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Journal of Experimental Botany, Vol. 59, No. 5, pp. 1023–1034, 2008

doi:10.1093/jxb/erm282

Advance Access publication 7 December, 2007

Advance Access publication 7 December, 2007 SPECIAL ISSUE RESEARCH PAPER Proteomic analysis of the

SPECIAL ISSUE RESEARCH PAPER

Proteomic analysis of the cyanobacterium of the Azolla symbiosis: identity, adaptation, and NifH modification

Martin Ekman 1 , Petter Tollba¨ ck 2 and Birgitta Bergman 1, *

1 Department of Botany, Stockholm University, SE-106 91 Stockholm, Sweden

2 Stockholm University Proteomics Facility, Department of Analytical Chemistry, Stockholm University, SE-106 91 Stockholm, Sweden

Received 24 June 2007; Revised 21 October 2007; Accepted 22 October 2007

Abstract

Cyanobacteria are able to form stable nitrogen-fixing symbioses with diverse eukaryotes. To extend our understanding of adaptations imposed by plant hosts, two-dimensional gel electrophoresis and mass spec- trometry (MS) were used for comparative protein expression profiling of a cyanobacterium (cyanobiont) dwelling in leaf cavities of the water-fern Azolla filiculoides. Homology-based protein identification us- ing peptide mass fingerprinting [matrix-assisted laser desorption ionization-time of flight (MALDI-TOF-MS)], tandem MS analyses, and sequence homology searches resulted in an identification success rate of 79% of proteins analysed in the unsequenced cyano- biont. Compared with a free-living strain, processes related to energy production, nitrogen and carbon metabolism, and stress-related functions were up- regulated in the cyanobiont while photosynthesis and metabolic turnover rates were down-regulated, stress- ing a slow heterotrophic mode of growth, as well as high heterocyst frequencies and nitrogen-fixing capacities. The first molecular data set on the nature of the NifH post-translational modification in cyano- bacteria was also obtained: peptide mass spectra of the protein demonstrated the presence of a 300– 400 Da protein modification localized to a specific 13 amino acid sequence, within the part of the protein that is ADP-ribosylated in other bacteria and close to the active site of nitrogenase. Furthermore, the distri- bution of the highest scoring database hits for the

identified proteins points to the possibility of using proteomic data in taxonomy.

Key words: Azolla, cyanobacteria, NifH modification, proteomics, symbiosis, taxonomy.

Introduction

Symbiotic associations between the water-fern Azolla and nitrogen-fixing cyanobacteria have gained attention through the centuries due to their potential as natural nitrogen fertilizers, especially in rice cultivation (van Hove and Lejeune, 2002). The ‘symbiotic organs’ of the Azolla plants are the comparatively large cavities that occupy each dorsal leaf of the plant. These cavities are in nature obligately infected by filamentous cyanobacteria (cyanobionts) and bacteria, both held within a mucilagi- nous sheath (Nierzwicki-Bauer et al. , 1989; Lechno- Yossef and Nierzwicki-Bauer, 2002). The cyanobiont is restricted to the Nostaceae and was originally designated Anabaena azollae (Strasburger, 1873). The taxonomic status is, however, still not clarified, and relatedness to the genera Nostoc and Anabaena, as well as to neither of these two genera, has been proposed (Meeks et al., 1988; Canini et al., 1992b; Baker et al. , 2005; Svenning et al., 2005). As in other plant–cyanobacterial symbioses, the plant host supplies the cyanobiont with fixed carbon, while the cyanobiont in return supplies the plant with the nitrogen needed via nitrogen fixation (Rai et al., 2000; Lechno-Yossef and Nierzwicki-Bauer, 2002). The benefits for the plant are therefore apparent, while less so for the

* To whom correspondence should be addressed. E-mail: Birgitta.Bergman@botan.su.se Abbreviations: ESI, electrospray ionization; FBA, fructose/tagatose bisphosphate aldolase; FBP, fructose-1,6-bisphosphatase; F6P, fructose-6-phosphate; FNR, ferredoxin NADP + reductase; G6P, glucose-6-phosphate; GPI, glucose-6-phosphate isomerase; GS, glutamine synthetase; MS, mass spectrometry; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight; OPP, oxidative pentose phosphate; 6PGD, 6-phosphogluconate dehydrogenase; PMF, peptide mass fingerprinting; Q-ToF, quadropole-time of flight; TCA, tricarboxylic acid.

ª The Author [2007]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

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cyanobiont which by itself has photosynthetic capacities. Attempts have been made to identify mechanisms used by the plant to maintain the cyanobiont as an efficient nitrogen fixer, but many aspects are still unknown. Unique to the Azolla symbiosis among cyanobacterial– plant symbioses is that a small cyanobiont ‘inoculum’ is sequestered into the reproductive organ (sporocarp) and

vertically transferred from one generation to the next (Peters and Meeks, 1989; Zheng et al., 1990; Perkins and Peters, 1993; Rai et al., 2002). In all other cynobacterial–plant symbioses, as well as Frankia and Rhizobium plant sym- bioses, de novo infection of each individual plantlet is required (Vessey et al., 2005; Bergman et al., 2007). There

is also no confirmed report of successful cultivation of the

cyanobiont outside the Azolla plants or reconstitution of the

symbiosis (Tang et al., 1990), suggesting that some crucial trait needed for survival as a free-living organism may have been lost during co-evolution of the partners, possibly via gene loss (due to redundancy) from the cyanobiont, or gene transfer to the nucleus of the host. Hence, in the context of the endosymbiotic theory, according to which chloroplasts evolved from an ancient monophyletic symbiosis between

a cyanobacterium and a pigment-free eukaryote, the cyano- biont of Azolla may be evolving towards becoming

a nitrogen-fixing ‘organelle’. This is also supported by

PCR-based DNA fingerprinting which shows that the association is highly specific and that each Azolla species associates with one specific cyanobacterial strain irrespec-

