Sampling focus: The Suboxic Zone

1. Fig. 1: The Black Sea Fig. 3: Black Sea Planctomycetes Diversity. DNA Samples from various density layers were amplified with Planctomycetes-specific primers (58f and 926r), and the entire insert sequenced. This phylogenetic tree, comparing our samples versus known Planctomycetes, indicate a plethora of uncharacterized bacteria. 15.8_JK624 86 15.8_JK616 15.8_JK632 15.8_JK625 15.8_JK528 15.8_JK588 15.8_JK622 15.8_JK584 15.8_JK587 15.8_JK594 15.8_JK609 Identified anammox and similar sequences 15.8_JK583 15.8_JK598 15.8_JK614 75 15.8_JK639 100 15.8_JK597 Density (depth) dependence: σ= 15.5 σ= 15.6 σ= 15.7 σ= 15.8 σ= 15.9 σ= 16.0 (Reference) Pirelulla-like sequences: found at all depths 100 15.8_JK615 15.8_JK529 15.7_JK707 70 15.8_JK586 10% 15.8_JK630 15.8_JK611 15.8_JK633 100 15.8_JK626 100 15.7_JK727 74 15.8_JK590 15.7_JK728 77 15.8_JK610 15.7_JK721 Pirellula 15.8_JK524 15.7_JK742 94 15.8_JK657 100 99 100 15.8_JK634 90 15.8_JK635 Pirellulastaleyi 80 75 70 96 83 74 15.8_JK636 100 100 100 15.8_JK613 Planctomycessp. 15.8_JK638 100 15.8_JK618 89 99 100 "Scalindua brodae" 15.8_JK629 100 79 "Scalindua sorokinii" 15.8_JK593 90 100 "Scalindua wagneri" 15.8_JK591 100 15.8_JK595 15.7_JK791 73 "Brocadia anammoxidans" 78 "Kuenenia stuttgartiensis" 15.8_JK619 100 15.8_JK620 78 15.8_JK608 97 Isosphaera 15.8_JK602 Gemmata 15.8_JK599 Sampling focus: The Suboxic Zone 100 15.7_JK697 15.8_JK600 100 15.7_JK776 15.8_JK596 98 Anammox enrichment 97 100 93 99 100 100 15.8_JK617 15.7_JK701 15.8_JK523 87 100 15.8_JK530 74 Gemmata Anammox enrichment 100 Isosphaera Planctomyces sp. 76 Pirellula staleyi Pirellula 75 100 Aquifex 96 "Kuenenia stuttgartiensis" "Brocadia anammoxidans" 15.7_JK735 98 "Scalindua wagneri" 100 100 "Scalindua brodae" 97 "Scalindua sorokinii" 71 Unknown group, showing some genera-level specificity to different densities 100 100 98 100 100 100 96 Fig. 2: Strong water column stratification results in anoxic conditions with a well defined suboxic zone, where both O2 and H2S are absent. We consider this an ideal location to sample for microbes that have not been well characterized (if at all). 100 75 10% 100 16.0_JK219 100 15.5_BO698 99 15.8_JK523 100 H' / H'max 15.9_JK445 100 15.6_JK871 15.5_BO699 15.9_JK487 15.9_JK312 100 0.2 0.4 0.6 0.8 95 15.8_JK617 15.5 15.6_JK826 16.0_JK235 88 15.9_JK415 15.6 100 100 15.8_JK613 100 16.0_JK221 100 15.9_JK416 15.6_JK843 15.5_BO719 100 15.7 15.5_BO681 Highly divergent group, typically found at the base of the suboxic zone. 15.5_BO704 100 Density, σ 15.5_BO726 100 15.5_BO715 100 15.5_BO720 88 15.6_JK852 15.8 15.5_BO694 92 15.9_JK448 100 16.0_JK211 100 86 100 16.0_JK94f 15.9 Aquifex 15.9_JK522 15.9_JK512 16.0_JK238 16.0_JK236 15.9_JK460 16.0 15.6_JK846 15.5_BO684 16.0_JK81f 15.8_JK619 15.5_BO703 15.8_JK602 15.9_JK485 15.6_JK831 15.6_JK798 15.5_BO708 15.9_JK417 0 1 2 3 4 15.8_JK636 - m N O M 15.8_JK630 3 15.9_JK461 15.6_JK854 0 0.1 0.2 0.3 0.4 16.0_JK215 15.9_JK420 13.6 15.8_JK618 m M 15.9_JK440 N O - Black Sea Central Gyre 2 14 15.6_JK800 15.8_JK638 NH4+ 16.0_JK207 15.9_JK501 14.4 NO3- 15.9_JK454 15.7_JK709 15.7_JK767 NO2- 15.7_JK789 14.8 15.6_JK808 N2/Ar Sample/Saturation Ratio 15.9_JK500 density, σ (depth proxy) 15.5_BO577 15.8_JK593 15.2 Suboxic zone Oxygen Sulfide 16.0_JK212 15.8_JK600 16.0_JK247 15.6 15.5_BO597 15.8_JK599 15.5_BO836 16.0_JK97 16 15.7_JK739 16.0_JK217 15.9_JK441 16.0_JK79 16.4 15.9_JK519 15.6_JK833 15.6_JK803 16.0_JK206 16.8 15.9_JK451 15.9_JK343 16.0_JK241 15.9_JK409 15.6_JK858 0 10 20 30 40 15.6_JK832 16.0_JK101 + m M 15.9_JK450 N H 4 16.0_JK189 15.9_JK412 16.0_JK201 15.9_JK506 15.8_JK530 16.0_JK245 1 1.005 1.01 1.015 1.02 16.0_JK83f 15.9_JK419 16.0_JK102 N2/Ar Sample/Saturation Ratio 15.9_JK442 The R/V Endeavor.Additional samples collected 3/26/05-4/5/05. Looking at Black Sea Microbial Communities to Understand Ancient Oceans John Kirkpatrick1, Brian Oakley2, Clara Fuchsman1, Sujatha Srinivasan2, James T. Staley2, James W. Murray1 1School of Oceanography and 2Department of Microbiology University of Washington, Seattle, WA