1 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, Spain
2 Departamento de Microbiología e Inmunología, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autónoma de México, Coyoacan 04510, Mexico DF, Mexico
Correspondence
Juan M García Lobo
jmglobo{at}medi.unican.es
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ABSTRACT |
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INTRODUCTION |
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The physiological role of the different mammalian aquaporins has been studied in detail, mostly by study of animal models deficient in individual aquaporins. These animals showed diverse phenotypes including lens cataract (Shiels & Bassnett, 1996), loss of the red cell Colton blood group antigens (Agre et al., 1995
), nephrogenic diabetes insipidus (Deen et al., 1994
) and incomplete renal fluid concentration (Agre, 2000
). In plants, aquaporins are involved in numerous processes such as transpiration, root water uptake, maintenance of cell turgor and inhibition of self-pollinization (Ikeda et al., 1997
; Johansson et al., 2000
). In contrast, the physiological role of aquaporins in bacteria is still undefined. Most of the available biological information on bacterial aquaporins derives from the study of the E. coli AqpZ, the first known bacterial aquaporin (Calamita et al., 1995
). The functional characterization of AqpZ demonstrated water selectivity without evidence of glycerol transport. Furthermore, cryoelectron microscopy of osmotically shocked cells demonstrated that AqpZ mediated water transport into and out of the cell (Delamarche et al., 1999
). In spite of the proven functionality of E. coli aquaporin and the availability of some structural studies on the protein (Borgnia et al., 1999
; Calamita et al., 1997
), the physiological importance of AqpZ was only apparent when the bacteria were cultured in hypo-osmolar medium and at maximum growth rates (Calamita et al., 1998
).
We have previously isolated and characterized the aquaporin of Brucella abortus, a facultative intracellular pathogen causing infertility and abortion in cattle and a debilitating disease in man. Amino acid sequence alignment between the B. abortus AqpX and other MIP family proteins revealed that B. abortus AqpX was 70 % identical to E. coli aquaporin AqpZ. AqpX expression in Xenopus oocytes and cryoelectron microscopy of E. coli aqpZ mutants transformed with the B. abortus aqpX gene confirmed that the aquaporin from B. abortus was an efficient water channel. Furthermore, it has been shown that AqpX was not able to transport glycerol (Rodriguez et al., 2000).
The present study was designed to characterize the expression of the B. abortus aqpX gene during the phases of the bacterial growth curve and under different osmotic conditions, and to investigate the physiological function of the AqpX water channel. Our studies showed that expression of the aqpX gene was induced in hypertonic conditions and it was enhanced during the mid-exponential growth phase. Additionally, we demonstrated that aqpX gene was not essential for bacterial survival. To our knowledge, these studies provide the first example of hypertonic induction of a bacterial aquaporin.
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METHODS |
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Plasmid constructions.
Plasmid pAQPX1 contains the B. abortus aqpX gene cloned in the high-copy-number vector pBluescript SK (Stratagene) (Rodriguez et al., 2000). A 2·3 kb fragment from pAQPX1 containing the putative promoter region and the first eight codons of the B. abortus aqpX gene was obtained by PCR, using synthetic oligonucleotides T3 (5'-ATTAACCCTCACTAAAGGGA-3') and ATG1 (5'-CGTAAGCTTCAGCCGATAATTTGTTCAA-3'). A second PCR using synthetic oligonucleotides TAA2 (5'-TGCAAGCTTTGCAATCATCTGGAAGGG-3') and T7 (5'-TAATACGACTCACTATAGGG-3') was carried out to amplify a 0·6 kb DNA fragment containing the last 12 codons of the aqpX gene and additional downstream sequence. The primers ATG1 and TAA2 both contain HindIII restriction sites. The first PCR product was digested with BamHI/HindIII and the second fragment was digested with EcoRV/HindIII. Both fragments were ligated together into pBluescript SK digested with BamHI/HincII, to generate plasmid pAQP2. This plasmid was linearized with HindIII and ligated with an 5·3 kb HindIII fragment, from pSU4111, containing a promoterless lacZ-Km cassette, to generate pSKaqpX : : lacZ-Km. The aqpX : : lacZ-Km fusion was obtained from plasmid pSKaqpX : : lacZ-Km as an 8·2 kb KpnI/XbaI fragment. This DNA fragment was blunt-ended with Klenow DNA polymerase and ligated into the EcoRV site of pKOK.4, a suicide vector mobilizable in Brucella. The resulting plasmid was named pAQPXlacZ-Km (Fig. 1
).
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Southern blot analysis.