tive of geographical origin (Zheng et al., 1999). In order to obtain further insights into adaptations required by a cyanobiont, potentially on its way to de- velop into a nitrogen-fixing ‘organelle’, an investigation of the proteome of the cyanobiont in Azolla filiculoides

was initiated. As the genome of this organism has not yet been sequenced, proteins were identified by a combination

of matrix-assisted laser desorption ionization-time of flight

mass spectrometry (MALDI-TOF MS), electrospray ioni- zation (ESI), tandem MS (MS/MS), and peptide homol- ogy software analyses, an approach which has previously been successfully used on other non-sequenced organisms such as Xenopus laevis (Liska et al., 2004) and Candida

magnoliae (Kim et al., 2004). As MS is also suitable for examining protein modifications, attempts were also made

to identify the modification of NifH, one of the subunits

of the nitrogen-fixing enzyme nitrogenase and therefore

a process (nitrogen fixation) of outmost importance for the symbiosis. The information gained is discussed in the context of taxonomy, adaptations, evolution, and co- development of the cyanobiont and its host.

Materials and methods

Growth conditions and isolation of the cyanobiont Azolla filiculoides plants were grown hydroponically in the greenhouse at the Department of Botany, Stockholm University.

The temperature was maintained at ;30 C and the light varied according to natural daylight with some addition of light from artificial sources. The cyanobiont was separated from the plant by the gentle rolling technique (Meeks, 1988). After crushing the plant

in a buffer containing 50 mM HEPES pH 7.8 and 1% polyvinyl-

pyrrolidone (PVP), using a roller, the cyanobacterial slurry obtained

was filtered through a nylon filter with a pore size of 100 lm. The cyanobacterial slurry was concentrated by centrifugation (1200 g, 10 min), placed on top of 40% Percoll (GE Healthcare, Uppsala, Sweden) in a centrifuge tube, and centrifuged for 5 min at 400 g.

The cyanobacteria gathered at the bottom of the tube, while chloroplasts, small-sized plant cell debris, and bacteria remained in the supernatant. The pellet was collected and centrifugation repeated with 80% Percoll for 15 min. This time the cyanobacteria stayed on top of the Percoll while plant starch granules formed a pellet. Approximately 90% pure cyanobacterial preparations were obtained

as evidenced by light microscopy.

Protein extraction Proteins were extracted as previously described (Ekman et al., 2006) by freezing the samples in liquid nitrogen and grinding with acid-washed sand, followed by sonication in sample buffer. The protein concentration in the samples was determined using the RCDC kit (Bio-Rad, Hercules, CA, USA).

2D gel electrophoresis

A 200 l g aliquot of protein was separated as previously described

(Ekman et al., 2006), first by isoelectric focusing using the

IPGphore system (GE Healthcare) and immobiline 18 cm gel strips

of pH 4–7, and secondly according to size in 10% acrylamide gels.

The gels were stained with SyproRuby (Bio-Rad) according to the manufacturer’s instructions, and a Typhoon 8600 laser scanner (GE

Healthcare) was used to visualize the gels.

In-gel digestion and mass spectrometry All protein spots selected from the 2D gels for analysis by MALDI- TOF MS were subjected to in-gel tryptic digestion, according to Gharahdaghi et al. (1999). In order to examine nitrogenase (NifH) modifications, the trypsin was replaced by GluC (V8 proteinase) of the same concentration. For double digests, GluC was added to the already trypsin-digested samples and incubated overnight. MALDI-TOF MS peptide mass spectra were obtained using a Voyager-DE STR mass spectrometer (Applied Biosystems, Foster City, CA, USA). As matrix, a-cyano-4-hydroxy cinnamic acid was used. ESI-MS/MS experiments were carried out using a Micromass Q-ToF (Waters, Milford, MA, USA) as previously described (Ekman et al. , 2006). Prior to analysis on the Q-ToF, salts and contaminants were removed from the samples by ZipTip purifica- tion and the peptides were eluted in water/acetonitrile (1:1 V:V) with 0.1% formic acid. A 1–3 l l aliquot of the sample was loaded into a nanospray tip (Proxeon, Odense, Denmark).

Data processing MALDI-TOF MS spectra were calibrated using internal trypsin autolysis peptides and the MoverZ software available online from Genomic Solutions (Ann Arbor, MI, USA). Obtained peptide mass lists were compared with sequences present in the NCBInr database using the Mascot (Perkins et al., 1999) search engine, available online from Matrix Science (Boston, MA, USA) with the following settings: taxonomy was set to all organisms; the mass

tolerance to 30 ppm; fixed modifications to alkylation of cysteine by carbamidomethylation; and variable modifications to oxidation

of methionine.

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MS/MS spectra were processed with MassLynx MaxEnt 3 before database searches in NCBInr, utilizing Mascot. As the Azolla cyanobiont is not represented in the available databases, this strategy relies on fragmentation of conserved parts of the proteins. Hence, additional information could be obtained by homology searches, allowing some individual amino acid substitutions. For this purpose, the peptides were sequenced de novo using the processed MS/MS spectra and the BioLynx peptide sequencing tool (Micromass). One to seven peptides/sample were fragmented, each resulting in 1–10 suggested sequences. All sequences, with 4–6 amino acid substitutions allowed, were submitted to MS-Homology from Protein Prospector, available online from the University of California, San Francisco (see http://donatello.ucsf.edu/index.html or http://prospector.ucsf.edu/). Data for which positive identifica- tions were obtained by common database search (cf below) were not included in the homology searches.

