APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 2006, p. 1891–1899
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 72, No. 3
Transcriptional and Functional Analysis of Oxalyl-Coenzyme A (CoA)
Decarboxylase and Formyl-CoA Transferase Genes
from Lactobacillus acidophilus
M. Andrea Azcarate-Peril,1,3Jose M. Bruno-Ba ´rcena,2† Hosni M. Hassan,2and Todd R. Klaenhammer1,2,3*
Departments of Food Science1and Microbiology2and Southeast Dairy Foods Research Center,3
North Carolina State University, Raleigh, North Carolina 27695-7624
Received 10 June 2005/Accepted 15 December 2005
Oxalic acid is found in dietary sources (such as coffee, tea, and chocolate) or is produced by the intestinal
microflora from metabolic precursors, like ascorbic acid. In the human intestine, oxalate may combine with
calcium, sodium, magnesium, or potassium to form less soluble salts, which can cause pathological disorders
such as hyperoxaluria, urolithiasis, and renal failure in humans. In this study, an operon containing genes
homologous to a formyl coenzyme A transferase gene (frc) and an oxalyl coenzyme A decarboxylase gene (oxc)
was identified in the genome of the probiotic bacterium Lactobacillus acidophilus. Physiological analysis of a
mutant harboring a deleted version of the frc gene confirmed that frc expression specifically improves survival
in the presence of oxalic acid at pH 3.5 compared with the survival of the wild-type strain. Moreover, the frc
mutant was unable to degrade oxalate. These genes, which have not previously been described in lactobacilli,
appear to be responsible for oxalate degradation in this organism. Transcriptional analysis using cDNA
microarrays and reverse transcription-quantitative PCR revealed that mildly acidic conditions were a prerequi-
site for frc and oxc transcription. As a consequence, oxalate-dependent induction of these genes occurred only
in cells first adapted to subinhibitory concentrations of oxalate and then exposed to pH 5.5. Where genome
information was available, other lactic acid bacteria were screened for frc and oxc genes. With the exception of
Lactobacillus gasseri and Bifidobacterium lactis, none of the other strains harbored genes for oxalate utilization.
Oxalic acid is a strong dicarboxylic acid (pKa
3.83) and a toxic compound that irritates tissues. This effect was
recognized in the 18th century, when oxalic acid was used for
cleaning and bleaching. Oxalate at extremely high concentrations
can cause death in humans and pathological disorders, including
hyperoxaluria (an oxalate level exceeding the normal range), pyr-
idoxine deficiency, urolithiasis (formation of calculi or uroliths),
renal failure, and other disorders (16). The toxicity of oxalate has
been related to its ability to generate reactive oxygen species
(through the Fenton reaction) as hydroxyl or carbonate radicals
during its interaction with hydrogen peroxide (30, 40). Oxalate
occurs widely in nature and in many foods, such as boiled carrots
(1.88 mg/g), tomatoes (0.04 mg/g), celery (0.17 mg/g), potatoes
(0.02 mg/g), and corn (0.03 mg/g), as well as in other dietary
sources, such as tea (0.11 mg/ml), coffee (0.05 mg/ml), and choc-
olate (1.17 mg/g) (15). Oxalic acid can also be produced by non-
enzymatic degradation or from metabolic precursors (like ascor-
bic acid) by the intestinal microflora (28). In the intestine, oxalate
may combine with calcium, sodium, magnesium, potassium, or
iron to form nonsoluble salts. It has been proposed that bacteria
that specifically degrade oxalate regulate the oxalate ho-
meostasis of the host by catabolizing free oxalate, reducing its
concentration in plasma and urine, and thereby preventing
adsorption. A recent clinical study demonstrated that there
1? 1.23; pKa
was a correlation between low rates of intestine colonization by
oxalate-degrading bacteria, specifically Oxalobacter formigenes,
and an increased risk of hyperoxaluria (39). This organism is
a natural inhabitant of the gastrointestinal tract (GIT) of
vertebrates, including humans, and it is the best-character-
ized microorganism of the intestinal microbiota with an
oxalate-degrading mechanism (11), which decarboxylates
oxalate, yielding formic acid and CO2. This reaction gener-
ates a proton gradient that contributes to the generation of
one ATP molecule when it is coupled with oxalate/formate
transport. Two enzymes involved in the catabolism of ox-
alate have been identified in O. formigenes. The first enzyme
is a formyl coenzyme A (CoA) transferase (EC 188.8.131.52),
encoded by frc, which activates the oxalate molecule by
cycling a CoA moiety from formyl-CoA (35). The second
enzyme is an oxalyl coenzyme A decarboxylase (EC 184.108.40.206),
encoded by oxc, which decarboxylates the activated oxalate
Lactobacillus acidophilus is a member of the lactic acid bac-
teria (LAB) that are used in the manufacture of fermented
milk products. LAB, especially bifidobacteria and lactobacilli,
constitute an important part of the human intestinal micro-
biota. The potential probiotic roles of these organisms have
been reviewed extensively (13, 29), and their beneficial effects
include reinforcement of natural defense mechanisms and pro-
tection against gastrointestinal disorders. Probiotics have been
successfully used to manage infant diarrhea, food allergies, and
inflammatory bowel disease (7). A recent study showed that
feeding a mixture of freeze-dried LAB led to a significant
reduction in urinary excretion in patients with idiopathic
calcium-oxalate urolithiasis and mild hyperoxaluria (10).
