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7-16-2008 Stable Carbon Isotope Discrimination by Form IC RubisCO from Rhodobacter sphaeroides Phaedra Thomas University of South Florida

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Stable Carbon Isotope Discrimination by Form IC RubisCO

from Rhodobacter sphaeroides

by

Phaedra Thomas

A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science Department of College of Arts and Sciences University of South Florida

Major Professor: Kathleen M. Scott, Ph.D. Valerie J. Harwood, Ph.D. John H. Paul, Ph.D.

Date of Approval: July 16, 2008

Keywords: , Calvin-Benson-Bassham cycle, alphaproteobacteria, , fractionation

© Copyright 2008, Phaedra Thomas

Acknowledgements

The funding for this graduate research was made possible by the NSF FGLSAMP

Bridge to the Doctorate Program at the University of South Florida (Award HRD #

0217675) and NSF Biological Oceanography (Award# OCE-0327488, to K.M.S).

Special thanks are given to the graduate researchers in Dr. Scott’s lab, as well as to Dr.

Robert Michener at the Boston University Stable Isotope Lab for analyzing our samples via , and to Dr. F. Robert Tabita for providing the Form IC RubisCO .

Table of Contents

List of Tables ii

List of Figures iii

Abstract iv

Chapter One: Introduction 1 The 1 Other Autotrophic Pathways 3 Elucidating autotrophic pathways from contemporary & ancient environmental samples 6 Stable carbon isotope ratios and δ13C values 7 Enzymatic isotope discrimination 10

Chapter Two: Isotope Discrimination by Form IC RubisCO 12 Experimental Procedures 16 Results 18 Discussion 20

Chapter Three: Conclusion 25 Experiments with Form ID RubisCO 25

References 27

Appendices 31 Appendix A: Table of RubisCO ε values from Ralstonia eutropha 32 Appendix B: Manuscript on form ID RubisCO ε value from Emiliania huxleyi 33 Appendix C: Manuscript on form ID RubisCO ε value from Skeletonema costatum 45 Appendix D: Abstract from Publication of Thiomicrospira crunogena XCL-2 genome 65 Appendix E: Abstract from Publication of Sulfurimonas denitrificans genome 68

i

List of Tables

Table 1 The distribution of autotrophic pathways among the domains of life 5

Table 2 RubisCO ε values determined from nine independent experiments with the Rhodobacter sphaeroides enzyme 19

Table 3 ε values from form I and II RubisCO 20

13 13 Table 4 δ CCO2 and δ Cbiomass values from autotrophic organisms whose RubisCO ε values have been determined 22

Table A-1 RubisCO ε values determined from two independent experiments with the IC enzyme from Ralstonia eutropha 33

Table B-1 ε values of different RubisCO forms 37

Table C-1 ε values of different forms of RubisCO that have been measured with kinetic isotope experiments 61

ii

List of Figures

Figure 1. The RubisCO reaction in the Calvin-Benson-Bassham (CBB) Cycle 2

Figure 2. Zero-point energy diagram of 12C - and 13C - 8

Figure 3. Minimum evolution tree of RubisCO large subunit (cbbL) nucleotide sequences 15

Figure 4. Changes in the concentration (‹) and δ13C („) of dissolved inorganic carbon (DIC) vs. time for Rhodobacter sphaeroides RubisCO 18

Figure 5. Natural log-transformed isotope ratios (R= 13C/12C) and concentrations of dissolved inorganic carbon (DIC) during by form IC RubisCO 19

Figure 6. Whole-organism isotopic discrimination 13 13 (δ CCO2 - δ Cbiomass) versus enzymatic isotopic discrimination (RubisCO ε values). 22

13 Figure 7. δ C values of atmospheric CO2 and biomass from organisms using different pathways of autotrophy 23

Figure B-1. Isotope fractionation by E. huxleyi RubisCO 39

Figure C-1. Isotope fractionation of DIC as CO2 is consumed by S. costatum RubisCO and spinach RubisCO 55

Figure C-2. The consumption of DIC over time by S. costatum RubisCO 56

Figure C-3. Radiometric assay of partially purified S. costatum RubisCO 57

Figure C-4. Phylogenetic tree of selected RubisCO large subunit genes (rbcL) 58

Figure D-1. PloS Biology Open Access License 67

Figure E-1. American Society for Microbiology permission document 70

iii

Stable Carbon Isotope Discrimination by Form IC RubisCO from Rhodobacter sphaeroides

Phaedra J. Thomas

ABSTRACT

Variations in the relative amounts of 12C and 13C in microbial biomass can be

used to infer the pathway(s) use to fix and assimilate dissolved inorganic

carbon. Discrimination against 13C by the enzymes catalyzing autotrophic carbon fixation is a major factor dictating the stable carbon isotopic composition (δ13C =

13 12 13 12 {[ C/ Csample/ C/ Cstandard] – 1} X 1000) of biomass. Six different forms of

1,5-bisphosphate carboxylase/ or RubisCO (IA, IB, IC, ID, II, and III), the

carboxylase of the Calvin-Benson-Bassham cycle (CBB), are utilized by and

autotrophic that rely on the CBB cycle for carbon fixation. To date, isotope

discrimination has been measured for form IA, IB, and II RubisCOs. Isotopic

discrimination, expressed as ε values (={[12k/13k] – 1} X 1000; 12k and 13k = rates of 12C and 13C fixation) range from 18 to 29‰, explaining the variation in biomass δ13C values

of autotrophs that utilize these enzymes. Isotope discrimination by form IC RubisCO has

not been measured, despite the presence of this enzyme in many of

ecological interest, including marine -oxidizing bacteria, some nitrifying and

nitrogen-fixing bacteria, and extremely metabolically versatile organisms such as

Rhodobacter sphaeroides. The purpose of this work is to determine the ε value for the

iv form IC RubisCO enzyme from R. sphaeroides. Under standard conditions (pH 7.5 and 5 mM DIC), form IC RubisCO had an ε value of 22.9‰. Sampling the full phylogenetic breadth of RubisCO enzymes for isotopic discrimination makes it possible to constrain the range of δ13C values of organisms fixing carbon via the Calvin-Benson-Bassham cycle. These results are helpful for determining the degree to which CBB cycle carbon fixation contributes to primary and secondary productivity in microbially-dominated food webs.

v

Chapter One

Introduction

The Calvin Cycle

The Calvin-Benson-Bassham (CBB) cycle is the most common carbon-fixing

pathway for autotrophic organisms. It is present in plants, algae, , and

proteobacteria, and has recently been found in firmicutes (3). The CBB cycle consists of

the dark reaction of in which CO2 is incorporated into organic compounds for the biosynthetic needs of the organism. It consists of three stages: carbon fixation, reduction, and regeneration (1).

RubisCO (Ribulose 1,5-bisphosphate carboxylase/oxygenase) is the carbon-fixing enzyme in the Calvin cycle (48). The RubisCO reaction involves the catalytic conversion of one of ribulose 1,5-bisphosphate (RuBP), via carboxylation, into two

of phosphoglyceric acid (PGA) (Figure 1). This comprises the carbon fixation

step of the Calvin cycle. The products of the RubisCO reaction can be shuttled into other

metabolic pathways and used to form amino acids and other precursors for biosynthesis.

RubisCO is a relatively nonselective enzyme that can utilize both carbon dioxide and

as substrates (18).

There are four main forms of RubisCO, form I, II, III, and IV that differ

substantially in their sequences (see below; 48). Form I RubisCO is

1 present in cyanobacteria, proteobacteria, and most plastids, and can be further subdivided, based on amino acid sequences, into forms IA, IB, IC, and ID. Form II is found in some proteobacteria and , and the Form III enzyme is present in some archaea. Form IV RubisCO is widespread in bacteria and is not active as a carboxylase (49).

Figure 1. The RubisCO reaction in the Calvin-Benson-Bassham (CBB) Cycle.

Catalytically active form I RubisCO consists of eight large subunits (encoded by the cbbL gene) and eight small subunits (encoded by the cbbS gene), while form II and III consist of a single type of subunit that is homologous to form I large subunits (48). The amino acid sequences of form I large subunits and II RubisCOs are quite divergent, with only ~23% sequence similarity (34). Within the form I RubisCOs, the amino acid sequences of the large subunits of form IA and IB RubisCOs are approximately 80% similar, as are the form IC and ID RubisCOs, while the IA/IB cluster has only

2 approximately 60% sequence similarity with the IC/ID cluster (34, 48). Consistent with

these differences in sequence, each form has different specificities for CO2 and O2. Form

I RubisCOs have a higher specificity for CO2 than O2, compared to the form II enzymes

(48, 49). However, despite these differences in the primary structures of the four main

forms, the responsible for the carboxylation of CO2 and the oxygenation of O2 is conserved across the various RubisCOs (48).

Other Autotrophic Pathways

There are three other autotrophic pathways present in microorganisms in addition to the CBB cycle. In the reverse citric acid cycle (rTCA), acetyl-CoA is formed by splitting citrate that was produced by reversing the citric acid cycle so that it operates in a

carboxylating, reductive direction (20). The acetyl-CoA is reductively carboxylated to

pyruvate, which is shuttled into other central metabolic pathways (20). The three key

enzymes responsible for the reverse rotation of the oxidative citric acid cycle are ATP

citrate , 2-oxoglutarate: ferredoxin , and fumarate (20).

The acetyl-CoA pathway (AcCoA) produces acetyl-coA via sequential reduction

of carbon dioxide. This molecule is reduced to the level of a methyl group while attached

to a , followed by acetyl-CoA -mediated condensation of this methyl

group with a second carbon that has been reduced from the level of carbon dioxide to

carbon monoxide via carbon monoxide (CODH) (40). The 3-

hydroxypropionate cycle (3-HPP), which is the most recently discovered pathway for

carbon fixation, functions by carboxylating acetyl-CoA and converting it to propionyl-

CoA with 3-hydroxyproprionate as an intermediate (52). The key enzyme for this cycle

3 is malonyl-CoA reductase, which reduces malonyl-CoA, an initial product of acetyl-CoA

carboxylation, to 3-hydroxypropionate (19). Propionyl-CoA undergoes carboxylation,

forming malyl-CoA, which further divides into acetyl-CoA and glyoxylate (52).

Autotrophic pathways cannot be predicted based on the host organism’s phylogeny (Table 1). The rTCA cycle is found in a variety of autotrophic bacteria and archaea, Chlorobium sp., sulfur-reducing Crenarchaeota (Thermoproteus and

Pyrobaculum), sulfate-reducing bacteria (Desulfobacter), as well as microaerophilic and hyperthermophilic hydrogen-oxidizing bacteria like Aquifex sp. and Hydrogenobacter sp.

(20). The acetyl-CoA pathway occurs in autotrophic sulfate-reducing bacteria, methanogens, and acetogenic bacteria (11). The acetyl-CoA pathway has also been studied in Spirochaetes like Treponema primitia (14). The 3-hydroxypropionate cycle operates in the green nonsulfur bacterium, Chloroflexus aurantiacus and autotrophic

Crenarchaeota (19).

The autotrophic pathways presented in Table 1 differ in two important ways: their sensitivity to oxygen (O2) and their energetic requirements. The rTCA cycle and the

acetyl-CoA pathway are sensitive to oxygen, due to the extreme oxygen sensitivity of

pyruvate: ferredoxin oxidoreductase, which is responsible for carboxylating acetyl-CoA

(rTCA and acetyl-CoA pathways), as well as CODH (acetyl-CoA pathway), which

explains why these alternative methods of fixing CO2 predominate in anaerobic

environments (4, 50). The Calvin cycle and the 3-hydroxyproprionate cycle are both less

sensitive to oxygen (Thauer, 2007).

4 Table 1. The distribution of autotrophic pathways among the domains of life. *Only divisions with autotrophic members are listed. †It has not been determined whether Treponema primitia is an autotroph. Abbreviations: Calvin-Benson-Bassham cycle = CBB, reductive citric acid cycle = rTCA, acetyl-CoA pathway = Ac-CoA, and 3- hydroxypropionate cycle = 3-HPP.

Domain Division* Pathway(s) Bacteria Proteobacteria CBB, rTCA

Cyanobacteria CBB (Eukarya - Plants) Chloroflexi 3-HPP

Chlorobi rTCA

Aquificae rTCA

Firmicutes CBB, Ac-CoA

Planctomycetes Ac-CoA

Spirochaetes Ac-CoA†

Archaea Crenarchaeota rTCA, 3-HPP

Euryarchaeota Ac-CoA, CBB?

