Nitrogenase Activity and nifH Expression in a Marine Intertidal Microbial Mat T.F. Steppe 1,2 and H.W. Paerl 1 (1) Institute of Marine Sciences, University of North Carolina at Chapel Hill, 3431 Arendell Street, Morehead City, NC 28557, USA (2) Center of Marine Biotechnology, UMBI, 701 E Pratt Street, Baltimore, MD 21202, USA Received: 6 November 2003 / Accepted: 24 February 2004 / Online publication: 17 June 2005 Abstract N 2 fixation, diazotrophic community composition, and organisms actively expressing genes for N 2 fixation were examined over at 3)year period (1997–1999) for inter- tidal microbial mats on a sand flat located in the Rachel Carson National Estuarine Research Reserve (RCNERR) (Beaufort, NC, USA). Specifically, diel variations of N 2 fixation in the mats from the RCNERR were examined. Three distinct diel patterns of nitrogenase activity (NA) were observed. NA responses to short-term inhibitions of photosynthesis corresponded to one of the three patterns. High rates of NA were observed during peak O 2 pro- duction periods for diel experiments during summer months. Different types of NA diel variations correspond to different stages of mat development. Chloramphenicol treatments indicated that the mechanism of protein synthesis supporting NA changed throughout the day. Analysis of mat DNA and RNA gave further evidence suggesting that in addition to cyanobacteria, other functional groups were responsible for the NA observed in the RCNERR mats. The role of microbial diversity in the N 2 fixation dynamics of these mats is discussed. Introduction Microbial mats are laminated structures that develop in marine, lacustrine, terrestrial, and thermal environments. In coastal areas, mats become established in the tidal zone as either semipermanent or ephemeral seasonal features [7, 33]. They are composed of a diverse assemblage of prokaryotes, including oxygenic phototrophs (cyano- bacteria and diatoms), anoxygenic phototrophs (green and purple sulfur bacteria), chemolithotrophs (sulfide oxidizers and nitrifiers), obligate anaerobic heterotrophs (sulfate-reducing bacteria), aerobic heterotrophs, and microaerophilic heterotrophs [7, 32]. The cyanobacteria, which provide the primary structure and color to mats, are the most conspicuous organisms. Cyanobacteria also provide most of the organic material that supports the growth and metabolism of other organisms. The array of metabolic and functional bacterial groups ensures that most, if not all, of the major biogeochemical cycling processes (i.e., C, S, N, and O) occur within a span of a few millimeters [16, 23]. The depletion in N relative to C and/or P is a general characteristic shared by the environments where mats develop [16]. As a result, N availability most often limits primary productivity and growth in mat communities [16, 18]. Mats maximize the inputs of new N by rapidly recycling and efficiently retaining the new N that is incorporated through uptake or N 2 fixation [2]. Recy- cling of mineralized N meets most biological N demand in mats [4]. Yet, optimization of N 2 fixation, which helps supplant N lost from the system and sustains new growth, is key to the development and continued survival of mats [16, 27]. The nitrogenase enzyme, which catalyzes the reduc- tion of N 2 into biologically utilizable NH 3 , is inactivated by O 2 [12, 19]. Generally, mat nitrogenase activity is attributed to diazotrophic cyanobacteria and explained in terms of how they have adapted to reconciling the chal- lenges the daily O 2 cycle presents [3, 6, 11, 15, 28, 33]. However, many other important mat bacterial groups including sulfate-reducing bacteria (SRB), microaero- philic bacteria, and anoxygenic phototrophs possess species and strains capable of fixing N 2 . Indeed, genetic and culturing analyses of several mat systems have shown that, in addition to cyanobacterial representatives, the diazotrophic assemblage encompasses myriad organisms possessing diverse metabolic capabilities [14, 29, 31, 35]. Although the potential contribution of these groups is Correspondence to: T.F. Steppe; E-mail: tsteppe@umbi.umd.edu DOI: 10.1007/s00248-004-0245-x d Volume 49, 315–324 (2005) d Ó Springer Science+Business Media, Inc. 2005 315