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Cultures of the thermophilic methanoarchaeon Methanothermobacter wolfeii (formerly Methanobacterium wolfei) (20, 21) spontaneously lyse upon hydrogen limitation (10). However, no phage-like particles have been detected in the culture supernatant or autolysate. A lytic enzyme was purified from the autolysate, and this enzyme has been shown to be a pseudomurein endoisopeptidase (9). M. wolfeii proved insensitive to the virulent phage M1 and its deletion mutant M2 of Methanothermobacter marburgensis (formerly Methanobacterium thermoautotrophicum) Marburg (P. Pfister, unpublished observation). The results of Southern hybridization suggested that there is a prophage in the chromosome of M. wolfeii, M100, which is homologous to phages M1 and M2 (14, 19; P. Pfister, unpublished data). The approximate location of M100 in the M. wolfeii chromosome was determined, and most of the prophage was shown to be located on a ca. 30-kb NotI-NheI fragment (19). Since attempts to detect free phage particles had failed, M100 was assumed to be defective. In contrast, infection of M. marburgensis Marburg with phages M1 and M2 consistently led to lysis of the host. The complete nucleotide sequence of M2 was established, and some of its open reading frames (ORFs) and the proteins they encode were characterized (15). In order to explore the relationships between the defective prophage M100, the autolysis phenomenon, and the M1 and M2 phages, we determined and analyzed the sequence of M100 and its flanking regions.

Cloning, PCR amplification, and nucleotide sequence determination of M100 and its flanking regions. Portions of M100 and its flanking regions were obtained from the M. wolfeii chromosome as overlapping clones and PCR fragments (data not shown). The methods used included the following: shotgun cloning, construction, and screening of a SuperCos1-based cosmid library; screening of a -ZAP Express genomic library (7); PCR amplification of either nonclonable portions or portions that were difficult to clone on both sides of M100; and PCR amplification of the regions across the restriction sites of fragments obtained by shotgun cloning. The nucleotide sequences of both strands were determined by primer walking and then assembled.

Properties of M100 DNA. The border of M100 is defined by the flanking 21-bp AT-only direct repeats, which probably represent the core of the putative attachment site (see below). The length of the M100 genome extends over 28,798 bp with an overall GC content of 45.4%. This is somewhat lower than the 48.3% GC determined for M. wolfeii by a melting point analysis (11). Sequence alignment of M100 and M2 (Fig. 1) reveals that, other than point mutations and small deletions or insertions, there is a large fragment of 2,793 bp, IFa, inserted into M100 between coordinates 5272 and 8066. The GC content of the IFa element is 33.4%, which is significantly lower than those of M100 and M100 without IFa (46.7%). Like M2, the GC content of the M100 sequence is not evenly distributed, with five low-GC (<40%) DNA regions of at least 300 nucleotides (nt) extending over parts of ORFs orf6, orf28, orf29, and orf30 and over the entire ORFs orf5, the IFa-encoded orfB, -C, and -D, and the putative attachment sites attL and attR (Fig. 1).

View larger version (28K): [in this window] [in a new window]   FIG. 1.   Schematic representation of the defective prophage with its flanking regions and comparison to that of phage M2. The ORFs are represented by boxes numbered as in Table 1, and their vertical placement indicates the gene location in one of the six possible reading frames. Homologous ORFs carry the same numbers, and the assigned functions for some ORFs of M2 are reported. MTW1215 and MTW1216 represent the two ORFs encoded by the chromosome sequences flanking M100. orfA to orfE are unique to M100. The experimentally determined pac locus for M2 is shown as a solid black triangle. The IFa fragment (black bar) and the DNA regions with at least 200 bp with >98% identity in M100 and M2 (grey bars) are indicated. Three arrowheads represent three contiguous copies of a direct repeat of 125 nt in the vicinity of the left end of the IFa fragment.

Coding capacity of M100 and comparison to that of M2. Putative ORFs were defined based on the following assumptions: an ORF should code for a polypeptide of at least 90 amino acids (aa) and be preceded by a ribosome binding site with at least 45% identity to the proposed consensus sequence 5'-AGGAGGTGATC-3' (2). Two exceptions were made; these exceptions were orf2, encoding a 85-aa polypeptide with a homologue of 95 aa in M2, and orf29, encoding a putative integrase, the ribosome binding site of which has only 36% identity to the consensus sequence (as in M2). Consequently, 34 genes were identified on the forward strand and 1 gene was identified on the reverse strand (Fig. 1 and Table 1).

View this table: [in this window] [in a new window]   TABLE 1.   General features of the putative ORFs encoded by M100 and comparison to the corresponding ORFs of M2 and to other proteins in databases

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Integration site of M100 in the chromosome of M. wolfeii. Screening for ORFs encoded by regions of the M. wolfeii chromosome flanking M100 revealed two genes encoding MTW1216 on the forward strand upstream of the putative attL site and MTW1215 on the reverse strand downstream of the putative attR site (Fig. 1). The two host genes share high similarity to their counterparts (i.e., mth1215 and mth1216) in the Methanobacterium thermautotrophicus H genome (18); therefore, the same numbers were used to designate the M. wolfeii homologues. At the amino acid level, MTW1216 exhibits 77% identity to MTH1216 (pantothenate metabolism flavoprotein) and MTW1215 is 81.9% identical to MTH1215 (fibrillarin-like pre-rRNA processing factor). MTW1215 and MTW1216 are transcribed convergently, as are MTH1215 and MTH1216. The MTH1215 gene in the M. thermautotrophicus H genome is located at nucleotides 1117886 to 1118560 on the direct strand, and the gene encoding MTH1216 is located at nucleotides 1119977 to 1118817 on the complementary strand (18). Remarkably, integration of M100 occurred exactly within the intergenic regions between these two host genes and between prophage genes orf28 (peiW) and orf29. Neither the host genes nor ORFs of the defective prophage were disrupted in terms of their transcription potential. Thus, the integration mode of M100 resembles that of the major integration pattern of coliphage  (4).

