Résumé de l'article:
Des expériences de "footprint" de la région opérateur avec du répresseur de Mu purifié
ont permis de déterminer la taille de la région opérateur qui s'étend sur 184 pb (Krause et
Higgins, 1986). Elle contient trois séquences opérateur, 01, 02 (qui contient le promoteur
pE), et 03 (qui contient le promoteur pCM), qui contiennent respectivement trois, quatre,
et deux séquences consensus asymétriques de fixation du répresseur CTTTTN3W3 (Krause et
Higgins, 1986; Vogel et al., 1991).
L'approche in vivo classique d'isolement de mutants opérateur consiste à sélectionner
des mutants phagiques virulents, c/s-dominants, capables de surmonter l'immunité et de se
multiplier dans une bactérie lysogène. Dans le cas de Mu, elle a fourni de rares mutations
dans le répresseur (voir plus haut la description des mutations vir de Mu) mais pas dans
l'opérateur (Geuskens et al., 1991, 1992). Une explication possible de cette observation est
que les mutations de la région opérateur qui empêchent la fixation du répresseur, affectent
aussi systématiquement la fixation de la transposase et donc bloquent la réplication du phage
et sa capacité de former des plages de lyse.
Nous avons donc choisi une autre stratégie de sélection pour isoler des mutations dans
la région opérateur. Pour sélectionner des mutations qui permettent l'expression
constitutive à partir du promoteur pE, nous avons utilisé le plasmide pJV313, un plasmide à
haut nombre de copies qui exprime, à partir de son promoteur naturel pCM, le répresseur
galactosidase. Ce plasmide a été introduit dans une souche clpP de manière à augmenter la
répression de pE (Desmet, Geuskens et Gama, résultats non publiés). Cette souche n'exprime
la p-galactosidase qu'à partir de pE. En isolant des bactéries capables de se développer sur le
lactose comme seule source de carbone, vu le haut nombre de copies du plasmide pJV313,
nous nous attendions à n'isoler que des mutations c/s-dominantes, conférant le phénotype
Lac+. Les colonies isolées grâce à cette sélection répondaient effectivement à ce critère. Les
opérateurs mutés ont été séquencés ce qui a révélé la présence d'une parmi plusieurs
mutations ponctuelles ou d'une délétion.
La nature et la localisation de ces mutations, qui toutes affectent la région 02 ou ses
abords immédiats, suggèrent que
02est le site le plus important soit dans la structuration
soit dans la stabilité du complexe opérateurs-répresseur. Les résultats suggèrent aussi
l'existence d'une hiérarchie dans les sites de fixation du répresseur localisés dans les
opérateurs 01 et 02. Enfin la caractérisation de ces mutations apporte de nouvelles lumières
sur l'importance relative des bases centrales (NNN) dans les contacts entre le répresseur et
la séquence consensus Cl i i i NNNWWW.
92
Article 4: Soumis à Genetics
In vivo Mutational analysis of bactériophage Mu
operators.
Lucie Desmet*, Marie-José Gama*, Jamal E. Laachouch*, loan Petrescu^
and Ariane Toussaint*»^
‘Laboratoire de Génétique des Procaryotes, Unité Transposition Bactérienne, Université
Libre de Bruxeiles, 65 rue des Chevaux, B1640 Rhode St Genèse, Belgium.
^ Laboratoire de Biochimie des Microorganismes, Université Joseph Fourier, BP 53,
F38041 Grenoble cedex, France.
