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Applied and Environmental Microbiology, September 2005, p. 5044-5049, Vol. 71, No. 9
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.9.5044-5049.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Maltotriose Utilization by Industrial Saccharomyces Strains: Characterization of a New Member of the -Glucoside Transporter Family

Madalena Salema-Oom,^1,2* Vera Valadão Pinto,¹ Paula Gonçalves,¹ and Isabel Spencer-Martins¹

Centro de Recursos Microbiológicos, Biotechnology Unit, Faculty of Sciences and Technology, New University of Lisbon, 2829-516 Caparica, Portugal,¹ Instituto Superior de Ciências da Saúde-Sul, 2829-511 Caparica, Portugal²

Received 27 January 2005/ Accepted 3 April 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
Maltotriose utilization by Saccharomyces cerevisiae and closely related yeasts is important to industrial processes based on starch hydrolysates, where the trisaccharide is present in significant concentrations and often is not completely consumed. We undertook an integrated study to better understand maltotriose metabolism in a mixture with glucose and maltose. Physiological data obtained for a particularly fast-growing distiller's strain (PYCC 5297) showed that, in contrast to what has been previously reported for other strains, maltotriose is essentially fermented. The respiratory quotient was, however, considerably higher for maltotriose (0.36) than for maltose (0.16) or glucose (0.11). To assess the role of transport in the sequential utilization of maltose and maltotriose, we investigated the presence of genes involved in maltotriose uptake in the type strain of Saccharomyces carlsbergensis (PYCC 4457). To this end, a previously constructed genomic library was used to identify maltotriose transporter genes by functional complementation of a strain devoid of known maltose transporters. One gene, clearly belonging to the MAL transporter family, was repeatedly isolated from the library. Sequence comparison showed that the novel gene (designated MTY1) shares 90% and 54% identity with MAL31 and AGT1, respectively. However, expression of Mty1p restores growth of the S. cerevisiae receptor strain on both maltose and maltotriose, whereas the closely related Mal31p supports growth on maltose only and Agt1p supports growth on a wider range of substrates, including maltose and maltotriose. Interestingly, Mty1p displays higher affinity for maltotriose than for maltose, a new feature among all the -glucoside transporters described so far.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Important biotechnological processes mediated by Saccharomyces species, such as brewing and baking, are based on the fermentation of starch hydrolysates. Maltose is the predominant sugar in these carbohydrate mixtures, which also contain glucose and maltotriose in considerable amounts. The great majority of yeast process strains consume both maltose and maltotriose only after glucose depletion. The repressive effect of glucose in the metabolism of alternative carbon sources has been extensively studied in Saccharomyces cerevisiae and is considered to limit the productivities of industrial fermentations, namely, in brewing. Moreover, most yeast strains use maltotriose only after maltose is exhausted, and very often the trisaccharide is not completely consumed (17). This can be deleterious to beer production, because it leads to lower ethanol yields and imparts sweetness to the final product. The specific features of maltotriose metabolism leading to incomplete or delayed utilization of this sugar by the yeast remain, in good part, elusive. There are controversial reports about the energetics of maltotriose utilization and the possible interactions between maltotriose and maltose uptake and metabolism. Although maltotriose is considered a fermentable sugar, which has been recently demonstrated for several brewer's and baker's strains (17), some authors observed that maltotriose is mainly respired, which might explain its incomplete consumption at the final stage of the oxygen-limited fermentation process (20, 25). Other authors suggest, however, that inhibition of maltotriose transport by maltose is the main problem (7, 21). Both maltose and maltotriose require a transporter to enter the cell and an intracellular -glucosidase to cleave them into glucose molecules. The hydrolase accepts both sugars as substrates, as well as other glucosides (1, 4), whereas there are six maltose transporters in S. cerevisiae (Mal21, Mal31, Mal61, Agt1, Mph2, and Mph3) but only three are capable of transporting maltotriose, the less specific -glucoside permeases encoded by the AGT1 gene and the recently characterized MPH2 and MPH3 genes (8). Each MAL locus includes, besides the gene encoding the specific proton symporter, two other genes encoding the -glucosidase (MALx2) and a transcriptional activator (MALx3). Agt1p is an inducible, wide-spectrum, -glucoside/proton symporter with high affinity for trehalose and sucrose, lower affinity for maltose and maltotriose (K_m of 18 mM), and even lower affinity for -methylglucoside, turanose, isomaltose, palatinose, and melezitose (13, 21, 23). The Malx1 proteins share at least 95% identity and show high affinity for maltose (K_m of 4 mM), also accepting turanose as a substrate (3, 4, 6). When studying sucrose transport in S. cerevisiae, Stambuk et al. (22) showed that the Mal21 permease could also transport sucrose, albeit with extremely low affinity (K_m of 120 mM). So far, no specific maltotriose transporter has been found, but there is genetic and kinetic evidence pointing to the presence in S. cerevisiae and closely related species (the so-called Saccharomyces sensu stricto group) of additional unidentified genes belonging to the -glucoside transporter family. Not only have Saccharomyces pastorianus brewing strains been shown to contain additional sequences homologous to both the MALx1 and AGT1 genes spread over the genome (15), but inhibition experiments using various sugars suggested the existence of different transporters with distinct substrate specificities (14, 16, 26).