* Corresponding author. Mailing address: Department of Food Sci-
ence, North Carolina State University, Box 7624, Raleigh, NC 27695.
Phone: (919) 515-2972. Fax: (919) 515-7124. E-mail: klaenhammer@ncsu
† Present address: Biomanufacturing Training and Education Cen-
ter (BTEC), North Carolina State University, Raleigh, NC 27695-7624.
The presence of an oxalyl-CoA decarboxylase gene in Bi-
fidobacterium lactis has recently been documented (12).
L. acidophilus NCFM has been widely used as a probiotic
organism for over 30 years in fluid milk, yogurt, infant formu-
las, and dried dietary supplements (34). In the present study,
genes potentially encoding a formyl-CoA transferase and an
oxalyl-CoA decarboxylase were identified in the L. acidophilus
NCFM genome (2). Predicted frc and oxc genes were transcrip-
tionally and functionally analyzed to reveal a pathway for ox-
alate catabolism in L. acidophilus.
MATERIALS AND METHODS
Bacterial strains and growth conditions. The bacterial strains and plasmids
used in this study are listed in Table 1. Escherichia coli was propagated at 37°C
in Luria-Bertani (Difco Laboratories Inc., Detroit, MI) broth with shaking.
When appropriate, E. coli cultures were plated onto brain heart infusion agar
(Difco) supplemented with 150 ?g/ml erythromycin. Lactobacilli were propa-
gated statically at 37°C in MRS broth (Difco) or on MRS broth supplemented
with 1.5% agar. Erythromycin (5 ?g/ml) and/or chloramphenicol (5 ?g/ml) was
added to MRS broth or agar when it was appropriate. The semidefined medium
(BM) contained 0.5% tryptone, 0.5% yeast extract, 0.5% meat extract, 0.25%
sodium chloride, 0.1% Tween 80, 0.02% MgSO4, 0.005% MnSO4, 0.004%
FeSO4, 0.2% ammonium citrate, 0.001% thiamine, 0.2% K2PO4, 0.01% CaCO3,
ammonium oxalate, and 0.1% glucose. For determination of the maximum spe-
cific growth rates of L. acidophilus strains, standardized inocula were added to
obtain an initial absorbance at 600 nm (A600) of approximately 0.1 (total volume,
200 ?l BM per well). Plates were incubated at 37°C, and growth was automati-
cally monitored by determining the changes in A600as a function of time using
a FLUOStar OPTIMA microtiter plate reader (BMG Labtech GmbH, Offen-
burg, Germany). The maximum specific growth rate was calculated from the
slope of a linear regression line during exponential growth with a correlation
coefficient (r2) of 0.99. Each point represented the mean of three independent
Standard DNA techniques. E. coli plasmid preparation was done by using a
QIAprep Spin Plasmid Minipreps kit (QIAGEN Inc., Valencia, CA). Chromo-
somal DNA from L. acidophilus was extracted by the method of Walker and
Klaenhammer (41). Restriction enzymes and T4 DNA ligase were obtained from
Roche Molecular Biochemicals (Indianapolis, IN) and New England Biolabs
(Beverly, MA), respectively, and were used according to the suppliers’ recom-
mendations. Standard protocols were used for ligation, restriction endonuclease
digestion, DNA modification, and transformation as described by Sambrook
et al. (33). Electrotransformation of L. acidophilus was carried out as de-
scribed previously (42). PCR was performed by using standard protocols.
Phylogenetic analysis and conserved domains. Protein sequences obtained
from the Entrez Protein Database at NCBI (http://www.ncbi.nlm.nih.gov/) were
aligned and utilized to generate an unrooted phylogram tree using the neighbor-
joining method (ClustalX software) (38).
Conserved domains in potential proteins encoded by the open reading frames
(ORFs) of interest were inferred from the amino acid sequences by using the
Protein Families Database of Alignments and HMMs (http://www.sanger.ac.uk
/Software/Pfam/) as well as Clusters of Orthologous Groups of Proteins (http:
RNA isolation, cDNA probe preparation, and microarray hybridization. RNA
isolation was carried out as described previously (5). Briefly, 10-ml aliquots of L.
acidophilus cultures were centrifuged at 3,148 ? g, and the cell pellets were
immediately frozen in a dry ice-ethanol bath. Cell pellets were thawed and
homogenized in 1 ml Trizol (Technologies, Rockville, MD) with a Mini-Bead-
beater-8 cell disruptor (Biospec Products, Bartlesville, OK). The phases were
separated by centrifugation (14,000 rpm, 15 min, 4°C). The aqueous phase was
removed and placed in a fresh tube, and 0.4 ml of Trizol and 0.2 ml of chloroform
were added. The mixture was vortexed for 15 s and centrifuged to separate the
phases. RNA was precipitated by adding 1 volume of isopropanol. Identical
amounts (25 ?g) of total RNA were aminoallyl labeled by reverse transcription
with random hexamers in the presence of aminoallyl dUTP (Sigma Chemical
Co.), using Superscript II reverse transcriptase (Life Technologies) at 42°C over-
night, followed by fluorescence labeling of aminoallylated cDNA with N-hydroxy-
succinimide-activated Cy3 or Cy5 esters (Amersham Pharmacia Biotech). Labeled
cDNA probes were purified using a PCR purification kit (QIAGEN). Coupling of
TABLE 1. Strains, plasmids, and primers used in this study
Strain, plasmid, or primerCharacteristics Reference
Escherichia coli EC1000
Lactobacillus acidophilus NCFM
Lactobacillus acidophilus NCK1392
Lactobacillus acidophilus NCK1728
Primers for gene replacementa
Primers for RT-QPCR
RepA?MC1000, Kmr; host for pORI28-based plasmids
Human intestinal isolate
NCFM containing pTRK669
NCFM containing deleted version of ORF LBA0395 (frc)
1.42 kb containing ORF_LBA395 cloned into the BglII/XbaI sites of pORI28
pFrc containing a 72-bp deletion introduced by inverted PCR and self-ligation
aDashes indicate introduction of restriction enzyme sites.
