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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 rvalue. 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 SI859A1 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 % 2 Center 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 1 Division 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 MnoxidaDon in the Columbia River Estuary and offshore plume Craig R. Anderson 1* , Richard E. Davis 1 , N. Shakira Bandolin 2 , Antonio M. BapIsta 2 and Bradley M. Tebo 1 *Corresponding author, Email: [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 (TRFLP) 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 MnoxidaIon assay 2. MPN analysis 3. Community proteome fracIonaIon 4. Subcellular protein fracIon MnoxidaIon assay 5. Size exclusion chromatography of protein fracIons posiIve for MnoxidaIon 6. Tandem mass spectrometry for protein idenIficaIon QPCR 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 hemeperoxidase have been idenIfied in 11 other sequenced genomes. Results and Discussion TRFLP 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 MnO 2 cell 1 day 1 . Evidence for the presence of the Mnoxidizing genera Auran&monas, Rhodobacter, Bacillus, and Shewanella was found in the TRFLP 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 hemeperoxidase from the Mnoxidizing genus Auran&monas (Table 1). Both of these proteins have been associated with bacterial MnoxidaIon before, specifically the peroxidase 1 . Figure 1: Locality map of CTD casts for sample collecIon during the MayJune 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 Mnoxidizing 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. 1 Anderson, 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 SI859A1 and Erythrobacter sp. strain SD21. Applied and Environmental Microbiology, 75 (12): 41304138. 2B 2A 2C 3A 3B
Transcript
Page 1: OS 2010 Anderson - stccmop.org · 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’  

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  

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