All of the carbon fixation pathways discussed require NADPH, ferredoxin, or other intracellular electron-shuttling cofactors as electron donors, except the acetyl-CoA pathway, which uses H2 as its donor and is the only known autotrophic pathway that yields metabolic energy instead of consuming it. This is because it directly couples the removal of electrons from H2, and transfers these electrons to CO2 for the creation of

membrane potential (11). The Calvin cycle is the most energetically expensive method

of fixing carbon, due to multiple dephosphorylation events necessary to regenerate RuBP

5 from 3-PGA, followed by the rTCA cycle, and acetyl-CoA pathway (28). Autotrophs that are primarily found in low-oxygen environments often use alternatives to the CBB cycle because they demand less energy to synthesize an equivalent amount of biomass.

However, in oxic environments, the CBB cycle dominates due to its relative stability under these conditions (28).

Elucidating autotrophic pathways from contemporary and ancient environmental samples

It is interesting to resolve which pathways are in operation in contemporary and ancient environments because it gives us insight into the history of carbon cycling.

Identifying the autotrophic pathways operating in ancient environments can help clarify whether the CBB cycle has always been the dominant carbon-fixing pathway.

In order to determine which pathways are operating in a particular contemporary environment, enzyme and nucleic acid assays can be used. If sufficient biomass is present, assays for enzymes diagnostic for the different pathways can be used; e.g.

RubisCO for the CBB cycle (48), ATP citrate lyase for the rTCA cycle (20), CODH for the acetyl-CoA pathway (40), and malonyl-CoA reductase for the 3-hydroxyproprionate cycle (19). If insufficient biomass is available for enzyme assays, nucleic acid-based approaches (e.g., Southern & Northern blots; PCR) can be utilized to identify which autotrophic CO2-fixing pathways are in operation. The cbbL or cbbM genes for form I or

II RubisCO can be used as indicators of Calvin Cycle presence in a target organism; genes encoding the enzymes diagnostic for other autotrophic pathways, (see above) can be utilized as markers for the presence of those pathways.

6 However, enzyme assays and nucleic acid-based techniques do not work for fossil

samples, as these macromolecules are not present in appreciable quantities. Instead,

isotope measurements can be used, since 12C and 13C contents persist throughout

geological time. Stable carbon isotope compositions of organic compounds extracted

from fossil sediments can help identify which autotrophic pathway dominated the input

of carbon into a particular ecosystem (42, 27).

Stable carbon isotope ratios and δ13C values

Three isotopes of carbon exist: two stable isotopes (12C and 13C), and radioactive

14C. 12C is more abundant in nature than 13C, which is ~1‰ of all carbon. The relative

amounts of 12C and 13C in a sample are expressed as δ13C values, in parts per thousand

13 (‰): δ C = ({Rsample/Rstandard} – 1) x 1000, where the limestone, PeeDee Belemnite, is

the standard (31, 27), and R = 13C/12C. A more negative δ13C value indicates that there is

less 13C in the sample (it is ‘isotopically depleted’). Conversely, a more positive δ13C indicates that more 13C is present in the sample (it is ‘isotopically enriched’) (31).

13 13 δ C values vary greatly. The δ C value of atmospheric CO2 is ~-8‰, while the

δ13C of inorganic carbon dissolved in seawater is between ~2‰ and ~1‰ (41, 21, 23).

Biomass δ13C values are more isotopically depleted than these inorganic carbon sources,

due to the relative weakness of bonds to 12C, compared to 13C. Since the zero-point

energy of bonds to 13C is lower than the zero-point energy of bonds to 12C, compounds containing 12C tend to react more quickly than those containing 13C (Figure 2) (29). As a

12 13 result, autotrophs fix CO2 more rapidly than CO2 and their biomass has more negative

13 δ C values (6). Since autotroph biomass is isotopically depleted relative to source CO2,

7 so is the biomass of the heterotrophs that consume them (17).

Figure 2. Zero-point energy diagram of 12C - and 13C - carbon dioxide.

The δ13C of C3 plants that use the CBB cycle is -18-30‰; this great degree of isotope depletion relative to atmospheric CO2 is largely due to substantial fractionation by RubisCO during carbon fixation (17). The δ13C of C4 plants is -8‰ to -20‰. These values are more positive than C3 plants because the enzymes responsible for the initial fixation of carbon into C4 compounds do not fractionate to the same degree as RubisCO does (17). The δ13C value of marine photoautotrophs such as algae is between -18 and -

28‰, but is generally more toward the isotopically enriched end of this range (13). They can be isotopically enriched due to a variety of factors, including carbon-concentrating mechanisms (CCMs) and diffusive limitation (DL) (22).

In the case of both CCMs and DL, the isotopic enrichment of intracellular biomass is due to isotopic enrichment of intracellular CO2. This isotopic enrichment of intracellular CO2 is due to low rate of CO2 efflux from the cells, compared to the rate of

8 influx and fixation. In the case of a cell with a CCM, the rate of influx is high, due to the

activities of multiple bicarbonate transporters. Intracellular bicarbonate is converted to

CO2 by . The majority of CO2 is fixed by RubisCO. The CO2 remaining is isotopically enriched. Cells with CCMs typically have mechanisms to prevent the efflux of this pool of intracellular CO2, which prevents the

from CO2 fractionation by RubisCO from being wiped away by rapid exchange of

intracellular CO2 with extracellular dissolved inorganic carbon (22). Likewise, cells experiencing DL have a very low rate of CO2 efflux, since diffusive limitation of CO2

supply to the cell results in extraordinarily low intracellular concentrations of CO2 (22).

The differences in δ13C values among C3, C4, and marine organisms make it possible to

trace carbon through food webs (17), drawing on the adage that you are what you eat.

The 13C-content of fossil organic carbon has been used as evidence for biological

carbon fixation. In some fossil samples, both organic and inorganic carbon are preserved,

providing a means to measure isotope discrimination between inorganic (δ13C = ~2‰)

and organic carbon (δ13C = -25‰), billions of years ago (42). The level of isotopic

discrimination between organic and inorganic carbon in these samples cannot be

achieved by a/biological processes, it is only possible via enzyme discrimination during

autotrophic carbon fixation (42). Based on the limited data available from cultures, the

level of isotope discrimination observed in these fossil biomass samples is consistent with

the CBB cycle and acetyl-CoA pathway. However, hypotheses about which autotrophic

pathways may be operating are weakened by limited sampling; it is still not possible to

predict, with confidence, the δ13C values expected for each pathway.

9 Enzymatic isotope discrimination

The ε value is a measure of isotope discrimination by an enzyme; in this case,

RubisCO. Biomass δ13C values from organisms using the CBB cycle are almost always more negative than source CO2 (39), but different RubisCO enzymes fractionate to

varying degrees due to slight differences in the structure of their active site (15). Isotope

fractionation is described as a discrimination factor (ε; = {[12k/13k] –1} X 1000, where 12k

and 13k = the rates of 12C and 13C fixation (15). Epsilon values (ε) for enzymes are

calculated by measuring the change in the isotopic composition of the pool as

an enzyme reaction progresses. More isotopically selective enzymes will leave behind

13 relatively more CO2, while less isotopically selective enzymes (with smaller ε values)

13 will leave behind less CO2.

For RubisCO enzymes, the isotopic composition of dissolved inorganic carbon

(DIC) is monitored; changes in the δ13C of DIC can be described using the Rayleigh

1/α –1 distillation equation: (R/R1) = (C/C1) , where R is the isotope ratio of the DIC, C is the

DIC concentration, and both R1 and C1 represent the corresponding quantities present at

the beginning of the experiment (45). The α is Rr/Rp; Rr is the isotope ratio of available

12 13 reactant, and Rp is the isotope ratio of the product, and is equal to k/ k (45). ε values

should affect the δ13C values of autotroph biomass. Large ε values should result in more negative biomass δ13C values, while small ε values should result in more positive

13 13 biomass δ C values, because a less isotopically selective enzyme should fix more CO2.

The ε values for forms IA, IB, and II RubisCO enzymes are currently known (ε = 18-

29‰). However, the ε values for forms IC and ID RubisCOs have not been measured,

10 which makes it impossible to predict the range of δ13C values expected for organisms using the CBB cycle, which really limits the interpretation of the δ13C values from contemporary and ancient samples.

The purpose of this research is to determine ε values for the form IC RubisCO enzymes. Form IC RubisCOs have not been explored yet and knowing their ε values will impact the fields of microbial ecology and biogeochemistry because it will help to constrain the range of δ13C values expected for organisms using the CBB cycle.

11

Chapter Two

Isotope Discrimination by Form IC RubisCO

It is of interest to ascertain which carbon-fixing pathways are operating in contemporary and ancient microbially-dominated habitats, as the pathways differ in energetic expense and cofactor requirements (11), which in turn influence the ecology of the organisms. It is not possible to collect biochemical and/or nucleic acid-based evidence for pathway presence in the case of fossil biomass samples. Variations in the relative amounts of 12C and 13C in autotrophic microbial biomass, expressed as δ13C

13 12 values (= {[Rsample/Rstandard] – 1} X 1000; Rsample = C/ Csample, Rstandard =

13 12 C/ CPeeDeeBelemnite), can be used to gather information about the metabolic pathway(s)

used by these organisms. For autotrophs, δ13C values can also be used to ascertain the

rate of CO2 exchange between the cell and the environment, to determine the source of

the CO2, and to elucidate environmental factors influencing carbon fixation (17). Indeed,

the broad range of δ13C values collected for autotrophic microorganisms (δ13C = -8‰ to -

35‰) has been utilized as evidence for the interplay of these factors. However, in order

for the influence of the above factors to be rigorously evaluated, the baseline

fractionation by the carboxylase(s) responsible for carbon fixation must be measured (8,

11, 13, 17, 33, 43).

12 Autotrophs that utilize the Calvin-Benson-Bassham cycle (CBB autotrophs), have

a diversity of RubisCO (ribulose 1,5-bisphosphate carboxylase/oxygenase) enzymes that

catalyze the carboxylation of ribulose 1,5-bisphosphate (RuBP) to form two molecules of

phosphoglyceric acid (PGA; 47). RubisCO exists in six different forms that are

catalytically active as carboxylases (IA, IB, IC, ID, II, and III; Figure 3). Form I

RubisCO is present in cyanobacteria (IA, IB), some proteobacteria (IA, IC), most

(IB, ID) (48), and the firmicute Sulfobacillus acidophilus (IC/ID) (3).

Catalytically active form I RubisCO consists of eight large subunits (encoded by the cbbL

gene) and eight small subunits (encoded by the cbbS gene). Form II is found in

proteobacteria and some dinoflagellates, and form III is present in some archaea (48).

Both form II and III enzymes consist of a single type of subunit, evolutionarily related to

the large subunits of form I RubisCO (48).

Given the divergent forms of RubisCO, it is not surprising that RubisCO enzymes

13 12 13 discriminate against CO2 to different degrees. Isotope discrimination (ε ={[ k/ k] – 1}

X 1000; 12k and 13k = rates of 12C and 13C fixation) (15) has been measured in a limited

number of form IA, IB, and II enzymes, and ranges from 18 to 29‰ (44, 15, 36, 37, 38,

13 46). Since the ε value is roughly equal to the difference between the δ C of the CO2

13 source from which the RubisCO is drawing (intracellular CO2) and the δ C of the CO2

that it fixes (15, 44), heterogeneity in RubisCO ε values is likely to be responsible for at least 10‰ of the δ13C scatter observed in CBB autotrophs. Prior to this study, it was

impossible to predict whether form IC RubisCO enzymes would have ε values similar to

those measured for form IA, IB, & II RubisCOs.

13 Our long-term objective is to sample the full phylogenetic breadth of RubisCO enzymes and be able to constrain the δ13C values expected for CBB autotrophs from

ancient and contemporary ecosystems. Isotope discrimination by form IC RubisCO has

not been measured, despite the presence of this enzyme in many proteobacteria of

ecological interest, including marine manganese-oxidizing bacteria (5, 32), some

nitrifying and nitrogen-fixing bacteria, and soil microorganisms like the extremely

metabolically versatile bacterium, Rhodobacter sphaeroides (48; Fig.3).

