FEMS Microbiology Letters 106 (1993) 147-156
© 1993 Federation of European Microbiological Societies 0378-1097/93/$06.00
Published by Elsevier
147
FEMSLE 05230
Serotype F double- and triple-converting phage
insertionally inactivate the Staphylococcus aureus
/3-toxin determinant by a common
molecular mechanism
J . D . C a r r o l l a, M . T . C a f f e r k e y b a n d D . C . C o l e m a n c
Department of Microbiology, The Moyne Institute, University of Dublin, Trinity College, Dublin, Ireland,
b Department of Clinical Microbiology, St. Jame's Hospital, Dublin, Ireland, and ¢ School of Dental Science, University of Dublin,
Trinity College, Dublin, Ireland
a
(Received 23 October 1992; accepted 28 October 1992)
Abstract: The precise molecular mechanism of Staphylococcus aureus fl-toxin inactivation by the serotype F triple-converting phage
~b42, ~bA1 and &A3 was investigated. Sequence analysis of the 4~42(attP) and Staphylococcus aureus (attB) attachment sites and
the left (attL) and right (attR) chromosomal/bacteriophage DNA junctions of individual lysogens, each harbouring a triple-con-
verting phage, revealed the presence of a common 14-bp core sequence in all four sites. These findings indicate that the genomes of
the triple-converting phage integrate into the 5'-end of the /3-toxin gene (h/b) by a site- and orientation-specific mechanism
identical to that previously described for the serotype F double-converting phage 4~13.
Key words: Staphylococcus aureus; /3-Toxin; Site-specific insertion; Triple-lysogenic conversion; Double-lysogenic conversion;
Bacteriophage
Introduction
Staphylococcus aureus fl-toxin expression can
be negatively affected by two types of serotype
F-converting bacteriophage following lysogenization [1-3]. T h e first types to be described were
double-converting phage, which effected the si-
Correspondence to: D. Coleman, Department of Oral Medicine
and Pathology, University of. Dublin, Trinity College, Dental
School Office, 30 Westland Row, Dublin 2, Republic of
Ireland.
multaneous positive conversion for fibrinolysin
expression [4,5]. T h e s e type of phage e n c o d e a
fibrinolysin determinant in their genomic D N A
[2]. M o r e recently, triple-converting p h a g e were
reported, which in addition to negatively converting /3-toxin expression also simultaneously mediate the positive-conversion for fibrinolysin and
enterotoxin A expression [2]. These p h a g e e n c o d e
determinants for b o t h fibrinolysin and enterotoxin A expression in their genomic D N A [2,6].
Studies f r o m this laboratory revealed that the
m e c h a n i s m of negative conversion o f / 3 - t o x i n ac-
148
tivity by the representative serotype F doubleconverting phage ~b13 and several triple-converting phage was due to the orientation-specific
insertion of circular phage DNA into similar or
closely linked sites within or adjacent to the S.
aureus /3-toxin structural gene, hlb [1,2]. More
detailed studies with the double-converting phage
~13 revealed that upon lysogenization the ~b13
genome inserted into the the 5'-end of hlb and
that the insertion site (attB) within hlb contained
a 14-bp core sequence in common with the attachment site (attP)-containing region of ~b13
DNA, which was duplicated at both ends of the
integrated linear prophage genome [3]. Thus, integration of q513 DNA as a linear molecule resulted from orientation- and site-specific recombination between the 14-bp core sequences present in the respective bacterial and phage attachment sites. The conservative nature of the insertion event is reflected by the structure of the two
bacterial chromosomal DNA/phage DNA junctions which were composed of composite copies
of the 14-bp core sequences, containing both
phage and bacterial sequences [3].
The purpose of this study was to determine
whether the precise molecular mechanism of hlb
inactivation by serotype F triple-converting phage
upon lysogenization was identical, or similar to,
the mechanism employed by the double-converting phage ~b13.
recombinant plasmids. Bacteria were routinely
cultured at 37°C in Trypticase soy broth (TSB,
Oxoid) for S. aureus or in L-broth (LB) [9] for E.
coli XL-1 and its derivatives in an orbital shaker
at 150 rpm. The corresponding agar media were
also used. Chemicals were purchased from Sigma
or BDH. Restriction endonucleases and other
enzymes were purchased from Boehringer,
Promega, Pharmacia or Stratagene and were used
according to the manufacturer's instructions.
