REGULAR PAPER

Identification and characterization of a carboxysomal c-carbonicanhydrase from the cyanobacterium Nostoc sp. PCC 7120

Charlotte de Araujo • Dewan Arefeen • Yohannes Tadesse • Benedict M. Long •

G. Dean Price • Roger S. Rowlett • Matthew S. Kimber • George S. Espie

Received: 8 May 2014 / Accepted: 19 May 2014 / Published online: 8 June 2014

� Springer Science+Business Media Dordrecht 2014

Abstract Carboxysomes are proteinaceous microcom-

partments that encapsulate carbonic anhydrase (CA) and

ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubi-

sco); carboxysomes, therefore, catalyze reversible HCO3-

dehydration and the subsequent fixation of CO2. The N-

and C-terminal domains of the b-carboxysome scaffold

protein CcmM participate in a network of protein–protein

interactions that are essential for carboxysome biogenesis,

organization, and function. The N-terminal domain of

CcmM in the thermophile Thermosynechococcus elongatus

BP-1 is also a catalytically active, redox regulated c-CA.

To experimentally determine if CcmM from a mesophilic

cyanobacterium is active, we cloned, expressed and puri-

fied recombinant, full-length CcmM from Nostoc sp. PCC

7120 as well as the N-terminal 209 amino acid c-CA-like

domain. Both recombinant proteins displayed ethoxyzola-

mide-sensitive CA activity in mass spectrometric assays, as

did the carboxysome-enriched TP fraction. NstCcmM209

was characterized as a moderately active and efficient c-

CA with a kcat of 2.0 9 104 s-1 and kcat/Km of

4.1 9 106 M-1 s-1 at 25 �C and pH 8, a pH optimum

between 8 and 9.5 and a temperature optimum spanning

25–35 �C. NstCcmM209 also catalyzed the hydrolysis of

the CO2 analog carbonyl sulfide. Circular dichroism and

intrinsic tryptophan fluorescence analysis demonstrated

that NstCcmM209 was progressively and irreversibly

denatured above 50 �C. NstCcmM209 activity was inhib-

ited by the reducing agent tris(hydroxymethyl)phosphine,

an effect that was fully reversed by a molar excess of

diamide, a thiol oxidizing agent, consistent with oxidative

activation being a universal regulatory mechanism of

CcmM orthologs. Immunogold electron microscopy and

Western blot analysis of TP pellets indicated that Rubisco

and CcmM co-localize and are concentrated in Nostoc sp.

PCC 7120 carboxysomes.

Keywords Carboxysome � CcmM � CO2 concentrating

mechanism � Carbonyl sulfide � Gamma-carbonic

anhydrase � Nostoc sp. PCC 7120

Introduction

Cyanobacteria possess a complex, biophysical CO2 con-

centrating mechanism (CCM) which is crucial for photo-

synthesis, growth, and survival. This adaptive mechanism

relies on light-energy-dependent membrane transport sys-

tems to acquire and concentrate HCO3- in the cytosol. The

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11120-014-0018-4) contains supplementarymaterial, which is available to authorized users.

C. de Araujo � D. Arefeen � G. S. Espie

Department of Cell & Systems Biology, University of Toronto,

Mississauga, ON, Canada

Y. Tadesse � G. S. Espie (&)

Department of Biology, University of Toronto, 3359

Mississauga Road, Mississauga, ON L5L 1C6, Canada

e-mail: george.espie@utoronto.ca

B. M. Long � G. D. Price

Division of Plant Science, Research School of Biology,

Australian National University, Canberra, ACT, Australia

R. S. Rowlett

Department of Chemistry, Colgate University, Hamilton, NY,

USA

M. S. Kimber

Department of Molecular and Cellular Biology, University of

Guelph, Guelph, ON, Canada

123

Photosynth Res (2014) 121:135–150

DOI 10.1007/s11120-014-0018-4

accumulated HCO3- subsequently enters the carboxysome

where it is dehydrated to CO2 by a specific carbonic

anhydrase (CA) and is then fixed by ribulose 1,5-bisphos-

phate carboxylase/oxygenase (Rubisco) to initiate the

photosynthetic carbon reduction cycle. By facilitating

HCO3-/CO2 equilibration solely within the confines of the

carboxysome, this CA effectively maintains a near-satu-

rating substrate concentration in close proximity to Rubi-

sco (Price et al. 2008). The importance of a carboxysomal

CA to the photoautotrophic lifestyle of cyanobacteria is

highlighted by the fact that the loss of this protein from the

carboxysome results in a conditional lethal phenotype that

can only be rescued by growth at supra-normal CO2

(Fukuzawa et al. 1992; So et al. 2002b; Yu et al. 1992). In

cyanobacteria with form 1A Rubisco (a-cyanobacteria), the

carboxysomal CA is CsoSCA (formerly CsoS3) a highly

divergent member of the b-class of CAs (Sawaya et al.

2006; So et al. 2004). In cyanobacteria with form 1B Ru-

bisco (b-cyanobacteria), like Synechocystis PCC 6803 and

Synechococcus PCC 7942, the carboxysomal CA has been

identified as a 30 kDa protein (CcaA) with closest

homology to the b-class of CAs that is widely distributed in

bacteria, eukaryotic algae, and plants (So et al. 2002b; Yu

et al. 1992; So and Espie 2005).

CcaA is recruited to the carboxysome as part of a larger

protein complex that also contains CcmM and CcmN (Cot

et al. 2008; Long et al. 2007). CcmM is the central com-

ponent of this complex, interacting with both CcaA and

CcmN directly through its N-terminal c-CA-like domain

(Cot et al. 2008; Long et al. 2007; Kinney et al. 2012). In

addition to the N-terminal domain, CcmM also possesses a

C-terminus comprised of a series of 3–5 RbcS-like domains

connected by flexible linkers. These domains interact with

RbcL and help recruit Rubisco to the carboxysome (Cot

et al. 2008; Long et al. 2007). CcmM is also expressed in

one or more ‘‘short forms’’ which arises from an internal

ribosome-binding site and contains only the RbcS-like

repeat domains (Cot et al. 2008; Long et al. 2007, 2010).

This variant interacts with RbcL to create an internal net-

work of protein interactions that organizes multiple Rubi-

sco molecules into an ordered array within the core of the

carboxysome. The CcmM/CcmN/CcaA complex also

appears capable of interacting with CcmK2 in the shell

through either the N-terminal domain of CcmM (Cot et al.

2008), or the extended C-terminus of CcmN (Kinney et al.

2012).The absence of carboxysomes and concomitant high

CO2 requiring phenotype of DccmM mutants attest to the

importance of this multi-domain protein to the structure

and function of b-carboxysomes (Berry et al. 2005;

Hackenberg et al. 2012; Ludwig et al. 2000; Marcus et al.

1992).

As mentioned, the N-terminal domain of CcmM has

detectable sequence homology to c-CAs, including 38 and

29 % identity, respectively, to the Methanosarcina ther-

mophila Cam (MtCam) and CamH enzymes (MtCamH)

(Alber et al. 1999; Alber and Ferry 1994, 1996; Zimmer-

man et al. 2010). However, in most instances, proteins with

similarity to Cam are determined to have no CA activity

(Pena et al. 2010; Zimmerman et al. 2010), and indeed,

previous work had shown that CcmMs from Synechocystis

PCC 6803 (SynCcmM) and Synechococcus PCC 7942

(SycCcmM) do not display CA activity under a variety of

assay conditions (Cot et al. 2008; So and Espie 2005).

However, the N-terminal 209 amino acids of CcmM of

Thermosynechococcus elongatus BP-1 (TeCcmM209) was

recently demonstrated to be catalytically active (Pena et al.

2010); this b-cyanobacterium lacks a ccaA homolog, and

CcmM, in this case, seems to take over the catalytic role of

the carboxysomal CA. The structure of TeCcmM209 is

characterized by the presence of a C-terminal a-helix

which is stabilized by a disulfide bond; this helix, and the

extended loop connecting the first pair of b-strands appears

to act as a redox switch, and was proposed to be the key

feature for predicting which CcmM’s retain CA activity

(Pena et al. 2010). While the presence of known functional

motifs in diverse CcmM homologs predicts that catalytic

activity is widespread (though not universal) among CcmM

homologs, no CA activity has been shown for a CcmM

from any cyanobacterium except T. elongatus. This is

problematic given that the only proven catalytically com-

petent cyanobacterial c-CA is from a thermophilic strain,

while CcmM homologs tested in two mesophilic cyano-

bacteria both proved to be catalytically incompetent. To

establish the enzymatic activity and role in the CCM for

CcmM in mesophilic cyanobacteria, we have examined the

sub-cellular localization, activity profile, and catalytic

properties of CcmM encoded by the diazotrophic cyano-

bacterium Nostoc sp. PCC 7120 (NstCcmM).

