Download An operon f rom Lactobacillus helveticus composed

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Microbiology (1996), 142, 3459-3468
An operon f rom Lactobacillus helveticus
composed of a proline iminopeptidase gene
(pep/) and two genes coding for putative
members of the ABC transporter family of
proteins
Pekka Varmanen,t Terhi Rantanen and Airi Palva
Author for correspondence: Airi Palva. Tel: +358 16 4188 277. Fax: +358 16 4384 550.
e-mail : [email protected]
Ag ricultura I Research
Centre of Finland, Food
Research Instit ute,
Jokioinen 31600, Finland
A proline iminopeptidase gene (pep/) of an industrial Lactobacillushelveticus
strain was cloned and found to be organized in an operon-like structure of
three open reading frames (ORFI, ORFZ and ORF3). ORFI was preceded by a
typical prokaryotic promoter region, and a putative transcription terminator
was found downstream of ORF3, identified as the pep/ gene. Using primerextension analyses, only one transcription start site, upstream of ORFI, was
identifiable in the predicted operon. Although the size of mRNA could not be
judged by Northern analysis either with ORFI-, ORFZ- or pep/-specific probes,
reverse transcription-PCR analyses further supported the operon structure of
the three genes. ORFl, ORFZ and ORF3 had coding capacities for 507,245 and
33.8 kDa proteins, respectively. The ORF3-encoded Pep1 protein showed 65 OO/
identity with the Pep1 proteins from Lactorbacillus delbrueckii subsp. bulgaricus
and Lactobacillus delbrueckii subsp. lactis. The ORFI-encoded protein had
significant homology with several members of the ABC transporter family but,
with two distinct putative ATP-binding sites, it would represent an unusual
type among the bacterial ABC transporters. ORFZ encoded a putative integral
membrane protein also characteristic of the ABC transporter family. The pep/
gene was overexpressed in Escherichia coli. Purified Pep1 hydrolysed only diand tripeptides with proline in the first position. Optimum Pep1 activity was
observed at pH 7.5 and 40 O C . A gel filtration analysis indicated that Pep1 is a
dimer of M, 53000. Pep1 was shown to be a metal-independent serine
peptidase having thiol groups at or near the active site. Kinetic studies with
proline-pnitroanilide as substrate revealed K,,, and Vmaxvalues of 0 8 mM and
350 mmol min-l mg-l, respectively, and a very high turnover number of
135000 s-l.
Keywords : Lactobacillus helveticus, proline iminopeptidase, ABC transporter
INTRODUCTION
Lactic acid bacteria (LAB) are limited in their capacity to
synthesize amino acids and, therefore, they need ex-
t Present
address: Valio Ltd, Research and Development, Helsinki,
Finland.
Abbreviations: DIG, digoxigenin-dUTP; LAB, lactic acid bacteria; PropNA, proline-p-nitroanilide; pHMB, p-hydroxymercuribenzoic acid; RTPCR, reverse transcription-PCR.
The EMBL accession number for the sequence of 3240 b p DNA fragment,
carrying the pep/ and ABC transporter genes, reported in this paper is
256283.
0002-0973 0 1996 SGM
ogenous nitrogen sources for optimal growth. When
growing in milk, LAB utilize complex proteolytic systems
for the degradation of milk proteins. In recent years, the
biochemical and genetic characterization of this system
has received considerable attention (for reviews, see Kok,
1990; Pritchard & Coolbear, 1993; Tan e t al., 1993;
Visser, 1993; Kok & de Vos, 1994). The proteolytic
system is composed of a cell-envelope-associated proteinase, membrane-bound transport systems, and several
cytoplasmic peptidases. The milk proteins contain a high
amount of proline (Fox, 1989), resulting in the generation
of proline-rich peptides during proteinase action (Kok &
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3459
P. V A R M A N E N , T. R A N T A N E N a n d A. P A L V A
de Vos, 1994). Dipeptides and oligopeptides containing
an N-terminal proline residue are usually not hydrolysed
by general-purpose amino-, di- or tripeptidases. Thus, for
degradation of the proline-rich peptides, several prol.inespecific peptidases with distinct substrate specificities
have been evolved in LAB. So far, activities such as
prolinase, prolidase, proline iminopeptidase, aminopeptidase P and X-prolyl dipeptidyl aminopeptidase have
been detected in various species of LAB (Baankreis &
Exterkate, 1991; Meyer-Barton e t al., 1993; Kok 8: de
Vos, 1994; Mars & Monnet, 1995; Stucky etal., 1995). It
has been previously reported that neither Lactobarillus
helveticm nor Lactobacilltls delbrueckii subsp. bdgarictls
(hereupon L. bulgaricus) exhibits proline iminopeptidase
activity (Kok 8: de Vos, 1994; Baankreis & Exterkate,
1991). However, two recent reports describe the characterization of an iminopeptidase gene (pep0 from L.
bulgaricus (Atlan e t al., 1994; Gilbert e t al., 1994). On the
other hand, a prolinase gene, cloned and characterized
from L. helvetictrs (Dudley & Steele, 1994; Varmanen e t
al., 1996), possesses an amino acid sequence with 31 YO
identity to the proline iminopeptidase from L. btllgaricus
(Atlan et al., 1994) and Lactobacillus delbrueckii subsp. r’actis
(Klein et al., 1994). Although the substrate specificities
and other properties of the proline iminopeptidase and
prolinase of these two Lactobacillus species show close
resemblance (Gilbert et al., 1994; Dudley & Steele, 1994;
Varmanen e t a/., 1996), the level of the amino acid
sequence homology does not support that these two
enzymes are counterparts.
