1 Université Catholique de Louvain, Place Croix du Sud, 2/12, B-1348 Louvain-la-Neuve, Belgium
2 National Institute of Occupational Health, Lersø Parkallé, DK-2100, Copenhagen, Denmark
Correspondence
Jacques Mahillon
Mahillon{at}mbla.ucl.ac.be
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ABSTRACT |
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The GenBank accession number for the sequence of pGIL01/GIL01 is AJ536073.
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INTRODUCTION |
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B. thuringiensis strains produce, during their sporulation, crystal toxins (delta-endotoxins) that are highly toxic to a number of insect larvae belonging to the orders Lepidoptera, Diptera and Coleoptera, but harmless to vertebrates. Classically, the numerous entomopathogenic B. thuringiensis strains, which have their own specific insecticidal activity, have been classified in different serotypes on the basis of their flagellar antigens. B. thuringiensis serovar (sv.) israelensis is active against dipteran species and is therefore one of the bioinsecticides of choice to control black flies and mosquitoes, both important vectors of human and animal diseases (for a recent review, see Glare & O'Callaghan, 2000).
B. thuringiensis sv. israelensis strain H14 has been reported to contain at least eight DNA molecules, including three small (5·4, 6·7 and 7·6 kb) and four large (128, 145, 240 and 350 kb) circular plasmids, and one linear molecule (G. Jensen & L. Andrup, unpublished results). The pathogenicity of this strain only depends on the presence of the 128 kb plasmid which encodes the Cry and Cyt toxins (González & Carlton, 1984). This megaplasmid, named pBtoxis, has been sequenced, allowing the discovery of new important toxic factors potentially involved in the insecticidal activity (Berry et al., 2002
). The 350 kb molecule, named pXO16, is a conjugative plasmid also observed in other strains of B. thuringiensis. It is responsible for an aggregation-mediated conjugation system, leading to a very high frequency of transfer (
100 %) (Jensen et al., 1995
). The sequence analysis of the three small circular plasmids, named pTX14-1, pTX14-2 and pTX14-3, revealed the presence of genes implicated in plasmid replication (rep) and mobilization (mob) (Madsen et al., 1993
). Interestingly, they each harbour a specific gene containing repetitive elements similar to collagen. However, the function of these genes, termed bcol for Bacillus-collagen-like genes, has not been established (Andrup et al., 2003
).
B. thuringiensis strains are naturally associated with a number of virulent (Colasito & Rogoff, 1969b) or temperate phages (Ackermann & Smirnoff, 1978
; Colasito & Rogoff, 1969a
). It has been shown that, during their lysogenic state, temperate phages are characterized by their integration into the host genome (chromosome or plasmids) (Kanda et al., 1998
). However, until now, no prophage existing as an autonomous plasmid in the host cell has been observed in B. thuringiensis.
In this study, we report the complete DNA sequence of a linear molecule originating from B. thuringiensis sv. israelensis strain H14. This 15 kb linear element, named pGIL01, actually corresponds to the prophage form of a temperate bacteriophage, GIL01, and this establishes the first molecular characterization of a linear prophage originating from B. thuringiensis.
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METHODS |
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Cloning strategy.
A combination of different strategies was used to sequence the entire pGIL01 molecule. Restriction fragments as well as PCR products were cloned into positive selection vectors [pZErO-2, pCR4-TOPO and pCR-XL-TOPO (Invitrogen)]. Sequences derived from these PCR products were confirmed by direct sequencing on phage GIL01 DNA. The remaining sequences of pGIL01/GIL01 were obtained by primer-walking along the phage DNA, including run-off sequencing of the extremities. Poly(dT) tailing of GIL01 used for the confirmation of the terminal sequences was performed with the calf thymus terminal transferase (Roche) according to the manufacturer's recommendations (Polo et al., 1998). Both extremities of the tailed DNA were amplified using poly(dA)35 in combination with internal primers. The detailed strategy can be obtained from the authors upon request.
