Autores: Jose Antonio Horcajadas ¹, Mara Monsalve¹, Fernando Rojo² and Margarita Salas¹

¹Centro de Biología Molecular `Severo Ochoa'' (CSIC-UAM) Universidad Autónoma, Cantoblanco, 28049-Madrid, Spain.

²Centro Nacional de Biotecnología (CSIC), Campus de la Universidad Autónoma de Madrid, Cantoblanco 28049-Madrid, Spain

Resumen: The transcription program of the Bacillus phage GA-1, a distant relative of phage φ29, has been studied. Transcription of the GA-1 genome occurred in two stages, early and late. Early genes were expressed from two promoters equivalent to the φ29 A2b and A2c promoters, whereas late transcription started at a site equivalent to the φ29 late A3 promoter

The activity of the GA-1 early A2b and A2c promoters diminished 10 minutes after infection, a time at which expression of the late promoter increased signi®cantly. The switch from early to late transcription required protein synthesis, suggesting the need for viral protein(s).

An open reading frame was found in the GA-1 genome coding for a protein showing a 53% similarity to φ29 regulatory protein p4, and was named p4G.

In φ29, protein p4 represses the early A2b and A2c promoters and activates the late A3 promoter by recruiting RNA polymerase to it. A binding site for protein p4G was localized upstream from the GA-1 late A3 promoter, overlapping with the early A2b promoter. In vitro, protein p4G prevented the binding of RNA polymerase to the GA-1 early A2b promoter but, unlike in φ29, had no effect on the expression of the lateA3 promoter: RNA polymerase could ef®ciently bind and initiate transcription from the A3 promoter in the absence of protein p4G.

Therefore, activation of late transcription occurs differently in GA-1 and φ29. We propose that protein p4G is an anti-repressor which inhibits the binding to the late promoter of an unknown repressor factor present in the host strain.

Keywords: transcription regulation; protein-DNA interactions; prokaryotic repressors; DNA binding; Bacillus

Introduction: The study of the life cycle of bacteriophages has provided invaluable information on the mechanisms of transcription regulation in prokaryotes. Several phages infecting Gram negative bacteria, such as lambda, T4, T7, Mu and others, have been studied in detail. Among them, phage lambda stands as the best-studied model of gene expression (Ptashne, 1986). In the case of phages infecting Gram positive bacteria, many fewer examples are known.

One of the most thoroughly studied is Bacillus subtilis phage φ29. A number of φ29 related phages have been isolated and their evolutionary relatedness has been established on the basis of serological properties, DNA physical maps, peptide maps, sequence of their 50-terminal inverted repeats (Yoshikawa et al., 1985, 1986), and comparison of DNA and amino acid sequences of selected DNA regions and proteins (Pecenkova & PacÏes, 1999). As in phage φ29, they all have linear double-stranded DNAs with a terminal protein covalently linked to their 50 ends. Within them, phage GA-1 is the most distantly related to φ29.

It has a different host speci®city, infecting Bacillus sp. G1R, but not B. subtilis (Arwert & Venema, 1974). Although it is expected that GA-1 and φ29 should regulate the transcription of their genomes in a similar way, their evolutionary distance is large enough so as to expect differences which could Present address: M. Monsalve, Dana Farber Cancer Institute, Smith Building, room 950, One Jimmy Fund Way, Boston, MA 02115, USA.

Abbreviations used: CTD, C-terminal domain; RNAP, RNA polymeras

Autores: Wilfred J. J. Meijer, José A. Horcajadas and Margarita Salas

Centro de Biología Molecular “Severo Ochoa” (CSIC-UAM), Universidad Autónoma, Canto Blanco, 28049 Madrid, Spain.

Introducción: The genus Bacillus incorporates many species of gram-positive, aerobic, endospore-forming bacteria that normally inhabit the soil or decaying plant material. In these habitats, a large variety of phages have been isolated that infect bacilli. All of these phages isolated so far have some common features

First, they all contain double-stranded DNA dsDNA), and second, the virions have prolate icosohedral heads and are tailed. Modern phage taxonomy is based on properties of the virion and its nucleic acid (see references 74 and 131).

The order of tailed phages, named Caudovirales, are classified into three families: Myoviridae (phages with contractile tails), Podoviridae (phages with short tails), and Siphoviridae (phages with long noncontractile tails).

For a general review on tailed bacteriophages, see reference 4. In addition to taxonomy based on properties of the virion and its nucleic acid, phages can be divided into three groups based on their infection cycle. The first group contains lytic phages that complete their life cycle within a well-defined period after infection and are unable to lysogenize their host. The second group is formed by the socalled pseudo-temperate phages.

These are virulent phages with an extended and irregular latent period. Although this stage mimics lysogeny, it does not involve a stable rophage.

The third group contains the temperate phages. The genomes of these phages are able to integrate into the host genome and can be maintained in this lysogenic stage for many generations. Generally, during this stage, the cells are immune to infection with the same phage ...