Quantitative protein analysis The relative quantity of all identified proteins of the Azolla cyanobiont was determined using the PDQuest software (Bio-Rad). Spot intensities were normalized to the total staining intensity of the gel in question. The quantification was based on four replica gels:

two gels of each of two separate protein extractions. For proteins whose identity had also been determined in the proteome of Nostoc PCC 73102 (Ran et al. , 2007), the volume ratio (i.e the ratio between the abundance in the Azolla cyanobiont and the abundance in Nostoc 73102) of the protein quantities relative to those determined in this organism was also calculated.

Results and discussion

In proteomics, proteins separated by two-dimensional gel electrophoresis are usually identified by peptide mass fingerprinting (PMF), which is a MALDI-TOF MS-based method that relies on matching of peptide masses to corresponding masses derived from theoretical digestions of protein sequences present in a database. Protein identification using this method requires that a large proportion of the protein in question has a sequence that is identical to that of a database entry. For instance, theoretical predictions by Wilkins and Williams (1997) proposed a requirement of 80% sequence identity. There- fore, the use of this method for unsequenced organisms usually results in a low protein identification success rate, e.g. 45% for Xenopus (Liska et al., 2004) and 42% for Zea mays (Chang et al., 2000). However, this rate may be improved by using tandem MS (MS/MS) by which proteins can be identified even if only a small part of the protein is completely conserved and only a few peptides match the database protein sequence (Shevchenko et al., 2001). Furthermore, by using the MS/MS spectra to predict amino acid sequences de novo, and allowing amino acid substitutions, homology searches using these predicted sequences may identify proteins with sequence identities to their closest homologue as low as 65% (Liska et al., 2004). Here, a combination of these three methods, PMF, MS/MS, and homology searches, was used to identify proteins in the Azolla cyanobiont, a strategy previously

Azolla cyanobiont proteome

1025

suggested as appropriate for identifying proteins of unsequenced organisms (Liska and Shevchenko, 2003).

Protein identification

In order to identify cellular mechanisms (adaptations) acting in the cyanobiont of the Azolla symbiosis, the cyanobacteria were extracted from the greenhouse-grown (cloned) A. filiculoides plants by the ‘gentle rolling technique’, followed by protein extraction and separation of the proteins by 2D gel electrophoresis. Approximately 300 protein spots were visualized when using a broad pH interval, 4–7 (Fig. 1A). The 52 most abundant proteins were selected for analyses by MALDI- TOF MS (Fig. 1A; Table 1). Of these, 32 spots were significantly identified (Mowse score >76) (Pappin et al., 1993) by their peptide mass fingerprints, resulting in 27 different proteins, as five of the proteins resolved into two spots. For another six proteins, the highest scoring hit in the database were cyanobacterial proteins with the expected mass and pI, but with a Mowse score of <76. Five of these were therefore analysed by MS/MS, and for three identities were confirmed; two (az21 and az50) by database searches using the MS/MS spectra and one (az26) via homology search. For the 14 remaining proteins, no cyanobacterial candidate was identified by PMF. Eight of these were analysed by MS/MS and six were identified; four by their MS/MS spectra (az04 and az30 by two peptides, az16 and az41 by one peptide) and two by homology search (az09 and az37). Hence, nine (3+6) of the 13 proteins analysed by MS/MS in combination with homology searches were identified. Some proteins with low peptide concentration, giving weak MALDI-TOF spectra, were not analysed by the less sensitive MS/MS. In total, 11 of the analysed proteins were not identified, giving a success rate for the MALDI-TOF analyses of 62% (32/52), and a success rate for MS/MS analyses in combination with homology searches of 70% (9/13). Since most MALDI-TOF-identified proteins would, if tested, have been identified by MS/MS, MS/MS would appear to be the method of choice when attempting to identify proteins in organisms with non-sequenced genomes, but is also considerably more time-consuming than MALDI-TOF MS.

Proteomics and taxonomy

As a relatively high percentage (62%) of the proteins were identified by PMF alone, a high percentage of the genome of the Azolla cyanobiont must be highly homologous to genomes present in the database. Indeed, all except two of the proteins identified showed highest homologies to genes/proteins of cyanobacteria of the same morphotype (filamentous and heterocystous; Section IV cyanobacteria; sensu Rippka et al. , 1979); the sequenced strains Nostoc punctiforme (i.e. Nostoc ATCC 29133 comparable with

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jxb.oxfordjournals.org Downloaded from 1026 Ekman et al. Fig. 1. Protein patterns of (A) the cyanobiont of

Fig. 1. Protein patterns of (A) the cyanobiont of Azolla and (B) the symbiotically competent, isolated, and free-living Nostoc PCC 73102. Numbers on gel A refer to Table 1; numbers on gel B refer to proteins in Nostoc PCC 73102 previously identified (see text for details; Ran et al., 2007).

Nostoc PCC 73102; 13), Anabaena variabilis (14), and Nostoc PCC 7120 (10), and the unsequenced Nostoc commune (1) and a Nostocaceae cyanobiont (1). One protein was identified via the genome of the unicellular cyanobacterium Synechosystis PCC 7942 (Table 1). These data show that the cyanobiont of A. filiculoides , and perhaps all Azolla cyanobionts, are phylogenetically closely related to other heterocystous cyanobacteria (Rippka et al. , 1979) such as Nostoc and Anabaena. Recent studies on 16S rDNA phylogeny of Nostoc and Anabaena and some symbiotic strains, including the cyanobiont of A. filiculoides , showed that the three sequenced Nostoc and Anabaena strains mentioned above are closely related, while the Azolla cyanobiont appeared in a separate branch constituting ‘true’ Anabaena strains (Svenning et al., 2005). The distribution of the highest scoring protein hits found here is in line with such a situation, i.e. the Azolla cyanobiont is approximately equidistant from the sequenced Nostoc/Anabaena strains. However, as none of these sequenced Nostoc/Anabaena strains belongs to the true Anabaena branch, a more detailed analysis of the phylogeny of the Azolla cyano- biont was not possible. The taxonomic uniqueness of the Azolla symbiosis among cyanobacteria in plant symbiosis is evident moreover when comparing proteomic-based phylogenetic data from the cyanobiont of Azolla with the corresponding data of the cyanobiont in the angiosperm Gunnera manicata as the latter are almost exclusively based on the N. punctiforme genome (Ekman et al. , 2006). This emphasizes a distinct genetic difference between the permanently associated cyanobiont of Azolla and that of the de novo infection-based Gunnera cyanobiont.