Aiming to better understand maltotriose utilization by industrial yeasts, we conducted a physiological characterization of process Saccharomyces strains and looked for new maltotriose transporter genes. A novel member of the -glucoside transporter family with specific biochemical properties is reported.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Yeast strains and plasmids.
A total of 21 strains, from different industrial sources, were used in this work (Table 1). S. cerevisiae PYCC 5297 (from Danisco, Copenhagen, Denmark) was used to characterize maltotriose metabolism. S. cerevisiae CMY1050 (MATa ura3-52 lys2-801 ade2-101 trp1-63 leu2 MAL12 MAL13) (19) was the Mal^– host strain used for the library screening.

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TABLE 1. Growth on maltotriose and presence of maltotriose transporter genes in Saccharomyces strains used in this study

S. cerevisiae strain FY1679 (isogenic to S288C) was used as a template to amplify the MAL31 and AGT1 genes by PCR, with the following gene specific primers: MAL31, 5'-CGGGATCCAAGTCCAAGCTGAACCACCAA and 5'-GATTTGCCTATCTCTAGACCA (underlined nucleotides correspond to an additional BamHI site and a naturally occurring XbaI site, respectively); AGT1, 5'-CGGGATCCAAGTCCAAGCTGAACCACCAA and 5'-GGAATTCAAAATCTCGAGCTAGCTGAGG (underlined nucleotides correspond to additional BamHI and EcoRI sites, respectively). The BamHI-XbaI and BamHI-EcoRI fragments, which included the coding and regulatory regions of the genes, were cloned in the episomal shuttle vector YEplac195 (9), yielding plasmids pMAL31 and pAGT1, respectively.

A 3.7-kb XbaI-KpnI fragment encompassing the MTY1 gene was obtained from one of the S. pastorianus library plasmids carrying an insert of approximately 6.1 kb. This fragment was subcloned in YEplac195, giving pMTY1.

Strains CMY1050/pMAL31, CMY1050/pAGT1, and CMY1050/pMTY1 are derivatives of strain CMY1050 harboring the plasmids pMAL31, pAGT1, and pMTY1, respectively (this work).

Genomic library screening.
An S. pastorianus PYCC 4457 (Saccharomyces carlsbergensis^T) genomic library (11) was used to isolate genes involved in maltotriose transport. Strain CMY1050 was transformed with 5 µg of library plasmid DNA by the lithium acetate method (10). The transformation mixture was first plated onto solidified yeast nitrogen base (YNB) medium with 2% (wt/vol) glucose as the sole carbon source. After 3 days of incubation at 30°C, URA⁺ transformants were obtained (several rounds of transformation yielded a total of 3 x 10⁴ transformants). These colonies were then replica plated onto YNB medium containing 2% (wt/vol) maltotriose as the sole carbon source. Seven transformants that had acquired the ability to grow on maltotriose were selected. Library plasmids were rescued from these transformants upon transformation of Escherichia coli SURE competent cells with total DNA.

Growth conditions.
Yeast strains were routinely grown in YNB medium (without amino acids) containing the indicated carbon source and the required supplements, at 30°C and 150 rpm, unless otherwise stated.