[a-35S]dATP (600 Ci mmo1-1) was purchased
from Amersham.
Materials and Methods
Oligonucleotides
Oligonucleotides for use in PCR experiments
were synthesized using a PCR Mate 391 synthesizer (Applied Biosystems Inc.) and purified
through NAP-10 (G25) columns (Pharmacia).
Bacterial strains, bacteriophage, plasmids, culture
conditions and enzymes
S. aureus strain 80CR3 [7], a restriction-deficient strain, was the host strain for triple-converting phage. This strain harbours a single copy of
the/3-toxin gene (hlb) located on a 2.2-kb HindIII
fragment as depicted in Fig. 1A. The 80CR3
lysogenic derivatives 42CR3-L (harbouring ~/,42),
A1CR3-L (harbouring ~bA1) and A3CR3-L
(harbouring ~bA3) have been described previously, as have the S. aureus serotype F triple-converting bacteriophage ~b42, ~bA1 and ~bA3 [2].
Escherichia coli strain XL-1, a recombination-impaired strain [8], was used as the host strain for
DNA procedures
Preparation of plasmid DNA from E. coli and
total genomic DNA from S. aureus was performed as described previously [1,2]. Cloning,
subcloning and transformation of E. coli were
performed using standard techniques [10]. DNA
sequencing was performed by the double-strand
dideoxy-mediated chain termination method [11].
DNA fragments to be sequenced were cloned
into pBluescript vectors (Stratagene) and were
sequenced using a T7 polymerase sequencing system kit (Promega) or a Sequenase 2.0 DNA sequencing kit (United States Biochemicals) with
[a-35S]dATP. DNA fragments amplified by PCR
were purified using Geneclean II (BIO 101 Inc.),
which was used according to the manufacturer's
instructions, and sequenced directly using the
PCR oligonucleotides as sequencing primers.
Polymerase chain reactions
The melting temperature (Tm) for each primer
was calculated using the equation Tm = (2(A + T)
+ 4(G + C))°C where A, T, G and C represent
the number of adenyl, thymidyl, guanyl and cytidyl
residues, respectively, in the primer. The annealing temperature for individual amplification reactions was calculated as 4°C less than the lower of
the two primer melting temperatures. Reaction
mixtures in a final volume of 100 ml in 0.5 ml
149
reaction tubes contained: 50 mM Tris. HCI, pH
9.0; 50 mM NaCI; 200 mM each Of dATP, dCTP,
dGTP and dTTP and 200 pmol of each primer.
Reaction mixtures were overlaid with 50 ml of
sterile mineral oil, UV-irradiated for 10 min in an
Amplirad UV cabinet (Genetic Research Instrumentation Ltd.), after which template DNA (50
ng) was added and the reaction mixture heated to
95°C for 5 min. Then, 2 units of Taq DNA
polymerase (Promega) were added and amplification reactions carried out in a Perkin Elmer Cetus D N A thermal cycler which was set for the
following conditions: 92°C, 1 min; annealing temperature, 1 min; 72°C, 1 min, for 30 cycles. Following thermal cycling, the mineral oil was removed from the reaction tubes, and the DNA
solution precipitated with ethanol and resuspended in TE buffer (10 mM Tris" HC1, 1 mM
EDTA at pH 8.0).
Results
Analysis o f S. aureus and bacteriophage attachment sites
In order to investigate the precise molecular
mechanism of S. aureus /3-toxin inactivation by
serotype F triple-converting phage it was necessary to analyse the structure of the bacterial and
phage DNA sequences involved in prophage integration during the process of lysogenization. Previous studies from this laboratory using cloned
hlb- and attP-containing DNA probes localized
the attachment-site-containing region of the
triple-converting phage 4~42 to a DNA region of
approximately 0.1 kb adjacent to the internal
E c o R I site of the attP-encoding plasmid pDC107
[2] (Fig. 1B). These studies also localized the
bacterial insertion site (attB) of 4~42 to a DNA
region of approximately 0.15 kb within the central
DdeI fragment of the 2.2-kb HindIII hlb-encoding fragment of S. aureus strain 80CR3 (Fig. 1A).