April, 2005 Abstract #987 • Abstract • Earth's oceans would have appeared foreign to the modern observer for much of our planet's history, as evidence shows that the oceans had oxic surface and anoxic deep layers from approximately 2 to 0.5 billion years ago (cf. Canfield, 1998; Anbar and Knoll, 2002). This is much like the current Black Sea, the world's largest anoxic basin. We are investigating the anaerobic bacterial communities of the Black Sea in order to better understand the ancient oceans. We are particularly interested in novel metabolisms, such as anaerobic ammonium oxidizing (anammox) bacteria, chemoautotrophs which can produce significant amounts of N2 gas. • Using samples collected from various depth horizons focused on the suboxic zone, we have constructed and sequenced 16S rDNA clone libraries using Planctomycetes-specific primers. This has allowed us to look at diversity within the Planctomycetes group, with several interesting results. • There are multiple unknown groups, many of which are highly divergent from known Planctomycetes. • One group of 16S rDNA environmental sequences (detected only with Planctomycetes primers) branches separately from known groups and is also found growing in a selective culture for anammox bacteria. • In addition, there are several other environmental sequences similar to known anammox bacteria. • The upper depths of the suboxic zone have a relatively low diversity, with the “basement” of the zone hosting a complex array of different groups. This coincides with the potential for various S-based metabolisms. • Altogether, this raises interesting questions about the genetic diversity and metabolism of the Planctomycetes in general and anammox bacteria in particular. It also leads us to believe that there is much yet unknown about the composition of early marine microbial communities, as well as their interactions with and contributions to the planetary environment. Fig. 4: While anammox-type sequences were detectable at all of the mid- and lower-depths, they dominated clone libraries at σθ = 15.8. This seems odd, because the anammox reaction requires NH4+, which approaches zero at σθ= 16.0. Project Outline As we are striving to better understand the different forms life has taken on Earth, we are particularly interested in metabolic diversity. Since N- and S-based chemistry have a wide variety of life-supporting reactions, we focused on a part of the Black Sea where there is a large potential for “atypical” biogeochemistry. This is the suboxic zone (cf. figure 2), a redoxcline spanning 10s of meters which is deficient in both oxygen and sulfide. Amongst others, anaerobic ammonium oxidizing (anammox) bacteria are known to exist here; these chemoautotrophs which can release significant amounts of N2 gas (cf. figure 3). Samples collected in 2003 on the R/V Knorr were analyzed to yield depth-specific information on the chemistry and microbiology of the water column. In order to characterize the microbial community, and relate it to the chemistry of the system, we have utilized both culture-independent and culture-based techniques. DNA extraction, cloning, and sequencing has given us the basic distributions and diversity of numerous bacteria. Here we have used specific primers (58f and 926r) to focus on the Planctomycetes, an unusual bacterial phylum characterized by intracellular membranes, a complete lack of peptidoglycan, and a diverse distribution. The results of these molecular studies are summarized in figures 4-6. Enrichment cultures, designed to select for different metabolisms based on the media composition, have also yielded some successes. Among these is anammox enrichment medium (sterile seawater with NH4+ and NO2-) which has produced an unknown strain of Planctomycetes (cf. figures 4,5). Fig. 5: Varying levels of diversity. This figure shows a diversity index (Shannon-Weaver, normalized for all depths by dividing by H’max); a higher number indicates increased diversity. σ = 15.8, shown above, is dominated by the anammox phylotype and has low