FEMS Microbiology Letters 222 (2003) 193^197
www.fems-microbiology.org
Molecular and biochemical analysis of the system regulating
the lytic/lysogenic cycle in the pneumococcal temperate phage MM1
Virginia Obrego¤n, Pedro Garc|¤a, Rubens Lo¤pez , Jose¤ L. Garc|¤a
Departamento de Microbiolog|¤a Molecular, Centro de Investigaciones Biolo¤gicas, CSIC, Vela¤zquez 144, 28006 Madrid, Spain
Received 28 January 2003 ; received in revised form 7 March 2003; accepted 31 March 2003
First published online 17 April 2003
Abstract
The temperate phage MM1 forms stable lysogens in Streptococcus pneumoniae. We report here the first characterization of the lysogenic
control region in Pneumococcus which contains two functional divergent promoters (PR and PL ). MM1 encodes a 14-kDa cI protein (CI)
that appears to be responsible for maintaining the lysogenic state in Pneumococcus since it prevents elongation of the transcripts
controlled by PR and PL .
7 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : Phage repressor; Lytic/lysogenic cycle; Temperate phage; Pneumococcus
1. Introduction
Streptococcus pneumoniae (the Pneumococcus) is a
Gram-positive human pathogen and the leading cause of
pneumonia, meningitis and bloodstream infections in the
elderly and one of the main responsible of middle ear
infections in children. A better knowledge of the molecular
biology of Pneumococcus has been achieved through the
study of pneumococcal phages [1]. The abundance of temperate phages in clinical isolates of Pneumococcus was
suggested some years ago [2] and it recently proposed
that they account for as much as 75% of the samples
analyzed [3].
We had isolated and partly characterized a temperate
phage (MM1), belonging to the Siphoviridae family, from
a clinical isolate (strain 949) of the multiply antibiotic-resistant Spain23F -1 S. pneumoniae clone [4]. This epidemic
clone is the best illustration of rapid spread of drug resistance [5], in this case originally detected in Spain and then
rapidly disseminated to other parts of the world. Most
recently, we have dissected the entire nucleotide (nt) sequence of this temperate phage, as well as the description
of its open reading frames (ORFs), the genome organiza-
* Corresponding author: Tel. +34-91-561-1800; Fax: +34-91-562-7518.
E-mail address : [email protected] (R. Lo¤pez).
tion, and functional assignments of gene products, based
on N-terminal amino acid sequencing and sequence similarities [6]. Temperate phages regulate their developmental fate by encoding a product (named CI) that works as a
transcriptional regulator. There are only few data available on the commitment between lytic and lysogenic state
in Gram-positive bacteria [7]. We report here the ¢rst molecular approach to characterize the elements involved in
the regulation of the lysogenic state in Pneumococcus.
2. Materials and methods
2.1. Bacteria, bacteriophages, plasmids, and growth
conditions
The bacterial strains and plasmids used in this study are
listed in Table 1. S. pneumoniae was grown in C medium
[8] supplemented with yeast extract (0.8 mg ml31 ; Difco
Laboratories) (C+Y medium) at 37‡C without shaking
and the growth was monitored with a Hach 2100N nephelometer. Escherichia coli was grown in Luria Bertani (LB)
medium at 37‡C with shaking. Phage MM1 was induced
from the lysogenic strain 949. At a cell concentration of
1.2U108 colony-forming units (CFU) ml31 , mitomycin C
was added to a ¢nal concentration of 75 ng ml31 , and the
culture was incubated in the dark at 37‡C until lysis occurred. The phages were precipitated with 0.5 M NaCl and
0378-1097 / 03 / $22.00 7 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
doi:10.1016/S0378-1097(03)00281-7
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194
10% (w/v) polyethylene glycol 6000 (w/v) and puri¢ed in a
two-step CsCl gradient as previously described [6].
2.2. DNA preparation, transformation procedures and
recombinant DNA techniques
The preparation of pneumococcal DNA has been described elsewhere [9]. Protein-free MM1 DNA was obtained by treatment of puri¢ed phage preparations with
sodium dodecyl sulfate (SDS) and proteinase K and
DNA^protein complexes were isolated as previously described [9]. Plasmid DNA was extracted from E. coli by
the rapid alkaline method [10]. DNA restriction or ampli¢ed fragments for cloning, probe preparation or sequencing were isolated from 0.7% (w/v) agarose gels with the
Geneclean II kit (Bio 101). Restriction endonucleases
(New England Biolabs, Beverly, MA, USA) and T4
DNA ligase (Amersham-Pharmacia-Biotech., AP) were
used as recommended by the suppliers. Polymerase chain
reaction (PCR) ampli¢cations were performed using 2 U
of AmpliTaq1 DNA polymerase (Perkin-Elmer Applied
Biosystems, Norwalk, CT, USA), 1 Wg of chromosomal
DNA (or 0.1 Wg of phage DNA), 1 WM of each deoxynucleoside triphosphate, and 2.5 mM of MgCl2 in the bu¡er
recommended by the manufacturer. Conditions for ampli¢cation were chosen according to the G+C content of the
corresponding oligonucleotide. The oligonucleotides used
are reported in Table 1. Transformation of E. coli DH5K
was carried out by the RbCl method [10]. Transformants
were selected on LB plates containing ampicillin (100 Wg
ml31 ). The transformation procedure for S. pneumoniae
has been described elsewhere [11]. Lincomycin-resistant
S. pneumoniae transformants were selected on C+Y plates
containing 0.6 Wg ml31 lincomycin.