The fact that proteomic-based taxonomic analyses are dependent on which genomes are present in the database is a limitation to this approach. However, given the recent technological development in high-throughput sequencing of genomes and the subsequent fast increase in numbers of sequenced genomes (especially microbial), the use of such proteomics data from unsequenced organisms in phylogenetic analyses has a high potential to become a useful complement to traditional methods. One protein was identified as a non-cyanobacterial protein, the ATP synthase of the liverwort Anthoceros formosae . The reason for this is probably that some proteins from the Azolla plant remained in the extract since ATP synthase is a highly abundant and also highly conserved plant protein, and that the Azolla protein sequence was not present in the database.

Protein profiles in the Azolla cyanobiont

The proteins identified in the Azolla cyanobiont were restricted to those most highly expressed (Fig. 1), and the majority of these proteins have functions related to fundamental processes in the cyanobacterial cell machin- ery, such as translation, stress response, ATP synthesis, and carbon and nitrogen metabolism (Table 1). The relative abundances of the SyproRuby-stained proteins identified in the 2D gels of the Azolla cyanobiont were determined using the PDQuest (Bio-Rad) software. Twenty-four (five of which resolved in two different spots) of the proteins identified in the Azolla cyanobiont (Table 1) were previously identified in an attempt to profile the proteome of Nostoc PCC 73102 (originally isolated from the cycad Macrozamia) when grown under

Azolla cyanobiont proteome

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Table 1. Proteins identified in the cyanobiont of the water-fern Azolla filiculoides

Gel ID a Annotation

Protein ID

MALDI- Matched %

MS/MS

Homology Theoretical Theoretical Observed Observed Organism

Volume ratio cyanobiont/ Nostoc PCC 73102 f

 

TOF

peptides c Sequence no. of

search e

mol. wt

pI

mol. wt

pI

Mowse

coverage peptides d

score b

az01

RNA polymerase beta prime subunit Heat shock protein hsp90 family Chaperonin Phosphotransferase system, fructose-specific IIC component Chaperonin GroEL Polynucleotide nucleotidyl- transferase Translation elongation Phosphoketolase Transketolase Chaperonin GroEL Chaperonin GroEL ATP synthase alpha ATP synthase beta ATP synthase beta

NP_485636

88

12/39

13

147

4.8

150

4.8

Nostoc PCC 7120

az02

BAB74022

92

11/28

18

76

4.9

75

4.8

Nostoc PCC 7120

az03

ZP_00107038

94

9/21

17

68

4.8

70

4.7

Nostoc PCC 73102 0.73 Anabaena variabilis 4.21**

az04

ZP_00158087 –

2

46

4.7

48

4.6

az05

ZP_00110155

88

9/17

15

59

4.9

60

4.9

Nostoc PCC 73102 Nostoc PCC 73102

1.68*

az06

ZP_00106148

91

10/23

16

78

5.2

88

5.1

0.68

az07

ZP_00107087

92

8/11

12

76

5.2

75

5.2

Nostoc PCC 73102 Nostoc PCC 7120 Anabaena variabilis Anabaena variabilis Anabaena variabilis Nostoc PCC 7120 Anabaena variabilis Anabaena variabilis Synechococcus

az08

BAB73549

76

8/18

15

79

5.6

75

5.5

1.30

az09

ZP_00163127.2 –

1

Yes

72

5.9

70

6.2

1.11

az10

ZP_00163108

82

7/14

19

58

5.1

61

5.1

1.87*

az11

ZP_00163108

95

8/16

21

58

5.1

60

5.2

1.87*

az12

NP_484049

78

7/15

12

54

5.1

55

5.1

2.45**

az13

ZP_00159432

122

11/27

27

52

5.0

50

5.0

1.70**

az14

ZP_00159433

125

13/32

17

52

4.9

50

4.9

1.70**

az15

Fructose-1,6-bisphosphatase

ZP_00163422

84

6/9

12

37

5.1

41

4.9

0.71*

az16

NADPH:quinone reductase Phosphoglycerate kinase GTPases translation elongation factors Anthoceros ATPase Not identified Glutamine synthetase ADP-glucose pyrophosphorylase Thioredoxin reductase

BAB74647

1

No

36

5.6

39

5.0

elongatus PCC 7942 Nostoc PCC 7120 Nostoc PCC 7120 Nostoc PCC 73102 0.93

az17

NP_488171

97

9/27

25

42

5.2

42

5.3

az18

ZP_00107088 111

12/32

40

45

5.5

44

5.4

az19

BAC55424

76

8/24

19

56

5.2

59

5.4

Anthoceros formosae

az20

 

az21

P00964

54

5/13

12

2

53

5.2

54

5.4

Nostoc PCC 7120 Anabaena variabilis

1.04

az22

ZP_00158969

88

6/9

17

49

5.7

50

5.5

az23

ZP_00161577

37

5/23

8

50

5.5

49

5.6

Anabaena variabilis Anabaena variabilis Nostoc PCC 73102 Nostoc PCC 73102 Nostoc PCC 73102

az24

Glucose-6-phosphate isomerase NP_485093

78

8/21

16

58

5.5

60

5.6

2.14*

az25

Uncharacterized flavoproteins Nitrogenase (NifK) Rubisco

ZP_00111402

80

8/21

18

65

5.6

65

5.7

az26

ZP_00112339

32

4/18

8

0

Yes

58

5.5

60

5.8

2.51*

az27

ZP_00108159

98

8/15

18

53

6.3

56

6.3

0.44*

az28

6-Phosphogluconate

ZP_00158100

78

7/18

22

52

5.9

48

6.2

Anabaena variabilis 2.47*

dehydrogenase

az29

Not identified

az30

Phosphoribulokinase

ZP_00109191 –

 