Materials and methods

Isolation of Nostoc sp. PCC 7120 carboxysomes

Nostoc sp. PCC 7120 (hereafter Nostoc PCC 7120) was

obtained from the Canadian Phycological Culture Collec-

tion, University of Waterloo, Waterloo, Canada as CPCC

387. The cells were maintained in unbuffered BG11

medium with constant shaking at 120 rpm. The cultures

were continuously illuminated using cool white and gro-

lux fluorescent lamps (25 lmol m-2 s-1, photosynthetic

photon flux density). For carboxysome isolation from 2 to

6 L of illuminated cells were grown to late log phase

(8 days) with 5 % v/v CO2 followed by 3 additional days

with air bubbling to fully induce the CCM. A carboxy-

some-enriched fraction (TP pellet) was obtained from

136 Photosynth Res (2014) 121:135–150

123

crude cell lysates using the Percoll-Mg2? technique as

described previously (Long et al. 2005, 2007).

Protein overexpression and polyclonal antibody

production

The ccmM gene (all0865) encoding a 555 amino acid

protein (NCBI Accession, NP_484908) was PCR amplified

from Nostoc PCC 7120 genomic DNA and cloned into

pET21b or pET28b (Novagen) using standard techniques.

The PCR primers employed for cloning and sequencing are

listed in Table S1. To confirm the presence of the gene of

interest, DNA sequencing was performed at The Centre for

Applied Genomics, The Hospital for Sick Children (Tor-

onto, Canada).

Recombinant E. coli BL21 DE3 cells or Rosetta Blue

DE3 cells were grown at 37 �C in shaker flasks, and

recombinant protein expression was induced with 1 mM

IPTG at 20 �C (16 h) or 37 �C (6 h). The cells were har-

vested by centrifugation, and the pellet was lysed on ice by

five 10 s bursts of ultrasound (50 % maximum setting,

Heat Systems Inc. sonicator W185) separated by 30 s

intervals on ice. Bacterial lysate was clarified by centrifu-

gation and passed through a 0.45 lm filter. The filtrate was

loaded onto an immobilized metal affinity column (IMAC,

Novagen). Weakly bound protein was removed by exten-

sive washing as prescribed in the manufacturer’s protocol.

His-tag-bound protein was eluted with 1 M imidazole,

0.5 M NaCl, 20 mM Tris–HCl, pH 7.9 and fractions con-

taining the purified proteins were pooled and subsequently

dialyzed against 500 mM NaCl, 1 mM EDTA, 20 mM

Tris–HCl, pH 8.0.

Protein inclusion bodies were solubilized in 100 mM

EPPS/NaOH, 20 mM MgSO4, 10 lM ZnSO4, 6.0 M urea,

pH 8.0. After the addition of solid urea, the slurry was

diluted to 5, 4, 3, or 2 M urea and stirred for 2 h at room

temperature. The slurries were centrifuged at 5,0009g for

15 min; the supernatant was collected and immediately

used for enzymatic analysis.

Protein purity was assessed by SDS-PAGE, and the identity

of the CcmM protein was verified by Western blot analysis.

Rabbit polyclonal antibodies were created in house against

two highly conserved regions of the SynCcmM protein. Syn-

thetic peptides corresponding to the N-terminal c-CA-like

domain (APGSSIRADEGTPFWIGGN) and the Rubisco

small subunit-like domain (HAGEYVRIIGIDRVGKRRVL)

were synthesized by Advanced SynTech Corporation (Tor-

onto, Canada) and used as the antigen. The IgG fraction from

immunized serum was separated using a Protein G affinity

column (Sigma). Reactivity of the antibodies (anti-CcmM NT

and anti-CcmM CT) was assessed using bona fide SynCcmM

and Synechocystis sp. PCC 6803 cell lysates. A third CcmM

antibody along with an anti CcmK antibody, raised against

SycCcmM and SycCcmK, respectively (Long et al. 2010), was

used in the analysis of carboxysome-enriched fractions. Anti

Rubisco antibody (anti-RbcL/S) was a gift from Dr. S. Whit-

ney, Australian National University. Protein concentrations

were determined by the Bradford method (Bio-Rad) using

bovine serum albumin as a standard.

Immunogold electron microscopy

Nostoc PCC 7120 cells (1 mL) were centrifuged at

10,000 rpm for 1 min. The pellet was suspended and fixed

with 1 % v/v glutaraldehyde (in 0.1 M Sorensen’s phos-

phate buffer) for 2 h at room temperature. The sample was

washed by centrifugation three times with Sorensen’s

phosphate buffer for 5 min. The cells were then dehydrated

using a graded ethanol series (50, 70, 95 % v/v) at room

temperature followed by 3 9 10 min washes with 100 %

ethanol. Samples were infiltrated with LR white resin in the

following ratios: 1:1 LR white (London Resin Co Ltd,

England) and 100 % ethanol for 30 min, 3:1 LR white

resin and 100 % ethanol for 1 h followed by 100 % LR

white for an hour at 4 �C. An additional overnight incu-

bation with 100 % LR white was performed, preceding a

final treatment for 4 h at 4 �C. Fractions of the pellet were

transferred to 00 gelatin capsules and covered with LR

white resin. Polymerization step was performed overnight

at 58 �C as prescribed previously (Lichtle et al. 1992).

Sections were cut with a Reichert Ultracut E microtome

and collected on 300-nm mesh nickel grids.

Post-embedding immunolabeling

Sample grids were incubated with blocking solution (100

lL of conditioned TBS supplemented with 1 mM CaCl2,

1 mM MgCl2, 0.1 % globulin free BSA, 0.4 % cold water

fish gelatin, pH 7.0) for 2 h. Grids were then transferred to

drops containing 100 lL of the primary antibody, either

anti-RbcL/S or anti-CcmM CT at a 1:100 (v/v) dilution and

incubated for 2 h. The grids were washed four times (2, 2,

4, and 4 min incubations) with TBS and then exposed to

100 lL of the secondary antibody at 1:100 (v/v) dilution

(Aurion Immuno Protein A gold, 15 nm) for an additional

2 h. Grids were re-washed three times for 3 min and

allowed to air dry. Sections were post-stained with satu-

rated uranyl acetate for 3 min and rinsed 10 times for

1 min with distilled water. The samples were viewed using

a Hitachi H7000 transmission electron microscope at an

accelerating voltage of 75 kV.

Carbonic anhydrase activity assays

CA activity was determined by three independent methods.

An aqueous inlet mass spectrometer (model MM 14-80 SC:

Photosynth Res (2014) 121:135–150 137

123

VG Gas Analysis, Middlewich, England) was used to

measure 18O exchange between 13C18O2 and H216O (Miller

et al. 1997; Silverman 1982), by following changes in13C16O16O (m/z = 45), 13C18O16O (m/z = 47), and13C18O18O (m/z = 49) over time in aqueous solution. The18O enrichment of the 13CO2 species (atom %) was cal-

culated as log18O atom % = log [([47] ? 2[49])/(2

([45] ? [47] ? [49]))) 9 100]. Standard assay conditions

employed 100 mM EPPS/NaOH buffer, pH 8, 20 mM

MgSO4, 10 lM ZnSO4, and 0.6 mM K213C18O3 as substrate

at 30 �C. The final protein concentration was 8 lg mL-1.

CA activity was also determined using the electrometric

assay (Wilbur and Anderson 1948). The time required for

the pH of a weakly buffered solution to change from 8.1 to

7.0 following the addition of CO2 was recorded in the

absence or presence of protein. CA activity per mg protein

was expressed as, Activity Units = (to - t)/t, where to and

t are the time required for the pH to change from 8.1 to 7.0

in absence and presence of the protein, respectively.

