Phage-borne factors and host LexA regulate the lytic...
Transcript of Phage-borne factors and host LexA regulate the lytic...
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Phage-borne factors and host LexA regulate the lytic switch in phage GIL01 1
Running title: Phage GIL01 is under LexA control 2
Nadine Fornelos1,2, Jaana K. H. Bamford2* and Jacques Mahillon1 3
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1 Laboratory of Food and Environmental Microbiology 6
Université catholique de Louvain 7
Croix du Sud, 2, Box L7.05.12 8
B-1348 Louvain-la-Neuve 9
Belgium 10
2 Department of Biological and Environmental Science and Nanoscience Center 11
University of Jyväskylä 12
P. O. Box 35 13
40014 Jyväskylä 14
Finland 15
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*Corresponding author. Mailing address: Department of Biological and Environmental Science 17
and Nanoscience Center, University of Jyväskylä, P. O. Box 35, 40014 Jyväskylä, Finland. 18
Phone: +358 14 260 4160. Fax: +358 14 260 2221. Email: [email protected] 19
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Keywords: prophage, lytic switch, clear plaque mutant, SOS response, LexA 22
Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Bacteriol. doi:10.1128/JB.05618-11 JB Accepts, published online ahead of print on 2 September 2011
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Abstract 23
The Bacillus thuringiensis temperate phage GIL01 does not integrate into the host chromosome 24
but exists stably as an independent linear replicon within the cell. Similar to the lambdoid 25
prophages, the lytic cycle of GIL01 is induced as part of the cellular SOS response to DNA 26
damage. However, no CI-like maintenance repressor has been detected in the phage genome, 27
suggesting that GIL01 uses a novel mechanism to maintain lysogeny. To gain insights into the 28
GIL01 regulatory circuit, we isolated and characterized a set of 17 clear plaque (cp) mutants that 29
are unable to lysogenize. Two phage-encoded proteins, gp1 and gp7, are required for stable 30
lysogen formation. Analysis of cp mutants also identified a 14-bp palindromic dinBox1 sequence 31
within the P1-P2 promoter region that resembles the known LexA binding site of Gram-positive 32
bacteria. Mutations at conserved positions in dinBox1 result in a cp phenotype. Genomic analysis 33
identified a total of three dinBox sites within GIL01 promoter regions. To investigate the 34
possibility that the host LexA regulates GIL01, phage induction was measured in a host carrying 35
a non-cleavable lexA (Ind-) mutation. GIL01 formed stable lysogens in this host but lytic growth 36
could not be induced by treatment with mitomycin C. Also, mitomycin C induced β-37
galactosidase expression from GIL01-lacZ promoter fusions and induction was similarly blocked 38
in the lexA (Ind-) host. These data support a model in which host LexA binds to dinBox 39
sequences in GIL01, repressing phage gene expression during lysogeny and providing the switch 40
necessary to enter lytic development. 41
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Introduction 42
Temperate phages are defined by their ability to infect a susceptible host cell and opt between 43
either of two developmental pathways, the lytic or the lysogenic cycle. In the lytic cycle, the 44
phage genome is replicated and expressed to produce the structural components necessary to 45
assemble capsids. Once engaged, this pathway inevitably ends with the death of the host cell and 46
the release of a crop of newly formed virions in the environment. In contrast, the lysogenic cycle 47
is defined by the tight repression of lytic behavior and the phage genome replicates along with 48
that of the host cell, either physically integrated into the host genome or as an independent 49
plasmid prophage. The quiescent prophage is thereby efficiently inherited as cells divide, until 50
the lytic cycle is reactivated in response to alterations in host cell physiology. This transition 51
requires the efficient interaction of regulatory components that control the passage from a highly 52
stable stand-by mode to multiple rounds of genome replication, vigorous gene expression, virion 53
assembly and host lysis. 54
In the well-studied bacteriophage λ, establishment of lysogeny relies on the synthesis of CII, a 55
transcriptional regulator that directs the expression of the lytic cycle repressor CI (15). Once 56
produced, CI dimers bind cooperatively at operator sites in the so-called switch region to prevent 57
transcription from the early promoters PL and PR and subsequently, due to the lack of early gene 58
products, from the late lytic promoters as well. Expression of CI alone is sufficient to maintain 59
lysogeny once it has been established (26). Despite an intrinsically stable quiescent state, the λ 60
prophage can efficiently switch to an alternate lytic cycle. The λ CI repressor functions 61
analogously to the bacterial SOS response master repressor LexA, to which it shares significant 62
sequence similarity (21, 45). LexA binds as a dimer to specific operator sites and negatively 63
regulates the expression of a host of genes that are essentially involved in DNA repair and cell 64
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survival (10, 18). Upon DNA damage, such as that inflicted by UV irradiation or mitomycin C 65
(MMC) treatment, the host recombinase RecA is converted to an activated form when it binds 66
single-stranded DNA. Activated RecA then acts as a co-protease to stimulate LexA auto-67
proteolysis and through a similar process, CI auto-proteolysis as well (43, 59). As a consequence, 68
LexA dissociates from its consensus sites to initiate the SOS response. Likewise, CI is no longer 69
able to bind its operator sites in the λ genome, leading to lytic gene expression and viral 70
proliferation. 71
Alternative mechanisms of regulation have been described in several SOS-inducible phages. The 72
repressors of coliphages 186 and N15, for example, are not RecA-sensitive but are instead 73
antagonized by phage-borne anti-repressors that are under LexA control (47, 61). In CTX�, the 74
filamentous phage that encodes cholera toxin in Vibrio cholerae, host LexA and phage repressor 75
function as both activators and repressors of gene expression to ensure the permanent production 76
and secretion of CTX� (36). 77
The Bacillus thuringiensis prophage GIL01 has been shown to be induced by DNA-damaging 78
treatments such as UV irradiation and MMC (69). The 15 kb linear prophage does not integrate 79
into the bacterial chromosome but instead, replicates independently within its host and 80
occasionally experiences induction of its lytic cycle. GIL01 shares a similar genome organization 81
and size with PRD1, the model tectivirus infecting Gram-negative hosts. However, unlike PRD1 82
and its close siblings, which are all lytic phages, GIL01 is able to enter a dormant state during 83
which its lytic functions are turned off. Two additional tectiviruses infecting B. thuringiensis 84
have also been described: Bam35, which is almost identical to GIL01 (55, 64) and has been the 85
object of many studies on the internal membrane that defines tectiviruses (20, 39-41), and 86
GIL16, which shares considerable sequence identity (83.6%) with GIL01 (67). A more distant 87
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relative, pBClin15, has been sequenced alongside with the B. cereus reference strain ATCC 88
14579 genome but is not yet known to be a prophage (29). Recently, the sequence of AP50, a 89
temperate phage highly specific to B. anthracis strains, added a new member to the subgroup of 90
Tectiviridae that proliferate among members of the B. cereus group (62). 91
By analogy to other temperate phages, it has been assumed that GIL01 controls its lysogenic 92
cycle by expressing a lytic gene repressor. A potential candidate for this function was predicted 93
to be encoded by open reading frame 6 (ORF6), by virtue of its N-terminal LexA-like DNA-94
binding domain (67). However, unlike the phage λ CI repressor, ORF6 lacks any recognizable 95
protease linker required for repressor cleavage or C-terminal dimerization domain. Furthermore, 96
spontaneous GIL01 clear plaque (cp) mutants – that are unable to establish the lysogenic cycle – 97
were previously mapped to ORF7 but no such mutations were found to affect ORF6 (67). In this 98
study, we demonstrate that ORF7 is indeed involved in the regulation of the phage cycle and that 99
it does so along with ORF1. Importantly, we also show that GIL01 is induced in response to SOS 100
signals due to the direct control by the host LexA repressor. LexA likely binds to a conserved 101
operator, termed dinBox1 (for damage-inducible Box), that when mutated commits GIL01 to 102
propagate exclusively as a virulent phage. dinBox1 lies within two promoters, P1 and P2, that 103
direct the expression of regulation and replication functions that are likely important for 104
lysogenic development/maintenance. Two additional LexA binding sites, dinBox2 and dinBox3, 105
were identified at promoter P3, regulating the downstream structure and lysis module. Curiously, 106
no cp mutants were ever isolated that carried mutations within these operator sites, possibly 107
indicating that dinBox2 and dinBox3 are not required for lysogenic regulation but are important 108
for lytic growth. Altogether, we identified one cis-acting element (dinBox1), two phage-borne 109
genes (ORF1 and ORF7) and a host regulator (LexA) that determine the passage from the latent 110
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state to lytic development in GIL01. The identification of these factors will ultimately help 111
elucidate the gene circuit that regulates GIL01 development. 112
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Materials and Methods 113
Bacterial strains, phages and plasmids 114
The natural host of phage GIL01 is the insect pathogen B. thuringiensis subspecies israelensis 115
strain NB31 (2). The permissive plasmid-free strain GBJ002 (31), derived from strain 4Q2 and 116
related to NB31 (32), was used to propagate GIL01. In order to work in identical genetic 117
backgrounds, GBJ002 lysogens were generated as follows. GIL01 filter-sterilized suspensions 118
were spotted onto 0.7% top agar lawns seeded with GBJ002. Plates were incubated overnight at 119
37°C. The center of a lysed spot was then recovered with a sterile inoculation loop and streaked 120
on LB agar. After 16 hours at 37°C, individual colonies had grown and were screened by 121
polymerase chain reaction (PCR) with GIL01-specific primers to confirm lysogeny. A positive 122
colony was selected (GBJ338) and retained for the study. 123
GIL01 clear plaque (cp) mutants were obtained spontaneously by infecting GBJ002 with wild-124
type GIL01 phage suspensions. Briefly, phage preparations from serial 10-fold dilutions were 125
added to 200 µl of mid-log-phase GBJ002 and incubated at room temperature for 30 minutes. 126
The infected cells were then plated on LB agar with 4 ml of molten 0.7% top agar. The plates 127
were incubated overnight at 37°C and visually screened for the presence of clear plaques. Single 128