It would have been more logical to determine
the molecular mechanism of/3-toxin inactivation
by 4~42 using S. aureus strain CN6708, which had
been used in similar studies with the double-converting phage ~b13 [2]. However, 4~42, which was
originally induced from strain PS42D, the propa-
gating strain for the S. aureus typing phage 42D
[2], would not infect strain CN6708. Therefore, it
was decided to perform the present studies with
S. aureus strain 80CR3, which could be readily
lysogenized with 4~42 [2].
Nucleotide sequence of the attachment sites of ch42
and S. aureus 80CR3
The nucleotide sequence of the attP-contain-
ing region of 4~42 extending approximately 380
bp from the E c o R I of plasmid pDC107 was determined (Fig. 1B and Fig. 2B). Comparison of
this sequence with the corresponding sequence of
the attP-containing region of the double-converting phage 613 [3] revealed that they were almost
identical, with two minor differences being evident (Fig. 2B). The attB-containing region of S.
aureus strain 80CR3 was isolated by polymerase
chain reaction (PCR) amplification of a DNA
fragment of approximately 280 bp from the 5'-end
of the hlb determinant encoding the N-terminus
of the/3-toxin protein. A pair of 17-met synthetic
oligonucleotide primers, termed primer A and
primer B, respectively (Table 1), were designed
for this purpose using the published nucleotide
sequence of the hlb determinant of S. aureus
strain CN6708 as a template (Fig. 2A) [3]. The
primers were homologous to sequences flanking
the 14-bp core sequence of the hlb gene (Fig.
2A). The nucleotide sequence of the amplified
fragment was determined and compared with the
corresponding sequence of the S. aureus strain
CN6708 hlb determinant. The two sequences were
almost identical except for three nucleotide mismatches which were present in the 80CR3 sequence (Fig. 2A). However, translational analysis
of both sequences revealed that the observed
nucleotide base mismatches were conservative.
Comparison of the 4~42 attP-containing sequence with the attB sequence of S. aureus strain
80CR3 revealed a 14-bp core sequence common
to both (Figs. 2A, B). Furthermore, this core
sequence was identical in sequence and juxtaposition to the core sequences previously identified in
the attP-containing DNA of 4~13 and in the
attB-containing DNA region of the S. aureus
strain CN6708 hlb determinant (Figs. 1, 2) [3].
These data suggested that 4~42 and ~b13 proba-
150
bly insert into the same site in the S. aureus hlb
determinant during lysogenization. To confirm
this suggestion it was necessary to examine the
nucleotide composition of the chromosomal/bacteriophage DNA junction fragments of a lysogenized derivative of S. aureus strain 80CR3 harbouring th42.
)
hlb
H
Nucleotide sequence of the chromosomal/bacteriophage DNA junctions of the c~42 lysogen
42CR3-L
Previous studies from this laboratory demonstrated that during lysogenization ~b42 integrates
into the chromosomally located hlb determinant
of S. aureus 80CR3 as a linear molecule (Fig. 1)
D
D
H
A
0.25 kb
attB
H
E
pDC~
H
H
L
j~..E
~
~
H
~rl
I
pDC 107
~
0.5 kb
¢42 attP
$13 attP
C
Left junction
HDE
Right junction
H
H
D
= i r a 0.5 kb
TGTATCCAAACTGG
~13 attL
~
HDE
~t~42attL
~13 attR
D
H
_#'__
TGTATCCAAACTGG
~,42 attR
151
[2]. The 2.2-kb hlb-containing HindlII fragment
of S. aureus 80CR3 was split in two by the
integration of the &42 genome, with the linear
phage genome separating the divided parts of the
hlb determinant. This resulted in the generation
of two junction fragments each composed of a
portion of the 5.0-kb attP-containing HindlII
fragment of ~b42 and the 2.2-kb hlb-containing
HindlII fragment of S. aureus 80CR3 (Figs. 1B,
C).
