47° 00’
46° 45’
46° 30’
46° 15’
46° 00’
125° 00’ 124° 45’ 124° 30’ 124° 15’ 124° 00’ 123° 45’ 123° 30’ 123° 15’ 123° 00’
47° 00’
46° 45’
46° 30’
46° 15’
46° 00’ 125° 00’ 124° 45’ 124° 30’ 124° 15’ 124° 00’ 123° 45’ 123° 30’ 123° 15’ 123° 00’
CTD139_22m
CTD128_51m CTD129_2m
CTD122_2m CTD122_19m CTD156_2m
CTD156_102m
CTD158_15m
CTD145_11m
CTD144_12m
Columbia River
Pacific Ocean
Astoria
Offshore Coastal Estuary
CTD122 – collected June 1st 2008, TransiIon from Neap to Spring Ide CTD144 – collected June 2nd 2008, Pre ETM CTD156 – collected June 3rd 2008, Spring Ide
Figure 2: Dynamics of microbial community transfer between coastal environments where discharge and mixing of river water was traced using salinity (2A and 2B) and microbial communiIes contained within those water bodies were fingerprinted using TRFLP and related using a UPGMA/Pearson product moment correlaIon cluster analysis (2C). Scale bar is the Pearson product moment correlaIon r-‐value. Numbers at nodes are copheneIc correlaIon coefficients.
Columbia River
Plume Environment
Deep Coastal
Coastal Surface
Offshore Surface
Estuary
0.2
CTD156_2m 25.5
CTD156_102m 34.0
PSU
CTD129_2m 30.8
CTD128_51m 33.9
CTD158_15m 32.2
CTD145_11m 15.7
CTD139_22m 32.2
CTD122_2m 2.0
CTD122_19m 33.4
CTD144_12m 0.3
90
100
100
100
100
100
86
91
76
River/ Estuary
Plume
Ocean
1st June 2008 17:00:00 PST
CTD122_2m 2.0 PSU
PSU 0.0
5.0
10.0
15.0
20.0
25.0
30.0
34.5
2nd June 2008 12:00:00 PST
~15 km
CTD145_11m 15.7 PSU
CTD144_12m 0.3 PSU
Salinity in the Columbia River estuary, coastal discharge and coastal NW Pacific
River mouth Estuary
2007 ETM samples
CTD122_2m
CTD122_19m
CTD156_2m
CTD156_102m
CTD144_12m
CTD8_In
CTD13_Out
% Auran'monas 16S rDNA copies (in proporDon to total Bacterial 16S rDNA gene copies)
Table 1: Summary of proteins idenIfied by funcIon from the Columbia River. Proteins from sub-‐cellular fracIons that were posiIve for manganese oxidaIon were separated using size exclusion chromatography and then MS/MS fragments generated by tandem mass spectrometry were indenIfied by querying against the both the NCBI database and th genome database of AuranImonas sp. strain SI85-‐9A1
Proteins Identified
Loosely Bound Outer Membrane Outer Membrane
CTD122 2m
CTD122 19m
CTD156 2m
CTD156 102m
CTD144 12m
CTD122 2m
CTD156 2m
CTD144 12m
Mn-oxidizing Associated 1 1 5 1 1 1
Motility 4 (4) 8 (2) 1 18 (1) 2 (6) 3 (6) 6 (2)
Transport and Receptors 4 (2) 1 (2) 4 (2) 22 4 2 (1)
Enzymes 2 2 (2) 1 (1) 1 21 1 (1) 1 1 (1)
DNA Binding/ Transcription/Translation 9 1 1
Cytochromes/Photosystem/ATP Generation 2 1 1 (1)
Carbon Fixation and Nitrogen 1 3 (1)
Miscellaneous / Hypothetical 11 (1) (1)
Numbers in parenthesis are proteins where the probability of correct idenIficaIon is below 70 %
2Center for Coastal Margin ObservaIon & PredicIon (CMOP) NaIonal Science FoundaIon Science & Technology Center School of Medicine Oregon Health & Science University 20000 NW Walker Road, Beaverton, OR, 97006 USA
1Division of Environmental and Biomolecular Systems Oregon Health & Science University 20000 NW Walker Road, Beaverton, OR, 97006 USA Telephone: 503 748 1992 Fax: 503 748 1464
Analysis of in situ Mn-‐oxidaDon in the Columbia River Estuary and offshore plume Craig R. Anderson1*, Richard E. Davis1, N. Shakira Bandolin2, Antonio M. BapIsta2 and Bradley M. Tebo1 *Corresponding author, E-‐mail: [email protected]
IntroducDon The Columbia River and its estuary are major sources of nutrients and trace metals for the enIre west coast of North America. As estuarine water is discharged off the coast it transports parIculate maner, dissolved nutrients and microorganisms forming nutrient rich and metabolically dynamic coastal plumes. Since manganese and manganese oxides have significant interacIons with major nutrient cycles, it is important to understand the biogeochemistry of manganese in this environment. The source of reduced manganese off the coast of Oregon has previously been traced to the Columbia River estuary but currently linle is known about manganese oxidaIon in this environment. This study aims to invesIgate the oxidaIve side of the manganese biogeochemical cycle from microbial community and proteomic perspecIves.