Chromosomal DNA from B. abortus 2308 Nxr, and from colonies putatively containing a gene replacement, was separately digested with EcoRI and HindIII, electrophoresed in a 0·9 % agarose gel, and transferred to positively charged nylon membranes (Roche Diagnostics) by capillarity. The blots were hybridized with a 3·6 kb EcoRI fragment containing the B. abortus aqpX gene, and a 5·3 kb HindIII fragment containing the lacZ-Km cassette. The probes were labelled with digoxigenin (Roche Diagnostics). After hybridization, the membranes were washed, incubated with alkaline-phosphatase-labelled anti-digoxigenin antibodies and developed using the luminescent substrate disodium 3-(4-methoxyspiro{1,2-dioxethane-3,2'-(5'-chloro)tricyclo[3.3.1.13,7]decan}-4-yl)phenylphosphate (CSPD, Roche Diagnostics).
-Galactosidase assays.
-Galactosidase assays were carried out with whole cells as described by Miller (1972)
. Saturated cultures in BB were diluted to reach OD600
0·1 and then grown at 37 °C as appropriate.
-Galactosidase activity was expressed as µmol 2-nitrophenyl
-D-galactopyranoside cleaved min-1 (mg protein)-1 (Miller, 1972
). Each assay was performed at least four times independently, and the results were averaged for display as bar graphs.
Primer extension.
Synthetic oligonucleotide primer E94 (5'-AACACCGAGGAAGCCGATACCCAG-3') was 5' end-labelled with T4 polynucleotide kinase and [-32P]ATP. Total RNA was prepared from B. abortus cultures with the High Pure RNA Isolation Kit (Roche Diagnostic). One microgram of RNA was used as template for the synthesis of cDNA with M-MLV (Moloney Murine Leukaemia Virus) reverse transcriptase (Gibco-BRL) from the E94 labelled primer. The products of the extension reactions were analysed in 6 % urea-polyacrylamide gels. A sequencing ladder using the same primer was run in the gel alongside the extension products to map the 5' end of the mRNA.
RT-PCR.
The reverse transcription assay was used to quantify the levels of aqpX mRNA in different osmolarity conditions. Briefly, 200 ng total RNA was mixed with 10 µl double-strength reverse transcriptase buffer containing 0·8 µM of each dNTP, 4 µM Aqpset. R primer (5'-GGCGAATTCTTCTGATTAATCTCGGCC-3'), starting 6 nt downstream from the aqpX stop codon, and 0·2 units µl-1 of RNAsin RNase inhibitor (Promega). The samples were incubated for 5 min at room temperature to allow the primer to anneal the template. For cDNA synthesis, 5 units RNasin and 10 units M-MLV reverse transcriptase were added and then the samples were incubated for 60 min at 42 °C. Amplification was performed by adding 5 µl of the reverse transcriptase reaction mixtures containing cDNA to 50 µl amplification buffer containing 0·2 mM of each dNTP, 0·25 µM of the forward and reverse primers and 1 unit Taq DNA polymerase (Qiagen). In these assays RT-AMP.R (5'-AGCAGGCCCTTCCAGA-3'), starting 10 nt upstream from the aqpX termination codon, was used as the reverse primer, and Aqpset.F (5'-TTCGGATCCCATGTTGAACAAATTA-3') as the forward primer. After 1 min incubation at 94 °C, samples were subjected to 35 amplification cycles (30 s at 94 °C, 30 s at 43 °C and 60 s at 72 °C), followed by a final incubation at 72 °C for 8 min. The reaction products were resolved in 1 % agarose gels run at 5 V cm-1 and quantified using the Molecular Analyst Software (Bio-Rad).
To normalize the aqpX cDNA bands, we used the gene for the translation initiation factor IF-1 of B. abortus, whose expression has recently been demonstrated to be constitutive (Eskra et al., 2001). The RT-PCR assay was performed as described above. The primer RT-IF.R (5'-TGAAGCGGTAGGTGATGCGG-3') was used for the reverse transcription assay. For the PCR amplification step we used the primers IF-1.F (5'-ATGGCGAAAGAAGAAGTCCT-3') and IF-1.R (5'-ACTAGAACCTTGTCACCGGC-3'), giving a specific product of 164 bp (Eskra et al., 2001
).