Fermentation and respiration rates.
Sugar metabolism was studied using the standard manometric method of Warburg (24) to measure CO₂ production and O₂ consumption rates. Experiments were started by the addition of 2% glucose, maltose, or maltotriose to exponential-phase glucose- or maltose-grown cells resuspended in 0.1 M potassium phosphate, pH 5. The experiments were performed at 30°C in duplicate. Values shown represent averages from at least two independent experiments.

Analytical procedures.
Glucose, maltose, and maltotriose concentrations in the culture supernatants were quantified by high-pressure liquid chromatography with an Aminex HPX-87H column (Bio-Rad), using an LKB2142 refractive index detector.

Sugar transport assays.
Initial uptake rates of sugar/proton symport activity were determined by computer recording the alkalification of an aqueous yeast cell suspension upon sugar addition (18), using a standard pH meter and home-designed software (four pH values recorded per second). The data obtained were fitted to a one- or two-component Michaelis-Menten kinetic model, using GRAPHPAD PRISM software.

Miscellaneous.
Both strands of the S. pastorianus genomic DNA insert in pMTY1 were sequenced by primer walking, using an ALF express automated sequencer apparatus (Amersham Pharmacia Biotech) and 5'-Cy5-labeled sequence-specific primers.

Strains were screened for the presence of the AGT1 gene by PCR amplification using the specific primers 5'-CAAGAAGAAGGCTGCCTCAAAA and 5'-TCCAATCGCTCACGTTTAGCAT. These primers were used to amplify an AGT1 gene-specific probe for Southern blot experiments (used to confirm negative results in the PCR assay). The MTY1 gene-specific probe was also amplified by PCR, using the specific primers 5'-TTGGTAGGTTTGACCTTTAC and 5'-AGATGCCATATTATATGCGT. This 249-bp-long probe hybridizes within the coding region of MTY1. The probes were labeled with [³²P]dATP (Amersham Life Science), using the Prime-a-Gene kit (Promega). Gels were blotted onto Hybond N nylon membranes (Amersham Life Science). Hybridization and washing conditions were as previously described (12).

Nucleotide sequence accession number.
The DNA sequence of the MTY1 open reading frame can be retrieved from the EMBL database under accession number AJ491328.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Maltotriose utilization in industrial Saccharomyces strains.
In a preliminary survey of 21 maltose-positive industrial Saccharomyces strains, we observed that around 80% of them were able to grow on maltotriose as the sole carbon and energy source, albeit at different rates (Table 1). A few strains grew more slowly, showing maximum specific growth rates of around 0.17 h^–1. For a second group of strains, specific growth rates ranged between 0.20 and 0.25 h^–1. S. cerevisiae PYCC 5297 was the best-performing strain, and its specific growth rate was practically the same (0.32 to 0.34 h^–1) irrespective of the carbon source (maltotriose, maltose, or glucose). Therefore, this strain was selected to clarify the energetic metabolism of maltotriose in comparison to glucose and maltose. Fermentation and respiration rates in resting cells of strain PYCC 5297 precultivated in either glucose or maltose were estimated, using a Warburg apparatus that allows direct measurement of the amount of gas produced/consumed during sugar metabolism. Table 2 shows total CO₂ production and O₂ consumption rates determined for each sugar. In preliminary experiments, we have observed that maltose- and maltotriose-grown cells yielded similar results with respect to maltose and maltotriose metabolism. Results are shown only for maltose-grown cells. The respiratory quotient (RQ) on maltotriose was significantly higher (0.36) than that on maltose (0.16) or glucose (0.11), although maltotriose was still essentially fermented by the strain studied. The higher RQ observed for maltotriose metabolism agrees with the lower glycolytic flux (Table 2). We have analyzed a different strain for which the specific growth rates on all three sugars were considerably lower (0.21 h^–1), and the results were similar (results not shown). The fermentation performance of strain PYCC 5297 was also evaluated in batch cultures with rich medium containing a mixture of glucose, maltose, and maltotriose at concentrations mimicking those in brewer's wort (70 g liter^–1 maltose, 15 g liter^–1 glucose, and 15 g liter^–1 maltotriose). The time course for consumption of these sugars followed the expected sequential pattern (results not shown). Glucose was consumed first, followed by maltose and maltotriose. Of note, the latter was consumed only after complete maltose depletion. We found that both maltose and maltotriose were transported in strain PYCC 5297 cultivated with either one of the saccharides but not in glucose grown-cells (results not shown). Although both sugars were transported by an H⁺/symport mechanism, a higher transport activity was consistently observed for maltotriose-grown cells in comparison to maltose-grown cells (Table 2), suggesting that maltotriose is a better inducer of maltose/maltotriose transporter proteins.

