Departamento de Biología Molecular, Facultad de Medicina, Universidad de Cantabria, Unidad asociada al Centro de Investigaciones Biológicas, CSIC, Cardenal Herrera Oria s/n, 39011 Santander, Spain1
UPRES-A CNRS 6026, Biologie Cellulaire et Reproduction, Equipe Canaux et Récepteurs Membranaires, Université de Rennes 1, Campus de Beaulieu, 35042 Rennes cedex, Bretagne, France2
Author for correspondence: Juan M. García-Lobo. Tel: +34 942 201948. Fax: +34 942 201945. e-mail: jmglobo{at}unican.es
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ABSTRACT |
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Keywords: aquaporin, water channel, Brucella abortus
The GenBank accession number for the nucleotide sequence reported in this paper is AF148066.
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INTRODUCTION |
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Aquaporins belong to the MIP (major intrinsic protein) superfamily of membrane proteins (Pao et al., 1991 ). All the members of this group are small proteins of about 250300 amino acids that share a common topology, consisting of a transmembrane domain formed by six hydrophobic
-helices. These
-helices form a hydrophilic channel through which polar substances can diffuse (Walz et al., 1997
). MIP proteins are classified into three groups according to their substrate specifity, namely aquaporins, glycerol facilitators and glyceroaquaporins. Aquaporins are ubiquitous proteins that transport water but not other small solutes (Verkman & Mitra, 2000
). The best known glycerol facilitator is the GlpF protein of Escherichia coli. GlpF does not transport water; however it is capable of transporting other small polyalcohols such as erythritol in addition to glycerol (Heller et al., 1980
). Glyceroaquaporins can transport both water and glycerol, and in some cases they may also mediate the transport of small molecules such as urea (Ishibashi et al., 1994
). Moreover, the transport of gas molecules (CO2) through aquaporin channels has also been discussed (Nakhoul et al., 1998
).
The genus Brucella is composed of several species of Gram-negative animal pathogens differing mainly in their preferred host. They belong to the -proteobacterial class, whose members often associate both with plants and with animals. Brucella abortus preferentially infects cattle and other ungulates. Infection of pregnant animals by B. abortus usually results in abortion and one clinical sign of such infection is the presence of erythritol in the placenta (Lowrie & Kennedy, 1972
; Smith et al., 1962
). This observation was correlated with the reported ability of E. coli GlpF to transport erythritol and was one of the reasons which led us to undertake the search for a MIP protein in Brucella. B. abortus is a facultative intracellular parasite; this lifestyle is very different from that of E. coli but close to that of Rhizobium, a symbiont of plant root nodules. The presence of the MIP protein nodulin-26 in the peribacteroid membrane of soybean root nodules infected with Rhizobium (Miao & Verma, 1993
) suggested a possible role of MIP proteins in the biology of intracellular parasites. Thus we considered that a MIP protein in Brucella could be involved either in erythritol transport or in the growth of the bacterium inside phagocytic vacuoles.
Here, we describe the cloning and characterization of an aquaporin, AqpX, from B. abortus. This finding adds to the previous description of an aquaporin in E. coli (Calamita et al., 1997 ). Functional studies of the role of AqpX in this different genus will be useful in determining the role of these proteins in bacterial physiology.
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METHODS |
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Plasmids.
Bluescript SK (Stratagene Cloning Systems) and pUC (Vieira & Messing, 1982 ) were used as general purpose cloning vectors in E. coli. The pGEM-T vector (Promega) was used for cloning of PCR fragments. The plasmid vector pXßG-ev1 (Preston et al., 1992
), which contains the ß-globin transcription control region including the 5' and 3' untranslated regions, was used for expression studies in Xenopus laevis oocytes. pUCGla, pUCAqpZ and pUCGlpF contained, respectively, the full-length coding sequence of the glyceroaquaporin from Lactococcus lactis (A. Froger, unpublished), aquaporin AqpZ from E. coli and the glycerol facilitator GlpF from E. coli inserted into pUC19 (Vieira & Messing, 1982
).