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RESULTS |
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Growth characteristics of the Brucella aqpX : : lacZ-Km mutant
B. abortus 2308 and the mutant B. abortus aqpX : : lacZ-Km were grown in BB (Fig. 2) and minimal media (data not shown) with different osmolarity conditions. When the optical density was measured, no significant differences were observed between the growth rate of the mutant and the wild-type strain under any condition. However, the number of c.f.u. of the B. abortus aqpX : : lacZ-Km strain decreased during growth in hypo-osmolar medium at the late stationary phase (Fig. 2a
, right panel). These results demonstrate that disruption of the B. abortus aqpX gene was not lethal and that the production of the aquaporin was apparently necessary for survival at the late stationary phase in hypo-osmolar medium only. On the other hand, no apparent differences in cell morphology were observed under the light microscope between the mutant and the wild-type cells under the different growth conditions (data not shown).
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DISCUSSION |
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Recently, we have cloned the gene and characterized functionally an aquaporin water channel in the intracellular facultative bacterium B. abortus (Rodriguez et al., 2000). To determine the function of this channel, an aqpX mutant carrying a single-copy aqpXlac fusion in the chromosome was constructed by fusing a lacZ-Km cassette with the promoter region of the aqpX gene. No significant differences in growth rates were found between the B. abortus 2308 wild-type strain and the B. abortus aqpX : : lacZ-Km mutant when grown in rich and minimal medium. This finding showed that the aqpX gene was not essential in B. abortus grown in isotonic media. However, the mutant showed decreased viability upon prolonged incubation in hypo-osmolar medium, in line with the observed osmotic regulation of the E. coli aqpZ gene (Calamita et al., 1995
). In the case of AqpZ, the decrease in cell viability was significant only when the E. coli AqpZ- strain was co-cultured with the wild-type parental strain in minimal media (Calamita, 2000
).
Experiments with the aqpX : : lacZ-Km transcriptional fusion showed that the aqpX gene was active throughout the growth curve, with an activity peak at the mid-exponential growth phase. This result was again similar to that reported for E. coli (Calamita, 2000; Calamita et al., 1998
), and indicated that the aqpX gene was subject to growth phase regulation. A recent report on the regulation of expression of the E. coli aqpZ gene using a single-copy chromosomal aqpZlacZ fusion indicated that expression of E. coli aqpZ increased in the stationary phase of growth and that this increase was dependent on the RpoS sigma factor (Soupene et al., 2002
).
Expression studies with our aqpX : : lacZ transcriptional fusion as well as the RT-PCR assays demonstrated that the expression of the aqpX gene was increased in hyperosmolar conditions (BB plus 125 mM NaCl). Surprisingly, the B. abortus gene showed a behaviour different from that of the E. coli aqpZ gene, whose expression was reported to increase significantly in hypo-osmolar conditions, and to decrease in hyperosmolar conditions (Calamita et al., 1998), or found to be unaffected by changes in osmolality (Soupene et al., 2002
).
We have also determined the transcriptional initiation point of the Brucella aqpX gene and found a nearby sequence with a reasonable similarity with the consensus E. coli 70 promoter sequence. The distance between the -10 box and the start of transcription is longer than usual in this putative promoter. This could be due to degradation of mRNA on its 5' side as suggested by the two extension products observed. Since the E. coli aqpZ gene and some osmotically regulated genes are transcribed from promoters active in the stationary phase, we also analysed the sequence for the presence of alternative promoters such as
38 (rpoS) (Lee & Gralla, 2002
) or gearbox promoters (Ballesteros et al., 1998
) with negative results. On the other hand, the translation start of AqpX is 169 bp away from the transcription start site. This long region of untranslated mRNA suggests the existence of some post-transcriptional mechanism of regulation of expression of the AqpX protein.
This study adds to the previous characterization of the E. coli aqpZ gene to clarify some aspects of bacterial aquaporin biology. A common finding in the two organisms was that the aqp gene was not essential either in Brucella or in E. coli, and that the aquaporin-defective mutants did not show a major phenotype in either bacterium. This observation is consistent with the irregular distribution of aquaporins in bacteria. Many bacteria do not possess aquaporin genes in their genomes; however, they are able to respond and to adapt to different osmotic conditions. This indicates that bacteria possess mechanisms to respond to water stress that are independent of the production of aquaporins. In spite of their dispensability, some bacteria have acquired aquaporins and evolved mechanisms for their regulated expression. The presence of regulatory mechanisms for the expression of these genes has to be interpreted as the result of some evolutionary advantage conferred by the possession of aquaporins. The differences observed between E. coli and B. abortus in these regulatory details indicate that the aquaporins play different roles in these bacteria, which have different lifestyles and evolutionary histories.
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ACKNOWLEDGEMENTS |
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Received 31 July 2003;
revised 22 August 2003;
accepted 2 September 2003.
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