clear plaques, isolated from independent experiments, were picked with micropipette tips and 129
subjected to three rounds of single plaque purification on host GBJ002. The wild-type pGIL01 130
genome sequence is available from the NCBI GenBank database under the accession number 131
AJ536073. 132
Strain GBJ499 (recA-) was constructed by disrupting the recA gene of strain GBJ002 with the 133
integrative vector pMutin4 (Bacillus Genetic Stock Center). pMutin4 is unable to replicate in 134
Bacillus and is therefore particularly well-suited for insertional mutagenesis in this host (66). 135
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Briefly, an internal fragment of recA (coordinates 3,719,072 through 3,720,009 in B. cereus 136
strain G9842, GenBank acc. number NC_011772) was amplified from strain GBJ002 with 137
primers RecAmutF (5’- GCGGCCGCTCTTCACCAATTCCGTGAT) and RecAmutR (5’- 138
GAATTCCAATTTGGTAAAGGTTCAATTA). The PCR product was digested with NotI and 139
EcoRI (restriction sites underlined), purified and cloned into NotI- and EcoRI-digested pMutin4 140
vector. The ligation was transformed into Escherichia coli (ER2925) and recombinant plasmids 141
were extracted and introduced into competent GBJ002. Bacillus transformants having integrated 142
the vector by homologous recombination were selected on 0.3 µg erythromycin ml-1. The 143
insertion of pMutin4 in the desired locus was confirmed by sequence analysis of recA. The 144
downstream gene, which encodes a phosphodiesterase (B1422), is separated from recA (B1421) 145
by a 500-bp non-coding region, including a Rho-independent terminator. It is therefore unlikely 146
that pMutin4 insertion in recA disrupts an operon. 147
Strain GBJ396 (lexAA96D) was constructed by substituting an Ala (GCT) residue at position 96 in 148
lexA with Asp (GAT). A 1,6-kb segment encompassing lexA (coordinates 3,623,569 through 149
3,625,169 in B. cereus strain G9842, GenBank acc. number NC_011772) was amplified from 150
strain GBJ002 with primers MutLex1 (5’- GGATCCTTGATATTCTTCAACAATAC) and 151
MutLex2 (5’- AAGCTTGTGTTAGAACACTTGCTGGA) and cloned into the pCR4-TOPO 152
vector (Invitrogen). The ligation was transformed into E. coli (TOP10) and recombinants were 153
extracted to undergo mutagenesis. A single nucleotide mutation was generated by using the 154
QuickChange II Site-Directed Mutagenesis kit (Stratagene) with primer pairs LexindF (5’- 155
GTCGGAAAAGTTACTGATGGTTTACCAATTACAGCG) and LexindR (5’- 156
CGCTGTAATTGGTAAACCATCAGTAACTTTTCCGAC) according to the manufacturer’s 157
instructions. DpnI-treated vector DNA was transformed into XL1-Blue E. coli cells and 158
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transformants were selected on 50 µg kanamycin ml-1. Mutants were confirmed by DNA 159
sequence analysis with the universal sequencing primers M13. Mutated DNA from one selected 160
clone was restricted with BamHI and HindIII (restriction sites underlined in MutLex1 and 161
MutLex2) and the lexA-containing fragment was cloned into BamHI- and HindIII-digested 162
pMutin4 plasmid DNA. The ligation was transformed into E. coli (ER2925) and recombinant 163
plasmids were extracted and introduced into competent GBJ002. Similarly to above, Bacillus 164
transformants having integrated the vector by homologous recombination were selected on 0.3 165
µg erythromycin ml-1. One of these recombinants was grown for several consecutive cycles of 166
four hours in liquid LB and samples were recovered at each cycle and plated on LB agar at 167
different dilutions. Single colonies were subjected to UV irradiation (wavelength 254 nm) for 10 168
seconds and left at room temperature for two days. UV-sensitive colonies were amplified with 169
primers MutLex1 and MutLex2 and the resulting PCR products were sequenced in order to 170
screen for clones that had undergone a second recombination event resulting in the lexAA96D 171
genotype. Lysogens GBJ500 and GBJ406 were generated by infecting the mutant strains 172
GBJ499 and GBJ396 with GIL01 as explained above. 173
E. coli strain ER2925 (New England Biolabs), a dam- and dcm- K12 derivative, was used to 174
propagate un-methylated constructs prior to transformation into GBJ002. pCR4-TOPO-derived 175
constructs were transformed into E. coli strain TOP10 (Invitrogen) or XL1-Blue (Startagene). 176
ORF1 and ORF7 were cloned into the multicopy plasmid pDG148, an E. coli – Bacillus spp. 177
shuttle vector with an isopropyl-β-D-thiogalactopyranoside (IPTG)-inducible Pspac promoter 178
upstream of the multiple cloning site (63). The pCR4-TOPO (Invitrogen) positive selection 179
vector was used to clone 5’-RACE generated fragments for sequencing as well as the amplified 180
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lexA gene and promoters P1-P2 segments for site-directed mutagenesis. pHT304-18Z is a low-181
copy-number shuttle vector used to generate lacZ fusions in B. cereus (1). 182
Growth conditions and phage induction 183
B. thuringiensis and E. coli strains were grown in modified Lysogeny Broth (LB) (6) (1% 184
tryptone, 0,5% yeast extract, 0,5% NaCl) at 37°C. B. thuringiensis liquid cultures were always 185
grown overnight with shaking at 200 rpm before being diluted 1:100 into LB and allowed to 186
grow exponentially. Transformation of B. thuringiensis and E. coli strains with the different 187
constructs was performed by electroporation (46, 57) and transformants were selected on LB 188
agar supplemented with 25 µg kanamycin ml-1 (pDG148-derived constructs), 50 µg kanamycin 189
ml-1 (pCR4-TOPO-derived constructs), 25 µg erythromycin ml-1 and 100 µg ampicillin ml-1 190
(pHT304-18Z-derived constructs in B. thuringiensis and E. coli, respectively) or 0.3 µg 191
erythromycin ml-1 and 100 µg ampicillin ml-1 (pMutin4-derived constructs in B. thuringiensis 192
and E. coli, respectively). 193
GIL01 was induced from GBJ338, GBJ500 and GBJ406 by UV irradiation and MMC treatment. 194
In both cases, exponential lysogenic cultures were pelleted and resuspended in 5 ml of 10 mM 195
MgSO4. For UV induction, the resuspended cells were irradiated at 254 nm for 10 seconds at a 196
distance of 10 cm. The irradiated cells were then incubated at 37°C and 200 rpm for 15 minutes, 197
pelleted and resuspended in liquid LB. Phage induction was allowed to proceed for 90 minutes at 198
37°C and 200 rpm before culture supernatants were recovered and filtered (0.45 µm). For MMC 199
induction, MMC was added to 5 ml of resuspended cells at a concentration of 50 ng ml-1. The 200
cells were then incubated at 37°C and 200 rpm for one hour before being pelleted and washed 201
twice with 10 mM MgSO4. The pellets were finally resuspended in liquid LB and phage 202
induction was allowed to proceed for 90 minutes at 37°C and 200 rpm before culture 203
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supernatants were recovered and filtered (0.45 µm). Control samples that were not irradiated or 204
MMC-induced were otherwise treated equally. GIL01 titers from induced and un-induced 205
cultures were estimated by infecting GBJ002 in soft agar (0.7%). 206
DNA, enzymes and reagents 207
Nucleotides and DNA modification enzymes were purchased from Roche and Fermentas. The 208
Phusion Flash High-Fidelity PCR Master Mix (Finnzymes) and GoTaq Flexi DNA polymerase 209
(Promega) were used in PCR amplifications. Oligonucleotides, antibiotics, mitomycin C, 210
isopropyl-β-D-thiogalactopyranoside (IPTG) and GenElute Plasmid Miniprep Kits were 211
purchased from Sigma-Aldrich. QIAquick Gel Extraction and PCR Purification Kits were 212
purchased from Qiagen. 213
Primer extension assays 214
Total RNA was extracted from mid-log-phase GBJ338 cultures with the Ambion RiboPureTM-215
Bacteria Kit and treated with DNase DNA-freeTM solution (Ambion). Transcription start sites 216
were determined with the 5’ RACE System for Rapid Amplification of cDNA Ends (Invitrogen) 217
according to the manufacturer’s instructions. Phage-specific primers hybridizing downstream of 218
P1-P2 (5’- TGTCCACTGTCATAATATCATTT, at coordinates 619-641) and P3 (5’- 219
TTGGAGATTATTTATTGACAAGA, at coordinates 5,233-5,255) were used to synthesize 220
cDNA strands from GIL01 transcripts. cDNAs were tailed and PCR-amplified before being 221
cloned into pCR4-TOPO and sequenced. 222
lacZ fusions and β-galactosidase assays 223
The region encompassing promoters P1 and P2 as well as dinBox1 (coordinates 41 through 413) 224
was amplified using primers pHT1 (5’- AAGCTTGACATGTAATAACATTTATG) and pHT4 225
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(5’- GGATCCTCTTTTTATGAACACGCA). The corresponding product was purified and 226
cloned into the selection vector pCR4-TOPO. The ligation was transformed in competent TOP10 227
cells and recombinants were extracted and restricted with enzymes HindIII and BamHI. The gel-228
purified fragments were then ligated into HindIII- and BamHI-digested pHT304-18Z to obtain 229
construct pDin1-2 in strain ER2925. A similar strategy was followed to generate a lacZ fusion 230
with promoter P3. The region encompassing the promoter sequence, dinBox2 and dinBox3 231
(coordinates 4,751 through 4,976) was amplified with primers pHT5 (5’- 232
AAGCTTAGACAAGCAAAACCGACA) and pHT6 (5’- 233
GTCGACTATGATTAGTTCCGTTAAAG), cloned into pCR4-TOPO, restricted with HindIII 234
and SalI and ligated to HindIII- and SalI-digested pHT304-18Z to obtain construct pDin3. Both 235
constructs pDin1-2 and pDin3 were confirmed by DNA sequence analysis and transformed to 236
competent GBJ338 cells. To generate pDin1-2mut, in which dinBox1 contains the same single 237
nucleotide mutation as in Bam35c (CGAACaagcGTTTT to CGAACaagcTTTTT), the 238
QuickChange II Site-Directed Mutagenesis kit (Stratagene) was used with primer pairs DinIndF 239
(5’- GTTAAAAAACGAACAAGCTTTTTATAAGTGTTCGG) and DinIndR (5’- 240
CCGAACACTTATAAAAAGCTTGTTCGTTTTTTAAC) on construct pCR4-TOPO containing 241
the Din1-2 insert. After verification of the desired mutation by sequence analysis, the same 242
cloning steps as for the generation of wild-type pDin1-2 were followed. 243
Bacillus transformants were grown in liquid cultures supplemented with 10 µg erythromycin ml-1 244
to exponential phase, at which point MMC 50 ng ml-1 was added to one-half of the cultures. 245
Cultures were further incubated for one additional hour and samples (20 µl) from each different 246
condition tested were taken. β-galactosidase activities were assayed as previously described (16) 247
with some modifications. Bacterial cells were permeabilized with 980 µl of buffer Z (containing 248
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50 mM β-mercaptoethanol) and 10 µl of toluene. After 5 minutes at 37°C, 200 µl of buffer Z 249
supplemented with ONPG (4 mg ml-1) were added to each sample. The treated samples were 250
incubated at 37°C for a time period ranging from 20 to 30 minutes and reactions were stopped by 251
adding 500 µl of Na2CO3 1M. The samples were centrifuged for 7 minutes at 13,000 rpm and 252
supernatants were collected. Optical densities were measured at a wavelength of 420 nm. The β-253
galactosidase activity values are expressed as Miller units and were calculated with the formula: 254
