Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Seville, 41012 Seville, Spain1
Sector of Organic Chemistry and Biochemistry, Department of Chemistry, University of Ioannina, 45110 Ioannina, Greece2
Author for correspondence: Antonio Ventosa. Tel: +34 95 455 67 65. Fax: +34 95 462 81 62. e-mail: ventosa{at}cica.es
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ABSTRACT |
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Keywords: -amylase, halophile, Halomonas
The EMBL accession number for the sequence reported in this paper is AJ239061.
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INTRODUCTION |
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While there have been numerous reports on extracellular amylases from non-halophilic bacteria, very limited information is available on amylases from halophilic species. Amylolytic activities have been reported in the moderately halophilic bacteria Acinetobacter sp. (Onishi & Hidaka, 1978 ), Nesterenkonia halobia (Onishi, 1972b
; Onishi & Sonoda, 1979
), Micrococcus varians subsp. halophilus (Kobayashi et al., 1986
), and other Micrococcus isolates (Khire, 1994
; Onishi, 1972a
). However, molecular characterization of these amylases is lacking. Apart from their biotechnological interest, the characterization of genes encoding amylase activity will be invaluable in elucidating their regulatory and secretion mechanisms, and the structure-function relationship of extracellular enzymes with optimal activity at high salt concentrations.
For this study we selected the moderate halophile Halomonas meridiana DSM 5425, which produces an extracellular -amylase that has been recently characterized biochemically (Coronado et al., 2000
). The enzyme was optimally active at 10% NaCl, although a remarkable activity was detected up to 30% salts, making it very attractive for a molecular characterization. Here we describe the isolation, cloning and sequencing of the
-amylase gene. The heterologous expression in Halomonas of the Bacillus licheniformis amyLI gene encoding a thermostable extracellular
-amylase is also reported.
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METHODS |
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The expression vectors pML122/123 (Gmr; Labes et al., 1990 ), and the plasmid vector pBluescript II KS (Ampr; Stratagene) were used for cloning. The broad-host-range cloning cosmid vector pVK102 (Kmr Tcr; Knauf & Nester, 1982
) was used to construct a genomic library of H. meridiana DSM 5425. Derivatives of pML123 and pVK102 were transferred from E. coli to Halomonas strains by triparental matings using pRK600 (Kessler et al., 1992
) as helper plasmid.
Preparation of cell fractions.
H. meridiana and mutant strains were grown in SW-5 medium supplemented with 0·5% (w/v) starch at 37 °C for 24 h. The supernatant was collected after centrifugation at 10000 r.p.m. and concentrated by centrifugation through Centricon and Centripep columns (Amicon). To obtain the periplasmic fraction, the method described by Harold & Heppel (1965) was followed. The cell extracts were obtained by ultrasonic treatment (Vibra-cell). If the amylase activity was not determined immediately, the samples were stored at -20 °C after addition of 1 mM CaCl2 and 10 µg ml-1 of the protease inhibitor PbSc (PefablocSC, Roche).
Amylase activity.
The presence of amylolytic activity on plates was routinely determined following the method described by Cowan (1991) , using SW-2 medium supplemented with 0·5% (w/v) soluble starch. After incubation at 37 °C for 7 d, the plates were flooded with 0·3% I2/0·6% KI solution: a clear zone around the growth indicated hydrolysis of starch. To determine the amylase activity in the different cell fractions, samples were assayed by automated reading in microtitre plates. Ten-microlitre samples were mixed with 50 µl amylase reagent from a diagnostic kit (Sigma) into microtitre plate vials, and incubated for 15 min at 37 °C. The reaction was stopped by the addition of 25 µl 1 M Na2CO3 solution. Enzymic activity was measured by following the formation of reaction products at 450 nm using an EL312e Microplate Reader (BIO-TEC Instruments).
Gel electrophoresis.
The method of Laemmli (1970) was used for SDS-PAGE. Proteins were stained with a silver nitrate solution (0·2%) (Sambrook et al., 1989
). High-MW protein markers (RPN 756, Amersham) were used as standards.
DNA manipulations.
DNA manipulations and isolation of plasmid DNA were performed by standard procedures (Sambrook et al., 1989 ). Southern blot analyses were carried out by using digoxigenin-labelled probes according to the instructions of the manufacturer (Roche).
Transposon mutagenesis.
Transposon mutagenesis was performed by conjugal transfer of pSUP102-Gm::Tn1732 from E. coli SM10 (Simon et al., 1983 ; Ubben & Schmitt, 1986
) to a spontaneous Rifr mutant of the H. meridiana wild-type strain. Matings were carried out by mixing the donor and recipient cultures at a ratio of 1:4 (100 µl donor, 400 µl recipient). After centrifugation, the pellet was washed with SW-2, resuspended in 100 µl SW-2 and placed on a 0·45 µm pore filter on SW-2 solid medium. After overnight incubation at 37 °C, cells were resuspended in 20% (v/v) sterile glycerol and, after appropriate dilutions, plated on SW-2 medium with rifampicin and kanamycin at a density resulting in about 100200 colonies per plate. For selection of H. meridiana transconjugants with the Amy- phenotype, colonies from these master plates were transferred with sterile toothpicks to SW-2 + Rif +Km plates containing 0·5% (w/v) soluble starch. The amylase activity on the plates was detected as described above.
