nitrogenase activity and nifh expression in a marine intertidal microbial mat

Nitrogenase Activity and nifH Expression in a Marine IntertidalMicrobial Mat

T.F. Steppe1,2 and H.W. Paerl1

(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

N2 fixation, diazotrophic community composition, andorganisms actively expressing genes for N2 fixation wereexamined over at 3)year period (1997–1999) for inter-tidal microbial mats on a sand flat located in the RachelCarson National Estuarine Research Reserve (RCNERR)(Beaufort, NC, USA). Specifically, diel variations of N2

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 ofphotosynthesis corresponded to one of the three patterns.High rates of NA were observed during peak O2 pro-duction periods for diel experiments during summermonths. Different types of NA diel variations correspondto different stages of mat development. Chloramphenicoltreatments indicated that the mechanism of proteinsynthesis supporting NA changed throughout the day.Analysis of mat DNA and RNA gave further evidencesuggesting that in addition to cyanobacteria, otherfunctional groups were responsible for the NA observedin the RCNERR mats. The role of microbial diversity inthe N2 fixation dynamics of these mats is discussed.

Introduction

Microbial mats are laminated structures that develop inmarine, lacustrine, terrestrial, and thermal environments.In coastal areas, mats become established in the tidal zoneas either semipermanent or ephemeral seasonal features[7, 33]. They are composed of a diverse assemblage ofprokaryotes, including oxygenic phototrophs (cyano-bacteria and diatoms), anoxygenic phototrophs (greenand purple sulfur bacteria), chemolithotrophs (sulfide

oxidizers and nitrifiers), obligate anaerobic heterotrophs(sulfate-reducing bacteria), aerobic heterotrophs, andmicroaerophilic heterotrophs [7, 32]. The cyanobacteria,which provide the primary structure and color to mats,are the most conspicuous organisms. Cyanobacteria alsoprovide most of the organic material that supports thegrowth and metabolism of other organisms. The array ofmetabolic and functional bacterial groups ensures thatmost, if not all, of the major biogeochemical cyclingprocesses (i.e., C, S, N, and O) occur within a span of afew millimeters [16, 23].

The depletion in N relative to C and/or P is a generalcharacteristic shared by the environments where matsdevelop [16]. As a result, N availability most often limitsprimary productivity and growth in mat communities[16, 18]. Mats maximize the inputs of new N by rapidlyrecycling and efficiently retaining the new N that isincorporated through uptake or N2 fixation [2]. Recy-cling of mineralized N meets most biological N demandin mats [4]. Yet, optimization of N2 fixation, which helpssupplant N lost from the system and sustains newgrowth, is key to the development and continued survivalof mats [16, 27].

The nitrogenase enzyme, which catalyzes the reduc-tion of N2 into biologically utilizable NH3, is inactivatedby O2 [12, 19]. Generally, mat nitrogenase activity isattributed to diazotrophic cyanobacteria and explained interms of how they have adapted to reconciling the chal-lenges the daily O2 cycle presents [3, 6, 11, 15, 28, 33].However, many other important mat bacterial groupsincluding sulfate-reducing bacteria (SRB), microaero-philic bacteria, and anoxygenic phototrophs possessspecies and strains capable of fixing N2. Indeed, geneticand culturing analyses of several mat systems have shownthat, in addition to cyanobacterial representatives, thediazotrophic assemblage encompasses myriad organismspossessing diverse metabolic capabilities [14, 29, 31, 35].Although the potential contribution of these groups isCorrespondence to: T.F. Steppe; E-mail: [email protected]

DOI: 10.1007/s00248-004-0245-x d Volume 49, 315–324 (2005) d � Springer Science+Business Media, Inc. 2005 315

recognized, no substantial evidence for their N2-fixingactivity has been obtained [3, 5, 22, 24]. Recently,though, Steppe and Paerl [30] have demonstrated thatSRB might be responsible for a significant amount ofnitrogenase activity in a mat system from coastal NorthCarolina, USA.

