Folia Microbiol. 51 (6), 535–541 (2006)
Analysis of the Presence of prtR Proteinase Gene
in Natural Isolates of Lactobacillus rhamnosus
I. PAŠTAR, D. FIRA, I. STRAHINIĆ, K. KRSTIĆ, J. BEGOVIĆ, L. TOPISIROVIĆ*, G. JOVANOVIĆ
Institute of Molecular Genetics and Genetic Engineering, 11010 Belgrade, Serbia and Montenegro
Received 7 February 2006
Revised version 16 May 2006
ABSTRACT. The region of the prtR gene coding for the active site of PrtR proteinase was detected in
natural isolates of lactobacilli, previously determined as Lactobacillus rhamnosus. This region was present
in all L. rhamnosus strains with proteolytic activity. The PCR primers used were constructed on the basis of
the sequence of the catalytic domain of the prtR proteinase gene. These primers generated in colony-PCR
procedure specific 611-bp product with DNA from natural isolates of L. rhamnosus. No PCR amplifications
using these primers were obtained for closely related bacteria of genus Lactobacillus, regardless of their pro-
teolytic activity. In addition, these primers could be used singly or in multiplex PCR together with the Lacto-
bacillus genus-specific primers. Compared with the other proteinases within the genus Lactobacillus (PrtP,
PrtB and PrtH) which retained the activity in cell-free proteinase extracts, PrtR proteinase showed proteoly-
tic activity only under in vivo conditions (whole cells of the producing strains).
cell-wall-bound extracellular proteinase(s) LAB lactic acid bacteria
Among LAB, genus Lactobacillus represents the largest group of microorganisms. Lactobacilli grow
under anaerobic conditions or under reduced oxygen level, predominantly at mesophilic to thermophilic tem-
perature range. Because of the complex nutritional requirements, they grow in all habitats rich in sacchari-
des, vitamins, peptides and free amino acids (Kandler and Weiss 1986). Lactobacilli are found in raw milk,
fermented milk products, meat and meat products, fermented vegetables, plant surfaces, fruits and silage,
and human and other mammal mucosa of either gastrointestinal or urogenital tract (see, e.g. Švec et al. 2005).
Industrially relevant species, e.g., Lactobacillus rhamnosus, L. paracasei, L. helveticus and L. delbrueckii
subsp. bulgaricus, are essential in both fundamental and applied research (see, e.g., Bujňáková et al. 2004;
Strompfová et al. 2004; Fajdiga et al. 2006; Horošová et al. 2006).
LAB have multiple amino acid auxotrophies. Some of them synthesize CEPs responsible for proteo-
lysis of the extracellular proteins into oligopeptides (Kojić et al. 1991; Kok and de Vos 1994; Kunji et al.
1996; Mierau et al. 1997; Paštar et al. 2003). Then, following the transport into the cell, different peptidases
hydrolyze oligopeptides to free amino acids. Four distinct types of genes encoding CEPs, prtP, prtB, prtH
and prtR, have been cloned and sequenced from either dairy or human vaginal LAB (Pastar et al. 2003; Sie-
zen 1999). The prtP gene has been cloned and sequenced from different Lactococcus lactis strains and from
L. paracasei NCDO 151 encoding proteinases with >95 % amino-acid sequence identity (Kunji et al. 1996).
The prtP gene is always accompanied by closely linked and the divergently oriented prtM gene encoding the
PrtP maturation protein (PrtM; Kunji et al. 1996; Miladinov et al. 2001). In thermophilic lactobacilli, genes
encoding PrtB and PrtH proteinases have been sequenced from strains L. delbrueckii subsp. bulgaricus
NCDO 1489 and L. helveticus CNRZ32, respectively (Gilbert et al. 1996; Pederson et al. 1999). Recently,
a novel type of CEP gene, prtR, from the human vaginal isolate L. rhamnosus BGT10 was identified, cloned
and sequenced (Pastar et al. 2003). These four types of CEPs belong to a superfamily of subtilisin-like serine
proteinases (also known as subtilases; Siezen and Leunissen 1997).
Comparison of CEPs showed that a number of different functional domains exist within the protein-
ase molecule (Paštar et al. 2003; Siezen 1999). Starting from the N-terminus, CEPs consist of the pre-pro
domain (PP-domain), the catalytic serine proteinase domain (PR-domain) that comprises the sequences homo-
logous to subtilases and an internal insert I-domain (with the exception of the PrtR), the A-domain, the
*Corresponding author; e-mail: email@example.com .
536 I. PASTAR et al. Vol. 51
B-domain, a helix spacer domain (H-domain) present in PrtP and PrtH, the cell-wall spacer (W-domain) and
a typical cell-wall anchor (AN-domain) present in PrtP and PrtR.
