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Previous Article | Next Article 
Applied and Environmental Microbiology, April 2001, p. 1445-1452, Vol. 67, No. 4
Department of Food Science, Cornell
University, Ithaca, New York 14853
Received 21 June 2000/Accepted 4 January 2001
Genetic and biochemical evidence for a defective xylan degradation
pathway was found linked to the xylose operon in three lactococcal
strains, Lactococcus lactis 210, L.
lactis IO-1, and L. lactis NRRL B-4449.
Immediately downstream of the xylulose kinase gene
(xylB) (K. A. Erlandson, J.-H. Park, W. El Khal,
H.-H. Kao, P. Basaran, S. Brydges, and C. A. Batt, Appl.
Environ. Microbiol. 66:3974-3980, 1999) are two open
reading frames encoding a mutarotase (xylM) and a xyloside
transporter (xynT) and a partial open reading frame
encoding a Xylan, a polymer with a
Previously, we reported the discovery of the genes necessary for xylose
metabolism (xylRAB) in three lactococcal strains, including
a non-xylose-fermenting (Xyl In this work, we report the subsequent discovery of the genetic
potential of both plant (Xyl+) and dairy
(Xyl Bacterial strains, plasmids, and cultivation.
Strains and
vectors are described in Table 1. All
strains were routinely cultivated at 30°C in M17 medium (Difco,
Detroit, Mich.) with 0.5% glucose or xylose. Escherichia
coli strains were cultured with aeration at 37°C in Luria broth
(LB; Sigma, St. Louis, Mo.) and with 100 µg of ampicillin (Sigma) per
ml or 50 µg of carbenicillin (Sigma) per ml as appropriate.
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.4.1445-1452.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Genetic Evidence for a Defective Xylan
Degradation Pathway in Lactococcus lactis
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-xylosidase (xynB). These are functions
previously unreported for lactococci or lactobacilli. The mutarotase
activity of the putative xylM gene product was confirmed by
overexpression of the L. lactis enzyme in Escherichia
coli and purification of recombinant XylM. We hypothesize that
the mutarotase links xylan degradation to xylose
metabolism due to the anomeric preference of xylose isomerase. In
addition, Northern hybridization experiments suggested that
the xylM and xynTB genes are cotranscribed with the xylRAB genes, responsible for xylose metabolism.
Although none of the three strains appeared to metabolize xylan or
xylobiose, they exhibited xylosidase activity, and L. lactis IO-1 and L. lactis NRRL B-4449 had functional mutarotases.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-1,4-linked backbone of xylose, is the major carbohydrate in the
hemicellulose portion of plant cell walls. One-third of all renewable
carbon is xylan (24). Although xylan metabolism has not
been previously reported for Lactobacillus and
Lactococcus species, xylan degraders include a wide range of
other microbes, including rumen organisms, plant pathogens, and soil
microbes (4, 10, 24, 36). Before the xylose backbone of
xylan is attacked, side chains such as acetyl and arabinofuranose are
removed by a variety of enzymes (4). Extracellular endo-1,4--xylanases then degrade the xylan to xylo-oligomers, which
are transported into the cell by permeases (5). Finally, intracellular -xylosidases hydrolyze the xylo-oligomers to xylose.
) dairy starter
culture strain (13). We also discussed the relationship between habitat and functional xylose metabolism. The dairy
fermentation environment, with its intense selection for rapid growth
and acid production, is very different from the plant niche that
Lactococcus lactis subsp. lactis strains
originally inhabited. The ability to metabolize xylose is not essential
for growth in dairy environments, and xylose-fermenting
(Xyl+) strains of the exclusively
dairy-associated L. lactis subsp. cremoris have
not been reported. However, the necessary genes (xylR,
xylA, and xylB) for the metabolism of the pentose
sugar xylose are still present in Xyl strains
of lactococci, including L. lactis subsp.
cremoris (3, 13).
