Molecular analysis of Methanobacterium phage ΨM2

Abstract

SummaryThe methanogenic archaeon Methanobacterium thermoautotrophicum Marburg is infected by the doublestranded DNA phage ⌿M2. The complete phage genome sequence of 26 111 bp was established. Thirty-one open reading frames (orfs), all of them organized in the same direction of transcription, were identified. On the basis of comparison of the deduced amino acid sequences to known proteins and by searching for conserved motifs, putative functions were assigned to the products of six orfs. These included three proteins involved in packaging DNA into the capsid, two putative phage structural proteins and a protein related to the Int family of site-specific recombinases. Analysis of the N-terminal amino acid sequences of three phageencoded proteins led to the identification of two genes encoding structural proteins and of peiP, the structural gene of pseudomurein endoisopeptidase. This enzyme is involved in the lysis of host cells, and it appears to belong to a novel enzyme family. peiP was overexpressed in Escherichia coli, and its product was shown to catalyse the in vitro lysis of M. thermoautotrophicum cells. Comparison of the phage ⌿M2 DNA sequence with parts of the sequence of the wild-type phage ⌿M1 suggests that ⌿M2 is a deletion derivative, which formed by homologous recombination between two copies of a direct repeat.

Pseudomurein endoisopeptidases PeiW and PeiP, two moderately related members of a novel family of proteases produced in Methanothermobacter strains

“…The cloned peiP gene [12] was recloned into the expression vector pET28a and the resultant construct was designated pME2508. The gene of the autolytic enzyme PeiW was ampli¢ed by PCR using the primers PeiWFor (5P-AGGTGATCATATGGAAGTGG-3P, NdeI site underlined) and PeiWRev (5P-AACAACTCGAGCATGTCTC-3P, XhoI site underlined) as a 0.9-kb NdeI^XhoI fragment from the M. wolfeii genomic DNA.…”

“…Clari¢ed crude extracts were obtained after 10 min centrifugation at 16 000Ug and 4³C. Without prior reactivation in a reduced bu¡er, they were directly used for enzymatic assays to monitor the cell wall-degrading activity as described [12].…”

“…All cell suspensions of M. marburgensis used were also preincubated as previously described [12]. The optimal temperatures were measured using 10 8 cells of M. marburgensis suspended in 1 ml 50 mM potassium phosphate bu¡er (pH 7.0).…”

“…In this respect, active recombinant pseudomurein endoisopeptidases o¡er signi¢cant advantages. However, currently available assays for the enzymes utilizing either whole cells [12] or puri¢ed cell walls [20] do have some limitations for quantitative measurements. An improved assay using synthetic peptides as substrates would signi¢cantly help to measure kinetic enzyme parameters and also allow the determination of minimal substrate requirements and substrate speci¢city.…”

“…Two lytic enzymes degrading the pseudomurein cell wall sacculi found in members of the order Methanobacteriales have been described in Methanothermobacter wolfeii (formerly Methanobacterium wolfei) [10,11] and in phage 8M2 of Methanothermobacter marburgensis (formerly Methanobacterium thermoautotrophicum Marburg) [11,12]. The former is responsible for autolysis of M. wolfeii under hydrogen limitation [13] and was recently shown to be encoded in the genome of defective prophage 8M100 [14].…”