Rhodobacter sphaeroides is an α-proteobacterium capable of nonoxygenic

photolithoautotrophic growth and photoheterotrophic growth (35). This organism has

two RubisCO enzymes: a form IC RubisCO and a form II RubisCO (12). In this

organism, the two forms of RubisCO are differentially expressed in response to a variety

of growth conditions, e.g. CO2 concentration (10). In order to be able to detect the

isotope signature of organisms using form IC RubisCO for carbon fixation, we measured

the ε value for R. sphaeroides RubisCO using the high-precision substrate depletion

method (15, 36, 43).

14

Rhodobacter sphaeroidesATCC17025 98 Stappia aggregataIAM12614 58 45 Paracoccus denitrificansPD1222 Xanthobacter autotrophicusPy2 99 100 Xanthobacter autotrophicus Methylibium petroleiphilumPM1 57 87 Ralstonia eutrophaH16 100 Ralstonia eutropha Burkholderia xenovoransLB400 100 Burkholderia phymatumSTM815 100 Bradyrhizobium japonicumUSDA110 IC 57 Bradyrhizobium japonicum

38 31 Oligotropha carboxidovorans Bradyrhizobium spBTAi1 92 20 Nitrobacter hamburgensisX14 Rhodopseudomonas palustrisCGA009 77 56 Rhodopseudomonas palustrisBisB18

98 54 Aurantimonas spSI859A1 100 Mnoxidizing bacteriumSI859A1

92 Roseovarius spHTCC2601 Acidiphilium cryptumJF5 Nitrosospira sp40KI

64 Emiliania huxleyi 99 59 Porphyridium aerugineum

74 Phaeodactylum tricornutum 100 Cylindrotheca sp ID 100 Thalassiosira pseudonana 99 Thalassiosira nordenskioeldii 100 Sulfobacillus acidophilus IC/ID 100 Nitrobacter winogradskyi 98 spENI11 Solemya velum 100 100 Prochlorococcus marinus MIT 9313 IA sp WH 8102 70 Chromatium vinosum 100 Spinacia oleracea 72 Trichodesmium erythraeum IB 63 Nostoc punctiforme 100 Synechococcus elongatus PCC 6301

57 Archaeoglobus fulgidus Methanococcus jannaschii III 99 Methanosarcina acetivorans 99 Thiobacillus denitrificans Thiomicrospira crunogena

100 Rhodobacter capsulatus II 100 Rhodobacter sphaeroides II

0.1

15 Figure 3. Minimum evolution tree of RubisCO large subunit (cbbL) nucleotide sequences. The nucleotide sequences obtained from GenBank were translated to amino acid sequences and aligned based on the amino acid sequences using BIOEDIT (16). The alignments were examined to make certain that active sites and other conserved regions were properly aligned. Nucleotide sequences were used to assemble a phylogenetic tree with MEGA 3.1 software, using the Kimura 2-parameter nucleotide model with 1000 replicates for calculating bootstrap values (24).

Experimental Procedures

Form IC enzyme was cloned from Rhodobacter sphaeroides, expressed in

Escherichia coli, and purified using standard HPLC protocols (25, 18). The purity of the

enzyme was monitored by sodium dodecyl sulfate polyacrylamide

(SDS-PAGE) and stained with coomassie blue (25).

RubisCO ε values were determined by using the substrate depletion method, in

which a buffered solution of RubisCO, carbonic anhydrase (CA), ribulose 1,5- bisphosphate (RuBP), and dissolved inorganic carbon is sealed in a gastight syringe (44).

The ε value is calculated from the changes in the δ13C value of the dissolved inorganic

carbon pool as it is consumed by RubisCO (44). RuBP was synthesized enzymatically to

minimize the concentration of inhibitory isomers present in commercially-available

RuBP (44, 46). Fresh RuBP was stored at –70°C and used within one week of synthesis.

Form IC RubisCO was prepared for the reaction by desalting it into RubisCO

buffer (25 mM MgCl2, 10 mM dithiothreitol, 50 mM Bicine, 5 mM NaHCO3, pH 7.5),

using PD-10 columns (Amersham Biosciences, NJ). 20 mL of reaction buffer (RubisCO

buffer without NaHCO3) was sparged with N2 and 1 mg of bovine carbonic anhydrase

(CA) was added. This solution was filter-sterilized and loaded into a glass gastight

syringe sealed with a septum (44). Filter-sterilized NaHCO3 (final concentration of 5

16 mM) and RubisCO (~1 mg/mL) were added to the reaction syringe and activated for 10-

15 minutes. The reaction was started by injecting fresh RuBP (~5 mM) into the syringe, and was maintained at 25°C.

Reaction progress was monitored by removing samples and injecting them into a gas chromatograph (9) to measure the dissolved inorganic carbon concentration (DIC, =

- -2 CO2 + HCO3 + CO3 ). Over the time course of the reaction, samples were removed

from the reaction syringe, acidified with 43% phosphoric acid, and injected into a

vacuum line to cryodistill the DIC (44). The cryodistilled DIC samples were sent to the

Boston University Stable Isotope Facility for measurement of the δ13C of the DIC.

ε values were derived from the DIC concentrations and the δ13C values of the DIC as calculated in Scott et al. (45). A modified version of the Rayleigh distillation equation was used:

(1/αC – 1) ln(RDIC) = {(1/αC) -1} x ln[DIC] + ln{(RDIC0/[DIC]0) } where

13 12 RDIC = C/ C of DIC at a particular timepoint

RDIC0 = RDIC at the first timepoint

[DIC] = concentration of DIC at a particular timepoint

[DIC]0 = [DIC] at the first timepoint

- C = RHCO3 /RCO2 from Mook et al., (30)

12 13 α = k /k = kinetic isotope effect for CO2

The ε values were calculated from the slope of this line (ε = (α - 1) ×1000), and data from multiple runs were averaged using Pitman Estimators (45).

17 Results

As expected, carbon fixation by form IC RubisCO of R. sphaeroides results in

isotopic enrichment of the remaining dissolved inorganic carbon (Figure 4, Figure 5).

The ε value for this enzyme is 22.9‰ with a 95% confidence interval of 21.4-24.7‰

(Table 2). Rhodobacter sphaeroides RubisCO is less isotopically selective than spinach

RubisCO (ε = 27.5‰ when incubated under identical conditions) (2).

10 6

8 5

6 4 4 C

13 3 δ 2 [DIC], mM 2 0 1 -2 -4 0

0123456 Time (hrs.)

Figure 4. Changes in the concentration (‹) and δ13C („) of dissolved inorganic carbon (DIC) vs. time for Rhodobacter sphaeroides RubisCO sealed in a sterile, gastight syringe in buffer with ribulose 1,5-bisphosphate and carbonic anhydrase (see Experimental Procedures for details).

18

-4.471

-4.479

lnR

-4.487

-4.495 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 lnDIC

Figure 5. Natural log-transformed isotope ratios (R= 13C/12C) and concentrations of dissolved inorganic carbon (DIC) during carbon fixation by form IC RubisCO. Results from nine independent experiments are shown, and each is depicted with a different symbol. The initial isotope ratio and concentration of DIC varied slightly between experiments; for clarity, data from all experiments have been normalized to have the same initial DIC concentration and isotope ratio.

Table 2. RubisCO ε values determined from nine independent experiments with one enzyme preparation of the R. sphaeroides enzyme

Experiment # ε value (‰) # of Timepoints

1 29.6 7 2 24.4 6 3 24.1 7 4 21.0 7 5 22.7 7 6 24.3 8 7 24.7 8 8 22.6 8 9 24.1 7

19 Discussion

This is the first determination of an ε value from a Form IC RubisCO, and it falls

within the range of ε values measured for other RubisCO enzymes (Table 3). Its ε value is statistically indistinguishable from form IA enzymes, form IB from the cyanobacterium

Anacystis nidulans, as well as from form II RubisCO from .

However, R. sphaeroides RubisCO has an ε value significantly higher than that of the

form II RubisCO from the gammaproteobacterial endosymbiont of the hydrothermal vent

tubeworm, , and significantly smaller than the ε value of spinach

RubisCO (Table 3).

Table 3. ε values from form I and II RubisCO enzymes

Species Form of RubisCO ε value (‰) {95% C.I.} Solemya velum symbiont IA 24.5

Prochlorococcus marinus MIT 9313 IA 24.0

Spinacia oleracea IB 29

Anacystis nidulans IB 22

Rhodobacter sphaeroides IC 22.9 {21.4-24.7} Riftia pachyptila symbiont II 19.5

Rhodospirillum rubrum II 22

The δ13C value of R. sphaeroides biomass, when it fixes carbon with form IC

RubisCO, can be predicted from the RubisCO ε value. Organisms that fix carbon with IC

enzymes, should they all fractionate similarly to the R. sphaeroides enzyme, are likely to

20 have biomass δ13C values similar to other CBB autotrophs whose RubisCO ε values have

13 been determined. When an organisms’ whole-cell isotopic discrimination (δ CCO2 -

13 δ Cbiomass) is plotted against its RubisCO ε value, it is apparent that organisms fixing

carbon via RubisCOs with larger ε values fractionate carbon to a greater extent (Table 4,

Figure 6). Since the R. sphaeroides RubisCO has an ε value similar to the enzyme from

R. rubrum, it is likely that whole-cell discrimination by R. sphaeroides is comparable to

that observed in R. rubrum when grown autotrophically. Indeed, biomass δ13C values for

R. sphaeroides can be predicted from the R. rubrum and R. sphaeroides ε values, as well

as R. rubrum whole-cell isotope discrimination:

εR rubrum = 22‰

εR sphaeroides = 22.9‰

13 13 For R. rubrum, δ CCO2 - δ Cbiomass = 12.3‰ Since εR sphaeroides - εR rubrum = 0.9‰, 13 13 For R. sphaeroides, predicted εbiomass = δ CCO2 - δ Cbiomass ≈12.3 + 0.9 = 13.2‰

13 If δ CCO2 ≈ -8‰, then 13 δ CR.sphaeroides ≈ -8‰ - 13.2‰ = -21.2‰

21 13 13 Table 4. δ CCO2 and δ Cbiomass values from autotrophic organisms whose RubisCO ε values have been determined

13 13 Species δ CCO2 (‰) δ Cbiomass (‰)

Riftia pachyptila symbiont -6.6 to -7.8 -9 to -16

Solemya velum symbiont -14 to -18 -30 to -35

C3 plants (Spinach) -8 -22 to -30

Rhodospirillum rubrum -11.3 -23.6

13 13 Figure 6. Whole-organism isotopic discrimination (δ CCO2 - δ Cbiomass) versus enzymatic isotopic discrimination (RubisCO ε values). The predicted εbiomass value for Rhodobacter sphaeroides assumes whole-cell fractionation similar to R. rubrum (see Discussion for details), and the predicted range (whiskers on graph) assumes changes in whole-organism isotopic discrimination similar to what has been observed in other organisms in response to changes in CO2 supply and/or demand.

22 A biomass δ13C value of -21.2‰ would place R. sphaeroides and other form IC autotrophs within the range of δ13C values observed for other CBB autotrophs, but would allow them to be distinguished from organisms using alternative carbon fixation pathways. Their δ13C values should be more negative than what is expected for organisms using the rTCA or 3-hydroxypropionate cycles for carbon fixation, but not the acetyl-CoA pathway (17; Figure 7).

13 Figure 7. δ C values of atmospheric CO2 and biomass from autotrophic organisms using the 3-hydroxyproprionate pathway (3-HPP), the reverse citric acid cycle (rTCA), the acetyl-CoA pathway, and the C3-Calvin Benson Bassham cycle (C3-CBB).

Future work with Form IC RubisCO will involve sampling the full phylogenetic breadth of Form IC RubisCOs to determine whether all Form ICs have ε values that fall

within the range observed for other RubisCO ε values. In order to sample the full

phylogenetic spectrum of this group, ε values could be measured for RubisCOs from, for

example, Burkholderia xenovorans LB400 and Roseovarius sp. HTCC 2601, as these two

enzymes are present in IC clades distinct from the clade containing R. sphaeroides

23 RubisCO (Figure 3). B. xenovorans LB400 is an aerobic soil microbe capable of degrading polychlorinated biphenyls (7). Burkholderia sp. have a great number of carbon genes that indicate the presence of different pathways for assimilating carbon, giving this strain an edge, ecologically (7). Roseovarius sp. HTCC 2601 is an aerobic, alphaproteobacterial isolate found in seawater collected from the Sargasso Sea, and is a member of the Roseobacter clade, which is important in the marine sulfur cycle

(26, 51). This clade is responsible for degrading dimethylsulfoniopropionate (DMSP) to methanethiol (51).