The purpose of the study was to clarify factorsinfluencing diel patterns and rates of N2 fixation and toelucidate some of the organisms responsible for N2 fix-ation. The results provide further evidence that differentdiel patterns and total daily NA in the RCNERR representvarious development stages of the mat and that NA ismediated by a diverse assemblage of organisms charac-terized by a wide range of metabolic capabilities.

Methods

Field Site. The RCNERR is a dredge spoil island nearBeaufort, North Carolina, USA (34�40¢N, 76�42¢W). Ithas been referred to as Bird Shoal (BS). The mats arelocated on a sand flat in the upper intertidal region. Theyare exposed from 7 to 12 h per daily tidal cycle,depending on tide levels and wind. In the phototrophiclayer, the dominant cyanobacteria genera include Mi-crocoleus, Lyngbya, and Oscillatoria. Phormidium, Ar-throspira-like, and unicellular genera are present to alesser extent. Pennate diatoms are usually found in theupper surface of the mats, but their relative abundance inrelation to the cyanobacteria varies greatly [18]. Duringthe summer, a pink to purple layer composed of an-oxygenic phototrophs occasionally develops below thecyanobacterial layer. Sediments below the photic zone areblack and sulfidic.

Rate Measurements. Several sections (20 · 35 ·5 cm) of microbial mat were collected in modified plastictrays and transported to the Institute of Marine Sciences(IMS) (Morehead City, NC) during the afternoon on theday prior to the experiment. The trays with the matsamples were placed in a tank with running seawater(�34 psu) and left exposed to natural illumination. Insitu tidal conditions were not simulated in the tanks.However, at every 18:30 and 06:30 hours the water in thetanks was drained and the mats were exposed for 1 hbefore the tanks were refilled. With respect to nitrogenfixation, mimicking in situ tidal conditions does affect themagnitude of rates, but not the overall daily pattern ofNA [2]. Rate measurements (see below) were made over a24-h cycle, starting at 06:00 hours under natural illumi-nation. See Table 1 for dates of experiments addressed inthis study.

The methods used in this study for measuringnitrogenase activity (NA) and net O2 production havebeen thoroughly described [15, 18, 31]. Replicates weretaken from each of the mat sections; n = 3–4 for NA

and n = 4–5 for net O2 production. NA was determinedby the acetylene reduction method, which indirectlymeasures nitrogen fixation by examining the conversionof acetylene (C2H2) to ethylene (C2H4). Subcores (�1cm2) are removed from the larger pieces and placed in a37-mL serum vial with 20 mL of seawater. A red, butylrubber stopper was placed in the vial, 4 mL of head-space removed, and 5 mL C2H2 injected. Vials wereincubated in the same tank containing the larger matpieces. NA measurements were made every 3 h from06:00 to 24:00, then every 2 h until 06:00 or 08:00. O2

measurements were made every 2 to 3 h from 06:00 to20:00. For net O2 production, 0.5 cm2 cores were placedin 10-mL serum vials and sealed with a red butylstopper. Care was taken to ensure no air bubbles re-mained in the vial by having a 25-gauge needle insertedinto the stopper while the vial is being sealed to relieveany air bubbles. Net O2 production was determined bymeasuring the change in O2 concentration during theincubation period. Vials were incubated in the sametank containing the larger mat pieces.