Putative attachment sites of M100. Sequence analysis revealed that M100 is flanked by direct repeats of a 21-bp pure-AT nucleotide sequence. This sequence is very likely the core of putative attachment sites where the crossing-over occurred during integration and/or excision. Therefore, the flanking regions of the core might be defined as hybrid attL and attR sites. Conversely, the sequences of attP [(pro)phage-encoded attachment site] and attB [chromosomal attachment site] can be derived, although their exact lengths remain to be determined (Fig. 2A). In the region between orf28 and orf29 of phage M2 (i.e., in the putative attP site of phage M2) and between MTH1216 and MTH1215 of M. thermautotrophicus H (i.e., in the putative attB site), similar AT-rich sequences with four and three mismatches, respectively, were identified (Fig. 2B). Interestingly, one stretch of sequence which is 100% identical to the core sequence but located in a different context was present in another region of the M. thermautotrophicus H genome (Fig. 2B). It is not known whether additional core-like sequences occur in the M. wolfeii genome. For phage , the core of the attachment sites consists of 15-bp AT-rich sequences (3). There are some similarities between the sequences of the  and M100 cores (Fig. 2C).

View larger version (22K): [in this window] [in a new window]   FIG. 2.   (A) Sequences of the attR, attL, attP, attB, and the N-terminal part of the putative integrase ORF29. Sequences originating from the M. wolfeii chromosome are shown in italics. The crossed lines represent the sequences of attP and attB derived from the sequences of attR and attL. The core of the putative attachment sites (grey box), the stop codon of MTW1215 (CTA on the reverse strand) (grey box), and the start codon of ORF29 (ATG on the forward strand) (black box) are indicated. The numbers 38~38 and 39~85 indicate the coordinates of the prophage M100 sequence. (B) DNA sequences of the corresponding regions in the phage M2 and M. thermautotrophicus H genomes as well as of another region in the M. thermautotrophicus H genome. The numbers refer to the coordinates of the sequences in the phage M2 or M. thermautotrophicus H genome. (C) DNA sequence of the core of coliphage  attachment sites. In panels B and C, nucleotides identical to those of the core of the M100 attachment sites are also highlighted.

Experimental identification of the autolytic enzyme pseudomurein endoisopeptidase PeiW. The deduced N-terminal sequence of the protein encoded by orf28 of prophage M100 is identical to that of the experimentally determined N-terminal sequence (18 aa) of the autolytic enzyme produced by M. wolfeii (15). This strongly suggests that the ORF28 protein is the pseudomurein endoisopeptidase PeiW that has been known for more than 10 years (9). The predicted molecular mass (33.4 kDa) is consistent with the observed 33 kDa for the autolytic enzyme (9).

Conclusions. The complete nucleotide sequences of phage M2 of M. marburgensis and of the defective prophage M100 of M. wolfeii and its flanking regions now allow us to make a thorough comparison between these two elements. In contrast to two other archaeal defective prophages in Halobacterium halobium (17) and in Sulfolobus sp. strain B12 (16), the sequence of M100 contains all the information necessary for synthesis of structural proteins homologous to those of M2. In addition to small insertions, deletions, and point mutations, downstream of the putative replication origin, M100 carries the inserted fragment IFa of 2,793 bp, which apparently originated from another source. The IFa element has none of the distinct characteristics usually found in IS sequences (13). It encodes three putative ORFs, of which two short ones (i.e., orfB and orfC) are similar to hypothetical ORFs of M. thermautotrophicus H. The largest ORF, orfD, has no homologues in the database, although PropSearch at the EMBL (6) yields some hits related to DNA metabolism. One of these hits is the potential transposase for IS1151 of Clostridium perfringens (5). Using the GAP program of GCG, the two proteins show 18% identity in a 522-aa overlap. Only the 5' part of the IFa DNA sequence, which is a duplication of the sequence harboring the putative origin of replication, shows traces of terminal redundancy, possibly derived from the packaging mechanism of phage M2 (8). Therefore, the rearrangement due to the insertion of this IFa in the DNA of M100 might have led to this defect. However, there is no evidence explaining the order of insertion of IFa into the M100 genome and integration of the M100 ancestor(s), if any, into the M. wolfeii chromosome. Alternatively, M100 might be defective due to the lack of a functional excisionase, which is usually required to excise (pro)- phage genomes from their host chromosomes, while M2 might recruit the putative excisionase function of the ORF5 encoded by plasmid pME2001 present in the host strain (12).

Nucleotide sequence accession number. The sequence is available in GenBank under accession number AF301375.