1892AZCARATE-PERIL ET AL.APPL. ENVIRON. MICROBIOL.
the Cy3 and Cy5 dyes to the aminoallyl dUTP-labeled cDNA and hybridization of
samples to microarrays were performed as described previously (5).
Data normalization and gene expression analysis. Fluorescence intensities
were acquired using a General Scanning ScanArray 4000 microarray scanner
(Packard Biochip BioScience, Biochip Technologies LLC, Massachusetts) and
were processed as TIFF images. Signal intensities were quantified using the
QuantArray 3.0 software package (Packard BioScience). Two independent ar-
rays (biological replicates) on slides containing each gene spotted in triplicate
(technical replicates) were hybridized reciprocally to Cy3- and Cy5-labeled
probes in each experiment (dye swap) as described previously (5). Spots were
analyzed by adaptive quantitation. The local background was subsequently sub-
tracted from the recorded spot intensities. Data were median normalized. The
median of the six ratios for each gene was recorded. The ratio of the average
absolute pixel value for the replicated spots of each gene with treatment to the
average absolute pixel value for the replicated spots of each gene without treat-
ment represented the fold change in gene expression. Confidence intervals and
P values for the fold changes were also calculated by using a two-sample t test as
described by Knudsen (25). P values of 0.05 or less were considered significant.
The microarray platform and data are available at the Gene Expression Omnibus
(http://www.ncbi.nlm.nih.gov/geo) under accession numbers GPL1401 (plat-
form), GSE2782 (series), and GSM60519 and GSM60522 (samples).
Construction of L. acidophilus frc mutant. A 1.42-kb fragment containing frc
was amplified using L. acidophilus NCFM chromosomal DNA as the template
and primers LFoX and RFoB (Table 1). The fragment was cloned in the inte-
grative vector pORI28 (26), generating pFrc. Subsequently, a 72-bp fragment of
the cloned gene was removed by inverse PCR amplification of pFrc (using
primers LfoE and RFoE) and posterior self-ligation of the created EcoRI site.
The resulting 3-kb plasmid, pTRK837, was then introduced by electroporation
into L. acidophilus NCFM harboring pTRK669 (32). Subsequent steps to facil-
itate the integration event and gene replacement were carried out by using the
protocols described previously (9, 32). The suspected integrants were confirmed
by PCR and Southern hybridization analysis, using standard procedures.
Survival of logarithmic-phase cells after acid challenge. To determine the acid
sensitivity of log-phase cells, cultures were grown to an A600of 0.25 to 0.3 (pH
?5.8) from a 2% inoculum (initial A600, ?0.05) in MRS broth. The cultures were
centrifuged at room temperature for 10 min at 3,148 ? g, and the cells were
resuspended in the same volume of MRS broth adjusted to pH 3.0, 3.5, or 4.0
with HCl, lactic acid, or 5% oxalic acid. After incubation for 2 h at 37°C, the
number of CFU was determined by serial dilution in 10% MRS broth and
enumeration on MRS agar using a Whitley automatic spiral plater (Don Whitley
Scientific Limited, West Yorkshire, England).
RT-QPCR. L. acidophilus was transferred three times in MRS broth or MRS
broth containing 0.05% ammonium oxalate (pH 6.7) and then transferred to
fresh media having the same composition. Cells were then grown to an A600of
0.3 and transferred to (i) fresh MRS broth, (ii) MRS broth containing 0.5%
oxgall (pH 6.5), (iii) MRS broth containing 0.5% ammonium oxalate (pH 6.8), or
(iv) MRS broth (pH 5.5; acidified with lactic acid) containing 0.5% ammonium
oxalate. Following incubation at 37°C, samples were taken at zero time and 1, 2,
4, and 6 h, and RNA was isolated, treated with DNase, quantified, and diluted to
a concentration of 50 ng/?l. Primers meeting the standard criteria for reverse
transcription-quantitative PCR (RT-QPCR) for the following genes were de-
signed using CloneManager 7, version 7.10 and Primer Designer 5, version 5.10
(Scientific & Educational Software, Cary, NC): LBA0394, LBA0395 (frc),
LBA0396 (oxc), LBA0397, LBA0892 (bsh1), and LBA1078 (bsh2) (Table 1).
RT and PCR were carried out with an iCycler iQ (Bio-Rad Laboratories Ltd.).
