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Applied and Environmental Microbiology, September 2000, p. 3974-3980, Vol. 66, No. 9
Department of Food Science, Cornell
University, Ithaca, New York 14853
Received 3 January 2000/Accepted 9 June 2000
Xylose metabolism, a variable phenotype in strains of
Lactococcus lactis, was studied and evidence was obtained
for the accumulation of mutations that inactivate the xyl
operon. The xylose metabolism operon (xylRAB) was sequenced
from three strains of lactococci. Fragments of 4.2, 4.2, and 5.4 kb
that included the xyl locus were sequenced from L. lactis subsp. lactis B-4449 (formerly
Lactobacillus xylosus), L. lactis subsp.
lactis IO-1, and L. lactis subsp.
lactis 210, respectively. The two environmental isolates,
L. lactis B-4449 and L. lactis IO-1, produce
active xylose isomerases and xylulokinases and can metabolize xylose.
L. lactis 210, a dairy starter culture strain, has neither
xylose isomerase nor xylulokinase activity and is Xyl Xylose metabolism has been described
for a wide array of microorganisms. Extensively characterized
xylose-metabolizing (Xyl+) bacteria include
Escherichia coli (24, 46),
Lactobacillus spp. (3, 4, 26-28),
Bacillus spp. (16, 33, 38), and Staphylococcus xylosus (44). Free xylose can be
transported via low-affinity symporters (XylE or XylT) or high-affinity
binding-protein dependent systems (XylFGH) (1, 6, 9, 41).
Xylose isomerase (xylA gene) then converts the aldose sugar
to xylulose, which is phosphorylated by xylulokinase (xylB
gene). Further metabolism of xylulose-5-phosphate occurs via the
pentose-phosphate or phosphoketolase pathways. Xylose metabolism is
induced by xylose, mediated via XylR. In Salmonella and
E. coli strains, XylR is an activator when xylose is present
(42, 46). In gram-positive organisms, XylR is a repressor
which is inactivated when xylose binds (13, 26, 27, 37, 44,
45). The catabolite repression of xylose metabolism by glucose is
mediated primarily through the CcpA protein (40).
Lactococcus lactis subsp. cremoris and almost all
L. lactis subsp. lactis strains cannot metabolize
xylose. These organisms have undergone intense selection for use in
dairy fermentations, but plants are thought to be their original
ecological niche because L. lactis subsp. lactis
isolates have been recovered from many different plants (22, 34,
35). Also, the plant isolate Lactobacillus xylosus has
been reclassified as L. lactis subsp. lactis
(39). However, L. lactis subsp.
cremoris strains are almost exclusively dairy associated
(22).
We discovered xylose metabolic genes from both plant (Xyl+)
and dairy (Xyl Bacterial strains, plasmids, and cultivation.
Strains and
vectors are described in the Table 1.
L. xylosus was reclassified as L. lactis ssp.
lactis based on immunological characteristics, 16S
sequences, and fatty acid composition (39) and is referred
to as L. lactis B-4449 here. All strains were routinely
cultivated at 30°C in M17 medium (Difco, Detroit, Mich.) with 0.5%
glucose or xylose. E. coli strains were cultured with aeration at 37°C in Luria broth (LB; Sigma, St. Louis, Mo.), with 100 µg of ampicillin (Sigma) or 50 µg of carbenicillin (Sigma) per ml
where appropriate.
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Dissolution of Xylose Metabolism in
Lactococcus lactis
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
.
Xylose isomerase and xylulokinase activities are induced by xylose and
repressed by glucose in the two Xyl+ strains. Sequence
comparisons revealed a number of point mutations in the
xylA, xylB, and xylR genes in
L. lactis 210, IO-1, and B-4449. None of these mutations,
with the exception of a premature stop codon in xylB, are
obviously lethal, since they lie outside of regions recognized as
critical for activity. Nevertheless, either cumulatively or because of
indirect affects on the structures of catalytic sites, these mutations
render some strains of L. lactis unable to metabolize xylose.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
) isolates of L. lactis. The
sequencing of xylRAB from L. lactis strains IO-1,
210, and B444-9 is described here. The induction of xylose isomerase
and xylulokinase activities from these three strains grown on different
carbon sources was measured. Activity measurements in conjunction with
the discovery of a xylR gene suggest that a positive
regulatory mechanism is present in lactococcal strains. Mutations which
could account for the lack of xylose isomerase and xylulokinase
activity were observed in L. lactis subsp. lactis
210. Further downstream of xylRAB are genes encoding a
mutarotase, xylanase, and xyloside transporter (K. A. Erlandson, S. Delamarre, and C. A. Batt, unpublished results), whose presence collectively suggests that ancestors of these strains were fully able
to degrade and metabolize xylan.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Bacterial strains and plasmids
Sequencing strategies.