1000*(A420/A595*time (minutes)*volume (0.02)). 255
pDG1, pDG7 and pDG1/7 plasmid constructions and complementation tests 256
ORF1 (coordinates 356-532) was amplified from GIL01 DNA with primers DG1F (5’- 257
GCTCTAGATGAGTAAACTACTGACTGCG) and DG1R (5’- 258
TGCGCATGCTTAATTGTCCAGTTTATTTT) and digested with XbaI and SphI (restriction 259
sites underlined). The restriction fragment was purified and ligated to XbaI- and SphI-digested 260
pDG148 plasmid DNA to generate pDG1. pDG7 was constructed similarly by using primer pairs 261
DG7F (5’- GCTCTAGATGCGTGACAAATTGCTCG) and DG7R (5’- 262
TGCGCATGCTCAGTCATCCTTCTTCCCC) (coordinates 4,564 through 4,716). An ORF1-263
ORF7 transcriptional unit was generated by amplifying ORF1 with primers DG1_rbsF (5’- 264
TGCAAGCTTGaaggtggtgCATATGTAAT) and DG1_rbsR (5’- 265
GCTCTAGATTAATTGTCCAGTTTATTTTTA) (coordinates 336 through 532, including the 266
ORF1 ribosome binding site shown in Fig. 1C in lower case), restricting the PCR product with 267
HindIII and XbaI, and cloning the purified product into HindIII- and XbaI-digested pDG148 268
plasmid resulting in construct pDG1_rbs. ORF7 was amplified with primers DG7_rbsF (5’- 269
GCTCTAGAaaggagggAAAAGGTATG) and DG7_rbsR (5’- 270
GCTCTAGATCAGTCATCCTTCTTCC) (coordinates 4,549 through 4,716, including the ORF7 271
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ribosome binding site shown in lower case). After digestion with XbaI and gel purification, the 272
fragment was ligated into XbaI-digested pDG1_rbs plasmid, resulting in construct pDG1/7. 273
pDG1, pDG7 and pDG1/7 ligations were transformed into competent E. coli (ER2925) and 274
recombinant plasmids were extracted and confirmed by DNA sequence analysis. Competent 275
GBJ002 was transformed with un-methylated pDG1, pDG7 and pDG1/7 (containing ORF7 in 276
the correct orientation) to yield the clones GBJ250, GBJ223 and GBJ467, respectively. Protein 277
expression was induced by adding IPTG to a final concentration of 1 mM to one-half of liquid 278
cultures in early exponential growth phase. Control cultures were grown without addition of 279
IPTG. After an additional 3-hours incubation at 37°C with shaking, 200 µl of induced cultures 280
were infected with three 10-fold serial dilutions of cp mutant phage stocks and plated with 281
molten top agar (0.7%) on LB plates supplemented with IPTG to a final concentration of 1 mM. 282
Un-induced cultures followed the same treatment but were plated on LB plates devoid of IPTG. 283
The plates were incubated overnight at 37°C and plaque counts were determined. Plate pictures 284
were taken with the Bio-Rad Molecular Imager Gel Doc XR System using the Quantity One 285
version 4.6.3. imaging software. 286
Sequencing and Sequence analyses 287
Constructs were sequenced at the Flanders Interuniversity Institute for Biotechnology (VIB) 288
Genetic Service Facility (University of Antwerp, Belgium) by double strand primer walking and 289
with individual quality control of sequence reads included. Cp mutants were amplified with 290
GIL01-specific primer pairs designed to cover the 15-kb genome in segments of 1 kb with 100-291
bp overlaps. PCR products were purified and sequenced with a BigDye® Terminator v3.1 Cycle 292
Sequencing Kit on an ABI Prism 3130xl Genetic Analyzer (both from Applied Biosystems). Cp 293
genomes were sequenced on both strands to provide 3- to 4-fold sequence coverage. Sequences 294
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were analyzed with the EMBOSS package at the Belgian EMBL node and the EMBL-EBI tools. 295
GBJ002 sequences were compared to the genome of strain G9842 (GenBank acc. number 296
NC_011772), to which GBJ002 shares most sequence identity. 297
Phylogenetic footprints discovery 298
To discover evolutionary conserved elements, we used the program footprint-discovery from 299
RSAT (Regulatory Sequence Analysis Tools) (65) and followed the protocol as described for the 300
study case 2 (17). This strategy has been systematically evaluated on E. coli and gives good 301
results at different taxonomical levels for well-conserved genes such as lexA (30). The algorithm 302
was run on the lexA and recA promoter sequences from the B. cereus reference strain ATCC 303
14579 (GenBank acc. number AE016877) and the GIL01 promoters. 304
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Results 305
The GIL01 genome has two major functional domains 306
Like most phage genomes sequenced to date, GIL01 contains closely packed genes with few 307
intergenic spaces. All GIL01 genes are transcribed in the same rightward direction and the ORFs 308
are frequently overlapping, suggesting that GIL01 genes are organized into several operons (Fig. 309
1A). Interestingly, genes with similar functions cluster together, forming two functional 310
modules: the left arm of approximately 4.8 kbp contains replication and regulatory genes, such as 311
the predicted terminal protein (ORF4), the DNA polymerase (ORF5) and two proteins with 312
predicted DNA-binding domains (ORF1 and ORF6). The larger downstream module (10 kbp) 313
contains all the structural and assembly genes as well as two endolysins (ORF26 and ORF30) 314
involved in cell wall degradation (68). Two non-coding segments stand out by their length and 315
position in the genome as containing potential promoter/regulatory elements. The first one lies in 316
the left extremity, upstream of ORF1 (355 bp), while the second one separates ORF8 from ORF9 317
(118 bp) (Fig. 1A). Considering this genetic organization, both non-coding segments were 318
examined for the presence of promoters using RACE PCR, a rapid method for the amplification 319
of 5’-cDNA ends. The technique relies upon amplifying RNA transcripts between a defined 320
internal site and the unknown 5’-extremity of interest, thus allowing for the identification of the 321
transcription start site. Using RNA extracted from a lysogenic culture in the exponential growth 322
phase, two transcription start sites were identified upstream of ORF1, at positions 175 and 278, 323
and one initiation site was identified in the intergenic region between ORF8 and ORF9, at 324
coordinate 4,915 (Fig. 1B). Centered approximately -10 and -35 nucleotides upstream of each 325
transcription start site are conserved sigma A-dependent promoter sequences (Fig. 1A and 1C). 326
The two potential promoters on the left were named P1 and P2 and the internal promoter, P3. It 327
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is interesting to note that the -10 hexamer in P1 is preceded by a TG dinucleotide, positioned 1 328
base upstream of the -10 sequence (Fig. 1C), a feature found in so-called extended -10 329
promoters. In E. coli and B. subtilis, the TGn extension allows for closer contacts between the 330
RNA polymerase sigma subunit and its promoters, especially when a well-conserved -35 box is 331
absent (5, 11, 34, 37, 58, 70). This could be the case for P1, where the -35 box does not begin 332
with T, the predominant base found at the beginning of the -35 hexamer in Gram-positive 333
bacteria (70). 334
In addition to the reported promoter sequences, potential ribosome binding sites (RBSs) were 335
identified 10 and 6 nucleotides upstream of the start codons of ORF1 and ORF9, respectively. 336
Also, Rho-independent transcription terminators were identified downstream of ORF8, where 337
transcription of the replication/regulation operon would be expected to terminate, and 338
downstream of ORF30, where transcription of the lysis/assembly operon is expected to terminate 339
(Fig. 1A). We confirmed that transcription does not span the regulatory region between ORF8 340
and ORF9 by performing RT-PCR with primer sets that span this region (data not shown). 341
Finally, lacZ reporter fusions were generated to the regions containing P1-P2 and P3 in order to 342
verify that these are biologically active promoters. As shown in Table 1, both promoter regions 343
directed the expression of β-galactosidase in the GIL01 B. thuringiensis host. Such 344
transcriptional organization and the corresponding gene distribution further strengthen the idea 345
that the GIL01 genome is transcribed as two major operons, one responsible for genome 346
replication and regulation, the other for virion structure, assembly and host cell lysis. 347
GIL01 clear plaque mutants map to three distinct loci 348
A chief objective of this study was to identify the components of the genetic circuit governing 349
GIL01 development. The only obvious candidates for transcription regulation were ORF1 and 350
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ORF6. PSI-BLAST searches identified a potential helix-turn-helix (HTH) DNA-binding motif 351
in ORF1 similar to the HTH found in the MerR super-family of transcription regulators. In 352
ORF6, the same database searches revealed a potential winged HTH DNA-binding domain 353
similar to that found in the N-terminus of LexA, the master repressor of the SOS regulon. We 354
reasoned that since GIL01 is induced in DNA-damaging conditions, most likely by way of the 355
cellular SOS response, it is potentially under the transcriptional control of a cleavable repressor 356
analogous to CI in phage λ. However, neither ORF1/ORF6 nor any other protein-coding region 357
in GIL01 displays the typical structure of the CI/LexA class of repressors: an N-terminal DNA-358
binding domain, followed by an Ala-Gly or Cys-Gly cleavage site and a C-terminal protease 359
domain. Yet, we did not rule out the existence of a distinct type of SOS-dependent induction 360
mechanism, such as the LexA-regulated anti-repressors found in coliphages 186 (8, 38) and N15 361
(47) or that GIL01 induction relies on a yet unknown SOS pathway. 362
To investigate these possibilities, we took advantage of knowledge acquired from a previous 363
study of spontaneous GIL01 clear plaque (cp) mutants defective for the lysogenic cycle (67). 364
Since ORF6 was a strong candidate for encoding a repressor of the lytic cycle, the first cp 365
mutants had been sequenced within and in the vicinity of ORF6. Unexpectedly, no mutations 366
were detected in ORF6. Instead, mutations were found in the downstream ORF7, the function of 367
which was unknown. ORF7 cp mutants displayed either a deletion or insertion of 11 nucleotides 368
in an almost perfect direct repeat (DR) (5’-CAAGTCGTAaG CAAGTCGTAtG) at coordinates 369
4,615 through 4,636. 370
In this study, a more extensive collection of cp mutants was assembled from multiple 371
independent experiments and analyzed. GIL01 cp mutants arose at a frequency of approximately 372
10-4 and displayed otherwise normal growth properties. In addition to the expected DR mutations 373
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in ORF7 (deletions in cp04 and cp18, insertions in cp25 and cp29), a mutant (cp08) carrying a 374
single nucleotide substitution in the second repeat, resulting in a premature terminator codon, 375
was also isolated. Another ORF7 mutant, cp27, has a single nucleotide insertion downstream of 376
the DR (Fig. 2A). In every instance, the mutation altered the predicted protein sequence, cp04, 377
cp08, cp18 , cp25 and cp29 all resulted in truncation of ORF7, while cp27 resulted in a modified 378