2.3. LytA activity assay
Pneumococcal cell walls were radioactively labeled with
[methyl-3 H]choline and the activity of pneumococcal LytA
amidase tested on these labeled walls as previously described [12]. Brie£y, pneumococcal cell walls were incubated with enzymatic extracts at 37‡C for 15 min and the
reaction was stopped upon the addition of formaldehyde
and bovine serum albumin. Non-digested murein was separated by centrifugation at 12 000Ug for 15 min, and the
digested (solubilized) substrate was quanti¢ed by measuring the reactivity in the supernatant. One unit of lytic
activity was de¢ned as the amount of enzyme that catalyzed the hydrolysis (solubilization) of 1 Wg (715 cpm) of
cell wall material in 15 min at 37‡C.
2.4. Miscellaneous methods
N-terminal amino acid sequence analyses were carried
out according to a published procedure [13]. SDS^polyacrylamide gel electrophoresis (PAGE) was performed on
gels containing 12.5% (w/v) polyacrylamide and the gels
were stained with Coomassie blue.
3. Results and discussion
3.1. Characterization of the promoters controlling the lytic/
lysogenic cycle
The most left region (from nt 1 to 5013) of the MM1
prophage [6] encompasses the lysogenic control region,
and contains four ORFs that are transcribed leftwards
(int, orf1, orf2, and cI). The Int protein was identi¢ed as
Table 1
Bacterial strains, plasmids and primers
Material
Bacterial strains
S. pneumoniae
949
E. coli
DH5K
C600
Plasmids
pLSE4
pUC18/19
pPL
pPR
pREP
Primersa
Xba-prom1
Sph-prom1
Xba-prom2
Sph-prom2
M1Rep5P
M1Rep3P
Description
Reference or source
Spanish/American 23F clone (Spain23F -1 strain)
[5]
P80 lacZvM15 gyrA96 thi-1 relA1 supE44 hsdR17 recA1 endA1 deoR v(lacZYA-argF) lacU169
thi-1 thr-1 leuB6 lacY1 tonA21 supE44
[10]
[10]
promoterless-lytAþ , TcR , LnR
ApR
PL -lytAþ , TcR , LnR
PR -lytAþ , TcR , LnR
cI, ApR
[14]
[17]
this study
this study
this study
(2477) 5P-TTGATACATCtAgaTTTCTGAAGC-3P
(2477) 5P-TTGATgCATgcACCTTTCTGAAGC-3P
(2785/c) 5P-GATATCTAGAATATGTGTTCCTCC-3P
(2785/c) 5P-GATATCTGGCATgcGTGTTCCTCC-3P
(2500/c) 5P-GCTctAGAAAGGTTGATGTATCAATG-3P
(2004) 5P-CTGGcATGcAAAAaGCTTCATCTGG-3P
this
this
this
this
this
this
study
study
study
study
study
study
a
Numbers indicate the position of the ¢rst nt of the primer in the sequence of the transposon Tn5252 (for primers Tn5P and Tn3P), or in the sequence
of MM1 DNA reported in this paper (for other primers), and /c means that the sequence corresponds to the complementary strand. Lowercase letters
indicate nt introduced to construct appropriate restriction sites (shown underlined).
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the MM1 integrase that belongs to the V phage family of
integrases [4]. The di¡erent functional clusters of the MM1
genome are apparently separated by intergenic regions
that may contain promoter sequences and/or regions capable of forming stable stem-loop structures, most probably representing transcription terminators. An interesting
¢nding was the existence of a 296-nt region located between genes cI and orf4 that contains two directly adjacent, outward-facing, putative promoter-like sequences
(PL : (2544/c) TTGTCA-19 nt-TAAAAT ; PR : (2696)
AAGCTA-20 nt-TTAAAT)) (/c means that the sequence
is located in the complementary strand). Interestingly, this
region contains three direct repeats of the sequence AACAACTAA, that most likely act as operators for regulatory purposes [6]. Moreover, this zone separates the two
gene clusters/operons that constitute the MM1 genome
and which are transcribed in opposite orientations. To
test the role of this region in regulation we used pLSE4,
a broad host range pneumococcal promoter-probe plasmid
that uses the lytA gene coding for the major pneumococcal
autolysin as reporter [14]. To clone the fragment contain-
195
ing the putative promoters in both orientations we prepared four oligonucleotides (Xba-prom1, Xba-prom2,
Sph-prom1, and Sph-prom2) as depicted in Fig. 1. Each
pair of oligonucleotides was designed to place PL , the
promoter of the cI gene (plasmid pPL ), or PR , the promoter of orf4 (plasmid pPR ), upstream of lytA in the correct orientation to be independently tested. The accuracy
of the constructions was con¢rmed by sequence determination. Extracts prepared from E. coli transformants containing either the resulting pPR or pPL plasmids were assayed on [3 H]choline-labeled pneumococcal cell walls and
the results demonstrated promoter activity for both PR
and PL sequences (Fig. 1).