Steady-state kinetic measurements of CA activity

Initial rates of CO2 hydration were determined by stopped-

flow spectroscopy (Hi-Tech SF-61DX2 spectrophotometer)

at 25 �C using the changing pH-indicator method (Khalifah

1971; Rowlett et al. 1991). Saturated solutions of CO2 were

prepared by bubbling CO2 gas (100 % v/v) into distilled

water in a vessel maintained at 25 �C. Dilutions of the CO2

solution were prepared by mixing with degassed water to

yield the desired CO2 concentration. Mixing was achieved

by coupling two gastight syringes with a luer-lock con-

nector. CO2 concentrations were calculated on the basis of

a 33.8 mM saturated solution at 25 �C (Rowlett et al.

1991). The following buffer/indicator combinations and

absorbance wavelengths were used: at pH 5.5–7.0, MES

(pKa = 6.1)/chlorophenol red (574 nm); pH 7.0–8.1,

MOPS (pKa = 7.2)/p-nitrophenol (400 nm); pH 8.0–9.5

Bicine (pKa = 8.3)/m-creosol purple (578 nm). Buffer

concentration during measurements was 40 mM. The

steady-state kinetic constant kcat, Km, and kcat/Km were

determined by fitting the observed initial rates to the

Michaelis—Menten equation as described previously

(Rowlett et al. 2002).

Alternative substrates

Carbonyl sulfide (COS) is an isoelectronic structural analog

of CO2 and it may, therefore, serve as an alternative sub-

strate for NstCcmM209. A COS stock solution was pre-

pared by sparging ice-cold acidified water with a

compressed gas mixture containing 1.05 % v/v COS in

nitrogen. Based on COS solubility in water, the stock

solution contained 0.631 mM COS (Miller et al. 1989; So

et al. 2002a). The capped solution was stored on ice and

periodically re-sparged during the day. COS can be directly

measured by aqueous inlet mass spectrometry at m/z = 60.

COS stock solution was injected into the mass spectrom-

eter cuvette containing 100 mM EPPS/NaOH pH 7,

20 mM MgSO4 at 30 �C to establish an initial concentra-

tion. The spontaneous hydrolysis of COS was then mea-

sured over time. A protein sample was subsequently

introduced into the reaction cuvette, and the effect of the

protein on the m/z = 60 signal was followed over time.

The ability of NstCcmM209 to hydrolyze p-

nitrophenylacetate was tested at room temperature (about

22 �C). The enzyme was incubated with 1.5 mM p-

nitrophenylacetate in the above buffer, pH 7.2, and the

change in A348 was followed over time.

Effect of temperature on the CA activity

The effect of temperature on NstCcmM209 CA activity

was determined by incubating protein samples at select

temperatures between 5 and 35 �C for 15 min and then

assaying for activity at the same temperature using the 18O

exchange method. CA activity above 35 �C could not be

reliably measured due to excessive permeability of the

membrane inlet. The thermostability of NstCcmM209 was

determined by incubating protein samples for 15 min at

select temperatures between 10 and 75 �C, and then

assaying for CA activity using the 18O exchange method

under standard conditions at 30 �C.

Fluorescence spectroscopy

A Fluoromax 4 (HORIBA Scientific) spectrometer equip-

ped with a Peltier unit for temperature control was used to

obtain intrinsic tryptophan fluorescence emission spectra

from protein samples in 20 mM Tris–HCl, pH 7.5–8.0.

Proteins solutions (1 lM) were excited at 290 nm (Trp

excitation, 1 nm band width), and emission spectra (6 nm

band width) were collected from 300 to 450 nm over a

range of temperatures from 10 to 95 �C. Protein solutions

were incubated for 60 s at each temperature increment

prior to recording the spectrum. Maximum fluorescence

emission was observed at 343 nm in all cases, and this

value was used for subsequent analysis.

Circular dichroism (CD) spectroscopy

The CD spectra (195–300 nm) of NstCcmM209 were

obtained using AVIV 250 CD spectrometer (Aviv Bio-

medical Inc., Lakeview, NJ, USA) in a quartz cuvette with

a 1mm path length. At each temperature from 10 to 80 �C,

the protein solution (10 lM) was scanned three times and

an average spectrum was determined.

138 Photosynth Res (2014) 121:135–150

123

Results

Identification and evaluation of candidate

carboxysomal carbonic anhydrases

The identity of the Nostoc PCC 7120 carboxysomal CA has

not been determined experimentally. Candidate CAs in the

proteome were identified by BlastP searches of the NCBI

database (http://www.ncbi.nlm.nih.gov) using the sequen-

ces of experimentally validated a, b, c, and d CAs as

search query. Proteins directly corresponding to previously

identified carboxysomal CAs, CcaA or CsoSCA, were not

found in the Nostoc PCC 7120 proteome when queried

with Synechocystis PCC 6803 CcaA (NCBI accession

AAC46375) or Halothiobacillus neapolitanus CsoSCA

(EEG96215), respectively. Similarly, no d-CA homolog

was found when the proteome was queried with T.

weissflogii CA1 (AAX08632). Four potential candidates

were identified; the a-CA, EcaA (NP_486969, all2929), the

b-CA, EcaB (NP_486950, all2910), and the c-CAs, CcmM

(NP_484908, all0865) and NP_485067 (alr1024), currently

annotated as a ferripyochelin-binding protein.

EcaA was previously identified as a catalytically inac-

tive a-CA homolog localized to the cell surface (So et al.

1998; Soltes-Rak et al. 1997). Analysis of the amino acid

sequence of EcaA using the TatP 1.0 algorithm (http://

www.cbs.dtu.dk/services/TaTP/) revealed the presence of

an N-terminal twin arginine translocation signal, RRQLL,

consistent with EcaA export to the periplasmic space. The

b-class CA homolog, EcaB, from Synechocystis PCC 6803

has been previously identified as a periplasmic protein in a

large-scale proteomic experiment (Fulda et al. 2000).

However, recombinant EcaB was unable to catalyze 18O

exchange, suggesting that it lacks CA activity (So et al.

1998). TatP analysis of the Nostoc PCC 7120 EcaB

sequence indicated the presence of an N-terminal twin

arginine signal sequence with an RRNFL motif, consistent

with a periplasmic localization.

The two candidate c-CA-like proteins identified in the

Nostoc PCC 7120 proteome are the 555 amino acid CcmM

(NP_484908), and the 202 amino acid polypeptide anno-

tated as ferripyochelin-binding protein (NP_485067).

NstCcmM209 (the first 209 amino acids) has 72, 70, 65,

and 62 % amino acid sequence identity to the same region

of TeCcmM, Synechococcus PCC 7002 CcmM, SycCcmM,

and SynCcmM, respectively, and 40 % identity to MtCam.

This protein has all of the residues currently known to be

required for c-CA activity, and the N- and C-terminal

motifs demonstrated to be necessary in TeCcmM for acti-

vation (Fig. 1). NP_485067, in contrast, is only 30 %

identical to TeCcmM209, and lacks the RbcS-like C-ter-

minal domains, the CcmM activation motifs, and lacks two

residues (E56, N185) demonstrably important for CA

activity. This protein is also only 25 % identical to both

MtCam and MtCamH. While NP_485067 is 40 % identical

to PgiCA, a recently characterized c-CA from the anaer-

obic eubacterium Porphyromonas gingivalis (Del Prete

et al. 2013), it should be noted that PgiCA is inhibited by

almost all anions, including chloride (KI = 0.9 mM), and it

is only in a perchlorate buffer that activity has been dem-

onstrated. The actual physiological efficacy of this enzyme

as a CA within the cell is, therefore, debatable. As

NP_485067 has none of the motifs associated with carb-

oxysome localization or activation, and is only distantly

related to established c-CAs, we do not consider it further.

CA activity in Nostoc is detectable

only in the carboxysomal fraction

The sensitive 18O exchange assay was not able to detect

CA activity on the external cell surface of intact Nostoc

PCC 7120 cells (200 lg Chl mL-1) or in equivalent cel-

lular lysate (Fig. 2a). Following Percoll-Mg2? fraction-

ation of cell lysates, however, a 2.6-fold enhancement in18O exchange was readily detected in the TP pellet

(Fig. 2a). This fraction is enriched in carboxysomes and

carboxysome fragments (Long et al. 2005, 2007). Other

fractions devoid of carboxysomes lacked enhanced 18O

exchange activity, suggesting that CA activity was pre-

dominately localized to the carboxysome fraction. 18O

exchange activity was inhibited by the classic CA inhibitor

ethoxyzolamide (EZ) and by denaturing conditions

(Fig. 2a, c) indicating the presence of a protein catalysis in

the TP pellet.

