Arsenic accumulation in cyanobacteria from a high

Cellular arsenic accumulation in a cyanobacteria
culture isolated from a high-As, low carbon
geothermal setting
Kimberly D. Myers, Christopher R. Omelon, & Philip C. Bennett
The University of Texas at Austin
ISEB 2014
November 18
ETGF
El Tatio Geyser
Field (ETGF)
Antofagasta Region, Chile
Atacama Desert
Andean Altiplano
Chemical Conditions at El Tatio
Basin
pH
T
(°C)
[Cl]
mM
[Na]
mM
[Si]
mM
[As]
mM
[HCO3-]
mM
Upper
7.0
80.5
204
169
5.8
0.60
0.02
Middle
6.9
85.0
163
132
4.2
0.45
0.05
Lower
7.4
78.0
155
157
3.1
0.35
0.06
² Circumneutral pH
² Very low dissolved inorganic carbon (DIC = CO2 + HCO3- + CO32-)
² Highest reported naturally occurring arsenic [As] as As(III), (V)
As(V) buﬀers pH in El Tatio waters
²  Neutral pH
²  DIC buﬀers most natural waters
Log activity mM
²  Trace [DIC] as CO2+ HCO32- (0.1-0.5 mM)
Stream pH is approximately
equal to pKa2 of arsenate
(6.94)
Landrum, 2007; Landrum et al. 2009
pH
Cyanobacteria
Important primary producers in hot
spring microbial mats
Oxygenic photosynthesis
6 CO2 + 6 H2O
light
Great Geyser cyanobacteria-‐55m 10 μm
C6H12O6 + 6 O2
Octopus Spring, YNP
RuBisCO: enzyme essential to
photosynthesis
El Tatio conditions known to limit
RuBisCO:
-Low [DIC]= 0.1-0.5 mM
-Temperature > 30°C
-High UV Badger et al. 2006 The Carbon Concentrating Mechanism (CCM)
Response to low [DIC]
Carbonic Anhydrase (CA)
HCO3-
CA
CO2 + OH-
Causes pH increase
pH buﬀering can
positively impact CCM
activity
Figure courtesy of C. Omelon
Silica - CCM of diatoms*
As(V) – Cyanobacteria?
*Milligan and Morel 2002 Arsenite Oxidation and Cyanobacterial Abundance
along a Low-DIC Transect
As(V)
454-‐pyrosequencing Negative association with As(III):
r2 = 0.64, P < 1.8 x 10-4 (n=15)
Positive association with As(V):
r2 = 0.31, P < 0.026
Myers et al.-‐in prep As(III)
El Tatio
[As(III)]
Cell death
Biomass increase
Average Response of Four Cyanobacteria Cultures Exposed to
0-20 mM As(III) or As(V) After 6 Days
Control
n=4
Std error of mean
Myers et al.-‐in prep Cyanobacteria culture T-031
Unclassified Nostoc-like sp.
Collection site:
-26°C
-Low DIC,
-0.35 mM As(V)
Subsection IV
Filamentous,
heterocystous,
binary fission
10 um 100
90
80
70
60
50
40
30
20
10
0
Closed microcosm experiment
AsIII AsV %DIC removed
Unbuffered media
0.8 mM DIC initially
0.5 mM As(III) and As(V)
40
2
N=10 Low error 4
Days
6
30
8
20
Δ Biomass
0
10
0
-10
2
4
-20
-30
Myers et al.-‐in prep 0
-40
Days
6
8
Carbon limited and As(V)-buﬀered conditions
10.5
100
90
10
9.5
70
9
60
50
8.5
40
8
30
pH
% DIC Removed
80
7.5
20
7
10
6.5
0
1
0mM As(V)
1.0mM As(V)
Myers et al.-‐in prep 3
5
Days
7
n=10
Std error of mean
Growth of Cyanobacteria with As(V)-buﬀering
•  Un-buffered treatment exhibits little growth on first day
•  As(V) buffered treatment diverts more DIC to growth
Myers et al.-‐in prep Zone of pH buﬀering corresponds to elevated O2 FIG. 5. In-‐situ microproﬁles of pH (¢) and dissolved oxygen concentraOon [O2] μmol/L (✚) of the top 1mm of a microbial mat at 75m along the Great Geyser transect, corresponding to site GG-‐75m (Table 1). Depth of 0 μm represents the water-‐microbial mat interface. Dashed lines on the photograph mark the upper 1 mm of a slice of a corresponding secOon of the sampled mat. Myers et al.-‐in prep Microbial Responses to As
1. 
2. 
3. 
4. 