Metabolic Inhibitors. The metabolic inhibitorsutilized in these experiments were 3-3,4-dichlorophe-nyl)1, 1-dimethylurea (DCMU) (2 · 10)5 M, final) andchloramphenicol (150 lg mL)1, final). Both inhibitorswere dissolved in 95% ethanol. Separate experimentsadding the ethanol without inhibitors showed there wasno effect (not shown). DCMU inhibits oxygenic photo-synthesis (photosytem II) and chloramphenicol inhibitsde novo protein synthesis. Serum vials with mat coreswere amended with chloramphenicol just prior to injec-tion of acetylene. Larger mat pieces were not amendedwith chloramphenicol. This treatment was included indiel experiments conducted in 1998 and 1999 in order toassess the relationship between NA and total proteinsynthesis. Two types of DCMU treatments were em-ployed, short-term and long-term (St-DCMU and Lt-DCMU, respectively). St-DCMU treatments representsamples to which DCMU was added to the serum vialjust prior to injection of acetylene. St-DCMU treatmentswere included in all experiments. Lt-DCMU treatedsamples comprised samples taken from mat piecesincubated with DCMU commencing sunset prior to thestart of the experiment. Lt-DCMU samples were incu-bated in a separate 3-L container, albeit under naturalillumination and in the same tank containing the largermat pieces with water flowing around the container tomaintain temperature. Water and DCMU in the Lt-DCMU container was changed every 12 h. St-DCMUtreatments assessed the effect of the short-term removalof O2 production on NA. Lt-DCMU treatments assessedthe effect of completely inhibiting O2 production throughthe entire light cycle. Lt-DCMU treatments were includedin diel experiments conducted in 1998 and 1999.

316 T.F. STEPPE AND H.W. PAERL: N2 FIXATION IN AN INTERTIDAL MAT

DNA Extractions and PCR. Small mat cores (�0.5cm2 · 0.6 cm) were collected from mat samples, placed in1.0 mL of TE buffer pH 8.0 (10 mM Tris, 1 mM EDTA),and stored at )80�C until extraction. Purified DNA wasobtained from mat pieces and SRB cultures according tothe procedures outlined in Steppe et al. [31]. NifH primersdescribed in Zehr and McReynolds [34] were used in allPCR experiments. The PCR conditions consisted of 30cycles of 94�C for 60 s, 54�C for 60 s, and 72�C for 90 s.

RNA Extractions and Reverse Transcription.. Smallmat cores (�0.5 cm2) were placed in 2.0-mL screw captubes. The tubes were frozen quickly in a dry ice/ethanolbath. The tubes were stored at )80�C until extraction.Tubes were removed from the freezer and allowed tothaw. All cores were washed four times with diethylpy-rocarbonate (DEPC)-treated TE buffer (pH 8.0). Afterwashing of the cores, 0.5 mL of glass beads (150–200 lm)and 1.0 ml RNAwiz (Ambion) were added. Tubes wereagitated using a ‘‘bead beater’’ for 3 min and then placedin a 70�C water bath for no more than 1 h. At the end ofthe hour, bead beating was repeated, the tube was cen-trifuged at 14,000 g for 1 min, and the supernatanttransferred to a new tube. RNAwiz instructions werefollowed. After the first extraction, the crude pellets weresuspended in 50 lL DEPC-treated dH2O. RNA was thenpurified twice more using the Totally RNA kit (Ambion)according to the manufacturer’s instructions. After thethird purification, the RNA solution was treated withDNAase. The RNA solution was purified a final timeusing RNAwiz. A total of four purification steps wereperformed. The final pellet was suspended in 50 lLDEPC-treated dH2O. To ensure there was no cross-contamination between different mat samples duringextraction, samples containing no biological material(NBM) were processed the same as tubes containing matpieces. The solution in the NBM tubes was used as one ofthe negative controls in subsequent RT-PCR reactions.

Reverse transcription cocktail components were (fi-nal reaction concentrations) 10–100 ng RNA, 50 mMTris-HCl (pH 8.3), 50 mM KCl, 5.0 mM, MgCl2,5.0 mM Spermidine, 5.0 mM DTT, 100 nM each dNTP,2.5 U AMV Reverse Transcriptase (Promega). Onehundred ng each of nifH Aero (R) and Cyano (R)primers [13] was used in each reaction. These primers areless degenerate then the Zehr and McReynolds [34]primers, but both are effective in amplifying diversegroup of organisms [14, 31]. After preparation of the RTcocktail, 47-lL aliquots were added to each reaction tube.RNA was added to the appropriate tubes. Negative con-trols included a reaction tube without reverse transcrip-tase; and a reaction tube seeded with negative extractioncontrol ‘‘RNA.’’ To further assess whether or not RT-PCR reactions were contaminated with DNA an addi-tional negative control was added. This control consistedT

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T.F. STEPPE AND H.W. PAERL: N2 FIXATION IN AN INTERTIDAL MAT 317

of purified RNA treated with RNase and then purifiedagain to remove RNase. The reverse transcription reac-tions were incubated at 48�C for 30 min. Five lL of thereverse transcription reaction was used to seed the tubesused for PCR experiments (see above).