A combination of strategies was used
to sequence the xyl operon genes (Table
2). First, the L. lactis
B-4449 xylose isomerase gene was sequenced in two fragments. Degenerate
xylA PCR primers (NH-1 and WG-1) were designed based on the
published N-terminal sequence of the L. xylosus XylA
(48) and an amino acid sequence (WGGREG, amino acids [aa]
188 to 193) conserved among bacteria including E. coli,
Salmonella enterica serovar Typhimurium, Lactobacillus pentosus, and Bacillus spp. A cloned portion of the
resulting 540-bp fragment was hybridized to a 2.4-kb
EcoRI/HindIII insert in a chromosomal
minilibrary. A portion of this fragment encoded the first 311 aa of
XylA. The sequences of the 3' end of the xylA gene and the
5' end of xylB were obtained via inverse PCR with primers 7 and 5.
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Inverse PCR. Chromosomal DNA was extracted from lactococci according to the method described by Kim and Batt (21). The chromosomal DNA was digested using a variety of restriction enzymes (New England Biolabs, Beverly, Mass.). The enzyme reactions were inactivated by incubation at 65°C for 15 min or by phenol extraction. Ligation reactions contained restricted DNA at a series of concentrations from 25 to 750 ng, together with 1 U of T4 DNA ligase (Gibco, Gaithersburg, Md.), 10 µl of 5× ligation buffer (Gibco), and sterile distilled H2O, for a final reaction volume of 50 µl. Ligations were incubated for 1 h at 37°C and heat inactivated at 65°C for 20 min. The resulting self-ligated DNA was then precipitated with NaCl and ethanol and then resuspended in sterile distilled H2O to give a final concentration of 5 ng/µl for use in PCR.
PCR amplification. PCR's were carried out in 1× PCR buffer (20 mM Tris-HCl [pH 8.3], 50 mM KCl) with a final volume of 50 µl. Reactions contained 1 µl of self-ligated chromosomal DNA or crude cell lysate prepared according to the method of Czajka and Batt (7), 50 pmol of each primer, 100 mM concentrations of each deoxynucleotide triphosphate, 1 U of AmpliTaq DNA Polymerase (Perkin-Elmer, Foster City, Calif.), and 1.5 mM MgCl2. A Hybaid TR1 or Perkin-Elmer 2400 thermocycler was programmed with a 4-min hold at 94°C, followed by 30 cycles of 1 min at 94°C, 1 min at 55 to 65°C (depending on the primer Tm), and 1 min at 72°C, followed in turn by a 10-min hold at 72°C. For inverse PCR the extension time at 72°C was increased to 2 min.
Cloning. The xylA genes from the three strains were cloned prior to sequencing. Standard (pUC19) and T-overhang (pGEM-T) vectors were used with a variety of host strains that allow blue-white screening, as indicated in Table 1. Ligations were performed according to the methods of Promega (Madison, Wis.) for pGEM-T or Sambrook et al. (36). Standard methods were used to prepare and transform competent cells (36).
Sequencing and sequence analysis.
The L. lactis
B444-9 xylA gene was sequenced manually according to the
double-stranded DNA method of Wang (49). Sequenase (v. 2.0;
Amersham, Piscataway, N.J.), [
-32P]dATP, and
dideoxynucleotides were used, together with the universal M13
primers (
40 and reverse) or internal primers. The other genes were
sequenced by the Cornell University BioResource Center using an ABI
Prism 373A Stretch automated sequencer. Several xylA PCR clones from each strain were analyzed to confirm the sequences. All
other sequences were obtained directly from PCR products. Sequences
were analyzed using several programs from the Lasergene software suite
(EditSeq v. 3.12, SeqMan v. 3.53, and MegAlign v. 3.05; DNASTAR, Inc.,
Madison, Wis.). We also performed BLASTX searches (2) of the
GenBank database with our nucleotide sequences to identify homologous genes.
