specific binding of maltose binding protein to pyrrole-derived bipyridium film using QCM. Langmuir. 18: 4892-4897.

Zhao, Y., D. A. Lukoyanov, Y. V. Toropov, K. Wu, J. P. Shapleigh, and C. P. Scholes. 2002. Catalytic function and local proton structure at the Type 2 copper of nitrite reductase: the correlation of enzymatic pH dependence, conserved residues and proton hyperfine structure. Biochemistry. 41:7464-7474.

Hosler, J., and J. P. Shapleigh. 2002. Aerobic respiration, Principles of. In G. Bitton (ed.), Encyclopedia of environmental microbiology. Wiley, New York, NY.

Bartnikas, T. B., Y. Wang, T. Bobo, A. Veselov, C. P. Scholes, and J. P. Shapleigh. 2002. Characterization of a member of the NnrR regulon in Rhodobacter sphaeroides 2.4.3 encoding a heme-copper protein. Microbiol. 148:825-833.

Naal, Z., J.-H. Park, S. Bernhard, J. P. Shapleigh, C. A. Batt, and H. D. Abruña. 2002. Amperometric TNT biosensor based on the oriented immobilization of a nitroreductase maltose binding protein fusion. Anal. Chem. 74:140 -148.

Mackenzie, C., M. Choudhary, F. W. Larimer, P. F. Predki, S. Stilwagen, J. P. Armitage, R. D. Barber, T. J. Donohue, J. P. Hosler, J. E. Newman, J. P. Shapleigh, R. E. Sockett, J. Zeilstra-Ryalls, and S. Kaplan. 2001. The home stretch, a first analysis of the nearly completed genome of Rhodobacter sphaeroides 2.4.1. Photosyn. Res. 70:19-41.

Jain, R., and J. P. Shapleigh. 2001. Characterization of nirV and a gene encoding a novel pseudoazurin in Rhodobacter sphaeroides 2.4.3. Microbiol. 147:2505-2515.

Shapleigh, J. P. 2000. The denitrifying prokaryotes. In M. Dworkin (ed.), The prokaryotes: An evolving electronic resource for the microbiological community. Springer-Verlag, New York, NY.

Mitchell, D. M., Y. Wang, J. O. Alben, and J. P. Shapleigh. 1998. Fourier transform infrared analysis of membranes of Rhodobacter sphaeroides 2.4.3 grown under microaerobic and denitrifying conditions. Biochim. Biophys. Acta. 1409:99-105.

Wu, Q., G. D. Storrier, K. R. Wu, J. P. Shapleigh, and H. D. Abruña. 1998. Electrocatalytic reduction of S-nitrosoglutathione at electrodes modified with an electropolymerized film of a pyrrole derived viologen system and their application to cellular S-nitrosoglutathione determinations. Anal. Biochem. 263:102-112.

Olesen, K.O., A. Veselov, Y. Zhao, Y. Wang, B. Danner, C.P. Scholes and J.P. Shapleigh. 1998. Spectroscopic, kinetic and electrochemical characterization of heterologously expressed wild type and mutant forms of copper-containing nitrite reductase from Rhodobacter sphaeroides 2.4.3. Biochem. 37:6086-6094.

Veselov, A., K. Olesen, A. Sienkiewicz, J.P. Shapleigh and C.P. Scholes. 1998. Electronic structural information from Q-band ENDOR on the Type 1 and Type 2 copper liganding environment in wild type and mutant products of copper-containing nitrite reductase. Biochem. 37:6095-6105.

Wu, Q., G.D. Storrier, F. Pariente, Y. Wang, J.P. Shapleigh and H.D. Abruña. 1997. A nitrite biosensor based on a maltose binding protein nitrite reductase fusion immobilized on an electropolymerized film of a pyrrole derived bipyridinium. Anal Chem. 69: 4856-4863.

Kwiatkowski, A.V., W.P. Laratta, A. Toffanin and J.P. Shapleigh. 1997. Analysis of the role of the nnrR gene product in the response of Rhodobacter sphaeroides 2.4.1 to exogenous nitric oxide. J. Bacteriol. 179:5618-5620.

Bartnikas, T.B., I.E. Tosques, W.P. Laratta, J. Shi and J.P. Shapleigh. 1997. Characterization of the region encoding the nitric oxide reductase of Rhodobacter sphaeroides 2.4.3. J. Bacteriol. 179:3534-3540.

Tosques, I.E., A.V. Kwiatkowski, J. Shi and J.P. Shapleigh. 1997. Characterization and regulation of the gene encoding nitrite reductase in Rhodobacter sphaeroides 2.4.3. J. Bacteriol. 179:1090-1095.

Hosler, J.P., J.P. Shapleigh, D.M. Mitchell, Y. Kim, M.A. Pressler, C. Georgiou, G.T. Babcock, J.O. Alben, S. Ferguson-Miller and R.B. Gennis. 1996. Polar residues in helix VIII of subunit I of cytochrome c oxidase influence the activity and the structure of the active site. Biochem. 35:10776-10783.

Kwiatkowski, A. and J.P. Shapleigh. 1996. Requirement of nitric oxide for induction of genes whose products are involved in nitric oxide metabolism in Rhodobacter sphaeroides 2.4.3. J. Biol. Chem. 271:24382-24388.