It would also be of great interest to measure the ε value of RubisCO from the firmicute, Sulfobacillus acidophilus. S. acidophilus is most peculiar because it is an

outlier from the Form IC and ID clades (Figure 3). Sulfobacillus species oxidize mineral sulfides and prefer environments with a high concentration of CO2, and their use of this

RubisCO substrate reveals much about the biogeochemical function of these acidophiles

(3). Elucidating the degree of isotopic discrimination by form IC enzymes, broadly

sampled, would make it possible to use stable carbon isotope analyses to learn more

about the role form IC RubisCOs play in the environment and the global .

24

Chapter Three

Conclusion

Rhodobacter sphaeroides IC RubisCO has an ε value of 22.9‰, which is within

the range measured for other RubisCOs. This implies that some CBB autotrophs using

form IC enzymes can be predicted to have δ13C values similar to those previously

measured in CBB autotrophs. Preliminary experiments have been conducted with form

IC RubisCO from Ralstonia eutropha, and this enzyme appears to have an ε value of

26.6‰ (see Appendix table A-1), though replicate experiments are necessary to verify

this measurement.

Experiments with Form ID RubisCO

ε values have also recently been collected for form ID RubisCOs from the phytoplankton species, Skeletonema costatum and Emiliania huxleyi. The ID RubisCOs have ε values that are lower than the ε value from R. sphaeroides RubisCO (ε = 11.1‰

for E. huxleyi; ε = 18.6‰ for S. costatum; see appendix). The diatom, S. costatum, and

, E. huxleyi, are a major part of the phytoplankton community and

contribute substantially to primary productivity in the oceans. The small ε values reveal

13 that their ID RubisCOs are less isotopically selective for CO2 compared to other

RubisCO enzymes. The ε values also provide a mechanism for the levels of isotopic

enrichment observed in these organisms, and marine organic carbon in general.

25

It is apparent that IC/ID RubisCOs have a broad range of ε values (11.1‰ to

22.9‰). Currently, it is impossible to predict ε values for RubisCO enzymes based on primary, secondary, tertiary, or quaternary structure. At this point, one must measure them, in the hope that at some point it will be possible to correlate ε values with primary structure or other features of these enzymes.

26

References

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29 40. Russell, M. J. and Martin, W. 2004. The rocky roots of the acetyl-CoA pathway. Trends in Biochemical Sciences 29(7):358-363. 41. Saurer, M., Siegwolf, R., Borella, S., and Schweingruber, F. 1998. Environmental information from stable isotopes in tree rings of Fagus sylvatica. In Beniston, M. and Innes, J. L. (eds). The Impacts of Climate Variability on Forests. Germany, Springer Press Berlin. pp.241–254. 42. Schidlowski, M., Hayes, J. M., and Kaplan, I. R. 1983. Isotopic inferences of ancient biogeochemistries: carbon, sulfur, hydrogen, and nitrogen. In Schopf, J. W. (ed). Earth’s Earliest : Its Origin and Evolution: Princeton, New Jersey, Princeton University Press. pp.149–186. 43. Scott, K. M. 2003. A δ13C-based carbon flux model for the hydrothermal vent chemoautotrophic symbiosis Riftia pachyptila predicts sizeable CO2 gradients at the host-symbiont interface. Environmental Microbiology 5(5):424-432. 44. Scott, K. M., Schwedock, J., Schrag, D. P., and Cavanaugh, C. M. 2004a. Influence of form IA RubisCO and environmental dissolved inorganic carbon on the δ13C of the clam-chemoautotroph symbiosis Solemya velum. Environmental Microbiology 6(12):1210-1219. 45. Scott, K. M., Lu, X., Cavanaugh, C. M., and Liu, J. 2004b. Optimal methods for estimating kinetic isotope effects from different forms of the Rayleigh distillation equation. Geochimica et Cosmochimica Acta 68(3):433-442. 46. Scott, K. M., Henn-Sax, M., Harmer, T. L., Longo, D. L., Frame, C. H., and Cavanaugh, C. M. 2007. Kinetic isotope effect and biochemical characterization of form IA RubisCO from the marine cyanobacterium Procholorococcus marinus MIT 9313. The American Society of Limnology and Oceanography 52(5):2199- 2204. 47. Tabita, F. R. 1988. Molecular and cellular regulation of autotrophic carbon dioxide fixation in microorganisms. Microbiological Reviews 52(2):155-189. 48. Tabita, F. R. 1999. Microbial ribulose 1,5-bisphosphate carboxylase/oxygenase: A different perspective. Photosynthesis Research 60:1-28. 49. Tabita, F. R., Satagopan, S., Hanson, T. E., Kreel, N. E., and Scott, S. S. 2008. Distinct form I, II, III, and IV Rubisco proteins from the three kingdoms of life provide clues about Rubisco evolution and structure/function relationships. Journal of Experimental Botany 59(7):1515-1524. 50. Thauer, R. K. 2007. A fifth pathway of carbon fixation. Science 318:1732-1733. 51. Tripp, H. J., Kitner, J. B., Schwalbach, M. S., Dacey, J. W. H., Wilhelm, L. J., and Giovannoni, S. J. 2008. SAR11 marine bacteria require exogenous reduced sulphur for growth. Nature 452:741-744. 52. van der Meer, M. T. J., Schouten, S., van Dongen, B. E., Rijpstra, W. I. C., Fuchs, G., Damsté, J. S. S., de Leeuw, J. W., and Ward, D. M. 2001. Biosynthetic controls on the 13C contents of organic components in the photoautotrophic bacterium Chloroflexus aurantiacus. The Journal of Biological Chemistry 276(14):10971-10976.

30

Appendices

31

Appendix A: Table of RubisCO ε values from Ralstonia eutropha

Table A-1. RubisCO ε values determined from two independent experiments with the IC enzyme from R. eutropha

Experiment # ε value (‰) # of Timepoints

1 27.1 7

2 26.2 8

Average ε value = 26.6 ‰

32

Appendix B: Manuscript on Form ID RubisCO ε value from Emiliania huxleyi

A New Low for RubisCO

(To be submitted to Science)

Amanda J. Boller1, Phaedra J. Thomas1, Colleen M. Cavanaugh2, and Kathleen M. Scott1*

1Biology Department, University of South Florida, Tampa, Florida 33620, USA. 2Department of Organismic and Evolutionary Biology, Harvard University, Cambridge Massachussetts 02138, USA.

*To whom correspondence should be addressed. E-mail: [email protected]

One-sentence summary

The first high-precision measurement of stable carbon isotopic discrimination by form ID RubisCO, responsible for a substantial portion of marine carbon fixation, is reported here and indicates that this enzyme has a shockingly small fractionation factor, providing a mechanistic explanation for 13C-enrichment in marine organic carbon.

33 Appendix B (Continued)

ABSTRACT

The 13C content of carbon is used to identify sources and sinks in the global

carbon cycle. One enduring mystery is why the 13C content of marine organic carbon is

relatively high. We tested the hypothesis that marine organic carbon 13C enrichment is due to reduced isotopic discrimination during carbon fixation by form ID RubisCOs

(ribulose 1,5-bisphosphate carboxylase/oxygenase), found in a substantial portion of marine algae responsible for oceanic carbon fixation. Here, form ID RubisCO from

13 coccolithophore Emiliania huxleyi discriminated substantially less against CO2 than other RubisCO enzymes (ε=11.1‰). Reduced discrimination by form ID RubisCO may be a major factor dictating the high 13C content of marine organic carbon, necessitating re-evaluation of how biological δ13C values are integrated into global primary

productivity models.

34 Appendix B (Continued)

Stable carbon isotope analyses are critical to the identification and modeling of

global carbon cycle sources and sinks. One key sink is marine carbon fixation (1), and

13 13 13 12 phytoplankton δ C values (δ C = [(Rsample/Rstd)-1] x1000, where R= C/ C) have been

used to untangle trophic links and to estimate in situ growth rates (2). However,

phytoplankton δ13C values vary widely (-16 to -36‰; (2), and the factors responsible for

this variation, as well as their relatively 13C-enriched δ13C values compared to terrestrial

C3 plants (3), are poorly understood. Given the substantial role that phytoplankton play

in the global carbon cycle, this conceptual gap compromises not only the interpretation of

phytoplankton δ13C values, but also introduces uncertainty into carbon cycle modeling

(4).

While some variation in phytoplankton δ13C values is clearly due to carbon concentrating mechanisms, C4 pathways, and diffusive limitation (5), the effect of

isotopic discrimination by different forms of RubisCO (ribulose 1,5-bisphosphate

carboxylase/oxygenase), the CO2 fixing enzyme of the Calvin-Benson-Bassham cycle,

has not been widely considered. Instead, δ13C analyses of oceanic primary productivity

typically assume that isotopic discrimination by phytoplankton RubisCO is similar in

extent to that of spinach RubisCO (e.g., (6-10). This assumption is untenable, however,

as prior studies have shown that different forms of RubisCO discriminate to a greater or lesser extent against 13C (4, 11-16). There are three known forms of RubisCO (I, II, and

III), which share as little as 25% in amino acid sequence identity, vary widely in KCO2

35 Appendix B (Continued)

and Vmax values, and display dramatic differences in tertiary and quaternary structure (17,

18). Form I enzymes are further subdivided into four subforms (IA – ID), whose amino acid sequences can differ by as much as 40% (17, 18). Marine algae fix carbon using at least four different RubisCO forms: IA in marine Synechococcus and Prochlorococcus spp., IB in algae with green plastids (and terrestrial plants), ID in ‘non-green’ algae

(, diatoms, rhodophytes, and some dinoflagellates), and II in peridinin- containing dinoflagellates (19).

Isotopic discrimination, expressed as ε values (ε = (RCO2/Rfixed – 1) x 1000), have

been measured for only three forms of RubisCO using high-precision methods (Table B-

1). While values vary considerably, RubisCO forms IA and B (ε = 22 - 29‰)

discriminate to a greater extent than form II (ε = 18 – 22‰) (4, 11-16). Given the

prevalence of form ID RubisCO in dominant marine primary producers, many of which

are used as model organisms for study in culture, determination of isotope discrimination

by this enzyme is critical for the interpretation of environmental and culture δ13C values,

and in turn for global modeling efforts, paleo-oceanography, and other studies using δ13C

values. Here, we characterized and determined the ε value of form ID RubisCO from a model marine alga, the coccolithophore Emiliania huxleyi CCMP 374.

Coccolithophores, whose massive blooms in the North Atlantic and Pacific

oceans are visible from space (20, 21), fix CO2 via form ID RubisCO (22) and play a

major role as primary producers and in jettisoning carbon to the ocean floor (23). The

36 Appendix B (Continued)

minute calcium carbonate plates (coccoliths) that cover coccolithophore cells enhance

inorganic and organic carbon export from surface waters by ballasting fecal pellets into

which they are packed (24, 25). In culture, the biomass δ13C values of Emiliania huxleyi vary widely, from -9.71 to -38.6‰ (6, 7, 10, 26, 27). While some of this heterogeneity is likely due to variation in study strains and growth conditions, these δ13C values cannot be

rigorously interpreted without a RubisCO ε value.