The reaction mixtures (final volume, 20 ?l) contained 2? QuantiTect SYBR
Green (10 ?l), each primer at a final concentration of 0.1 ?M, a Quanti Tect RT
mixture (0.2 ?l), RNase-free H2O (1.8 ?l), and 4 ?l of template. The conditions
for the RT and amplification reactions were one cycle at 50°C for 30 min and one
cycle at 95°C for 15 min, followed by 40 cycles of 15 s at 94°C, 30 s at 49°C, and
30 s at 72°C for data acquisition. A melting curve analysis was conducted at 65°C,
with increments set at 1°C for 10 s (31 cycles). Serial dilutions (from 102to 1010
molecules) of a known PCR product (using the 16S primers [Table 1]) were
included in each run to establish a standard curve. Each sample was included in
triplicate in each run. Data were analyzed using the iCycler iQ software (version
3.0; Bio-Rad Laboratories Ltd.). The user-defined “PCR base line subtracted”
and “threshold cycle calculation” options were used to obtain the number of
threshold cycles per well. The linear equation for the standard curve (i.e., for
preparations containing known quantities of DNA) was then used to interpolate
the numbers of copies present in the unknown samples. The correlation coeffi-
cients for the standards were 0.99.
A reliable quantitative RT-PCR method requires correction for experimen-
tal variations in individual reverse transcription and PCR steps, since differ-
ences in the efficiency of each can result in a concentration of cDNA that does
not correspond to the starting amount of RNA (14). For this study, the 16S
rRNA gene was used for normalization.
Oxalate degradation activity. Lactobacillus strains were transferred three
times in BM broth without citrate (BMcit) containing 1% glucose plus 3.5 mM
ammonium oxalate. After this, 100 ?l of cells was inoculated into the same
medium, grown to an A600of 0.6, centrifuged, and resuspended in BMcit
containing 0.1% glucose plus 35 mM ammonium oxalate (32 mM oxalate).
The initial pH of BMcitwas 6.5, and the pH was allowed to naturally fall
during the 90-h incubation. Samples were taken over time, centrifuged, neu-
tralized to obtain pH values between 5 and 7 (according to the manufacturer’s
instructions) with 1 N sodium hydroxide, and stored at ?20°C. The oxalate
concentrations in the supernatants were measured in triplicate using a diag-
nostic oxalate kit (Trinity Biotech, County Wicklow, Ireland). In this assay,
oxalate is oxidized to carbon dioxide and hydrogen peroxide by oxalate
oxidase. Hydrogen peroxide, 3-methyl-2-benzothiozolinone hydrazone, and
3-(dimethylamino)benzoic acid, in the presence of peroxidase, yield an in-
damine dye which has a maximum absorbance at 590 nm.
Analysis of the chromosomal region containing frc and oxc.
The genome sequence of L. acidophilus NCFM (2) revealed the
presence of an operon putatively involved in oxalate catabolism
(Fig. 1). The predicted operon consisted of two genes: the formyl-
CoA transferase gene (LBA0395, frc) and the oxalyl-CoA decar-
boxylase gene (LBA0396, oxc). High-energy rho-independent ter-
minators were predicted to be downstream of LBA0397 (?G,
?11.4 kcal/mol) and frc (?G, ?14.6 kcal/mol). Additionally, a
typical ribosome binding sequence (AGAAGG; 7 nucleotides
from the start codon) and a putative promoter were located
upstream of oxc (data not shown).
frc encoded a 445-amino-acid (aa) protein that was very
similar to a predicted acyl-CoA transferase/carnitin dehy-
dratase from Lactobacillus gasseri NCK334 (accession num-
ber ZP_00046082) and a putative formyl-CoA transferase
from E. coli K-12 (accession number NP_416872). A con-
served domain (pfam02515) belonging to a new family of CoA
FIG. 1. Formyl-CoA transferase and oxalyl-CoA decarboxylase genes in L. acidophilus NCFM. Putative rho-independent terminators (lollipop
symbols) and their corresponding free energies (in kcal/mol) are indicated. Potential promoter regions for ORFs LBA0396 and LBA0397 are
indicated by bent arrows.
VOL. 72, 2006 OXALATE DEGRADATION BY LACTOBACILLUS ACIDOPHILUS 1893
transferases is present in this protein. Most CoA transferases
belong to two well-known enzyme families, but recently a third
family of CoA transferases was described (17). The members of
this enzyme family include oxalyl-CoA transferase, succinyl-CoA:
(R)-benzylsuccinate CoA transferase, (E)-cinnamoyl-CoA:(R)-
phenyllactate CoA transferase, and butyrobetainyl-CoA:(R)-car-
nitine CoA transferase. Additionally, the NCFM frc product
exhibited 44% identity (61% similarity) with the protein encoded
by O. formigenes frc. The gene encoding formyl-CoA transferase
(35) was the first member of family III of CoA transferases to be
oxc encoded a 569-aa protein similar to the oxalyl-CoA de-
carboxylases (EC 220.127.116.11) from O. formigenes (53% identity and
71% similarity) (27) and B. lactis (46% identity and 63% sim-
ilarity) (12). The protein encoded by oxc has a conserved do-
main that is present in thiamine pyrophosphate (TPP)-requir-
ing enzymes (COG0028). This domain is also present in several
other enzymes, including acetolactate synthase, pyruvate de-
hydrogenase (cytochrome), glyoxylate carboligase, and phos-
phonopyruvate decarboxylase. In the oxc product, an N-termi-
nal TPP-binding domain (pfam02776) starts at residue 20 and
spans 171 aa, and the central TPP domain (pfam00205) starts
at residue 210 and spans 154 aa.
The potential product of the gene downstream of frc,
LBA0394, was a 395-aa protein which was virtually identical
(90% identity; E value, 0.0) to the predicted acyl-CoA trans-
ferase from L. gasseri and exhibited 44% identity to the formyl-
CoA transferase from E. coli K-12 and 44% identity to putative
protein F (accession number BAA16242) encoded by a bile
acid-inducible operon in E. coli. Interestingly, the frc product
exhibited 30% identity (48% similarity) with the putative prod-
uct of LBA0394. The latter, however, did not exhibit significant
similarity to the formyl-CoA transferase from O. formigenes,
indicating that although LBA0394 might encode a CoA trans-
ferase, the enzyme is not necessarily a formyl-CoA transferase.