C-terminus (Fig. 2B). 379
Additionally, a class of GIL01 cp mutants was isolated that lacked mutations in ORF7, 380
suggesting that mutations in other regions of the GIL01 genome might account for their cp 381
phenotype. This class of mutants all contained single nucleotide substitutions, deletions or 382
insertions within ORF1 (Fig. 2A) or they contained mutations immediately upstream of ORF1 383
between promoters P1 and P2 (Fig. 1C and 3A). Interestingly, mutations in ORF1 cluster in two 384
regions: the predicted HTH DNA-binding motif (cp32, cp33 and cp36) or the most C-terminal 385
residues (cp06 and cp10) (Fig. 2B). Mutations in the non-coding region upstream of ORF1, 386
hereafter termed dinBox1, resulted in base substitutions (cp16, cp19, cp23 and cp26) or single 387
nucleotide deletions (cp17 and cp37) at either of two nucleotide positions within a 14-bp 388
sequence that bears strong similarity to the LexA binding site in Gram-positive bacteria (Fig. 389
3B). As outlined below, a single mutation in dinBox1 is sufficient to abolish the lysogenic 390
pathway of GIL01. 391
To rule out the possibility that additional mutations, outside the sequenced areas, contributed to, 392
or were responsible for, the observed cp phenotype, we sequenced the entire GIL01 genome of 393
cp26 (mutated in dinBox1), cp27/cp29 (mutated in ORF7) and cp32/cp33 (mutated in ORF1). 394
cp26 (dinBox1), cp27 (ORF7) and cp33 (ORF1) only displayed the initially identified nucleotide 395
substitutions (cp26 and cp33) and insertion (cp27). cp29 and cp32, however, each contained one 396
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additional mutation. In cp29, a single nucleotide substitution changed a Gln to Lys 397
(CAA→AAA) at residue 19 of ORF29, the function of which is currently unknown. cp32 398
contained an additional deletion of three nucleotides at codon 64 within ORF19 (function 399
unknown), resulting in the in-frame deletion of a single Ile residue. Yet, cp29 and cp32 display 400
the same infectious behavior as their cognate cp27 and cp33 mutants, which are solely mutated 401
in ORF7 and ORF1, respectively (see complementation experiments below and Table 2). This 402
strongly suggests that ORF18 and ORF29 are not involved in the regulatory network of GIL01. 403
Taken together, our results indicate that dinBox1, ORF1 and ORF7 are critical for the 404
establishment or maintenance of the GIL01 lysogenic cycle. 405
Ultimately, we wished to classify cp mutants according to their degree of virulence on lysogen 406
and non-lysogen hosts. However, none of the cp mutants tested produced plaques on lawns of the 407
lysogen strain GBJ338, indicating that GIL01 lysogens are immune to infection by cp mutants. 408
This could be due to the activity of phage- and/or host-encoded immunity factors that shift 409
GIL01 towards lysogenic development. Alternatively, we cannot rule out the possibility that 410
infection was barred by a yet unidentified phage-borne super-infection exclusion system. 411
gp1 and gp7 regulate lysogenic development 412
Since mutations in either ORF1 or ORF7 commit GIL01 to multiply exclusively as a lytic phage, 413
we investigated more closely the roles of their respective gene products, gp1 and gp7, in GIL01 414
development. The corresponding genes were cloned into the E. coli - B. subtilis shuttle vector 415
pDG148, placing them under the transcriptional control of the IPTG-inducible Pspac promoter. 416
Three different plasmids were generated, whereby gp1 and gp7 were expressed individually or 417
together as a single transcriptional unit. The permissive B. thuringiensis host GBJ002 was 418
transformed with the resulting plasmids, generating strains GBJ250 (gp1), GBJ223 (gp7) and 419
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GBJ467 (gp1/gp7). Each of these strains were induced for protein expression and subjected to 420
infection with GIL01 cp mutants in dinBox1, ORF1 or ORF7. The results of these experiments 421
are summarized in Table 2. Expression of gp1 alone reduced the infective titers of ORF1 mutants 422
cp32 and cp33 approximately 10-fold. Surprisingly, gp1 expression also reduced the plating 423
efficiency of dinBox1 and ORF7 mutants, although to a lesser extent (2-fold and 5-fold PFU 424
reduction, respectively). This was not the case when gp7 was expressed in the host strain. In this 425
case, a reduction in phage titers was only observed when induced GBJ223 cells were infected 426
with the ORF7 mutants cp27 and cp29. Although titers only showed a 2-fold decrease, the 427
plaques were now turbid and considerably smaller than those seen on the un-induced strain 428
GBJ223 (Fig. 4). This observation was less remarkable when ORF1 mutants were plated on 429
induced cultures of GBJ250, as ORF1 mutants tend to produce smaller plaques in general. 430
Infection of these same induced cell lines with wild-type GIL01 yielded titer reductions that 431
varied between 2-fold and 10-fold when compared to infections of un-induced cultures, while 432
plaque morphology remained unaltered (data not shown). This noticeable reduction in titers, as 433
well as the considerable experiment-to-experiment variability in titers, could be explained by 434
variations in the cellular concentrations of gp1 and gp7 generated with our plasmid-borne, IPTG-435
inducible system. Wild-type GIL01 probably expresses balanced levels of gp1 and gp7 inside the 436
host cell and disrupting that balance is likely to affect phage behavior in many ways. These data 437
are consistent with the idea that gp1 and gp7, and possibly the proper expression rates of these 438
two factors, play important roles in the choice between lytic and lysogenic development. 439
Given the above results, we investigated whether there was any effect due to the simultaneous 440
expression of both gp1 and gp7 in the cell. There was indeed a substantial reduction in phage 441
titers when induced GBJ467 cells were infected with dinBox1 mutants and no plaques at all were 442
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observed with ORF1 mutants (Table 2). In contrast, no difference in plating efficiency was 443
observed between infections of induced GBJ250 and GBJ467 with ORF7 mutants. Hence, co-444
expressing gp1 and gp7 in the same strain enhances complementation of dinBox1 and ORF1 445
mutants but does not result in enhanced rescue of ORF7 mutants. 446
Since complementation was associated with sharp reductions in infective titers, we wished to 447
verify that expression of gp1 and/or gp7 conferred immunity by preventing the infecting mutant 448
from propagating, rather than by blocking infection altogether. In order to investigate the fate of 449
the cp mutants post-infection, bacterial samples were recovered from infected spots and streaked 450
on fresh plates containing IPTG. PCR reactions with GIL01-specific primers were performed on 451
isolated colonies in which gp1 and/or gp7 were expressed and which had been infected with 452
mutants cp26 (dinBox1), cp27 (ORF7) and cp33 (ORF1). Induced GBJ223 colonies were only 453
found to be positive for GIL01 when infected with cp27, showing that expression of gp7 454
complements the missing immunity function of the corresponding cp mutant. GIL01-positive 455
colonies were also obtained from GBJ250 spot infections with cp33. Similar results were 456
obtained when induced GBJ467 was infected with each of the three cp mutants. These results are 457
in agreement with the infection patterns described above (Table 2) and altogether, they support 458
the idea that gp1 and gp7 act in immunity against infection and, together with the dinBox1 459
element, play important roles in the regulatory network of GIL01. 460
dinBox1 is a conserved LexA box 461
dinBox1 showed strong similarity to the consensus LexA binding site (CGAACn4GTTCG) in B. 462
subtilis (13, 30, 72). Its sequence, CGAACaagcGTTTT, is centered at positions +17 and -86 with 463
respect to the P1 and P2 transcription start sites, respectively (Fig. 1C). Cp mutants at dinBox1 464
were unable to enter lysogeny and mutations always involved changes to strongly conserved 465
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positions within the SOS box. Interestingly, Bam35c, a cp mutant of phage Bam35 (55), only 466
differs from GIL01 at a few nucleotide positions, one of which is located within the critical bases 467
for LexA binding to dinBox1 (Fig. 3A). In agreement with this finding, experimental data for the 468
LexA box sequence requirements in B. subtilis shows that changes to any one of the five outer 469
nucleotides can considerably reduce or prevent LexA binding (24). In addition to conserved 470
dinBox1, two motifs with similar characteristics were found near promoter P3. dinBox2 and 471
dinBox3 are centered at -22 and +11 relative to the start of transcription (Fig. 1C). It is worth 472
noting that, while numerous mutations were isolated in dinBox1, none of the cp mutants isolated 473
in this study carried mutations in dinBox2 or dinBox3. 474
To further consolidate our observations, we searched for SOS-box sequences in the upstream 475
regions of SOS genes in B. cereus strain G9842, which is closest to strain GBJ002 in DNA 476
sequence. We compared the LexA operators found upstream of genes involved in transcriptional 477
regulation (lexA), recombination (recA and ruvAB) and DNA repair (uvrAB) to the consensus 478
sequence for LexA binding in B. subtilis and to the three dinBox sequences identified in GIL01. 479
As shown in Fig. 3B, the B. cereus SOS boxes and the three GIL01 dinBox sequences closely 480
match the B. subtilis consensus SOS box. Additionally, an in silico approach using the program 481
RSAT (17) identified the same three dinBox sites found by visual analysis of the GIL01 482
promoter sequences (Fig. 3B). Lastly, alignment of the B. cereus LexA protein sequence with the 483
Gram-positive consensus LexA sequence reveals strong conservation of the N-terminal regions 484
that make close contacts with DNA (Fig. 3C). Taken together, these observations strongly 485
suggest that the dinBox sequences identified in GIL01 function as LexA binding sites in the B. 486
cereus group of bacteria. 487
GIL01 is induced in DNA-damaging conditions in a LexA-dependent manner 488
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In a previous study, we reported that GIL01 was induced by DNA-damaging agents such as 489
mitomycin C (MMC), nalidixic acid or exposure to UV irradiation (69). Such treatments are 490
known to elicit the cellular SOS response in a RecA-dependent manner (71). To investigate if 491
RecA is involved in the induction cycle of GIL01, we disrupted the B. thuringiensis recA gene 492
through insertional mutagenesis (66). The resulting strain, GBJ499, was particularly sensitive to 493
UV and MMC treatment and displayed a slower growth rate than the wild-type strain (data not 494
shown). GBJ499 was infected with GIL01 to generate the lysogen GBJ500. Phage titers 495
produced by spontaneous induction of GBJ500 were reduced approximately 10-fold compared to 496
the wild-type lysogen GBJ338 (Table 3). In addition, whereas GIL01 was strongly induced after 497