DNA purification and sequencing, and other DNA manipulations.
Standard DNA purification and manipulation methods were performed essentially as described by Sambrook et al. (1989) . Purification of genomic DNA was performed by the guanidinium thiocyanate method (Pitcher et al., 1989
). Plasmid DNA prepared by standard alkaline lysis was further purified using either the MiniPrep Express Matrix (Bio101) or the Plasmid Midi kit (Qiagen). DNA from agarose gels was purified using the Qiaquick Gel Extraction kit (Qiagen). DNA sequencing was performed on double-strand templates by cycle sequencing using Texas-red-labelled primers in an automatic Vistra sequencer.
Southern blot analysis.
Chromosomal DNA from B. abortus was partially or totally digested with EcoRI and HindIII, electrophoresed in a 0·9% agarose gel and transferred by capillarity to positively charged nylon membranes (Roche Diagnostics). The blots were hybridized with a 399 bp internal sequence fragment of aqpX labelled with digoxigenin (Roche Diagnostics) and washed three times with 0·15 M sodium chloride, 0·015 M sodium citrate, pH 7·2 (SSC) at 42 °C, once with 0·1xSSC at 42 °C, and once more with 0·1xSSC containing 0·1% SDS at 65 °C. Washed blots were incubated with alkaline phosphatase labelled anti-digoxigenin antibody and developed using the luminescent substrate disodium-3-(4-methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.13.7]decan}-4-yl)phenyl phosphate (CSPD; Roche Diagnostics).
For chromosomal mapping of the gene, DNA was isolated and digested in agarose blocks with restriction endonucleases PacI and SpeI. Fragments were separated by PFGE and hybridized as described by Jumas-Bilak et al. (1998) .
Expression and water transport studies in Xenopus oocytes.
For expression in oocytes, appropriate inserts were cloned into the plasmid vector pXßG-ev1 (Preston et al., 1992 ). Capped cRNAs were synthesized in vitro using T3 RNA polymerase after plasmid linearization with XbaI. Defolliculated X. laevis oocytes (stage VVI) were injected with 50 nl water or up to 200 ng sample cRNAs and incubated in modified Barths solution (osmolality 200 mosM) at 18 °C (Le Caherec et al., 1996
). After incubation for 25 d, the oocytes were transferred to hypoosmotic modified Barths solution (osmolality 70 mosM) at 18 °C. Oocyte swelling was monitored by videomicroscopy and the coefficient of osmotic water permeability (Pf) was determined (Preston et al., 1992
). To calculate the Arrhenius activation energy (Ea), the Pf was measured for four different temperatures between 16 and 32 °C.
Glycerol transport.
The capability of the different gene products to transport glycerol was assayed in the E. coli strain OSBR1 (glpF). Bacterial cultures were grown overnight at 30 °C in M9 medium supplemented with maltose (10 mM). Cultures were then diluted to an OD600 of 0·08 in the same medium and allowed to grow at 30 °C to an OD600 of 0·3. Cells were harvested, pelleted and washed twice with M9 medium. Assays were performed at room temperature with 6x108 cells in a final volume of 500 µl M9 medium containing 1 µM [U-14C]glycerol (Amersham) at a final specific activity of 1·77 GBq mmol-1. After 1 min incubation, cells were vacuum-filtered through 0·45 µm pore diameter cellulose nitrate membrane filters (Whatman), washed with 2 ml cold M9 medium and their radioactivity counted.
Cryoelectron microscopy.
Overnight cultures of E. coli SK46 containing the appropriate plasmid in M9 maltose medium were diluted in M9 glucose and grown at 37 °C to the exponential phase of growth (OD600 0·8) in M9 medium supplemented with glucose. Bacteria were then rapidly pelleted and resuspended in M9 medium (osmolality 240 mosM) at room temperature. A 2·5 µl drop of the cell suspension was placed directly on a copper grid coated with a thin carbon film, upon which osmotic challenges were performed. Osmotic up-shocks were induced by rapidly mixing 2·5 µl 1·2 M sucrose-M9 solution with the cell suspension on the grid (final osmolality 1000 mosM). After 10 s, the grid was briefly blotted with filter paper and plunged into liquid ethane held at liquid nitrogen temperature. Specimens were examined at -170 °C in a Philips CM12 microscope with a Gatan model 626 cryoholder (Delamarche et al., 1999 ). Micrographs were recorded on Kodak SO 163 film under low-dose conditions at a nominal magnification ofx6300.
