Laboratoire de Microbiologie1 and Laboratoire de Biologie du Développement2, Université des Sciences et Technologies de Lille, F-59655 Villeneuve dAscq Cedex, France
Author for correspondence: Valérie Leclère. Tel: +32 3 20 43 46 68. Fax: +32 3 20 43 65 04. e-mail: Valerie.Leclere{at}univ-lille1.fr
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ABSTRACT |
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Keywords: Vibrionaceae, MnSOD, FeSOD
Abbreviations: CDM, chemically defined medium; DIP, 2,2'-dipyridyl; EDDA, ethylenediamine di-(o-hydroxyphenylacetic acid); PQ, paraquat; SOD, superoxide dismutase
The GenBank accession numbers for the sequences reported in this paper are AF317226 and AF317227.
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INTRODUCTION |
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The SODs are classified according to the metal ion cofactor required for their activity: the copper-zinc type (Cu/ZnSOD), the manganese type (MnSOD), the iron type (FeSOD) (Fridovich, 1986 ) and the most recently described nickel type (NiSOD) (Youn et al., 1996
). Bacteria contain one to three SOD enzymes, which can be expressed simultaneously. The facultative anaerobe Escherichia coli possesses three SODs which differ in their location and temporal expression. Both FeSOD and MnSOD are cytoplasmic. FeSOD is produced at a constant rate under aerobic and anaerobic conditions, but MnSOD is only synthesized aerobically and its presence is modulated by exposure to oxygen or intracellular
or upon changes in growth phase (Demple, 1991
; Compan & Touati, 1993
). The third E. coli SOD, containing Cu/Zn, is located within the periplasmic space (Benov et al., 1995
).
Bacteria belonging to the genus Aeromonas are Gram-negative facultatively anaerobic rods displaying catalase and oxidase activities. They are currently classified in the family Vibrionaceae, but a separate family has been proposed on the basis of the 16S rRNA cataloguing and rRNADNA hybridization results (Colwell et al., 1986 ). They are commonly found in aquatic environments and increasingly in ready-to-eat foods (Kirov, 1997
). They are considered as emerging opportunistic pathogens associated with gastroenteritis and enterocolitis in humans (Merino et al., 1995
).
According to the description of the family Vibrionaceae in Bergeys Manual of Systematic Bacteriology (Baumann & Schubert, 1984 ), Aeromonas expresses a single FeSOD and this feature tends to discriminate between the families Vibrionaceae and Enterobacteriaceae.
In this study we identified and characterized the genes encoding SODs from Aeromonas hydrophila and compared their expression to that of the E. coli SODs.
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METHODS |
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Preparation of bacterial extracts and assays.
The cells were harvested by centrifugation at 12000 g for 15 min at 4 °C, washed with and suspended in 50 mM sodium phosphate buffer pH 7·8, then stored at -20 °C. Cells were disrupted by sonication, and after centrifugation at 12000 g for 15 min at 4 °C, the supernatants (crude extracts) were stored at 4 °C for immediate use or frozen at -20 °C.
Protein concentration was determined using a Bio-Rad DC protein assay kit. Total SOD activity from mid-exponential-phase crude extracts was estimated using the xanthine/xanthine oxidase procedure (Beauchamp & Fridovich, 1971 ). The amount of SOD required to inhibit the reduction rate of nitro blue tetrazolium by 50% was defined as one unit of activity.
The number and nature of SODs were determined by the PAGE method already described (Leclère et al., 1999 ). Triplicate gels were soaked in riboflavine containing 5 mM H2O2 or 2 mM KCN to differentiate between Fe- and MnSOD (Droillard et al., 1989
).
Cell fractionation by osmotic shock.
Bacterial cells harvested after 30 h growth in LB medium were washed twice with 10 mM Tris. The cells were suspended in 10 mM Tris/20% sucrose and stirred at room temperature for 15 min, then suspended in the same buffer without sucrose and stirred again for 15 min in ice. The periplasmic fraction was obtained by centrifugation at 15000 g for 30 min at 4 °C and the cells were disrupted by sonication to get the cytosolic fraction.