UV or MMC treatment of the wild-type lysogen strain, there was no increase in phage titers 498
when GBJ500 is similarly induced (Table 3, lines 2 and 5). These results indicate that GIL01 499
induction is RecA-dependent and potentially linked to the B. thuringiensis SOS response 500
pathway. To investigate this hypothesis, the role of cellular LexA in the lytic switch was 501
assessed. We were unable to isolate a lexA null mutant of B. thuringiensis. This result was not 502
entirely unexpected considering that LexA represses the expression of cell division inhibitors in 503
other bacterial groups (sulA in E. coli and yneA in B. subtilis). In E. coli, lexA null mutants are 504
not viable unless sulA is first inactivated (52). In B. subtilis, lexA-deficient cells grow into long 505
filaments (25) as a consequence of YneA upregulation (33). The B. cereus LexA is 70% identical 506
to its counterpart in B. subtilis and most of the shared residues lie in the N-terminal DNA-507
binding domain (Fig. 3C). Most importantly, B. cereus LexA contains an Ala96-Gly97 peptide 508
sequence that in B. subtilis and E. coli (at Ala91-Gly92 in both organisms) is the site of RecA-509
stimulated auto-proteolysis (28, 51). Certain amino acid changes at the Ala-Gly cleavage site are 510
known to abolish LexA proteolysis (19, 42). We used a similar approach to construct a non-511
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cleavable lexA mutant by allele exchange in B. thuringiensis, replacing the Ala codon with an 512
Asp codon (lexAA96D). As expected, the resulting strain, GBJ396, was hyper-sensitive to DNA 513
damage conditions, displaying a 300-fold reduction in survival after treatment with MMC, 514
compared to a 9-fold reduction in survival of MMC-treated wild-type cells. Furthermore, a lexA-515
lacZ reporter fusion, when introduced into GBJ002 (lexA+), showed an expected 4-fold increase 516
in lacZ expression after treatment with MMC. However, there was no increase in lacZ expression 517
after SOS treatment of GBJ396 (lexAA96D) carrying the same lexA-lacZ reporter plasmid, 518
indicating that LexA was not proteolyzed and therefore not released from its operator sites by 519
SOS induction signals (data not shown). The lexAA96D mutant was infected with GIL01 to 520
generate the lysogen strain GBJ406. Similar to what we observed for the recA- lysogen, GIL01 521
induction from GBJ406 was strongly reduced in comparison to the wild-type host strain (Table 522
3, lines 3 and 6). The data obtained with both recA and lexA mutants show that GIL01 is induced 523
as part of the host SOS response pathway. 524
In order to assess if promoters P1-P2 are regulated by LexA, we determined whether a 373-bp 525
region encompassing both promoters and dinBox1 could direct transcription of a promoter-less 526
lacZ gene upon SOS induction. The 373-bp segment was cloned into the low-copy-number 527
pHT304-18Z plasmid (1), transcriptionally fusing the 5’ end of ORF1 to the lacZ reporter gene. 528
The construct was transformed to GBJ338 (lexA+) and GBJ338 (lexAA96D) and β-galactosidase 529
expression from the P1.P2-lacZ fusion was measured with and without SOS induction by MMC. 530
Table 1 shows that expression from promoters P1-P2 was induced approximately 4-fold in the 531
wild-type host in SOS conditions whereas no increase in β-galactosidase levels was observed in 532
the lexAA96D strain. Moreover, β-galactosidase expression from a P1.P2-lacZ fusion in which 533
dinBox1 was mutated at a critical residue was no longer repressed in either GBJ338 (lexA+) or 534
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GBJ338 (lexAA96D) and MMC treatment did not further increase LacZ expression. Taken 535
together, these data strongly indicate that LexA regulates P1-P2 and that dinBox1 is necessary 536
for this LexA-mediated regulation. Parallel experiments with promoter P3, in which a 226-bp 537
fragment including dinBox2 and dinBox3 was fused to lacZ, yielded a 9-fold increase in β-538
galactosidase activity in the wild-type host after MMC treatment. Once again, the increase in 539
lacZ expression levels after MMC treatment was eliminated when the same experiment was 540
performed in a host carrying the lexAA96D mutation. These experiments demonstrate that host 541
LexA regulates promoters P1-P2 and P3, most likely by binding to the dinBox sequences and 542
preventing transcription initiation. 543
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Discussion 544
In this study we used a genetic approach to identify central components of the system regulating 545
the temperate state in phage GIL01. We provide evidence that the host SOS repressor LexA is 546
directly involved in the switch but that phage-borne factors gp1 and gp7 are also necessary for 547
lysogeny. LexA is likely to act by binding to dinBox1, a conserved SOS box lying between two 548
newly identified promoters upstream of lysogeny and replication genes. Two additional 549
conserved LexA binding sites were also identified upstream of structure and lysis genes, further 550
suggesting that LexA plays a central role in GIL01 development. Our results lead to the 551
conclusion that GIL01 uses a combination of host- and phage-derived regulators to control its 552
cycle. 553
At first glance, GIL01 does not appear to display the characteristic modular organization usually 554
encountered in temperate phages. Its 15 kbp genome is smaller than most sequenced prophage 555
genomes and all 30 ORFs are transcribed in the same rightward direction, a rarely observed 556
feature. In addition, only a few genes have attributed functions, making it difficult to define the 557
boundaries of gene clusters involved in related functions. Most notably, a lysogeny module is not 558
readily apparent as there are only two gene candidates, ORF1 and ORF6, predicted to code for 559
DNA-binding proteins that could function as transcription regulators. Nevertheless, the 560
identification of promoters in large non-coding segments and Rho-independent terminators at the 561
end of transcriptional units revealed a functional organization into two major modules (Fig. 1A). 562
Two adjacent promoters, P1 and P2, at the left extremity of the phage genome, control 563
transcription of lysogeny and replication functions whereas a probable single internal promoter, 564
P3, directs the expression of structural and lytic genes. Such transcriptional organization 565
typically allows for complex, multi-layered regulation of gene transcription. It would, for 566
instance, permit the expression of regulatory factors and phage DNA polymerase to levels that 567
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are required for lysogeny while keeping lytic genes fully repressed. Conversely, upon perception 568
of an inducing signal, synthesis of terminal protein and DNA polymerase would be significantly 569
enhanced along with induction of structural and lytic genes. 570
A similar overall genome organization can be found in the B. subtilis lytic phage φ29, with the 571
notable difference that early and late genes are divergently transcribed. Tandem promoters A2b 572
and A2c jointly direct the expression of the leftward regulation and replication genes, which are 573
similarly distributed in GIL01 (49). Both promoters are strong and their repression by two early 574
factors coincides with late gene transcription activation from the flanking divergent promoter 575
A3, a determining step in the switch from early to late transcription (11, 50). The coupling of 576
tandem promoters to the same operator is frequently observed in bacteria (examples are the 577
rRNA operons (23, 73), crp gene (22) and fis operon (53)) and offers a certain flexibility for 578
differential gene regulation. In the autonomously replicating coliphage N15 for example, two 579
adjacent promoters direct gene expression from the anti-immunity locus immA and their 580
differential control is important for the choice between lysis and lysogeny (56). Similarly 581
arranged promoters regulate anti-immunity operons in distinct phages such as P1, P4 and P7 582
(56), suggesting that this regulatory strategy is widespread among temperate phages. It is 583
therefore conceivable that in GIL01, promoters P1 and P2 display distinct strengths, are subject 584
to variable levels of regulation or give rise to different transcripts. 585
GIL01 lytic development is induced by DNA-damaging treatments that elicit the cellular SOS 586
response and yet, GIL01 does not code for a cleavable CI-like repressor. Instead, we found that 587
regulation is mediated by host LexA since the phage lytic pathway was no longer induced in a 588
non-cleavable lexA (Ind-) background. Accordingly, three putative LexA operators that bore a 589
strong resemblance to the consensus LexA binding site in Gram-positive bacteria were identified 590
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between promoters P1 and P2 (dinBox1) and at P3 (dinBox2 and dinBox3). We showed that the 591
SOS induction of both promoter regions was blocked by a non-cleavable lexA (Ind-) host 592
mutation. Moreover, a single nucleotide mutation at a strongly conserved position within 593
dinBox1 completely eliminates transcription repression at P1-P2. Although additional 594
experiments will be needed in order to confirm LexA binding to dinBox1, these data lend 595
credence to the idea that host LexA is directly involved in the GIL01 control circuit. 596
Whereas numerous cp mutants were isolated in dinBox1, no mutations were ever detected in the 597
downstream dinBox2 or dinBox3. This suggests that dinBox2 and dinBox3 are either essential for 598
phage development or, if such mutants are indeed viable, they cannot be detected as clear plaque 599
formers. Also dinBox2 and dinBox3 could perform an additional function in the regulatory 600
system such as in LexA-mediated DNA looping between dinBox2/3 and the dinBox1 promoter 601
region. In this case, a cp mutant might not be obtained unless both boxes were simultaneously 602
mutated, which is likely to occur at too low a frequency to be detected. In any event, LexA 603
binding to both sites provides the basis for a plausible model in which a cleavable repressor 604
would be immediately responsible for lytic gene expression. However, it is unlikely that LexA is 605
the sole regulator of the switch as this would lead to inefficient GIL01 induction. Due to the 606
negative auto-regulation of lexA transcription, normal LexA levels are restored once DNA 607
damage inside the cell has been repaired (7, 44). LexA would then be able to bind its operators in 608
GIL01 again and interrupt lytic development. In phage lambda, for instance, this is prevented by 609
strong repression of CI synthesis once lytic development commences (60). It is therefore 610
conceivable that LexA only plays an ancillary role in the regulatory network by controlling the 611
expression of phage-borne repressor(s) and activator(s), these in turn being responsible for the 612
lytic switch per se. This hypothesis is strongly supported by the isolation of GIL01 cp mutants 613