2

39

5.4

41

5.6

Nostoc PCC 73102 Nostoc PCC 73102

0.40**

az31

Fructose/tagatose

ZP_00110670

95

7/12

17

39

5.5

39

5.4

0.78

bisphosphate aldolase

az32

Fructose/tagatose

ZP_00110670

94

7/12

16

39

5.2

38

5.3

Nostoc PCC 73102

0.78

az33

bisphosphate aldolase Not identified Not identified Nitrogenase reductase (NifH) P0A3S1

0

No

az34

az35

127

14/32

46

32

5.1

30

5.4

Anabaena variabilis 9.10**

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Ekman et al.

Table 1. (Continued )

 

Gel ID a Annotation

Protein ID

MALDI- Matched %

MS/MS

Homology Theoretical Theoretical Observed Observed Organism

Volume ratio cyanobiont/ Nostoc PCC 73102 f

 

TOF

peptides c Sequence no. of coverage peptides d

search e

mol. wt

pI

mol. wt

pI

Mowse

 

score b

az36

Nitrogenase reductase (NifH) (modified) Glutathione S-transferase Not identified Not identified Peroxiredoxin Peroxiredoxin Superoxide dismutase Ferredoxin-NADP(+) reductase Ferredoxin-NADP(+) reductase Phycocyanin

P0A3S1

102

16/47

53

32

5.1

31

5.3

Anabaena variabilis 3.46*

az37

ZP_00111757 –

 

1

Yes

29

5.0

31

4.8

Nostoc PCC 73102

az38

 

0

No

az39

az40

ZP_00108523

89

5/6

20

24

4.9

26

4.9

Nostoc PCC 73102 Anabaena variabilis Nostoc commune Nostoc PCC 7120 Nostoc PCC 7120 Nostocaceae cyanobiont AE1 Anabaena variabilis

 

az41

BAB76340

1

No

23

4.9

22

4.8

az42

AAF25009

100

7/16

32

22

5.5

22

5.4

0.74

az43

CAA51088

115

8/12

24

34

6.3

31

6.4

2.95**

az44

CAA51088

94

7/12

19

34

6.3

31

6.6

2.95**

az45

AAO31788

88

5/12

54

11

9.7

15

6.6

0.68

az46

Glycyl-tRNA synthetase, beta subunit Chaperonin clpB2 Aconitate hydratase Not identified Carboxysome shell protein Nucleoside-diphosphate-sugar epimerases Not identified

ZP_00161122

40

6/25

8

80

5.1

78

5.3

az47

Q8YM56

86

12/30

10

99

5.4

90

5.3

Nostoc PCC 7120 Anabaena variabilis

az48

ZP_00160026

81

10/18

15

95

5.2

95

5.4

0.89

az49

az50

ZP_00162489

68

4/10

32

2

11

6.1

40

6.4

Anabaena variabilis

0.73

az51

ZP_00159658

63

5/11

16

44

5.9

39

6.3

Anabaena variabilis

az52

 

a Protein IDs as denoted in Fig. 1.

b For proteins identified by MALDI-TOF MS, Mowse score, number of matched peptide, and percentage sequence coverage is reported. If the highest scoring database hit was cyanobacterial protein, these are reported, even if the Mowse score was below the significance limit of 76.

c The two numbers indicate matched peptides and all peptides in the spectrum.
d

e A successful identification by homology search is reported with ‘yes’ and unsuccessful with ‘no’.

For proteins identified by database searches of their MS/MS spectra, the number of peptides matching the protein is reported.

f

Volume ratios are in relation to the same proteins identified in free-living Nostoc PCC 73102 and these are marked with (*) when the difference was significant at the 95% level and with (**) when significant at the 99% level (Students t -test). When a protein resolved in two different spots, their volumes were summed.

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free-living diazotrophic conditions (Ran et al., 2007). Sixteen (four of which resolved in two different spots) of these cyanobiont proteins were identified based on sequence similarity to proteins of other cyanobacteria, but their closest homologue in the Nostoc PCC 73102 genome had been identified in this study of the Nostoc PCC 73102 proteome. The abundances of these 24 proteins (plus

a modified form of NifH, see below) could therefore be

compared with the corresponding proteins in the cultured Nostoc PCC 73102 (Fig. 1B) and the relative volume ratio calculated (Table 1). The discussion below will focus on these proteins and less on those only identified in the

cyanobiont (Table 1).

Nitrogen fixation and assimilation

Two of the most abundant protein spots in the Azolla cyanobiont 2D gels were identified as NifH (az35 and az36; Fig. 1), the small subunit of the nitrogenase

complex that functions as a reductase (Fe-protein). As one of these spots (az36) was slightly more acidic and of

a higher mass, this may represent a modified, probably

inactive, form of NifH. Up to 25% of NifH in the cyanobiont of Azolla may therefore be inactive. However, the active non-modified form of NifH showed a >9 times higher level in the cyanobiont compared with the cultured nitrogen-fixing Nostoc PCC 73102. Likewise, NifK (az26), one of the larger subunits of the nitrogenase enzyme complex, was 2.5 times more abundant in the cyanobiont. These findings reflect the higher heterocyst frequency in the Azolla cyanobiont, being ; 20%, com- pared with 5–10% in free-living cyanobacteria (Adams, 2000). Indeed, N 2 fixation rates in the Azolla cyanobiont may be 4–18 times higher than in free-living cyanobac- teria (Watanabe, 1982) and increase with heterocyst frequencies (Braun-Howland et al., 1988; Canini et al.,

1990).