Table B-1. ε values of different RubisCO forms, *ε = RCO2/Rfixed – 1) x 1000

Form Organisms ε value (‰)* Reference

IA Marine cyanobacteria 24 – 22.4 (4, 16) and gammaproteobacteria IB Terrestrial plants and 21 – 30.3 (11, 13, 15) freshwater cyanobacteria ID Marine coccolithophore 11.1 This study Emiliana huxleyi II Alpha- and 17.8 – 23.0 (12-15) gammaproteobacteria

RubisCO KCO2 and ε values

In order to obtain sufficient Emiliana huxleyi Form ID RubisCO for determination of kinetic parameters and isotope fractionation (~20 mg per ε value experiment), 100 L of

E. huxleyi CCMP 374 cultures were harvested and RubisCO was partially purified from

sonicated cells via NH4SO4 precipitation (28). Carboxylase activity was verified to be

37 Appendix B (Continued)

due exclusively to RubisCO based on incubations in the presence and absence of the

substrate ribulose 1,5-bisphosphate (RuBP; (28). To characterize E. huxleyi RubisCO

activity, its Michaelis-Menten constants (KCO2 and Vmax) were measured radiometrically

as in (29). The ε value of E. huxleyi RubisCO was measured by the high-precision

substrate depletion method, in which the concentration and isotopic composition of

dissolved inorganic carbon are measured as they are consumed by RubisCO (4, 16, 28).

The ε value was calculated using a modified version of the Rayleigh distillation equation

and Pitman estimators to calculate the least-biased average (28, 30).

E. huxleyi RubisCO Michaelis-Menten constants (KCO2 = 111 +/- 40 μM; Vmax =

146 +/- 102 nmol/min×mg, calculated from five independent experiments) were quite

different than those measured for other form ID enzymes from diatoms and rhodophytes

(KCO2 = 5 – 60 μM; Vmax = 670 – 1670 nmol/min×mg; (31, 32). The larger KCO2 value

for the Emiliania huxleyi RubisCO may necessitate a carbon concentrating mechanism

with active transport of dissolved inorganic carbon into the cells, since the concentration

of CO2 in seawater is only ~20 μM (33). The lower Vmax value reflects the labile nature

of this enzyme.

E. huxleyi form ID RubisCO had an astonishingly low ε value of 11.1‰ (95% CI:

9.8 - 12.6‰; Figure B-1), substantially smaller than any other RubisCO ε measured to date (Table B-1). ε values from replicate experiments were very consistent, falling within a 3.1‰ range (Figure B-1). Spinach form IB RubisCO, used as a control and

38 Appendix B (Continued)

incubated under identical conditions to those used for the E. huxleyi enzyme, had an

ε value of 27.5‰ (95% CI: 24.0 – 30.9‰; Figure B-1), similar to previously reported

values (4, 11, 13). This is the first high-precision ε value to be measured for any eukaryotic algae of ecosystem-level importance; the peculiarity of its ε value highlights how little we know about the RubisCO enzymes responsible for marine primary productivity.

Figure B-1. Isotope fractionation by E. huxleyi RubisCO. R is the isotope ratio (13C/12C) of dissolved inorganic carbon (DIC) and [DIC] is its concentration. Solid symbols (▲, ♦, ■, and ●) correspond to independent incubations of E. huxleyi RubisCO, with ε = 10.8‰, 11.1‰, 11.8‰, and 13.9‰, respectively. Open symbols (□, ○, and ∆) correspond to independent incubations of spinach RubisCO, with ε = 28.3‰, 28.2‰, and 27.2‰, respectively.

39 Appendix B (Continued)

Implications for interpreting δ13C values from environmental samples and

phytoplankton cultures

This remarkably small RubisCO ε value (11.1‰) suggests a novel explanation for the isotopically enriched δ13C values typically observed for marine phytoplankton and

the food webs they support. An enzymatic basis for these values must now be

considered. Indeed, phytoplankton collected from environmental samples demonstrate

whole-cell isotope discrimination values (εp, = [RCO2/Rbiomass – 1] × 1000) that span this

RubisCO ε value. Samples collected from marine environments worldwide have values of εp ranging from 7 to 19‰ (7, 34, 35). For environmental samples dominated by E.

huxleyi, reported εp values are 0-14‰ (calculated directly from biomass; (36) and 7-19‰

(calculated from E. huxleyi alkanones, assuming a constant fractionation between

alkanones and biomass; (7). Using the (incorrect) assumption of a RubisCO ε value of

29‰, these εp values suggest the isotope-enriching effects of carbon concentrating

mechanisms or C4 pathways. However, given the small RubisCO ε value measured here,

it is not necessary to invoke these mechanisms to explain 13C-enriched biomass.

Similar to ocean samples, εp values from E. huxleyi culture studies are small and

reasonably consistent with the measured RubisCO ε value. In many of these studies, the

13 13 13 δ C of dissolved inorganic carbon (δ CDIC), and not the δ CCO2, is reported. Therefore,

to calculate εp from these culture studies, the equilibrium isotope effect between

- dissolved CO2 and HCO3 (=εbc, = 1000 ×[{RCO2/RHCO3-}- 1]; = -10‰ at 15°C; (37) was

40 Appendix B (Continued)

13 13 used to calculate the δ CCO2 from δ CDIC. Based on these studies, εp ranges from 2-

18‰ for Emiliania huxleyi (6, 10, 38, 39). One potential factor that could be influencing the εp value is the form of inorganic carbon transported, since there is an equilibrium

- isotope effect between HCO3 and CO2, which causes CO2 to be isotopically depleted

- relative to HCO3 (37). It is clear that E. huxleyi alters its pattern of inorganic carbon

uptake in response to growth conditions (40). Perhaps smaller εp values are the result of

- reliance on extracellular HCO3 , while larger εp values may result from CO2 utilization.

Deciphering the form(s) of dissolved inorganic carbon taken up by this organism under

different growth conditions is instrumental in developing a mechanistic understanding for

the changes in dissolved inorganic carbon abundance and composition that accompany E.

huxleyi blooms, which in turn determine whether these blooms are sources or sinks of

atmospheric and dissolved CO2.

The unexpectedly low ε value of E. huxleyi RubisCO highlights the necessity of collecting RubisCO ε values from other organisms, in order to uncover any phylogenetic patterns in isotope discrimination. At this point, given the small numbers of enzymes examined (2 form IA’s; 2 form IB’s; 1 form ID; 2 form II’s) it is irresponsible to suggest

‘typical’ ε values for different forms of RubisCO, or even for RubisCO enzymes in general. Particularly with respect to the interpretation of marine δ13C values, and to

refine models of the global carbon cycle that rely on these δ13C values, it is necessary to

determine whether form ID RubisCO enzymes from other algae of ecosystem-level

41 Appendix B (Continued)

importance (e.g., diatoms, rhodophytes) have low ε values. Factors such as carbon concentrating mechanisms, inorganic carbon supply and demand, and C4 pathways

clearly exert an influence on the δ13C values of algal biomass (38, 41-43), but more

measurements of RubisCO ε values are critical to discern their significance.

42 Appendix B (Continued)

References

1. W. H. Schlesinger. 1997. in Biogeochemistry: An analysis of global change W. H. Schlesinger, Ed. (Academic Press, San Diego) pp. 291-341. 2. K. H. Freeman. 2001. in Stable Isotope Geochemistry J. W. Valley, D. R. Cole, Eds. (Mineralogical Society of America, Washington, DC) pp. 579-605. 3. R. H. Michener, D.M. Schell. 1994. in Stable isotopes in ecology and environmental science K. Lajtha, R.H. Michener, Ed. (Blackwell Scientific Publications, Cambridge, MA) pp. 138-157. 4. K. M. Scott, J. Schwedock, D. P. Schrag, C. M. Cavanaugh. 2004. Environ Microbiol 6:1210. 5. R. Goericke, J. P. Montoya, B. Fry. 1994. in Stable Isotopes in Ecology and Environmental Science K. Lajtha, R. H. Michener, Eds. (Blackwell Scientific Publications, Boston) pp. 187-221. 6. P. A. Thompson, S. E. Calvert. 1995. Limnol Oceanogr 40:673. 7. R. R. Bidigare et al. 1997. Global biogeochemical cycles 11:279. 8. E. A. Laws, P. A. Thompson, B. N. Popp, R. R. Bidigare. 1998. Limnology and oceanography 43:136. 9. E. A. Laws, B. N. Popp, N. Cassar, J. Tanimoto. 2002. Functional Plant Biology 29:323. 10. B. Rost, I. Zondervan, U. Riebesell. 2002. Limnol Oceanogr 47:120. 11. C. A. Roeske, M. H. O'Leary. 1984. Biochemistry 23:6275. 12. C. A. Roeske, M. H. O'Leary. 1985. Biochemistry 24:1603. 13. R. D. Guy, M. L. Fogel, J. A. Berry. 1993. Plant Physiol 101:37. 14. J. J. Robinson et al. 2003. Limnol Oceanogr 48:48. 15. D. B. McNevin, M. R. Badger, H. J. Kane, G. D. Farquhar. 2006. Funct. Plant Biol 33: 1115. 16. K. M. Scott, M. Henn-Sax, D. Longo, C. M. Cavanaugh. 2007. Limnol Oceanogr 52:2199. 17. G. M. Watson, F. R. Tabita. 1997. FEMS Microbiol Lett 146:13. 18. F. R. Tabita et al. 2007. Microbiol and Molec Biol Rev 71:576. 19. F. R. Tabita. 1999. Photosynth Res 60:1. 20. Y. Dandonneau, Y. Montel, J. Blanchot, J. Giraudeau, J. Neveux. 2006. Deep-Sea Res. I 53:689. 21. H. Siegel, T. Ohde, M. Gerth, G. Lavik, T. Leipe. 2007. Continental Shelf Res. 27:258. 22. M. V. S. Puerta, T. R. Bachvaroff, C. F. Delwiche. 2005. DNA Research 12:151. 23. E. Paasche. 2002. Phycologia 40:503. 24. S. Honjo. 1976. Mar. Micropaleontol. 1:65. 25. P. Ziveri, B. deBernardi, K. Baumann, H. M. Stoll, P. G. Mortyn. 2007. Deep Sea Res. II 54:659.

43 Appendix B (Continued)

26. A. M. Johnston. 1996. Mar. Ecol. Progr. Ser. 132:257. 27. U. Reibesell, A. T. Revill, D. G. Holdsworth, J. K. Volkman. 2000, Geochim Cosmochim Acta 64:4179. 28. Materials and methods are available as supporting material on Science Online. 29. J. Schwedock et al. 2004. Arch Microbiol 182:18. 30. K. M. Scott, X. Lu, C. M. Cavanaugh, J. Liu. 2004. Geochim Cosmochim Acta 68:433. 31. B. A. Read, F. R. Tabita. 1992. Biochemistry 31:519. 32. K. Horken, F. R. Tabita. 1999. Archives of Biochemistry and Biophysics 361:183. 33. R. E. Zeebe, D. Wolf-Gladrow. 2003. CO2 in seawater: Equilibrium, kinetics, isotopes. D. Halpern, Ed., Elsevier Oceanography Series (Elsevier, New York) pp. 346. 34. I. Bentaleb et al. 1998. Journal of Marine Systems 17:39. 35. H. Kukert, U. Riebesell. 1998. Mar Ecol Prog Ser 173:127. 36. A. Benthien et al. 2007. Geochimica et cosmochimica acta 71:1528. 37. W. G. Mook, J. C. Bommerson, W. H. Staverman. 1974. Earth Plan Sci Let 22:169. 38. K. R. Hinga, M. A. Arthur, M. E. Q. Pilson, D. Whitaker. 1994. Global biogeochemical cycles 8:91. 39. U. Riebesell, A. T. Revill, D. G. Holdsworth, J. K. Volkman. 2000. Geochim Cosmochim Acta 24:4179. 40. B. Rost, U. Riebesell, D. Sultemeyer. 2006. Limnol Oceanogr 51:12. 41. E. A. Laws, R. R. Bidigare, B. N. Popp. 1997. Limnol Oceanogr 42:1552. 42. A. S. Fielding et al. 1998. Can J Bot 76:1098. 43. J. R. Reinfelder, A. M. L. Kraepiel, F. M. M. Morel. 2000. Nature 407:996.

44

Appendix C: Manuscript on Form ID RubisCO ε value from Skeletonema costatum

Isotopically less selective RubisCO from the diatom Skeletonema costatum

A.J. Boller1, P.J. Thomas1, C.M. Cavanaugh2, and K.M. Scott1

1University of South Florida, Tampa, Florida 33620; 2Harvard University, Cambridge, Massachusetts 02138

Acknowledgments

Funding for this project was received from NSF Biological Oceanography OCE-

0327488 (to K.M.S). Special thanks are given to undergraduate researchers Kelly

Fitzpatrick and Michelle Echevarria who contributed to growing cell cultures as well as data collection. Thanks are also given to Sidney K. Pierce and his experimental writing class for editorial assistance.