) isolates of L. lactis to
metabolize xylan, a function encoded by the xylM,
xynT, and xynB genes immediately downstream of
the xylRAB locus. The sequencing of xylM and
xynTB from L. lactis NRRL B-4449, L. lactis IO-1, and L. lactis 210 and the measurement of
mutarotase and xylanolytic activities are described here. We propose a
new function linking xylose and xylan metabolism: the mutarotase
encoded by the xylM gene.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Bacterial strains and plasmids
Cloning of xylM
The xylM gene
was obtained previously from L. lactis 210 (13) via an inverse PCR. The FNDE-XM and RBAM-XM primers
(Table 2) were designed to amplify
xylM from L. lactis NRRL B-4449 and L. lactis IO-1 for cloning prior to sequencing. The PCR
products were initially ligated to pGEM-T (Table 1) and then
transformed into E. coli DH5
F'. The
xylM gene was also subcloned in pET19d for use in the
Novagen (Madison, Wis.) E. coli BL21 (DE3)
overexpression system. Ligations were performed as described by Promega
(Madison, Wis.) for pGEM-T or Sambrook et al. (28).
Standard methods were used to prepare and transform competent cells
(28).
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Inverse PCR. To obtain the L. lactis 210 xynT and xynB genes downstream of xylM, an inverse PCR with the r-xylM and Ip1225 primers (Table 2) was performed as described previously (13). Briefly, ligation reaction mixtures contained Csp6I (New England Biolabs, Beverly, Mass.)-digested chromosomal DNA at concentrations of 25 to 750 ng. The resulting self-ligated DNA was used at a final concentration of 5 ng/µl in the PCR.
PCR primers were designed from the L. lactis 210 sequence to obtain the xynT and xynB genes from L. lactis IO-1 and L. lactis NRRL B-4449.PCR amplification. PCRs were carried out with PCR buffer (20 mM Tris-HCl [pH 8.3], 50 mM KCl) in a final volume of 50 µl. Reaction mixtures contained 1 µl of self-ligated chromosomal DNA or crude cell lysate prepared as described by Czajka and Batt (11), 50 pmol of each primer, 100 mM (each) deoxynucleoside triphosphate, 1 U of AmpliTaq DNA polymerase (Perkin-Elmer, Foster City, Calif.), and 1.5 mM MgCl2. A Perkin-Elmer 2400 thermocycler was programmed with a 4-min hold at 94°C; 30 cycles of 1 min at 94°C, 1 min at 55 to 65°C (depending on the primer melting temperature), and 1 min at 72°C; and a 10-min hold at 72°C. For inverse PCR, the extension time at 72°C was increased to 2 min.
Sequencing and sequence analysis. Genes were sequenced at the Cornell University BioResource Center using an ABI Prism 373A Stretch automated sequencer. Sequences were analyzed using several programs from the Lasergene software suite (DNASTAR, Inc., Madison, Wis.): EditSeq version 3.12, SeqMan version 3.53, and MegAlign version 3.05. We also performed BLAST X searches (1) of the GenBank database with our nucleotide sequences to identify homologous genes.
Overexpression and purification of XylM.
A 200-ml culture of
E. coli BL21(pLbx10XM) was grown in LB supplemented with
50 µg of carbenicillin/ml at 37°C with aeration until it reached an
optical density at 600 nm (OD600) of 0.65. Isopropyl--D-thiogalactopyranoside (IPTG) was
added to a final concentration of 0.2 mM, and the culture was incubated
for an additional 2.5 h at 30°C with aeration. The cell pellet
was harvested by centrifugation and frozen at 
70°C until
purification of the enzyme was performed. Mutarotase labeled with a
histidine tag was extracted and batch purified under native conditions
using Ni-nitrilotriacetic acid (NTA) Superflow resin according to the instructions of the manufacturer (Qiagen, Chatsworth, Calif.). The
protein was further concentrated with a Centricon-10 spin column
(Amicon, Beverly, Mass.) and resuspended in 10 mM Tris-HCl (pH 7.5).
Purified XylM was run on a sodium dodecyl sulfate (SDS)-polyacrylamide gel to estimate its molecular weight (28).