The nitrogen fixed into ammonia is rapidly assimilated

by glutamine synthetase (GS) into glutamine in free-living cyanobacteria (Flores and Herrero, 2005). As seen in Fig. 1 (Table 1), the total GS protein (az21) levels were similar

in the A. filiculoides cyanobiont and in the cultured Nostoc

PCC 73102. This is in contrast to previous studies reporting cyanobiont GS protein levels in Azolla caro- liniana to be 5–40%, and glnA (encoding GS) transcrip- tion levels ;10% of the levels in free-living Nostoc and Anabaena strains (Nierzwicki-Bauer and Haselkorn, 1986; Lee et al., 1988; Peters and Meeks, 1989). Nonetheless, given the high frequency of heterocysts in the cyanobiont of Azolla, and that heterocysts normally have twice the amount of GS protein compared with vegetative cells under free-living conditions (Bergman et al., 1985), the GS protein levels detected still indicate a reduction of the GS level in the cyanobiont heterocysts. Similar reductions have previously been observed in heterocysts of

Azolla cyanobiont proteome

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cyanobionts in other plant–cyanobacterial symbioses, e.g. Gunnera, Anthoceros, lichens, and diatoms (Rai et al., 2000). It is also possible that nitrogen assimilation in the cyanobiont is mainly regulated via the GS activity, which would not be reflected in the GS protein abundance. Moreover, it has been shown that although the cyanobiont releases N for the benefit of the host, ; 60% of the nitrogen fixed is retained by the A. caroliniana cyanobiont (Peters and Meeks, 1989), which is a higher percentage than that reported for cyanobacteria in other symbioses (Rai et al., 2000). This would require a substantial GS activity to be maintained given the pronounced up- regulation of the nitrogenase enzyme levels of the Azolla cyanobiont detected here (Table 1).

Energy production and conversion

N 2 fixation is an energetically costly process in terms of reducing equivalents and ATP. In the Azolla cyanobiont, it has been suggested that nitrogenase activity is mainly supported by ATP produced via cyclic photophosphory- lation (Peters and Meeks, 1989). Furthermore, high re- spiratory rates in heterocysts lower the oxygen concentration (Fay, 1992). As both these processes in- volve ATP synthase (az12–az14), the 2-fold higher levels of this protein complex observed in the cyanobiont (Table 1) may be associated with the high levels of nitrogenase and frequency of heterocysts. An important enzyme in the production of reducing equivalents in organisms performing oxygenic photosyn- thesis is ferredoxin NADP + reductase (FNR) (az43 and az44; Table 1), which catalyses the terminal step of the photosynthetic electron transport chain. However, it has also been shown that N 2 -fixing non-photosynthetic hetero- cysts may contain up to 14 times more FNR than the photosynthetic vegetative cells (Razquin et al., 1996) and that FNR has two promoters, one of which is specific for heterocysts (regulated by NtcA) (Valladares et al. , 1999). The low rates of photosynthesis in the Azolla cyanobiont (see below) imply that the four times higher levels of this enzyme in symbiosis may be associated with a high symbiotic heterocyst frequency. In symbiosis, FNR activities may therefore mainly be related to electron transfer reactions required to provide reducing equiva- lents to nitrogenase and respiratory electron transport (Schmetterer, 1994) and also to cyclic electron transport in the heterocysts, as such a function has been observed for this enzyme in chloroplasts (Zhang et al., 2001).

Photosynthesis and CO 2 fixation

The levels of the two enzymes specific for the Calvin cycle, Rubisco (az27) and phosphoribulokinase (az30), were lower in the Azolla cyanobiont than in the cultured Nostoc PCC 73102 (Table 1). These findings agree with previous data showing a reduction in CO 2 fixation activity

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1030 Ekman et al.

(Kaplan and Peters, 1988) and Rubisco mRNA levels (5– 7 times lower) (Braun-Howland and Nierzwicki-Bauer, 1990) in the Azolla cyanobiont. The relatively high level of the proteins still persisting is, however, also in accordance with the finding that Azolla cyanobionts removed from the symbiosis fix carbon at a rate compa- rable with that of free-living cyanobacteria (Ray et al. , 1979; Kaplan and Peters, 1988). As the cyanobiont is not light limited (Peters and Meeks, 1989), some symbiosis- specific mechanism appears to regulate both the synthesis and activity of photosynthetic components. Fructose-1,6-bisphosphatase (FBP) (az15), fructose/ tagatose bisphosphate aldolase (FBA) (az31–az32), trans- ketolase (az09), and phosphoketolase (az08) are other enzymes functioning in CO 2 fixation. However, since they also participate in catabolic processes, the interpretation of the abundance of these proteins is discussed below.

Carbohydrate uptake and metabolism

With a reduced photosynthetic activity in the cyanobiont, energy requirements will have to be met by carbohydrates supplied by the host plant. Indeed, a phosphotransferase system fructose-specific IIC component (az04) was among the most highly expressed proteins in the 2D gels of the mixotrophically grown cyanobiont of Azolla, and was ;4 times more abundant than its homologue in free-living Nostoc PCC 73102 (Fig. 1A, B; Table 1). It has been suggested that the carbohydrate transferred is mainly sucrose (Kaplan and Peters, 1988), and the transport system identified here is potentially therefore a hexose transporter. 6-Phosphogluconate dehydrogenase (6PGD) (az28) was also strongly up-regulated in the cyanobiont. The same is the case in the Nostoc cyanobiont of G. manicata (Ekman et al., 2006). The activity of this enzyme is specific for the oxidative pentose phosphate (OPP) pathway, which is the major route of carbon catabolism in cyanobacteria and also in providing reductant to nitrogenase (Summers et al. , 1995). The up-regulation of 6PGD therefore implies a high demand for reductant by N 2 fixation, and by the mixotrophic growth mode of the cyanobiont. Given this, the up-regulation of the enzyme glucose-6-phosphate isomerase (GPI) (az24) is probably less associated with the glycolytic conversion of glucose-6-phosphate (G6P) into fructose-6-phosphate (F6P) than with the opposite reaction, conversion of F6P into G6P, the latter sub- sequently oxidized in the OPP pathway. Furthermore, this enzyme is needed if host-derived sugars, supplied as sucrose or fructose, are to be catabolized via the OPP pathway. That the strong up-regulation of GPI seen is related to carbon flow through the OPP pathway is supported by the fact that when free-living Nostoc PCC 73102 was supplied with fructose, the up-regulation of this protein was less pronounced ( ;30%, M Ekman et al. ,

