Journal of Bacteriology, August 2001, p. 4668-4673, Vol. 183, No. 15
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.15.4668-4673.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Transcriptional Analysis and Regulation of
Expression of the ScrFI Restriction-Modification
System of Lactococcus lactis subsp.
cremoris UC503
Derek
Butler and
Gerald F.
Fitzgerald*
National Food Biotechnology Centre
and Department of Microbiology,
University College Cork, Cork, Ireland
Received 11 January 2001/Accepted 2 May 2001
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ABSTRACT |
ScrFI is a type II restriction-modification system
from Lactococcus lactis which recognizes
the nucleotide sequence 5'-CC
NGG-3', cleaving at the
point indicated by the arrow, and it comprises an endonuclease gene
that is flanked on either side by genes encoding two
5-methylcytosine methylases. An open reading frame
(orfX) of unknown function is located immediately
upstream of these genes. In this study Northern analysis was performed,
and it revealed that orfX, scrFIBM, and
scrFIR are cotranscribed as a single polygenic mRNA
molecule, while scrFIAM is transcribed independently. 5' extension analysis indicated that the start site for the
scrFIAM promoter was a thymine located 4 bp downstream
of the 
10 motif. The transcriptional start site for the
orfX promoter was also found to be a thymine which is
more atypically located 24 bp downstream of the 10 motif
proximal to the start codon. A helix-turn-helix motif was identified at
the N-terminal end of one of the methylases (M.ScrFIA). In order to determine if this motif played a
role in regulation of the ScrFI locus,
M.ScrFIA was purified. It was then employed in gel
retardation assays using fragments containing the two promoters found
on the ScrFI operon, one located upstream of
orfX and the other located just upstream of
scrFIAM. M.ScrFIA was found to bind to
the promoter region upstream of the gene encoding it, indicating that
it may have a regulatory role. In further studies the two putative
promoters were introduced into a vector (pAK80) upstream of a
promoterless lacZ gene, and cloned fragments of the
ScrFI locus were introduced in trans with
each of these promoter constructs to investigate the effect on promoter activity. These results implicated M.ScrFIA in
regulation of both promoters on the ScrFI locus.
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TEXT |
The potentially lethal endonuclease
activity characteristic of all restriction-modification (R/M) systems
necessitates maintenance of the inherent coordination between the
restriction and methylation activities. Consequently, the genetic
elements of R/M systems are usually tightly regulated, and the
regulatory components are generally found close to the functional
genes. This organization facilitates transfer of these elements as a
single genetic unit. Two modes of control are commonly found; in some
cases expression is directed by the product of a small open reading
frame (ORF) which is separate from the functional genes of the operon
(10, 16, 22). In other instances, the systems are
controlled by a helix-turn-helix (H-T-H) motif situated at the
N-terminal end of a methylase which regulates the system by interacting
with the promoter region (5). To date, 11 R/M systems have
been characterized to the DNA sequence level in Lactococcus,
and the recognition sites of two more systems have been established,
although the LlaI system is the only one for which a
regulatory mechanism has been deduced (15, 16). Expression
of this system is modulated by the product of a 254-bp ORF positioned
at the start of the operon.
It has previously been demonstrated that the ScrFI
system of Lactococcus lactis subsp. cremoris
UC503 (Table 1) is encoded by an operon
consisting of four ORFs (Fig. 1)
(4, 23, 24). The entire operon has been sequenced and has
previously been published (accession no. LI2227) (24). An
endonuclease gene capable of encoding a 34-kDa protein is flanked by
two 5-methylcytosine methylase genes, designated scrFIAM and
scrFIBM, encoding 44.5- and 41.8-kDa proteins, respectively.
Although the methylases recognize the same target sequence and exhibit
significant sequence similarity, there is no definitive evidence to
suggest that the two genes are the result of a recent duplication
event. The difference between the methylases is illustrated by the fact
that while M.ScrFIB exhibits significant sequence identity
with M.ScrFIA (29.7%), it actually exhibits greater
sequence identity with M.SssI (39.5%) (23). The orfX gene (size of putative
product, 18.5 kDa; accession no. LI2227) upstream of these genes does
not show significant homology to any other ORF in the gene database,
and its function is not known yet (24).
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FIG. 1.