Xylan-degrading activity. Cell pellets and medium supernatants were harvested from 100-ml cultures of the three lactococcal strains grown in M17 medium-xylose to an OD600 of 0.4 to 1.0. The supernatants were concentrated approximately 20-fold by ultrafiltration (Millipore; 10,000-molecular-weight cutoff). The cell pellets were resuspended in 3 ml of 50 mM potassium phosphate buffer (pH 6), French pressed at 10,000 lb/in2, and centrifuged at 5,000 × g for 10 min to separate the cytoplasmic extracts from the membrane fractions. The membrane fractions were resuspended in 50 mM potassium phosphate buffer (pH 6).
The xylanase reaction mixtures consisted of 50 to 200 µl of sample and 100 µl of birch wood xylan (Sigma; 1% [wt/vol] in potassium phosphate buffer) brought to a final volume of 400 µl with 50 mM potassium phosphate buffer (pH 6). After overnight incubation at 37°C, the reaction mixtures were mixed with 1 ml of dinitrosalicylic acid reagent (22) and boiled for 15 min. The amount of reducing sugar released was measured at an absorbance of 600 nm using an LKB Biochrom Ultrospec II and compared against a standard curve prepared using various concentrations of xylose. One unit of enzyme activity was defined as the amount of enzyme required to release 1 µmol of reducing sugar per min. Xylosidase activity was determined by measuring the hydrolysis of p-nitrophenyl--D-xylopyranoside.
The xylosidase reactions comprised 50- or 150-µl samples and 100 µl
of p-nitrophenyl--D-xylopyranoside (Sigma; 20 mM) brought to a final volume of 500 µl with 50 mM potassium phosphate buffer (pH 6). The reaction mixtures were incubated
at 37°C, and the reactions were terminated with 500 µl of a stop
reagent (1 M Na2CO3
[pH 9.0]). The amount of p-nitrophenol released was
determined spectrophotometrically at 420 nm. An enzyme activity unit
was defined as the amount of enzyme that produced 1 µmol of
p-nitrophenol per min.
Mutarotase assay.
The and anomers of xylose can be
easily discriminated via 13C nuclear magnetic
resonance (NMR) spectroscopy, so this technique was performed to
compare the rate of xylose mutarotation in cell extracts and the rate
of spontaneous (nonenzymatic) mutarotation. Samples were prepared as
follows. Cultures (100 ml) were grown to an OD600
of 0.4 to 1.0 in LB or M17 medium containing 0.5% glucose or xylose.
The cell pellets were washed, resuspended in 3 ml of 10 mM
Tris-HCl (pH 7.5), and then sonicated. Assay samples were prepared
immediately before measurement by resuspending 16 mg of xylose (99%
anomer; Sigma) in NMR buffer (10 mM Tris-HCl [pH 7.5], 15%
D2O [Sigma]) and adding 5 to 400 µl of cell
extract to a final volume of 800 µl. Spontaneous mutarotation was
evaluated with 16 mg of xylose dissolved in 800 µl of NMR buffer.
-anomer peak at 95 ppm and a
-anomer peak at 91 ppm. Mutarotation rates were calculated based on
the rate of change of the -anomer peak amplitude relative to the sum
of the amplitudes for the two anomers. The enzymatic mutarotation was defined as the sample mutarotation rate minus the spontaneous mutarotation rate. Data were normalized based on protein concentrations.
Northern hybridizations. RNA was isolated from xylose-grown and glucose-grown L. lactis using the hot phenol method of van der Vossen et al. (37). The RNA was precipitated with LiCl as described by Zantinge and Wessels (38). The formamide-denatured RNA was electrophoresed through a 1% agarose gel containing 2.2 M formaldehyde and then transferred by capillary action to a nylon membrane using 20× SSPE (1× SSPE is 0.18 M NaCl, 10 mM NaH2PO4, and 1 mM EDTA [pH 7.7]) as described by Sambrook et al. (28). After the RNA was fixed by baking the membrane at 80°C for 2 h, the membrane was prehybridized for 3 h at 68°C in modified Church buffer (33). Hybridization was carried out overnight at 68°C with digoxigenin-labeled DNA probes in modified Church buffer. The xylB and xylM probes were prepared by PCR incorporation of digoxigenin-dUTP using the F210xb-R210xb and FNDE-XM-RBAM-XM primer pairs, respectively. A Boehringer Mannheim Biochemicals (Indianapolis, Ind.) Genius kit was used for detection.