Computer-assisted sequence analysis and comparison.
We used the BLAST programs (Altschul et al., 1997 ) for sequence comparison against the databases at the NCBI website (http://www.ncbi.nlm.nih.gov/BLAST/). The sequence alignment was done with the CLUSTAL W (Thompson et al., 1994
) program at the Pole Bioinformatique Lyonnais website (http://pbil.ibcp.fr). Prediction of protein hydrophobicity and transmembrane regions was performed with the TMPRED program at the swiss EMBnet web site (http://www.ch.embnet.org/pages/services.html).
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RESULTS |
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Glycerol transport
The ability of B. abortus AqpX to facilitate glycerol uptake was assayed in the E. coli glpF strain OSBR1 containing the plasmid pUCAqpX, which contains the aqpX ORF cloned into the plasmid pUC18. This strain did not transport any radioactive glycerol, whilst the positive controls, E. coli OSBR1(pUCGlpF) and E. coli OSBR1(pUCGla) incorporated significant amounts of radioactivity under the same assay conditions (Fig. 5).
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DISCUSSION |
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The aqpX gene from B. abortus was cloned and its sequence revealed a close homology with aqpZ from E. coli. The gene was flanked by three copies of a pallindromic repeated sequence of Brucella (Halling & Bricker, 1994 ). The function of these sequences, similar to ERIC sequences of E. coli (Hulton et al., 1991
), has not yet been determined, and the meaning of this association is also unknown. We have been unable to identify sequences similar to consensus transcription promoters or terminators. However, as previously reported for the E. coli gene, we suspect that the B. abortus aqpX gene forms a single transcriptional unit. The deduced amino acid sequence of AqpX has already suggested that the protein is a water channel belonging to the MIP protein family (Froger et al., 1998
). Expression studies in Xenopus oocytes and in an aqpZ null E. coli strain clearly demonstrated that B. abortus AqpX was able to form active water channels in the cell membrane. The specificity for water was confirmed using E. coli mutants defective in glycerol transport containing plasmids expressing AqpX. The primers used in the PCR experiment to clone the aqpX gene were designed to recognize the conserved regions of all MIP proteins, including aquaporins and glycerol facilitators. However, we never obtained any amplification fragment corresponding to a glycerol facilitator using B. abortus DNA as the template. Furthermore, secondary hybridization bands were never observed when Brucella chromosomal DNA was hybridized with probes from the B. abortus aqpX gene under low-stringency conditions. Similar hybridization experiments using probes from the E. coli aqpZ and glpF genes were equally negative (data not shown). These negative results seem to indicate that there is no GlpF-like protein in B. abortus, an unexpected finding which leaves unsolved the question relating to the molecular transport pathway for both glycerol and erythritol into B. abortus. The possibility of a phosphotransferase-linked transport system was discarded, at least for erythritol, since it is known that the first step in erythritol catabolism is the phosphorylation of the polyalcohol by a kinase whose gene has been recently identified (Sangari et al., 2000
; Sperry & Robertson, 1975
).
One of the possible roles for a MIP protein in a bacterium living inside a vacuole is to contribute to the acquisition of nutrients. In the case of B. abortus AqpX, this option should be disregarded given its specificity for the transport of water. Thus, a role specifically linked to water transport, such as adaptation to variation in intravacuolar pH or osmolarity, should be expected for this aquaporin. The construction of aqpX mutants will be of great help to determine the biological role of the aquaporin of Brucella.
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ACKNOWLEDGEMENTS |
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Received 15 June 2000;
revised 2 August 2000;
accepted 18 August 2000.