diversity. At deeper density interfaces, approaching the bottom of the suboxic zone and the onset of sulfide, Planctomycete diversity increases dramatically. Further investigation will help us determine what sort of N and / or S metabolisms are related to these various groups of bacteria. (Note that these results are PCR based, and so may reflect the biases of that technique. Samples have all been screened for chimeras using Bellerophon [see Huber et al., 2004] and also RDP’s Chimera Check.) • Conclusions • "Life as we know it" is defined not by 3 dimensions, but by 4. If we as Astrobiologists want to overcome the inherent scientific difficulty of being in one place at one time, one way to start is by thinking about not only other places, but other times. By considering the unusual (yet historically important) environment of the Black Sea, we can gain insight not only into Earth's past but also on the variety of forms and functions that life takes. Here we have presented the results of a first-pass assessment of the microbes living in the Black Sea. Among other observations, we can say that: • There is a very large amount of diversity in the phylum Planctomycetes; more unknown bacteria in this location, in fact, than previously known and characterized worldwide. • The Planctomycetes of some depths (such as σ = 15.7, 15.8) appear to be dominated by a few strains, possibly due to the favorability of the anammox reaction in certain chemoclines. • A few strains of Planctomycetes are ubiquitous throughout the suboxic zone. This includes one type of bacteria, previously unknown, which grows in inorganic anammox enrichment media. • There appears to be increased diversity at the bottom of the suboxic zone; we hypothesize that there might be a correlation to the viability of S-based metabolisms at those depths. • Now that we have gathered essential 16S rDNA data on various depths of the Black Sea, along with fresh samples, we can attempt to answer questions raised by our previous work. These include: • What are the dominant species? We are working on Fluorescent In-Situ Hybridization (FISH) techniques to identify and count specific strains of bacteria. • How active are these bacteria? We have incubated and collected samples spiked with 14C-bicarbonate in order to measure rates of chemosynthesis. • Which species or groups are primary producers? We have collected samples for a combined 14C and FISH analysis to determine which kinds of bacteria are fixing carbon. • What are the bacteria doing? We have many samples growing in selective cultures, including those for anammox, heterotrophic denitrification, thiodentrification, and sulfite reduction, amongst others. Fig. 3: Chemical gradients and the suboxic zone This graph shows the various chemical distributions around the suboxic zone. Ammonium is produced at depth and is consumed (along with nitrate) in the suboxic zone; both reach negligible values around σθ = 16.0. This depth corresponds to an N2 gas maximum, relative to saturation. Nitrite shows a more complex profile, with maxima at σθ = 15.0 and 15.9. The suboxic zoneis shaded (cf. figure 2). Note the relative scale bars. Anammox bacteria are known to live in the suboxic zone, and survive autotrophically by producing N2 gas. We are attempting to understand their importance in the complex interplay of various N species (and their isotopes). The relevant reaction is: Anammox Reaction: NH4+ + NO2- N2(g) + H2O References Anbar, A. D. , and A. H. Knoll. Proterozoic Ocean Chemistry and Evolution: A Bioinorganic Bridge? Science297, 1137-1142 (2002). Canfield, D.E. A new model for Proterozoic ocean chemistry. Nature396, 450-453 (1998). Huber, T. , G. Faulkner and P. Hugenholtz. Bellerophon; a program to detect chimeric sequences in multiple sequence alignments. Bioinformatics20 2317-2319 (2004). Acknowledgements Thanks to Billy Brazelton for all of his advice; and also to Mark Rider and Audrey Harris for their laboratory assistance. This work was funded by NSF Microbial Observatories 0132101 and NSF-IGERT grant DGE-9870713.