Vol. 70: 245–259, 2013 AQUATIC MICROBIAL ECOLOGY Published online October 16 doi: 10.3354/ame01654 Aquat Microb Ecol
FREEREE ACCESSCCESS FEATURE ARTICLE
Community composition of bacteria involved in fixed nitrogen loss in the water column of two major oxygen minimum zones in the ocean
Amal Jayakumar*, Xuefeng Peng, Bess B. Ward
Department of Geosciences, Princeton University, Princeton, New Jersey 08544, USA
ABSTRACT: The community composition of bacteria involved in nitrogen (N) transformations in the oxy- gen minimum zones (OMZ) may be related to the rates of fixed N loss in these systems. The abundance of both denitrifying and anammox bacteria and the assemblage composition of denitrifiers were investi- gated in the Eastern Tropical South Pacific and the Arabian Sea using assays based on bacterial molecu- lar markers. Quantitative PCR was used to investi- gate the abundance and distribution of genes encod- ing nitrite reductase (nirK and nirS) in denitrifying bacteria and hydrazine oxidase (hzo) and 16S rRNA genes in anammox bacteria. All of these genes had depth distributions with maxima associated with the secondary nitrite maximum in low-oxygen waters. NirS was much more abundant than nirK and much Nitrogen removal genes (yellow curve) peaked at the top of more abundant than the 16S rRNA gene from anam- the OMZ (red curve shows O2 concentration). The nirS mox bacteria. The ratio of hzo:16S rRNA was low and assemblage differed between oxic (upper array) and OMZ variable, implying greater unexplored diversity in (lower array) waters and between oceanic regions. the hzo gene. Assemblage composition of the abun- Image: Bess Ward dant nirS-type denitrifiers was evaluated using a functional gene microarray. Of the nirS archetypes represented on the microarray, very few occurred specifically in one region or depth interval, but the assemblages varied significantly. Community com- INTRODUCTION position of denitrifiers based on microarray analysis of the nirS gene was most different between geo- The Arabian Sea (AS) and the Eastern Tropical graphical regions. Within each region, the surface South Pacific (ETSP) ocean regions are known as layer and OMZ assemblages clustered distinctly. oxygen minimum zones (OMZ) (Paulmier & Ruiz- Thus, in addition to spatial and temporal variation in denitrification and anammox rates, microbial abun- Pino 2009) because they are associated with subsur- dance and community composition also vary face oxygen depleted depths, in which the oxygen between OMZ regions and depths. concentration is essentially zero (Revsbech et al. 2009). Depth intervals where oxygen is completely KEY WORDS: Denitrifying bacteria · Oxygen depleted, allowing the functioning of anaerobic minimum zone · Community composition microbial pathways including denitrification and Resale or republication not permitted without anammox, can extend for hundreds of meters and are written consent of the publisher
*Email: [email protected] © Inter-Research 2013 · www.int-res.com 246 Aquat Microb Ecol 70: 245–259, 2013
often referred to as the oxygen depleted zone (ODZ). yield widely varying estimates of their contributions Both denitrification and anammox occur in waters to the total microbial abundance (Hamersley et al. with oxygen concentrations below detection limits 2007, Galan et al. 2009, Jayakumar et al. 2009a, Lam (10 − 90 nM; Dalsgaard et al. 2012). We refer to the et al. 2009, Ward et al. 2009, Casciotti et al. 2011). We oceanic regions as OMZs and the depth interval of investigated the distribution of the microbes respon- essentially zero oxygen as the ODZ. sible for the N loss rates using a suite of mostly func- The water columns of OMZs have characteristic tional genes related to the N transformations. Quan- chemical distributions, usually including 2 nitrite titative PCR was used to estimate the distribution of maxima and 2 nitrous oxide maxima (Bange 2008, denitrifiers and anammox bacterial cells based on Devol 2008). In the AS, high-resolution profiles of single copy genes, and the community composition of − NO2 and N2O show multiple maxima and minima, denitrifiers was investigated using a functional gene suggesting the interleaving of water masses with microarray based on nirS, the gene encoding the different oxygen histories (Nicholls et al. 2007). iron-type nitrite reductase. We expected that depth Although the distribution of the N loss processes is distributions of the functional genes would be consis- known with much lower resolution, the highest tent with the chemical distributions that define the rates of anammox appear to be associated with the OMZ and therefore would be similar between geo- upper regions of the low oxygen water, just below graphical regions. We hypothesized, however, that the depth at which oxygen reaches zero levels the community composition of denitrifiers would dif- (Bulow et al. 2010, Hamersley et al. 2007, Dalsgaard fer between oxic and anoxic depths and between et al. 2012). Denitrification is more variable and geographical regions. shows no obvious depth pattern in published reports to date. In an extensive study of the coastal region of the ETSP, Dalsgaard et al. (2012) found MATERIALS AND METHODS that anammox and denitrification had somewhat opposite distributions; where one rate was high, the Study area and sample collection other was low. Thus, it is not possible at present to explain the reproducibility