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RESULTS |
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The ability of the plasmids to restore resistance to PQ to the SOD- double mutant of E. coli was studied by growth measurements in the presence of 100 µM PQ (Table 2). A. hydrophila was naturally resistant to PQ at this concentration. Clones EcA125 and EcA126 showed the same PQ resistance as E. coli QC868, while E. coli QC871 (SOD-) did not grow under these conditions.
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The 3·6 kb insert of pVLAh126 contained a complete promoter and coding sequence (sodA) for a protein of 204 amino acids sharing 55% identity with E. coli MnSOD (114/206 residues). A transcription initiation sequence (+1; AACA), an AT-rich region (-13; ATTAAT) and a ShineDalgarno box (+5; GAGG) were found upstream of the ATG (+14). Three potential -35 boxes (TTGA/TCA) could also be found within the sequence, but the role of these sequences has not been studied. An inverted 11 nt repeated sequence centred on nucleotide 661 with a probable hairpin structure could correspond to the rho-independent terminator sequence of the mRNA. The ORF of about 700 bp encoded a protein with a theoretical pI of 6·07 and a molecular mass of 22·3. Amino acids important for the ligand binding were conserved (G77, G78, F85, Q150 and D151) and the 12 first amino acids constituted a potential signal peptide with the most likely cleavage site between A11 and Y12.
Growth-phase-dependent expression of the MnSOD
When E. coli QC868 was grown in LB medium under high aeration to mid-exponential phase, both FeSOD and MnSOD were detected on PAGE. EcA126 expressed the MnSOD under the same experimental conditions. A further assay, using A. hydrophila samples withdrawn hourly during 30 h, showed that total SOD activity from crude extracts remained unchanged: 5·92±1·62 units SOD (mg total protein)-1 at 3 h (mid-exponential phase) and 5·96±1·15 units SOD mg-1 at 16 h (stationary phase). On PAGE, the FeSOD was expressed whatever the growth phase whereas the MnSOD only appeared on gels after 16 h culture, corresponding to the stationary phase (Fig. 2a).
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Effect of iron deficiency on MnSOD synthesis
A. hydrophila was grown in LB medium in which iron was sequestered by either chemical (DIP, EDDA) or biological (Desferal) chelators. Whatever the chelator used, an identical result was obtained: the lack of iron induced the expression of MnSOD during mid-exponential phase, since the MnSOD was detectable on PAGE gels after 3 h growth (data not shown).
Effect of PQ on expression of the MnSOD
To test the induction of the sodA gene, A. hydrophila, E. coli EcA126 and E. coli QC868 (SOD+) were grown in the presence of different concentrations of PQ (10 to 103 µM). PQ at any concentration did not affect the growth rate or the final biomass and the MnSOD was not detectable on PAGE gels before the stationary phase (data not shown). The total SOD activity assayed during stationary phase (16 h) remained unchanged for A. hydrophila and slightly enhanced for pVLAh126 cloned in E. coli QC871 (strain EcA126), whereas it was strongly stimulated for E. coli QC868 (Table 3).
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DISCUSSION |
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A positive effect of the iron-dependent regulatory Fur protein has been observed in the expression of the sodB gene of E. coli (Niederhoffer et al., 1990 ) and recently the site of Fur regulation has been found in the promoter region. The site functioned as an extended -10 promoter containing a TGN sequence near the TA-rich region (Dubrac & Touati, 2000
). Nevertheless the A. hydrophila FeSOD seemed to be constitutively expressed since the environmental changes such as iron limitation tested in this study did not affect its synthesis; moreover, the A. hydrophila sodB promoter did not show the TGN sequence and so could be unaffected by the Fur regulator.