Running head: Bactériophage Mu Operators
Keywords: phage Mu, repressor, operator
Corresponding author:
Ariane Toussaint
Laboratoire de Génétique des procaryotes
Université Libre de Bruxelles
65 rue des Chevaux
B1640 Rhode St Genèse, Belgium
Tel: 32 2 650 97 41
Fax: 32 2 650 97 44
ABSTRACT
In bacteria lysogénie for bactériophage Mu, the phage repressor binds to a tripartite
operator région, 01, 02, 03, to repress the lytic promoter pE, located in 02, and negatively
autoregulate its own synthesis at the pCM promoter located in 03. We isolated and
characterized operator mutations which lead to at least partial derepression of pE. Their
location reveals a hierarchy. between operator subsites and between the repressor binding
cts45 cts71 cts25 cts62
L I D Q
13 KSIWCSPQEIMAADGMPGSVAGVHYRANVQGWTKRKKEGVKGGKAVEYDVMSMPTKEREQVIAHLGLSTPDTGAQANEKQ 92
1 MELWVSPKECANLPGLPKTSAGVIYVAKKQGWQNRTRAGVKGGKAIEYNANSLPVEAKAALLLRQGEIETSLGYFEIARP 80
V7////////A yCHZ] V///////M
H3
W
[ YZZZ2t
B1 H1 H2
DNA binding
Figure 1. Alignement of the repressor and transposase N-terminal régions.
Residues 13 to 92 of Mu repressor hâve been aligned on transposase residues 1 to 80 using the GCG Bestfit program. The repressor cts
mutations (VOGEL et al., 1991) are shown at their positions. B, H, T, and W strand for b-sheet, a-helices, turn and wing respectively. The DNA
INTRODUCTION
Bactériophage Mu is a transposable temperate phage. Upon infection of a suitable host
(here E.coli K12), the injected viral DNA intégrâtes at random in the host genome. Although
this remains to be demonstrated, it is usually assumed that it is after this first intégration
event that the décision is made between remaining a silent, repressed prophage or entering
the lytic cycle. In the first case, repressor is expressed from the pCM promoter and binds a
tripartite operator, blocking transcription from the early promoter pE. Promoters pCM and
pE initiate overlapping transcripts in opposite directions, generating mRNA with
complementary 5' ends. In the second case, transcription from pE leads to the expression of
the transposition proteins pA (the transposase) and pB (a DNA dépendent ATPase which
favors inter-molecular transposition events) and other early lytic proteins. Viral DNA
copies accumulate at random positions in the host genome by successive rounds of réplicative
transposition (for review see SYMONDS étal. 1987, TOUSSAINT et al. 1994, HANIFORD and
CHACONAS 1992, MIZUUCHI 1992). The 250-bp Mu early regulatory région plays a
crucial rôle in both cases. In the prophage State this région is tightiy bound by repressor.
During réplication it is transiently bound by transposase to allow for the synapsis of the
first réplicative transposition intermediate (for review see HANIFORD and CHACONAS
1992, MIZUUCHI 1992) and hence is called lAS (for internai activating sequence). The
repressor and transposase N-terminal régions (around 70 amino acids) are homologous
(44% identity, HARSHEY et al. 1985) and define the operator binding moiety of the
proteins.
The operator région, as defined by footprinting experiments with purified repressor,
is 184 bp long (KRAUSE and HIGGINS 1986). It contains three operators, 01, 02 and 03
with respectively three, four and two asymmetrical
11bp consensus repressor binding
sites (CTTTTN3W3, KRAUSE and HIGGINS 1986, VOGEL étal. 1991). It aiso contains the pE
(in 02) and pCM (in 03) promoters. Operators sites 01 and 02 overlap with the lAS
transposase binding sequence (LEUNG et al., 1989). Binding of Mu repressor to the
operators is stable and cooperative (VOGEL et al. 1991) and is facilitated by the host IHF
(GAMA étal. 1992, ALAZARD étal. 1992) and H-NS (FALCONI étal. 1991) histone-like
proteins. Binding of transposase is transient and the influence of histone-like proteins on
this binding has not been directiy investigated.
The three dimentional structure of the transposase N-terminal domain was recently
solved and the lAS binding domain consiste of a novel class of winged helix-turn-helix motif
(CLUBB et al., 1994). In view of the high similarity between repressor and transposase in
that région and the location of ail 4 types of thermosensitive DNA binding repressor
mutations in that région. Mu repressor is very likely to display the same structural
features (see Figure 1).