Schematic representation of the molecular organization
of the ScrFI R/M locus, indicating the position of the
restriction endonuclease gene (scrFIR) flanked by two
5-methylcytosine methylase genes. An ORF of unknown function
(orfX) and two ribosomal protein genes
(rpmF and rpmG) are situated upstream of
scrFIBM. Promoter sequences are indicated by P. (Modified from reference 6.)
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The mechanism by which the genes involved in the ScrFI
system are regulated was investigated in this study. This is of
particular interest since the difficulties encountered during attempted
cloning of the complete active locus may have been due to interference with the system's natural regulation (24). Northern and
5' extension analyses were performed to determine the number and sizes
of the ScrFI transcripts. In addition, the activities of the
two promoter motifs were analyzed by performing expression analysis.
Transcriptional analysis of the ScrFI locus.
Previously, analysis of the ScrFI locus revealed that there
are two putative promoter sequences present, one upstream of
orfX and the other upstream of scrFIAM. Secondary
structures in the form of a stem-loop situated between rpmG
and orfX and another secondary structure just downstream of
scrFIAM were also noted (Fig. 1) (6).
Northern analysis was performed to elucidate the manner in which the
locus was transcribed. Aliquots of total RNA were extracted from
lactococcal cultures in the early exponential phase as outlined by
Keilhauer et al. (13) (the procedure was modified by the addition of lysozyme to aid cell lysis), and the aliquots were resuspended in diethyl pyrocarbonate-treated water (17).
RNA samples were treated with DNase and with RNase inhibitor (Roche Diagnostics, Lewes, East Sussex, United Kingdom), denatured at 70°C
for 10 min, and loaded with formamide-containing dye onto a 1.2%
formaldehyde gel (1). RNA size standards from Promega (Madison, Wis.) were included to enable estimation of the sizes of the
observed transcripts. Capillary blotting to
Hybond-N+ nylon membranes (Amersham, Little
Chalfont, Buckinghamshire, United Kingdom) was performed as described
by Sambrook et al. (17). PCR products, used as probes,
were labeled with 32P by using a Prime-a-Gene kit
(Promega). Overnight hybridization was done in a 0.5 M sodium phosphate
(pH 7.0)-5% sodium dodecyl sulfate (SDS) buffer at 50°C, after
which the blots were washed with 2× SSC-0.1% SDS and then with 0.2×
SSC-0.1% SDS and 0.1× SSC-0.1% SDS, at temperatures ranging from
55 to 65°C depending on the required stringency (1× SSC is 0.15 M
NaCl plus 0.015 M sodium citrate).
A blot containing total RNA from L. lactis subsp.
cremoris UC503 was probed with DNA corresponding to each of
the ORFs on the locus. In this manner it was deduced that two
transcripts were produced. scrFIAM was transcribed
individually on a 1.3-kb transcript, while orfX, scrFIBM,
and scrFIR were transcribed together on a single 2.6-kb
polycistronic mRNA molecule (Fig. 2).
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FIG. 2.
Northern analysis of the ScrFI locus
using RNA from L. lactis UC503. The probes used were
orfX (lane orfX), scrFIBM (lane BM),
scrFIR (lane R), and scrFIAM (lane AM).
The sizes of the transcripts are indicated on the left and right.
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5' extension analysis was also performed using primers positioned 100 bp downstream of each of the putative promoters. This analysis
indicated that the transcriptional start site for the promoter just
upstream of scrFIAM was a thymine 4 bp downstream of the
10 motif (Fig. 3). The transcriptional
start site for the orfX promoter was a thymine that is more
atypically located 24 bp downstream of the 10 motif proximal to the
start codon (Fig. 4). The increased
distance may be attributed to the stem-loop structure located between
the 10 motif and the position determined for the start site as RNA
hairpins can cause premature termination by reverse transcriptase
(25). The smearing directly upstream of the position of
the stem-loop structure could represent transcripts initiating at
adjacent positions; these positions are located a more typical distance
from the 10 hexamer.
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FIG. 3.
(a) 5' extension analysis of the promoter region
upstream of scrFIAM of the ScrFI
locus of L. lactis subsp. cremoris UC503.
The arrow indicates the position of the extension product. (b) Sequence
of an extended region between scrFIR and
scrFIAM, showing the regulatory signals. The
transcriptional start site is indicated by a solid triangle. RBS,
ribosome-binding site.
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FIG. 4.
