Area de Microbiología, Facultad de Medicina, Universidad de Oviedo, Julián Clavería s. n., 33006 Oviedo, Spain1
Centro Nacional de Biotecnología, Campus de la Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain2
Author for correspondence: Juan Suárez. Fax +34 985 103148. e-mail jsuarez{at}correo.uniovi.es
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Abstract |
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Main text |
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The genetic switch of bacteriophage A2, which infects industrially relevant strains of Lactobacillus casei (Herrero et al., 1994 ), has a unique operator region (Op). Op is at the centre of a regulatory circuit governing the commitment of the phage between lytic or lysogenic development (Fig. 1A
) (Ladero et al., 1998
, 1999
; García et al., 1999
). The A2 Op region comprises two divergently oriented promoters: PL, which promotes transcription from cI, and PR, which directs expression of cro and the replication cassette (Ladero et al., 1998
; Moscoso & Suárez, 2000
). Interspersed in this segment, three similar, although not identical, 20 bp operator subsites (O1, O2 and O3) have been identified. The CI protein, which confers host immunity against phage A2, binds specifically to Op. At low concentrations, it interacts selectively with O1 and O2 (which overlap PR). This results in displacement of the RNA polymerase (
A-RNAP) from this promoter and enhances its positioning onto PL (García et al., 1999
). At higher concentrations, CI also occupies O3 (which overlaps PL) with the subsequent exclusion of
A-RNAP from the PL promoter. It is likely, therefore, that this is the mechanism by which CI represses in vivo the lytic development of phage A2, while promoting and maintaining its lysogenic state.
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The Cro protein of phage A2 was purified as described previously (Ladero et al., 1999 ). DNA extraction, analysis and purification of DNA fragments were carried out by standard methods (Sambrook et al., 1989
). In vitro run-off transcription, electrophoretic mobility shift and DNase I footprint assays were performed as described previously (García et al., 1999
).
The Cro protein of lambdoid phages is known to inhibit the transcription of cI by binding to OR3, which overlaps PRM (Ptashne, 1986 ). We hypothesized that the Cro analogue from A2 might play a similar role at PL, taking into account that Cro binds to the operator region and that it shows a higher affinity for O3 than for O1 and O2 (Ladero et al., 1999
). To address this question, we used a DNA fragment containing the switch region and an in vitro-transcription assay using
A-RNAP to follow the expression of PL in the presence of increasing concentrations of Cro. The addition of Cro reduced the utilization of PL in a concentration-dependent manner until no expression was detected (Fig. 1B
). As a control, it was determined that even the highest concentration of Cro used in the experiment did not affect the expression of an unrelated promoter (data not shown).
Having demonstrated that Cro represses transcription from the PL promoter, we studied its interaction with RNAP through electrophoretic mobility shift assay (EMSA) experiments (Fig. 2). The phage A2 switch region, obtained as an
-32P-labelled DNA segment, was incubated with Cro,
A-RNAP or
A-RNAP and increasing concentrations of Cro. ProteinDNA complexes were separated by non-denaturing PAGE and analysed by autoradiography. The DNA fragment formed two complexes with Cro: complex I, corresponding to its binding to O3, and complex II, in which it is bound to O1 and/or O2 (Fig. 2
, lane 3) (Ladero et al., 1999
). Similarly, two
A-RNAPDNA complexes (RPI and RPII) (Fig. 2
, lane 2) (García et al., 1999
) were observed. The RPI complex is probably made of a single
A-RNAP molecule bound to the DNA segment, while RPII would contain two
A-RNAP molecules, one bound to each promoter. The addition of one Cro promoter per
A-RNAP molecule to the pre-formed
A-RNAPDNA complex resulted in
45% loss in the intensity of the RPII band and an equivalent increase in the intensity of the RPI band, or, possibly, of a band that migrates slightly slower than RPI (named RPI*, see below) (Fig. 2
, lane 4). In the presence of a 2·5-fold excess of Cro per
A-RNAP molecule, the amount of
A-RNAP bound to both promoters became dramatically reduced (
5%) (Fig. 2
, lane 6), although complete displacement was not reached even at a 10-fold excess of Cro per
A-RNAP molecule (Fig. 2
, lane 8). Under these conditions, complex II formed by Cro bound to the three operator sequences became increasingly abundant. In parallel, a diffuse fast moving proteinDNA complex (termed II*) that migrated between both CroDNA complexes accumulated. These data can be best explained by assuming that Cro initially displaces
A-RNAP from one of the promoters to originate RPI*, which is postulated to be formed by the binding of one molecule of both
A-RNAP and Cro to the DNA fragment. Furthermore, although the nature of complex II* is unknown, it is proposed that it involves a loose interaction of
A-RNAP with the DNACro complex, because the II* diffuse complex is only formed when
A-RNAP is present in the reaction mixture (an explanation for the fast migration of complex II* in gels is provided below).