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deficient for gp1 or gp7. Both proteins are required for lysogeny and their absence can be 614
complemented by expressing the missing functions in trans. Interestingly, cross-615
complementation is observed with gp1 whereas it is not seen with gp7. The observed differences 616
could be attributed to distinct and/or independent roles of gp1 and gp7, gp1 potentially having a 617
broader activity range. In addition, since the expression system used in this study does not 618
provide any insight into the protein concentrations generated, it is quite probable that they differ 619
from the intracellular levels produced by GIL01 during infection. This could also explain why 620
wild-type GIL01 is not fully repressed when infecting cells expressing both gp1 and gp7. One 621
would expect the wild-type phage cycle to be hindered by the two factors known to be involved 622
in lysogeny and yet, titers are only slightly reduced. This observation suggests there is the need 623
for precise levels of both factors and probably, a determined timing of expression, too. 624
gp1 displays a DNA-binding domain similar to the one found in the MerR (repressor of mercury 625
resistance operons) super-family of transcription regulators. MerR itself is known to mediate 626
both repression and activation of transcription by virtue of its ability to distort the DNA helix (3, 627
4). A role for gp1 as a modulator of DNA structure is an attractive idea as it is a common theme 628
among transcription regulators. gp7, on the other hand, has no predicted function or recognizable 629
sequence motif. It might cooperate with gp1 in the same regulatory process or it could act 630
independently, at a completely different level of immunity regulation. 631
Temperate phages that are under direct control of host LexA utilize a variety of regulatory 632
mechanisms. Coliphages 186 and N15 code for LexA-regulated anti-repressors (8, 47) and gene 633
homologues that are preceded by LexA binding sites have been identified in other phages (9, 12, 634
27). A unique example where LexA exerts a dual function is provided by the Vibrio cholerae 635
filamentous phage CTX. The phage-encoded repressor RstR acts together with host LexA to 636
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activate and repress gene transcription (36, 54). Such a mechanism allows the phage to respond 637
to small variations in RstR-LexA levels and switch from a low particle formation mode to higher 638
yields and vice versa. This regulatory system appears to be well suited for a filamentous phage, 639
the production of which is maintained throughout the cell cycle. 640
GIL01 cp mutants were isolated in three distinct loci – dinBox1, ORF1 and ORF7 – each of them 641
necessary for the lysogenic cycle. Surprisingly, none of the isolated mutants produced plaques on 642
lysogens, a result that we expected to see with at least the operator mutants in dinBox1. There are 643
many possible explanations for the observed immunity effect. One is that GIL01 codes for an as 644
yet unidentified super-infection exclusion system that blocks entry of newly infecting phage. A 645
more attractive explanation, however, would be that dinBox1 is not the only operator needed for 646
lysogeny. If other regulatory sites are present on the phage genome, potentially responding to 647
gp1 and/or gp7, these sites could constitute themselves a LexA-independent pathway for 648
regulation and compensate for the loss of the dinBox1 pathway. In this case, a mutant super-649
infecting phage would still be sensitive to repression upon infection of a lysogen, provided that 650
the appropriate immunity factors are readily available in the cell. It is noteworthy that even 651
though the dinBox1, ORF1 and ORF7 alleles were independently isolated multiple times, they 652
probably do not represent a saturated screen of all possible mutations leading to the cp 653
phenotype. We therefore do not exclude the possibility that yet unidentified factors also 654
participate in GIL01 regulation. 655
GIL01 is regulated by a multi-component system composed of at least host LexA, gp1 and gp7. 656
The combination of host- and phage-encoded functions in the lytic switch has already been seen 657
in other phages and enables a fine regulation of lytic functions. LexA is used as an internal 658
sensor of stress and allows for the rapid escape from potentially deleterious damage to the host. 659
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The addition of phage-borne functions could modulate the switch by conferring more precision 660
to the system or by adding a further layer of control. GIL01 has developed an elaborate network 661
to control its development and future studies will address how the different components 662
identified in this study intervene in the switch. 663
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Acknowledgements 664
We are grateful to B. Clément and S. Degand for their contribution to the early stages of this 665
work and to A. Toussaint and A. Aertsen for stimulating discussions. We thank R. Janky for 666
continuous encouragement and skilful advice with RSAT and R. Leplae for assistance with 667
protein analysis. We are particularly indebted to H. Kimsey for insightful observations and 668
comments on the manuscript. We wish to thank C. Condon for kindly providing the pDG148 669
vector. This work was supported by grants from the National Fund for Scientific Research and 670
the Université catholique de Louvain (grants to NF and JM). Part of this work was also 671
supported by the Finnish Centre of Excellence Program of the Academy of Finland (grant 672
1129648 to JKHB), an EMBO short-term fellowship and the National Research Fund in 673
Luxembourg, co-funded under the Marie Curie Actions of the European Commission (FP7-674
COFUND) (grants to NF). 675
676
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References 677
1. Agaisse, H. and D. Lereclus. 1994. Structural and functional analysis of the promoter region 678 involved in full expression of the cryIIIA toxin gene of Bacillus thuringiensis. Mol. Microbiol. 679 13:97-107. 680
2. Andrup, L., J. Damgaard, and K. Wassermann. 1993. Mobilization of small plasmids in 681 Bacillus thuringiensis subsp. israelensis is accompanied by specific aggregation. J. Bacteriol. 682 175:6530-6536. 683
3. Ansari, A. Z., J. E. Bradner, and T. V. O'Halloran. 1995. DNA-bend modulation in a 684 repressor-to-activator switching mechanism. Nature 374:371-375. 685
4. Ansari, A. Z., M. L. Chael, and T. V. O'Halloran. 1992. Allosteric underwinding of DNA is 686 a critical step in positive control of transcription by Hg-MerR. Nature 355:87-89. 687
5. Barne, K. A., J. A. Bown, S. J. Busby, and S. D. Minchin. 1997. Region 2.5 of the 688 Escherichia coli RNA polymerase sigma70 subunit is responsible for the recognition of the 689 'extended-10' motif at promoters. EMBO J. 16:4034-4040. 690
6. Bertani, G. 1951. Studies on lysogenesis. I. The mode of phage liberation by lysogenic 691 Escherichia coli. J. Bacteriol. 62:293-300. 692
7. Brent, R. and M. Ptashne. 1980. The lexA gene product represses its own promoter. Proc. 693 Natl. Acad. Sci. U. S. A. 77:1932-1936. 694
8. Brumby, A. M., I. Lamont, I. B. Dodd, and J. B. Egan. 1996. Defining the SOS operon of 695 coliphage 186. Virology 219:105-114. 696
9. Bunny, K., J. Liu, and J. Roth. 2002. Phenotypes of lexA mutations in Salmonella enterica: 697 evidence for a lethal lexA null phenotype due to the Fels-2 prophage. J. Bacteriol. 184:6235-698 6249. 699
10. Butala, M., D. Zgur-Bertok, and S. J. Busby. 2009. The bacterial LexA transcriptional 700 repressor. Cell Mol. Life Sci. 66:82-93. 701
11. Camacho, A. and M. Salas. 2001. Mechanism for the switch of phi29 DNA early to late 702 transcription by regulatory protein p4 and histone-like protein p6. EMBO J. 20:6060-6070. 703
12. Casjens, S. R., E. B. Gilcrease, W. M. Huang, K. L. Bunny, M. L. Pedulla, M. E. Ford, 704 J. M. Houtz, G. F. Hatfull, and R. W. Hendrix. 2004. The pKO2 linear plasmid prophage of 705 Klebsiella oxytoca. J. Bacteriol. 186:1818-1832. 706
13. Cheo, D. L., K. W. Bayles, and R. E. Yasbin. 1991. Cloning and characterization of DNA 707 damage-inducible promoter regions from Bacillus subtilis. J. Bacteriol. 173:1696-1703. 708
on May 17, 2018 by guest
http://jb.asm.org/
Dow
nloaded from
35
14. Chu, W. Y., Y. F. Huang, C. C. Huang, Y. S. Cheng, C. K. Huang, and Y. J. Oyang. 709 2009. ProteDNA: a sequence-based predictor of sequence-specific DNA-binding residues in 710 transcription factors. Nucleic Acids Res. 37:W396-401. 711
15. Court, D., L. Green, and H. Echols. 1975. Positive and negative regulation by the cII and 712 cIII gene products of bacteriophage lambda. Virology 63:484-491. 713
16. Cutting, S. M. and P. B. Vander Horn. 1990. Genetic Analysis, p. 27-74. In C. R. Harwood 714 and S. M. Cutting (ed.), Molecular Biological Methods for Bacillus. John Wiley, Chichester. 715
17. Defrance, M., R. Janky, O. Sand, and J. van Helden. 2008. Using RSAT oligo-analysis 716 and dyad-analysis tools to discover regulatory signals in nucleic sequences. Nat. Protoc. 3:1589-717 1603. 718
18. Erill, I., S. Campoy, and J. Barbe. 2007. Aeons of distress: an evolutionary perspective on 719 the bacterial SOS response. FEMS Microbiol. Rev. 31:637-656. 720
19. Fabret, C., S. D. Ehrlich, and P. Noirot. 2002. A new mutation delivery system for 721 genome-scale approaches in Bacillus subtilis. Mol. Microbiol. 46:25-36. 722
20. Gaidelyte, A., V. Cvirkaite-Krupovic, R. Daugelavicius, J. K. Bamford, and D. H. 723 Bamford. 2006. The entry mechanism of membrane-containing phage Bam35 infecting Bacillus 724 thuringiensis. J. Bacteriol. 188:5925-5934. 725
21. Gicquel-Sanzey, B. and P. Cossart. 1982. Homologies between different procaryotic DNA-726 binding regulatory proteins and between their sites of action. EMBO J. 1:591-595. 727
22. Gonzalez-Gil, G., R. Kahmann, and G. Muskhelishvili. 1998. Regulation of crp 728 transcription by oscillation between distinct nucleoprotein complexes. EMBO J. 17:2877-2885. 729
23. Gourse, R. L., H. A. de Boer, and M. Nomura. 1986. DNA determinants of rRNA 730 synthesis in E. coli: growth rate dependent regulation, feedback inhibition, upstream activation, 731 antitermination. Cell 44:197-205. 732
24. Groban, E. S., M. B. Johnson, P. Banky, P. G. Burnett, G. L. Calderon, E. C. Dwyer, S. 733 N. Fuller, B. Gebre, L. M. King, I. N. Sheren, L. D. Von Mutius, T. M. O'Gara, and C. M. 734 Lovett. 2005. Binding of the Bacillus subtilis LexA protein to the SOS operator. Nucleic Acids 735 Res. 33:6287-6295. 736
25. Haijema, B. J., D. van Sinderen, K. Winterling, J. Kooistra, G. Venema, and L. W. 737 Hamoen. 1996. Regulated expression of the dinR and recA genes during competence 738 development and SOS induction in Bacillus subtilis. Mol. Microbiol. 22:75-85. 739
26. Hendrix, R. W., J. W. Roberts, F. W. Stahl, and R. A. Weisberg (eds.). 1983. Lambda II. 740 Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 741
on May 17, 2018 by guest
http://jb.asm.org/
Dow
nloaded from
36
27. Hertwig, S., I. Klein, V. Schmidt, S. Beck, J. A. Hammerl, and B. Appel. 2003. Sequence 742 analysis of the genome of the temperate Yersinia enterocolitica phage PY54. J. Mol. Biol. 743 331:605-622. 744