Methods Five samples were collected for both DNA and protein extracIon and another 5 for just DNA extracIon (Fig. 1). DNA was extracted from cells filtered from 0.5 to 15L volumes water samples. Microbial community fingerprints were created using terminal restricIon fragment length polymorphism (T-‐RFLP) and then compared using cluster analysis. For protein analysis, microbial cells from 40L water samples were concentrated with tangenIal flow filtraIon and then analyzed with the following methods:
1. Whole cell Mn-‐oxidaIon assay 2. MPN analysis 3. Community proteome fracIonaIon 4. Sub-‐cellular protein fracIon Mn-‐oxidaIon assay 5. Size exclusion chromatography of protein fracIons posiIve
for Mn-‐oxidaIon 6. Tandem mass spectrometry for protein idenIficaIon
Q-‐PCR was used to quanIfy the gene copies of heme-‐peroxidase, Auran&monas SSU rRNA and total bacterial SSU rRNA in the samples collected. Auran&monas only accounted for 1.7% of heme-‐peroxidase genes quanIfied suggesIng that peroxidase driven manganese oxidaIon capabiliIes are widespread throughout other organisms in this environment. Orthologs of the Auran&monas Mn-‐oxidizing heme-‐peroxidase have been idenIfied in 11 other sequenced genomes.
Results and Discussion T-‐RFLP cluster analysis and salinity maps indicate that bacterial communiIes from within the estuary are transported off the coast and mix with the ocean communiIes (Fig. 2). As a whole, these communiIes can produce up to 10 fM MnO2 cell-‐1 day-‐1. Evidence for the presence of the Mn-‐oxidizing genera Auran&monas, Rhodobacter, Bacillus, and Shewanella was found in the T-‐RFLP fingerprints (data not shown).
A total of 105 proteins were idenIfied from the Mn oxidaIon acIve outer membrane including a mulI-‐copper oxidase (MCO) and a heme-‐peroxidase from the Mn-‐oxidizing genus Auran&monas (Table 1). Both of these proteins have been associated with bacterial Mn-‐oxidaIon before, specifically the peroxidase1.
Figure 1: Locality map of CTD casts for sample collecIon during the May-‐June 2008 R/V Wecoma cruise. Samples were collected using a seabird CTD system equipped with an SBE Carousel sampler with 12 10L Niskin sampling bonles.
Figure 3: QuanIficaIon of Auran&monas sequences in proporIon with total bacterial sequences from the Estuary, Plume and surface offshore samples (A) and Mn-‐oxidizing peroxidase (Mop) (B). Graphs include data from previous years samples for comparison. Clearly Auran&monas cannot account for all of the peroxidsae present in the environment.
2007 ETM samples
CTD122_2m
CTD122_19m
CTD156_2m
CTD156_102m
CTD144_12m
CTD8_In
CTD13_Out
Mop
Auran&monas 16S rDNA
Mop copies versus Auran'monas 16S rDNA gene copies
Acknowledgements and References: Many thanks the Chief ScienIst on the cruise, Tawnya Peterson, and to the captain and crew of the R/V Wecoma. 1Anderson, C. R., Johnson, H. A., Caputo, N., Davis, R. E., Tebo, B. M. 2009. Mn(II) oxidaIon is catalyzed by heme peroxidase in Auran&monas manganoxidans strain SI85-‐9A1 and Erythrobacter sp. strain SD-‐21. Applied and Environmental Microbiology, 75 (12): 4130-‐4138.
2B 2A
2C
3A
3B