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From the data reported here and in previous articles, it appears that the general features of the lysis/lysogeny decision of phage A2 resemble those of the genetic switch (Hochschild et al., 1986
; Ptashne, 1986
) but its implementation and regulation seems to be simpler (García et al., 1999
; Ladero et al., 1999
; this work). It appears that the commitment between the two life cycles of phage A2 is taken very early after infection. Host RNAP presumably binds to both promoters, PL and PR, in the genetic switch region of the phage genome, since the space between the promoters easily admits two RNAP molecules coexisting in it, as has been shown in vitro (Fig. 2
, lane 2) (García et al., 1999
). This results in RNA synthesis from both operons; the constitutive transcription of the lysogenic operon, governed by PL, is noticeable (Ladero et al., 1998
). This contrasts with the case of phage
, where CII is needed for the initial synthesis of cI mRNA and where the N protein allows efficient read-through of the terminators placed in the early lytic region.
Presumably, the constitutive synthesis of CI has to be counterbalanced by an efficient production of Cro in order to direct a significant proportion of the infectious events towards the lytic route. This necessity might explain why the transcripts arising from PR are at least 10 times more abundant than those generated from PL (García et al., 1999 ). In this context, it must be noted that the in vitro affinity constants of Cro and CI for the genetic switch of phage A2 are very similar (7 and 6 nM, respectively) (García et al., 1999
; Ladero et al., 1999
). Thus, the in vivo function of these repressors in determining the commitment of the phage is determined primarily by their ability to distinguish between the O1O2 and the O3 operator sequences. CI binds preferentially to the O1 and O2 subsites, shutting off transcription from PR, while enhancing expression from PL so that lysogeny becomes established (García et al., 1999
).
Conversely, Cro binds preferentially to the O3 subsite. This results in repression of transcription from PL (Fig. 1B) and abolition of CI production. However, the data reported here suggest that RNAP remains bound to the PR promoter, originating a ternary DNACro (at PL)RNAP (at PR) complex, which accounts for the appearance of RPI* in EMSA (Fig. 2
, lane 4). Under these conditions, Cro (and replication proteins) accumulate(s). As a consequence, the phage will enter the lytic cycle. As the concentration of Cro rises, it will progressively interact with the remaining, low affinity, subsites (O2/O1). This would promote displacement of
A-RNAP from PR, which is detected by the generation of the II and II* complexes in EMSA (Fig. 2
, lanes 68) and repression of cro transcription. The biological meaning of this process might be that the unbinding of
A-RNAP from PR would indirectly enhance phage maturation by shutting off early gene expression.
In conclusion, it appears that the physiological differences induced by CI and Cro which lead towards the lysogenic versus the lytic cycle may be attributable to (i) the different degree of utilization of the promoters PL and PR, (ii) the different affinity of the repressors for the operator subsites and (iii) the subsequent displacement of A-RNAP from its cognate promoters induced by them.
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Acknowledgments |
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References |
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Received 1 March 2002;
accepted 10 July 2002.