28. Horii, T., T. Ogawa, T. Nakatani, T. Hase, H. Matsubara, and H. Ogawa. 1981. 745 Regulation of SOS functions: purification of E. coli LexA protein and determination of its 746 specific site cleaved by the RecA protein. Cell 27:515-522. 747
29. Ivanova, N., A. Sorokin, I. Anderson, N. Galleron, B. Candelon, V. Kapatral, A. 748 Bhattacharyya, G. Reznik, N. Mikhailova, A. Lapidus, L. Chu, M. Mazur, E. Goltsman, N. 749 Larsen, M. D'Souza, T. Walunas, Y. Grechkin, G. Pusch, R. Haselkorn, M. Fonstein, S. D. 750 Ehrlich, R. Overbeek, and N. Kyrpides. 2003. Genome sequence of Bacillus cereus and 751 comparative analysis with Bacillus anthracis. Nature 423:87-91. 752
30. Janky, R. and J. van Helden. 2008. Evaluation of phylogenetic footprint discovery for 753 predicting bacterial cis-regulatory elements and revealing their evolution. BMC Bioinformatics 754 9:37. 755
31. Jensen, G. B., L. Andrup, A. Wilcks, L. Smidt, and O. M. Poulsen. 1996. The 756 aggregation-mediated conjugation system of Bacillus thuringiensis subsp. israelensis: host range 757 and kinetics of transfer. Curr. Microbiol. 33:228-236. 758
32. Jensen, G. B., A. Wilcks, S. S. Petersen, J. Damgaard, J. A. Baum, and L. Andrup. 759 1995. The genetic basis of the aggregation system in Bacillus thuringiensis subsp. israelensis is 760 located on the large conjugative plasmid pXO16. J. Bacteriol. 177:2914-2917. 761
33. Kawai, Y., S. Moriya, and N. Ogasawara. 2003. Identification of a protein, YneA, 762 responsible for cell division suppression during the SOS response in Bacillus subtilis. Mol. 763 Microbiol. 47:1113-1122. 764
34. Keilty, S. and M. Rosenberg. 1987. Constitutive function of a positively regulated promoter 765 reveals new sequences essential for activity. J. Biol. Chem. 262:6389-6395. 766
35. Kelley, L. A. and M. J. Sternberg. 2009. Protein structure prediction on the Web: a case 767 study using the Phyre server. Nat. Protoc. 4:363-371. 768
36. Kimsey, H. H. and M. K. Waldor. 2009. Vibrio cholerae LexA coordinates CTX prophage 769 gene expression. J. Bacteriol. 191:6788-6795. 770
37. Kumar, A., R. A. Malloch, N. Fujita, D. A. Smillie, A. Ishihama, and R. S. Hayward. 771 1993. The minus 35-recognition region of Escherichia coli sigma 70 is inessential for initiation 772 of transcription at an "extended minus 10" promoter. J. Mol. Biol. 232:406-418. 773
38. Lamont, I., A. M. Brumby, and J. B. Egan. 1989. UV induction of coliphage 186: 774 prophage induction as an SOS function. Proc. Natl. Acad. Sci. U. S. A. 86:5492-5496. 775
on May 17, 2018 by guest
http://jb.asm.org/
Dow
nloaded from
37
39. Laurinavicius, S., D. H. Bamford, and P. Somerharju. 2007. Transbilayer distribution of 776 phospholipids in bacteriophage membranes. Biochim. Biophys. Acta 1768:2568-2577. 777
40. Laurinavicius, S., R. Kakela, P. Somerharju, and D. H. Bamford. 2004. Phospholipid 778 molecular species profiles of tectiviruses infecting Gram-negative and Gram-positive hosts. 779 Virology 322:328-336. 780
41. Laurinmaki, P. A., J. T. Huiskonen, D. H. Bamford, and S. J. Butcher. 2005. Membrane 781 proteins modulate the bilayer curvature in the bacterial virus Bam35. Structure 13:1819-1828. 782
42. Lin, L. L. and J. W. Little. 1988. Isolation and characterization of noncleavable (Ind-) 783 mutants of the LexA repressor of Escherichia coli K-12. J. Bacteriol. 170:2163-2173. 784
43. Little, J. W. 1984. Autodigestion of lexA and phage lambda repressors. Proc. Natl. Acad. 785 Sci. U. S. A. 81:1375-1379. 786
44. Little, J. W. and J. E. Harper. 1979. Identification of the lexA gene product of Escherichia 787 coli K-12. Proc. Natl. Acad. Sci. U. S. A. 76:6147-6151. 788
45. Luo, Y., R. A. Pfuetzner, S. Mosimann, M. Paetzel, E. A. Frey, M. Cherney, B. Kim, J. 789 W. Little, and N. C. Strynadka. 2001. Crystal structure of LexA: a conformational switch for 790 regulation of self-cleavage. Cell 106:585-594. 791
46. Mahillon, J. and D. Lereclus. 2000. Electroporation of Bacillus thuringiensis and Bacillus 792 cereus, p. 242-252. In J. Teissié and N. Eynard (ed.), Electrotransformation of Bacteria. Springer 793 Lab Manual, Springer-Verlag, Germany. 794
47. Mardanov, A. V. and N. V. Ravin. 2007. The antirepressor needed for induction of linear 795 plasmid-prophage N15 belongs to the SOS regulon. J. Bacteriol. 189:6333-6338. 796
48. Mazon, G., I. Erill, S. Campoy, P. Cortes, E. Forano, and J. Barbe. 2004. Reconstruction 797 of the evolutionary history of the LexA-binding sequence. Microbiology 150:3783-3795. 798
49. Meijer, W. J., J. A. Horcajadas, and M. Salas. 2001. Phi29 family of phages. Microbiol. 799 Mol. Biol. Rev. 65:261-87 ; second page, table of contents. 800
50. Mencia, M., M. Monsalve, M. Salas, and F. Rojo. 1996. Transcriptional activator of phage 801 phi 29 late promoter: mapping of residues involved in interaction with RNA polymerase and in 802 DNA bending. Mol. Microbiol. 20:273-282. 803
51. Miller, M. C., J. B. Resnick, B. T. Smith, and C. M. Lovett Jr. 1996. The Bacillus subtilis 804 dinR gene codes for the analogue of Escherichia coli LexA. Purification and characterization of 805 the DinR protein. J. Biol. Chem. 271:33502-33508. 806
on May 17, 2018 by guest
http://jb.asm.org/
Dow
nloaded from
38
52. Mount, D. W. 1977. A mutant of Escherichia coli showing constitutive expression of the 807 lysogenic induction and error-prone DNA repair pathways. Proc. Natl. Acad. Sci. U. S. A. 808 74:300-304. 809
53. Nasser, W., M. Rochman, and G. Muskhelishvili. 2002. Transcriptional regulation of fis 810 operon involves a module of multiple coupled promoters. EMBO J. 21:715-724. 811
54. Quinones, M., H. H. Kimsey, and M. K. Waldor. 2005. LexA cleavage is required for 812 CTX prophage induction. Mol. Cell 17:291-300. 813
55. Ravantti, J. J., A. Gaidelyte, D. H. Bamford, and J. K. Bamford. 2003. Comparative 814 analysis of bacterial viruses Bam35, infecting a gram-positive host, and PRD1, infecting gram-815 negative hosts, demonstrates a viral lineage. Virology 313:401-414. 816
56. Ravin, N. V., A. N. Svarchevsky, and G. Deho. 1999. The anti-immunity system of phage-817 plasmid N15: identification of the antirepressor gene and its control by a small processed RNA. 818 Mol. Microbiol. 34:980-994. 819
57. Sambrook, J. and D. Russell. 2001. Molecular Cloning: a Laboratory Manual. 3rd ed. Cold 820 Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 821
58. Sanderson, A., J. E. Mitchell, S. D. Minchin, and S. J. Busby. 2003. Substitutions in the 822 Escherichia coli RNA polymerase sigma70 factor that affect recognition of extended -10 823 elements at promoters. FEBS Lett. 544:199-205. 824
59. Sauer, R. T., M. J. Ross, and M. Ptashne. 1982. Cleavage of the lambda and P22 825 repressors by recA protein. J. Biol. Chem. 257:4458-4462. 826
60. Schubert, R. A., I. B. Dodd, J. B. Egan, and K. E. Shearwin. 2007. Cro's role in the CI 827 Cro bistable switch is critical for {lambda}'s transition from lysogeny to lytic development. 828 Genes Dev. 21:2461-2472. 829
61. Shearwin, K. E., A. M. Brumby, and J. B. Egan. 1998. The Tum protein of coliphage 186 830 is an antirepressor. J. Biol. Chem. 273:5708-5715. 831
62. Sozhamannan, S., M. McKinstry, S. M. Lentz, M. Jalasvuori, F. McAfee, A. Smith, J. 832 Dabbs, H. W. Ackermann, J. K. Bamford, A. Mateczun, and T. D. Read. 2008. Molecular 833 characterization of a variant of Bacillus anthracis-specific phage AP50 with improved 834 bacteriolytic activity. Appl. Environ. Microbiol. 74:6792-6796. 835
63. Stragier, P., C. Bonamy, and C. Karmazyn-Campelli. 1988. Processing of a sporulation 836 sigma factor in Bacillus subtilis: how morphological structure could control gene expression. 837 Cell 52:697-704. 838
on May 17, 2018 by guest
http://jb.asm.org/
Dow
nloaded from
39
64. Stromsten, N. J., S. D. Benson, R. M. Burnett, D. H. Bamford, and J. K. Bamford. 2003. 839 The Bacillus thuringiensis linear double-stranded DNA phage Bam35, which is highly similar to 840 the Bacillus cereus linear plasmid pBClin15, has a prophage state. J. Bacteriol. 185:6985-6989. 841
65. Thomas-Chollier, M., O. Sand, J. V. Turatsinze, R. Janky, M. Defrance, E. Vervisch, S. 842 Brohee, and J. van Helden. 2008. RSAT: regulatory sequence analysis tools. Nucleic Acids 843 Res. 36:W119-27. 844
66. Vagner, V., E. Dervyn, and S. D. Ehrlich. 1998. A vector for systematic gene inactivation 845 in Bacillus subtilis. Microbiology 144 ( Pt 11):3097-3104. 846
67. Verheust, C., N. Fornelos, and J. Mahillon. 2005. GIL16, a new gram-positive tectiviral 847 phage related to the Bacillus thuringiensis GIL01 and the Bacillus cereus pBClin15 elements. J. 848 Bacteriol. 187:1966-1973. 849
68. Verheust, C., N. Fornelos, and J. Mahillon. 2004. The Bacillus thuringiensis phage GIL01 850 encodes two enzymes with peptidoglycan hydrolase activity. FEMS Microbiol. Lett. 237:289-851 295. 852
69. Verheust, C., G. Jensen, and J. Mahillon. 2003. pGIL01, a linear tectiviral plasmid 853 prophage originating from Bacillus thuringiensis serovar israelensis. Microbiology 149:2083-854 2092. 855
70. Voskuil, M. I. and G. H. Chambliss. 1998. The -16 region of Bacillus subtilis and other 856 gram-positive bacterial promoters. Nucleic Acids Res. 26:3584-3590. 857
71. Walker, G. C. 1984. Mutagenesis and inducible responses to deoxyribonucleic acid damage 858 in Escherichia coli. Microbiol. Rev. 48:60-93. 859
72. Winterling, K. W., D. Chafin, J. J. Hayes, J. Sun, A. S. Levine, R. E. Yasbin, and R. 860 Woodgate. 1998. The Bacillus subtilis DinR binding site: redefinition of the consensus 861 sequence. J. Bacteriol. 180:2201-2211. 862
73. Young, R. A. and J. A. Steitz. 1979. Tandem promoters direct E. coli ribosomal RNA 863 synthesis. Cell 17:225-234. 864
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Figure legends 865
Fig. 1. Partial physical map of phage GIL01 with SOS-regulated promoters. (A) Predicted 866
open reading frames (ORFs) are depicted as open boxes and left-to-right arrows indicate the 867
direction of transcription of the left and right functional domains. Probable gene functions are 868
indicated above certain ORFs. Putative Rho-independent transcription terminators are shown as 869
open stem-loops downstream of ORF8 and ORF30. Promoters are shown as filled arrowheads 870
and each promoter region is enlarged below the map. -35 and -10 boxes are depicted as black 871
rectangles and angled arrows represent transcription start sites. Grey boxes show the location of 872
conserved LexA binding sites with regard to the three promoters. (B) Transcription start site 873
mapping of GIL01 ORF1 and ORF9 by 5’-RACE. cDNAs were prepared from GIL01 mRNA 874
and were tailed with dCTP and PCR-amplified. The purified products were cloned into pCR4-875
TOPO and sequenced using the universal primers M13. The electropherograms show the 876
transcription start sites (indicated with an arrow; sequences correspond to the GIL01 negative 877
strand) fused to their oligo-dC tails. The respective promoters from which transcription is 878
initiated are indicated for each electropherogram. Note that for the transcription start at P3, an 879
additional thymine residue that is not found in the GIL01 sequence has been inserted upstream of 880