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Autores: José A. Horcajadas,1 Wilfried J. J. Meijer,1 Fernando Rojo,2 Margarita Salas1 1Centro de Biología Molecular “Severo Ochoa” (CSIC-UAM)1 and Centro Nacional de Biotecnología (CSIC), 2Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain

Resumen: Bacteriophage GA-1, which infects Bacillus sp. strain G1R, is evolutionarily related to  φ29, which infects Bacillus subtilis. We report the characterization of several GA-1 promoters located at either end of its linear genome. Some of them are unique for GA-1 and drive the expression of open reading frames that have no counterparts in the genome of φ29 or related phages.

These unique promoters are active at early infection times and are repressed at late times. In vitro transcription reactions revealed that the purified GA-1-encoded protein p6 represses the activity of these promoters, although the amount of p6 required to repress transcription was different for each promoter. The level of protein p6 produced in vivo increases rapidly during the first stage of the infection cycle. The protein p6 concentration may serve to modulate the expression of these early promoters as infection proceeds

Introducción: A large variety of phages that infect bacteria of the genus Bacillus have been characterized. Particular attention has been given to the so-called φ 29 family of phages that infect different Bacillus species. The genome of these lytic phages consists of a small linear double-stranded DNA of about 20 kbp, with a terminal protein covalently linked to the 5 ´ends that plays a key role in the initiation of phage DNA replication. On the basis of serological properties, DNA physical maps, peptide maps, and partial or complete DNA sequences (26, 36, 37), the φ 29 family of phages has been classified into three groups. The first group includes phages φ 29, PZA, φ 15, and BS32; the second one includes B103, Nf, and M2Y; and the third group has phage GA-1 as its sole member. Among them, phage φ29 has been extensively characterized, being one of the best-studied bacteriophages of gram-positive bacteria. Its mechanism of DNA replication and its regulation of transcription have been reviewed previously (19, 28, 29). Within this family of phages, GA-1 is the one most distantly related to φ 29; this has stimulated the study of its mechanisms of DNA replication (6, 7, 8, 12) and transcription regulation (11).

The DNA sequences of the complete genomes of phages φ 29 (34), PZA (23), B103 (25), and GA-1 (19) have been determined. The GA-1 genome has a size of 21,129 bp, which is larger than those of φ 29 (19,285 bp), PZA (19,366 bp), and B103 (18,630 bp). In most aspects, the genomes of phages φ 29, B103, and GA-1 are similarly organized. In φ 29 (group I) and B103 (group II), the genes expressed soon after infection (early genes) are clustered in two operons located at each end of the genome. The late genes are located in a single operon that is positioned at the central part of the genome.

As shown schematically in Fig. 1, the late genes of GA-1 (genes 7 through 16) are also present in a single operon located in the central part of the genome. As in φ 29 and B103, the late GA-1 operon is flanked on its left side by an early operon that contains genes necessary for DNA replication and for transcriptional regulation (genes 6 through 2).

These genes are expressed from the early promoters A2b and A2c (11). The right region of the GA-1 genome contains open reading frames (ORFs) whose deduced protein sequences are homologous to those of the φ 29 early genes 17 and 16.7, which are involved in DNA replication (5, 17, 18).

However, both ends of the GA-1 genome contain a number of sequences and ORFs that have no counterparts in φ 29 or in any of the other related phages characterized (19). Therefore, the proteins that are probably encoded by these ORFs are unique for GA-1. Several putative promoters that could be responsible for the expression of these unique ORFs were identified. In this work we characterized these promoters, analyzing their expression patterns throughout the infection cycle.

We also analyzed the role of GA-1 protein p6 in the regulation of the early promoters in vitro. Identification of the early promoters A1a, A1b, A1c, C2, and C1b. Analysis of the 2.8-kb region on the left side of the GA-1 genome led to the identification of at least three possible promoters (Fig. 1), all of which contain typical -35 and -10 boxes for the σ A RNA polymerase. Promoter A1b is homologous to the φ 29 A1 promoter, which is responsible for theexpression of a small RNA (named pRNA) required for theencapsidation of the viral genome into the proheads (2, 9). The two other promoters, A1a and A1c, are not present in the genomes of other related phages whose sequences are known.

Promoter A1a is located upstream of a putative operon containing ORFs M, N, and O, and promoter A1c is located upstream of another putative operon containing ORFs P, Q, R, S, and T. These ORFs account for part of the difference in size of GA-1 DNA and φ 29 (GA-1 DNA ca. 2 kb larger than φ 29). Two other promoters, named C2 and C1b, can be predicted in the right region of the GA-1 genome. Promoter C2 is homologous to the φ 29 C2 promoter that drives the expression of the operon containing genes 17 and 16.7. In φ 29, an additional weak promoter, named C1, is present within gene 16.7. In the case of GA-1, the second predicted promoter in this region maps upstream of gene 16.7. Therefore, this promoter is not equivalent to φ 29 C1, and we have named it C1b.

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