Cloning, Sequencing, and Phylogenetic Analy-

sis.. Amplification products were gel-purified andcloned into the pCR 2.1 sequencing vector using the TACloning Kit (Invitrogen). The UNC-Chapel Hill Auto-mated Sequencing Facility completed the sequencing on aModel 373A DNA Sequencer (PE-Applied Biosystems)using the Taq DyeDeoxy Terminator Cycle SequencingKit (PE-Applied Biosystems). Sequences were edited (e.g.,primer sequences removed) and manually aligned usingthe GCG Genetics Suite (Genetics Computer Group,1994). Phylogenetic analyses were done using Phylip 3.5[9] based on protein distances. Protein distances wereobtained using the Dayhoff protein matrix. Phylogenetictrees were constructed using neighbor joining.

Statistical Analyses. Statistical analyses wereperformed using a software program, Statview (SASInstitute). Either a one- or two-way analysis of variance(ANOVA) was run on all results. A posteriori multiplecomparisons of means was achieved with the Bonferroniprocedure, p = 0.05. Within each experiment, compari-sons were made within treatments over a diel cycle, be-tween treatments within individual incubation period,between treatments during daytime hours (06:00 to18:00), and between treatments during nighttime hours(21:00 to 06:00).

Results

Diel Patterns. During the study, three basic diel NApatterns (types I–III) were observed (Fig. 1). The typeswere characterized by the time period of maximumnitrogenase activity, (10:00 to 15:00, 06:00 to 09:00, and21:00 to 06:00), total amount of NA or average NA forall time periods, and the ratio of daytime (06:00 to21:00) to nighttime (21:00 to 06:00) NA (Table 1).Types I and III have been described for the RCNERR inprevious studies [3, 15, 18]. Type I (Fig. 1A) is generallyobserved from late fall to early spring. Maximum NA isobserved during the day but is low compared to max-imum NA rates in the summer. Type II has been doc-umented for mats on Shackleford Banks, <3 km fromRCNER [1, 5], but has not been described for theRCNERR mats. Type II (Fig. 1B) may be summarized asmaximum NA during the sunrise period (06:00 to09:00) with decreased, but substantial, NA during theday (09:00 to £ 20:00) and at night (21:00 to 06:00).This pattern of diel variation was observed mostlyduring the summer of 1997 and once in the summer of

1998. Table 1 provides a summary for the diel experi-ments. Type III (Fig. 1C) is the more typical summer tolate fall signature where maximum NA is observed atnight and NA is almost completely repressed during theday [3, 15, 18]; a distinct temporal separation of pho-tosynthesis and N2 fixation.

NA responses to inhibiting total photosynthesisthrough dark incubations or only inhibiting photosystemII (oxygenic photosynthesis) using DCMU correspondedto diel variation types. For type I (Fig. 1A), DCMU anddark treatments both significantly (P < 0.05) repressedNA compared to the control during the day. For type II(Fig. 1B), dark incubations significantly inhibited NAfrom 06:00 to 18:00. DCMU treatments stimulated NAduring the sunrise period, but had no effect to slightlyrepressing NA for the remainder of daylight. For type III(Fig. 1C), dark incubations either had no effect or slightlyreduced NA from 06:00 to 18:00. DCMU treatments ei-ther stimulated NA throughout the day or had little effectfrom 09:00 onward.

