Tài liệu Báo cáo khoa học: Identification, sequencing, and localization of a new carbonic anhydrase

Identification, sequencing, and localization of a new

carbonic anhydrase transcript from the hydrothermal

vent tubeworm Riftia pachyptila

Sophie Sanchez, Ann C. Andersen, Ste´phane Hourdez and Franc¸ois H. Lallier

Equipe Ecophysiologie: Adaptation et Evolution Mole´ culaires, UMR 7144 CNRS UPMC, Station Biologique, Roscoff, France

Vestimentiferan tubeworms (Polychaeta; Siboglinidae)

often represent a major component of the endemic

fauna at hydrothermal vents and cold seeps. These

annelid worms are devoid of mouth, digestive tract,

and anus [1], relying completely on their autotrophic

sulfide-oxidizing symbionts to fulfill their metabolic

needs [2]. These symbionts are located deep inside the

body of the host, in a specialized organ called the

trophosome. This location, remote from the environ￾ment that contains all the necessary nutrients for the

bacteria, implies that the tubeworm host needs to

transport oxygen, hydrogen sulfide and inorganic car￾bon compounds in large quantities for the bacteria to

produce organic matter [3].

CO2 is acquired from the environment by diffusion

through the branchial plume [4,5], the respiratory￾exchange organ, where it is immediately converted

into bicarbonate through high activities of carbonic

Keywords

chemoautotrophy; differential expression;

messenger RNA; symbiosis; Siboglinidae

Correspondence

F. H. Lallier, Equipe Ecophysiologie:

Adaptation et Evolution Mole´ culaires,

UMR 7144 CNRS UPMC, Station

Biologique, Place Georges Teissier,

BP 74, 29682 Roscoff Cedex, France

Fax: +33 29829 2324

Tel: +33 29829 2311

E-mail: [email protected]

Database

Nucleotide sequence data are available in

the GenBank database under the accession

numbers EF490380 (RpCAbr) and EF490381

(RpCAbr2)

(Received 22 March 2007, revised 24 July

2007, accepted 20 August 2007)

doi:10.1111/j.1742-4658.2007.06050.x

The vestimentiferan annelid Riftia pachyptila forms dense populations at

hydrothermal vents along the East Pacific Rise at a depth of 2600 m. It

harbors CO2-assimilating sulfide-oxidizing bacteria that provide all of its

nutrition. To find specific host transcripts that could be important for the

functioning of this symbiosis, we used a subtractive suppression hybridiza￾tion approach to identify plume- or trophosome-specific proteins. We

demonstrated the existence of carbonic anhydrase transcripts, a protein

endowed with an essential role in generating the influx of CO2 required by

the symbionts. One of the transcripts was previously known and sequenced.

Our quantification analyses showed a higher expression of this transcript in

the trophosome compared to the branchial plume or the body wall. A sec￾ond transcript, with 69.7% nucleotide identity compared to the previous

one, was almost only expressed in the branchial plume. Fluorescent in situ

hybridization confirmed the coexpression of the two transcripts in the bran￾chial plume in contrast with the trophosome where only one transcript

could be detected. An alignment of these translated carbonic anhydrase

cDNAs with vertebrate and nonvertebrate carbonic anhydrase protein

sequences revealed the conservation of most amino acids involved in the

catalytic site. According to the phylogenetic analyses, the two R. pachyptila

transcripts clustered together but not all nonvertebrate sequences grouped

together. Complete sequencing of the new carbonic anhydrase transcript

revealed the existence of two slightly divergent isoforms probably coded by

two different genes.

Abbreviations

BP, bootstrap value; CA, carbonic anhydrase; FISH, fluorescent in situ hybridization; HB, hybridization buffer; IRES, internal ribosome entry

site; MP, maximum parsimony; NJ, Neighbour-joining; RpCAtr, Riftia pachyptila carbonic anhydrase trophosome; RpCAbr, Riftia pachyptila

carbonic anhydrase branchial plume; SSH, subtractive suppression hybridization.

FEBS Journal 274 (2007) 5311–5324 ª 2007 The Authors Journal compilation ª 2007 FEBS 5311