We designed a pair of primers on the highly conserved sequences specific for the catalytic PR-do-
main of L. rhamnosus PrtR proteinase. These primers were used, singly or in a multiplex PCR, for the
screening of a collection of natural isolates of L. rhamnosus showing proteolytic activity. Besides that, we
have also examined the activity of cell-free proteinase extracts obtained from the strains of lactobacilli that
produce different extracellular proteinases.
MATERIALS AND METHODS
Bacterial strains, media and culture conditions. The bacterial strains used are listed in Table I.
Human vaginal Lactobacillus strains were isolated from the vagina of healthy women, while the strains
BGRI7, BGZL7, BGZL18, BGZL19 and BGZL20 were natural isolates from soft home-made cheese manu-
factured in a village located at the Zlatar mountain (Serbia & Montenegro); the rest of the strains were
obtained from different laboratory collections. The strains were isolated by using standard microbiological
procedures and identified by using the API 50 CHL system (bio-Mérieux, France). The strains were generally
grown and maintained in MRS broth (Merck, Germany) at 30 °C (L. paracasei subsp. paracasei) or at 37 °C
(L. rhamnosus, L. helveticus and L. delbrueckii subsp. bulgaricus). Agar plates were prepared by addition of
agar (1.5 %, W/V) to MRS broth. For the test of proteolytic activity, the cells were grown on milk–citrate
agar (MCA) plates with 4.4 % reconstituted non-fat skim milk (RSM), 0.5 % glucose, 0.8 % trisodium citr-
ate, 0.1 % yeast extract and 1.5 % agar (all W/V in water).
Table I. Bacterial strains used
Strain Relevant characteristic Reference
human vaginal isolate, proteinase positive
natural isolate from soft homemade cheese,
natural isolate from soft homemade cheese, prtP +
yogurt, prtB +
intestinal isolate, prtH +
Kojic et al. 1991 L. paracasei subsp. paracasei BGHN14
L. delbrueckii subsp. bulgaricus BGPF1 Fira et al. 2001
Banina et al. 1998a L. helveticus BGRA43
aThis strain was formerly known as L. acidophilus but the extensive analysis (including SDS-PAGE electrophoresis, RAPD and pro-
bing for the prt gene) classified this strain as L. helveticus (unpublished results).
Proteinase activity assay. Proteolytic activities of Lactobacillus strains were assayed according to
Kojić et al. 1991, 1995). For enzyme assays, the strains were grown on MCA plates for 2 d at 30 °C (L. para-
casei ssp. paracasei) or at 37 °C (L. rhamnosus, L. helveticus and L. delbrueckii subsp. bulgaricus) prior to
cell collection. Collected fresh cells (10 mg, cell concentration ≈ 10/pL, i.e. 1010 cells per mL) were resus-
2006 PRESENCE OF prtR PROTEINASE GENE IN NATURAL ISOLATES OF L. rhamnosus 537
pended in 100 mmol/L sodium phosphate buffer (pH 6.5). The cell suspension was mixed with substrate
dissolved in the same buffer at a 1 : 1 (V/V) ratio. As substrate, the β-casein fraction (Sigma, Germany) was
used. After a 3-h incubation at 30 °C cells were pelleted by centrifugation (12 000 g, 5 min), the clear super-
natant fluid was taken off and samples were prepared for SDS-PAGE. To obtain the crude proteinase extract
the cells were extracted twice in 100 mmol/L sodium phosphate buffer (pH 6.5) by centrifugation and extracts
were collected. Protein concentration was measured according to Lowry. Substrate (5 mg/mL) was mixed
with crude extracts in a 1 : 1 (V/V) ratio and incubated at optimum temperatures for each strain. After incu-
bation, samples from the reaction mixture were analyzed by using SDS-PAGE.
Samples for SDS-PAGE were prepared by heating (2 min, 100 °C) with an equal volume of
125 mmol/L Tris-HCl (pH 6.8) containing (in %) SDS 4 (W/V), glycerol 20 (W/V), 2-mercaptoethanol
10 (V/V), and bromophenol blue 0.07 (W/V). The products of hydrolysis were analyzed by SDS-PAGE.
Samples were loaded on 15 % (W/V) acrylamide gel; gels were run on vertical slab electrophoresis cells for
20 h at 10 mA constant current, stained with Coomassie Brilliant Blue R250 (Serva, Germany) and de-
stained in a mixture of methanol (20 %) and acetic acid (7 %) in water.
Specific primers and DNA amplification. Lactobacillus-specific primers P1 16S and P2 16S (Tm =
54 °C), were designed to recognize the conserved region of the 16S rRNA gene (Table II). Two specific pri-
mers (synthesized by Genosys–Sigma), prti2 and PRT1592R (Tm = 58 °C), were designed to target the con-
served region surrounding the PrtR active-site residues encoded by the L. rhamnosus proteinase gene prtR.