Xylose isomerase assays. The cysteine carbazole method was used to measure xylose isomerase activity as indicated by the appearance of xylulose (10). Reagents were purchased from Sigma. Stationary phase M17 xylose- or glucose-grown cultures (100 ml) were harvested, resuspended in 4 ml of 50 mM Tris-HCl (pH 7.5) containing 1 mM dithiothreitol, and lysed by sonication. Sample absorbances were measured at 500 nm with an LKB Biochrom Ultraspec II spectrophotometer (Biochrom, Cambridge, United Kingdom) after 20 min at room temperature. One unit of enzyme activity is expressed as 1 nmol of D-xylulose produced per min.
Xylulokinase assays. Xylulose formation was measured using a method modified from Shamana and Sanderson (43) that couples xylulokinase to pyruvate kinase and L-lactate dehydrogenase. Cell lysates (containing 80 mg of protein) were prepared by sonication as described above. The oxidation of NADH was monitored by measuring the absorbance at 340 nm with a Beckman DU 640 spectrophotometer (Beckman Instruments, Fullerton, Calif.). One unit of enzyme activity was expressed as 1 nmol of D-xylulose consumed per min.
Nucleotide sequence accession numbers. The GenBank accession numbers for the L. lactis B444-9, L. lactis IO-1, and L. lactis 210 xylRAB genes are AF092042, AF092041, and AF092040, respectively.
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RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Three xylose metabolism genes (xylRAB) were sequenced
from three lactic acid bacteria, including one Xyl
strain. A combination of PCR products generated with degenerate conserved, homologous, and inverse primers was assembled to give a
total of 4.2 kb of sequence from L. lactis subsp.
lactis IO-1 and L. lactis subsp.
lactis B444-9 and 5.4 kb of sequence from L. lactis subsp. lactis 210 (Fig.
1).
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Xylose isomerase.
Xylose isomerase is encoded by a 1,320-bp
gene in L. lactis strains IO-1, 210, and B444-9. Figure
2 shows the IO-1 XylA sequence with
conserved and similar residues indicated based on an alignment of 22 group II xylose isomerase sequences, including our three sequences.
These xylose isomerases are grouped together because of their catalytic
metal preference and size, which ranges from 438 to 465 aa (25,
48, 50). A total of 96 aa (21%) are identical for all 22 bacteria. An additional 118 residues (26%) are similar or identical
for 90% of the strains. All of the group II XylAs conserved 17 active-site residues (Fig. 2) determined for the group I
Streptomyces rubiginosus XylA (50-52). Although
there are conserved residues throughout the group II protein, the most
highly conserved region, aa 179 to 345, is in the middle of the
protein. This region includes 12 S. rubiginosus active-site
residues. The carboxyl terminus of XylA is the least conserved among
the group II proteins. The XylA sequences of L. lactis
B444-9, L. lactis IO-1, and L. lactis 210 are
very similar (Fig. 2). Only 2 aa differ between the Xyl+
strains L. lactis IO-1 and L. lactis B444-9
(Lys407
Glu and His416Tyr). These residues are not conserved among
the other XylAs. Strain 210 differs from IO-1 at six residues:
Met202Arg, Asp218Tyr, Ser247Ala, Ala275Val, Ser388Thr,
and Lys407Glu. Three of the six residues (i.e., residues 202, 218, and 247) are similar in the other group II XylAs, and residue 275 is
conserved in 20 of the 22.
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, conserved S. rubiginosus
active site residue, according to Whitaker (Xylose isomerase activity. Xylose isomerase activities for crude extracts of L. lactis strains IO-1, B444-9, and 210 were measured as described in Materials and Methods. The specific activity for IO-1 cells grown on xylose was 464 U/mg of protein, a value almost fourfold greater than that for B444-9 (127 U/mg of protein). Glucose-grown IO-1 and B444-9 xylose isomerase activities averaged 12.8 U/mg of protein. L. lactis 210 had little xylose isomerase activity whether grown on xylose (<6 U/mg of protein) or glucose (<12 U/mg of protein).
