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Expression of Mycobacteriophage Ms6 Lysis Genes Is Driven by Two ⁷⁰-Like Promoters and Is Dependent on a Transcription Termination Signal Present in the Leader RNA

Laboratório de Microbiologia Molecular, Centro de Patogénese Molecular, Faculdade de Farmácia, Universidade de Lisboa, Lisbon, Portugal

Received 11 December 2000/ Accepted 20 February 2002

A mycobacteriophage Ms6 strong promoter region (P_lys) was isolated by using transcriptional fusions with the lacZ reporter gene. Two tandem ⁷⁰-like promoter sequences (P1 and P2) were found in this region. DNA sequencing of the promoter downstream region revealed a 214-bp leader sequence followed by five adjacent coding regions of 231 bp (ORF1), 1,152 bp (ORF2), 996 bp (ORF3), 231 bp (ORF4), and 372 (ORF5). ORF1 has the potential to encode a 77-amino-acid protein which revealed similarity to mycobacteriophage TM4 gp90, a predicted protein with unknown function. ORF2 encodes a 384-amino-acid protein which is related to several bacteriophage amidases. This protein induced cell lysis upon addition of chloroform, confirming its mureinolytic activity. ORF3 encodes a 332-amino-acid protein which is related to TM4 gp30, a protein with sequence similarity to amidases. ORF4 encodes a 77-amino-acid holin-like protein with significant similarity to the holin of Lactococcus lactis r1t bacteriophage. ORF5 encodes a 124-amino-acid protein which is related to mycobacteriophage L5 gp30, a protein with unknown function. These data indicate that the promoter region P_lys drives the transcription of the Ms6 lysis genes. An intrinsic transcription termination signal was identified in the leader sequence. Experiments using lacZ fusions showed that ß-galactosidase synthesis is inhibited when this transcription termination signal is present in the leader sequence. In conclusion, mycobacteriophage Ms6 cell lysis genes are expressed by their own promoter region, independently of virion structure and assembly protein genes. Moreover, an antitermination mechanism might be involved in their transcription regulation.

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