of the observed chemical The data presented here were obtained from sam- distributions in terms of the measured N transfor- ples collected from 12 stations in the ETSP (RV mation rates. After chemistry and transformation ‘Knorr’, October 2005) and 6 stations in the AS rates, a third piece of the puzzle is the distribution (Stns 1, 2 and 3, RV ‘Roger Revelle’, September 2007; and community composition of the microbes Stns 1, 2, and 23, RV ‘Sagar Kanya’, September 2004). responsible for the rate transformations. At the in Station locations are shown in Fig. 1, and details are situ temperatures and substrate concentrations of given in Table S1 in the Supplement at www. int-res. the OMZ waters, the generation times of the micro- com/articles/ suppl/a070 p245_ supp.pdf . bial assemblage are on the order of a few days for Water-column sampling and temperature/salinity denitrifiers (Ward et al. 2008) and likely a few profiling were carried out using a Sea-Bird Elec- weeks for anammox bacteria (van der Star et al. tronics CTD (conductivity-temperature-depth)- 2008). The response times of the biomass are thus rosette sampling system, Model SBE9, fitted with longer than the response times of the processes Niskin/ Go-Flo bottles. Previous work in both themselves. Anammox appears to occur ubiqui- regions had identified strong correlations between tously at relatively low and steady rates, while den- nitrite and oxygen concentrations (Codispoti et al. itrification may occur slowly or rapidly in response 1986, Morrison et al. 1999), so we used nitrite con- to episodic organic matter inputs (Ward et al. 2008, centration as a proxy to select sampling depths Dalsgaard et al. 2012). Thus, the distribution of the within the OMZ. Water samples from an initial cast microbes might represent some damped average were quickly analyzed for nitrite content. Nutrient integration over temporally varying rates of biomass analyses were carried out onboard ship using a production. SKALAR auto analyzer or manually using spec- Earlier investigations of total microbial abundance trophotometry for rapid nitrite assays (Grasshof et reported microbial cell number maxima coincident al. 1983), and dissolved oxygen (DO) concentrations with the main secondary nitrite maximum (SNM) in were taken from the CTD system, which in turn the OMZ (Spinrad et al. 1989, Ducklow 1993, was calibrated with data determined by the titri- Jayakumar et al. 2009a). Quantitative PCR-based metric Winkler method (Carpenter 1965) on discrete abundances of denitrifying and anammox bacteria water samples. Jayakumar et al.: Bacterial community composition in oxygen minimum zones 247
al. (2009b). For hzo and nirK quantification, Ex-taq Premix perfect SYBR Green Real-time master mix (Takara) was used. Primers, amplification conditions and master mix kits used for each qPCR assay are provided in Table S2 in the Supplement. After run- ning test assays, specific kits were chosen based on amplification efficiency for a particular gene to max- imize reproducibility with a single product. The effi- ciency of the qPCR reactions were calculated using the slopes of the standard curves: for Total nirS, Dom nirS, AMX 16S and nirK, the efficiencies ranged between 85 and 106%, and for hzo, the efficiency ranged between 60 and 65%. The amplified products were visualized after electrophoresis in 1.0% aga - rose gels stained with ethidium bromide. Standards for PCR quantification of each fragment were pre- pared by amplifying a constructed plasmid contain- ing the respective gene fragment, followed by quan- tification and serial dilution. Assays for each gene at all depths were carried out within a single assay plate (Smith et al. 2006). Each assay included triplicates of the no-template control (NTC), a no-primer control (NPR), ≥4 standards and triplicates of known quantity of the environmental DNA samples (20 to 25 ng). DNA was quantified using PicoGreen fluorescence (Molecular Probes) calibrated with several dilutions of phage lambda standards, and qPCR was performed using a Strata- gene MX3000P (Agilent Technologies). Automatic analysis settings were used to determine the thresh- old cycle (Ct) values. The copy numbers were calcu- lated as follows: copy number = (ng × number mole−1) Fig. 1. Station locations for (A) Eastern Tropical South / (bp × ng g−1 × g mole−1 of bp) and then converted to Pacific (2005) and (B) Arabian Sea (2004, 2007) samples copy number per milliliter of seawater filtered, assuming 100% extraction efficiency. To maintain continuity and consistency among Nucleic acid manipulations and quantitative qPCR assays between the AS samples and the ETSP PCR analysis samples for a particular gene, a subset of samples from the previous run was included in the subse- Particulate material from 5 to 10 l seawater was col- quent assay, and a new dilution series for standard lected onto Sterivex capsules (0.2 μm filter, Millipore) curves was used in every assay. Plasmid DNA was with a peristaltic pump. Immediately after filtration, quantified prior to every dilution series to account for the capsule filters were quick-frozen in liquid nitro- DNA loss that occurs upon repeated freeze-thaw gen and stored at −80°C until the DNA could be cycles. extracted. Methods of DNA extraction, PCR amplification and qPCR using SYBR Green for nirS, Dom nirS (a nirS nirS microarray sequence that was very abundant in clone libraries obtained previously from the AS by Jayakumar et al. The array (BC014) was developed following the 2004, 2009a) and anammox 16S rRNA genes (Schmid archetype array approach described and employed et al. 2003) have been described previously. Stan- previously (e.g. Ward & Bouskill 2011, Bulow et al. dardization and verification of specificity for qPCR 2008) with 90-mer oligonucleotide probes. Each assays was performed as described by Jayakumar et probe included a nirS-specific 70-mer region and a 248 Aquat Microb Ecol 70: 245–259, 2013