In this paper, we show that L. he1veticu.r also possesses an
iminopeptidase, in addition to the prolinase, by cloning its
gene with the aid of a L. bukaricus pep1 probe. Furthermore, DNA sequence and mRNA analyses ofpep1 and
two ABC transporter genes, all organized in an operonlike structure in L. helveticus 53/7, are described. In
addition, overproduction, purification and enzymic
characterization of the recombinant L. helveticus Pep1 are
reported.
METHODS
Bacterial strains, plasmids and culture conditions. L. helveticus
strain 53/7 is an industrial starter from the collection of Valio
Ltd (Helsinki, Finland). L. helveticus was routinely grown in
MRS or Whey broth at 42OC without shaking. Whey broth
comprised 50 g whey permeate (Valio), 20 g casein hydrolysate
(Valio) and 10 g yeast extract per 1. For a pH-controlled
cultivation, a Biostat M bioreactor was used. The pH was
allowed to decrease to 5.6 at which point it was maintained by
addition of 1 M NH,. L. bulgaricus B14 (Federal Dairy Research
Centre, Kiel, Germany) was grown in MRS broth at 37 “C.
Escherichia coli strains DH5aF’ (Woodcock e t al., 1989), JMl05
(Pharmacia) and NM514 (Amersham) were grown in Luria
broth. Erythromycin (0.3 mg ml-l) and ampicillin (0-05 mg
ml-l) were added when using the vectors pJDC9 (Chen &
Morrison, 1987) and pKK223-3 (Pharmacia), respectively, in
E. coli.
Constructionand screening of a L. helveticus genomic library.
Chromosomal DNA was isolated essentially as described by
Vidgren e t al. (1992) without a guanidine hydrochloride
3460
treatment, followed by partial digestion with HaeIII. A 3-6 kb
fragment pool was selected for a L. helvetictls genomic library
established in AgtlO using A-DNA in vitro packaging and cDNA
rapid cloning modules (AgtlO; Amersham). Instead of the
Amersham cDNA rapid adaptor ligation module, a RiboClone
EcoRI Linker System (Promega) was employed. The titration
and plating of the phage were performed according to the
manufacturers instructions. A cloning efficiency of
1.16 x lo7 p.f.u. (pginsert DNA)-l was reached. The L. helveticus
57/3 genomic library contained 9.5 x lo5 p.f.u., of which
80-90% carried an insert. The diluted library was plated and
approximately 7500 plaques were transferred to positively
charged nylon membranes (Boehringer Mannheim) and
screened by DNA hybridization. For the hybridization probe, a
1-2 kb fragment, carrying the pepI gene from the L. bulgaricus
chromosomal DNA (Atlan e t al., 1994), was amplified by PCR
using the primer pair 5’-GTCGGTCGACAGACTGGCACGTCATAGAC-3’ and 5’-GGTTGAATTCGGTTCAGCGAGCATGTC-3’. Labelling of the probe was with digoxigenindUTP (DIG, Boehringer Mannheim). Hybridization was at
60 “C overnight, followed by washes with 0 . 5 ~SSC, 0.1 %
SDS at 50 OC. Isolation of phage DNA was according to the
instructions of the cDNA rapid cloning module. Alkaline lysis
(Sambrook e t al., 1989) was used for plasmid isolations from E .
coli with the DNA purification systems Magic (Promega) and
Flexi-Prep (Pharmacia). Other recombinant DNA techniques
were performed essentially as described by Sambrook e t al.
(1989).
DNA synthesis. The oligonucleotides were synthesized with an
Applied Biosystems DNA/RNA synthesizer model 392 and
purified by ethanol precipitation or with NAP-10 columns
(Pharmacia). For DNA synthesis, performed by PCR amplification, reaction conditions recommended by the manufacturer
of DynaZyme DNA polymerase (Finnzymes) were used.
Nucleotide sequencing and sequence analysis. Sequencing
was performed on an ALF DNA Sequencer (Pharmacia). The
dideoxy sequencing reactions (Sanger e t al., 1977) were performed according to the AutoRead Sequencing Kit manual
(Pharmacia). Both DNA strands were sequenced using pUC19specific primers and sequence-specific oligonucleotides for
primer walking. DNA sequences were assembled and analysed
with the PC/GENE set of programs (release 6.85 ;IntelliGenetics).
The PROSITE program of PC/GENE was used to detect specific
sites and signatures in protein sequences. Hydropathy analyses
were by the method of Kyte & Doolittle (1982) with the SOAP
program of PC/GENE. For homology searches, the databases of
EMBL and SWISS-PROT were used both as a CD-ROM
version (release 15.0, June 1995) and directly by e-mail with the
EMBL BLITZ server.
RNA methods. Total RNA was isolated from L. helveticus cells
essentially as described by Palva e t al. (1988) and Vesanto e t al.
(1994). RNA agarose gel electrophoresis and Northern blot
were performed as described previously (Hames & Higgins,
1985). Hybridization probes were labelled with [a-32P]dCTP
(> 3000 Ci mmol-’/lll TBq mmol-l; Amersham) by the
protocol of a random-primed DNA labelling kit (Amersham) or
with nonradioactive DIG. DIG luminescent kit (Boehringer
Mannheim) was used for hybrid detection with the DIGlabelled probe. The primer extensions were performed using an
ALF DNA Sequencer essentially as described by Myohanen &
Wahlfors (1993) and Vesanto e t al. (1995). The antisense
fluorescein-labelled oligonucleotides used in primer extensions
were 5’-TTCCGGCATATTTTGGATAG-3’,5’-AGCGTCGCAAATATCGTG-3’ and 5’-TTTGTGCTAATTCGTCCAG-
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PepI-ABC transporter operon from L. heluetictls
3', being complementary to nucleotides at positions 317-337,
1698-1715 and 2319-2337, respectively (Fig. 2).
accumulation during 5 h by SDS-PAGE and measurement of
PepI activity against the Pro-pNA substrate.