NstCcmM has CA activity localized within the N-

terminal domain

The full-length ccmM gene product with an N-terminal T7

affinity tag, T7-NstCcmM555, was overexpressed in

E. coli. CA activity was detected in crude cell lysates but

not in cell lysates that lacked the expression plasmid.

However, CA activity was not observed following centri-

fugation of the lysate. Most of T7-NstCcmM555 was

subsequently found in insoluble inclusion bodies. Treat-

ment of the inclusion bodies with urea solubilized T7-

NstCcmM555 and yielded a protein with strong 18O

exchange activity, diagnostic for CA (Fig. 2b). 18O

exchange activity in the urea-treated protein fraction

obtained from control E. coli cells that harbored the

pET21b null construct alone was similar to uncatalyzed

control levels (Fig. 2b). Complete removal of urea from

T7-NstCcmM555 resulted in protein precipitation and loss

of catalytic activity. Similar outcomes were observed for

an N-terminal 6X-His-tagged version of NstCcmM555 (not

shown).

Photosynth Res (2014) 121:135–150 139

123

To circumvent these problems, a 6X-His-tagged version

of CcmM containing the first 209 amino acids

(NstCcmM209) was constructed and purified using IMAC.

NstCcmM209 proved to be highly soluble (at 3–5 mg/mL)

and showed 18O exchange activity (Fig. 2c) comparable to

that observed for T7-NstCcmM555. NstCcmM209 and

TeCcmM209 SS*=

TeCcmM209 1 ------MAVQ---------------SYAA-PP-TPWSRDLAEPEIAPTAYVHSFSNLIGDNstCcmM209 1 ------MAVR---------------STAA-PP-TPWSRSLAEAQIHESAFVHPFSNIIGDMtCam 1 QEITVDEFSN---------------IREN-PV-TPWNPEPSAPVIDPTAYIDPQASVIGEMtCamH 1 -------------------------MKRNFKMHLP-NPHKQHPKVSKRAWISETALIIGNPgiCA 1 ---------MAQRENSDYLTTKMALIQSV-R--------GFTPIIGEDTFLAENATIVGD

TeCcmM209 SS # #* * *# + *

TeCcmM209 38 VRIKDYVHIAPGTSIRADE-GTPFHIGSRT-NIQDGVVIHGLQQ----------GRVIGDNstCcmM209 38 VHIGANVIIAPGTSIRADE-GTPFHIGENT-NIQDGVVIHGLEQ----------GRVVGDMtCam 44 VTIGANVMVSPMASIRSDE-GMPIFVGDRS-NVQDGVVLHALETINEEGEPIEDNIVEVDMtCamH 35 VSIADDVFVGPNAVLRADEPGSSITV-HRGCNVQDNVVVHSLSH--S-------------PgiCA 43 VVMGKGCSVWFNAVLRGDV--NSIRIGDNV-NIQDGSILHTLYQ----------------

TeCcmM209 SS = + +

TeCcmM209 86 DGQEYSVW-IGDNVSITHMALIHGPAYIGDGCFIGFRSTVFN-ARVGAGC-VVMMHVLI-NstCcmM209 86 DNKEYSVW-VGSSASLTHMALIHGPAYVGDNSFIGFRSTVFN-AKVGAGC-IVMMHALI-MtCam 102 -GKEYAVY-IGNNVSLAHQSQVHGPAAVGDDTFIGMQAFVFK-SKVGNNC-VLEPRSAA-MtCamH 79 ---E--VL-IGKNTSLAHSCIVHGPCRIGEDCFIGFGAVVFD-CNIGKDTLVLH-KSIV-PgiCA 84 ----KSTIEIGDNVSVGHNVVIHGA-KICDYALIGMGAVVLDHVVVGEGA-IVAAGSVVL

TeCcmM209 SS * *

TeCcmM209 142 QDVEIPPGKYVPSGMVITTQQQADRLPNVEE--SDIHFAQ-HVVGINEALLSGY-Q--CANstCcmM209 142 KDVEVPPGKYVPSGAIITNQKQADRLPDVQP--QDRDFAH-HVIGINQALRAGY-L--CAMtCam 157 IGVTIPDGRYIPAGMVVTSQAEADKLPEVTDDYAYSHTNE-AVVYVNVHLAEGY-K--ETMtCamH 129 RGVDISSGRMVPDGTVITRQDCADALEDITK--DLTEFKR-SVVKANIDLVEGYIRLREEPgiCA 138 TGTQIEPNSIYA-GAP------ARFIKKVDP--EQSREMNFRIAHNYRMYASWF-KDESS

TeCcmM209 SS

TeCcmM209 196 ENIACIAPIRNELQ 209NstCcmM209 196 ADSKCIAPLRNDQV 209MtCam 213 S------------- 213MtCamH 187 S------------- 187PgiCA 188 E-------IDNP-- 192

Fig. 1 Structure-based multiple sequence alignment of the currently

known, catalytically active c-CAs: TeCcmM209, Thermosynecho-

coccus elongatus BP1 CcmM (NCBI Accession NP_681734);

NstCcmM209, Nostoc PCC 7120 CcmM (NP_484908) this study;

MtCam, Methanosarcina thermophilia Cam (P40881); MtCamH, M.

thermophilia CamH (ACQ57353), and PgiCA, Porphyromonas

gingivalis W83 PgiCA (NP_905402). Sequences were aligned using

T-Coffee—Expresso (http://www.tcoffee.org) and PDB (http://www.

rcsb.org) model structures 3kwcF (TeCcmM209 & NstCcmM209),

1qreA (MtCam), 3ou9A (MtCamH), and 3vnpC (PgiCA). The SS of

TeCcmM209 is shown; b, b-sheet; a, a-helix. Symbols: red inverse,

80 % or greater sequence identity; yellow black, 80 % or greater

sequence similarity; bold and bold underline strain specific sequences

relevant to catalysis; hash mark structurally relevant residues identi-

fied in MtCam; asterisk catalytically relevant residues identified in

MtCam; ?, metal ligands identified in MtCam and TeCcmM209; C C,

Cys194–Cys200 disulfide bond identified in TeCcmM209 as catalyt-

ically relevant; =, source of Trp fluorescence in NstCcmM 209

140 Photosynth Res (2014) 121:135–150

123

NstCcmM555 CA activity were both also significantly

impaired by EZ (Fig. 2c). Consequently, we used the 6X-

His-tagged version of NstCcmM209 as a functional sur-

rogate for the catalytic activity of the full-length CcmM555

protein.

Electrometric assays indicated that NstCcmM209 cata-

lyzed CO2 hydration at a rate of 2,560 units mg-1 protein

(Table 1). On a protein basis, this level of activity was

approximately 6 % of the activity of the highly active

bovine a-CA II (Table 1). Under air-oxidizing conditions,

addition of 10 mM of the thiol oxidizing agent diamide

enhanced the CA activity of NstCcmM209 by 10 %.

Conversely, when NstCcmM209 was incubated with the

phosphine reducing reagent tris(hydroxymethyl)phosphine

(THP) CA activity at 4 �C was reduced by 88.4 %

(Table 1), and by 60 % at 30 �C as determined by the 18O

exchange assay (Fig. 2d). This inhibitory effect of 10 mM

THP could be fully reversed by subsequent treatment with

a molar excess (20 mM) of diamide (Table 1). These

results suggest that NstCcmM209 is activated under oxi-

dizing conditions and substantively inhibited under reduc-

ing conditions.

Kinetic characterization of NstCcmM209

CA activity of recombinant NstCcmM209 has been dem-

onstrated by both mass spectrometric and electrometric

assays (Fig. 2; Table 1). However, kinetic parameters

describing the catalytic features of this b-carboxysomal c-

CA are lacking. Thus, we have conducted an initial

investigation to determine steady-state kinetic parameters

by stopped-flow spectroscopy. Figure 3 shows the pH

dependence of kcat, and kcat/Km for the CO2 hydration

reaction at 25 �C. The rate constant kcat increases with

increasing pH to a maximal value of 1.9 ± 0.1 ms-1, and

is apparently controlled by a single ionization with a pKa

value of 6.83 ± 0.08 (Fig. 3a). The rate constant kcat/Km

appears (Fig. 3b) to be approximately log-linear with a

slope of 0.58 ± 0.03, suggesting that this rate constant is

controlled by two or more ionizations in the pH range. The

data can be satisfactorily fitted to a two-pKa model with

amplitudes of 2.8 ± 0.8 and 92 ± 246 lM-1 s-1 con-

trolled by pKa values of 7.3 ± 0.2 and 10 ± 1, respec-

tively. The second amplitude and pKa are poorly defined

because the apparent pKa value lies outside the experi-

mental range of data. However, the two-pKa dependence of

kcat/Km is qualitatively similar to that reported for MtCam.