LBA0397, upstream of oxc, encodes a 639-aa protein having
the conserved COG0488 domain Uup, which corresponds to
ATPase components of ABC transporters with duplicated
ATPase domains (21). High levels of identity (more than 75%)
were observed with nearly equivalent proteins in L. gasseri and
Other members of the lactic acid bacteria were screened in
silico for frc- and oxc-related genes. L. gasseri NCK334 (acces-
sion number ZP_00046991) and B. lactis DSM 10140 (formerly
Bifidobacterium animalis) (12) harbored genes for oxalate uti-
lization, whereas Lactobacillus plantarum WCFS1 (24) and L.
johnsonii NCC553 (31) did not. Figure 2 shows the phylo-
genetic relationships of several putative oxalyl-CoA decarboxy-
lases from organisms whose protein sequences were available.
As expected, the decarboxylases from L. gasseri and L. acido-
philus clustered together and, interestingly, clustered closer to
the enzyme from B. lactis.
Transcriptional analysis of the oxc operon using microar-
rays. Antiport of oxalate/formate in O. formigenes is coupled
to oxalate decarboxylation and generates a proton motive
gradient (1). We were not able to identify a putative oxalate
permease/antiporter by in silico analysis of the L. acido-
philus genome. Therefore, microarray experiments were con-
ducted in an attempt to identify a candidate that might be
responsible for the specific transport of oxalate into the cell.
During growth of L. acidophilus in MRS medium, the pH
of a culture starting at pH 6.5 typically decreases to less than
4.0 due to fermentation and lactic acid production. In a
previous study (Gene Expression Omnibus accession num-
bers GPL1401 [platform] and GSE1976 [series]) (5), a
whole-genome array containing 97.4% of the NCFM anno-
tated genes was used to identify genes that were differen-
tially expressed when log-phase cells were exposed to MRS
medium at pH 5.5 and pH 4.5 acidified with lactic acid. After
exposure to pH 5.5 (adjusted with lactic acid) for 30 min, we
observed induction of frc (3.2-fold) and oxc (4.5-fold) en-
coding the putative formyl-CoA transferase and oxalyl-CoA
decarboxylase, respectively. No statistically significant dif-
ferences in the levels of expression of frc or oxc were ob-
served between the control (pH 6.8) and the samples ex-
posed to pH 4.5.
In the present study, we studied gene expression at pH 6.8
in an attempt to separate the specific effect of the oxalate
salt from the effect of the pH. The L. acidophilus whole-
genome array was used to analyze the global gene expres-
sion after cells were exposed to 1% (70 mM) ammonium
oxalate for 30 min at pH 6.8. A summary of the results of our
previous study (in which log-phase cultures were exposed to
pH 5.5 and pH 4.5 with no oxalate) and the present study is
shown in Fig. 3. In the presence of 1% oxalate at pH 6.8, 16
genes were significantly upregulated (P ? 0.05 and a ratio of
?2.0) (Table 2), and 315 genes were downregulated (P ?
0.05 and a ratio of ?0.5). Both the frc and oxc genes were
downregulated under these conditions. The most upregu-
lated genes were a cadmium/manganese transport ATPase
gene (LBA1234; upregulated 9.6-fold) and the genes encod-
ing two uncharacterized membrane proteins (LBA1119 and
LBA1690; upregulated 5.9- and 4.8-fold, respectively). Interest-
ingly, ORFs LBA0038, LBA0039, LBA0040, and LBA0041 were
FIG. 2. Unrooted phylogram tree of oxalyl-CoA decarboxylase se-
quences from diverse organisms. Proteins were aligned by CLUSTALX.
Alignments were used for tree reconstruction. The organisms used were
L. acidophilus NCFM, L. gasseri ATCC 3323 (GenBank accession
number ZP_00046991), B. lactis (BAD11779), Bradyrhizobium japoni-
cum USDA110 (BAC48422.1), E. coli CFT073 (NP_754791.1), Myco-
bacterium tuberculosis CDC1551 (NP_334536.1), O. formigenes (P40149),
Mycobacterium bovis (NP_853789), Oryza sativa (BAB33274.1), Schizosac-
charomyces pombe (CAA22176), Mycobacterium leprae (CAA15478), Sac-
1894AZCARATE-PERIL ET AL.APPL. ENVIRON. MICROBIOL.
upregulated between 1.4- and 2.4-fold. These four genes appear
to form an operon. LBA0041 is predicted to encode a putative
adenosylcobalamin-dependent ribonucleoside triphosphate re-
ductase. ORFs LBA0038, LBA0039, and LBA0040 are poorly
characterized; however, the LBA0040 product is similar to a pu-
tative ATP:cob(I)alamin adenosyltransferase (23), the enzyme
responsible for the last step in the activation of vitamin B12
(cyanocobalamin) to coenzyme B12(adenosylcobalamin). A rela-
tionship between altered oxalate metabolism and B vitamin defi-
ciency has been documented (3, 18), which resulted in some
interest in why these genes are upregulated in the presence of
Transcriptional analysis of the oxc operon by RT-QPCR.