Proteinase K, exonuclease III and lambda exonuclease treatments of GIL01.
DNA preparations of GIL01 (see below), performed without proteinase K pretreatment, were incubated with variable amounts of proteinase K (0·01 and 0·1 mg ml-1) for 4 h at 37 °C. The effect of the protease was analysed by running the GIL01 DNA preparation on a 0·8 % agarose gel (0·5xTAE; TAE, 40 mM Tris/acetate, 1 mM EDTA). GIL01 preparations were treated with exonuclease III (MBI, Fermentas) or lambda exonuclease (Roche), according to the manufacturer's recommendations. After incubation at 37 °C for 1090 min, the reaction was stopped by chilling on ice. The samples were analysed by electrophoresis on a 0·8 % agarose gel (0·5xTAE).
Phage DNA extraction.
DNA extraction from phage particles was performed using the protocol described by Santos (1991) and modified as follows. To each millilitre of phage suspension treated with DNase (5 µg ml-1) and RNase (10 µg ml-1) for 30 min at 37 °C, 20 µl of a filtered sterilized 2 M solution of ZnCl2 was added and the solution was incubated for 5 min at 37 °C. After centrifugation for 1 min at 10 000 r.p.m., the supernatant was removed and the pellet was resuspended in 500 µl TES buffer (0·1 M Tris/HCl, pH 8; 0·1 M EDTA; 0·3 % SDS) and incubated at 60 °C for 15 min. After incubation for 90 min at 37 °C with 20 µl proteinase K (stock solution of 20 mg ml-1), 60 µl of a 3 M potassium acetate solution (pH 5·2) was added (incubation on ice for 1015 min). This solution was first treated with phenol/chloroform/isoamyl alcohol (25 : 24 : 1, by vol.) and then with chloroform/isoamyl alcohol (24 : 1, v/v), was precipitated with 2-propanol, and finally washed with 70 % ethanol, dried and recovered in 1020 µl distilled water.
Treatment of phage GIL01 with organic solvents.
The GIL01 suspension (2 ml) was mixed with various amounts of chloroform (10, 40 and 80 µl) or ether (10, 40, 80, 160 and 320 µl) and incubated at room temperature for 15 min. The aqueous phase was then titrated for surviving phages. A control sample without solvent was treated simultaneously.
Mitomycin C, nalidixic acid and UV treatments.
An exponential culture (20 ml) was centrifuged and the pellet was resuspended in 5 ml of 0·01 M MgSO4. Mitomycin C and nalidixic acid were added to the solution at final concentrations of 1 and 40 µg ml-1, respectively. The solution was incubated for 30 min at 30 °C, and then washed twice with 5 ml of 0·01 M MgSO4. The bacterial cells were resuspended in 20 ml fresh LB medium and incubated for 4 h at 30 °C. After centrifugation, the filtered supernatant (0·45 µm filter) was analysed by spot test and/or by titration.
For UV treatment, the suspension was placed in a 9 cm glass Petri dish and irradiated with 254 nm UV light, for 520 s. This treatment produced between 90 and 95 % cell lethality. The irradiated suspension was then incubated for 15 min at 30 °C, and centrifuged. The bacterial cells were resuspended in 20 ml fresh LB medium, incubated for 4 h at 30 °C and, after centrifugation, the titre of the filtered supernatant was analysed.
PCR amplification from phage plaques.
Samples of top agar (1 mm in diameter) corresponding to a phage plaque, or a bacterial lawn for the negative control, were recovered from a titration plate, and diluted in 20 µl of 0·9 % NaCl. PCRs were then performed on 2 µl of those samples using primers matching pGIL01. Primer 3 (5'-CGGTTGCTTTGCCGTATG-3') and primer 4 (5'-GGCGAACAACATGCTTTGGG-3') allowed the amplification of a 1·4 kb fragment.
Bioinformatics.