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TABLE 2. Respiratory and fermentative characteristics of S. cerevisiae PYCC 5297a

Isolation of genes involved in maltotriose transport.
It has often been suggested that insufficient maltotriose uptake capacity could account for the incomplete or late consumption of this sugar in industrial processes using starch hydrolysates as raw material. However, only a few industrial strains have been genotyped for the presence of genes encoding maltose and maltotriose transporters. In one such a survey, MALx1 genes were found to be ubiquitous, being present in at least one locus, but the same was not true for AGT1-like genes (15). To investigate a possible correlation between the presence of the AGT1 gene and the ability to assimilate maltotriose, we screened the set of industrial strains listed in Table 1 for the presence of this gene, using PCR with specific primers. Although four of the strains were unable to grow on maltotriose, only one baking strain (PYCC 5295) was shown to lack the AGT1 gene-specific sequences (Table 1). We also examined AGT1 expression in all four maltotriose-negative strains. For one of the AGT1⁺ strains (PYCC 5291), no AGT1 mRNA could be detected, whereas the other two expressed the gene, although at very low levels. These results are compatible with Agt1p being the main maltotriose transporter in S. cerevisiae.

To search for additional maltotriose transporters, we used a functional complementation approach. A genomic library constructed from the type strain of S. carlsbergensis (S. pastorianus) PYCC 4457 (11), a brewing strain able to grow on both maltose and maltotriose, was used to identify genes capable of restoring growth on maltotriose of a strain devoid of AGT1 and MALx1 permease genes (CMY1050). In PYCC 4457, both maltose and maltotriose are transported by H⁺/symporters, with approximate K_m values of 3 mM for maltose and 24 mM for maltotriose.

Transformation of strain CMY1050 yielded seven transformants that were able to grow both on maltotriose and on maltose. It was readily shown that growth on maltotriose was dependent on the presence of a library plasmid, following retransformation of CMY1050 with each of the seven plasmids rescued. Restriction analysis and partial sequencing of the DNA inserts revealed that all the fragments map to the same genomic region encompassing a typical MAL locus. The seven fragments partially overlap each other and encode the permease and either the glucosidase or the MAL transcriptional activator. A 3.7-kb XbaI-KpnI fragment, obtained from the largest insert and containing the complete structural and regulatory regions of the transporter gene, was subcloned in the same vector. Sequencing of the entire open reading frame unveiled a novel gene, encoding a member of the -glucoside transporter family, highly homologous to both MALx1 and AGT1. The transporter gene thus isolated was designated MTY1 (for maltotriose transport in yeast). Surprisingly, although strain S. pastorianus PYCC 4457 contains the AGT1 gene, and this gene was found to be present in its genomic library by PCR (Table 1), it was not retrieved by functional complementation.

Using a MTY1 gene-specific probe, showing no cross hybridization with either MAL31 or AGT1 by Southern blot analysis, we found that MTY1 is probably present in five out of the eight industrial strains examined (Table 1).

Molecular characterization of the novel transporter Mty1.
Mty1p is predicted to be an integral membrane protein of the major facilitator superfamily of sugar transporters, with 12 transmembrane domains and 615 amino acids, one more than Malx1p. It shows 90% and 54% sequence identity to Mal31p and Agt1p, respectively (Fig. 1). Alignment of the three proteins has shown that amino acid differences between Mty1p and Malx1p are distributed throughout all transmembrane regions, whereas the 60 amino acids at the N- and C-terminal ends are virtually identical. Transmembrane domains 2, 3, 7, 9, and 11, as well as the section between Asn333 and Val359 in the central cytoplasmic loop, are the least homologous regions when the new gene is compared to all the other previously known -glucoside transporters. Moreover, in the same section, Mty1p includes an extra amino acid (Ser347), whereas a lysine was found at the same position for both Agt1p and Mph2p. Interestingly, 13 of the 58 amino acids that together distinguish Mty1p from Mal31p are present in Agt1p. Out of these 13, 2 are also common to Mph2p (Leu344 and Ser350).