Table II. Sequences and positions of the primers used
Primer Locus Position Reference sequence Length, bp Sequence (5´→ 3´ )
GGA ATC TTC CAC AAT GGA CG
TGA CGG GCG GTG TGT ACA AG
CAA CAC CGG GAC CAC GGT G
TCG TTG GAT CAA TCG CCG TGG
Primers were used for proteinase gene detection employing as a template chromosomal DNA isolated from
the lactobacilli (Table I), or by direct DNA amplification (colony PCR). Chromosomal DNA from Lacto-
bacillus strains was isolated according to Hopwood et al. (1985). Colony PCR was performed as a direct
DNA amplification of whole bacterial cells grown in Lactobacillus-selective media (MRS), omitting DNA
extraction. Briefly, fresh bacterial colonies grown on MRS agar were picked with the sterile tip (Gilson P20)
and the cells adhering to the tip were dispersed in 0.5-mL PCR tubes containing the complete PCR reaction
mix. DNA amplifications were done singly or in a multiplex PCR with a GeneAmp PCR System 2700 ther-
mal cycler (Applied Biosystems, USA) by using Taq polymerase (Pharmacia, Austria). All PCR amplifica-
tions were done in tubes containing 25 µL of the complete reaction mixture (Taq buffer 1×, Taq polymerase
1 U, MgCl2 1.5 mmol/L, dNTPs 200 µmol/L each nucleotide, primers 1.5 µmol/L each) under the follow-
ing thermal cycling conditions: 1 cycle (5 min at 94 °C), then 30 cycles (1 min at 94 °C, 1 min at 58 °C
(prti2/PRT1592R) or 1 min at 50 °C in multiplex PCR with combination prti2/PRT1592R + P1 16S/P2 16S
and 1 min at 72 °C), and the last cycle (5 min at 72 °C). Generally, each reaction was analyzed by electro-
phoresis on 1 % agarose gel run at 7 V/cm.
RESULTS AND DISCUSSION
Proteinase of L. rhamnosus. The proteolytic activity and distribution of the ptrR gene in L. rham-
nosus strains (Table I) was determined. For all strains tested, efficient β-casein hydrolysis was obtained at
pH 6.5 (shown only for BGT51-K, BGT54-K, BGT389, BGT391 and BGT394 strains; Fig. 1).
We also examined all strains by multiplex PCR employing the Lactobacillus genus-specific 16S rRNA
primers, P1 16S and P2 16S and the L. rhamnosus prtR gene specific primers, prti2 and PRT1592R (see
Table II). As prtR codes for PrtR proteinase, which does not contain the insert I-domain otherwise present in
the catalytic PR-domain of PrtP, PrtH and PrtB CEPs, the prtR-specific primers were constructed to target
the conserved catalytic region (PR-domain) framing the sequence containing in other proteinases the I-domain.
As a template isolated DNA or whole bacterial cells taken from colony were used. Since the results were the
same we carried out the same examination as colony PCR. All 20 strains that efficiently degraded β-casein
538 I. PASTAR et al. Vol. 51
at pH 6.5 contained a 1048-bp band specific for 16S rRNA and a 611-bp band specific for the prtR gene
(results presented for 12 of 20 strains; Fig. 2). The control Lactobacillus strains (that produce other types of
proteinases, L. paracasei subsp. paracasei BGHN14, L. delbrueckii subsp. bulgaricus BGPF1 and L. helve-
ticus BGRA43) displayed only the 16S rRNA-specific band
and did not contain the prtR gene (data not shown). In addi-
tion, no prtR-specific DNA amplification was obtained with
other chosen natural isolates from different species of lacto-
These results suggested that primers designed to detect
the region of the prtR gene encoding the fragment of the PR-
domain are specific for L. rhamnosus proteinase since they
generated under the given PCR conditions a single 611-bp
band (see Materials and Methods). These primers can be used
for direct DNA amplification (colony PCR) singly or in a mul-
tiplex PCR. It should be noted here that all proteinase-posi-
tive strains carrying the prtR gene were previously identified
by using API 50 CHL system as L. rhamnosus.
Release of proteinases from the cell surface. In order to
confirm the release of different CEPs from the cell surface, we
determined the activity of cell-free proteinase extract (Fig. 3).