3.2. Expression of the CI protein
The ¢rst ORF downstream of PL corresponds to gene cI
that codes for a protein sharing extended similarity to
well-characterized repressors identi¢ed in other temperate
phages [6]. To further demonstrate that cI codes for a
repressor protein, this gene was cloned into plasmid
Fig. 1. Cloning of the repressor cI gene and the promoters PR and PL controlling the lysogenic/lytic cycle. At the top of the ¢gure we show a schematic
representation of the proposed organization of the genome of MM1 [6]. Genes involved in the lytic/lysogenic cycle are indicated in black and stripped
rectangles. Promoters were cloned upstream of the lytA reporter gene into plasmid pLSE4 (right), and the repressor cI gene was cloned into plasmid
pUC18 (left). Abbreviations : H, HindIII; S, SphI; X, XbaI; ApR , LnR , and TcR represent genes coding for resistance to ampicillin, lincomycin, and tetracycline, respectively.
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pUC18 under the control of the Plac promoter. To do that,
the cI gene was ampli¢ed using primers MM1Rep5P and
MM1Rep3P, digested with HindIII and XbaI and ligated
to pUC18 previously digested with the same enzymes to
originate plasmid pREP. Crude sonicated extracts prepared from E. coli DH5K containing the recombinant
plasmid pREP induced with isopropyl-L-D-thiogalactopyranoside were analyzed by SDS^PAGE and revealed the
presence of an overproduced 14-kDa protein (Fig. 2).
The size of the protein corresponds to that deduced
from the nucleotide sequence of cI (13 814 Da) and analysis of the N-terminal sequence of this protein yielded
M-F-E-T-F-E-K that perfectly matched the seven ¢rst
amino acid residues of the CI protein (not shown). Plasmid pREP was used to transform E. coli C600 (pPR) and
E. coli C600 (pPL) to analyze the e¡ect of CI on the
promoters PR and PL by testing the inhibitory e¡ect on
the expression of the reporter lytA gene, that is under the
control of either of these promoters, on the degradation of
the labeled cell wall substrate. The results reported in Table 2 demonstrate that CI is a strong repressor of the
activity of both promoters since residual activity of the
amidase is only 2^3% of that found in the absence of
the CI protein. To our knowledge, this is the ¢rst report
on a lysogenic control protein in S. pneumoniae that
should govern the genetic switch from the lysogenic to
the lytic cycle.
Regulation of the lytic/lysogenic cycle of V phage infecting E. coli has been analyzed in detail [15] whereas this
type of regulation has been little studied in Gram-positive
systems. An interesting and recent contribution to this
topic has been the study on the regulation of lysogeny
of Lactobacillus casei by phage A2 [7] where the activity
of the CI protein regulates the lytic and lysogenic pathways of the A2 phage. We have demonstrated here that
there are two promoter activities (PR and PL ) in opposite
Table 2
Enzymatic assays of the activities of the promoters PR and PL
Straina
E.
E.
E.
E.
E.
E.
coli
coli
coli
coli
coli
coli
Sp act
(U mg31 )
C600
C600
C600
C600
C600
C600
(pPR)
(pUC18, pPR)
(pREP, pPR)
(pPL)
(pUC18, pPL)
(pREP, pPL)
7 300
7 500
210
13 150
13 230
580
Residual activity
(%)
100
100
3
100
100
4
a
Crude extracts prepared from di¡erent E. coli transformed strains were
assayed on [3 H]choline-labeled pneumococcal cell walls for enzymatic
activity, as described in Section 2.
orientations that are probably involved in the regulation
of the lytic/lysogenic cycle of phage MM1. Besides, we
have cloned and expressed here the product of gene cI,
highly similar to de¢ned phage repressor proteins like
the CI of P NIH1.1 that infects Streptococcus pyogenes
[16], that was capable of inhibiting the activity of PL .
The development of these molecular tools is essential to
pave the way to study the competing transcription events
occurring during the early decision for lysis or lysogeny in
an important system like Pneumococcus where the presence of temperate phages appears to a¡ect up to the 70%
of clinical strains freshly isolated from humans.
Acknowledgements
The advice and improvements suggested by E. Garc|¤a
during the preparation of this work are gratefully acknowledged. We thank the technical assistance of E.
Cano and M. Carrasco. This work was supported by grant
BCM2000-1002 from the Direccio¤n General de Investigacio¤n Cient|¤¢ca y Te¤cnica and by the grant provided by the
Programa de Grupos Estrate¤gicos de la Comunidad
Auto¤noma de Madrid.
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Fig. 2. Analysis by SDS^PAGE of the expression of the CI protein.
Lanes 2 and 3 correspond, respectively, to crude extracts prepared from
E. coli DH5K (pUC18) and E. coli DH5K (pREP). Molecular size
markers are shown in lane 1. Arrow indicates the position of CI.
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