Xylulokinase.
L. lactis strains IO-1 and B444-9 have a
1,506-bp xylB gene located 66 bp downstream of the
xylA translation stop codon. The L. lactis 210 xylB gene is 67 bp downstream of xylA.
Xylulokinase sequences, which range in size from 475 to 511 aa, are
available from 14 of the 22 XylA group II organisms. Figure
3 displays the IO-1 XylB sequence with
conserved and similar residues indicated, based on an alignment of the
14 sequences. The XylB sequences are less conserved than the XylA
sequences, because only 45 residues (10%) are identical for all 14 strains. An additional 62 aa are conserved and 51 aa are similar for
85% of the strains. Similar and identical residues are found
throughout the XylB protein, but homologous regions with clusters of
conserved residues are small and concentrated in the N-terminal half of
the protein.
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1 frameshift in a run of six (instead
of seven) adenine residues. Resequencing this region confirmed the
frameshift. Premature termination of translation is the likely
explanation for the lack of significant xylulokinase activity of strain
210 (see below). If the stop codon were ignored, only seven residues
(aa 200 to 202, 230, 290, 448, and 496) differ between L. lactis strains IO-1, B444-9, and 210 (Fig. 3). None of these
differences correspond to conserved XylB residues. Aside from the stop
codon, strain 210 differs from the Xyl+ strains at residue
290 (cysteine instead of tyrosine).
Xylulokinase activity. Xylulokinase activities for L. lactis strains IO-1, 210, and B444-9 were determined using a coupled enzyme assay as described in Materials and Methods. As with the xylose isomerase activities, the xylulokinase activity of IO-1 surpassed that of B444-9. The magnitude of the difference was much less, however, with 53.8 U/mg of protein for IO-1 compared to 41.4 U/mg of protein for B444-9. The enzyme activities for glucose-grown strains IO-1 or B-4449 were two to three times lower than when grown on xylose (24.9 and 12.4 U/mg, respectively). L. lactis 210 had a very low level of xylulokinase activity whether grown on xylose (7.8 U/mg) or on glucose (6.8 U/mg).
Xylose regulator.
In all three strains, the 1,280 bp of
sequence upstream of xylA included a 950-bp open reading
frame, divergently oriented and 104 bp upstream from xylA,
which we have termed xylR. The XylR protein is not
homologous to XylRs from other gram-positive bacteria such as
Bacillus megaterium and L. pentosus. Instead, it
is moderately homologous to proteins such as RhaS (21% identical and
40% similar over 250 aa), the E. coli rhamnose metabolism regulator in the AraC/XylS family. This family includes other carbohydrate operon regulators such as melR (from E. coli), rafR (Pediococcus pentosaceus), and
xylR (E. coli) which regulate melibiose, raffinose, and xylose metabolism, respectively. Gallegos et al. (12) have recently redefined this family to include over 100 proteins, with a C-terminal 99-aa stretch of homology constituting the
DNA-binding domain of the regulator proteins. This region contains two
helix-turn-helix (HTH) motifs. Although our XylR is only moderately
homologous to individual AraC family proteins, a PFSCAN search
(15, 29) of the PROSITE profile database revealed that it
was significantly homologous to the 99-aa AraC family consensus region
(Fig. 4). The IO-1 XylR sequence matched
37 of the 99 residues and was similar to 17 residues. Gallegos et al. identified 17 residues in the 99-aa stretch that are highly conserved (shared by more than 60% of the family member sequences). Thirteen of
these residues are conserved by the IO-1 XylR.