The chromosomal/bacteriophage DNA junctions of a ~b42 Iysogenized derivative of S. aureus
strain 80CR3, termed 42CR3-L [2], were isolated
by PCR amplification using specific 17-mer
oligonucleotide primers. A separate pair of
primers was used to amplify each junction fragment. One primer from each pair was homologous to hlb sequences flanking the 14-bp core
sequence of the hlb determinant and the other
was homologous to &42 nucleotide sequences
flanking the 14-bp core sequence of ~b42 attPcontaining DNA. Primers A and D (Table 1)
were used to amplify approximately 240 bp of
DNA from the left junction and primers B and C
(Table 1) were used to amplify approximately 280
bp of DNA from the right junction. The hlb or
~b42 sequences to which the primers were complementary are shown in Figs. 2A and B. No PCR
products were obtained when similar experiments
were performed with the phage-free parental
strain 80CR3. Examination of the nucleotide sequences of both PCR products revealed a 14-bp
sequence, in each case, identical to the 14-bp
core sequences present in the ~b42 attP-containing DNA and the amplified attB-containing hlb
DNA of S. aureus strain 80CR3 (Figs. 2, 3). The
14-bp core sequence of the left junction was
termed attL and that of the right junction was
termed attR (Fig. 3). The sequence of the left
junction immediately 5' to attL was identical to
the corresponding 5' sequence flanking the attB
sequence of the hlb gene of S. aureus 80CR3,
whereas the sequence of the left junction immediately 3' to attL was identical to the correspond-
Fig. 1. (A) Structure of the 2.2-kb HindlII fragment encoding the/3-toxin gene (hlb) of S. aureus strains 80CR3 and CN6708 [2,3].
The arrowed line in the upper part of the figure represents the position and shows the direction of transcription of the open
reading frame encoding E-toxin. The location and the nucleotide sequence of the 14-bp attB core region is shown in the lower
portion of the figure. The unshaded portion of the figure refers to a 0.15-kb sequence of bacterial DNA to which the insertion site
(attB) of the double-converting phage 613 was localized in S. aureus strain CN6708 [3], and to a similar 0.15-kb sequence in the
corresponding genomic DNA of S. aureus strain 80CR3 in the case of the triple-converting phage 642, 6A1 and 6A3, respectively
[2,3]. (B) Restriction maps of the cloned attP-containing HindlII fragments of the double- and triple-converting phage 613
(pDCll0) and 642 (pDC107), respectively [2]. The unshaded portion of both HindlII fragments refers to a 0.15-kb sequence of
DNA in each case to which the attP sites of 613 and 642, respectively, were previously localized by restriction mapping and
hybridization analysis [2]. The actual location and nucleotide sequence of the 14-bp attP core sequence of 613 [3] and 642,
respectively, is shown in the lower portion of the figure. The minor disparity between the mapped location for attP and the position
determined by sequence analysis, for both phage, is probably due to small inaccuracies in the measurement of the sizes of
restriction fragments encountered when originally mapping the attP sites [2,3]. The arrowed line in the upper portion of the figure
refers to the 380-bp attP-sequence of 642 DNA that was sequenced in the present study. (C) Restriction maps of the two HindlII
fragments generated at the chromosomal/bacteriophage DNA junctions of the S. aureus strains CN6708 (upper portion of figure)
and 80CR3 (lower portion of figure) following lysogenization with 613 and 642, respectively. For both lysogens the chromosomally
located 2.2-kb hlb-containing HindlII fragment (A) has been split in two by the integration of phage DNA, resulting in two
composite junction fragments each containing a part of the 2.2-kb hlb-containing HindlII fragment and part of the attP-containing
HindlII fragment of phage DNA (Fig. 2B), with the linear phage genome separating the two disrupted hlb portions. The composite
HindlII fragments containing the 5' (left junction) and 3' (right junction) ends of the hlb-containing HindlIl fragment are
represented by shaded horizontal boxes. Black shading represents bacterial DNA and lighter shading phage DNA. The linear
phage genome separating the two composite HindlII junction fragments is represented by the cross-hatched dotted line, in each
case. The nucleotide sequence and locations of the 14-bp core sequences constituting the actual chromosomal/bacteriophage DNA
junctions are shown. The left junction of each lysogen was termed attL and the right junction attR. The asymmetrical nature of the
attB and attP core sequences ensures prophage integration occurs in one direction only. Because of this orientation specificity and
because the right-hand portions (as drawn in B) of the attP-containing HindlII fragments of 613 and 642 have similar structures,
the left composite HindlII junction fragments of lysogens harbouring 613 and 642, respectively, are of a similar size and structure.