Acid induction of frc and oxc was evaluated in the presence and
FIG. 3. Transcriptional response of frc and oxc to pH 5.5, pH 4.5, and 1% (70 mM) ammonium oxalate (pH 6.8) in MRS broth after 30 min.
The solid rectangles indicate ?twofold-higher expression, the cross-hatched rectangles indicate a ?twofold reduction in expression (P ? 0.05), and
the open rectangles indicate values of gene expression that are not statistically different from values obtained under the control conditions (L.
acidophilus incubated in fresh MRS broth for 30 min). Plus and minus signs indicate that the experiment was carried out in the presence and in
the absence of oxalate, respectively. The proposed metabolic pathway of oxalate decarboxylation by L. acidophilus is also shown. The structures
of the compounds were obtained from the website http://www.genome.jp/kegg/kegg2.html.
TABLE 2. Genes upregulated in response to 1% (70 mM) ammonium oxalate at pH 6.8 in L. acidophilus NCFM
PTS system IIab
Putative inner membrane protein
Cadmium/manganese transport ATPase
Putative membrane protein
aArray ratios obtained from two biological replicates and two technical replicates for each condition were averaged.
bPTS, phosphotransferase system.
VOL. 72, 2006OXALATE DEGRADATION BY LACTOBACILLUS ACIDOPHILUS1895
absence of ammonium oxalate as an inducer of expression of
the operon. Primers meeting RT-QPCR criteria were designed
for LBA0394, frc, oxc, and LBA0397. L. acidophilus was
adapted to oxalate by three consecutive transfers in MRS broth
containing 0.05% ammonium oxalate, a noninhibiting concen-
tration. Cells preexposed or not exposed to this compound
were then transferred to MRS broth at pH 5.5 (adjusted with
lactic acid) containing ammonium oxalate, and samples were
taken over time. When L. acidophilus cells were first propa-
gated in the presence of ammonium oxalate and then exposed
to pH 5.5 plus 0.5% ammonium oxalate, both frc expression
and oxc expression increased dramatically to levels that ap-
proached fourfold induction (Fig. 4A). In the absence of ox-
alate adaptation, exposure to pH 5.5 plus oxalate (Fig. 4B)
again resulted in induction of both genes, but oxc was ex-
pressed significantly more highly (two- to fourfold) than frc
(one- to twofold). It is not clear why the levels of expression of
frc and oxc differed under these conditions, particularly since
both genes are predicted to be in the same operon and exhib-
ited similar expression levels when they were highly induced
(Fig. 4A). Expression of ORFs LBA0394 and LBA0397 re-
mained constant under these conditions.
were also resuspended in MRS medium containing 0.5% ammo-
nium oxalate at pH 6.8. Under these conditions, none of the
genes examined in this study (frc, oxc, bsh1, bsh2, LBA0394,
and LBA0397) were induced (data not shown).
LBA0394, the ORF immediately downstream of frc, showed
some homology to a bile-inducible protein. The NCFM genome
contains genes encoding two bile salt hydrolases, LBA0872 (bsh1)
and LBA1078 (bsh2). Therefore, we designed RT-QPCR primers
for bsh1 and bsh2 and examined the expression of these genes
after exposure of the cells to 0.5% oxalate at pH 5.5 or exposure
to oxgall (0.5%). Neither bsh1 nor bsh2 was induced under these
conditions, and expression of LBA0394 remained basal and con-
Inactivation of frc and mutant analysis. Integrative plasmid
pORI28, a pWV01-derived vector (26), was used to replace frc
with the deleted version of the same gene by using the proto-
cols described previously (9, 32). PCR and Southern hybrid-
ization experiments using an internal fragment of frc as the
probe confirmed that the frc gene was replaced with the de-
leted version in NCK1728 (data not shown).
The survival of log-phase cells (A600, 0.3) of wild-type L.
acidophilus NCFM and the survival of the frc mutant were
compared at pH 3.0, 3.5, and 4.0 by using hydrochloric acid
(HCl), lactic acid, or oxalic acid to acidify MRS broth (Fig. 5).
No differences between the parent and the frc mutant were
observed when HCl or lactic acid was used to acidify the
culture medium. Additionally, no differences in survival were
observed in the presence of 5% oxalic acid, at pH 4.0 (?50%
survival) or pH 3.0 (?0.01% survival). However, the frc mutant
was significantly more sensitive to 5% (wt/vol) oxalic acid after
2 h of exposure at pH 3.5. The Henderson-Hasselbalch equa-
tion for oxalic acid predicts that at pH 4.0 most of the oxalate
is dissociated (pKa
At pH 3.5 a larger amount would be undissociated. When
combined with a higher concentration of the acid (5%), this
would increase the amount of protonated acid available to
diffuse into and acidify the cell. At pH 3.0, the combination of
a low pH (closer to the pKa
high concentration of acid was lethal for both the wild type and
the frc mutant.
oxalate was also examined (Fig. 6). A semidefined medium (BM
with an initial pH of 6.5) was used since addition of the oxalate
salt caused formation of a precipitate in MRS broth. The maxi-
mum specific growth rate was 0.70 h?1in BM containing 0.1%
glucose, with or without 0.1% oxalate. The maximum specific
growth rate decreased somewhat in the presence of 0.5% am-
monium oxalate (0.48 h?1), and the maximum cell density was
noticeably lower. In the absence of glucose, growth occurred,
but the growth rate and maximum cell density were substan-
tially lower. BM is a semidefined medium containing complex
sources of nutrients, such as tryptone, yeast extract, and meat
extract, which can support limited growth until any residual
carbohydrate is exhausted. Interestingly, when 0.5% ammo-
2? 3.83) and hence unable to enter the cell.