95
In order to better understand repressor-operator interactions and the features that
distinguish repressor and transposase binding to that région, it is important to isolate and
characterize operator mutations which modify these interactions. The classical in vivo
approach, i.e. the isolation of virulent mutant Mu phages able to overcome Mu immunity and
multipiy in a Mu lysogen, unexpectedly provided very rare repressor mutations but no
operator mutations (GEUSKENS et al. 1991, 1992). One possible explanation for that
observation is that mutations in the operator that prevent repressor binding (O^) aiso
simultaneousiy affect transposase binding and hence block phage DNA réplication and the
ability to form plaques.
Therefore, in order to hâve a first évaluation of the relative importance of the
different operator subsites and their nucléotides for interaction with repressor, we set up a
different in vivo sélection to isolate mutations which allow for constitutive expression from
the early pE promoter. The characterization of these mutations confirmed their location in
the operator région, suggests the existence of a hierarchy in the repressor binding sites
located in the
01and
02operators and sheds more light on the relative importance of some
MATERIALS AND METHODS
Bacterial strains. Ail the bacterial strains used in this study were derived from
MC4100 (CASADABAN 1976) which was lysogenized with Mucfs62 (HOWE 1973) or Muc"^
(TAYLOR 1963). MC4100/j/mA;:Tn10 (GAMA étal. 1992) was constructed by transducing
the D82himA::Tn10 mutation from MC253 (M. Chandler's collection) into MC4100 with
phage PI. The pJV300, 304 and 313 plasmids hâve been described (VOGEL étal. 1991,
GEUSKENS et al. 1992). They carry the left end of Mu, with the c"^, cts62 and
cts62,sts62-1 mutations respectively, up to the pE promoter, which is fused to the lac operon (see
results for more details).
Media. Bacteria were grown in Luria-Bertani broth (LB) (Miller, 1972) and titrated on
LB agar plates (MILLER, 1972) containing LB supplemented with 1.2% Difco agar. Minimal
medium was 132 (GLANSDORFF étal., 1965) supplemented with 0.2% lactose. Kanamycin
(Kn, 25 pg ml‘^) and ampicillin (Ap, 25 pg ml*"') were included when appropriate. Mac
Conkey-lactose was from Difco.
»
Chemicals and enzymes. Enzymes were purchased from Boehringer-Mannheim and used
as recommended by the suppliers. ‘
Methods. General methods used in the manipulatin of phages were those of BUKHARI and
LJUNDQUIST (1977).
p-galactosidase was assayed as described by MILLER (1972).
*
Small-scale extraction of plasmid DNA and transformation of that DNA into competent cells
were performed as described by MANIATIS étal. (1982).
Repressor in crude bacterial extracts was assayed as described by GEUSKENS et al. (1991)
A 981
976
I
1004 1011
02
Hind m
GGAATTTACCAAAAAGCAGCmACAAAAAaCTTTTCA&AÆTArCTTTTTAGTAAGC
il II
G (7.4) A(L5) A T (4.1, 4.2, 6.1,8.4, L3)
(1.5, L8)
T) 880 01
repressor translation initiation
ACACCAi^ ATTGACTTTTCAGTATTATTCTTTTCTATAAAGTTACTTTTCAAAAT|TTlAAAC'rCC’r
111111 M 1111 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _
ACACCAP. ATTGAC^
IHF 02
1005
TATTTATCA ÆGCGTTAATCAGTAA rCAAAGGAATTTACCAAAAAGCAGC rTTAc; AA I^AGCTT
M
TT
deleted région ^
rTCAGTAAltatct: 'TTTAGTAAGCTAGCTA\gtttttacacttagttaaattgctaactttat
IMM IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIMIIIIIIIIIIIIIIII
TATCT'; 'TTTAGTAAGCTAGCTA\GTTTTTACACTTAGTTAAATTGCTAACTTTAT
03
Hind III site
AGATT^CAAAACTTAGGAGGGTTTTTAAATGTGTTCCAACGAAAAGGCCCGTGATTGGCATCGT
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIMIIMIIIIMIIIMIIIIIIII
AGATTACAAAACTTAGGAGGGTTTTTAAATGTGTTCCAACGAAAAGGCCCGTGATTGGCATCGT
-35
A. Point mutations. The coordinates are calculated from the first nucléotide at the Mu left
(or c) end (PRESS et al. 1987). The Hind\\\ restriction site is marked by a shaded box. The
horizontal arrows delineate the consensus repressor binding sequences. Among the mutants
analyzed eonly 4.1 and 4.2 originated from the same mutagenized culture and could be
siblings.