Analyses of DNA and protein sequences were performed with either the GCG (genetics computer group) or the EMBOSS packages at the Belgian EMBL Node (BEN). The sequence of pGIL01/GIL01 has been deposited in the reference databases under accession number AJ536073.
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RESULTS |
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pGIL01 is a 14 931 bp linear molecule
Several complementary approaches were combined to determine the entire DNA sequence of pGIL01 (see Methods). The size of pGIL01 was determined to be 14 931 bp. As illustrated in Fig. 1(b), its genome is flanked by 73 bp terminal inverted repeats sharing more than 75 % identity. Detailed analysis of the pGIL01 sequence also revealed the existence of 30 potential ORFs. The main features of these ORFs are listed in Table 2
. Interestingly, most ORFs were relatively short, all pointing in the same direction, with small intergenic spaces (Fig. 1a
).
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ORF13 displays 23 % identity (Fig. 2b) to the DNA-packaging protein of PR4 and PRD1 (P9 protein), two closely related bacteriophages belonging to the Tectiviridae family. These proteins are necessary for filling new virus particles with DNA (Mindich et al., 1982
).
The amino acid sequences of ORF25 and ORF30 are related to various lytic enzymes (muramidase and autolysin precursors, and N-acetylmuramidases) found in bacteria (B. thuringiensis, Lactobacillus plantarum, Streptococcus mutans, Streptococcus pyogenes and Clostridium tetani) and phages (bacteriophage Bastille, bacteriophage phi-gle and bacteriophage phi-105) (3238 % identity for ORF25 and up to 53 % identity for ORF30). The similarity is mainly observed in the N-terminal region of the enzymes, which, most probably, contains the active site (Buist et al., 1995). In bacteria, these lytic enzymes are implicated in peptidoglycan recycling, cell separation, formation of flagella, or sporulation (Buist et al., 1995
; Heidrich et al., 2001
). They are also found in several phages where they allow the phage particle to enter or to escape the cell through (partial) wall lysis (Young et al., 2000
).
Interestingly, ORF1 showed distant similarity (3140 % identity) to putative excisionases (Mycobacterium tuberculosis, Corynebacterium glutamicum) and DNA-binding proteins (Mycobacterium leprae) reported in bacterial genomes (data not shown). Where documented, these proteins are known to help phage integrases in the process of prophage excision from the bacterial chromosome. However, in the case of pGIL01, no integrase-like ORF could be identified.
pGIL01 corresponds to the prophage state of a temperate phage, GIL01
Based on the pGIL01 sequence analysis indicating relevant homologies to different phage proteins, it was postulated that pGIL01 could correspond to the prophage form of a lysogenic phage. To verify the presence of particles of this putative phage (named GIL01), titration analysis was performed on strain GBJ002, a H-14 derivative cured of all plasmids, including pGIL01 (and therefore supposedly sensitive to GIL01 infections). As predicted, typical small and turbid plaques were observed on plate cultures of GBJ002, suggesting the presence of phage particles in the AND508 culture supernatant. Furthermore, no plaques were observed when strain AND508 was plated out, presumably because of phage immunity. The titre of phage suspensions, corresponding to the supernatant of an exponential culture of AND508, was estimated to be 8x105 p.f.u. ml-1.
To identify these virus particles, PCRs, using primers specific to pGIL01, were performed on individual plaques, as well as on the GBJ002 bacterial lawn. A 1·4 kb fragment was observed for both PCRs performed on the phage plaques and on the AND508 DNA preparation. However, the GBJ002 DNA preparation did not produce any PCR product (data not shown).
Finally, potential GBJ002 lysogens were isolated from the turbid plaques and tested for the presence of the pGIL01 prophage. When PCRs were performed on the DNA extracted from these bacteria, they showed the same 1·4 kb fragment, supporting the transfer of pGIL01 from strain AND508 to strain GBJ002 (data not shown).