Reverse transcription-PCR (RT-PCR) was performed as
follows. Three cDNA preparations were first synthesized with
RT (AMV reverse transcriptase, Promega) as described for the
primer extension using 20 pg total RNA isolated from L.
helvetictls after growth of 4 h. The oligonucleotides used in the
first-strand synthesis were P1, 5'-GCCTCTAATTCTTTCACCC-3', P2, 5'-TCGGCAGAGTTGAGCGC-3' and P3, 5'CCAGGAGATAAATCAACGC-3', being complementary to
nucleotides at positions 2431-2449,2011-2027 and 1618-1636,
respectively (Fig. 2). After reverse transcription, the three
cDNA samples were phenol extracted, precipitated and dissolved in 16 p1 H,O. Amplifications by PCR were performed
with 2 pl cDNA samples. The oligonucletide P4, 5'-CATTGCGGGACTCTATCC-3', binding to nucleotides 306-323
(Fig. 2), was used as the upstream primer for the cDNA
obtained with P1. The oligonucleotide P5, 5'-GCTGATGACAAAGCATAATGTG-3', binding to nucleotides 1305-1 326
(Fig. 2) was used as the upstream primer for cDNAs obtained
with P2 or P3. As a control for the absence of contaminating
DNA, 5 pg total RNA was directly amplified by PCR using
oligonucleotide pairs P1 /P4, P2/P5 and P3/P5.
Purification of Pepl. The crude extract of E . coli JM105,
carrying the pepI construct in pKK223-3, was prepared essentially as described by Vesanto e t al. (1995). Briefly, the cells,
grown in 250 ml L-broth, were harvested 5 h after 1 mM IPTG
induction ofpepI expression. After lysing the cells and removal
of the cell debris, the cell-free extract was fractionated by 50 and
80 % (NH&SO, precipitations in two steps, respectively. The
80 YOprecipitate was collected by centrifugation (4OC, 6400 g,
30 min), solubilized in 20 mM Tris/HCl, pH 7.5 and dialysed
first against deionized water (2 x 30 min) and then overnight
against 20 mM Tris/HCl, pH 7.5.
Peptidase activity assays. From liquid cultures, peptidase
activities in L. helvetiem and E. coli were determined as follows :
cells were harvested by centrifugation at 7000g for 15 min,
frozen in liquid nitrogen, thawed, washed once with 50mM
HEPES (pH 7*0), resuspended in one-third of the original
volume of the same buffer, followed by sonication on ice using
an Ultrasonic 2000 sonicator (B. Braun) with intervals of 15 s
until the cells were disrupted. After removal of the cell debris,
peptidase activities were determined according to the method of
El Soda & Desmazeaud (1982) using 16.4 mM L-proline-pnitroanilide (Sigma) as the substrate in 50 mM HEPES (pH 7.0).
The enzyme activity measurements with purified PepI were
performed at 40 "C using Pro-pNA as the substrate in 50 mM
HEPES (pH 7.5). The release ofp-nitroanilide was followed at
410 nm (Perkin Elmer Lambda Bio UV/VIS spectrophotometer). A molar extinction coefficient of 8800 M-' cm-' was
used to calculate the specific enzyme activity as units
(mg protein)-'.
Hydrolysis of non-chromogenic peptides (Sigma; Bachem) was
determined by two alternative methods : the iminopeptidase
activity (proline-liberating) was determined using the modified
method of Troll & Lindsley (1955) as described by Baankreis &
Exterkate (1991). The hydrolysis of peptides not containing
proline as the amino-terminal residue was assayed by measuring
the amount of liberated a-amino termini from hydrolysis of
peptide substrates using the Cd/ninhydrin method as described
by Doi e t al. (1981). The buffer and the incubation temperature
used were 50 mM HEPES (pH 7.5) and 40 "C. All activity
assays were performed with at least two parallel samples. The
protein content was determined by the Bio-Rad protein assay
using BSA as the standard.
Construction of an overexpression system for the L.
helveticus Pep1 in E. coli. The pepI gene was synthesized by
PCR using the primer pair 5'-TTAAGAATTCGTTGTTCATTTTTAAGTAGG-3' and 5'-AAATAAGCTTACAAACGCAGTGAAAGAATG-3'. The 1 kb PCR product, carrying
the structural gene of pepI with its ribosome-binding site, was
ligated as an EcoRI-HindIII-fragment with the pKK223-3
vector and transferred into the E. coli JMl05 host by electroporation. The expression of pepI was characterized by
growing the cells to a density of 50 Klett units (filter 66),
followed by 1 mM IPTG induction and estimation of product
The dialysed sample (10.2 ml) was filtered through a 0.45 pm
membrane filter (Millex HA ; Millipore) and applied to a D E A E
Sepharose FastFlow column (Pharmacia Biotech), equilibrated
with 20 mM Tris/HCl, p H 7.5. Proteins were eluted in a linear
gradient of 0.15-0.4 M NaCl in starting buffer (flow rate 3 ml
min-l, fraction size 5 ml, gradient volume 780 ml), and the
fractions were tested for Pro-pNA-hydrolysing activity. One
active fraction (5 ml) was concentrated 10-fold by ultrafiltration
through a 30 kDa cut-off membrane (Amicon). In the second
purification step, the concentrated fraction (500 pl) was applied
to a gel filtration column (Superdex 75 HR 10/30, Pharmacia)
equilibrated with 25 mM Tris/HCl, p H 7.5, containing 0.15 M
NaC1. Proteins were eluted with the same buffer (flow rate
0.4 ml min-l, fraction size 1 ml, gradient volume 28 ml) and
assayed for Pro-pNA-hydrolysing activity.