45 Appendix C (Continued)

ABSTRACT

Form ID ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) is present

in several major oceanic primary producers that grow in large blooms. These large

blooms may be one of the causes of 13C enriched marine carbon because these organisms use an isotopically less selective form of RubisCO for carbon fixation. The purpose of this study is to determine the isotopic discrimination and KCO2 of Skeletonema costatum

RubisCO, and to compare its large subunit gene (rbcL) with form ID rbcL genes from other organisms. We found the isotopic discrimination of S. costatum RubisCO is 18.5‰ and its KCO2 is 90 (+/- 20) μM. Comparatively, this form ID RubisCO is isotopically less

selective than forms IA and IB RubisCOs (22-29‰), which are also present in some

oceanic primary producers. However, the ID RubisCO from the coccolithophore,

Emiliana huxleyi (11.1‰) is the most non-selective RubisCO measured. Since ID

RubisCOs are less selective against 13C, the isotopic discrimination by form ID RubisCO must be considered when making carbon cycle models or biological food chain models

using 13C values. Consequently, current carbon models could miscalculate global carbon

fixation.

46 Appendix C (Continued)

Diatoms are major primary producers in the ocean and contribute at least a quarter

of inorganic carbon fixed each year (10). Most diatoms live in pelagic marine and

freshwater ecosystems, but can also be found at the water-sediment interface. In areas of

nutrient upwelling, large blooms of diatoms quickly dominate the phytoplankton

communities (18) because they contain high proportions of growth machinery that allows

for exponential growth (1). Because of their larger cell size and abundance, diatoms form

the base of bloom associated food webs. Skeletonema costatum is a common, global

member of the plankton community in temperate areas, and therefore, is a good candidate

for further investigation into diatom carbon fixation kinetics.

At least five forms of ribulose-1,5-bisphosphate carboxylase/oxygenase

(RubisCO) catalyze carbon fixation in the marine organisms. Form IB RubisCO is the

most extensively studied because it is present in eukaryotic green chloroplasts from algae

to land plants. Other forms of RubisCO, prevalent in marine organisms, include form ID

RubisCO, which is present in eukaryotic organisms with non-green chloroplasts, such as

coccolithophores, rhodophytes, and diatoms (31), and form IA, IC, and II RubisCO,

which are present in diverse marine prokaryotes (4, 21, 30). Different forms of RubisCO

are found in diverse intracellular environments, and hence have evolved varying

structural and kinetic properties to fit their individual conditions (29). However, most

carbon cycle models are based on properties associated with IB RubisCO, even though

other forms of RubisCO are major contributors to carbon fixation, particularly in the marine environment.

47 Appendix C (Continued)

Stable carbon isotope compositions (δ13C values) of phytoplankton biomass have

been used to infer the physical and physiological factors influencing carbon fixation in the ocean (15). δ13C values are determined by measuring amounts of 13C and 12C in

biomass and comparing the ratio to a limestone standard (19). The main influence of δ13C

values measured in phytoplankton biomass is the selectivity of 13C by the RubisCO.

However, physical factors, such as dissolved inorganic carbon (DIC) pool composition

and nutrient availability, as well as physiological factors, such as growth rate and type of

carbon concentrating mechanism (CCM) utilized, can also influence the δ13C value of

marine phytoplankton (15). However, the effects of different factors on the δ13C biomass

values are difficult to interpret without the initial baseline isotopic selectivity from

RubisCO. The isotopic selectivity of very few RubisCOs have been measured to date;

therefore, other forms of RubisCO need to be examined to be able to correctly identify

factors which play a role in biomass composition.

Most biological models assume RubisCOs isotopic discrimination (ε value; =

RCO2/Rfixed – 1) x 1000)) is ~25‰ (12). This model value is reasonable when using ε

values from terrestrial plants, cyanobacteria, and bacterial chemolithoautotrophs, with ε

values ranging from 18-29‰ (14, 21, 22, 23, 26). However, the very isotopically non-

selective ID RubisCO from the coccolithophore, Emiliana huxleyi, differs greatly from

the model value (ε = 11.8‰) (2).

48 Appendix C (Continued)

If all ID RubisCO are isotopically non-selective, carbon models based on an

isotopic discrimination of 25‰ will underestimate the amount of 13C cycled, therefore

miscalculating total carbon fixation. Therefore, to determine if form ID RubisCOs have

similar carbon fixation kinetic parameters, the kinetic isotopic effect (KIE) and the KCO2 of S. costatum RubisCO was measured. Skeletonema costatum RubisCO large subunit

gene (rbcL) was also sequenced and compared to other ID RubisCOs from various organisms.

Methods

Cell Culture Methods

Skeletonema costatum cultures were purchased from the Provasoli-Guillard

National Center for Culture of Marine Phytoplankton (CCMP1332; West Boothbay

Harbor, Maine). The cells were grown in f/2 medium with silica (13) at 18-22°C, under

constant illumination. One L of f/2 media was inoculated with 5 mL of cell culture. After one week of growth, the 1 L culture was added to 4 L of fresh media for another week of growth with aeration.

RubisCO purification from S. costatum

RubisCO was extracted and partially purified from frozen cell cultures using ammonium sulfate precipitation. For all purification steps, samples were kept at 4°C.

Cells were harvested from the 5L cultures by centrifugation (5000g, 15min), flash-frozen

in liquid nitrogen, and stored at -80°C. Cell pellets were resuspended in lysis buffer

-1 -1 -1 -1 [20mmol L Tris pH 7.5, 10mmol L MgCl2, 5mmol L NaHCO3, 1mmol L EDTA,

49 Appendix C (Continued)

and 1mmol L-1 dithiothreitol (DTT)] per gram of pellet, sonicated twice for 30 sec with

glass beads, and centrifuged (5000g, 15 min). (NH4)2SO4 was added to the supernatant to

a final concentration of 30% saturation, and proteins were allowed to precipitate for 15

min. After centrifugation (5000g, 15 min), the pellet was discarded and the supernatant

was brought to 50% (NH4)2SO4 saturation. Proteins that precipitated at 30-50%

(NH4)2SO4 saturation were collected by centrifugation and dissolved in BBMD buffer

-1 -1 -1 -1 (50mmol L Bicine pH7.5, 25mmol L MgCl2, 5mmol L NaHCO3, and 1mmol L

DTT). The partially purified proteins were desalted with an anion exchange column

(HiPrep 26/10 desalting column; Amersham Biosciences, New Jersey, USA) equilibrated and eluted with BBMD buffer. The elutant was monitored at 280nm and peaks that had an absorbance of 0.5 or higher were collected (<15ml).

RubisCO activity assays

RubisCO activity in partially purified protein extracts was monitored by the

14 incorporation of CO2 into 3-phosphoglycerate (PGA). Purified RubisCO extracts were

-1 -1 -1 added to the assay buffer [50 mmol L Bicine, pH 8.0, 10 mmol L MgCl2, 1 mmol L

-1 -1 14 EDTA, 5mmol L DTT, and 25 mmol L NaH CO3 55 mCi/mmol bicarbonate (MP

Biomedicals, Irvine CA)] in a 1:2 ratio and pre-incubated for 15 min. Reactions were

started by the addition of 1 mmol L-1 RuBP (Sigma R0878, USA). At 3- 30 sec intervals,

one third of the sample was removed from the reaction mix and added to acetic acid to

stop the reaction. After the samples were dried by sparging with air, scintillation cocktail

(Fisher Scientific, USA) was added to the samples. Assays lacking RuBP were performed

50 Appendix C (Continued)

as a control for background CO2 fixation.

RubisCO isotope discrimination by kinetic isotope effect (KIE)

Using the partially purified protein from S. costatum, the kinetic isotope effect

(KIE) of RubisCO was measured at pH 7.5 using the high-precision substrate depletion

method (7, 26). Previous KIE were performed at pH 8.5 (14, 22), however, the optimal

pH for partially purified S. costatum RubisCO is pH 7.5. To test the effects of the altered

parameter on KIE’s, control experiments using spinach RubisCO were also conducted.

The reaction was prepared by sparging BBMD buffer with N2 to minimize CO2 and O2 concentration, and 1mg/25mL of bovine erythrocyte carbonic anhydrase (CA;

Sigma 3934, USA) was added to maintain DIC at chemical and isotopic equilibrium.

Approximately 5mL of partially purified RubisCO was added to the BBMD buffer, filter sterilized (0.45µm), and loaded into a heat-sterilized, septum-sealed 25 mL glass gastight syringe with a stir bar. Ribulose 1,5-bisphosphate (RuBP), substrate for RubisCO, was enzymatically synthesized from ribose 5- using spinach phosphoriboisomerase

(PRI; Sigma P9752-5KU) and purified (PRK)(26). After allowing

the RubisCO to pre-incubate for 15 min at 25°C, filter-sterilized (0.22 µm) RuBP (~100

mmoles) was injected into the gastight syringe to begin the reaction.

13 The concentration and δ C value were measured as the CO2 was consumed by

the RubisCO reaction. Samples were removed at 8 time intervals from the reaction

syringe based on the decrease of the DIC concentration. Triplicate samples were acidified

in a gas tight syringe with 43% phosphoric acid (1:4 ratio) to terminate the reaction and

51 Appendix C (Continued)

convert the DIC to CO2. The [DIC] of the triplicate samples was measured using a gas

chromatograph (HP/Agilent 5890A, USA) (7). The remaining sample was injected into

gas-tight syringes with 43% phosphoric acid (1:1 ratio). Using a vacuum line, the CO2 was cryodistilled from the sample and sent to Boston University Stable Carbon Isotope

Laboratory (Robert Michener) to determine their δ13C values using a gas inlet mass

spectrometer (7).

Five independent KIE’s were performed using partially purified S. costatum

RubisCO and three independent reactions were completed using spinach RubisCO

(10mg; Sigma R8000), for a positive control. The average ε values for the KIE’s were

calculated using the Pitman estimator with a 95% confidence interval (25). To ensure

alternate carboxylases were not fixing CO2, KIE experiments with S. costatum RubisCO

were also run without RuBP and without RuBP + 5 mmol L-1 3-PGA for negative

controls (Sigma P8877, USA).

KCO2 measurements

The KCO2 and Vmax of S. costatum RubisCO were measured radiometrically (17,

24). The assay buffer (50 mM Bicine, pH 7.5, 30 mM MgCl2, 0.4 mM RuBP, 1 mM

DTT) was prepared with low CO2 and O2 concentrations as in Schwedock et al., (2004).

Eight different incubations were conducted with DIC ranging from 0.2 to 14 mM in glass vials primed with stir bars and sealed under a N2 headspace with gas-tight septa. Before

14 the reaction was started, 25 mmol NaH CO3 (SA = 55 mCi/mmol bicarbonate) along

with bovine erythrocyte carbonic anhydrase (40 μg/mL; Sigma C3934), to maintain CO2

52 Appendix C (Continued)

and HCO3 in chemical equilibrium, was added to each vial. Partially purified S. costatum

RubisCO in BBMD buffer that lacked RuBP was added to assay buffer, immediately

before the reaction was started. To begin a reaction, RuBP was injected into a vial, and

samples were removed with a gastight syringe at 1 min intervals over a 4 min time

course. These samples were immediately injected into scintillation vials containing

14 14 glacial acetic acid to remove all CO2 and collect acid stable C. The samples were

sparged with air and left for approximately 6 hours to ensure samples were completely

dry before scintillation cocktail was added. The initial activity of each incubation was

measured by injecting 10μl samples into scintillation cocktail containing

14 phenylethylamine (sx10-1000 Fisher Scientific, USA) to trap the NaH CO3. Both acid

stable 14C samples and the initial activities were measured via scintillation counting. Five independent experiments were conducted, and carbon fixation followed the Michaelis-

Menten response curve. KCO2 and Vmax values for the five experiments were estimated from the carbon fixation rates using direct linear plots (9).