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FIG. 1. Relationship between Mty1p and other transporters belonging to the -glucoside transporter family in S. cerevisiae. The dendrogram was obtained using Megalign software version 3.14 (Clustal method). Sequences were retrieved from GenBank.

Sugar uptake by the Mty1 transporter.
In order to compare the biochemical properties of the Mty1 transporter and other maltose and maltotriose transporters, namely, Mal31p and Agt1p, the respective genes were cloned in the same yeast episomal vector (YEplac195) used to construct pMTY1. Both the MAL31 and AGT1 genes, including upstream and downstream regulatory regions, were amplified by PCR from strain FY1679, using specific gene primers. The three recombinant plasmids were used to transform the S. cerevisiae Mal^– strain CMY1050. Expression of Mty1p restores growth of CMY1050 on both maltose and maltotriose culture media (Fig. 2), whereas the highly homologous Mal31p supports growth only on maltose. This showed a difference in substrate specificity that clearly distinguishes Mty1p from Mal31p. The specificity of Mty1p is also apparently different from that of Agt1p, since only the latter supports growth on -methylglucoside (results not shown). However, all three -glucoside transporters operate as proton symporters. The strains expressing each single -glucoside permease were used to investigate in more detail the differences in kinetics and specificity, by measuring proton symport activity with different sugars as substrates (Fig. 3). In these experiments, a relatively high concentration (40 mM) of each sugar was used to allow detection of lower-affinity transport systems (e.g., sucrose via Mal31p). The broadest range of sugars, including trehalose, sucrose, -methylglucoside, turanose, maltose, and maltotriose, was accepted by the general -glucoside transporter (Agt1). The strain carrying Mal31p transported only maltose, turanose, and trehalose, although the last very weakly. These results are in agreement with those of other authors (13, 21). Mty1p showed an intermediate specificity and is capable of transporting maltose and maltotriose, as well as turanose and trehalose, but neither sucrose nor -methylglucoside. Another feature that distinguishes Mty1p from Mal31p is the relative affinity for maltose and maltotriose. The kinetic parameters determined for CMY1050/pMTY1 revealed a much lower affinity for maltose (K_m of 61 to 88 mM) than for maltotriose (K_m of 16 to 27 mM). This characteristic has not been reported for any other -glucoside transporter known so far.

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FIG. 2. Growth of CMY1050 transformants carrying different plasmid-borne -glucoside transporter genes in maltose (A) or maltotriose (B) medium.

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FIG. 3. Proton symport activities, using 40 mM of different sugars as substrates, in strains expressing single -glucoside permeases. A. Strain CMY1050/pMTY1. B. Strain CMY1050/pMAL31. C. Strain CMY1050/pAGT1. Alkalification indicates proton uptake by the cells in response to sugar addition at the time indicated by the arrow. TR, trehalose; MT, maltotriose; M, maltose; T, turanose; M, -methylglucoside. S, sucrose.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The reasons leading to incomplete or delayed consumption of maltotriose in industrial starch-based processes remain controversial. Maltotriose, like maltose, must be internalized by the process yeast strain and hydrolyzed into glucose molecules before being catabolized via the glycolytic pathway. Since the intracellular -glucosidase apparently hydrolyzes both sugars at the same rate, it has been strongly advocated that the uptake step is responsible for the undesirable behavior. The distiller's strain used in our studies (PYCC 5297) is able to grow on maltotriose as the sole carbon and energy source. However, it transports maltotriose at a much lower rate than maltose, which could indeed result in the diminished flux through glycolysis observed during maltotriose metabolism compared to that of maltose or glucose. As a consequence of the reduced glycolytic flux, there would be an increase in respirative metabolism, just as we observed in PYCC 5297. Similar observations when using maltotriose have been described for other industrial strains (25). Very likely, the relative fractions of fermented and respired maltotriose are strain dependent, varying with the number of functional maltotriose transporters in each strain. Therefore, our results are in line with the hypothesis that the sugar uptake step is of crucial importance for the performance of industrial yeasts on maltotriose-rich substrates. Increasing the maltotriose uptake rates as well as derepressing maltotriose metabolism in the presence of glucose should result in a significant enhancement of maltotriose utilization.