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Ser substitution
outside of the AraC family consensus region and an Phe269Ile
substitution very near the C terminus of the consensus region. L. lactis B-4449 has an Asp147His substitution. Also, due to an
additional adenosine at the end of a run of six adenosines, the 5 aa at
the C terminus of XylR are not conserved in the L. lactis
210. The strain 210 xylR gene encodes 2 aa after the
frameshift before a premature stop codon (indicated in Fig. 4) results
in a protein that is 3 aa shorter than that of L. lactis
strains IO-1 and B-4449.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Xylose metabolism has been described and characterized in lactic acid bacteria, but it is rare in L. lactis. We have discovered a metabolic pathway for the metabolism of xylose to xylulose-5-phosphate in L. lactis. Xylose metabolism requires the products of the xylR, xylA, and xylB genes. Although the three strains we studied possess sequences for these xyl genes, xylose metabolism in L. lactis is a function of the source of the strains. L. lactis subsp. lactis 210 is a dairy starter culture strain and cannot metabolize xylose, whereas L. lactis subsp. lactis B-4449 and L. lactis subsp. lactis IO-1 (which were isolated from a vegetation sample and a kitchen drain in Japan, respectively) can metabolize xylose. Recently, other Xyl+ L. lactis strains have been isolated from minimally processed fruit and vegetable products, which further supports the link between functional xylose metabolism and habitat (20).
Other than their differences in habitat, L. lactis B-4449, L. lactis IO-1, and L. lactis 210 are closely related. The ribotype patterns of L. lactis strains IO-1 and 210 have similarity values to the L. lactis B-4449 pattern of 0.69 and 0.72, respectively (data not shown). The L-lactate dehydrogenase sequences of the three strains also differ by less than 1% compared to 12 and 17% dissimilarities to Streptococcus thermophilus and Lactobacillus casei, respectively (11).
Although the xylose isomerases of our two Xyl+ strains differed by only 2 aa, the specific activity of IO-1 was almost fourfold greater than that of B-4449 when both strains were grown on xylose. The two residues are not conserved among the other XylAs nor are they in obvious proximity to any active-site residues. Northern analysis indicates that the xylA gene is transcribed at similar levels in both IO-1 and B-4449 (data not shown). Also, although the xylA gene is transcribed, L. lactis 210 did not have significant xylose isomerase activity. Its sequence differs by only 6 aa from IO-1. Three of these residues, R202, Y218, and V275, are located in highly conserved regions and differ from the group II XylA consensus (L/M202, D/E218, and A/S275; Fig. 2). The nucleotide sequence of the xylR-xylA intergenic region is identical in strains IO-1, 210, and B-4449, so it is more likely that differences in activity among the three strains are due to differences in protein structure rather than transcription levels. However, all of the conserved xylose isomerase active-site residues identified (50) are intact in L. lactis IO-1, L. lactis 210, and L. lactis B-4449. Site-directed mutagenesis studies are in progress in our laboratory to characterize the functional importance of the differences between the L. lactis strain IO-1, 210, and B-4449 XylAs.
Differences in xylulokinase activities were also measured among L. lactis strains IO-1, 210, and B-4449. The xylulokinase activity of L. lactis 210 is very low regardless of the carbon source (7.8 U/mg of protein when xylose grown versus 6.8 U/mg when glucose grown). Although the strain 210 xylB gene is transcribed (Erlandson et al., unpublished), a premature translation stop codon (corresponding to residue 154) would almost certainly prevent formation of active xylulokinase.
As with xylose isomerase, xylose-grown IO-1 had more xylulokinase
activity than B-4449. Their XylB sequences differed at six residues,
none of which are conserved among the compared XylBs. XylB is part of a
large, extensively characterized ATPase superfamily of proteins which
includes other sugar kinases, eukaryotic heat shock and stress-70
proteins, and actin (17, 18). There are 127 structurally
equivalent residues shared among the family members which fold into two
symmetrical domains (each with a 
topology) to
form an ATP- and substrate-binding cleft (17). Using the
algorithm of Rost and Sander (32), the secondary structure of XylB was predicted (data not shown). The sequence differences of
IO-1 versus B-4449 did not map to a -sheet or -helix. However, residue 230 (lysine in IO-1, glutamate in B-4449) is 10 residues from
Asp239 (Asp245 of E. coli glycerol kinase), a key residue conserved throughout the superfamily that is thought to act as a
general catalytic base (30). Residue 230 is also 3 aa from a
sequence block thought to be involved in hinge motion during protein
conformational change (5). In addition, residue 448 (phenylalanine in IO-1, leucine in L. xylosus) is located
six residues after a second sequence block putatively involved in hinge
motion (5). Therefore, differences in the predicted
three-dimensional structure of the IO-1 XylB could account for its
greater activity relative to B-4449.