′ 20-mer control region (5 -GTA CTA CTA GCC TAG where ri represents the ratio of Cy3 to Cy5, and s the GCT AG-3′) bound to a glass slide. The design and standard deviation of the 3 Cy3:Cy5 ratios. The raw spotting of the probes has been described previously microarray image was checked to ensure that fea- (Bulow et al. 2008). The 164 nirS archetype probes tures filtered out actually exhibited anomalous represent ~2000 sequences from a wide range of hybridization signals. Then, a normalized fluores- environments, which were available in the public cence ratio (FRn) for each archetype was calculated databases at the time the array was designed in by dividing the fluorescence signal of the archetype November 2009. The probe accession numbers and by the highest fluorescence signal within the same sequences are listed in Table S3 in the Supplement. array. Then, the FRn of each archetype from the duplicate arrays were averaged. The relative fluores- cence ratio (RFR) of each archetype was calculated as Target preparation, microarray hybridization the contribution of FRn of the archetype to the sum of and data analysis FRn of all nirS archetypes on the array and averaged for duplicate arrays from each sample. The original Array analysis was performed as described previ- array data are available at Gene Expression Omnibus ously (Ward & Bouskill 2011) using whole DNA (www. ncbi. nlm. nih. gov/ projects/ geo/) at the Natio - extracts. Briefly, the genomic DNA was digested with nal Center for Biotechnology Information under GEO HinfI and then labeled with amino-allyl-dUTP (Am - Accession No. GSE50787 http:// www. ncbi. nlm. nih. bion, now Life Technologies, www. life techno logies. gov/ geo/query/acc.cgi?acc=GSE50787. com) during linear amplification using random octa - mers and a Klenow polymerase (Invitrogen). The reaction contained 3.9 mM d(AGC)TP, 0.4 mM dTTP Statistical analysis and 4.8 mM dUaa and was amplified for 3 h at 37°C. The Klenow product was purified by precipitation The array data were analyzed using the vegan and conjugated with Cy3. The Cy3-labelled target package in R (CRAN website; www.R-project.org) (1000 ng) was combined with 2× hybridization buffer (Borcard et al. 2011). Archetypes contributing <1% of (1× final concentration; Agilent) and 0.25 pmol of a the total signal were removed from further analysis. Cy5-labelled complementary 20-mer standard oligo- RFR values > 1% were transformed (arcsin [square nucleotide and incubated at 95°C for 5 min before root]) to normalize the proportional data. Environ- being cooled to room temperature. Targets were mental data were transformed (square root) and then hybridized to duplicate arrays by overnight incuba- standardized around zero (decostand in vegan). The tion at 64°C and washed. The arrays were scanned transformed data were used in all diversity and ordi- with a laser scanner (Molecular Devices 4300) and nation analyses according to Borcard et al. (2011). The analyzed with Gene Pix Pro 6.0 software (Molecular null hypothesis that the nirS community composition Devices). Quantification of hybridization signals was did not differ between surface depths and OMZ performed as described previously (Ward & Bouskill depths or between regions was tested using multi-re- 2011) with the following modifications. For each sponse permutation procedure (MRPP) (Zimmerman channel (532 nm [Cy3] and 635 nm [Cy5]), the aver- et al. 1985) with a significance level of 5%. age background fluorescence was recalculated after excluding background fluorescence values greater than the upper whisker of all of the background fluo- RESULTS rescence values. The upper whisker is defined as the 75th percentile plus 1.5 times the difference between Station characteristics the 25th and 75th percentiles. Such a filtering pro- cess was applied within each block on a microarray The hydrography and chemical distributions at to account for variability in background fluorescence some of these stations have been published previ- between blocks within an array. Another filter was ously (Table S1 in our Supplement; Ward et al. applied to remove anomalous values of Cy3:Cy5 2008, 2009). Stn 9 ETSP (coastal) and Stn 1 AS ratios among the triplicate features. This filter (AS2007-1; open ocean OMZ) provide the greatest excluded any feature with a test statistic Z > 1.9 (CI = contrast and contain the most complete qPCR pro- 80%), where Z is calculated as follows: files, so they are plotted here (Fig. 2). Data are pre-
ri sented in Tables S1 & S2 for the samples analyzed Zi = ,i = 1, 2, or 3 (1) s 3 in the present study. The AS stations were located Jayakumar et al.: Bacterial community composition in oxygen minimum zones 249
A Temperature (ºC) Nitrate (µM) B nirK (copies ml–1) 04010 20 30 025050 100 150 200
Salinity Anammox 16S Δ hzo (both ×105 copies ml–1) 34.4 34.6 34.8 35.0 0.0 0.5 1.0 1.5 2.0