Abbreviations: H, HindlII; D, Ddel; E, EcoRI.
152
A.
~D~n ORF >
TTGTTGTAAGCTATATAAAAGGAGTGATA,~TGGTGAAAAAAACAAAATCCAATTCACTAAAAAAAGTTGCAACACTT
-20
-I0
+i
+I0
+20
+30
+40
+50
Ddel
S' __PrirnerA.L:)
G•ATTAGCAAATTTATTATTAGTTGGTGCACTTACTGACAATAGTGC•AAAG•CGAATCTAAGAAAGATGATACTGATTT
+60
+70
+80
+90
+I00
+ii0
+120
+130
GAAGTTAGTTAGTCATAACGTTTATATGTTATCGACCGTTTTGTATCCAAACTGGGGGCAATATAAACGCG•TGATTTAA
-- --+140
+150
+160
+170
+180
+190
+200
+210
TCGGACAATCTTCTTATATTAAAAATAATGATGTCGTAATATTCAATGAAGCATTTGATAATGGTGCATCAGACAAATTA
+220
+230
+240
250
+260
+270
+280
+290
C
T
T
TTAAGTAATGTG~M~AAAAGAATATCCTTA~CAAACAC~TGTACTCGG~GTTCTCAATCAGG~TGGGACAAAACTGAAGG
+300
+310
+320
+330
+340
+350
+360
+370
3'
Primer,_8.1~l
5'
TAGCTACTCATCAACTGTTGCAGAAGATGGTGGCGTAGCGATTGTAAGTAAATATCCTATT/~AGAAAAAATCCAGCATG
+380
+390
+400
+410
+420
+430
+440
+450
B,
S'
AAAAACAACTAAAAT
-90
GGTTTCTATGTATC
-I0
Primer C
(-)3'
T T T A G A A A G T A T G T A A T T T A G G G A C C CAT T A G G G A C T C C A A A C C C A A T A A A T A C T G T
-80
-70
-60
-50
-40
-30
T GTTACAA
-20
C A A A C T G G A G A C T T T T A A C A T A A A A T T A C T TAT CAT T C A A A A A G T A A A A C A G C A T A A T A T C A A G G T
+I
+I0
+20
+30
+40
+50
+60
TTATAAC TTTAT CATTAT C AATAATACCTCATATAAAATAAAAT
T T T A G G G A C TTT T T A G G G A C T T T A A A T T T A A A A T T A
+70
+80
+90
+I00
+Ii0
+120
+130
+140
Primer D .(.t)-- __S'
CAAGTTTAATAGAAACAT
+150
+160
CAAAATAAT CACATGT T T GTGT GGAATGTACAC C CCAAAAGCTAGACTGAAAAATC
TATTTT
+170
+180
+190
+200
+210
+220
TTGAGGT GTAT T TTTATAGG TAAATATAATAAAT TAGAG TAGACAAC T CAG AATTC
+230
+240
+250
+260
+270
Ec:oRi
Fig. 2. (A) Nucleotide sequence of the 5'-end of the hlb determinant of S. aureus strain CN6708 encoding the N-terminal 150
amino acids of/3-toxin. The data are taken from the nucleotide sequence of hlb as reported by Coleman et al. [3] ( E M B L Data
Library accession n u m b e r X61716). Sequences are numbered from the start of the hlb gene; the first base of the hlb sequence is
denoted + 1 and the base immediately to the left is denoted - 1. The 14-bp core sequence constituting attB is doubly underlined.