Library construction.
An H. meridiana gene bank was constructed in the broad-host-range cosmid pVK102. H. meridiana genomic DNA was partially digested with HindIII, and DNA fragments in the size range 2330 kb were separated in sucrose gradients and cloned into the HindIII site of pVK102. In vitro packaging of the recombinant molecules was performed with a commercially available extract (Amersham) as recommended by the manufacturer. Aliquots of the packaging reaction mixture were used to infect cells of E. coli HB101 that were then plated onto LB agar plates containing tetracycline to select recombinant clones. Randomly selected clones were found to contain DNA inserts ranging from 20 to 30 kb in size.
DNA sequencing.
DNA sequencing was performed by MWG-Biotech using an automatic DNA sequencer (LiCor). DNA sequence was analysed with the GCG Sequence Analysis Software Package (Genetics Computer Group) and the BLAST program of the National Center for Biotechnology Information (NCBI). Protein analyses and alignments were performed by using the ProtParam program from the ExPASy (Expert Protein Analysis Systems) of the Swiss Institute of Bioinformatics and the CLUSTAL program of EBI (European Bioinformatics Institute), respectively.
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RESULTS |
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The Amy- mutants were identified as those unable to show amylase activity when grown on SW-2 plates supplemented with starch. Out of 5850 Kmr colonies screened, 13 did not show clear haloes when iodine solution was added to the plates. These Amy- mutants were named I to XIII. To confirm the transposon insertion in these mutants, chromosomal DNA of each strain was digested with SalI (which does not cut Tn1732) and an internal fragment of Tn1732 was used as a probe. All mutants were shown to have a unique insertion of Tn1732 in a DNA fragment of approximately 15 kb (type I mutants), except mutant II (type II), which contained the transposon within a fragment ranging from 6 to 11 kb in size (data not shown).
To determine if any of the isolated mutants was defective in amylase synthesis, the amylolytic activity in their different cell fractions was tested and compared to that of the wild-type strain (Table 1). In the wild-type strain, most of the activity was associated with the supernatant, but activity was also detected in the periplasmic fraction. As expected, none of the mutants showed extracellular amylolytic activity. However, type I mutants showed amylase activity in the periplasmic fraction (although this was lower than that of the parental strain), suggesting that these mutants were able to synthesize the enzyme. In contrast, amylase activity was not detected in the periplasmic fraction of mutant II. Amylolytic activity was not detected in cell extracts of either type of mutants or the wild-type strain (Table 1
).
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Mutant II is affected in the -amylase synthesis gene
To identify the gene disrupted by Tn1732 in mutant II, the region flanking the left end of the transposon insertion in this mutant was isolated and sequenced. A preliminary hybridization experiment using Tn1732 as a probe against mutant DNA independently digested with several restriction enzymes showed that a DraI site is located very close to the Tn1732 insertion in mutant II. Chromosomal DNA of mutant II was digested with DraI and PstI, and ligated to EcoRV/PstI-digested pKS. The ligation mixture was used to transform E. coli DH5. From several Kmr Ampr colonies, plasmid pMJC27 was isolated, which carried a 2·2 kb DraI/EcoRV-PstI insert (Fig. 1a
). A restriction analysis of this plasmid showed a 1·85 kb EcoRI-PstI region, corresponding to the left end of Tn1732, and a 0·3 kb DraI/EcoRV-EcoRI fragment of DNA from mutant II, which was sequenced. A computer-assisted search in the databases revealed a high degree of homology to the genes encoding
-amylases from different micro-organisms (data not shown). This clearly demonstrated that mutant II was defective in the gene encoding the
-amylase from H. meridiana.
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Sequence analysis of the -amylase gene (amyH)
Hybridization analysis using the 0·3 kb DNA insert of pMJC27 as a probe against pMJC26 localized the amyH gene in a 0·7 kb DraI-EcoRV fragment, included in a larger 6·25 SacI-EcoRV region (Fig. 1b). Two SacI-DraI segments of pMJC26, of 4·1 kb and 2·3 kb, were subcloned in pKS, generating pHS182 and pHS183, respectively (Fig. 1b
). These plasmids were used for sequencing. A total of 1·6 kb DNA spanning the 0·7 kb DraI-EcoRV fragment was sequenced (EMBL accession no. AJ239061). A primer complementary to the 5' end of pHS183 was generated and used to sequence the DNA including the DraI site with pMJC26 as a template. This ruled out the possibility of a second DraI site at the junction between pHS182 and pHS183. Computer-assisted analysis of the sequence revealed the presence of one ORF that corresponded to the amyH gene. The gene starts with a TTG codon at position 201 and ends with a TAA codon at position 1572. It encodes a 457-residue protein with a deduced molecular mass of 50 kDa. Database searches revealed the product encoded by amyH to have extensive sequence similarity to
-amylases from Gram-negative and Gram-positive bacteria (Fig. 2
). The best alignment (55% identity) was obtained with the thermolabile
-amylase from the Antarctic psychrophilic Alteromonas (Pseudoalteromonas) haloplanktis A23 (Feller et al., 1992
), followed by the
-amylases from the facultatively anaerobic Aeromonas hydrophila (53%; Chang et al., 1993
), Bacillus sp. (49%; EMBL accession no. AB006823), the actinomycete Thermomonospora curvata (48%; Petricek et al., 1992
) and Streptomyces griseus (48%; Vigal et al., 1991
). Moreover, a considerable degree of homology (about 48% identity) was found with
-amylases from insects, such as Tribolium castaneum (Hickey et al., 1987
), Aedes atropalpus (EMBL accession no. U01209) and Drosophila melanogaster (Inomata et al., 1995
), and mammals, such as mouse (Hagenbuchle et al., 1980
).