Long-Term Effects of DCMU. Results similar toprevious work were observed for the Lt-DCMU treat-

Figure 1. Representative experiments demonstrating differentdiel NA variation types of RCNERR mats and responses to short-term manipulations of photosynthesis. (A) Type I, (B) type II,and (C) type III. Error bars represent standard deviation.


ments [3]. Three responses to the Lt-DCMU wereobserved (Fig. 2). The first response was characterizedby a dramatic stimulation of NA during the sunriseperiod followed by a more gradual decrease in Lt-DCMU NA during the rest of the day (Fig. 2A). Thesecond response was characterized by a similar stimu-lation during the sunrise period, but with a dramaticdecrease in Lt-DCMU NA during the subsequentincubations (Fig. 2B). In the third response, Lt-DCMUNA appeared to follow the irradiance curve throughoutthe day (Fig. 2C). Figure 2 also demonstrates how NAresponded to the different DCMU treatments. Lt-DCMU incubations generally stimulated NA more thanthe St-DCMU additions, even during the sunrise per-iod.

Effect of Chloramphenicol (CAP). CAP’s effect onNA appeared to be time-dependent. Three representativeexperiments are shown in Fig. 3. In all experiments, CAPhad little to no effect during the sunrise period. However,CAP inhibited virtually all (>90%) NA from 09:00 to20:00. At night, CAP repressed NA (50–75%), but not asmuch during the day.

Diazotrophic Community Composition. Analysesof translated, partial nifH sequences were made to iden-tify organisms within the mat having the ability to fix N2,and to detect organisms expressing nitrogenase genes.Sequences obtained from Sept. 1997, July 1998, and July1999 are presented in Fig. 4. Sequences clustered withone of three groups: nonheterocystous cyanobacteria,beta/gamma Proteobacteria, and obligately anaerobicbacteria such as delta sulfate reducers. All cyanobacterialsequences, except BS0799 D26, obtained showed themost similarity to either Lyngbya lagerheimii or Phormi-dium spp. nifH sequences. By Blast comparison to Gen-Bank, BS0799 D26 was most similar to Gloeothece andSynechocystis spp., unicellular cyanobacteria. One se-quence grouped with the beta/gamma proteobacterialnifH sequences. However, most sequences clustered withthose derived from obligate anaerobes, such as delta SRBand Clostridia pasteurianum. Several of the nifH se-quences in the ‘‘anaerobe’’ group closely aligned with thegreen sulfur bacterium, Chlorobium tepidum.

RT-PCR was performed on RNA extracted from matpieces collected at selected times during two diel exper-

Figure 2. Comparisons of effects of St-DCMU and Lt-DCMUtreatments on daytime NA. (A) 06/03/98, (B) 07/29/98, (C) 10/14/98. Error bars represent standard deviation.

Figure 3. Effect of chloramphenicol on NA. Arrows point to timepoint when samples were collected for RNA extraction and sub-sequent nifH RT-PCR analysis. ‘‘+’’ or ‘‘)’’ indicates a positive ornegative nifH RT-PCR. Error bars represent standard deviation.


iments, 07/29/98 (two time points) and 07/10/99 (fourtime points) (Fig. 3). Positive RT-PCR signals were ob-tained for five of the six extractions. Table 2 provides thedate, the time of core collection, and with what majorgroup (based on Fig. 4) the sequence clustered. Thirteendistinct sequences were obtained for both experiments.Although multiple clones were sequenced for each timepoint (10 to 13), many were identical (100% similarity).For example, 10 clones were sequenced for the 06:30 timepoint on 07/29/98, and all 10 possessed the same nifHsequence. Several clones matched sequences obtainedfrom genomic DNA. However, most were uniquesequences. The RT-PCR sequence numbers are listedbeside their sequence names in Table 2. Several of thesame sequences were detected for multiple time points

within the same diel period. For example, sequence RT05was detected for three time points during the 07/09/98diel experiment. Only three cyanobacterial nifH se-quences were obtained with RT-PCR. Two sequences(RT04 and RT13) either matched or were highly similar(>99% at the amino acid level) to the Lyngbya lagerheimiigroup of sequences. RT01 exhibited the most similarity toTrichodesmium spp. sequences and was obtained throughRT-PCR and not PCR. The remaining sequences clus-tered with the anaerobic nifH group.