Determination of pH and temperature optima. The effect of
pH on PepI activity was determined in the range pH 4-1 0 using
a buffer system containing either boric acid, MES, HEPES or
maleic acid (50 mM each). Reactions were incubated at 37 "C for
5 min and stopped with 0.5 ml 3 0 % (v/v) acetic acid. The
absorbance of the samples was measured at 410nm. The
optimum temperature was estimated by the same method in the
temperature range &70 "C with 50 mM HEPES (pH 7.5).
Inhibition studies. Potential inhibitors were added to final
concentrations of 0.1 and 1 mM. Reaction mixtures with the
purified PepI were preincubated in 50 mM HEPES (pH 7.5) at
4 "C for 30 min. Reactivation was performed by adding the
reactivating agent to the reaction mixture followed by incubation at 4 "C for an additional 10 min. After addition of the
substrate (Pro-pNA), enzyme activity was determined at 40 "C.
Molecular mass determination. The molecular mass of the
native PepI was determined by gel filtration chromatography
(Superdex 75) equilibrated with 25 mM Tris/HCl, pH 7.5,
containing 0.15 M NaC1. The column was calibrated with
catalase (232 kDa), aldolase (158 kDa), albumin (68 kDa),
ovalbumin (43 kDa) and chymotrypsinogen (25 kDa). SDSPAGE was performed with an 11 YO polyacrylamide gel
(Laemmli, 1970). The gel was stained with Coomassie Brilliant
Blue R250. LMW-SDS proteins (Pharmacia) were used as
marker standards.
RESULTS
Cloning of the proline iminopeptidase region from
L. helveticus 5317
T h e homologous counterpart o f t h e L. bzllgarictls pep1
gene (Atlan e t al., 1994) was identified from the chromosomal DNA of L. helveticus 53/7 by Southern
hybridization using a 1.2 k b pep1-specific p r o b e derived
by PCR from L. btllgaricus chromosomal DNA (data n o t
shown). T h e probe hybridized to restriction fragments
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P. V A R M A N E N , T. R A N T A N E N a n d A. P A L V A
H P
E
OW1
E
SP
ORE2
A
pepl
-7
1 kb
-
gtlOlcl23
gtlOldll
gtlOld4
gtlOldj0
p-107
pKTHZlO8
pIzTIi2109
pKTH2110
Fig. 7. Partial restriction map of the L. helveticus 53/7 pepl
region. The positions and orientations of ORF1, ORF2 and pep/
are indicated by arrows. The inserts of the AgtlO clones
gtlOk123, gtlO/clll, gtlOkl4 and gt10k130 and the inserts of
the plasmids pKTH2107, pKTH2108, pKTH2109 and pKTH2110
are shown below. Restriction enzymes: El EcoRI; HI Hindlll; P,
Pstl; 5, Sall. The open bar with a Hindlll site refers to a DNA
segment from AgtlO in pKTH2107.
apart from those detected by a L. helvetictls prolinase
(Varmanen e t al., 1996) probe (data not shown). To clone
the corresponding pep1 gene, a AgtlO-based genomic
library of L. helvetictls 53/7 in E. coli was screened with the
above pep1 probe by plaque hybridization. Four
hybridization-positive clones, gtlO/cl4, gtlO/clll, gtlO/
c123 and gt10/c130 were chosen for further characterization. Phage DNAs of the clones were isolated and
digested with various restriction enzymes and further
analysed
by
Southern
hybridization.
Positive
hybridization signals were obtained with 1.2 and 1.6 kb
EcoRI fragments from c14 and c l l l , respectively, with a
4.5 kb HikdIII fragment of c123 and with a 0.7 kb EcoRIHind111 fragment of c130 (Fig. 1). For sequencing, these
fragments were subcloned with p JDC9 and designated
pKTH2109, pKTH2108, pKTH2107 and pKTH2110,
respectively. A partial restriction enzyme map of the pep1
region in L. helvetictls is shown in Fig. 1.
Nucleotide and amino acid sequence analyses
DNA sequencing of the inserts of pKTH2'108,
pKTH2109 and pKTH2110 and partial sequencing of
that of pKTH2107 revealed a 3240 bp region of the
chromosomal DNA of L. helvetictls carrying three open
reading frames (ORF1, ORF2 and ORF3) of 1332, 651
and 882 bp, respectively (Fig. 2). ORFl starts with the
GTG codon, ORF2 and ORF3 with ATG. All ORFs are
preceded by a putative ribosome-binding site (Fig. 2).
ORFl contains 444 codons and has a coding capacity for
a 50.7 kDa protein. Conserved -35 and -10 regions
(TAGACA-N,,-TACAAT) of a putative promoter were
found 52 and 29 nucleotides upstream of the ORFl start
codon, respectively. The start codon of ORF2 is located
three nucleotides downstream of the stop codon of ORFl.