The sensitivity of GIL01 to organic solvents was then investigated. GIL01 turned out to be very sensitive to chloroform. For a ratio of 25 : 1 (phage suspension versus solvent), all the GIL01 present in the phage suspension was inactivated. Conversely, ether had a more limited effect on GIL01: with a ratio of 6·25 : 1, less than 10 % of the phage particles were inactivated.
UV irradiation, mitomycin C and nalidixic acid induce GIL01 phages
Different DNA-damaging treatments, known to induce the lytic cycle of temperate phages, were tested on strain AND508 (see Methods). The addition of mitomycin C and nalidixic acid, at final concentrations of 1 and 40 µg ml-1, respectively, increased the GIL01 titre of a culture supernatant of AND508 by a factor of 102103. GIL01 was also induced by UV irradiation since a 104-fold increase of the titre could be obtained with UV doses producing between 90 and 95 % cell lethality (data not shown). These experiments suggested the existence of a specific correlation between the bacterial SOS response and GIL01 induction.
pGIL01 and GIL01 DNA are protected by proteins at their 5'' extremities
Several Gram-positive linear phages (e.g. the 29-like family) belong to the group of molecules called invertrons, which have a protein at their 5' extremities (Salas, 1991
). These terminal proteins are essential for the replication process. Similar proteinDNA structures are also observed in adenoviruses, in the Tectiviridae virus PRD1 and in several bacterial and fungal linear plasmids. Experiments using proteases and exonucleases were therefore set up to characterize the structure of the GIL01 extremities.
Proteins present at the termini of a linear molecule generally prevent its migration through an electrophoresis gel. Their removal by proteolysis during DNA extraction is expected to restore its migration ability. The fact that GIL01 DNA migration was dependent on proteinase K treatment (Fig. 3, lane 2) suggested that proteins were bound to the DNA molecule. Furthermore, subsequent additions of proteinase K (0·01 and 0·1 mg ml-1) to GIL01 DNA preparations obtained without protease pretreatment restored its migration (Fig. 3
, lanes 3 and 4).
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To complete this analysis, a subset of five positive strains was further investigated by PCR, using primer pairs defining a series of overlapping sections of pGIL01. However, some of the pGIL01-related PCR fragments obtained for strains B16, B23, DBT012, BGSC4D14 and Bt5 displayed different sizes than their corresponding pGIL01 fragments (data not shown), suggesting that these molecules were slightly different from pGIL01. To gather more insights into these pGIL01-related molecules, additional experiments were undertaken on strain B16. This strain supernatant produced small and turbid plaques when tested on the GIL01-sensitive strain GBJ002, and PCR analysis confirmed that the corresponding viral particles, named GIL16, were GIL01-related. Comparative RFLP analyses were undertaken on the B16 and AND508 phages using DNA preparations from phage mutants producing clear plaques (named cpGIL16 and cpGIL01, respectively). As shown in Fig. 5, although both phage DNA had a similar size (lanes 1 and 2), their restriction patterns indicated the presence of important variations. Whereas the restriction profile of the GIL01 DNA perfectly matched that deduced from the pGIL01 sequence, that of GIL16 turned out to be different.
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Linear molecules of apparently the same size as pGIL01 were also reported in the type strain of B. cereus (ATCC 14579T) (Carlson et al., 1992) and in strain SCE2 of Paenibacillus polymyxa (Rosado & Seldin, 1993
). However, hybridization, using pGIL01 as probe, performed on these strains displayed no cross-reaction between these plasmids, suggesting that they are not closely related (data not shown).
GIL01 host range
The activity of GIL01 was tested on 44 strains of the B. cereus sensu lato group (30 B. cereus sensu stricto, 3 B. thuringiensis, 3 B. weihenstephanensis, 7 B. mycoides and 1 B. pseudomycoides) which did not harbour pGIL01-related elements, as indicated by hybridization and PCR experiments. Surprisingly, none of the strains tested was sensitive to GIL01, and, under these experimental conditions at least, its host range seemed to be restricted to H14-derived strains cured of pGIL01.