Discussion

Diel NA Variations and Mat Development. Three dis-tinct diel NA patterns were observed in this study. Each

Figure 4. NifH phylogenetic tree.Tree topology was obtained usingDayhoff PAM matrix and NeighborJoining in Phylip utilizingtranslations of an �327-bp sectionof the dinitrogenase reductase gene(nifH). *Sequences derived fromRNA that matched sequencesderived from DNA. Bootstrap values>50 that support a particularbranching are listed above theappropriate branches. Sequencesobtained from the RCNERR matsamples through PCR or RT-PCRnot previously published are inbold. GenBank Accession numbersfor this study: AY137208–AY137239.


type was characterized by its responses to short-termmanipulations of photosynthesis, period of maximumNA, and total daily NA (Fig. 1 and Table 1). Similar toearlier mat NA studies, the observed diel NA cycles andrates suggest a successional or seasonal development inthe RCNERR mats and imply strategies for how the matsoptimize N2 fixation to meet new N requirements [1, 2,15, 33]. Based on photosynthetic rates [15, this study],during the late fall to early spring (type I), requirementsfor new N and daily NA are comparatively low. The matsare characterized by low growth or photosynthetic rates[15]. During periods of growth (type II) (typically latespring to early summer), the requirement for new N ishighest. Maintaining relatively high N2 fixation ratesduring daylight increases N input and could alloworganisms to meet urgent N demand. During midsum-mer to early fall (type III), N requirements for newgrowth decrease as the mats move into a more stationaryor maintenance phase [4, 15, 28]. At this time, photo-synthetic rates are still relatively high, but NA is observedmainly during the night. Total NA for type III is de-creased to type II.

The type II diel NA cycles persisted through thesummer of 1997. However, this may in part be explainedby major storms, Hurricanes Bertha and Fran in July andSeptember of 1996, which likely affected sediment move-ment and mat development. In August of 1996, there waslittle to no mat development on RCNERR. After Hurri-cane Fran impacted the region in early September of 1996,mats became more prevalent. Presumably, the hurricanereworked the sediment so that mat development acceler-ated. From July through September (1997), the ratio ofnighttime NA to daytime NA in the RCNERR mats in-creased, which supports our argument that different dielcycles represent different successional stages of the mat.

There is evidence suggesting diel NA patterns arerelated to alterations in the phototrophic community

[18]. Pinckney et al. [18] monitored the relative abun-dance of the phototrophic community in four sites overthe course of a year on the RCNERR tidal flat. Each siteproved representative of specific stages of a mat devel-opment. The relative abundance of diatoms and cyano-bacteria fluctuated at each site during course of the year,but the relative abundance of diatoms (ratio of fuco-xanthin to chl a) was highest in less developed mats.Furthermore, over the course of the year, the ratio ofdaytime to nighttime NA corresponded to the relativeabundance of cyanobacteria to diatoms at the mostdeveloped mat site. When the ratio of nighttime NA todaytime NA increased, so too did the ratio of cyano-bacteria to diatoms.

Previously, the progression of NA diel patterns fromtype I to type III was largely attributed to increased O2

production [3, 15, 25, 33]. Results from this studyindicate that whereas O2 generation represses daytimeNA (Figs. 1, 2), elevated or high O2 production rates donot preclude relatively high daytime NA rates or nec-essarily indicate that most NA occurs during the nightor low photosynthetic periods (Table 1). As a result,increased O2 production, singularly, cannot account forthe switch.