Cloning and sequencing of the RubisCO large subunit gene (rbcL)

DNA was purified from S. costatum cells using the CTAB method (11). Primers for the S. costatum rbcL gene were designed from a partial rbcL sequence (6) and purchased from Invitrogen (USA). Primers used to replicate the first half of the gene (1bp

- 840 bp) include the forward primer (GGGTTACTGGGATGCTTCATACAC) and reverse primer (CCAACAGCTTTAGCATACTCAGCAC). The primers used to replicate the second half of the gene (560bp - 1428bp) include forward primer

53 Appendix C (Continued)

(GGAAGGTATTAACCGTGCATCAGC) and reverse primer

(TCTGTTTGCAGTTGGTGTTTCAGC). The two RubisCO gene sequences were replicated with PCR, using the above primers, and the PCR product was ligated into the pCR2.1 vector using TA Cloning Kit (Invitrogen, 45-0046; California, USA). The plasmids with RubisCO gene inserts were transformed into One Shot TOP10 cells and grown on plates with Luria broth agar with 100µg/ml ampicillin, 50 µg/ml kanamycin, and spread with X-gal. Colonies which had the plasmid + insert were grown in liquid LB overnight and the plasmids were purified using the QIAprep Spin Miniprep Kit (Qiagen,

12125; California, USA). Three different purified plasmids for each of the two RubisCO gene inserts were sent for sequencing (Macrogen, Maryland, USA). A consensus sequence was made using repetitive nucleotides from the three sequenced plasmid inserts.

Phylogenetic Analysis

A phylogenetic analysis of the large subunit RubisCO gene (rbcL) from S. costatum and other form ID rbcL genes from diverse organisms was accomplished using a maximum parsimony tree with rbcL sequences obtained from NCBI- BLAST nucleotide searches. Sequences were aligned and a maximum parsimony tree, with 1500 replicates, was constructed using MEGA 4.0 software (28).

Results

The five independent incubations of S. costatum RubisCO had ε values of 19.0‰,

18.6‰, 19.7‰, 15.9‰ and 20.2‰, respectively (Figure C-1). The average ε value calculated using the Pitman estimator was 18.5‰ (95% CI: 17.0-19.9‰). Three spinach

54 Appendix C (Continued)

RubisCO incubations had ε values of 28.3‰, 28.2‰, and 27.2‰, with a Pitman average

ε value of 27.5‰ (95% CI: 24.0 – 30.9‰; Figure C-1) that was within range of

previously published values. In order to further characterize S. costatum RubisCO

activity, its KCO2 was measured radiometrically in oxygen-free incubations. The KCO2 and

Vmax were calculated from five independent experiments and found to be 90 (+/- 20) μM and 15.6 (+/- 5.8) nmol/min* mg, respectively.

-4.467 S.costatum 1 S.costatum 2 -4.47 S.costatum 3 -4.473 S.costatum 4

C ) -4.476 S.costatum 5 12 Spinach 4 C / -4.479 13 Spinach 2 -4.482

lnR ( ( lnR Spinach 3 -4.485

-4.488 -4.491 0.80.91 1.11.21.31.41.5

-1 ln[DIC mmol L ]

Figure C-1. Isotopic fractionation of dissolved inorganic carbon (DIC) as CO2 is consumed by S. costatum RubisCO and spinach RubisCO. The slope of the line is used to estimate the ε value for each organism.

Since partially purified extracts of S. costatum RubisCO were used in the KIE,

controls for alternative carboxylase activity were run. KIE incubations with RuBP

showed a decrease in [DIC] over time, which is consistent with S. costatum RubisCO

catalyzing the carbon fixation in these assays (Figure C-2). Conversely, control

experiments, which did not contain RuBP, showed no change of [DIC] in the reaction

55 Appendix C (Continued) syringe. DIC also was constant in the RuBP-free incubations that contained 3-PGA. A more sensitive bicarbonate 14C activity assay performed on partially purified enzyme also showed carboxlyation occurring only in the presence of RuBP. Therefore, RubisCO was the only active carboxylase in the partially purified extracts (Figure C-3).

6

5

4

3 S. costatum 1

DIC [mM] S. costatum 2 2 S. costatum 3 S. costatum 4 1 S. costatum 5 no RuBP no RuBP + PGA 0 0246810 Time (hr)

Figure C-2. The consumption of DIC over time by S. costatum RubisCO. Incubations that included RuBP (solid symbols) were used to calculate the ε value of S. costatum RubisCO. In absence of RuBP, (open symbols) no DIC was consumed even with 3- PGA included in the incubation.

56 Appendix C (Continued)

10 With RuBP ) 8 Without RuBP

6

4

Carbon Fixation (nmol Fixation Carbon 2

0

02468101214 Time (min)

Figure C-3. Radiometric assay of partially purified S. costatum RubisCO. Assays were conducted in the presence and absence of RuBP to confirm that all detectable carboxylase activity was due to RubisCO.

Since the isotopic discrimination of form ID RubisCOs differ, a phylogenetic analysis was completed to identify if the ID RubisCO rbcL genes are divergent. In the maximum parsimony tree, the rbcL genes formed clades, which included diatoms, coccolithophores, and rhodophytes (Figure C-4). Higher bootstrap values at the base of the clades differentiate the rbcL genes based on their phylogenetic groups. Furthermore, the rhodophyte rbcL genes group with the diatoms genes, while the coccolithophore rbcL genes formed a separate branch. The form IC rbcL genes formed a separate group away from the ID RubisCO genes.

57 Appendix C (Continued)

99 Thalassiosira pseudonana 75 Skeletonema costatum 84 Cylindrotheca sp. Diatoms Phaeodactylum tricornutum 99 73 Lyrella hennedyi 95 Thalassionema frauenfeldii Purpureofilum apyrenoidigerum Gelidium pusillum 89 Rhodophytes 92 Polysiphonia stricta 100 Polysiphonia morrowii 57 Emiliania huxleyi Isochrysis sp. Coccolithophores 99 Chrysochromulina hirta 37 Platychrysis sp. Ralstonia eutropha

100 Rhodobacter sphaeroides IC RubisCO

Figure C-4. Phylogenetic analysis of selected RubisCO large subunit genes (rbcL). Sequences were aligned using the maximum parsimony method (MEGA software). The numbers next to the branches represents the number of times, of the 1500 bootstrap trials, that the associated taxa clustered together as shown.

Discussion

Skeletonema costatum RubisCO is less selective against 13C; consequently, providing a basis for enriched 13C marine carbon biomass. The carbon isotope compositions of S. costatum biomass correlate with the selectivity of its RubisCO, having

δ13C values ranging from -16.8 to -27.6‰. (3, 16). The higher (-16.8‰) δ13C biomass values can be attributed primarily to carbon fixation of bicarbonate carbon by the less

- selective RubisCO. At marine pH values, bicarbonate (HCO3 ) is the main form of DIC

58 Appendix C (Continued)

in the ocean; however, RubisCO fixes DIC in the form of CO2 (5). To cope with the

- paucity of CO2 in the environment, some marine autotrophs will convert HCO3 into CO2

- using carbonic anhydrase (CA). Organisms, that use HCO3 as a main carbon source, have

13 - higher δ C biomass values because HCO3 is isotopically enriched compared CO2.

Even though S. costatum RubisCO is less isotopically selective, its biomass has less 13C than expected, with δ13C values as low as -27.6‰ (3). Low δ13C values could be

attributed to S. costatum RubisCO using a carbon source depleted in 13C, such as

isotopically depleted CO2 (-8‰). Furthermore, carbon-concentrating mechanisms (CCM)

could also play a role in low biomass δ13C values. In diatoms, the internal DIC

concentration can reach 3.5 times higher than external DIC concentrations (20). This high

internal concentration is not achievable strictly though CO2 diffusion, which is indicative

of a CCM; however, the exact CCM used by S. costatum is unknown. The CCM may

- consist of active transport of CO2 or HCO3 into the cell as well as CA to carry out the interconversion of the two substrates (20). The CCM could actively transport less 13C into the cell for RubisCO to fix, also causing low δ13C biomass values in S. costatum. Once the exact mechanism of S. costatum’s CCM is elucidated, the isotopic effect associated with the CCM can be investigated.

Because of their bloom life style, organisms, such as diatoms and coccolithophores that have a less selective form ID RubisCO, can greatly alter the δ13C of

oceanic biomass. In areas of nutrient upwelling, large blooms of phytoplankton can

trigger the larval spawning of grazing zooplankton (27). Consuming organisms with ID

59 Appendix C (Continued)

RubisCO causes the zooplankton biomass to become rich in 13C. Furthermore, diatoms

are very efficient at exporting carbon to the sea floor because the cells are larger and

heavier, which enhance sinking rates; therefore, increasing the amount of 13C in deep

waters (8). An increase in oceanic biomass δ13C values can be caused by zooplankton

grazing and increased vertical carbon flux of diatoms.

With rising atmospheric CO2 values, it is essential to have accurate carbon cycle

modeling. Although isotopic discrimination by different forms of RubisCO vary between

13 organisms, most carbon models assume that all RubisCOs discriminate against CO2 to the same degree (12). Form ID RubisCOs, including S. costatum and E. huxleyi, are isotopically less selective than other forms of RubisCO (Table C-1), calling for changes in current modeling equations. However, rhodophytes, another major phylogenetic group with ID RubisCO, also need to be investigated to understand the full range of isotopic fractionation by ID RubisCO. Collecting isotopic fractionation data from RubisCO of diverse organisms will allow for more inclusive carbon models that will generate better estimations of global carbon fixation.

60 Appendix C (Continued)

Table C-1. Epsilon values of different forms of RubisCO that have been measured with kinetic isotope experiments.

Taxon Form of RubisCO ε value (‰)

Solemya velum symbiont IA 24.5

Prochlorococcus marinus MIT9313 IA 24.0

Spinacia oleracea IB 29.0

Anacystis nidulans IB 22.0

Rhodobacter sphaeroides IC 22.9

Skeletonema costatum ID 18.6

Emiliana huxleyi ID 11.1

Riftia pachyptila symbiont II 19.5

Rhodospirillum rubrum II 22.0

61 Appendix C (Continued)

References

1. Arrigo, K. R. 2005. Marine microorganisms and global nutrient cycles. Nature 437:349-355. 2. Boller, A. J., Thomas, P. J., Cavanaugh, C. M., and Scott, K. M. in prep. A New Low for RubisCO. 3. Burkhardt, S., U. Riebesell, and I. Zondervan. 1999. Stable carbon isotope fractionation by marine phytoplankton in response to daylength, growth rate, and CO2 availability. Marine Ecology-Progress Series 184:31-41. 4. Caspi, R., M. G. Haygood, and B. M. Tebo. 1996. Unusual ribulose 1,5- bisphosphate carboxylase/oxygenase genes from a marine manganese oxidizing bacterium. Microbiology 142:2549-2559. 5. Cooper, T. G., and D. Filmer. 1969. The active species of "CO2" utilized by ribulose diphosphate carboxylase. Journal of Biological Chemistry 244:1081- 1083. 6. Daugbjerg, N., Andersen, R.A. 1997. A molecular phylogeny of the heterokont algae based on analyses of -encoded rbcL sequence data. J. Phycol. 33:1031-1041. 7. Dobrinski, K. P., D. L. Longo, and K. M. Scott. 2005. A hydrothermal vent chemolithoautotroph with a carbon concentrating mechanism. J Bacteriol 187:5761-5766. 8. Dugdale, R. C., and F. P. Wilkerson. 1998. Silicate regulation of new production in the equatorial Pacific upwelling. Nature 391:270-273. 9. Eisenthal, R., and A. Cornish-Bowden. 1974. The direct linear plot. Biochemistry Journal 139:715-720. 10. Falkowski, P.G. and Raven, J.A. 1997. Aquatic Photosynthesis. In Carbon acquisition and Assimilation. Boston: Blackwell Scientific Publication, pp. 128- 162. 11. Feil, W. S., and H. Feil. 2004. Bacterial genomic DNA isolation using CTAB. DOE Joint Genome Institute (JGI). 12. Goericke, R., J. P. Montoya, and B. Fry. 1994. Physiology of isotopic fractionation in algae and cyanobacteria, pp. 187-221. In K. Lajtha and R. H. Michener (eds.), Stable Isotopes in Ecology and Environmental Science. Blackwell Scientific Publications. 13. Guillard, R. R., and J. H. Ryther. 1962. Studies of Marine Planktonic Diatoms .1. Cyclotella Nana Hustedt, and Detonula Confervacea (Cleve) Gran. Canadian Journal of Microbiology 8:229. 14. Guy, R. D., M. L. Fogel, and J. A. Berry. 1993. Photosynthetic fractionation of the stable isotopes of oxygen and carbon. Plant Physiol 101:37-47. 15. Hayes, J. M. 2001. Fractionation of carbon and hydrogen isotopes in biosynthetic processes, pp. 225-277. In J. W. Valley and D. R. Cole [eds.], Stable Isotope Geochemistry. The Mineralogical Society of America.

