Organization and transcriptional analysis of the Listeria phage A511 late gene region comprising the major capsid and tail sheath protein genes cps and tsh.

摘要

A511 is a broad-host-range, virulent myovirus for Listeria monocytogenes. The genes encoding major structural proteins of the capsid (cps) and tail sheath (tsh) were mapped to a 10.15-kb late gene fragment. We have determined the complete nucleotide sequence of this region and confirmed the identities of Cps (48.7 kDa) and Tsh (61.3 kDa) by N-terminal amino acid sequencing of both proteins. In addition, nine other open reading frames were identified. On the basis of amino acid sequence homologies to known phage-encoded proteins, some putative functions and locations could be assigned to some of the deduced gene products. We present evidence that the cps product is proteolytically cleaved between Lys-23 and Ser-24 to yield the 444-residue polypeptide found in the mature viral capsid. We also found that the N-terminal methionine is absent from the mature tail sheath protein. cps and tsh are late genes; mRNAs first appear 15 to 20 min after infection of L. monocytogenes. Northern (RNA) hybridizations of total late mRNA with specific oligonucleotide probes were used to determine the sizes of respective transcripts. Primer extension analyses enabled the positive identification of six late promoters, which were found to differ from those identified in the chromosome of Listeria spp. The bulk of transcripts from cps and tsh arise from two phage promoters with identical 13-nucleotide sequences (TGCTAGATTATAG [core region underlined]) in the -10 region which we speculate determines specific and timed expression of these genes. A 123-nucleotide leader sequence at the 5' end of the cps transcript was predicted to form a strong secondary structure (deltaG=-40.7 kcal [-170.3 kJ]/mol). Out results show that the strongly expressed A511 cps and tsh genes are included in two separate gene clusters and are independently regulated at the transcriptional level.