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DISCUSSION |
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One of the most notable characteristics of tectiviruses resides in their double-layer capsid: the dsDNA is located within a lipid-containing membrane vesicle covered by a rigid protein capsid (Bamford & Mindich, 1982). This lipid layer, derived from the host phospholipid pool (Davis et al., 1982
), plays a key role during infection. It is transformed into a tubular structure that penetrates the host cell, allowing the injection of the viral genome (Bamford & Mindich, 1982
; Grahn et al., 2002
).
Although members of the Tectiviridae family are found in both Gram-negative (PRD1, PR3, PR4, PR5, PR722 and L17) and Gram-positive hosts (AP50 and Bam35, see below), the best-studied members are those infecting Gram-negative bacteria. These phages are very closely related to each other and PRD1 is considered as the family model (Bamford et al., 1981). PRD1 can infect a large variety of Gram-negative hosts, including E. coli and Salmonella typhimurium, harbouring conjugative plasmids of the P, N and W incompatibility groups, which code for the phage receptor (Bradley & Rutherford, 1975
). The genome of PRD1 itself consists of a 14 925 bp linear dsDNA protected by proteins covalently attached at the 5' ends (Bamford et al., 1983
). As for adenoviruses (Challberg et al., 1980
) and the Bacillus phage
29 family (Meijer et al., 2001
), the DNA polymerase catalyses the formation of a covalent bond between the first 5' nucleotide and the OH group of a specific amino acid of the terminal protein, which will then serve as primer (de Jong & van der Vliet, 1999
; Salas, 1991
).
In Bacillus spp., two potential Tectiviridae phages have been reported. Phage AP50 (Nagy, 1974) and Bam35 (Ackermann et al., 1978
) were isolated from B. anthracis and from strain Nr.35 of B. thuringiensis sv. alesti, respectively. These bacteriophages were only partially characterized. While AP50 produced turbid plaques and had an outer diameter of 80 nm, Bam35 made clear plaques and measured 63 nm in diameter. They were both sensitive to lipid solvents, such as chloroform and ether, and were highly resistant to UV.
pGIL01/GIL01 harboured six ORFs showing significant similarity to known phage proteins. The 735 residues of ORF5 showed homology to several DNA polymerases of the B-type family requiring the terminal protein as primer to initiate the replication process. Analysis of the pGIL01 nucleotide sequence revealed that, in addition to the presence of the three regions conserved in the B-type family, this putative polymerase also contains the specific residues involved in the protein-priming mechanism (Fig. 2a) (Meijer et al., 2001
), suggesting that its replication process may occur via a similar mechanism to those of the PRD1 and
29 phages.
By analogy to the genetic organization of PRD1, 29 and other linear molecules, ORF4, located upstream of the DNA polymerase gene, is a good candidate for encoding the terminal protein of pGIL01. Moreover, the size of this potential protein (245 aa) is similar to that of the PRD1 and
29 terminal protein (259 and 266 aa, respectively). However, ORF4 did not share any significant homology with any other terminal protein. Experimental evidence is thus required to ascertain this putative function.
As in the case of other lysogenic phages, it is likely that pGIL01 ensures its cell immunity by expressing a phage repressor during its lysogenic cycle. The sequence derived from ORF6 showed homology to several LexA repressors. In the case of UV irradiation or other DNA-damaging treatments, RecA acts as a co-protease and cleaves the LexA repressor, resulting in acceleration of the replication and repair process. It has been shown that, for several prophages, the induction mechanism appears to be mediated by DNA-damaging treatments, in the same way as for the bacterial SOS response. A very similar SOS regulatory network to that of E. coli has been identified for the Bacillus group. In Bacillus subtilis, for instance, the induction of the SOS responses is dependent on the coordinate expression of more than 20 din (damage inducible) genes (Dubnau & Lovett, 2002). In the case of GIL01, experiments using mitomycin C, nalidixic acid and UV irradiation caused an increase in the phage titre by 50- to 1000-fold. Whether or not ORF6 corresponds to the bona fide phage repressor participating in the bacterial SOS induction remains to be confirmed.