The hlb sequences to which the P C R primers A and B (Table 1) were complementary are underlined above and below by dotted
lines; ( + ) and ( - ) denote whether the primers were complementary to the coding or non-coding strands, respectively. Primers A
and B were used to amplify the corresponding attB-containing D N A from S. aureus strain 80CR3, the sequence of which was
identical to the S. aureus strain CN6708 attB-containing sequence apart from three conservative single nucleotide substitutions
corresponding to nucleotide positions +321, +339 and +354. These nucleotides are underlined and the corresponding nucleotides
present in the 80CR3 attB-containing sequence are indicated above the underlined nucleotides. The location of a site for the
restriction endonuclease DdeI is indicated. This corresponds to the left-hand DdeI site depicted in Fig. 1 and in (B). Nucleotide
sequence of 4'42 D N A containing the attP site. The 14-bp core sequence constituting attP is doubly underlined and the two pairs
of 8 bp direct repeats flanking the core sequence are singly underlined. Sequences are numbered from the centre of the core; the
base immediately to the right of the core centre is denoted + 1 and the base immediately to the left of the core centre is denoted
- 1. Sequence was determined from the E c o R I site of plasmid pDC107 (Fig. 1B). The phage sequences to which the P C R primers
C and D were complementary are underlined above and below by dotted lines; ( + ) and ( - ) indicate that the primers were
complementary to the coding or non-coding strands, respectively. The underlined nucleotides at positions - 80 and + 227 indicate
differences between the 4'42 attP-containing sequence and the corresponding attP-containing sequence of the double-converting
phage 4'13 [3]; (EMBL Data Library accession n u m b e r X61717). The thymidine nucleotide at position - 8 0 of the 4,42 sequence
was occupied by a guanosine residue in the corresponding 4,13 sequence and an additional thymidyl nucleotide was present at
position + 227 in the 4,42 sequence compared with the corresponding 4,13 sequence. The E M B L Data Library accession number
for the 4,42 attP-containing sequence is X67739.
153
Table 1
Composition of oligonucleotide primers used to amplify the attB-containing DNA from S. aureus strain 80CR3 and the left and
right chromosomal/bacteriophage DNA junctions from the 80CR3 lysogens 42CR3-L, A1CR3-L and A3CR3-L
hlb primers a
Phage a t t P primers a
Primer A: 5'-TGATACTGATTTGAAGT-3'
Primer B: 5'-TCGCTACGCCACCATCT-3'
Primer C: 5'-CATTAGGGACTCCAAAC-3'
Primer D: 5'-AACATGTGATTATTTTG-3'
a Refers to primers which were complementary to hlb or 4,42 sequences respectively (Fig. 2A, B). Primers A + D and B + C were
used to PCR amplify the left and right chromosomal/bacteriophage DNA junctions, respectively, of lysogens harbouring
triple-converting phage.
ing 3' sequence flanking the attP sequence of
4,42 (Fig. 3). In addition, the sequence of the
right junction immediately 5' to attR was identical to the corresponding 5' sequence flanking the
attP sequence of 4,42, and the sequence of the
right junction 3' to attR was identical to the
corresponding 3' sequence flanking the attB sequence of the hlb gene of S. aureus 80CR3 (Fig.
3).
Nucleotide sequence of the chromosomal~bacteriophage DNA junctions of the lysogens A I C R 3 - L
and A3CR3-L
Similar experiments to those described above
were performed with lysogenized derivatives of S.
aureus strain 80CR3 harbouring the serotype F
triple-converting phage (hA1 and 4,A3, which
were originally isolated from clinical isolates of S.
aureus [2]. Previous studies demonstrated that the
organization of the attP-containing DNA of both
(hA1 and 4,A3 was very similar to that of 4,13 and
4,42 [2]. Furthermore, 4,A1 and 4,A3 genomic
DNA, respectively, inserted into the 5'-end of the
S. aureus hlb determinant during lysogenization
at an identical or closely linked site(s) to that
used by 4,13 and 4,42 [2].