Nucleotide sequence accession numbers. The xylM and xynTB gene sequences for L. lactis NRRL B-4449, L. lactis IO-1, and L. lactis 210 can be found in GenBank under accession numbers AF092042, AF092041, and AF092040, respectively.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Two xylan metabolism genes (xynTB) and one novel
mutarotase gene (xylM) were sequenced from three lactic acid
bacteria, including one Xyl strain. A
combination of PCR products generated with traditional and inverse
primers was assembled to give 3.3, 3.0, and 3.3 kb of sequence
downstream of xylB from L. lactis NRRL
B-4449, L. lactis IO-1, and L. lactis 210, respectively (Fig. 1).
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Mutarotase.
A gap of only 2 nucleotides (nt) exists between
the stop codon of xylB and the start codon of another
1,005-bp open reading frame (ORF). The translated ORF is homologous
(averages of 30% identical and 46% similar) over its entire length to
galactose mutarotases from E. coli, Haemophilus
influenzae, Acinetobacter calcoaceticus, and
Streptococcus thermophilus. Mutarotases catalyze the
interconversion of the and anomers of carbohydrates. Figure 2 shows an alignment of the L. lactis 210, IO-1, and NRRL B-4449 XylM sequences to the
sequences of seven other mutarotases ranging in size from 335 to 381 amino acids. Approximately 5% (26) of the amino acids are
identical for all 10 strains. Thirty-one additional residues (10%) are
conserved or similar among the mutarotases. The L. lactis
210, IO-1, and NRRL B-4449 XylM sequences differ at only eight
positions. Our XylM sequences are most similar (40%) to S. thermophilus GalM. Although promoter prediction by a
neural network (26; M. G. Reese, N. L. Harris,
and F. H. Eeckman, Electronic Proc. 1996. Pacific Symp.
Biocomput., abstr., 1996) suggests two possible transcription
start sites for our xylM, located 34 or 45 nt upstream of
the start codon (data not shown), we also have evidence linking
xylM transcription to xylAB transcription (see Northern hybridization results below).
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Mutarotase activity.
Concentrated crude cell extracts of
xylose-grown L. lactis NRRL B-4449, IO-1, and 210 were
tested for mutarotase activity using 13C NMR. The
results are shown in Table 3. The
specific mutarotase activities of L. lactis IO-1 and
L. lactis NRRL B-4449 extracts were 2.9 and 0.9 U/mg of
protein, respectively. L. lactis 210 did not have
mutarotase activity above the base (spontaneous) rate. Growth on
glucose decreased the L. lactis NRRL B-4449 specific mutarotase activity 29-fold, to almost undetectable levels. The L. lactis NRRL B-4449 mutarotase gene was
overexpressed in E. coli BL21 under the control of the
inducible T7lac promoter. Concentrated cell extracts
prepared from induced cells exhibited high levels of mutarotase
activity (38.7 U/mg of protein) compared to those in control BL21 cells
transformed with pET19d. Histidine-tagged mutarotase was purified as
described in Materials and Methods. The purified mutarotase
(including 10 histidine residues) had an apparent molecular
mass of approximately 40 kDa, as estimated by SDS-polyacrylamide
gel electrophoresis (PAGE) (Fig. 3).
Molecular masses of 37 to 40 kDa have been reported for galactose
mutarotases (15, 23). The purified protein had a specific
mutarotase activity of 67 U/mg (Table 3).
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Xyloside transport.