Fig. 2 CA activity assays. CA activity was detected as acceleration

in the rate of loss of 18O from 13C18O labeled substrate following the

addition of sample to the reaction vessel under standard conditions:

100 mM EPPS, pH 8.0, 20 mM MgSO4, 10 lM ZnSO4, 30 �C,

600 lM K213C18O3. a As labeled: uncatalyzed control in the absence

of protein, Nostoc whole-cell lysate, denatured (boiled) TP pellet and

carboxysome-enriched TP pellet derived from Nostoc PCC 7120 cell

lysate, b purified T7- NstCcmM555 (230 lg mL-1) in the absence/

presence of 5 mM EZ or the null construct (pET28b) (141 lg mL-1).

c Purified NstCcmM209 (8 lg mL-1) in the absence/presence of

5 mM EZ. Bovine CA (4 lg mL-1) served as a positive control,

d purified NstCcmM209 (8 lg mL-1) in the absence/presence of

10 mM THP

Table 1 Electrometric determination of NstCcmM209 CA activity

under oxidizing and reducing conditions

Protein CA activity, units/mg

proteina%

Control

NstCcmM209 2,560 100.0

NstCcmM209 ? 10 mM diamide 2,814 109.9

NstCcmM209 ? 10 mM THP 296 11.6

NstCcmM209 ? 10 mM

THP ? 20 mM diamide

2,875 112.3

Bovine CA 45,608 1,781.6

a n = 3

Photosynth Res (2014) 121:135–150 141

123

The maximal kcat value and the low-pH amplitude of kcat/

Km values fall between the values previously reported for

the Zn2?-containing forms of MtCam and MtCamH

(Zimmerman et al. 2010), but are more than an order of

magnitude lower than those reported for PgiCA (Del Prete

et al. 2013) (Table 2).

Alternative substrates for CcmM

In addition to the reversible hydration of CO2, a-CA enzymes

are known to possess other catalytic activities including

phosphatase and esterase activities with 4-nitrophenyl phos-

phate or 4-nitrophenyl acetate as substrate (Pocker and

Sarkanen 1978). However, b- or c-CAs generally do not seem

to catalyze these reactions (Heinhorst et al. 2006; Innocenti

and Supuran 2010; Innocenti et al. 2004). Similarly,

NstCcmM209 did not display esterase activity against

4-nitrophenyl acetate (not shown). However, like a-CAs

(Chengelis and Neal 1979; Miller et al. 1989; Protoschill-

Krebs and Kesselmeier 1992), NstCcmM209 did catalyze the

decomposition of COS in a substrate and protein concentra-

tion-dependent manner while heat denatured enzyme lost this

ability (Fig. 4) indicating that native protein structure was

essential for expression of this activity. The uncatalyzed

hydrolysis of COS proceeds through an acid-labile mono-

thiocarbonate intermediate that slowly decomposes to CO2

and H2S (Miller et al. 1989). Acidification of the reaction

medium following the consumption of COS by NstCcmM209

did not result in any COS reappearance as would be expected

if there had been a build-up of the reaction intermediate. Our

initial data, therefore, indicate that NstCcmM209 catalyzes

the complete hydrolysis of COS.

Fig. 3 pH dependence of the steady-state kinetic parameters kcat (a),

and kcat/Km (b) for the CO2 hydration reaction at 25 �C catalyzed by

NstCcmM209. Data for kcat were fit to the logarithmic form of the

equation k(obs) = k(max)/(1 ? [H?]/Ka), where k(obs) is the observed

value of kcat, k(max) is the maximal value of kcat at high pH, and Ka

relates to the ionization constant controlling the reaction. For ak(max) = 1.93 9 104 s-1 ± 0.15, pKa = 6.83 ± 0.08. Data for kcat/

Km were fit to a log-linear function with slope 0.58 ± 0.03 (solid line)

and to the logarithmic form of the equation kobs ¼ k1

1þ½Hþ�

Ka1

þ k2

1þ½Hþ�

Ka2

,

where kobs is the value of kcat/Km at any given pH, k1

(2.9 9 106 ± 0.8 M-1 s-1) and k2 (92 9 106 ± 246 M-1 s-1) are

the low and high-pH amplitudes of kcat/Km, and Ka1 and Ka2 are the

controlling acid dissociation constants (pKa1 = 7.3 ± 0.2;

pKa2 = 10 ± 1) (dashed line)

Table 2 Comparison of steady-state kinetic parameters for the CO2

hydration reaction

Enzyme kcat (s-1) Km

(mM)

kcat/Km

(M-1 s-1)

pH/

(�C)

NstCcmM209a 2.2 9 104 5.2 4.1 9 106 8.0/25

NstCcmM209a 1.9 9 104 10.2 1.9 9 106 7.5/25

Zn-MtCamb 11.6 9 104 15.6 7.5 9 106 7.5/25

Zn-MtCamHb 0.05 9 104 3.1 0.16 9 106 7.5/25

PgiCAc 41 9 104 7.6 5.4 9 107 7.5/20

CsoSCAd 8.9 9 104 3.2 2.8 9 107 8.0/25

Data was obtained using stopped-flow spectroscopy at the indicated

pH and temperature and are derived from a this study, b Zimmerman

et al. (2010), c Del Prete et al. (2013) and d Heinhorst et al. (2006)

Fig. 4 Time courses of COS consumption mediated by

NstCcmM209. Dissolved COS (5.5 lM) was introduced into the

reaction chamber of an aqueous inlet mass spectrometer, and COS

disappearance was measured over time at m/z = 60 in the absence or

presence (upward arrow) of NstCcmM209 (45–354 lg mL-1). Fol-

lowing COS consumption the reaction medium was acidified to pH 4

with HCl (asterisk) to determine residual [COS]

142 Photosynth Res (2014) 121:135–150

123

Effect of temperature on catalysis

The effect of temperature (5–35 �C) on the catalytic

activity of NstCcmM209 was assessed using the 18O

exchange assay (Fig. 5a). The data were normalized to the

activity (100 %) obtained at 35 �C. At the lowest temper-

ature examined (5 �C), catalytic activity was depressed by

around 90 % relative to the control (Fig. 5a). As antici-

pated, NstCcmM209 activity was increased with increasing

temperature with a Q10 value[2. Maximum activity in our

experiments was achieved across a range from 25 to 35 �C.

At 25 �C, NstCcmM209 maintained maximum activity for

at least 6 h. Instrument limitations precluded assays at

higher temperatures. Regardless, NstCcmM209 responded

dynamically to changes in temperature over a physiological

range that is relevant to the growth of Nostoc PCC 7120.

Thermostability

The thermostability of NstCcmM209 was characterized

using the 18O exchange CA activity assay (Fig. 5a).

Enzyme samples were initially incubated for 15 min at a

selected temperature between 10 and 75 �C, followed by

activity measurements under standard conditions at 30 �C.

The 18O exchange data were normalized (100 %) to the

rate observed for samples incubated at 10 �C. Exposure of

NstCcmM209 to temperatures between 10 and 45 �C prior

to assay at 30 �C had little effect on the activity of the

protein (Fig. 5a), indicating that the reduced activity at

temperatures below 25 �C does not reflect irreversible

changes to protein structure. A precipitous loss of catalytic

activity was observed for proteins pre-treated at tempera-

tures above 50 �C, with no activity being recovered from

proteins exposed to 75 �C (Fig. 5a). These data suggest

that NstCcmM209 undergoes thermal denaturation begin-

ning around 50 �C and is irreversibly denatured at 70 �C

and above.

