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.