Anaerobic respiration (AsV
AsIII)
Arr
Assimilation (arsenolipids, arsenosugars)
Arsenite oxidation (AsIII
AsV)
Aox
Detoxification (AsV
AsIII)
Ars
Silver & Phung, 2005; Mukhopadhyay et al., 2002; Stolz et al 2006
Cyanobacteria
•  Cellular detoxification using Ars (Synechocystis sp. PCC 6803)
•  Cellular As(V) accumulation
•  As(V) accumulation, increased pigment content, and
stimulated growth in Nostoc sp. and Anabaena sp.
(Lopez-Maury et al. 2003, 2009; Bhattacharya & Pal 2010; Thiel 1988; Ferrari et al. 2013)
As K-‐edge XANES •  Cultures washed, pelleted, suspended on ﬁlter paper and analyzed for As speciaOon [(III), (V)] by synchrotron radiaOon •  Only As(V) observed •  Yet to be determined if As(V) is adached to cell membrane or stored within the cell Summary of results •  Cyanobacteria are more abundant in As(V)-‐
dominated stream areas throughout El TaOo •  Cyanobacteria are more sensiOve to As(III) in unbuﬀered condiOons •  Cells accumulate As(V), which sOmulates carbon uptake and growth in DIC limited condiOons ImplicaOons •  AccumulaOon and retenOon in cells makes inorganic As(V) available for buﬀering •  As(V)-‐buﬀering leads to increased carbon assimilaOon and growth, advantageous to microbial communiOes in DIC-‐limited streams at El TaOo •  May explain why previous work has observed As(V) accumulaOon (they possess the cellular machinery for ars—why not use it??) Acknowledgments
Thanks to:
The Bennett Lab Group, especially
Chris Omelon, Megan Franks, and Phil
Bennett
Funding:
The Jackson School of Geosciences,
UT Austin
NSF (SGER: EAR0085576)
Current and Previous work by:
Philip C. Bennett (PhD) and PI of NSF
(SGER: EAR0085576)
Annette S. Engel (PhD)
Megan Franks (PhD)
Christopher R. Omelon (PhD)
Jeff Landrum (MS)
Suzanne Pierce (PhD)
Kim Myers: [email protected]
References Badger, M. R., G. D. Price, B. M. Long and F. J. Woodger (2006). "The environmental plasOcity and ecological genomics of the cyanobacterial CO2 concentraOng mechanism." J Exp Bot 57(2): 249-‐265. Bhadacharya, P. and R. Pal (2010). "Response of cyanobacteria to arsenic toxicity." Journal of Applied Phycology 23(2): 293-‐299. Ferrari, S. G., P. G. Silva, D. M. González, J. A. Navoni and H. J. Silva (2013). "Arsenic tolerance of cyanobacterial strains with potenOal use in biotechnology." Revista ArgenOna de Microbiología 45(3): 174-‐179. Landrum, J. T. (2007). Fate and Transport of Arsenic and AnOmony in the El TaOo Geyser Field, Chile. Masters of Science in Geological Sciences, The University of Texas at AusOn. Landrum, J. T., P. C. Benned, A. S. Engel, M. A. Alsina, P. A. Pastén and K. Milliken (2009). "ParOOoning geochemistry of arsenic and anOmony, El TaOo Geyser Field, Chile." Applied Geochemistry 24(4): 664-‐676. Lopez-‐Maury, L., F. J. Florencio and J. C. Reyes (2003). "Arsenic Sensing and Resistance System in the Cyanobacterium SynechocysOs sp. Strain PCC 6803." Journal of Bacteriology 185(18): 5363-‐5371. Lopez-‐Maury, L., A. M. Sanchez-‐Riego, J. C. Reyes and F. J. Florencio (2009). "The glutathione/glutaredoxin system is essenOal for arsenate reducOon in SynechocysOs sp. strain PCC 6803." J Bacteriol 191(11): 3534-‐3543. Milligan, A. J. and F. M. Morel (2002). "A proton buﬀering role for silica in diatoms." Science 297(5588): 1848-‐1850. Mukhopadhyay, R., B. P. Rosen, L. T. Phung and S. Silver (2002). "Microbial arsenic: from geocycles to genes and enzymes." FEMS Microbiol Rev 26: 311-‐325. Silver, S. and L. T. Phung (2005). "Genes and enzymes involved in bacterial oxidaOon and reducOon of inorganic arsenic." Appl Environ Microbiol 71(2): 599-‐608. Stolz, J. F., P. Basu, J. M. SanOni and R. S. Oremland (2006). "Arsenic and selenium in microbial metabolism." Annu Rev Microbiol 60: 107-‐130. Thiel, T. (1988). "Phosphate transport and arsenate resistance in the cyanobacterium Anabaena variabilis." Journal of Bacteriology 170: 1143-‐1147.

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