A 1,485-bp ORF, located 53 to 54 nt
downstream of the xylM stop codon, encompasses a putative
xyloside transport gene, xynT. XynT appears to be a
hydrophobic protein (232 hydrophobic residues out of 494) with at least
11 transmembrane domains, as predicted by a Kyte-Doolittle
hydrophobicity plot (18a) (data not shown). XynT is
homologous to a variety of di- and trisaccharide transporters, with 40 to 46% amino acid similarity to E. coli glucuronide
permease (UidB), lactose permease of E. coli and S. thermophilus (LacY), Pediococcus raffinose
carrier protein (RafF), and melibiose carrier protein of
Salmonella enterica serovar Typhimurium (MelB). Our prediction that the product of xynT could be involved in
xyloside transport is also based upon its location upstream of xylan
metabolism genes (see below). XynT is also 44% similar to a putative
Lactobacillus pentosus proton symporter (XylP) that has
recently been demonstrated to transport -xylosides (9).
The xynT gene is not homologous to the xylT
(Lactobacillus brevis) or xylE (E. coli) xylose transporter genes.
Xylosidase.
The L. lactis 210 downstream
xylM inverse PCR product contained one additional 806-bp
ORF, beginning 24 nt downstream of xynT. We have sequenced
457 and 770 nt of this ORF from L. lactis IO-1 and L. lactis NRRL B-4449, respectively. The product of the ORF has
strong similarity to the first 264 amino acids of -1,4-xylosidases from Bacillus subtilis (GenBank accession number
G69735), Bacillus pumilus (P07129), and E. coli
(P77713). It is also quite similar to broad-specificity
Selenomonas ruminatum xylosidase-arabinosidase Xsa (GenBank
accession number AAB97967). Xylosidases are typically
intracellular enzymes that further break down xylosides to xylose. The
product of our ORF, which we have named xynB, is 57%
identical and 66% similar to Selemonas Xsa and 52%
identical and 64% similar to B. subtilis XynB (data not
shown). To a lesser degree, our XynB is also homologous to
endoarabinases from B. subtilis and Pseudomonas
fluorescens.
Xylan-degrading activity.
L. lactis IO-1 and
L. lactis NRRL B-4449 did not show significant increased
growth in M17 medium (a carbon-limited complex medium) plus xylan
versus in M17 medium alone (data not shown). Also, IO-1 did not grow
better in M17medium-0.25% xylobiose than in M17 medium alone (data
not shown). To determine if these strains possess any xylan-degrading
activity, xylanase and xylosidase assays were performed with culture
supernatants, cytoplasmic extracts, and membrane fractions. Although no
xylanase activity was detected in the supernatants, the cytoplasmic
extracts of L. lactis IO-1 and L. lactis
NRRL B-4449 produced enzymatic activities of 1 × 103 and 6 × 104
U mg of protein1, respectively.
Xylosidase activity was detected in the cytoplasmic extracts and the
membrane fractions of all three strains. The specific activities of
-xylosidases in the cytoplasmic extracts were 0.36, 0.003, and 0.01 U mg1 for L. lactis IO-1,
L. lactis 210, and L. lactis NRRL
B-4449, respectively.
Transcription analysis.