B. Délétion mutation. The 01, 02, 03 and IHF binding sites are boxed (open boxes for
operators, hatched box for IHF) according to footprinitng data published earlier (Krause and
Higgins 1986, Alazard et al. 1992). Coordinates are as in A. The délétion in mutant 1.3 is
shown by the thick horizontal arrow. The pE and pCM promoters are shown with their -35
and -10 postitions. The new promoter generated after the délétion could hâve its -35
position at the border of the délétion (TTGACT starting at position 887) and its -10 in the
1022-1032 région.
C. The plasmid used for selecting operator mutations. The left end of Mu up to the pCM
promoter was inserted in pRS551, a pBR322 dérivative (SIMONS étal. 1987), at the EcoRI
site (VOGEL et al. 1991). In this construct the Mu c gene is expressed from the pCM prmoter
97
RESULTS
Sélection of mutations allowing for constitutive expression from pE.
Using plasmid pRS551 (a pBR322 dérivative, SIMONS et al 1987), VOGEL et al.
(1991) constructed plasmids which contain the left end of Mu, including the repressor gene
and the whole regulatory région. In these constructs, repressor is expressed from its
naturel promoter pCM and p_E is fused to the lac operon (see Figure 2C). In bacteria which
hâve a chromosomal délétion of the lac operon, the plasmids should allow for sélection of
cis-dominant constitutive expression from pE, by isolating bacteria able to grow with
lactose as the sole carbon source.
The pRS551 dérivatives pJV300, pJV304 and pJV313 were introduced into MC4100.
The three plasmids respectively carry the wiid type, cts62 and cfs62,sfs62-1 repressor
genes. The cts62 mutation is a thermosensitive mutation which confers thermoinducibility
to Mucfs62 prophages and to pE-initiated lacZ expression from pJV304. The sfs mutation
(for suppression of température sensitivity) is an amber mutation which truncate the
repressor C-terminal end by 18 amino acids. Prophages with a cfs62,sfs62-1 mutated
repressor gene are no more induced at high température. Moreover, contrary to the cts62
repressor which binds Mu operator DNA very poorly at 42°C, the purified cfs62,sfs62-1
repressor binds operator DNA at 42°C almost as efficiently as wild type repressor (VOGEL
et al., submitted for publication). Although there is no clear understanding of the molecular
basis of the Sts phenotype yet, ail the available experimental résulta suggest that it behaves
as a "super-repressor" (VOGEL et al., submitted for publication). Ail these strains grew
well on minimal lactose medium at ail températures. This is most likely due to the fact that,
on the plasmids used, pE is not fully repressed (VOGEL et a/.,1991). Since there are
indications that the host Clp protease destabilizes Mu repressor (GEUSKENS et al. 1992, our
unpublished résulta, SHAPIRO 1993), we transformed the three plasmids in a
MC4100c/pP::Cm strain. In that genetic background, repression of lacZ expression on
pJV313 (with the cts.sts c gene) was indeed strong enough to prevent growth on minimal
lactose medium.
Several independent cultures of MC4100c/pP::Cm/pJV313 were grown in LB,
mutagenized with ethyl-methane-sulfonate (EMS, which cause primarily AT->GC
transitions) and plated on minimal lactose plates which were incubated at 30°C. Control
plates of the same cultures not treated with EMS revealed that the overall mutation
frequency was high (around 10'^) and the same in ail cultures. Consequently, it is not clear
whether the mutants recovered arose from induced or spontaneous mutations.
Isolated colonies were purified on MacConkey lactose medium at 30°C; ail colonies
were red. Then 63 mutant plasmids recovered from 12 independent mutagenised cultures
were transformed into MC4100(Mucfs62) in order to check for the cis-dominance of the
Each Slot has been loaded with 5 pi of a bacterial crude extract.The gel is
12