We describe the isolation and characterization of a gene (MTY1) encoding a novel maltotriose transporter from a lager brewing yeast (S. pastorianus). This gene belongs to the family of -glucoside transporters that comprises three distinct lineages, defined by the MAL, MPH, and AGT1 genes (Fig. 1).

The Mty1 protein is very closely related to the Mal proteins, sharing 90% identity with Malx1p, which suggests a common origin. The new gene variant may have originated in brewing yeasts, due to the abundance of -glucosides in beer wort. The fact that sequences homologous to the MTY1 and AGT1 genes were detected in other industrial yeasts (15; this work) is consistent with this hypothesis. Gene duplication and natural selection could favor genetic changes leading to the acquisition of novel functions (maltotriose transport) and of different relative specificities to maltose and maltotriose. Sequencing of the DNA insert containing the MTY1 gene unveiled the usual organization of a MAL locus: the genes encoding the -glucosidase and the MAL transcriptional activator were found upstream and downstream of the MTY1 coding sequence, respectively. Of note, AGT1 was also identified in a partially functional allele of the MAL1 locus (5, 13).

The most relevant novel characteristic of the Mty1 permease is its extended specificity compared with the specificity of the highly homologous Mal31p. Like Agt1p and Mph2p, it accepts maltotriose and trehalose as substrates, but unlike these two proteins, Mty1p displays a higher affinity for maltotriose than for maltose, a unique feature among members of the -glucoside transporter family. Therefore, maltose transport through Mty1p may contribute to the low-affinity component detected in some strains, as was already suggested for Agt1p (21). It should be stressed that in our work the kinetic parameters were determined by H⁺ influx, thereby avoiding the experimental artifact of nonspecific binding of labeled sugar (2), which is particularly relevant when the use of high sugar concentrations is required, as in this case.

Protein sequence alignment revealed that Mty1p and Malx1p differ in 58 amino acids. Some of these differences must account for the observed change in biochemical properties. Interestingly, 13 amino acids out of those 58 are present in Agt1p, and only 2 (Leu344 and Ser350) of these 13 are conserved in Mph2p, another member of the same transporter family. The observation that Mal21p can transport sucrose with a K_m of 120 mM, in a strain that harbors solely this maltose transporter (22), might explain why Day and coworkers (7) observed maltotriose transport mediated by Mal61p in a strain overexpressing this permease. We did not detect any maltotriose, trehalose, or -methylglucoside symport activities in strain CMY1050/pMAL31 expressing solely the MAL31 gene, even using 40 mM sugar (Fig. 3). Furthermore, this strain grows neither on maltotriose nor on -methylglucoside, confirming that maltose permeases encoded by the MALx1 genes do not transport maltotriose to a degree that can support growth on this sugar as the sole carbon and energy source (4, 13, 23). Moreover, competition assays carried out with industrial strain PYCC 5297 grown on maltose (i.e., under inducing conditions) showed that transport of radiolabeled maltose was not inhibited by 100 mM maltotriose (results not shown). This indicates that maltose permeases do not transport maltotriose at a physiologically significant rate. Knowing that the intracellular -glucosidase is able to hydrolyze both maltose and maltotriose, the fact that 20% of the maltose-positive industrial strains are unable to grow on maltotriose favors the same conclusion, i.e., that to grow on maltotriose, the yeast requires additional transporters such as Agt1p and Mty1p. The diversity of -glucoside transporters found in industrial strains subjected to high concentrations of maltose and maltotriose highlights, in our view, the important role of substrate transport in the yeast fitness for the fermentation conditions.

 
We thank C. A. Michels (Queens College, New York, N.Y.) for the generous supply of strain CMY1050.

This work was financially supported by EC project BIO4-CT95-0107 and by the Fundação para a Ciência e a Tecnologia, Portugal (Praxis project P/BIA/11104/1998).

 
* Corresponding author. Mailing address: Instituto Superior de Ciências da Saúde-Sul, 2829-511 Caparica, Portugal. Phone: 351 21 2948530. Fax: 351 21 2948530. E-mail: moom{at}egasmoniz.edu.pt.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Applied and Environmental Microbiology, September 2005, p. 5044-5049, Vol. 71, No. 9
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.9.5044-5049.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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