the dC tail. (C) Detailed nucleotide sequence of promoters depicted in (A). Transcription start 881
sites detected in (B) are indicated with angled arrows (P1, P2 and P3). Putative ribosome 882
binding sites (RBSs) are shown in lower case, start codons in bold and SOS boxes are shaded 883
grey. A TG dinucleotide motif characteristic of -10 extended promoters is double-underlined 884
within P1. Genome coordinates are indicated above each sequence. 885
Fig. 2. Clear plaque (cp) mutants in ORF1 and ORF7. Gene (A) and protein (B) sequences of 886
cp mutants were aligned and compared to their respective wild-type sequences. Nucleotide 887
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coordinates (A) and the corresponding protein sizes (B) are indicated for each sequence. Start 888
and stop codons are shown in bold and mutations appear on a light grey background. (A) The 889
direct repeat (DR) in ORF7 is highlighted against a black background in the wild-type sequence. 890
(B) ORF1 harbors a putative helix-turn-helix DNA-binding motif in its N-terminal domain and 891
the two α helices, α1 and α2, are shown boxed in black in the wild-type sequence. α1 and α2 892
were predicted using the softwares Phyre (35) and ProteDNA (14). 893
Fig. 3. dinBox1 is a conserved LexA operator. (A) A class of GIL01 cp mutants harbor single 894
nucleotide mutations (shaded grey) in dinBox1, a 14-bp predicted SOS box located between 895
promoters P1 and P2 (see Fig. 1A and 1C). The Bam35c operator differs from GIL01 by one 896
single nucleotide and this difference is believed to account for the opposed propagation styles of 897
both phages (see text for details). The GIL01 nucleotide coordinates are indicated above the 898
wild-type sequence. (B) Putative LexA operators were detected upstream of SOS genes in strain 899
B. cereus G9842 (GenBank acc. number NC_011772) and compared to the consensus sequence 900
for LexA binding in B. subtilis (24). In addition to dinBox1, two more potential SOS boxes, 901
dinBox2 and dinBox3 were identified within promoter P3 (see Fig. 1A and 1C). The outer 902
nucleotides, shown in capital letters, are highly conserved in SOS boxes of Gram-positive 903
bacteria and particularly important for LexA binding (24). (C) The N-terminal DNA binding 904
region of the B. cereus LexA protein is compared to the consensus sequence determined for 905
Gram-positive bacteria (48). The three α helices directly involved in DNA binding are 906
underlined and amino acid differences are shaded grey (24). 907
Fig. 4. gp7 is involved in immunity against infection. GBJ223 was grown exponentially and 908
gp7 expression was induced by adding IPTG 1 mM to one-half of the culture. Un-induced and 909
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induced cultures were then allowed to grow for three more hours. Cells were then infected with 910
cp29, a mutant in ORF7 (see Fig. 2), and plated. 911
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Table 1. P1-P2 and P3 promoter activities in lexA+ and lexAA96D backgrounds. 1 2
Promoter fusion lexA allele Mean β-galactosidase activity (Miller units ± SD)a
-MMC +MMC
P1-P2 lexA+
lexAA96D 75 ± 13 91 ± 5
303 ± 106 78 ± 5
P1-P2 (dinBox1mut) lexA+
lexAA96D 345 ± 28 339 ± 19
351 ± 10 326 ± 12
P3 lexA+
lexAA96D 3 ± 1 3 ± 1
26 ± 10 6 ± 2
3 alacZ fusions to P1-P2, P1-P2 mutated in dinBox1 (CGAACaagcTTTTT) and P3 4
promoters were generated and β-galactosidase activity was measured in both lexA+ and 5
lexAA96D lysogens in normal growth (-MMC) and in SOS-inducing (+MMC) conditions. 6
Cultures were grown for 3 hours at 37°C when MMC50 was added to one-half of the 7
cells and growth was allowed to proceed for one additional hour. Cells were then 8
permeabilized and assayed for β-galactosidase. Activity values are the average of three 9
independent experiments. 10
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Table 2: Functions of defective gp1 and gp7 can be complemented 1 2
cp mutants (relevant mutation)
Host (expressed protein(s))
cp23/cp26 (dinBox1)
cp32/cp33 (ORF1)
cp27/cp29 (ORF7)
Ratio of plating efficiencies (PFU on host +IPTG/
PFU on host -IPTG)a
GBJ250 (gp1) 0.5 0.1b 0.2
GBJ223 (gp7) 1 1b 0.5c
GBJ467 (gp1/gp7) 0.2 <10-8 0.2c
aThe GIL01-cured host strain (GBJ002) was transformed with plasmids expressing either 3
gp1 (GBJ250), gp7 (GBJ223) or gp1 and gp7 (GBJ467). The resulting strains were 4
grown exponentially before IPTG 1mM was added to one-half of the culture and growth 5
was allowed to proceed for three additional hours. Un-induced and induced cultures 6
were then infected with GIL01 cp mutants in dinBox1 (cp23/cp26), ORF1 (cp32/cp33) or 7
ORF7 (cp27/cp29). Two different cp mutants at each locus were tested independently 8
and similar infection patterns were obtained for each pair of mutants. The results shown 9
are representative of four independent experiments and give the ratio between titers 10
produced on un-induced cells and titers counted on the same induced strains. 11
bcp32 and cp33 form small clear plaques on the cured host strain. There was no 12
systematic change in plaque morphology between induced and un-induced cells infected 13
with ORF1 mutants. 14
cThe plaques observed on induced lawns were turbid and significantly smaller than 15
those produced on the un-induced lawns (see Fig. 4). 16
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Table 3: GIL01 induction is recA- and lexA-dependent 1 2 Phage titera
Strain - MMC + MMCb Ratio
GBJ338 (GBJ002 (GIL01)) 3.1 x 107 1.9 x 109 61.3
GBJ500 (GBJ338 recA::pMutin4) 4.0 x 106 3.0 x 106 0.8
GBJ406 (GBJ338 lexAA96D) 1.5 x 105 1.8 x 105 1.2
-UV +UVc
GBJ338 (GBJ002 (GIL01)) 4.0 x 107 2.7 x 109 67.5
GBJ500 (GBJ338 recA::pMutin4) 1.1 x 106 2.0 x 106 1.8
GBJ406 (GBJ338 lexAA96D) 6.7 x 104 5.3 x 104 0.8
aPhage titers were determined by plating serial dilutions of GIL01 on the cured host 3
GBJ002. Results are the average from three independent experiments. 4
bThe MMC concentration used was 50 ng ml-1 and cultures were induced for one hour. 5
cCells were irradiated at 254 nm for 10 seconds. 6
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0.3 kb 4.8 kb 15 kb
1 2 4 5
P1 P2 P3
6 93 8 107
replication & regulation structure & lysis
11 27 2826 29 30
-35 -10 -35 -10 -35 -10
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GAAAAGTGTGACAAGATATTCCAAGGGTGCTATAATAGGAAATGAGTTAAAAAACGAACAAGCGTTTTAT -35 -10 dinBox1 AAGTGTTCGGTTTTTGTAACATAACTGTAATATAAAAACATTTGCATTACTGGTAACTTTCGGGTAATAT
P1 131
AAGTGTTCGGTTTTTGTAACATAACTGTAATATAAAAACATTTGCATTACTGGTAACTTTCGGGTAATAT -35 -10 TGGTAGCAGAGAGGGACAAACGGGACAAATCGAGACACGATACATACTCTCTATACAAGTGAAAAGaagg tggtgCATATGTAATATG
P2
358 (ORF1)
CACACGTGTGACGTTATGCGGAACACTCGTTCGTATTATAGTATACATATAGCGAACAAACATTCGATaagg -35 dinBox2 -10 dinBox3
P3 4867
4952 (ORF9)agtgtGACAAAGTG
4952 (ORF9)
on May 17, 2018 by guest
http://jb.asm.org/
Dow
nloaded from
(A) ORF1
wt atgagtaaactactgactgcgcaagaggttgcagacttattgcgtgttcataaaaagactatcc cp06 atgagtaaactactgactgcgcaagaggttgcagacttattgcgtgttcataaaaagactatcc
356
cp06 atgagtaaactactgactgcgcaagaggttgcagacttattgcgtgttcataaaaagactatcccp10 atgagtaaactactgactgcgcaagaggttgcagacttattgcgtgttcataaaaagactatcc cp32 atgagtaaactactgactgcgcaagaggttgcagacttattgcgtattcataaaaagactatcc cp33 atgagtaaactactgactgcgcaagaggttgtagacttattgcgtgttcataaaaagactatcc cp36 atgagtaaactactgactgcgcaagaggttgcagacttattgcgtgttcataaaaagactatcc
wt atagaatgatacattctggaaagttggacgcttcaaaagtagcaaacaaattcctcattaaagag g gg g gg g g g gcp06 atagaatgatacattctggaaagttggacgcttcaaaagtagcaaacaaattcctcattaaaga cp10 atagaatgatacattctggaaagttggacgcttcaaaagtagcaaacaaattcctcattaaaga cp32 atagaatgatacattctggaaagttggacgcttcaaaagtagcaaacaaattcctcattaaaga cp33 atagaatgatacattctggaaagttggacgcttcaaaagtagcaaacaaattcctcattaaaga cp36 atagaatgatacattttggaaagttggacgcttcaaaagtagcaaacaaattcctcattaaaga
wt ggaagatgcaaaa-ggactattggaaaataaaaataaactggacaattaa--------------cp06 ggaagatgcaaaa-ggactattggaaaataaaa-taaactggacaattaataggaggaatattc cp10 ggaagatgcaaaaaggactattggaaaataa---------------------------------cp32 ggaagatgcaaaa-ggactattggaaaataaaaataaactggacaattaa--------------cp33 ggaagatgcaaaa-ggactattggaaaataaaaataaactggacaattaa--------------cp36 ggaagatgcaaaa-ggactattggaaaataaaaataaactggacaattaa-------------- wt -------------------------- 532 cp06 aatggaaaacaatactaaacaattaa 571 cp10 -------------------------- 514 cp32 -------------------------- 532 cp33 -------------------------- 532 cp36 -------------------------- 532cp36 532
on May 17, 2018 by guest
http://jb.asm.org/
Dow
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ORF7
wt atgcgtgacaaattgctcgactttatcatcgaactatcacaatctagcaaacaagtcgtaag-- cp08 atgcgtgacaaattgctcgactttatcatcgaactatcacaatctagcaaacaagtcgtaag-- cp27 atgcgtgacaaattgctcgactttatcatcgaactatcacaatctagcaaacaagtcgtaag-- cp04/18 atgcgtgacaaattgctcgactttatcatcgaactatcacaatctagcaaa------------- cp25/29 atgcgtgacaaattgctcgactttatcatcgaactatcacaatctagcaaacaagtcgtaagca
4564
cp25/29 atgcgtgacaaattgctcgactttatcatcgaactatcacaatctagcaaacaagtcgtaagca
wt --------caagtcgtatgtcattgatagactgatgcaagtaacaaaa-gaagactacaaggaa cp08 --------caagtcgtaa----------------------------------------------cp27 --------caagtcgtatgtcattgatagactgatgcaagtaacaaaaagaagactacaaggaa cp04/18 --------caagtcgtatgtcattga--------------------------------------cp25/29 agtcgtaa--------------------------------------------------------cp25/29 agtcgtaa
wt ctagaaaagaatgtggaggggaagaaggatgactga 4716 cp08 ------------------------------------ 4635 cp27 ctagaaaagaatgtggaggggaagaaggatga---- 4713 cp04/18 ------------------------------------ 4632 cp25/29 ------------------------------------ 4635 cp 5/ 9 635
on May 17, 2018 by guest
http://jb.asm.org/
Dow
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(B) gp1 wt MSKLLTAQEVADLLRVHKKTIHRMIHSGKLDASKVANKFLIKEEDAKGLLENKNKLDN------------- 58 cp06 MSKLLTAQEVADLLRVHKKTIHRMIHSGKLDASKVANKFLIKEEDAKGLLENKINWTINRRNIQWKTILNN 71
α1 α2
cp10 MSKLLTAQEVADLLRVHKKTIHRMIHSGKLDASKVANKFLIKEEDAKRTIGK------------------- 52cp32 MSKLLTAQEVADLLRIHKKTIHRMIHSGKLDASKVANKFLIKEEDAKGLLENKNKLDN------------- 58 cp33 MSKLLTAQEVVDLLRVHKKTIHRMIHSGKLDASKVANKFLIKEEDAKGLLENKNKLDN------------- 58 cp36 MSKLLTAQEVADLLRVHKKTIHRMIHFGKLDASKVANKFLIKEEDAKGLLENKNKLDN------------- 58
gp7gp7 wt MRDKLLDFIIELSQSSKQVVSKSYVIDRLMQVTKEDYKELEKNVEGKKDD 50 cp08 MRDKLLDFIIELSQSSKQVVSKS--------------------------- 23 cp27 MRDKLLDFIIELSQSSKQVVSKSYVIDRLMQVTKRRLQGTRKECGGEEG- 49 cp04/18 MRDKLLDFIIELSQSSKQVVCH---------------------------- 22 cp25/29 MRDKLLDFIIELSQSSKQVVSKS--------------------------- 23
on May 17, 2018 by guest
http://jb.asm.org/
Dow
nloaded from
(A) dinBox1 wt CGAACaagcGTTTT
185 198 g
cp16 CCAACaagcGTTTT cp17 CGAACaagcG-TTT cp19 CGAACaagcGATTT cp23 CAAACaagcGTTTT cp26 CGAACaagcGCTTT cp37 CGAACaagcG TTTcp37 CGAACaagcG-TTT Bam35c CGAACaagcTTTTT (B) B. subtilis cons. CGAACatatGTTCG lexA CGAACttatGTTTGrecA CGAACatttATTCG uvrAB CGAACagatATTCG ruvAB AGAACatttGTTGG dinBox1 CGAACaagcGTTTT di B 2 GG C G CGdinBox2 GGAACactcGTTCGdinBox3 CGAACaaacATTCG
(C) α1 α2 α3 B. cereus G9842 5 MEKLTKRQQDILDFIKLKVQEKGYPPSVREIGQAVGLASSSTVHGHLSRLEEKGYIRRDP 64 Gram-positive cons. 1 MSKLTKRQREILDVIKASVQSKGYPPSVREIGQAVGLASSSTVHGHLSRLERKGYIRRDP 60
on May 17, 2018 by guest
http://jb.asm.org/
Dow
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