ARTICLE

Top Abstract Article References

Bacteriophages are recognized to be the main source of disruption in industrial food fermentations (5). The temperate phage A2 infects strains of Lactobacillus casei and Lactobacillus paracasei of industrial relevance. The virions present isometric heads and noncontractile tails. The phage genome is a 44.02-kb double-stranded DNA molecule with 3'-protruding cohesive ends (6, 7). A2 can be recovered from lysogens through mitomycin C induction, suggesting that the phage repressor becomes inactivated by proteolytic cleavage during the mitomycin-induced SOS response as it occurs with bacteriophage  (17). The  repressor binds to promoters P_L and P_R, which results in repression of the genes that lead to the lytic development. One consequence of this regulation is that lysogens are immune to superinfection by the same or related viruses. In addition, cI is autogenously regulated through differential binding to three adjacent operator sites.

Characterization of A2 clear plaque deletion mutants. The gene that encodes the viral repressor was localized through the selection of deletion mutants unable to lysogenize L. casei ATCC 393. To get them, phage suspensions were treated with 10 mM sodium pyrophosphate, pH 7.4, at 37°C for 30 min, which resulted in survival of 7 × 10⁴ phage. Appropriate dilutions were plated onto MCM (4), and the surviving phage was collected, suspended in SM buffer, and subjected to new rounds of treatment until a plateau was reached at around 10% survival. Phage from isolated plaques, obtained after each round, was repurified, and their DNA restriction patterns were compared with that of the wild type. The deletions ranged from 0.5 to 3.5 kb and mapped in three EcoRI fragments that defined two regions of the genome, comprising up to 7.9 kb dispensable for lytic development (Fig. 1). The phage whose deletion was located in the center of the physical map showed a clear plaque phenotype and was unable to lysogenize its hosts. In contrast, lysogenization was easily obtained with the mutants lacking segments in the right arm of the genome.

View larger version (7K): [in this window] [in a new window]   Fig. 1.   EcoRI restriction map of A2 DNA with indication of dispensable regions for lytic development (thick lines in parentheses). Below the map is shown the organization of the region that controls the phage cycles. The arrows indicate the relative sizes and directions of transcription of the indicated genes.

Structural characterization of the repressor region of bacteriophage A2. Since the deletions located at the center of the A2 physical map resulted in impairment of lysogenization, we started the analysis of this region by cloning and sequencing it. In this sequence, four open reading frames (orfA to orfD), which read in opposite directions, were found (Fig. 1). The products of orfB and orfC were hypothesized to be the functional homologs of the  proteins CI and Cro, respectively. This was based on their sizes (224 and 81 amino acids for ORFB and ORFC, respectively, which correspond to 25,277- and 9,180-Da polypeptides), their transcription in opposite directions, with an intergenic region of 161 nucleotides (Fig. 2), and the similarities shown by ORFB (pI, 4.56) to phage repressors and also to regulatory proteins involved in SOS induction (Fig. 3). The NH₂-terminal end of ORFB presents a helix-turn-helix motif, which is possibly involved in binding of a specific DNA target, while its carboxy-terminal part shows a domain for protease RecA recognition. It includes conserved Ser and Lys residues and the Ala-Gly motif (marked with asterisks in Fig. 3), in front of which cleavage has been reported to occur in the  repressor, as the first step towards lytic development of the prophage (18).

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Nucleotide sequence of the region between cI and the putative cro. Potential ribosomal binding sites (RBS), consensus promoter sequences (35 and extended 10 boxes), the starts of translation, and the inverted repeats found are indicated.

View larger version (28K): [in this window] [in a new window]   Fig. 3.   Amino acid sequence alignment of ORFB with the CI proteins of phages  (16), 80 (12), and r1t (11); the putative repressor of Tuc2009 (19); and the SOS response-related proteins LexA (8) and DinR (14). The DNA binding motifs of the amino termini are boxed. Conserved amino acids are shown in boldface, and the RecA cleavage point is marked with asterisks.

Expression of orfB confers immunity to A2 superinfection. If orfB coded for the repressor, it should confer superinfection immunity against A2 when cloned into L. casei cells. To test this possibility, a 0.8-kb DNA segment containing orfB was amplified by using a primer that included its putative promoter sequence and a converging one located just after the stop codon, into which EcoRI restriction sites were introduced. The amplified DNA segment was purified, EcoRI cleaved, and ligated to pEM40 digested with the same enzyme to generate pEM40::orfB. This plasmid is a pUC18 derivative that contains an erythromycin resistance gene for selection in gram-positive bacteria. It does not replicate in L. casei but carries the integrase gene and the attP sequence of A2, which allows its insertion into a tRNA gene of several lactobacilli (1). Challenge of several independently obtained pEM40::orfB transformants with bacteriophage A2 resulted in complete immunity to superinfection (no plaques were produced by a phage suspension with a titer on the untransformed host of 10¹⁰ PFU/ml). This is consistent with the suggested function of orfB as the gene that encodes the A2 repressor (cI).

cI-specific transcripts produced during the lytic and lysogenic cycles of A2. As a final test of cI identity, its transcription pattern was investigated. A Northern blot of total RNA from L. casei ATCC 393 (without infection and at several times postinfection) and from one A2 lysogen derivative was probed with a PCR-generated P-labelled DNA fragment that exactly spans cI. Two transcripts, of 0.8 and 1.4 kb, were observed in the lysogen (Fig. 4A). The size of the first fits with the distance between the putative cI promoter and the rho-independent terminator identified 3' of that gene. The 1.4-kb transcript most probably corresponds to cI plus orfA. In productively infected cultures of L. casei ATCC 393 (Fig. 4B), the same pattern of cI-specific RNAs was found at early times postinfection (from 15 to 25 min) and fading afterwards (the eclipse period of the phage under the propagation conditions used lasts about 120 min).

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Northern blot analysis of the cI transcripts. (A) Equal amounts of total RNA from uninfected L. casei (lane 1) and from an A2 lysogen (lane 2) were used. (B) Total RNA from L. casei at various times postinfection (in minutes) with bacteriophage A2. The numbers beside the size standards are in kilobases.

Nucleotide sequence accession number. The sequence of the repressor region of bacteriophage A2 has been submitted to EMBL under accession no. Y12813.