ORF2 contains 217 codons and has a coding capacity for
a 24.5 kDa protein. The start codon of ORF3 and the stop
codon of ORF2 are separated by eight nucleotides. The
ORF3 contains 294 codons and has a coding capacity for a
33.8 kDa protein. ORF3 is followed by an inverted repeat
structure one nucleotide downstream of two stop codons.
3462
This hairpin with a AG of -50 kJ mol-' consists of a
stem of 9 bp and a loop of 6 bp and is a putative rhoindependent transcription terminator.
The nucleotide and deduced amino acid sequences of
ORF3 were shown to be 64 and 65 % identical with those
of the pep1 gene and its product from L. btl&arictl.r,
respectively. Thus, ORF3 was confirmed to be the
structural pep1 gene of L. helvetiem. The similarity of the
L. helvetictls Pep1 protein with PepIs from L. delbrtleckii
subsp. lactis (Klein e t al., 1994) and L. bz4Lgarictl.r (Atlan e t
al., 1994) and with the L. helvetictls prolinase (Dudley &
Steele, 1994; Varmanen e t al., 1996) was shown to be 75,
75 and 39 YO,respectively.
Analysis of the predicted amino acid sequence of the
ORF1-encoded 50-7 kDa protein revealed the presence of
two ATP/GTP-binding site P-loops also known as
Walker A motifs (Walker e t al., 1982; Higgins, 1992) ; one
in the N-terminal half of the protein (residues 29-36, Fig.
2) and the other in the C-terminal part (residues 263-270,
Fig. 2). In addition, the Walker B motifs (Walker e t al.,
1982; Higgins, 1992), also involved in ATP-binding,
were found in the N- and C-terminal halves (residues
143-147 and 382-386). According to the homology
searches performed, the 50.7 kDa protein showed homology to several members of the family of ABC transporters. Surprisingly, the highest levels of relatedness of
the N- and C-terminal halves of the 50.7 kDa protein were
to different proteins. The highest similarities of the Nterminal half was found with Nod1 (50 YO)
from RhiTobitlm
legtlminosartlm (Evans & Downie, 1986), MalK (44 YO)
from E. coli (Gilson e t al., 1982) and LacK (42 %) from
Agrobacteritlm radiobacter (Williams e t al., 1992). Instead,
the C-terminal half of the 50-7 kDa protein shared the
highest similarities with OppF (43 YO)from Lactococctls
lactis (Tynkkynen e t al., 1993), GlnQ (40 YO)from Bacilltls
stearothermophiltls (Wu & Welker, 1991) and PhnC (37 YO)
from E. coli (Makino e t al., 1991). The similarity between
the N- and C-terminal parts of the 50.7 kDa protein was
38%. Thus, the ORF1-encoded protein seems to be
formed of two different ATP-binding domains fused into
a single polypeptide, which represents an unusual ATPbinding protein type among the prokaryotic ABC transporters. Hydropathy analysis revealed that the 24.5 kDa
protein, encoded by ORF2, contains stretches of hydrophobic amino acids being able to form five transmembrane
helices (data not shown). Thus, the 24.5 kDa protein
represents the transmembrane protein of this putative
ABC transporter.
Expression of pep/ in L. helveticus
Expression of the pep1 gene in L. helvetiem 53/3 was
studied in a pH-controlled culture in Whey broth by
measuring peptidase activity against the L-proline-pnitroanilide substrate and the level of pep1 transcripts as
the function of growth (data not shown). The amount of
proline-specific enzyme activity increased up to the early
stationary phase reaching the highest level 8-10 h after
inoculation (Klett,, values 532-530). During the rest of
the growth analysed, the activity against the Pro-pNA
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PepI-ABC transporter operon from L. heluetictls
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Fig. 2. The nucleotide and deduced amino acid sequences of the ABC transporter-pep/ region. ORFl and ORF2 refer t o the
ATP-binding protein and the transmembrane protein of the ABC transpoi-ter, respectively.The predicted -10 and -35
regions of the promoter are underlined. The 5' end of the common transcript of the ABC transporter genes and pep/,
found by primer extension, is marked with a vertical arrow. RBS, predicted ribosome-binding site. The deduced
transcription terminator downstream of ORF3 (pep/) is shown with dotted arrows. The Walker motifs, A (WA) and B
(WB), characteristicof many nucleotide-binding proteins, are underlined. The conserved residues of the active site region
of prolyl oligopeptidases are in bold and marked with asterisks.
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3463
P. V A R M A N E N , T. R A N T A N E N a n d A. P i i L V A
mRNA
cDNA 1
cDNA 2
cDNA 3
ORFI
ORF2 pep1
PCR 1
PCR 2
PCR 3
fig. 4. SDS-PAGE analysis o f Pep1 expression and purification in
E. coli. Lanes: 1, molecular mass markers; 2 and 3, crude
extracts isolated 5 h after IPTG-induction from E. coli JM105
with the pKK223-3 vector, and from E. coli JM105 with pKK2233 carrying the pep/ gene, respectively; 4, purified Pep1 (2 pg).
Gel electrophoresis was with an 11 % (w/v) polyacrylamide gel
followed by staining with Coomassie Brilliant Blue R-250.