ORFs 25 and 30 displayed homologies to different peptidoglycan lytic enzymes. For PRD1, two lytic enzymes have been reported. Protein P15, a 1,4--N-acetylmuramidase, which is connected to the phage membrane, causes the host cell lysis and the release of the viruses (Caldentey et al., 1994
). Protein P7 carries a conserved transglycosylase domain and is the main muralytic enzyme involved in PRD1 entry. However, P7 is not absolutely essential for infectivity, probably due to the presence of another lytic enzyme (Grahn et al., 2002
; Rydman & Bamford, 2002
). In the case of phage
29, the membrane protein holin introduces pores in the cell membrane to permeabilize it, allowing the peptidoglycan hydrolase to degrade the cell wall (Meijer et al., 2001
). In other cases where no muralytic enzyme is encoded, the lytic enzyme acts as a cell wall antibiotic inhibiting the synthesis of the peptidoglycan (Bernhardt et al., 2001
). In GIL01, no ORF sequences displayed similarity to known holin proteins, nor to inhibitors of peptidoglycan synthesis.
ORF13 shared similarity to the P9 protein of PRD1, which corresponds to the DNA-packaging protein implicated in maturation of PRD1 particles. The presence of an ATP-binding consensus motif (GxxGxGKxxxxxxxL) on gene IX of PRD1, which is also observed on ORF13 of GIL01, suggests that protein P9 may be the packaging ATPase providing the energy required for DNA encapsidation (Bamford et al., 1991; Mindich et al., 1982
).
The role of the putative excisionase (ORF1) in the translocation and/or structural rearrangements of the pGIL01/GIL01 genomes remains very speculative. Indeed, no site-specific recombinase (tyrosine or serine recombinases) could be identified in pGIL01. Such proteins are generally associated with excisionases in other plasmid or phage integration/excision processes.
GIL01 exhibited an extremely narrow host specificity since it seemed to be restricted to strains of serotype H14 of B. thuringiensis cured of pGIL01, such as GBJ002. Despite this GIL01-limited host range, pGIL01 variants were observed in five other B. cereus sensu lato strains (B16, B23, DBT012, BGSC4D14 and Bt5). It is therefore plausible that pGIL01 used alternative ways for transfer.
The PRD1 host spectrum depends on the presence in its Gram-negative hosts of conjugative plasmids from the P, N and W incompatibility groups (such as the broad-host-range IncP-type RP4 plasmid). The plasmid functions are only required in the early stage of infection, the phage DNA entry. In fact, the plasmid protein complex involved in the mating pair formation (Mpf, a type IV secretion system) is a membrane-associated structure facilitating the DNA transfer through the membranes into the recipient cell (Grahn et al., 2000). It remains to be seen whether GIL01 also takes advantage of a Gram-positive cell wall- and/or a membrane-associated complex involved in genetic transfer or bacterial pathogenesis.
No hybridization between pGIL01 and the linear molecules present in the type strain of B. cereus sensu stricto (ATCC 14579T) (Carlson et al., 1992) and in P. polymyxa (Rosado & Seldin, 1993
) could be observed. Although the nature of these extrachromosomal DNA remains uncertain, it is possible that they also belong to the Tectiviridae family, which would further extend the variation spectrum of this interesting family of bacteriophages. Finally, it is also important to note that, in addition to pGIL01, the coliphage N15 is the only prophage existing as an autonomous linear plasmid reported so far (Rybchin & Svarchevsky, 1999
). However, the occurrence of extrachromosomal linear molecules has been systematically underestimated due to the use of inappropriate protocols for plasmid DNA extraction. A more thorough search for linear extrachromosomal molecules might provide new linear prophage candidates, both in Gram-negative and Gram-positive bacteria.
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ACKNOWLEDGEMENTS |
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Received 18 February 2003;
revised 11 April 2003;
accepted 28 April 2003.