1of oxalate [pKa
1? 1.23]) and the
FIG. 4. Transcriptional analysis of the oxc operon in L. acidophilus
cells at pH 5.5. (A) Cells were first transferred in MRS broth (pH 6.8)
containing noninhibitory concentrations of ammonium oxalate (pre-
adapted). Solid bars, frc; cross-hatched bars, oxc. Gene induction was
monitored over time after cells were placed in MRS broth containing
0.5% ammonium oxalate at pH 5.5. (B) Gene induction for cells in
MRS broth at pH 6.8 (nonadapted). Experiments were carried out in
triplicate. The error bars indicate standard deviations.
1896 AZCARATE-PERIL ET AL.APPL. ENVIRON. MICROBIOL.
nium oxalate was added to the media without added glucose, a
increased to a value similar to that observed in the presence of
0.1% oxalate. The reasons for this delay in growth are unknown.
The frc mutant showed similar patterns of growth under these
conditions, indicating that initiation of growth at the oxalate con-
Finally, oxalate utilization was measured for both NCFM
and the frc mutant (Fig. 7). The Lactobacillus strains were
transferred three times in broth containing a noninhibitory
concentration of ammonium oxalate (0.05%) to ensure high
levels of expression of the oxalate genes, oxc and frc. For the
assay, cells were inoculated into the same medium, propagated
to the mid-log phase (A600, ?0.6), and then centrifuged and
resuspended in broth containing 0.1% glucose and 0.5% (35
mM) ammonium oxalate. The concentration of oxalate in the
culture supernatant decreased significantly for the control (up
to 24%) but not for the frc mutant, for which the oxalate
concentration decreased only 6%. Most of the oxalate degra-
dation occurred during the first 16 h. The results indicated that
L. acidophilus NCFM was able to degrade oxalate, and Frc
participated in this process.
FIG. 5. Survival of log-phase cells of L. acidophilus NCFM and the frc mutant after challenge with MRS broth adjusted to pH 4.0, 3.5, and 3.0
with HCl, lactic acid, or oxalic acid for 2 h. The values are the averages for six separate incubations. The error bars indicate standard deviations.
FIG. 6. Growth curves for L. acidophilus NCFM in semidefined
BM containing different concentrations of ammonium oxalate. Cell
growth was evaluated in BM in the presence of 0.1% glucose (■), in
the presence of glucose plus 0.1% ammonium oxalate (Œ) or 0.5%
ammonium oxalate (?), in the absence of glucose (?), or in the
absence of glucose plus 0.1% ammonium oxalate (‚) or 0.5% ammo-
nium oxalate (ƒ). Each point represents the mean of three indepen-
dent experiments. The error bars indicate standard deviations.
FIG. 7. Oxalate-degrading activity of L. acidophilus. Strain NCFM
(■) and the frc mutant (F) were consecutively transferred in BMcit
containing a noninhibitory concentration of oxalate (0.05%; 3.5 mM)
and then exposed to 0.5% (32 mM) oxalate in broth. Samples were
taken over time, and the oxalate concentration in the supernatants was
measured. Each point represents the mean of three independent ex-
periments. The error bars indicate standard deviations.
VOL. 72, 2006 OXALATE DEGRADATION BY LACTOBACILLUS ACIDOPHILUS1897
Studies using a whole-genome microarray of L. acidophilus
NCFM (5) showed that there was induction of two ORFs,
LBA0395 and LBA0396, at a mildly acidic pH, pH 5.5. Com-
parative analysis of these genes and the adjacent genes with the
available sequences in the GenBank database resulted in iden-
tification of genes encoding a formyl-CoA transferase (frc) and
an oxalyl-CoA decarboxylase (oxc), which were highly similar
to frc and oxc genes which direct oxalate degradation by O.
formigenes. In L. acidophilus, frc and oxc appear to form an
operon as the two genes are flanked by two predicted termi-
nators. RT-QPCR and microarray experiments showed that
oxalate (at a pH above 5.8) did not directly induce the expres-
sion of frc and oxc. However, when L. acidophilus was repeat-
edly transferred in broth containing noninhibitory concentra-
tions of ammonium oxalate and subsequently exposed to pH
5.5 plus oxalate, the expression of oxc and frc was dramatically
increased. Moreover, when frc was inactivated and the mutant
was exposed to an acidic pH, the strain became more suscep-
tible to oxalic acid specifically at pH 3.5, indicating that frc is
involved in the degradation of oxalate by L. acidophilus. In this
regard, unlike the wild-type strain, the frc mutant was unable to
The concept of autochthonous microorganisms in the GIT
has been discussed by several authors (for a review see refer-
ence 36). In fact, Tannock proposed a concise definition based
on three important characteristics: a long-term association
with the host, a stable population in a particular region of the
gut, and a demonstrated ecological function. Oxalate occurs
widely in nature, and oxalate-rich foods are important sources
of oxalate in the diet. Bacteria that specifically degrade oxalate
in the GIT can regulate oxalate homeostasis by both prevent-
ing absorption and catabolizing free oxalate. Consequently, the
ability to detoxify this compound potentially suggests a new
ecological function for L. acidophilus.