unpublished results). In free-living cyanobacteria, a higher proportion of the fructose is catabolized via glycolysis and the incomplete tricarboxylic acid (TCA) cycle to meet the higher amount of biosynthetic precursors needed to achieve the higher rates of nitrogen assimilation (Meeks and Elhai, 2002). On the other hand, the level of aconitase (az48) was not significantly changed in the cyanobiont, which may indicate that glycolysis and the TCA cycle are active to some degree in the cyanobiont. The abundance of this protein was still only a fraction of that of most other proteins identified in the Calvin cycle and the OPP pathway in both organisms (Fig. 1), once again stressing the limited role of the TCA cycle in cyanobacteria. The abundance of FBA, transketolase, and phospho- ketolase in the cyanobiont was not significantly different from the levels in Nostoc PCC 73102, while that of FBP was 30% lower. All four are necessary for both the cyclic OPP pathway and CO 2 fixation via the Calvin cycle. Given the symbiotic down-regulation of the Calvin cycle, the fact that these enzymes are still expressed at a high level further emphasizes the role of the OPP pathway in symbiosis. However, as regulation of metabolic pathways is often reflected in activities rather than enzyme levels, it is also possible that the protein levels would be high even without a highly active OPP pathway. A different FBP isozyme was found to be up-regulated under conditions of nitrogen limitation in N. punctiforme, and was suggested to function mainly in the cyclic OPP pathway of heterocysts (Summers and Meeks, 1996). The reduced levels of the FBP identified here may therefore potentially be associated with a principal function in the Calvin cycle of vegetative cells.

Stress responses

Some of the most abundant proteins in the 2D gels of the Azolla cyanobiont and Nostoc PCC 73102 were proteins with functions related to protein assembly, modification, and degradation (chaperones; az03, az05, and az10–az11), and stress-related proteins, such as superoxide dismutase (az42) and peroxiredoxins (az40 and az41) (Table 1). Some of these have different abundances in the two organisms, possibly reflecting the radically different growth conditions experienced in symbiosis. It is also known that specific peroxiredoxins are expressed in rhizobia living in symbiosis with legumes but not when free-living (Dombrecht et al., 2005). Furthermore, the intracellular localization of superoxide dismutase in heterocysts of the Azolla cyanobiont suggested a role in protecting nitrogenase from superoxide radicals generated via respiration (Canini et al., 1992a). Besides experienc- ing stress when entering and accommodating to a sym- biotic lifestyle in a plant, the high heterocyst differentiation and nitrogen fixation rate elicited is per se a stress response elicited by N deprivation. Stress proteins were

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likewise up-regulated in the cyanobiont of G. manicata (Ekman et al. 2006). Down-regulation of polynucleotide nucleotidyltransfer- ase (az06), which is involved in RNA degradation, suggests a reduced RNA turnover rate possibly associated with the lowered cell division rates in cyanobionts in plant symbioses. Also, a translation elongation factor (az18), even if highly expressed in both organisms, occurred in lower relative quantities in the cyanobiont.

Nitrogenase modification

In addition to protein identification, mass spectrometry has also been widely used for identifying post-translational modifications of proteins (Larsen and Roepstorff, 2000; Reinders et al., 2004) and, as discussed, a large proportion of the Azolla cyanobiont NifH protein appeared to be modified. The mechanism behind the modification of nitrogenase in cyanobacteria is still unknown and is not due to ADP-ribosylation in the heterocystous cyanobac- terium A. variabilis (Durner et al., 1994) as in several other N 2 -fixing bacteria. In the unicellular cyanobacterium Gleothece, NifH was proposed to be modified by several palmitoylations, or palmitoylation in combination with other modifications (Gallon et al., 2000), but the reported size difference of >2000 Da between the unmodified and modified protein is considerably larger than that identified here in the cyanobiont of Azolla . Attempts were therefore made to identify the nitrogenase modification by compar- ing the peptide mass spectra of trypsin-digested unmodi- fied and modified NifH of the Azolla cyanobiont. Theoretically, the peptide carrying the modification would have a higher mass in the modified protein compared with the unmodified protein, and the mass difference would allow identification as well as location of the modification. Indeed, one peptide peak was consistently missing from the spectra of the modified protein (Fig. 2A, B), potentially corresponding to the peptide being modified. However, no new peptide of higher mass carrying the modification was observed. The identity of the disappear- ing peptide was confirmed by analysing the modified and non-modified protein spectra of NifH of Nostoc PCC 73102, in which a peptide of the same mass disappeared (Fig. 2C, D). The disappearing peptide corresponded to the amino acid sequence CVESGGPEPGVGCAGR of NifH. When Blasting the NifH sequence of the Nostoc PCC 73102 against the NCBInr database, it is clear that this is the most conserved sequence of the protein, being 100% identical in most nitrogenase proteins sequenced. Furthermore, the ADP-ribosylation of Rhodospirillum has been shown to be located on Arg101 (Pope et al., 1985), which corresponds to the arginine at the end of this sequence. Trypsin cleaves at arginine, and a modification of this amino acid would block this cleavage and result in a peptide of ; 6000 Da, which is too large to be analysed