The far-UV CD spectrum provides a convenient means

to query the effect of temperature on global secondary

structure (SS) (Fig. 5c). As a reference, the structure of the

closely related TeCcmM209 has 3 a-helices and 22 b-

strands, comprising 18 and 28 % of the protein, respectively

a b

c d

Fig. 5 The effect of temperature on NstCcmM209 catalytic activity,

thermostability, and SS. a Effect of assay temperature (5–35 �C) on

NstCcmM209 activity was measured by mass spectrometry under

standard conditions (filled squares). CA activity at 35 �C was set to

100 % (n = 3). Also shown is the effect of temperature pre-treatment

(10–75 �C) on thermostable NstCcmM209 activity as assayed by

mass spectrometry at 30 �C (open circles). The activity for the 10 �C

pre-treatment was set to 100 % (n = 3). Dashed black curve,

thermostability profile of MtCam (Alber and Ferry 1996). b Effect

of temperature on endogenous tryptophan fluorescence yield.

NstCcmM209 protein solutions (1 lM, 20 mM Tris–HCl, pH 8.0)

were incubated at a range of temperatures between 10 and 80 �C. At

each temperature tryptophan fluorescence yield was determined at

343 nm (n = 3). This thermal melt curve was normalized to the

fluorescence yield measured at 10 �C. c Effect of temperature

(10–80 �C) on the far-UV CD spectra of NstCcmM209 (10 lM,

20 mM Tris–HCl, pH 8.0). The spectra are the average of 3 scans

each. Spectra are arranged from the bottom (10 �C) to top (80 �C)

alternating between a solid line and a dotted line d Temperature

induced changes in a-helical content of NstCcm209 (10 lM, 20 mM

Tris–HCl, pH 8.0) inferred from CD measurements at 222 nm

Photosynth Res (2014) 121:135–150 143

123

(Pena et al. 2010) (Fig. 1). At 10 �C, the CD spectrum of

NstCcmM209 is dominated by a large swing in mean molar

ellipticity from a high positive value at 195 nm to a low

negative value at 206–208 nm. The positive band at 195 nm

and negative bands at 208 and 222 nm are characteristic of

a-helical proteins, while proteins with well-defined b-sheets

typically have a positive band at 195 nm and a minimum at

218 nm (Greenfield 2007; Kelly et al. 2005). The latter

features are not resolved in our spectra; however, the

atypical phi-psi angles seen in left-handed b-helices along

with the large numbers of identically structured turns may

result in spectral features that differ from those seen with

more typical, twisted b-sheets. The CD spectra change little

between 10 and 45 �C, but above 50 �C show a progressive

loss of mean molar ellipticity and a shifting of the minimum

upstream, indicative of protein unfolding (Fig. 5c, d)

(Greenfield 2007). The loss of CD signals at 208 and

222 nm in particular indicates a rapid decline in a-helical

content above 50 �C (Fig. 5d) and provides a mechanistic

explanation for the loss of CA activity in NstCcmM209

exposed to temperatures over 50 �C (Fig. 5a). Near com-

plete denaturation of NstCcmM209 SS was observed at

70 �C (Fig. 5c, d), with the presence of protein aggregates

indicated from an increase in molar ellipticity observed

between 240 and 260 nm.

We also used intrinsic tryptophan fluorescence to follow

structural changes in NstCcmM209 as a function of tem-

perature. Tryptophan fluorescence yield decreases as a

result of increasing solvent quenching and, therefore, pro-

vides information on the extent to which this hydrophobic

residue is buried by native protein structure. NstCcmM209

has two tryptophan residues. Trp13 is located in the b1-b2

loop within a conserved motif (APPTPWS, Fig. 1). This

loop extends to within 1.5 nm of the active site and also

makes contact with the aC helix (Pena et al. 2010). Trp93

is located on b13 of the left-handed b-helix and appears to

be shielded by the b11-b12 loop and, therefore, is protected

from the solvent. Trp13 and/or Trp93 were increasingly

solvent exposed as the temperature increased from 10 to

55 �C (Fig. 5b). There is an abrupt transition in the fluo-

rescence yield curve at 55 �C suggesting a major reorga-

nization of the protein. The fact that tryptophan

fluorescence remained relatively constant from 60 �C

onward suggests that these residues are fully solvent

exposed and disordered.

CcmM and Rubisco are present in Nostoc

carboxysomes

SDS-PAGE and Western blot analysis indicated that purified

NstCcmM209 (MW = 24.9 kDa) and T7-NstCcmM555

(MW = 60.7 kDa) immunologically cross-reacted with an

antibody (anti-CcmM NT) directed against the N-terminal

domain of CcmM (Fig. 6a). A second antibody, anti-CcmM

CT (directed against the C-terminal region of CcmM), was

also tested against Nostoc PCC 7120 cell lysate and E. coli

cell lysate expressing recombinant 6X-His-NstCcmM555

(Fig. 6b). Two protein bands were observed to cross-react in

the Nostoc PCC 7120 lysate with approximate MWs of 59

and 35 kD. The first protein corresponds to the predicted

size of the full-length NstCcmM555 (59.4 kD), while the

second protein corresponds to the C-terminal ‘‘short form’’

of CcmM (35.4 kD) that is translated from an internal

ribosome-binding site within the ccmM transcript (Long

et al. 2010). No other protein bands were observed. The

same two immune-reactive bands were also observed in

E. coli lysate harboring the ccmM expression plasmid, but

not in the null control.

The presence of CcmM in the TP pellet was verified by

SDS-PAGE analysis which revealed 23 major protein

bands (Fig. 7). TP pellets were also analyzed by 2D LC–

MS/MS and a number of carboxysomal proteins were

positively identified as significant matches to proteins from

the Nostoc PCC 7120 proteome database (Table S2). These

included RbcL, RbcS, CcmM555, CcmK1, and CcmK2.

The other proteins identified included hypothetical pro-

teins, transcriptional regulator proteins, and a set of 30S

and 50S ribosomal proteins, similar to the contaminants

found in TP pellets from Synechococcus PCC 7942 (Long

et al. 2007). CcmL, CcmN, and CcmO were not among the

identified proteins.

Western blot analysis of the TP pellet confirmed the

presence of RbcL, CcmM555, and the 35.4 kDa short form

of CcmM (Fig. 7). RbcS was detected as a faint band using

anti-RbcL/S (Fig. 7). A single protein around the mass

expected for CcmK was identified using an antibody to the

shell protein CcmK2 raised against the Synechococcus

PCC 7942 protein. The CcmM short form has been pre-

viously identified in Nst-TP pellets by Western analysis

and its identity verified by Edman sequencing of the

N-terminal 7 amino acids (Long et al. 2007).

The cellular distribution of Rubisco and CcmM was then

analyzed by immunogold electron microscopy using either

anti-RbcL/S or anti-CcmM CT. A total of 100 medial cell

sections were analyzed in each instance. On average, each

cross section of an air-grown Nostoc PCC 7120 cell had 1.6

carboxysomes (range of 0 to 7) distributed through the cell.

Nostoc PCC 7120 carboxysomes are typically larger (400-

600 nm on the long axis) and more irregularly shaped (Fig.

S1) than the smaller, polygonal carboxysomes (100-

200 nm in diameter) found in many unicellular cyanobac-

teria (Espie and Kimber 2011; Long et al. 2007; Rae et al.

2013). When Nostoc PCC 7120 cells were labeled with

anti-RbcL/S, 49 % of the gold particles were found local-

ized within carboxysomes (Table 3; Fig. S1) while the

remaining gold particles were distributed throughout the

144 Photosynth Res (2014) 121:135–150

123

cytoplasm and sporadically over the thylakoid membranes.

Gold particle density was 15.2 times higher in the carb-

oxysome than in the rest of the cell, indicating that Rubisco

is highly concentrated within these structures, in agreement

with the observations of Jager and Bergman (1990). When

Nostoc PCC 7120 cells were labeled with anti-CcmM CT

about one-third of the gold particles were located in visu-

ally identified carboxysomes (Table 3; Fig. S1). Again, the

remainder of the gold particles was distributed throughout

the cell. Similar to Rubisco, CcmM was also found to be

concentrated (ninefold) within the carboxysome relative to

the cytoplasm (Table 3). In general, CcmM seemed to be

distributed throughout the carboxysome lumen and was not

preferentially associated with the shell. This antibody

detects both CcmM555 and the short form and cannot,

therefore, resolve the specific location of either form within

the carboxysome.