While the PCR results are not quantitative, the nifHsequences obtained during this and a previous study [35]provide some insight into the dynamics of the majordiazotrophic groups in the RCNERR mat. To date, mostnifH sequences obtained from the RCNERR mats werefrom samples collected 11/93, 09/10/97, and 07/09/99. Onall three dates different diel NA variations were observed[15, this study]. The nifH sequences from all three datesclustered within the anaerobic, cyanobacterial, or beta/gamma prokaryotic groups. Most sequences clusteredwithin the anaerobic group. The few cyanobacterial nifHsequences were similar to the Lyngbya lagerheimmi nifHsequence. The sequence types show that the same major

Table 2. Time course of nifH RT-PCR results

Diel 07/29–30/98 Diel 07/09–10/98

07–29 07–30 07–09 07–09 07–09 07–1006:30 02:30 06:30 15:30 21:30 4:30

Cyanobacteria RT01 RT01 — — RT04 RT04RT13

— RT02* — RTO5 RT05RT03* RT06*

RT07RT08

Anaerobes RT09*RT10 RT10RT11RT12

Results are grouped according to the diel experiment, the time mat samples were collected for RNA extraction, and the nifH phylogenetic group the RT-PCRderived sequence clustered with based on Fig. 4.*Sequence matched a nifH sequence obtained through PCR of DNA extraction. Ten clones were sequenced for each time point.


diazotrophic groups the PCR primers are able to discernappear to be present in the RCNERR from year to yearand season to season. Although this may solely be anartifact due to primer bias, the effectiveness of theprimers in discerning different organisms and even dif-ferent groups of diazotrophs (see Fig. 4) from differentenvironments leads us to believe this is not the case [14,31, 35]. If so, this suggests large-scale shifts in the majordiazotrophic groups may not appear to be associatedwith different diel variations. However, the PCR resultsdo not preclude the possibility that individual diazo-trophic populations within the major groups and/or theirrelative contributions vary seasonally or during variousstages of mat development [10].

Ecophysiology of Diel NA Variations. The resultsfrom the CAP treatments explain why high daytime NAwas observed for some diel experiments and not forothers. In the early morning (06:00 to 09:00), inhibition ofprotein synthesis slightly repressed or had no effect onNA. In contrast, chloramphenicol additions inhibitedvirtually all NA from 09:00 to 20:00. Based on Fig. 3, itmay be concluded that high daytime NA during thesummer required protein synthesis. Therefore, when thereare high rates of daytime NA concomitant with high ratesof photosynthesis (i.e., type II), a diazotroph or group ofdiazotrophs is able to maintain a level of protein expres-sion that allows NA. At night, chloramphenicol additionssignificantly reduced NA, but did not completely inhibitNA. Protein synthesis was required to support much ofthe nighttime NA. Because CAP’s effect on NA wasdependent largely on the time of day, it may be surmisedthat the degree to which NA is directly coupled to de novoprotein synthesis changes over a diel cycle (Fig. 4).

Culture studies have demonstrated that diazotrophicmat cyanobacteria possess sufficient metabolic plasticityto explain NA rates, diel variations, and CAP data ob-served in situ. A Lyngbya sp. isolated from the RCNERRmat and grown in a 12-h light/dark cycle exhibited atemporal separation of NA and photosynthesis [3]. An-other Lyngbya sp. isolated from the Shackleford matsdemonstrated the ability to contemporaneously conductNA and photosynthesis, although NA was lower duringthe day than at night [19]. An Oscillatoria sp. isolatedfrom the Mellum (N. Germany) mats grown in alter-nating light/dark cycles conducted most NA during thelight–dark transition periods, and also showed the abilityto conduct aerobic NA [25, 26].

However, a broad diversity of diazotrophs may befound in the RCNERR mats [14, 35, this study]. Fur-thermore, physiological and RT-PCR data suggest sig-nificant N2 fixation by SRB or other noncyanobacteria[30, this study]. This contradicts the notion that noncy-anobacterial diazotrophs are not likely to contribute sig-nificantly to NA rates observed in mats [6, 28]. Detection

of specific nifH transcripts in this study does notunequivocally indicate organisms were actively fixing N2

for several reasons. First, the possibility of posttransla-tional modification of nitrogenase existed. Second, nifHexpression was not correlated with NA. Third, the relativeabundance of nifH genes is not known. Finally, sequencesobtained for each time point represent sequences fromone relatively small sample of the mat (�0.5 cm2).Therefore, the sequences obtained may not be a repre-sentative sample of the organisms expressing nifH. De-spite the methodological limitations, the RT-PCR andphysiological rate data presented here and previously [30]demonstrate four main points: (1) an assortment oforganisms representing various metabolic capabilities wasat least expressing nitrogenase (Fig. 4), (2) nifH tran-scription for these organisms is not solely confined to thenighttime, (3) noncyanobacterial diazotrophs such as SRBare capable of generating ATP to meet N2 fixation de-mands [30], and (4) there may be shifts during the day ofdifferent species transcribing nifH during the day.