Efficient hydrolysis of the substrate was detected with strains
L. paracasei subsp. paracasei HN14, L. delbrueckii subsp.
bulgaricus PF1 and L. helveticus BGRA43 containing PrtP,
PrtB and PrtH proteinases, respectively. The crude protein-
ase extract from the strain L. rhamnosus EN1, however, did
not show significant hydrolysis even after a 4-h incubation
(Fig. 3D). The active extract of PrtR proteinase from the
strain EN1 and other strains from that species could not be
obtained after repeated procedures for isolation, suggesting
that, in comparison with the other CEPs, PrtR proteinase does
not have the same mechanism of release from the cell sur-
Fig. 2. Amplification products after multiplex PCR of L. rhamnosus natural isolates (lanes 1–12) proteolytically
active at pH 6.5 (all carry the prtR gene); pairs of primers used: Lactobacillus genus-specific – 16S rRNA (1048 bp),
L. rhamnosus prtR-specific (611 bp); 1 – BGT266, 2 – BGT390, 3 – BGT391, 4 – BGT392, 5 – BGT394, 6 –
BGT805, 7 – BGT54-K, 8 – BGT43, 9 – BGT110, 10 – BGT393, 11 – BGZL19, 12 – BGT51-K; L – 1 kb plus DNA
Fig. 1. Hydrolysis of β-casein at pH 6.5 by
the whole cells of natural isolates of L. rham-
nosus; S – starting substrate (β-casein), 1 –
BGT51-K, 2 – BGT54-K, 3 – BGT389, 4 –
BGT391, 5 – BGT394.
2006 PRESENCE OF prtR PROTEINASE GENE IN NATURAL ISOLATES OF L. rhamnosus 539
Screening of the presence of the prtR proteinase gene in natural isolates of lactobacilli showed that
this gene was present only in strains of L. rhamnosus. A comparative PCR analysis of the prtR gene region
encoding the part of PrtR subtilisin-like serine proteinase catalytic domain in L. rhamnosus and in other
available Lactobacillus species revealed a sufficient difference among them that could be used to identify
the DNA sequence unique to L. rhamnosus proteinase. According to this, different types of extracellular pro-
teinases may be present in different species of the Lactobacillus genus, even though, except for some pre-
liminary results (Banina et al. 1998; Fira et al. 2001), the species specificity of the prtH gene for L. helveticus
and the prtB gene for L. delbrueckii subsp. bulgaricus strains was not studied extensively to date. We tested
fourteen L. rhamnosus strains for proteolytic activity of the whole cells (Paštar et al. 2003). All strains
(Table I) efficiently degraded β-casein at pH 6.5 and carried the prtR gene, suggesting that this proteinase gene
could be characteristic for the L. rhamnosus species. Furthermore, Southern blot analysis showed that under
high-stringency conditions (65 °C) the probe from the catalytic domain of L. rhamnosus BGT10 prtR hybri-
dized to a 2.3-kb XbaI DNA fragment in all tested strains; this band is specific for the prtR gene since this
region lacks the sequence that encodes the I-domain (Pastar et al. 2003). The results presented in this work
also suggest that the presence of the prtR proteinase gene in strains of L. rhamnosus could be species-de-
Fig. 3. Activity of cell-free proteinase extracts of lactobacilli. A: Lactobacillus paracasei subsp. paracasei HN14; B: L. delbrueckii subsp.
bulgaricus PF1; C: L. helveticus BGRA43; D: L. rhamnosus EN1. The samples were taken from reaction mixture for SDS-PAGE
analysis at following intervals: A, B, C: lane 1 – 15 min, 2 – 30 min; 3 – 1 h; 4 – 2 h; 5 – 3 h; 6 – 4 h; D: lane 1 – 3 h, 2 – 4 h. Total
protein concentrations were 2 mg/mL for all four crude proteinase extracts; S – starting substrate (β-casein).
Regarding the results published to date, all proteinase-positive strains of L. paracasei should carry
the prtP gene encoding the PrtP proteinase and, in close proximity, the divergently oriented prtM gene res-
ponsible for production of the PrtP maturation protein PrtM (Kok and de Vos 1994; Kunji et al. 1996). How-
ever, the 95 % identical prtP gene and the homologous prtM gene are characteristic for L. lactis strains
(Kunji et al. 1996); however, in lactococci they are located on plasmids of different size. It has been also
shown that the release of the active PrtP proteinase from the cell envelope is a process accomplished by
540 I. PASTAR et al. Vol. 51 Download full-text
autodigestion of the enzyme at the C-terminus in the abscence of Ca2+ ions. In comparison with PrtP, PrtB
and PrtH, proteinase PrtR cannot be released from the cell surface by the standard experimental procedure
successfully used for other CEPs. The release by autodigestion in the absence of Ca2+ ions that was confir-
med for PrtP proteinase (Kok and de Vos 1994; Kunji et al. 1996) has been obviously found in strains pro-
ducing PrtB and PrtH proteinase. This is not the case of the PrtR proteinase from L. rhamnosus: different
structure of the enzyme, particularly the absence of the insert I-domain within the catalytic domain of the pro-
teinase (Pastar et al. 2003), may contribute to different release process.
This work was supported by the Ministry of Science and Environmental Protection grant no. 1442.
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