62 Appendix C (Continued)

16. Hinga, K. R., M. A. Arthur, M. E. Q. Pilson, and D. Whitaker. 1994. Carbon isotope fractionation by marine phytoplankton in culture: The effects of CO2 concentration, pH, temperature, and species. Global biogeochemical cycles 8:91- 102. 17. Horken, K., and F. R. Tabita. 1999. Closely related form I ribulose bisphosphate carboxylase/oxygenase molecules that possess different CO2/O2 substrate specificities. Archives of Biochemistry and Biophysics 361:183-194. 18. Nielsen, T. G., and B. Hansen. 1995. Plankton community structure and carbon cycling on the western coast of Greenland during and after the sedimentation of a diatom bloom. Marine Ecology Progress Series 125:239-257. 19. O'leary, M. H., S. Madhavan, and P. Paneth. 1992. Physical and chemical basis of carbon isotope fractionation in plants. Plant, Cell, and Environment 15:1099-1104. 20. Roberts, K., E. Granum, R. C. Leegood, and J. A. Raven. 2007. Carbon acquisition by diatoms. Photosynth Research 93:79-88. 21. Robinson, J. J. et. al. 2003. Kinetic isotope effect and characterization of form II RubisCO from the chemoautotrophic endosymbionts of the hydrothermal vent tubeworm Riftia pachyptila. Limnol Oceanogr 48:48-54. 22. Roeske, C. A., and M. H. O'leary. 1984. Carbon isotope effects on the enzyme- catalyzed carboxylation of ribulose bisphosphate. Biochemistry 23:6275-6284. 23. Roeske, C. A., and M. H. O'leary. 1985. Carbon isotope effect on carboxylation of ribulose bisphosphate catalyzed by ribulosebisphosphate carboxylase from Rhodospirillum rubrum. Biochemistry 24:1603-1607. 24. Schwedock, J. et. al. 2004. Characterization and expression of genes from the RubisCO gene cluster of the chemoautotrophic symbiont of Solemya velum: cbbLSQO. Arch Microbiol 182:18-29. 25. Scott, K. M., X. Lu, C. M. Cavanaugh, and J. Liu. 2004a. Optimal methods for estimating kinetic isotope effects from different forms of the Rayleigh distillation equation. Geochim Cosmochim Acta 68:433-442. 26. Scott, K. M., J. Schwedock, D. P. Schrag, and C. M. Cavanaugh. 2004b. Influence of form IA RubisCO and environmental dissolved inorganic carbon on the δ13C of the clam-bacterial chemoautotrophic symbiosis Solemya velum. Environ Microbiol 6:1210-1219. 27. Starr, M., J. C. Therriault, G. Y. Conan, M. Comeau, and G. Robichaud. 1994. Larval Release in a Sub-Euphotic Zone Invertebrate Triggered by Sinking Phytoplankton Particles. Journal of Plankton Research 16:1137-1147. 28. Tamura, K., J. Dudley, M. Nei, and S. Kumar. 2007. MEGA 4: Molecular Evolutionary Genetics Analysis (MEGA) Software Version 4.0. Molecular Biology and Evolution pp. 1596-1599.

63 Appendix C (Continued)

29. Tcherkez, G. G. B., G. D. Farquhar, and T. J. Andrews. 2006. Despite slow and confused substrate specificity, all ribulose bisphosphate carboxylases may be nearly perfectly optimized. Proceedings of the National Academy of Sciences of the United States of America 103:7246-7251. 30. Watson, G. M. F. and F. R. Tabita. 1996. Regulation, unique gene organization, and unusual primary structure of carbon fixation genes from a marine phycoerythrin-containing cyanobacterium. Plant Molecular Biology 32:1103- 1115. 31. Watson, G. M. F. and F. R. Tabita. 1997. Microbial ribulose 1,5-bisphosphate carboxylase/oxygenase: A molecule for phylogenetic and enzymological investigation. Fems Microbiology Letters 146:13-22.

64

Appendix D: Abstract from Publication of Thiomicrospira crunogena XCL-2 genome

The Genome of Deep-Sea Vent Chemolithoautotroph Thiomicrospira crunogena

XCL-2

Kathleen M. Scott*1, Stefan M. Sievert2, Fereniki N. Abril1, Lois A. Ball1, Chantell J. Barrett1, Rodrigo A. Blake1, Amanda J. Boller1, Patrick S. G. Chain3,4, Justine A. Clark1, Carisa R. Davis1, Chris Detter4, Kimberly F. Do1, Kimberly P. Dobrinski1, Brandon I. Faza1, Kelly A. Fitzpatrick1, Sharyn K. Freyermuth5, Tara L. Harmer6, Loren J. Hauser7, Michael Hügler2, Cheryl A. Kerfeld8, Martin G. Klotz9, William W. Kong1, Miriam Land7, Alla Lapidus4, Frank W. Larimer7, Dana L. Longo1, Susan Lucas4, Stephanie A. Malfatti3,4, Steven E. Massey1, Darlene D. Martin1, Zoe McCuddin10, Folker Meyer11, Jessica L. Moore1, Luis H. Ocampo Jr.1, John H. Paul12, Ian T. Paulsen13, Douglas K. Reep1, Qinghu Ren13, Rachel L. Ross1, Priscila Y. Sato1, Phaedra Thomas1, Lance E. Tinkham1, and Gary T. Zeruth1

Biology Department, University of South Florida, Tampa, Florida USA1; Biology Department, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts USA2; Lawrence Livermore National Laboratory, Livermore, California USA3; Joint Genome Institute, Walnut Creek, California USA4; Department of Biochemistry, University of Missouri, Columbia, Missouri USA5; Division of Natural Sciences and Mathematics, The Richard Stockton College of New Jersey, Pomona, New Jersey USA6; Oak Ridge National Laboratory, Oak Ridge, Tennessee USA7; Molecular Biology Institute, University of California, Los Angeles, California USA8; University of Louisville, Louisville USA9; The Monsanto Company, Ankeny, IA USA10; Center for Biotechnology, Bielefeld University, Germany11; College of Marine Science, University of South Florida, St. Petersburg, Florida USA12; The Institute for Genomic Research, Rockville, Maryland USA13

*Corresponding author. Mailing address: 4202 East Fowler Avenue; SCA 110; Tampa, FL 33620. Phone: (813) 974-5173. Fax: (813) 974-3263. E-mail: [email protected].

Citation: Scott, K. M., Sievert, S. M., Abril, F. N., Ball, L. A., Barrett, C. J., et. al. 2006. The genome of deep-sea vent chemolithoautotroph Thiomicrospira crunogena XCL-2. PloS Biology 4(12):e383. DOI: 10.1371/ journal.pbio.0040383.

65 Appendix D (Continued)

ABSTRACT

Presented here is the complete genome sequence of Thiomicrospira crunogena

XCL-2, representative of ubiquitous chemolithoautotrophic sulfur-oxidizing bacteria isolated from deep-sea hydrothermal vents. This gammaproteobacterium has a single chromosome (2,427,734 bp), and its genome illustrates many of the adaptations that have enabled it to thrive at vents globally. It has 14 methyl-accepting chemotaxis protein genes, including four that may assist in positioning it in the redoxcline. A relative abundance of CDSs encoding regulatory proteins likely control the expression of genes encoding carboxysomes, multiple dissolved inorganic nitrogen and phosphate transporters, as well as a phosphonate operon, which provide this species with a variety

of options for acquiring these substrates from the environment. T. crunogena XCL-2 is unusual among obligate sulfur oxidizing bacteria in relying on the Sox system for the oxidation of reduced sulfur compounds. The genome has characteristics consistent with an obligately chemolithoautotrophic lifestyle, including few transporters predicted to have organic allocrits, and Calvin-Benson-Bassham cycle CDSs scattered throughout the genome.

66 Appendix D (Continued)

Figure D-1. PloS Biology Open Access License for use of the above abstract.

67

Appendix E: Abstract from Publication of Sulfurimonas denitrificans genome

Genome of the Epsilonproteobacterial Chemolithoautotroph Sulfurimonas

denitrificans

Stefan M. Sievert‡*1, Kathleen M. Scott‡*2, Martin G. Klotz3, Patrick S. G. Chain4,5, Loren J. Hauser6, James Hemp7, Michael Hügler1,8, Miriam Land6, Alla Lapidus5, Frank W. Larimer6, Susan Lucas5, Stephanie A. Malfatti4,5, Folker Meyer9, Ian T. Paulsen10#, Qinghu Ren10, Jörg Simon11, and the USF Genomics Class2, ∆

Biology Department, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts1; Biology Department, University of South Florida, Tampa, Florida2; Departments of Biology and Microbiology & Immunology, University of Louisville, Louisville, Kentucky3; Lawrence Livermore National Laboratory, Livermore, California4; Joint Genome Institute, Walnut Creek, California5; Oak Ridge National Laboratory, Oak Ridge, Tennessee6; Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois7; Leibniz-Institut für Meereswissenschaften, Kiel, Germany8; Mathematics and Computer Science Division, Argonne National Laboratory, Argonne, Illinois9; The Institute for Genomic Research, Rockville, Maryland10; Institute of Molecular Biosciences, Johann Wolfgang Goethe University, Frankfurt am Main, Germany11

∆Kathryn Bailey, Erik Diaz, Kelly Ann Fitzpatrick, Bryan Glover, Natasha Gwatney, Asja Korajkic, Amy Long, Jennifer M. Mobberley, Shara N. Pantry, Geoffrey Pazder, Sean Peterson, Joshua D. Quintanilla, Robert Sprinkle, Jacqueline Stephens, Phaedra Thomas, Roy Vaughn, M Joriane Weber, Lauren L. Wooten

‡SMS and KMS contributed equally to this work.

*Corresponding authors. SMS: Mailing address: WHOI; Watson Building 207, MS#52; Woods Hole, MA 02543. Phone: (508) 289-2305. Fax: (508) 457-2076. E-mail: [email protected]. KMS: Mailing address: 4202 East Fowler Avenue, SCA 110; Tampa, FL 33620. Phone: (813) 974-5173. Fax (813) 974-3263. E-mail: [email protected]

Citation: Sievert, S. M., Scott, K. M., Klotz, M. G., Chain, P. S. G., Hauser, L. J., et. al. 2008. Genome of the epsilonproteobacterial chemolithoautotroph Sulfurimonas denitrificans. Applied and Environmental Microbiology. 74(4):1145-1156. American Society for Microbiology. All rights reserved.

68 Appendix E (Continued)

ABSTRACT

Sulfur-oxidizing epsilonproteobacteria are common in a variety of sulfidogenic

environments. These autotrophic and mixotrophic sulfur-oxidizing bacteria are believed to contribute substantially to the oxidative portion of the global sulfur cycle. In order to better understand the ecology and roles of sulfur-oxidizing epsilonproteobacteria, in

particular the widespread genus Sulfurimonas, in biogeochemical cycles, the genome of

Sulfurimonas denitrificans DSM1251 was sequenced. This genome has many features,

including a larger size (2.2 Mbp), that suggest a greater degree of metabolic versatility or

responsiveness to the environment than most of the other sequenced

epsilonproteobacteria. A branched electron transport chain is apparent, with genes

encoding complexes for the oxidation of hydrogen, reduced sulfur compounds, and

formate, and the reduction of nitrate and oxygen. Genes are present for a complete,

autotrophic reductive citric acid cycle. Many genes are present that could facilitate

growth in the spatially and temporally heterogeneous sediment habitat from where

Sulfurimonas denitrificans was originally isolated. Many resistance-nodulation-

development-family transporter genes (11 total) are present, several of which are

predicted to encode heavy metal efflux transporters. An elaborate arsenal of sensory and

regulatory protein-encoding genes is in place, as well as genes necessary to prevent and

respond to oxidative stress.

69 Appendix E (Continued)

Figure E-1. American Society for Microbiology permission document for use of the above abstract from the AEM publication.

70