Role of Diversity within the RCNERR Mats. On adaily basis, the new N from N2 fixation that may be usedto meet primary production needs may not be the largestN source utilized by phototrophs in mats [4]. However,the new N that N2 fixation provides at least facilitates matdevelopment and maintenance by offsetting any N that islost through diffusion, erosion, grazing, and denitrifica-tion. For this purpose, N2 fixation is an important pro-cess for supporting RCNERR mats. The RCNERR matsare intertidal and experience both temperate and sub-tropical climates. As a result, they are subjected to a greatrange of micro- and macroenvironmental conditions. Forexample, air temperatures can range from �)3 to >37�C.Internal porewater salinities range from ambient (34 psu)to over twice that during the summer when low tidesoccur midday. Changes in ambient environmental con-ditions and periodic flooding may likewise have a largeimpact on the internal microenvironment of mats [8].Steep concentration gradients of chemical species such asO2, H2S, and NH4

+ define the internal microenvironmentof mats [4, 21, 32]. The concentration magnitudes (i.e.,gradient steepness) of O2, NH4

+, and H2S are dynamicand may vary dramatically hourly, daily, and seasonally.In order for any diazotroph to fix N2, certain intracellularparameters must be met (e.g., low pO2 and DIN con-centrations, ATP levels above what is required for cellularmaintenance, tolerable temperature range, tolerablesalinity range). The ranges that circumscribe favorablegrowth and N2 fixing conditions for individual diazo-trophs can vary greatly, though, such that conditionspermitting N2 fixation by one organism select againstactivity by another. As an example, some mat hetero-trophs can fix N2 under microaerophilic conditions [14]while diazotrophic Desulfovibrio spp. are obligately


anaerobic [20]. For the RCNERR mats, micro- andmacroenvironmental conditions that favor growth andN2 fixation by one group of organisms can shift towardconditions that favor another group. Therefore, a highlydiverse diazotrophic assemblage possessing myriad met-abolic traits within a habitat subjected to great fluctua-tions in micro- and macroenvironmental conditions suchas the RCNERR mats increases the likelihood that theinput of new N via N2 fixation is maintained.

Summary

Several diel N2 fixation patterns in the RCNERR matswere observed. The differences in patterns were reflectedin the NA responses to different experimental manipu-lations. Chloramphenicol treatments demonstrate thateven within a diel cycle, NA is supported by differentmechanisms of total protein synthesis. Overall, the dif-ferences in diel patterns of N2 fixation within the RCN-ERR mats likely arise from the complex and dynamicinteractions between environmental conditions, thecomposition and activity of the photosynthetic commu-nity, biogeochemical cycling and gradients, and the dia-zotrophic community composition. The nifH PCR andRT-PCR data further demonstrate that a diverse assem-blage of diazotrophs actively expressed the genes neces-sary to fix N2. This suggests that diazotrophicassemblages composed of organisms with diverse meta-bolic traits (i.e., photosynthesis, heterotrophy, optimalgrowth conditions) function to maintain a consistentinput of new N by allowing N2 fixation to proceed in theface of changes in macro- and microenvironmentalconditions. Through a well-designed and concertedapplication of rate measurements, microscopic and che-motaxonomic analysis, and molecular genetic techniques,a clearer picture of the ecophysiology of N2 fixationwithin the RCNERR and other mats will emerge.

Acknowledgments

We acknowledge Tom Nanni and John Fear for technicalassistance. This work was supported by NSF LEXEN980895 and NSF Microbial Observatories 0132528.

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nitrogenase activity and nifh expression in a marine intertidal microbial mat

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