In order to determine the
transcriptional relationships of our xylose and xylan genes, we
performed Northern hybridizations with xylB and
xylM as probes. Figure 4 shows
the autoradiograph (top panel) and also schematically depicts
our results (bottom panel) with RNA extracted from xylose-grown
cultures of L. lactis IO-1, 210, and NRRL B-4449. The
xylB probe overlaps the 3' end of xylA, while the
xylM probe contains the complete ORF and does not overlap
any other genes. Three different xyl and xyn
transcripts were produced in xylose-grown L. lactis 210, IO-1, and NRRL B-4449. Both probes hybridized to 7.4-, 5.0-, and 2.7-kb transcripts. The 7.4-kb transcript is long enough to
encompass xylABM and xynTB. The 5.0-kb transcript
could include either xylABM or xylBMxynT, due to
the overlapping probes. Similarly, the 2.7-kb transcript could include
xylAB or xylBM. These transcripts were present at much higher levels in L. lactis 210 than in L. lactis IO-1 or NRRL B-4449 (Fig. 4, top panel), perhaps
accumulating because the inducing signal (xylose) could not be
metabolized. In contrast, the three transcripts were not detectable in
glucose-grown cells even after very long exposure times (Fig. 4, top
panel). Although neural network analysis (26; Reese et
al., Electronic Proc. 1996 Pacific Symp. Biocomput.) suggests the
existence of potential promoters upstream of each of the xyl
and xyn genes (data not shown), it appears from the Northern
hybridization data that the xyl genes are cotranscribed and
may also be cotranscribed with the xyn genes.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Many lactic acid bacteria are found in plant environments, but xylan metabolism has not been described for these bacteria. Xylose metabolism has been described for and characterized in lactic acid bacteria but is rare in L. lactis. We have discovered a metabolic pathway with the potential to metabolize xylan to xylose and to xylulose-5-phosphate in L. lactis. This xylan pathway is encoded by six genes clustered together at a single locus: xylRABM-xynTB.
Although L. lactis IO-1, L. lactis 210, and
L. lactis NRRL B-4449 were not able to grow on xylan or
xylobiose, we found ORFs encoding a putative xyloside transporter
(xynT) and part of a xylosidase (xynB). Our XynB
is highly homologous to characterized -1,4-xylosidases from B. subtilis, B. pumilus, and Butyrivibrio fibrisolvens, as well as a xylosidase-arabinosidase from the rumen bacterium Selenomonas. Our partial ORF encodes over one-half
(268 amino acids) of a typical xylosidase (517 to 535 amino acids). We
identified the remainder of the xynB sequence in the
low-redundancy genomic sequence database of L. lactis
IL-1403 (Institut National de la Recherche Agronomique [INRA],
Jouy en Josas, France), and its similarity to other xylosidase genes
was confirmed by a BLASTX search.
L. lactis IO-1 exhibited a significant level of
intracellular xylosidase activity compared to other known xylosidase
producers. Specific xylosidase activities of 1.2 U
mg1, 0.21 U mg1, and
0.09 U mg1 have been previously reported for
Aspergillus nidulans (18), Bacillus
sp. strain K-1 (25), and Clostridium
cellulolyticum (29). Low xylosidase activities were
also detected in the cytoplasmic extracts of L. lactis 210 and L. lactis NRRL B-4449.
We further tested the xylanolytic activity with a general xylanase
assay measuring the release of reducing sugars from xylan. The initial
breakdown of xylan involves extracellular xylanase activity, but this
activity was not detected in the culture supernatants of the three
lactococcal strains tested. Instead, we detected activities in the
cytoplasmic extracts of L. lactis IO-1 and L. lactis NRRL B-4449, but they were not significant compared to those of other known xylanases. Xylanase specific activities as high as
9.3 U mg1 and 4.8 U mg1
have been reported for Aspergillus ochraceus 42 (2) and Bacillus sp. strain K-1
(25), respectively. We suspected that the xylanase activity was in fact a xylosidase activity encoded by xynB.
We have not identified a putative xylanase gene in L. lactis, but the genomic sequence of L. lactis IL-1403
revealed the presence of a gene highly homologous to the
endo-1,4--xylanase D gene from a different species. This gene (nt
281456 f to 282572 f) is located far from the xylose-xyloside
operon (nt 1543238 r to 1547484 r) that we identified. However, even if
Lactococcus has the genetic potential to effectively degrade
xylan, our three strains, including the plant environmental isolates
L. lactis NRRL B-4449 and L. lactis IO-1,
seem to have lost that function. It is possible that L. lactis has evolved to exploit the xylanase secretion of other
bacteria in the environment, transporting the resulting
xylo-oligosaccharides for further metabolism.