Azolla cyanobiont proteome

1031

is too large to be analysed Azolla cyanobiont proteome 1031 Fig. 2. MALDI-TOF spectra of trypsin-digested

Fig. 2. MALDI-TOF spectra of trypsin-digested NifH of (A) the Azolla cyanobiont and (C) Nostoc PCC 73102; and the modified NifH of (B) the Azolla cyanobiont and (D) Nostoc PCC 73102. The peptides of mass 1588.7, corresponding to the sequence CVESGGPEPGVGCAGR, are missing from the two modified proteins, as indicated by arrows.

in the reflective mode of MALDI-TOF. As a consequence,

a second enzyme was used, GluC, which cleaves at

glutamic acid (when in bicarbonate buffer). However, again

the peptide corresponding to the same part of the protein (amino acid sequence SGGPEPGVGCAGRGIITAIN- FLEE) in the Azolla cyanobiont and in Nostoc PCC 73102 disappeared in the modified protein, but no other peptide appeared (Fig 3). When the protein was cleaved with both trypsin and GluC, once again a peptide correspond- ing to the same part of the protein (amino acid sequence SGGPEPGVGCAGR) disappeared (Fig. 4; Table 2). There may be several explanations for the absence of the modified peptide in the mass spectra, even when using GluC, or both enzymes. It has been shown that some modifications may interfere with the ionization step in MALDI-TOF MS or, alternatively, the modified protein was not eluted from the gel after in-gel digestion. Attempts were therefore made to elute the whole protein from the gel in order to be able to cleave the protein in solution. This would also allow for determination of the exact difference in mass between the modified and the non-modified protein, thereby identifying a candidate modification whose identity could be confirmed by chemically de-modifying the protein. However, the elu- tion was not successful without using SDS, which is incompatible with MALDI-TOF MS analysis. Currently, optimization of this procedure is under way. However, it

1032 Ekman et al.

is shown for the first time that the NifH protein modification in cyanobacteria is localized within the 13 amino acid sequence SGGPEPGVGCAGR, which in turn is positioned close to the active site of the enzyme (Georgiadis et al., 1992). Based on migration in the 2D gels, the modification has a mass of 300–400 Da (Table 3), making it too small for ADP-ribosylation (541 Da). In bacteria, palmitoylation (238 Da) is usually observed only on lysine and on N-terminal cysteine after cleavage of signal peptide (FindMod tool, Expasy Proteomics Server; available online from the Swiss Institute of Bioinfor- matics, Basel, Switzerland), making this modification also less likely. It is clear that the NifH modification of the Azolla cyanobiont is located within the part of the protein that is ADP-ribosylated in other bacteria and that it has a mass of 300–400 Da, but the identity of the modification remains to be determined.

the identity of the modification remains to be determined. Fig. 3. MALDI-TOF spectra of GluC-digested NifH

Fig. 3. MALDI-TOF spectra of GluC-digested NifH of (A) the Azolla cyanobiont and (C) Nosto c PCC 73102; and modified NfH of (B) the Azolla cyanobiont and (D) Nostoc PCC 73102. The peptides of mass 2501.18, corresponding to the sequence SGGPEPGVGCAGRGIITAIN- FLEE, are missing from the two modified proteins, as indicated by arrows.

Conclusions

The versatility of the proteomic approach was evident, as it was possible to identify several adaptations to symbiosis in the Azolla cyanobiont, to perform a basic analysis of the taxonomic affiliation of the Azolla cyanobiont, and to clarify important aspects of the NifH modification in cyanobacteria. First, the adaptations found reflect a metab- olism in the cyanobiont largely devoted to production of fixed nitrogen (for the benefit of the plant) and include the differential expression of a number of key proteins previously not known to be affected in cyanobacterial symbioses. This may in turn be a consequence of the long co-evolution between the plant and the cyanobiont. Secondly, the information obtained with regard to the taxonomic affiliation of the Azolla cyanobiont was in line with previous findings (Svenning et al., 2005), suggesting that proteomics-based taxonomic analyses may become

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Fig. 4. MALDI-TOF spectra of GluC–trypsin double-digested NifH of (A) the Azolla cyanobiont and (C) Nostoc PCC 73102; and modified NifH of (B) the Azolla cyanobiont and (D) Nostoc PCC 73102. The peptides of mass 1200.563, corresponding to the sequence SGGPEPGVGCAGR, are missing from the two modified proteins, indicated by arrows in (B) and (D).

Table 2. Peptides obtained after cleavage of NifH of the cyanobiont of Azolla with trypsin, GluC, or both in combination

Enzyme

Cleaves at

Modified peptide sequence

Modified peptide mass

Trypsin

C-terminal side of K or R C-terminal side of E C-terminal side of K or R and also E

CVESGGPEPGVGCAGR SGGPEPGVGCAGRGIITAINFLEE 1. SGGPEPGVGCAGR 2. GIITAINFLEE

1588.69

GluC (bicarbonate)

2401.18

Trypsin+GluC

1200.54

 

1219.66

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Table 3. Mass differences between the two NifH proteins of Nostoc PCC 73102 estimated by PDQuest software from four different 2D gels

Gel no.

Mass

Mass NifH

Mass

NifH (kDa)

modified (kDa)

difference (kDa)

1

33.6

34.0

0.4

2

32.2

32.6

0.4

3

32.7

33.1

0.4

4

33.1

34.4

0.3

a useful complement to traditional taxonomic methods. Thirdly, the first molecular data set was identified on the nature of the NifH modification in cyanobacteria, a mole- cule of 300–400 Da located at a 13 amino acid sequence positioned close to the active site of nitrogenase. It is also clear that although an organism with a non-sequenced genome was analysed, the present approach generated highly valuable information.

Acknowledgements

Funding from the Swedish Research Council (to BB) and the European Science Foundation CYANOFIX Programme (to ME) is gratefully acknowledged.

References

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