The chromosomal/bacteriophage DNA junctions of the S. aureus lysogens A1CR3-L
(harbouring 4,A1) and A3CR3-L (harbouring
4,A3) were isolated by PCR amplification as described for 4,42, and the nucleotide sequences of
the amplified products determined. These were
identical to the corresponding sequences obtained with the 4,42 lysogen 42CR3-L (Fig. 3).
Left junction
+170
Right junction
+i0
-i0
+190
CNe~0,-L GACCG~T~ [ TG~ATCCAAA~TG~] Aj,~TTT----~J-3---~GTT~TA_
. [~G~TC~A~C~G~ ] ~ A A ~ .
~42
42c,,-. ~ " C ~ T [ ~ T A T C C ~ A A = ~ G ] " J ' C ~
.........
GG~T~C~" [ ~ " " A ~
]~""~"
AICR3-L
GACCGTTT[TGTATCCAAACTGG]AGACTTTT---ff-~-'---GcTTT~TA
hlb
~toxin O R F
attL
--"
'"
~
Linearprophage genorne
[~G~.~c..Ac~G
attR
h/b
13-toxinO R F
Fig. 3. Nucleotide sequences of the left and right chromosomal/bacteriophage DNA junctions from S. aureus lysogens harbouring
serotype F converting phage. The lysogens 42CR3-L, A1CR3-L and A3CR3-L were derivatives of S. aureus strain 80CR3
lysogenized with the triple-converting phage 4,42, 4,A1 and 4,A3, respectively. The lysogen CN6708-L was a derivative of S. aureus
CN6708 lysogenized with the double-converting phage 4,13 [3]. The data for CN6708-L have been described previously [3] and are
included here for comparative purposes. For the lysogens harbouring triple-converting phage, sequences were determined from
PCR products obtained following amplification of the right and left junctions of each lysogen, respectively, using the specific
oligonucleotide primers shown in Table 1. The 14-bp core sequences constituting attL and attR, for each lysogen respectively, are
shown within brackets. The positions of hlb and phage attP-associated sequences are shown and numbered according to the
sequences to which they correspond in Figs. 2A and 2B, respectively. The dashed line between the left and right junction of each
lysogen represents the remainder of the prophage genome in each case. ORF refers to sequences belonging to the open reading
frame encoding/3-toxin.
154
Discussion
Serotype F converting phage are probably the
most common type of phage found in clinical
isolates of S. aureus [12,13]. The ability of these
phage to control the expression of several extracellular proteins and to mediate transfer of virulence factors between strains, leading to the development of strains with enhanced virulence potential and altered phage immunity [2,12,14],
probably accounts for the prevalence and success
of these entities. Thus, the high incidence of
enterotoxin A expression by methicillin-resistant
S. aureus isolates from Dublin hospitals is directly
attributable to the prevalence of triple-converting
phage in these organisms [14].
Sequence analysis of the attL and attR composite attachment sites of the ~b42 lysogen
42CR3-L, the attB region of S. aureus strain
80CR3 and the attP region of ~b42 demonstrated
that ~b42 integrative recombination occurred
within a 14-bp core sequence present in both
bacterial and phage genomes. The precise duplication of this sequence in the attL and attR sites
at the chromosomal/bacteriophage D N A junctions of a 4)42 lysogen indicates that ~b42 integrative recombination is site- and orientation-specific
and conservative. The asymmetrical structure of
the core, which directs orientation specificity during prophage integration, is a common feature of
many integrative phage [15]. Furthermore, the
sequence of all four attachment sites is identical
to the corresponding attL and attR composite
attachment sites of the ~b13 lysogen CN6708-L,
the attB region of S. aureus strain CN6708 and
the attP region of the serotype F double-converting phage ~b13 [3], demonstrating unequivocally
that a common molecular mechanism of S. aureus
/3-toxin inactivation is employed by ~b13 and ~b42.
Analysis of the composite attachment sites from
S. aureus 80CR3 lysogens harbouring the serotype
F triple-converting phage ~bA1 and ~bA3 demonstrated that these phage also integrate into the
hlb determinant in a manner indistinguishable
from that of 4)13 and ~b42. These findings confirm previous studies from this laboratory, which
on the basis of hybridization analysis of genomic
D N A from several S. aureus lysogens using attP-
and hlb-specific probes, indicated that both double- and triple-converting serotype F phage inserted into a single site, or closely linked sites, in
the hlb determinant [2].