Maskus, M., F. Pariente, Q. Wu, A. Toffanin, J.P. Shapleigh and H.D. Abruña. 1996. Electrocatalytic reduction of nitric oxide at electrodes modified with electropolymerized films of [Cr(v-tpy)2]+3 and their applications to cellular NO determinations. Anal. Chem. 68:3128-3124.

Mitchell, D.M., J.P. Shapleigh, A.M. Archer, J.O. Alben and R.B. Gennis. 1996. A pH-dependent polarity change at the binuclear center of reduced cytochrome c oxidase detected by FTIR difference spectroscopy of the CO adduct. Biochem. 35:9446-9450.

Toffanin, A., Q. Wu, M. Maskus, S. Casella, H.D. Abruña and J.P. Shapleigh. 1996. Characterization of the gene encoding nitrite reductase and the physiological consequences of its expression in the nondenitrifying Rhizobium "hedysari" strain HCNT1. Appl. Environ. Microbiol. 62:4019-4025.

Tosques, I.E., J. Shi and J.P. Shapleigh. 1996. Cloning and characterization of nnrR, whose product is required for the expression of proteins involved in nitric oxide metabolism in Rhodobacter sphaeroides 2.4.3. J. Bacteriol. 178:4958-4964.

Fetter, J.R., J. Qian, J. Shapleigh, J.W. Thomas, A. Garcia- Horsman, E. Schmidt, J. Hosler, G.T. Babcock, R.B. Gennis and S. Ferguson-Miller. 1995. Possible proton relay pathways in cytochrome c oxidase. Proc. Natl. Acad. Sci. USA. 92:1604-1608.

Yun, C.H., B. Barquera, K. Iba, K.I. Takamiya, J. Shapleigh, A.R. Crofts and R.B. Gennis. 1994. Deletion of the gene encoding cytochrome b-562 from Rhodobacter sphaeroides. FEMS Microbiol. Lett. 120:105-110.

Hosler, J.P., J.P. Shapleigh, M.M.J. Tecklenburg, J.W. Thomas, Y. Kim, M. Espe, J. Fetter, G.T. Babcock, J.O. Alben, R. Gennis and S. Ferguson-Miller. 1994. A loop between transmembrane helices IX and X of subunit I of cytochrome c oxidase caps the heme a-heme a3-CuB center. Biochem. 33:1194-1201.

Garcia-Horsman, J.A., E. Berry, J.P. Shapleigh, J.O. Alben and R.B. Gennis. 1993. A novel cytochrome c oxidase from Rhodobacter sphaeroides that lacks CuA. Biochem. 33:3113-3119.

Hosler, J.P., S. Ferguson-Miller, M.W. Calhoun, J.W. Thomas, J. Hill, L. Lemieux, J. Ma, C. Georgiou, J. Fetter, J. Shapleigh, M.M.J. Tecklenburg, G.T. Babcock and R.B. Gennis. 1993. Insight into the active site structure and function of cytochrome oxidase by analysis of site-directed mutants of bacterial cytochrome aa3 and cytochrome bo. J. Bioenergetics and Biomembranes. 25:121-136.

Shapleigh, J.P. and R.B. Gennis. 1992. Cloning, sequencing and deletion from the chromosome of the gene encoding subunit I of the aa3-type cytochrome c oxidase of Rhodobacter sphaeroides. Mol. Microbiol. 6:635-642.

Shapleigh, J.P., J.J. Hill, J.O. Alben and R.B. Gennis. 1992. Spectroscopic and genetic evidence for two heme-Cu containing oxidases in Rhodobacter sphaeroides. J. Bacteriol. 174:2338-2343.

Shapleigh, J.P., J.P. Hosler, M.M.J. Tecklenburg, Y. Kim, G.T. Babcock, R.B. Gennis and S. Ferguson-Miller. 1992. Definition of the catalytic site of cytochrome c oxidase: The specific ligands of heme a and the heme a3-CuB center. Proc. Natl. Acad. Sci. USA. 89:4786-4790.

Casella, S., J.P. Shapleigh, F. Lupi and W.J. Payne. 1988. Nitrite reduction in bacteroids of Rhizobium "hedysari" strain HCNT1. Arch. Microbiol. 149:384-388.

Shapleigh, J.P., K.J.P. Davies and W.J. Payne. 1987. Detergent inhibition of nitric oxide reductase. Biochim. Biophys. Acta. 911:334-340.

Casella, S., J.P. Shapleigh and W.J. Payne. 1986. Nitrite reduction in Rhizobium "hedysari" strain HCNT1. Arch. Microbiol. 146:233-238.

Shapleigh, J.P. and W.J. Payne. 1985. Differentiation of c,d1 cytochrome and copper nitrite reductase production in denitrifiers. FEMS Microbiol Lett. 26:275-279.

Shapleigh, J.P. and W.J. Payne. 1985. Nitric oxide-dependent proton translocation in various denitrifiers. J. Bacteriol. 163:837-840.

Payne, W.J., M.A. Grant, J.P. Shapleigh and P. Hoffman. 1982. Nitrogen oxide reduction in Wolinella succinogenes and Campylobacter species. J. Bacteriol. 152:915-918.

click here for a PubMed listing of Dr. Shapleigh's publications