(a) 5' extension analysis of the orfX
promoter region of the ScrFI locus of L.
lactis subsp. cremoris UC503. The arrow
indicates the position of the extension product. A deviation
from the correct sequence published by Twomey et al. (28)
is indicated by the underlined base. (b) Sequence of an extended region
upstream of orfX, showing the regulatory signals. The
transitional start site is indicated by a solid triangle. A direct
repeat upstream of the 35 hexamer is indicated by boldface type. RBS,
ribosome-binding site.
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No hybridization to the 2.6-kb transcript was observed with the
scrFIAM probe, and it was therefore assumed that
transcription of this mRNA molecule terminated within the intergenic
region between scrFIR and scrFIAM. Termination
was possibly via a Rho-dependent event since no stem-loop structures
with strong 
G values were identified within this region. The
possibility that the 1.3-kb RNA molecule is a processed transcript
arising from cleavage of a larger RNA molecule originating at the
orfX promoter cannot be discounted. Nevertheless, assuming
that the locus is transcribed as two separate mRNA molecules, this
result provides for independent regulation of both the
scrFIR and scrFIAM genes.
M.ScrFIA binding assays.
To date, the
mechanisms by which expression of type II R/M systems is controlled
fall into two distinct groups, and it has been proposed that these
systems may actually be characterized in accordance with their
mechanisms of regulation (10, 11). Two of the best-studied
examples, the EcoRII and BamHI systems, illustrate the two modes of regulation. The BamHI system is
regulated by the product of a third ORF found close to the normal
restriction and modification genes. The encoded protein,
designated C.BamHI, was found to control expression of
the other two genes in the locus (18, 19). It was
determined that C.BamHI acts as a transcriptional regulator
by way of an H-T-H motif, a feature common to all C proteins identified
to date (10, 19). The second transcriptional regulatory
mechanism which has been reported is typified by the EcoRII
system (5). In this case an H-T-H motif identified at the
N terminus of M.EcoRII has been shown to bind specifically to the promoter region between the methylase and endonuclease genes and
to subsequently modulate expression of these genes (20).
The presence of both a small ORF, which could potentially code for a
protein equivalent to a C protein, and a highly probable H-T-H motif
(90% possibility of being a functional DNA-binding domain according to
the algorithm of Dodd and Egan [3]) at the N terminus of
M.ScrFIA suggested the possibility that both mechanisms of
control of the ScrFI locus are present. The autoregulatory promoter-binding methylases M.MspI (21) and,
more significantly, M.SsoII (11, 12), which
shows up to 70% similarity with scrFIAM, also have
similarly positioned H-T-H motifs. To establish if the M.ScrFIA H-T-H had a DNA-binding function, the protein was
overexpressed and purified by using the QIAexpress system
(Qiagen Ltd., Surrey, United Kingdom). The intragenic region between
scrFIR and scrFIAM was amplified, and the 300-bp
product was labeled with polynucleotide kinase by using
[
-32P]ATP (Amersham) and used in retardation
assays with the purified M.ScrFIA protein. DNA-binding
assays were performed in 20-µl reaction mixtures containing 50 mM
Tris (pH 8.0), 10% (vol/vol) glycerol, 1 mM EDTA, 5 mM
MgCl2, 500 mM KCl, 2 mM dithiothreitol, 50 µg of bovine serum albumin/ml, 75 µg of poly(dI-dC)/ml, probe
(approximately 0.3 ng), and up to 4 pmol of purified
M.ScrFIA. Incubation was for 15 min at room temperature;
this was followed by addition of 5 µl of 50% glycerol, and then the
samples were loaded onto a 4% polyacrylamide gel containing 2.5%
glycerol. Gels were electrophoresed in TAE buffer (0.04 M Tris-acetate
[pH 7.5], 2 mM EDTA) at 120 V for 3 h, dried, and exposed
overnight at 70°C to X-Omat film (Kodak, Rochester, N.Y.).
From these assays (Fig. 5) it can be
deduced that M.ScrFIA does bind to the intragenic region
upstream of scrFIAM, which contains the promoter motif.