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Detection of the -amylase in SDS-polyacrylamide gels
To visualize the amyH-encoded protein, supernatant fractions were prepared from the wild-type and mutant II strains, and proteins were electrophoretically separated in an SDS/10% polyacrylamide gel (Fig. 3). After silver staining, a protein band with an apparent molecular mass of 49 kDa was detected in the supernatant of the wild-type strain. As expected, this protein was absent from the supernatant of mutant II. The electrophoretic estimation of the molecular mass of the
-amylase agrees well with the value predicted from the deduced primary structure of the mature protein.
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DISCUSSION |
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The cloning and characterization of the H. meridiana amyH gene in this study will enable investigations of the molecular adaptations required by the enzyme to be active at high salt concentrations. Moreover, the gene has a potential application for the construction of expression and secretion vectors for the production of heterologous proteins by moderate halophiles (Ventosa et al., 1998 ).
Amino acid sequence comparison revealed that AmyH belongs to the already proposed family of -amylases composed of the enzymes from Alteromonas haloplanktis, Thermomonospora curvata, streptomycetes, insects and mammals (Janecek, 1994
). Janecek (1994)
suggested a separate evolutionary origin of this group from the two other groups of amylases from plants, fungi and yeasts. Therefore, our results include in the same branch of evolution the two
-amylases from extremophilic bacteria Alteromonas haloplanktis and H. meridiana. The enzymes from Alteromonas haloplanktis and Aeromonas hydrophila were assigned to family 13 of Henrissats classification of glycosyl hydrolases (Henrissat & Bairoch, 1993
). Therefore, on the basis of amino acid homology, AmyH may be considered also a member of this family.
The AmyH protein contains the four highly conserved regions in amylase enzymes (Nakajima et al., 1986 ). The invariant amino acid residues are also all conserved in the AmyH sequence. Some of these residues in the consensus sequences have been determined to play a role in the amylolytic activity. The sites responsible for substrate binding proposed by Matsuura et al. (1984)
for Taka-amylase A from Aspergillus oryzae are present in the H. meridiana amylase. Furthermore, the residues Asn106 and His198 reported to be involved in Ca2+ binding (Machius et al., 1995
) are fully conserved. In addition, the two strictly conserved residues likely to be involved in chloride binding (Arg192 and Asn281) are also present in AmyH.
Although the AmyH protein excreted by H. meridiana shared a high homology with the -amylases shown in Fig. 2
, it displays optimal activities at high salt concentration (Coronado et al., 2000
). Extracellular enzymes produced by halophilic micro-organisms have to be adapted to high salinity. It has been suggested that at least part of this adaptation involves an abundance of acidic residues (Lanyi, 1974
) that, as judged by the examination of the first crystal structures of proteins from a halophilic organism (Haloarcula marismortui) are distributed over the protein surface (Elcock & McCammon, 1998
). In agreement with this, the amylases from the moderate halophile H. meridiana and the halophilic archaeon Natronococcus sp., as well as the serine protease from the halophilic archaeon Natrialba asiatica, were shown to be very acidic proteins. This fact could partially explain the halotolerance exhibited by these enzymes. However, the molecular basis of the halotolerance of the characterized amylase is probably much more complex and is currently under investigation.
The ability of moderate halophiles to grow under extreme salt conditions makes them potentially useful for the production of heterologous proteins. An additional advantage is that most of them have very simple nutritional requirements, being able to use a wide range of compounds as the sole source of carbon and energy (Kushner & Kamekura, 1988 ; Ventosa et al., 1998
). In addition, for the production of stable salt-tolerant enzymes from extremophiles it is important to use halophilic micro-organisms as hosts, since the correct protein folding at high salt concentrations, specific post-translational modification, and protein secretion in extreme conditions are essential for a correct enzyme function. The correct heterologous expression and secretion by Halomonas of the thermostable
-amylase from B. licheniformis is a promising starting point for the use of moderately halophilic bacteria as cell factories.
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ACKNOWLEDGEMENTS |
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Received 2 November 1999;
revised 30 November 1999;
accepted 24 December 1999.