The common mechanism of integration exhibited by serotype F double- and triple-converting
phage reflects the conserved organization of the
attP-associated sequences in both groups of
phage. This is reflected not only by the presence
of identical attB core sequences but by the presence of identical pairs of direct repeat sequences
flanking the core (Fig. 2B) [3]. Similar repeat
sequences have been observed flanking the core
sequences of the S. aureus phage L54a and ~bll
[16,17]. The precise function of the direct repeats
is poorly understood, but they are thought to be
involved in integrase binding during lysogenization [16].
The findings of the present study suggest that
serotype F double- and triple-converting phage
are closely related and may have evolved from a
common origin. Further studies from this !aboratory have found that the organization and expression of integrase determinants by both groups of
phage is also very similar (D. Carroll, M. Kehoe
and D. Coleman, in preparation), confirming this
suggestion.
References
1 Coleman, D.C., Arbuthnott, J.P., Pomeroy, H. and Birkbeck, T.H. (1986) Microb. Pathog. 1, 549-564.
2 Coleman,D.C., Sullivan, D.J., Russell, R.J., Arbuthnott,
J.P., Carey, B.F. and Pomeroy,H.P. (1989) J. Gen. Microbiol. 135, 1679-1697.
3 Coleman, D., Knights, J., Russell, R., Shanley, D., Birkbeck, T.H., Dougan, G. and Charles, I. (1991) Mol. Microbiol. 5, 933-939.
4 Winkler, K.C., De Waart, J., Grootsen, C., Zegers, B.J.,
Tellier, N.F. and Vertergt, C.D. (1965) J. Gen. Microbiol.
39, 321-333.
5 Kondo, I., Ito, S. and Yoshizawa, Y. (1981) In: Staphlylococci and StaphylococcalInfections (Jeljaszewicz, J., Ed.),
pp. 357-361. Gustav Fisher Verlag, Stuttgart.
6 Betley, M.J. and Mekalanos, J.J. (1985) Science 229, 185187.
7 Stobberingh,E.E. and Winkler, K.C. (1977)J. Gen. Microbiol. 99, 359-367.
8 Bullock, W.O., Fernandez, J.M. and Short, J.M. (1987)
Biotechniques 5, 376.
155
9 Lennox, E.S. (1955) Virology 1, 190-206.
10 Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Handbook, 2nd edn., Cold
Spring Harbor Laboratory, Cold Spring Harbor, NY.
11 Sanger, F., Nicklen, S., and Coulson, A.R. (1977) Proc.
Natl. Acad. Sci. USA 74, 5463-5467.
12 Coleman, D.C. (1990) In: Bacterial Protein Toxins (Rappuoli, R., Alouf, J.E., Falmagne, P., Fehrenbach, F.J.,
Freer, J., Gross, R., Jeljaszewicz, J., Montecucco, C.,
Tomasi, M., Wadstr6m, T. and Witholt, B., Eds.), Zentralblatt fiir Bakteriologie, Mikrobiologie und Hygiene 19, pp.
333-340. Gustav Fisher Verlag, Stuttgart.
13 Parker, M.T. (1983) In: Staphylococci and Staphylococcal
Infections, Vol. 1 (Easmon, C.S.F. and Adlam, C., Eds.),
pp. 33-62. Academic Press, London.
14 Humphreys, H., Keane, C.T., Hone, R., Pomeroy, H.,
Russell, R.J., Arbuthnott, J.P. and Coleman, D.C. (1989)
J. Med. Microbiol. 28, 163-172.
15 Sadowski, P. (1986) J. Bacteriol. 165, 341-347.
16 Lee, C.Y. and Iandolo, J.J. (1986) Proc. Natl. Acad. Sci.
USA 83, 5474-5478.
17 Lee, C.Y. and Iandolo, J.J. (1988) J. Bacteriol. 170, 24092411.