Titration of the quantity of protein added to the binding assays with
respect to the DNA quantity present revealed up to four DNA-methylase
complexes, which probably indicates that four M.ScrFIA
molecules bound to the DNA segment (three bands are visible on the
autoradiogram in Fig. 5; overexposure of the retardation gel revealed
an additional fourth bound band). This implies that more than one
molecule of the methylase binds to the promoter region, which
correlates with an analogous observation for M.SsoII, for
which a similar band pattern was observed (11). In order
to determine more precisely the location of the M.ScrFIA binding site(s) within the scrFIAM promoter region, a series
of subfragments was generated by PCR (Fig.
6). The results of binding assays in
which these subfragments were used indicated that the M.ScrFIA binding site is located on a 30-bp segment (Fig.
6). This region contains a putative symmetrical target sequence.
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FIG. 5.
Gel retardation assay results, showing binding between
the M.ScrFIA protein and the scrFIAM
promoter region. A 0.3-ng portion of 32P-labeled
fragment harboring scrFIAM promoter DNA (300-bp region)
was used as the probe. Lanes 1 to 7, 0, 0.1, 0.2, 0.5, 1.0, 2.0, and
4.0 pmol of M.ScrFIA, respectively; lane 8, negative
control containing a 32P-labeled 300-bp lactococcal DNA
fragment from the origin of replication region of pCI372 plus 4 pmol of
M.ScrFIA.
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FIG. 6.
Schematic diagram of the scrFIAM promoter
region. The transcriptional signals of the promoter are indicated. A to
G represent the 32P-labeled PCR-generated fragments used in
the gel retardation assays. The positions of these fragments as found
in the GenBank sequence are indicated.
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When this assay was repeated by using the same purified protein but DNA
from the intergenic region upstream of orfX (including the
promoter), no binding was observed despite the fact that numerous binding reactions were performed under various binding conditions. The
intergenic region upstream of orfX does not contain a
similar putative symmetrical target sequence, although a 10-nucleotide direct repeat is present upstream of the 35 motif (Fig. 4b). Nevertheless, it is still possible that M.ScrFIA is involved
in regulating this promoter but that additional external factors not
present in the assay mixtures are required for binding to occur in
vitro. These results imply that M.ScrFIA plays a role in
regulation of the promoter upstream of scrFIAM only.
Analysis of promoter strength.
Two promoters were identified
on the ScrFI locus by comparison with other lactococcal
promoter sequences and also by transcriptional analysis. In order to
determine the strength of the promoter upstream of scrFIAM,
a 300-bp fragment containing this promoter region was cloned into the
vector pAK80 immediately upstream of a promoterless lacZ
gene. This fragment included the entire intergenic region between
scrFIR and scrFIAM, and the resultant construct
was designated pCI921. Introduction of pCI921 into MG1363 resulted in a
blue colony phenotype indicative of 
-galactosidase (-Gal)
activity, confirming that the intergenic region contains an active
promoter. The resultant culture was designated L. lactis
subsp. cremoris DB002. -Gal activity was measured as
described by Miller (14), with the following
modifications. Cultures were grown in M17 and harvested in the mid-log
phase. Cells were harvested by centrifugation and concentrated up to
10-fold in Z buffer. One-half milliliter of bacterial suspension was
mixed with 12.5 µl of 0.1% SDS and 25 µl of chloroform in a vortex
mixer for 10 s. After 5 min of incubation in a 30°C water bath,
100 µl of
o-nitrophenyl--D-galactopyranoside (4 mg/ml of A medium [14]) was added, and the suspension
was vortexed for 2 s and incubated further at 30°C. Reactions
were stopped by adding 250 µl of 1 M sodium carbonate. After
centrifugation, A420 and
A550 values were measured for the
supernatant. If the A550 exceeded
0.050, the sample was centrifuged again and remeasured. -Gal
activity (in Miller units) was calculated as follows: (522 × A420)/(t × v × OD600), where t
is time (in minutes), v is the volume of culture used in the
assay (in milliliters), and OD600 is the optical
density of the culture at 600 nm. The -Gal assays gave an activity
of 138.5 Miller units for this promoter (Table 2).
To determine the strength of the promoter identified just upstream of
orfX, a 252-bp fragment containing this promoter region was
introduced into pAK80 to create a construct designated pCI922. Introduction of pCI922 into MG1363 resulted in a culture which was
designated L. lactis subsp. cremoris DB003.
-Gal assays performed with MG1363 containing pCI922 gave an activity
of 132 Miller units (Table 2).
Investigation of the in trans effect of segments of
the ScrFI locus on promoter activity.