Discussion

Carboxysomes are specialized microcompartments that

support the photoautotrophic growth of cyanobacteria under

normal environmental conditions. These structures are

encapsulated by a unique protein shell in which both Rubisco

and its substrate, CO2, are concentrated. Once encapsulated,

Rubisco is a permanent resident of the carboxysome (Chen

et al. 2013) where it mediates CO2 fixation. In contrast,

carboxysomal CO2 must be constantly generated from the

cytosolic pool of HCO3-, which is itself created by the active

transport of inorganic carbon from the environment into the

cell. Crucial to the function of the cyanobacterial CCM is a

carboxysome localized, luminal CA that generates the CO2

required to sustain Rubisco at near maximal rates of fixation.

The behavior and catalytic characteristics of the carbox-

ysomal CA are, therefore, important for understanding both

the CCM and carboxysome function and factors that limit

primary productivity. In contrast to a-cyanobacteria which

have only CsoSCA, b-cyanobacteria have two dis-

tinct carboxysomal CAs (CcaA and CcmM) that have been

identified and characterized to various degrees. Given the

diversity of b-cyanobacteria, however, we cannot yet

exclude the possibility that there are additional, unidentified

carboxysomal CAs within this lineage. Identification of b-

carboxysomal CAs is complicated by our inability to isolate

b-carboxysomes in pure form. Experimental investigations

have also been confined primarily to three unicellular model

organisms, which represent only a small fraction of the

genetic diversity of b-cyanobacteria. To address these lim-

itations, we undertook research to identify and characterize

the carboxysomal CA from the filamentous, diazotrophic,

and mesophilic cyanobacterium Nostoc sp PCC 7120.

Fig. 6 Western blot analysis using polyclonal antibodies directed

against a the N-terminal c-CA domain of CcmM: lane 1 molecular

weight markers; lane 2 purified 6X-His NstCcmM209

(0.89 lg mL-1); lane 3 purified 6X-His NstCcmM555

(0.102 lg mL-1); and b C-terminal domain of CcmM: lane 1

molecular weight markers; lane 2 pET21b vector null construct

(0.110 lg mL-1) or 6X-His NstCcmM209, lane 3 purified 6X-His

NstCcmM555 (140 lg mL-1); lane 4, E. coli cell lysate expressing

6X-His NstCcmM555; lane 5 molecular weight markers; lane 6, cell

lysate (200 lg mL-1) from low inorganic-carbon-acclimated Nostoc

PCC 7120

Photosynth Res (2014) 121:135–150 145

123

CcmM is a b-carboxysomal c-carbonic anhydrase

Full length, recombinant NstCcmM555 catalyzed the

reversible hydration of CO2 and by all criteria is a func-

tional CA. CA activity was readily detected in the carb-

oxysome-enriched TP pellet which was shown by Western

blot and 2D-LC–MS/MS to contain CcmM, along with

Rubisco. Immunogold electron microscopy provided fur-

ther evidence that CcmM is concentrated within the carb-

oxysome and co-localizes with Rubisco. On the other hand,

other candidate CAs (EcaA, EcaB, NP_485067) were

systematically excluded as the carboxysomal CA based on

bioinformatics analysis and the results of previous studies.

Consequently, the collective evidence indicates that CcmM

is the sole functional carboxysomal CA of the mesophilic

cyanobacterium Nostoc PCC 7120 and that CcmM cata-

lytic function is not restricted to thermophilic

cyanobacteria.

Based on gold particle distribution, CcmM was also

found in the cytoplasm in unexpectedly large amounts and

in proximity to thylakoid and plasma membranes (Table 3;

Fig. S1). Western blots of Nostoc PCC 7120 cell lysates

using the same anti-CcmM CT antibody as used in the

immunogold experiments indicated the presence of only

two immune-reactive proteins that corresponded to the

CcmM555 and the 35 kDa short form of CcmM. Thus, a

non-specific antibody reaction complex cannot account for

the observed distribution of CcmM in the cytoplasm of

Nostoc PCC 7120 cells. Two large-scale proteomics

experiments have also noted the presence of CcmM (but

not Rubisco) in isolated outer membrane fractions from

both Nostoc PCC 7120 and Synechocystis PCC 6803

(Huang et al. 2004; Moslavac et al. 2005), while both RbcL

and the CcmM short form were found in peripheral

membrane fractions from Synechocystis PCC 6803 using

TrxA-linked/mass spectrometric identification method

(Mata-Cabana et al. 2007). These findings provide some

evidence for a membrane-associated CcmM in addition to

the carboxysome-localized CcmM. In their transmission

electron microscope study of the architecture and biogen-

esis of carboxysomes from the closely related Anabaena

PCC 7119, Orus et al. (2001) observed a variety of carb-

oxysome shapes and textures ranging from electron-trans-

lucent to electron-intermediate and mature, electron-dense

structures. In our experiments, preservation of immature

carboxysomes may have been compromised due to the

reduced amount of fixative used to preserve antigenicity of

the thin sections employed for the immunogold experi-

ments. Consequently, some of the cytoplasmic CcmM may

be associated with developing carboxysomes whose

structures are poorly resolved under our conditions. In

1 2 3 4250150100

75

50

37

2520

15

10

CcmM555

CcmMshort

CcmK2

RbcL

RbcS

Fig. 7 SDS-PAGE (lane 1) and Western blot analysis (lane 2, 3, 4) of

the carboxysome-enriched TP pellet obtained from Nostoc PCC 7120

cell lysate following Percoll-Mg2? fractionation. Lane 1 Bio-Rad

stain-free acrylamide gel showing protein-banding pattern from

enriched carboxysome preparations (TP pellets). Lanes 2–4, Western

blots of SDS-PAGE separated TP pellet probed with; lane 2, anti-

CcmK2; lane 3 anti-CcmM; lane 4 anti-RbcL/S. Blots for lane 4 are

for different loadings of protein for RbcL and RbcS due to the relative

difficulty in detecting RbcS. In this case the antibodies were raised

against the homologous proteins from Synechococcus sp. PCC 7942.

Molecular weight markers are indicated. CcmK2 appears as an

indistinct band below RbcS with Coomassie staining and is not visible

on Bio-Rad stain-free gels due to the absence of tryptophan residues.

Additional proteins identified from TP pellets are indicated in Table

S2

Table 3 Immuno-localization of Rubisco and CcmM in Nostoc PCC

7120

Parametera Anti-

Rubisco

Anti-C-terminal

CcmM

Carboxysome

Gold particles in cbx 1,274 501

Total cbx area (lm2) 11.3 18.9

Average cbx area (lm2)/section 0.11 0.19

Cbx: gold particles (lm2) 112.6 26.4

Cbx/section 1.42 1.77

Cytosol

Gold particles cytosol 1,674 874

Total cytosol area (lm2) 225.3 290.0

Average cytosol area (lm2)/section 2.3 2.9

Cytosol: gold particles (lm2) 7.4 3.0

Total gold particles 2,948 1,375

Cytosol refers to the area of the section minus carboxysome area and

includes the thylakoid membranes, carboxysome = cbxa A total of 100 medial cell sections were analyzed for gold particle

distribution in each case. The sections contained 142 (anti Rubisco)

and 177 (anti-CcmM) carboxysomes respectively. In control experi-

ments lacking antibody, gold particle density was \0.1/lm2 and was

subtracted from the experimental value

146 Photosynth Res (2014) 121:135–150

123

support of this is the observation that nearly 50 % of the

immunogold labeled Rubisco was also found in the cyto-

plasm. The presence of Rubisco outside the mature carb-

oxysome has been noted frequently (Friedberg et al. 1993)

and for Nostoc PCC 7120 in particular (Jager and Bergman

1990). Recently Chen et al. (2013), using live cell fluo-

rescence microscopy, have shown that the first step in the

biogenesis of Synechococcus sp. PCC 7942 carboxysomes

involves the growth of un-encapsulated Rubisco aggregates

in the cytoplasm, providing direct evidence for a second

cellular reservoir for Rubisco outside the mature carboxy-

some. Depending on the study and the cyanobacterial

species investigated, the cytosolic Rubisco content ranges

from about 5–65 % of the total (Agarwal et al. 2009;

McKay et al. 1993). The co-occurrence of CcmM and

Rubisco in the cytoplasm is consistent with a Rubisco-

centric model for b-carboxysome biogenesis (Chen et al.

2013; Rae et al. 2013; Long et al. 2007, 2010) which

proposes that b-carboxysomes are formed around a Rubi-

sco-CcmM organizing complex that is subsequently

encapsulated by shell proteins. Additional experiments are

required, however, to determine if cytosolic CcmM and

Rubisco actually co-localize and form these complexes in

the cytoplasm of Nostoc PCC 7120 cells.