Fig. 3. Identification of ORF1-, ORF2- and pepl-specific mRNA
using RT-PCR. (a) Schematic presentation of the RT-PCR
analysis of ORFl-, ORF2- and pep/-specific mRNA. The putative
mRNA is indicated by the dotted arrow. cDNA1, cDNA2 and
cDNA3 refer t o the reverse transcription products obtained with
primers P1, P2 and P3, respectively. PCR1, PCR2 and PCR3 refer
t o the amplification products when using cDNA1, cDNA2 and
cDNA3 as the templates for the primer pairs Pl/P5, P2/P5 and
P3/P4, respectively. The sequences of the primers used are
described in Methods. (b) Agarose gel electrophoresis of RT-PCR
and control PCR samples. Lanes: 1, part of Pstl-digested A-DNA;
2, 4, and 6,PCR-amplification products from cDNA3, cDNA2 and
cDNA1, respectively; 3, 5, and 7, the control PCR reactions o f L.
helveticus total RNA with primer pairs P3/P4, P2/P5 and Pl/P5,
respectively. An equal volume from each PCR reaction was
applied.
the total RNA using the minus-strand primers P1 , P2 and
P3, designed to detect either apepl (P1)- or an ORF2 (P2,
P3)-specific region. The resulting cDNAs were then
amplified by PCR using the plus-strand primers P4 and
P5, specific to O RFl and ORF2 (Fig. 3a), respectively. All
three RT-PCR reactions performed resulted in a PCR
product, PCRl, PCR2 or PCR3, with the expected sizes of
1140,720 or 1330 bp, respectively (Fig. 3), thus indicating
the presence of a common transcript for the entire operon.
The absence of contaminating genomic DNA in the RNA
samples used in RT-PCR was confirmed by the absence of
PCR products when the same total RNA was directly used
as the template in PCR with the same primers as used for
RT-PCR (Fig. 3b).
substrate remained relatively constant. To measure the
steady-state level ofpep1 transcripts by Northern and dot
blots, total RNAs were isolated from L.helveticzrs cell
samples taken 4, 6, 8, 12 and 16 h after inoculation,
corresponding to Klett66 values of 120,427, 532, 530 and
485, respectively. Surprisingly, no clear-cut hybridization
signals could be detected either with the DIG- or 32Plabelled pep1 probes apart from a weak and diffuse band
observed with the 4 h sample (data not shown).
The primer-extension mapping of the 5’ end of ABC
transporter-pep1 mRNA was performed with different
primers, designed downstream of the possible transcription start sites at the ABC transporter-pep1 operon, by
using the same total RNA samples as in the Northern
analyses. A primer-extension signal was obtained only
with the oligonucleotide complementary to the 5’ end of
ORFl (nucleotides 317-337, Fig. 2). The transcription
start site was thus localized 16 nucleotides upstream of the
start codon of ORFl (data not shown). These results
further confirmed that the pep1 gene is co-transcribed with
the ABC transporter genes under the growth conditions
used.
mRNA analyses
Overexpression and purification of Pep1
Repeated attempts to determine the size of the ORF1-,
ORF2- and pepl-specific mRNA(s) by Northern blot,
using their respective probes, failed. Only when very
large amounts of total RNA (100 pg) were used, a faint
signal was detected from the RNA samples isolated from
the cells grown 4 h after inoculation (data not shown).
However, the signal was very diffuse, preventing the
determination of transcript sizes. Similar results were
obtained with both probes. Instead, ORF1-, ORF2- and
pepl-specific mRNAs could be detected using RT-PCR
(Fig. 3). The first-strand syntheses were performed with
The overexpression construct was created by joining pep1
as a 1.0 kb PCR fragment, amplified from the chromosomal DNA of L. helvetictls, with the pKK223-3 vector
(resulting in pKTH2111) followed by transformation of
E. coli. After IPTG-induction, the amount and activity
level of PepI increased in E. d i throughout the entire
growth phase of 5 h as judged by SDS-PAGE (Fig. 4) and
by determination of enzyme activity. The yield of PepI
was approximately 20% of the total cellular proteins.
Using two chromatographic steps, PepI was purified to
homogeneity. The results of the purification scheme are
3464
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pepl-ABC transporter operon from L. helveticm
Table 1. Purification of the L. helveticus 53/7 Pep1 from E. coli JM105
Purification step
Total protein
(mg)
Total activity
(units)*
Specific activity
[units (mg protein)-']t
Purification
factor (-fold)
Yield
activity (%)
Cell-free extract
80 % (NH,),SO,
DEAE-Sepharose
Superdex 75
72.9
62.0
15.3
2.8
3005.1
1923.2
1080.0
329.6
41.20
31-02
70.60
117.70
1.0
0.8
1-7
2.8
100
64
36
11
* One unit corresponds to the release of 1 pmol p-nitroaniline min-'.
j- Specific activity was calculated as units (mg protein)-', where a molar extinction coefficient of 8800 M cm-' was used for p-nitroaniline.
Temperature ("C)
40
60 70
100-
s
n
80 -
v
>r
.-+
mg40a
0I
4
I
I
8
6
I
I
10 11
PH
Fig. 5. Effect o f temperature and pH o n the proline
iminopeptidase acitivity. Open and filled circles refer t o Pep1
activity profiles as a function o f temperature and pH,
respectively.
summarized in Table 1. The cell-free extract was first
fractionated with ammonium sulphate precipitation in
two saturation steps (50 and 80 YO).
In the first chromatographic step, the dialysed and filtered sample was applied
onto DEAE-Sepharose anion-exchange column and
eluted by a 0.15-0.4 M NaCl gradient. Pro-pNAhydrolysing activity was eluted at 0.26 M NaC1. During
the second step, the active fractions were pooled, concentrated and purified by gel filtration (Superdex 75). This
yielded a preparation with PepI activity that was determined to be electrophoretically pure (Fig. 4).