Other oxalate-degrading bacteria isolated from the human
GIT include Eubacterium lentum (22) and Enterococcus faeca-
lis (20). Hokama et al. isolated an oxalate-degrading E. faecalis
strain from human stools and identified the formyl-CoA trans-
ferase and oxalyl-CoA decarboxylase by Western blotting using
antibodies against Frc and Oxc from O. formigenes. Campieri
et al. (10) measured oxalate degradation in patients with idio-
patic calcium-oxalate urolithiasis that was treated with 8 ?
1011LAB (including L. acidophilus, L. plantarum, Lactobacil-
lus brevis, Streptococcus thermophilus, and Bifidobacterium in-
fantis). They observed a reduction in the excreted oxalate in
the patients and showed that L. acidophilus and S. thermophilus
could reduce oxalate concentrations in vitro, even when their
growth was partially inhibited by this compound. However, the
genes responsible for oxalate degradation by these micro-
organisms were not identified. More recently, an oxalyl-CoA
decarboxylase gene was identified in B. lactis, and the oxalate-
degrading activity of the enzyme was confirmed by a capillary
electrophoresis-based method (12). Therefore, oxalate catab-
olism in the GIT may be an important property of some com-
mensal and probiotic bacteria.
In other oxalate-degrading organisms, such as O. formigenes,
the utilization of oxalate is coupled to energy produced by the
antiport of oxalate and formate. By in silico analysis, we were
not able to identify a putative permease/antiporter that might
incorporate dissociated oxalate into the cell. It is commonly
known that the nondissociated forms of organic acids, such as
oxalic acid, can freely diffuse through the cytoplasmic mem-
brane. This might explain the apparent absence of a specific
transporter for oxalic acid in the genome of NCFM. The concen-
tration of nondissociated oxalate (pKa
entering the cell will increase under acidic conditions, such as
those encountered in the digestive tract, where the pH values
range from 1 to 7. In the stomach, the pH values range from 1 to
3; in the large intestine, the pH values range from 5 to 7; and in
the duodenum, the pH values range from 6 to 6.5. As an alter-
native hypothesis, an oxalate transporter may be involved, as
three genes predicted to encode membrane proteins were
strongly upregulated in the presence of ammonium oxalate. Gene
expression studies in the presence of oxalate at pH 6.8, which
separated the specific effect of the oxalate salt from the effect of
the low pH, resulted in identification of a cadmium/manganese
transport ATPase gene as the most upregulated gene (9.6-fold)
under these conditions. The predicted protein encoded by
LBA1234 has two conserved domains, pfam00122 (E1-E2
ATPases are primary active transporters that form phospho in-
termediates during the catalytic cycle. They are classified as P1 to
P4 based on the primary structure and potential transmembrane
segments (4). E1-E2 ATPases transport divalent cations, and ox-
alate is a divalent cation. Hence, LBA1234 might be the trans-
porter responsible for the translocation of oxalate into the cell.
Two other uncharacterized membrane proteins (LBA1119 and
LBA1690) were also upregulated, but they did not have any
features that could be used for putative identification.
Since oxalate is normally present in the human GIT, the
ability to degrade this compound may provide a selective ad-
vantage to certain members of the intestinal microbiota. Ad-
ditionally, since other microorganisms present in the intestine
produce the enzymes for oxalate degradation, we speculate
that the ability to decarboxylate oxalyl-CoA was acquired by
L. acidophilus via horizontal gene transfer. A number of ob-
servations support this hypothesis. The gene upstream of
LBA0394 is similar to a gene encoding a transcriptional regu-
lator, and the gene downstream of LBA0397 encodes a puta-
tive AT-rich DNA binding protein. The region comprising
ORFs LBA0394 to LBA0397, including frc and oxc, is on the
complementary strand, and the G?C contents of frc (38.4%)
and oxc (40.2%) are notably higher than the average G?C
content of the NCFM genome (34.71%). Several studies have
reported the occurrence of natural transformation events due
to additive integration of DNA, based on two flanking regions
with high DNA similarity that initiate the recombination pro-
cess (for a review see reference 37). It is notable that the region
containing ORFs LBA0394 to LBA0397 is flanked by DNA
regions that are highly similar to the equivalent segment in the
L. johnsonii genome (31), even though oxalate genes are not
present in this bacterium.
The efficacy of probiotics as a means to prevent and/or treat
urogenital infections and recurrent bladder cancer has been
scientifically accepted in the past two decades. More recently,
encouraging results were obtained in a clinical trial of
O. fomigenes with patients suffering from hyperoxaluria type I,
an inherited, life-threatening disease characterized by recur-
1? 1.23; pKa
1898AZCARATE-PERIL ET AL.APPL. ENVIRON. MICROBIOL.
rent oxalate stone formation, nephrocalcinosis, and eventual Download full-text
liver and kidney failure (19). Further characterization of ox-
alate-degrading probiotic bacteria and efforts to promote the
expression, activity, and release of the enzymes involved may
lead to a complementary method to manage oxalate-related
kidney disease via oral microbial supplements. This is a par-
ticularly exciting use of probiotic bacteria, because high levels
of these organisms can be safely consumed in food (109
CFU/g) or dietary supplements (1010CFU/g).
This work was partially supported by the Southeast Dairy Foods
Research Center, Dairy Management, Inc., the North Carolina Dairy
Foundation, and Danisco USA, Inc.
We thank Evelyn Durmaz and B. Logan Buck for helpful discussions
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VOL. 72, 2006OXALATE DEGRADATION BY LACTOBACILLUS ACIDOPHILUS1899