The sequenced insert of pVLAh126 contained a 700 bp gene encoding a MnSOD displaying 55% identity with the E. coli MnSOD (Takeda & Avila, 1986 ). The amino acids implicated in the metal binding, the Y34 playing a catalytic role (Hunter et al., 1997
), and those pinpointed as potential discriminators between the iron and manganese proteins (G77, G78, F85, N149, Q150, D151 and V189) (Parker & Blake, 1988
), were conserved. A conservative change between the K29 responsible for electrostatic steering of the substrate and an arginine residue was observed. The amino-terminal part of the protein (65 residues) shared high homology (71%) with that of E. coli. Nevertheless, although 9 out of the 12 first residues were conserved (MSHTLPLALYAY for A. hydrophila vs MSYTLPSLPYAY for E. coli), the A. hydrophila MnSOD contained a potential signal sequence lacking in E. coli Fe- and MnSODs (Nielsen et al., 1997
). A similar potential sequence suggesting a periplasmic location of the enzyme has been described in Acinetobacter calcoaceticus MnSOD (23 amino-terminal residues) (Geißdörfer et al., 1997
). In the fish pathogen Aeromonas salmonicida a periplasmic MnSOD has also been detected (Barnes et al., 1996
) and a role in the pathogenicity was proposed by the authors. This SOD may play a role in the defence against external reactive oxygen species like the periplasmic Cu/ZnSOD found in E. coli (Benov & Frodovich, 1996
), Legionella pneumophila (Saint-John & Steinmann, 1996
), Haemophilus influenzae and H. parainfluenzae (Kroll et al., 1991
, 1995
). Recently, the molecular analysis of genetic differences between virulent and avirulent strains of A. hydrophila isolated from diseased fish did not indicate SODs as virulence factors (Zhang et al., 2000
).
As mentioned by Barnes et al. (1996) for A. salmonicida, the MnSOD of A. hydrophila was induced when no iron was available in the medium. In E. coli, sodA was regulated by the Fur repressor binding to iron boxes, consensus sequences NATA/TAT (Escolar et al., 1998
), present in the promoter. These iron-regulatory sequences were not found in the region 300 bp upstream of the sodA gene of A. hydrophila although a Fur protein was present in the wild-type A. hydrophila 495A2 (Barghouthi et al., 1991
). So it remains unclear whether the regulation is Fur-dependent, and further investigation is necessary to determine the mechanism of regulation of sodA by iron.
In LB medium, the A. hydrophila MnSOD was only expressed in the stationary phase under high aeration, even with an excess of iron (data not shown). These results are in accordance with the high expression of MnSOD in response to elevated oxygen levels and upon changes in growth phase in E. coli (Compan & Touati, 1993 ). Similarly increased levels of SOD activity during stationary phase were observed in other bacterial genera (Saint-John & Steinmann, 1996
; Inaoka et al., 1998
; Clements et al., 1999
). The authors suggested that enhanced SOD levels are connected with survival of the bacterial cells under stressed conditions. The expression of a MnSOD can also be related to quorum sensing, as was recently demonstrated for Pseudomonas aeruginosa (Bollinger et al., 2001
).
The sodA gene of A. hydrophila was not regulated by addition of PQ to the medium even in E. coli EcA126 although PQ enters the cells, supported by the fact that the expression was strongly stimulated by PQ in E. coli QC868. This non-regulation by PQ may not be so surprising considering the periplasmic location of the MnSOD in A. hydrophila and the increased flux of intracellular superoxide caused by PQ (Hassan, 1984 ). Physiological studies are necessary to determine the precise function of the MnSOD for Aeromonas.
Lastly, considering the particular conditions of the MnSOD expression in A. hydrophila described in this paper, together with the previously reported occurrence of this enzyme in A. salmonicida (Barnes et al., 1996 ) it now appears clear that the presence of a unique SOD in Vibrionaceae should no longer be taken into account to distinguish this family from Enterobacteriaceae.
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ACKNOWLEDGEMENTS |
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Received 26 March 2001;
revised 4 June 2001;
accepted 13 July 2001.