Characterization of the purified Pep1 protein
The L. helveticas PepI had a pH optimum at pH 7.5 and a
sharp temperature optimum at 40 OC (Fig. 5). At pH values
greater than 7.5, the activity of PepI sharply decreased.
Table 2 shows the hydrolytic activity of PepI against 31
peptides. Of the dipeptides tested, PepI hydrolysed only
peptides with proline at the first position. Of the longer
peptides tested only Pro-Gly-Gly was very weakly hydrolysed. PepI was completely inhibited by Zn2+, Cu2+ and
Cd2+at concentrations of 0.1 and 1 mM (data not shown).
No significant inhibition was found with Mg2+, Ca2+,
Table 2. Activity of the L. helveticus prolineiminopeptidase against different substrates
......................................................................................................................................................... .
Hydrolysis of peptides was analysed using Cd/ninhydrin and
proline-specific ninhydrin assays (see Methods). Activity against
Pro-Ala was arbitrarily taken as 100%. Values for the following
substrates were under the detection limit : Ala-Phe, Ala-Val, ArgAsp, Ile-Gln, Ile-Val, Leu-Gly, Leu-Leu, Leu-Pro, Lys- Ala, LysGlu, Lys-Lys, Lys-Tyr, Met- Ala, Phe-Gly, Phe-Leu, Val-Gly,
Val-Leu, Leu-Leu-Leu, Ile-Pro-Ile, Thr-Lys-Pro, Val-Gly-Gly,
Val-Pro-Leu, Pro-Phe-Gly-Lys, Pro-His-Pro-Phe-His-Phe-PheVal-Tyr-Lys.
I
Substrate
Pro-Ala
Pro-Ile
Pro-Leu
Pro-Phe
Pro-Val
Pro-Pro
Pro-G1y-G1y
Relative activity (%)
100
21
28
31
38
9
1
Co2+ and Mn2+ at the same concentrations. The PepI
activity was also strongly inhibited by 0.1 mM and 1 mM
3,4-dichloroisocoumarin and p-hydroxymercurybenzoate
(pHMB), whereas PMSF had no significant effect (data
not shown). The PepI activity inhibited by pHMB was
fully restored by DTT (data not shown). The affinity of
the L. helvetiem PepI for the Pro-pNA substrate (0.22.0 mM final concentration) was studied by measuring the
initial rate of the reaction with a continuous assay in
thermostatted cuvettes at 40 "C and pH 7.5. A Lineweaver-Burk plot of the rate of hydrolysis indicates that
the K , and Vmax
values for Pro-pNA are 0.84 mM and
345 mmol min-' mg-l, respectively, indicating a turnover
number of approximately 135000 s-', By gel filtration,
the molecular mass for the native enzyme was calculated
to be around 53 kDa, indicating that the enzyme probably
consists of two subunits.
DISCUSSION
In this work, we have cloned the gene encoding proline
iminopeptidase in L. helveticzls with DNA hybridization
using the pep1 gene from L. btllgarictls (Atlan e t al., 1994)
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3465
P. V A R M A N E N , T. R A N T A N E N a n d A. P A L V A
as the probe. Although the PepI proteins of L. helvetictls,
L. bzllgarictls (Atlan e t al., 1994) and L. delbrzleckii subsp.
lactis (Klein e t al., 1994) were revealed to share a relatively
high amino acid sequence similarity (75%), the genetic
organization of the L. helveticaspep1 locus turned out to be
totally different from that of L. btllgarictls and L. delbrtleckii
subsp. lactis. The L. helvetictlspepl is preceded by two open
reading frames (ORF1 and ORF2), which encode a
putative ABC transporter and seem to form an operon
with the pep1 gene. A well-conserved promoter is found
upstream of ORFl and the only putative transcription
terminator locates downstream of pep1 (Fig. 2). The pep1
gene is, however, preceded by a putative weak promoter
with a moderately conserved - 35/ - 10 region (TTAACT-N,,-TAATAT, position 2128, Fig. 2). Nevertheless,
in the 5' end mapping by primer extension, using the total
RNA samples from different growth phases, a signal
could be detected neither with a primer designed to bind
downstream of this putative pep1 promoter (position
2319-2337, Fig. 2) nor with a primer binding downstream
of the start site of ORF2 (position 1698-1715, Fig. 2).
Instead, the only primer-extension product obtained
confirmed the location of the predicted promoter region
preceding ORFl . Furthermore, the pep1 gene, cloned
under its own putative promoter, was not expressed in E.
coli (data not shown). The operon structure was further
confirmed by the results of RT-PCR studies, which
revealed that at least part of mRNAs derived from the
ORFl promoter are transcribed through the pep1 gene
(Fig. 3). Unfortunately, the mRNA size determinations
by Northern blots were unsuccessful, due to an extremely
low concentration of ORF1-, ORF2- and pepl-specific
transcript(s) in total RNA. According to the results
shown, wc can, howcvcr, conclude that in the growth
conditions used the L. belveticzlspepl gene is expressed as
a polycistronic transcriptional unit with the two upstream
ABC transporter genes. In L. delbrtleckii subsp. lactis and
subsp. bzllgariczls, the pepl gene is most probably monocistronic, since it is flanked by divergent ORFs encoding
proteins not identical with the L. helvetictls ABC transporter and the ORFs downstream of pep1 (this study;
Atlan e t al., 1994; Klein e t al., 1994).
Analysis of the substrate specificity of the purified PepI
revealed that the L. helveticas 53/7 PepI is a prolinespecific peptidase with a narrow specificity, similar to that
reported for the PepIs from L. btllgarictls (Gilbert et al.,
1994) and Lactobacilltls casei (Habibi-Najafi & Lee, 1995).