We hypothesize that the ORF downstream of xynB encodes a
xyloside transporter, XynT. Previous characterization of oligoxyloside transport has implicated an ATP-binding cassette (ABC) transport system. When an ATP-binding component of an ABC transport system, msiK, is inactivated in Streptomyces
lividans, the transport of cellobiose and xylobiose is abolished
(17). Genes encoding two potential membrane
components of an ABC xyloside transporter, xynB and
xynC, have been sequenced from Thermoanaerobacterium thermosulfurigenes (GenBank accession number U50952).
Although our XynT is not homologous to these ABC proteins, several
pieces of evidence support its putative function. First, XynT is
homologous to known di- and trisaccharide transporters as well as an
-xyloside transporter (encoded by the xylP gene) recently
described by Chaillou et al. (9). Second, xynT
is located between the xylose mutarotase and xylosidase genes,
downstream of the xylose metabolic locus. B. subtilis has a
similar organizational relationship for the xyl and
xyn loci
(xynC-xynB-xylR-xylA-xylB0)
(16). Third, almost half of the XynT amino acid residues
(232 of 494) are hydrophobic, indicative of a membrane-associated
protein. Another gene located further downstream of xynB and
showing high homology to D-xylose, hexose, or
monosaccharide transporter genes is included in the lactococcal
genome database (INRA). This finding further supports our
hypothesis that XynT is involved in xyloside rather than xylose transport.
We discovered a new function related to xylan metabolism and encoded
immediately downstream of xylB by xylM. This
mutarotase function could link together xylose metabolism and xylan
metabolism. The first enzyme in the xylose utilization pathway, xylose
isomerase, has a marked preference for
-D-xylose over
-D-xylose (14, 32). However, the
end product of xylan breakdown is -D-xylose. Although xylose mutarotation occurs spontaneously in solution, the
actual rates in vivo are not known. The spontaneous rate of galactose
mutarotation was shown to be insufficient for E. coli growth
on lactose, because a deletion of the mutarotase gene (galM) resulted in very slow growth (7). If a bacterium
transported and metabolized only free xylose, a mutarotase would not be
necessary. If a bacterium metabolized xylan or xylo-oligosides, a
xylose mutarotase could give an additional competitive advantage
because of the relatively high initial concentration of -xylose. The xylM gene could be part of the multicistronic xylose
operon because of its close proximity to the stop codon of
xylB. In addition, xylA and xylM
probes both hybridized to a 7-kb mRNA transcript (among other
transcripts), large enough to encompass xylA,
xylB, and xylM.
Recently, other mutarotase sequences from L. lactis subsp. lactis ATCC 7962 and L. lactis subsp. cremoris MG1363 have been deposited in GenBank (accession numbers U60828 and AJ011653, respectively). These mutarotases are reportedly involved in lactose and galactose metabolism via the Leloir pathway. Our XylM sequence is approximately 28% identical and 46% similar to these GalM sequences. The dramatically increased xylose mutarotase activity that we observed upon overexpression and purification of L. lactis NRRL B-4449 XylM confirmed the existence of a mutarotase separate from galactose mutarotases.
In conclusion, we have discovered the genetic potential in
L. lactis for two related metabolic capacities, xylose
utilization and xylan utilization, encoded by the xylRABM
and xynTB genes. Based on transcription analysis,
these genes are organized in what could be a single
xylABM-xynTB 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 NRRL B-4449 and
L. lactis IO-1 retain the ability to metabolize xylose. Despite the functioning xylose metabolism and xylosidase capability of
L. lactis IO-1 and L. lactis NRRL
B-4449, these two strains could not grow on xylan or xylobiose. Further
studies are necessary to elucidate the metabolic defect. Finally, we
propose a new enzyme associated with xylan metabolism, a mutarotase
encoded by xylM, that may speed the further metabolism of
xylose released from xylan.
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ACKNOWLEDGMENTS |
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We thank David Wilson and Diana Irwin for help with the xylosidase and xylanase assays. We thank Alexei Sorokin (INRA, Jouy en Josas, France) for providing us with information about the genomic sequence of L. lactis IL-1403.
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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 Corporation, Cincinnati, Ohio.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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