To further
delineate the extent of the involvement of the ORFs present in the
ScrFI locus in regulation of the scrFIAM
promoter, the construct pCI934 (containing the entire ScrFI
locus) was transformed into DB002, which resulted in a significant loss
of -Gal activity (the activity decreased from 138.5 to 20 Miller
units) (Table 2). Subclones of the ScrFI locus were
subsequently transformed into DB002 to determine the smallest fragment
of the locus sufficient to decrease the -Gal activity. A construct
designated pCI925 containing the scrFIAM promoter region and
the scrFIAM gene with an internal deletion which removed
part of the H-T-H motif was found to have no effect on the -Gal
activity, while a similar construct with the complete
scrFIAM gene, pCI926, gave decreased -Gal activity (Table
2). This is consistent with the gel retardation assay results which
indicated that there is a binding interaction between
M.ScrFIA and the promoter region preceding
scrFIAM.
Introduction of pCI934 into DB003 (which harbors pCI922 [i.e., the
orfX promoter cloned in pAK80]) resulted in downregulation of the promoter's activity (the activity decreased from 132 to 30 Miller units) (Table 2). Interestingly, introduction of pCI945 (containing orfX and scrFIBM) into DB003 resulted
in no change in -Gal activity, indicating that orfX
itself does not have any noticeable effect on the promoter (Table 2).
This result implies that the product of orfX is not involved
in regulating transcription of the ScrFI locus. As with
DB002, any construct containing a complete scrFIAM gene
resulted in a decrease in -Gal activity.
Transformation of pCI921 and pCI922 into L. lactis subsp.
cremoris UC503 was performed, and transformants containing
each of these constructs were assayed for -Gal activity. A decrease in -Gal activity was observed when either pCI921 or pCI922 was introduced into UC503; the -Gal activity level decreased to about 80% of that observed when either construct was in a MG1363 background.
Somewhat unexpectedly considering the results of the binding
experiments in which M.ScrFIA downregulated only its own
promoter region, it was found in these experiments that the protein
downregulated expression of both promoters. The reason for this
apparent discrepancy is not obvious. As indicated above, it is possible
that the failure to show M.ScrFIA binding to the
orfX promoter may have been due to some deficiency in the
assay conditions and that M.ScrFIA requires some external
factor not present under the reaction conditions to bind the
orfX promoter. However, it is also noteworthy that the
-Gal assays were performed with M.ScrFIA expressed from a multicopy gene cloned on the vector pCI372. Thus, the concentration of
this protein within the cell was significantly higher than would be the
case in the wild-type situation. Therefore, this may have favored a
level of interaction between M.ScrFIA and the orfX promoter that was not truly reflective of the situation
in the wild-type cell. The results of the -Gal assays performed when
pCI921 and pCI922 were introduced into a UC503 background support this
theory. A single copy of scrFIAM is present in the UC503
genome, which probably results in a lower concentration of
M.ScrFIA than would be the case if multiple copies of the
gene were present. The less dramatic drop in -Gal activity (only
20%) observed may be attributed to the lower concentration of
M.ScrFIA.
Conclusion.
In conclusion, transcriptional analysis revealed
that orfX, scrFIBM, and scrFIR are cotranscribed
as a single 2.6-kb polygenic mRNA molecule, while scrFIAM is
transcribed independently on a 1.3-kb transcript. In addition, the
results obtained with respect to the promoter upstream of
orfX are ambiguous as M.ScrFIA does appear to
downregulate its activity but cannot be shown to interact directly with
the promoter in binding assays. However, binding of M.ScrFIA
to the promoter region upstream of scrFIAM and the subsequent downregulation of promoter activity show that
M.ScrFIA is similar to M.EcoRII-like methylases
from a regulatory viewpoint.
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ACKNOWLEDGMENTS |
This research has been funded by grant aid under the Food
Sub-Programme of the Operational Programme for Industrial Development, administered by the Department of Agriculture and Food, and is partially financed by the European Regional Development Fund.
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FOOTNOTES |
*
Corresponding author. Mailing address: National Food
Biotechnology Centre, University College Cork, Cork, Ireland. Phone: 353 21 902730. Fax: 353 21 903101. E-mail:
g.fitzgerald{at}ucc.ie.
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Journal of Bacteriology, August 2001, p. 4668-4673, Vol. 183, No. 15
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.15.4668-4673.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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