The L. btllgariczls PepI has also been reported to have
partial or weak hydrolysing activity against several
dipeptides not containing a proline residue (Gilbert e t al. ,
1994). A few of these substrates (Ala-Phe, Leu-Gly) were
also tested with our PepI, which, however, did not
hydrolyse them at a detectable level. It has also been
reported that the L. bzllgarictls PepI is equally active
against Pro-Ala and Pro-Gly-Gly (Gilbert e t al., 1994 ),
whereas a 100-fold difference was observed with the
method used in our study (Table 2). It is not known
whether these differences are due to real differencies in the
enzymic properties of PepIs from L. helvetictls and L.
btllgariczls (Gilbert e t al., 1994), or whether they are due to
3466
methodological differences in the measurements of enzyme activities. The effect of various chemicals against
PepI activity was similar to that reported for the L.
btllgarictls PepI. The PepI activity is strongly inhibited by
3,4-dichloroisocoumarin and pHMB. The serine protease
nature of the L. helvetictls PepI is also confirmed by the
presence of the active site region (GQSWGG; at position
103-108, Fig. 2), typical for prolyl oligopeptidases
(Rawlings e t al., 1991) and identical with that of the two
L. delbrtleckii PepIs. It is noteworthy that PMSF had only
a minor effect on PepI activity. The inhibition profiles
with divalent cations are similar for the L. helveticzls and L.
btllgarictls PepIs (this study; Gilbert e t al., 1994) whereas
the L. casei PepI was not strongly inhibited by Cu2+and
Zn2+(Habibi-Najafi & Lee, 1995). The temperature and
pH optima for L. helvetictls PepI (40 "C and pH 7.5) are,
instead, identical to those reported for the L. casei Pep1
(Habibi-Najafi & Lee, 1995), whereas the optimum
pH for the L. btllgarictls PepI has been shown to be
between pH 6.0-7.0, and the enzyme was reported to be
unstable at temperatures above 35 "C. The molecular
mass determination of the native L. helveticzls PepI
indicates that the enzyme probably consists of two
subunits. A monomeric and a trimeric form has been
reported for PepIs of L. casei and L. btllgariczls (HabibiNajafi & Lee, 1995; Gilbert e t al., 1994), respectively. To
our knowledge, the turnover number of the L. helveticzls
PepI with the Pro-pNA substrate (135000 s-'), and that
deduced for L. btllgaricus PepI, substantially exceed all
those reported for Lactobacilltls peptidases so far. The
extremely high turnover number of PepI may also explain
the need for a very small amount of pep1 transcripts. The
low expression of pep1 is also supported by its codon
usage which suggests a low level of translation. Since we
were unable to distinguish the PepI activity from that of
prolinase (Varmanen e t al., 1996) in L. helveticzls lysates,
the ratio and role of these two proline-specific peptidases
under the growth conditions used remains to be
elucidated.
The ORFl -encoded 50.7 kDa protein shared significant
homology with several members of the family of ABC
transporters. If ORFl does indeed encode such a transporter, then its two putative ATP-binding domains are
apparently evolved through an ancient gene duplication
or fusion of adjacent genes, thus representing an exception
to the more common organization of prokaryotic ABC
transporters, where the individual domains are expressed
as separate polypeptides (Higgins, 1992). In prokaryotes,
only four other ATP-binding proteins from the carbohydrate metabolism of E. coli could be found to possess a
similar double ATP-binding domain structure to that of
the 50.7 kDa protein. These were the transporters of
ribose (RbsA) (Buckel e t al., 1986), methylgalactoside
(MglA) (Hogg e t al., 1991), L-arabinose (AraG) (Scripture
e t al., 1987) and D-xylose (XylG) (Sofia e t al., 1994) of
which the RbsA shares the highest similarity with the
50.7 kDa protein (37 Y O ) ;these all belong to bindingprotein-dependent transport systems. The periplasmic
binding protein has been considered to be a unique
feature of all bacterial ABC transporters involved in the
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PePI-ABC transporter operon from L. helveticus
import of substrates (Fath & Kolter, 1993). However, a
gene encoding such a substrate-binding protein was not
present in our operon, suggesting that, despite the
similarity of the genetic organization of these five ATPbinding proteins, the putative ABC transporter identified
in our study may have an export function. This is also
supported by the fact that the signature present in all
transmembrane proteins of import-specific ABC transporter systems (Saurin e t al., 1994) could not be found in
the 24.5 kDa putative transmembrane protein encoded by
ORF2. Further support for this contention is provided by
the observation that the 50.7 kDa protein shares the
highest homology with NodI, a transport protein
involved in nodulation and probably in the export of a
modified /3-1,4-linked N-acetylglucosamine oligosaccharide (Evans & Downie, 1986).
It is unknown whether the common operon structure of
the putative ABC transporter genes and pep1 also indicates
an interaction of the encoded functions. The structural
relationship may be of significance, since the majority
of the peptidase genes characterized so far from LAB
seem to form a monocistronic transcriptional unit.
Interestingly, the L. helveticus prolinase gene is also
connected to a putative ABC exporter via the partially
overlapping promoter regions (Varmanen e t al., 1996).
ACKNOWLEDGEMENTS
This work was supported by the Ministry of Agriculture and
Forestry of Finland. The authors are grateful t o Mrs Anneli
Virta for the running of the ALF Sequencer and Ms Jaana
Jalava for technical assistance.
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Received 15 May 1996; revised 12 August 1996; accepted 16 August
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