Microbial Ecology of Fe (hydr)oxide Mats and Basaltic Rock from Vailulu'u Seamount, American Samoa

Abstract

Microbial community analysis of a deep-sea volcanic and hydrothermal system at Vailulu'u Seamount yielded 89 new organisms and three detailed 16S-rRNA gene clone libraries (one rock and two microbial mats). Proteobacterial communities dominate in most environments, but important differences are found between microbial mats from distinctly different geochemical environments and for the rock surface. Many cultured organisms are metabolically and functionally diverse, displaying at least two of the tested functions: heterotrophy, Fe(II) and Mn(II) oxidation, and siderophore-production. Metabolic versatility of microorganisms is suggested as an important trait allowing diverse populations of bacteria to adapt to these environments.

Keywords:

• biogeochemical cycling
• community structure
• deep biosphere
• iron-oxidizing bacteria
• molecular ecology

The authors would like to thank the Captain and crew of the R/V Ka'imikai O' Kanaloa as well as the pilots and support staff for the HURL Pisces IV/V submersibles. Additional thanks to Daniel Rogers and Katrina Edwards for the protocol used for “F-plates,” Rick Davis for his help with Dotur, Ryan Anderson at the Nano3 facility at UCSD for his assistance on the SEM and last but not least to Brad Bailey for his help in American Samoa and in the laboratory. The authors would also like to acknowledge the thoughtful comments of Dave Emerson and two anonymous reviewers. We would like to thank our funding sources for this work including the NSF Microbial Observatories and Biogeosciences programs (MCB-0348668 and OCE-0433692), NSF Ocean Sciences (OCE0526285), and the Agouron Institute. Any opinions, findings and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of these agencies.

Notes

¹per L of ultrapure H₂O: 50 g NaCl, 0.94 g casamino acids, 0.625g proteose peptone #3, 0.125 g yeast extract, 0.375 g sodium citrate, 2.5 g MgSO₄· 7H₂O, 0.00625 g Fe(NH₄)₂(SO₄)₂· 6H₂O, 15 g agar, adjusted to pH∼ 8.2 to balance addition of acidified FeSO₄ solution after autoclaving. After autoclaving at 121 °C for 30 min.: 1ml vitamin mix, 1 ml trace element mix (Pfennig and Lippert 1966), 1 ml filter sterilized 400 mM FeSO₄.

²per L of ultrapure H₂O: 27.5 g NaCl, 5.38 g MgCl, 0.72 g KCl, 0.2 g NaHCO₃, 1.4 g CaCl₂, 1 g NH₄Cl, 0.05 g K₂HPO₄, 15 g agar, pH 7.3, and 1ml trace element mix added after autoclaving at 121°C for 30 min and cooling to ∼ 50°C.

³Per L of ultrapure H₂O: 10 g Bacto Tryptone, 5 g Yeast Extract, 10 g NaCl, adjusted to pH 7.0.

⁴27f (5′-AGA GTTT GAT CMT GGC TCA G-′3); 1492r (5′-TAC GGY TAC CTT GTT ACG ACT T-′3) PCR conditions: 95°C = 3 min.; 35 x (95°C = 30 sec., 50°C = 30 sec., 72°C = 1 min.); 72°C = 7 min. Each 50 μ l PCR reaction contained 1.5 μ M of each primer, 1.25 units Taq DNA polymerase (Roche), 1x PCR buffer (Roche), a 200 μ M concentration of each deoxynucleoside triphosphate (Invitrogen) and 0.2 mg/ml bovine serum albumin. PCR amplification was originally tried at a variety of annealing temperatures. However we were unable to amplify consistently at temperatures higher than 50°C.

⁵Each sequencing reaction included 2 μ l of BigDye Terminator v3.1 (Applied Biosystems), 5 pmol primer and 4–6 μ l of PCR reaction (for isolates) or 3 μ l of plasmid (for clones) in a total volume of 8 μ l. The sequencing reaction consisted of: 96°C = 10 sec; 28 x (96°C = 10 sec., 50°C = 5 sec., 60°C = 4 min. After completion 40 μ l of 75% isopropanol were added to the reactions and incubated at room temperature for 15 minutes followed by a 30-minute centrifugation at 2,000 x g. The supernatant was removed and the samples were dried by inverted centrifugation for 1 minute at 700 x g prior to sequencing.

⁶M13f (5′-GTA AAA CGA CGG CCA G-3′), M13r (5′-CAG GAA ACA GCT ATG AC-3′), 1074r (5′-CAC GAG CTG ACG ACA GCC AT-′3).

⁷ http://hcv.lanl.gov/content/sequence/ELIMDUPES/elimdupes.html.

⁸Per 800 ml of distilled water: 8 g NaCl, 0.2 g KCl, 1.44 g Na₂HPO₄ and 0.24 g KH₂PO₄, adjusted to pH 7.4, filled up to 1 L.