Xylulokinase and xylose isomerase activities decreased substantially when L. lactis strains IO-1 and B-4449 were grown on glucose. Catabolite repression by glucose is a phenomenon common to all xylose metabolizers (13, 14, 31, 33). We could not find cis-acting cre sites upstream of xylA or xylB. However, it is likely that lactococci have a homologue to the cre sequences identified in Bacillus spp., L. pentosus, and S. xylosus that bound the catabolite regulator CcpA (19, 26, 40, 45).
The XylR sequences of L. lactis strains IO-1, 210, and B-4449 are moderately homologous to AraC/XylS family proteins, including the E. coli and Haemophilus influenzae XylR activator proteins. The AraC/XylS family profile (PROSITE entry PS01124) consists of 99 aa, usually toward the C-terminal end of the protein, encompassing two HTH motifs. The region is thought to constitute the DNA-binding domain of the proteins. Our XylR is homologous over the entire AraC family profile, corresponding to aa 174 to 272 of the IO-1 sequence (Fig. 4). The xylR genes from all other gram-positive bacteria (B. subtilis, B. megaterium, B. licheniformis, L. pentosus, and S. xylosus) fall into a different family of transcriptional regulators and sugar kinases, the ROK family (47). The repressor proteins in the ROK family (PROSITE entry PS01125) have an HTH motif in the N-terminal portion of the protein and a 300-residue domain with a central glycine-rich region, although the rest of the consensus pattern is not highly conserved. The ROK XylR protein has been shown to bind a tandem repeat, designated xylO, near the xylA transcription start site (8, 13, 23, 27, 37, 45). This xylO site does not appear to be present upstream of xylA in our three strains. It is unusual that our XylR is homologous to the AraC family proteins because all of these proteins are activators, except the E. coli cellibiose repressor (CelD) and the bifunctional E. coli AraC arabinose and Yersinia pestis YbtA pesticin and yersiniabactin regulators (which activate or repress depending on the effectors). In general, xylose regulation is positive in the case of gram-negative organisms but negative in gram-positive organisms (16, 23, 42, 46). Our sequence homology suggests a positive regulatory mechanism different from that previously described for gram-positive xylose metabolism.
What will be the fate of the xyl genes in dairy L. lactis strains? We speculate that the xylose metabolic genes in dairy L. lactis strains are becoming pseudogenes because there is no selective pressure to maintain xylose metabolism in the dairy environment. Deleterious mutations in the xyl genes have accumulated in L. lactis 210, and dairy starter L. lactis subsp. cremoris strains have even more extensive mutations to the xyl genes than L. lactis subsp. lactis 210 (data not shown). Interestingly, L. lactis subsp. cremoris strains have never been isolated from plant or other environmental samples, despite thorough searching (22, 34, 35). It is also possible that the xyl genes are evolving new catalytic functions in dairy lactococcal strains. To examine this, the activity of the isomerase and kinase enzymes would need to be tested against alternative substrates and/or mutants isolated with increased activity against these substrates.
In conclusion, we have discovered the genetic potential in L. lactis for xylose metabolism encoded by the xylRAB
genes. These genes are organized in what could be a single
xylAB operon, potentially regulated by xylR. The
Xyl dairy starter culture strain L. lactis 210 has experienced a loss of function in an environment no longer
selective for xylose metabolism. Plant environmental isolates such as
L. lactis B-4449 and L. lactis IO-1 retain the
ability to metabolize xylose.
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FOOTNOTES |
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* Corresponding author. Mailing address: Cornell University, Department of Food Science, 312 Stocking Hall, Ithaca, NY 14853. Phone: (607) 255-2896. Fax: (607) 255-8741. E-mail: cab10{at}cornell.edu.
Present address: Procter and Gamble Co., Cincinnati, Ohio.
Present address: Nestle S.A., Paris, France.
§ Present address: Department of Environmental and Occupational Health, National Cheng Kung University Medical College, Tainan, Taiwan, Republic of China.
Present address: Department of Food Science and Technology,
Cornell University, Geneva, N.Y.
# Present address: Department of Microbiology and Immunology, Johns Hopkins University, Baltimore, Md.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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