The presence of CcmM in the cytoplasm could potentially

thwart CCM function by prematurely dissipating the cyto-

solic pool of HCO3- (Price and Badger 1989). Indeed, Nostoc

PCC 7120 has substantial capacity to concentrate HCO3-

with a cytosolic pool as high as 60 mM and a [Ci]in/[Ci]out as

high as 360 (McGinn et al. 1997). However, we did not detect

CA activity in concentrated whole-cell lysates. Only when

the TP pellet was separated from other cytoplasmic compo-

nents did the CA activity of the carboxysome become

apparent (Fig. 2). The activity of NstCcmM209 was signifi-

cantly diminished in the presence of the phosphine reducing

agent THP (Table 1; Fig. 2). Thus, the highly reducing

environment of the cytoplasm, particularly in the light, may

prohibit CcmM catalysis outside the mature carboxysome

(Pena et al. 2010) thereby preventing a short circuit in the

CCM. Pena et al. (2010) have shown that reversible Cys194–

Cys200 disulfide/thiol formation mediates the oxidative

activation/reductive inhibition of TeCcmM 209. These resi-

dues are conserved in NstCcmM and provide a potential

mechanism to explain the redox activation/inhibition of this

enzyme. Chen et al. (2013) have recently shown that the

carboxysome remains a reducing environment as it assem-

bles, and only switches to being oxidizing late in its bio-

genesis following recruitment of the shell proteins. Our initial

data indicate that this could be the case for NstCcmM and

suggest that redox activation of the carboxysomal c-CA may

be a general feature of b-carboxysomes that rely on CcmM

catalysis. A detailed analysis of the redox regulation of

NstCcmM209 is currently in preparation.

Kinetic and biophysical characterization of CcmM

Kinetic analysis of NstCcmM209 was conducted in an air-

oxidizing environment as experimental evidence indicated

that sustained, near-maximum activity was maintained

under these conditions (Table 1). The kinetic parameters

for CO2 hydration, kcat and kcat/Km, increased with

increasing pH, indicating that an unprotonated form of

NstCcmM209 is required for activity, consistent with the

widely accepted metal-hydroxide mechanism of CA

catalysis (Alber et al. 1999; Rowlett et al. 2002). The

overall rate-pH profile for the kinetic parameters was

similar to those previously reported for MtCam though the

absolute values were consistently lower (Alber et al. 1999)

(Fig. 3; Table 2), while the NstCcmM209 kinetic parame-

ters were 10–40-fold higher than those reported for

MtCamH (Zimmerman et al. 2010). Under similar condi-

tions, the kcat and kcat/Km for CO2 hydration of

NstCcmM209 were four-fold and 6.8-fold lower than the

corresponding values for CsoSCA from the a-carboxysome

(Heinhorst et al. 2006). Possibly, this similarity in catalytic

values reflects the common function both enzymes play in

the CCM, though the differing size of the carboxysomes

(a-carboxysomes are considerably smaller), organization

(CsoSCA associates tightly with the shell) and Rubisco:

CA stoichiometry complicates direct comparisons. Like

NstCcmM209, CsoSCA is inhibited by thiol reducing

agents (So et al. 2004) though the underlying mechanism is

unknown; it is, therefore, possible that oxidative activation

of carboxysomal CAs is a general phenomenon.

The rate constant kcat is typically controlled by the pKa

of the proton shuttle residue when proton transfer is rate-

limiting; this is typical of most CAs (Rowlett et al. 2002).

For NstCcmM209, the pKa controlling kcat is 6.83 ± 0.08,

which is consistent with Glu78 performing this role. Glu78

in NstCcmM209 is analogous to Glu84 in MtCam (Fig. 1),

which has been shown through mutagenesis to be the

proton shuttle residue in that enzyme (Tripp and Ferry

2000).

The pH-rate profile of kcat/Km for NstCcmM209 is

apparently log-linear, and is not consistent with a single

controlling pKa. The data suggest that two or more pKa

values control kcat/Km in NstCcmM209. In MtCam, kcat/Km

is controlled by two-pKa values with two distinct ampli-

tudes for kcat/Km at low and high pH. We have fit the data

of Fig. 3 in the same fashion, although the high-pH

amplitude and pKa values are not well determined. The

low-pH pKa (7.3 ± 0.2) should correspond to the pKa value

of the metal-bound water molecule, and this value is

qualitatively similar to that of MtCam (Tripp and Ferry

2000).

In addition to the reversible hydration of CO2, our initial

investigation shows that NstCcmM209 also catalyzes the

Photosynth Res (2014) 121:135–150 147

123

hydrolysis of COS, the most abundant sulfur-containing

compound in the atmosphere and a greenhouse gas. This is

the first example of this activity in a c-CA. The carbox-

ysomal b-CA, CcaA, from Synechocystis PCC 6803 (So

et al. 2002a) and a-CAs from diverse organisms including

rat, cow, and the green alga Chlamydomonas also catalyze

COS hydrolysis (Chengelis and Neal 1979; Miller et al.

1989; Protoschill-Krebs and Kesselmeier 1992) suggesting

that this reaction may be a general biochemical feature of

all CAs arising from a common reaction mechanism, as is

the case for the CO2 hydration reaction. COS is also an

alternative substrate, but not an activator, of Rubisco

yielding 3-phosphoglycerate and 1-thio-3-phosphoglycer-

ate as products of the thiocarboxylation of RuBP (Lorimer

and Pierce 1989). The presence of both CA and Rubisco in

carboxysomes suggests that this bacterial organelle may,

therefore, play a role in the biological degradation of COS.

The kinetic effectiveness of carboxysomes in this process

is yet to be demonstrated, but given the sheer number and

global distribution of cyanobacteria, carboxysomes could

be an important environmental sink for COS.

NstCcmM209 displayed a broad pH optimum (8–9.5)

for CO2 hydration activity. Evaluation of enzyme activity

at different temperatures by the 18O exchange assay also

revealed a broad temperature optimum (25–35 �C), which

spans the optimal temperature range for the growth of

Nostoc PCC 7120. Enzyme activity and protein SS were

reversibly stable to thermal treatment between 5 and 45 �C,

a range broader than likely experienced during the normal

physiological growth of Nostoc PCC 7120. A precipitous

loss of activity occurred following temperature treatments

above 50 �C. CD spectra and intrinsic fluorescence emis-

sion spectra revealed a significant loss in a-helical content

and exposure of buried Trp residues that tracked the loss in

activity. These results demonstrate that irreversible thermal

denaturation of NstCcmM209 occurred above 50 �C that

accounts directly for the loss in enzyme activity and may,

therefore, be one factor that limits the growth of Nostoc

PCC 7120 to mesophilic temperatures. By comparison, the

thermal denaturation activity curve for MtCam is shifted

about 10 �C higher (Alber and Ferry 1996), (Fig. 5) con-

sistent with the moderate thermophilic nature of M. ther-

mophila (optimum growth temperature of 55 �C). A similar

shift in the thermal denaturation activity curve of TeCcmM

might, therefore, be anticipated as this enzyme also

evolved and functions in a moderately thermophilic envi-

ronment, unlike NstCcmM. However, we have not found

substantive structural or compositional differences between

the two enzymes to account for any difference in thermo-

tolerance. Further experimentation and analysis are

required to resolve this issue.

CcmM is a characteristic protein of all b-carboxysomes

with an established role as a structural protein and as a

binding partner for CcaA, the conventional carboxysomal

CA in the model organisms Synechocystis sp. PCC 6803

and Synechococcus sp. PCC 7942. Our demonstration that

NstCcmM and TeCcmM (Pena et al. 2010) are both func-

tional c-CAs indicate that the use of CcmM as the carb-

oxysomal CA is likely widespread in cyanobacterial strains

that lack a ccaA gene. Many strains, however, encode what

appear to be a functional CcaA and a functional CcmM

(Pena et al. 2010). The advantage of expressing two dis-

tinct CA enzymes within the same b-carboxysome is

unknown and is currently under investigation.

Acknowledgments This study was supported by grants from the

Natural Sciences and Engineering Research Council of Canada

(NSERC) to G. S. E. and M. S. K. and an NSERC CGSD to C. A.; by

a grant from the Australian Research Council to B. M. L. and G.

D. P., and by a grant from the U.S.A. National Science Foundation,

MCB-1157332, to R. S. R.

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