Characterization of the type IV restriction modification system BspLU11III from Bacillus sp. LU11.

We report the characterization and cloning of the genes for an unusual type IV restriction-modification system, BspLU11III, from Bacillus sp. LU11. The system consists of two methyltransferases and one endonuclease, which also possesses methyltransferase activity. The three genes of the restriction-modification system, bsplu11IIIMa, bsplu11IIIMb and bsplu11IIIR, are closely linked and tandemly arranged. The corresponding enzymes recognize the dsDNA sequence 5'-GGGAC-3'/5'-GTCCC-3', with M.BspLU11IIIa modifying the A (underlined) of one strand and M.BspLU11IIIb the inner C (underlined) of the other strand. R.BspLU11III has both endonuclease and adenine-specific methyltransferase activities and is able to protect the DNA against cleavage by itself. In contrast to all type IV restriction-modification systems described so far, which have only one adenine-specific methyltransferase, BspLU11III is the first type IV restriction-modification system that includes two methyltransferases, one of them being cytosine specific.

Cytosine-specific type II DNA methyltransferases. A conserved enzyme core with variable target-recognizing domains.

Comparisons of the amino acid sequences of m5C DNA methyltransferases (Mtases) from 11 prokaryotes and one eukaryote reveal a very similar organization. Among all the enzymes one can distinguish highly conserved "core" sequences and "variable" regions. The core sequences apparently mediate steps of the methylation reaction that are common to all the enzymes. The major variable region has been shown in our previous studies on multispecific phage Mtases to contain the target-recognizing domains (TRDs) of these enzymes. Here we have compared the amino acid sequences of various TRDs from phage Mtases. This has revealed the presence of both highly conserved and variable amino acids. We postulate that the conserved residues represent a "consensus" sequence defining a TRD, whereas the specificity of the TRD is determined by the variable residues. We have observed similarity between this consensus sequence and sequences in the variable region of the monospecific Mtases. We predict that the regions thus identified represent part of the TRDs of monospecific Mtases.

Sequential headful packaging and fate of the cleaved DNA ends in bacteriophage SPP1.

The virulent Bacillus subtilis bacteriophage SPP1 packages its DNA from a precursor concatemer by a headful mechanism. Following disruption of mature virions with chelating agents the chromosome end produced by the headful cut remains stably bound to the phage tail. Cleavage of this tail-chromosome complex with restriction endonucleases that recognize single asymmetric positions within the SPP1 genome yields several distinct classes of DNA molecules whose size reflects the packaging cycle they were generated from. A continuous decrease in the number of molecules within each class derived from successive encapsidation rounds indicates that there are several packaging series which end after each headful packaging cycle. The frequency of molecules in each packaging class follows the distribution expected for a sequential mechanism initiated unidirectionally at a defined position in the genome (pac). The heterogeneity of the DNA fragment sizes within each class reveals an imprecision in headful cleavage of approximately 2.5 kb (5.6% of the genome size). The number of encapsidation events in a packaging series (processivity) was observed to increase with time during the infection process. DNA ejection through the tail can be induced in vitro by a variety of mild denaturing conditions. The first DNA extremity to exit the virion is invariably the same that was observed to be bound to the tail, implying that the viral chromosome is ejected with a specific polarity to penetrate the host. In mature virions a short segment of this chromosome end (55 to 67 bp equivalent to 187 to 288 A) is fixed to the tail area proximal to the head (connector). Upon ejection this extremity is the first to move along the tail tube to exit from the virion through the region where the tail spike was attached.

M.BssHII, a multispecific cytosine-C5-DNA-methyltransferase with unusual target recognizing properties.

A new multispecific cytosine-C5-DNA-methyltransferase (C5-MTase), M.BssHII, was identified in Bacillus stearothermophilus H3. The M.BssHII gene was cloned and sequenced. The amino acid sequence deduced shows the characteristic building plan of a C5-MTase. By sequencing bisulfite-treated DNA methylated by M.BssHII and by restriction enzyme analysis, we defined the following methylation targets of M.BssHII: ACGCGT/CCGCGG (MluI/SacII), PuGCGCPy (HaeII), PuCCGGPy (Cfr10I) and GCGCGC (BssHII). The relative location of the specificity determinants in the C5-MTase was derived from the analysis of M.BssHII derivatives carrying deletions within the variable region "V" and chimeric C5-Mtases constructed between M.BssHII and the related monospecific enzyme M.phi3TII. Four of the M.BssHII specificities (MluI, SacII, Cfr10I and BssHII) could be associated with amino acid segments within the variable region "V". The determinant for HaeII activity had to be assigned to sequences defining the enzyme core, the first example of a C5-MTase in which a sequence-specific methylation potential is mediated by structures outside of the variable region. Another intriguing result came from the analysis of one particular chimera made between M.BssHII and M.phi3TII. This construct showed a relaxation of the methylation capacity, both with respect to the target recognized and the targeting of methylation within this sequence.

Molecular analysis of the Bacillus subtilis bacteriophage SPP1 region encompassing genes 1 to 6. The products of gene 1 and gene 2 are required for pac cleavage.

Packaging of Bacillus subtilis phage SPP1 DNA into viral capsids is initiated at a specific DNA site termed pac. Using an in vivo assay for pac cleavage, we show that initiation of DNA synthesis and DNA packaging are uncoupled. When the DNA products of pac cleavage were analyzed, we could detect the pac end that was destined to be packaged, but we failed to detect the other end of the cleavage reaction. SPP1 conditional lethal mutants, which map adjacent to pac, were analyzed with our assay. This revealed that the products of gene 1 and gene 2 are essential for pac cleavage. SPP1 mutants that are affected in the genes necessary for viral capsid formation (gene 41) or involved in headful cleavage (gene 6) remain proficient in pac site cleavage. Analysis of the nucleotide sequence (2.769 x 10(3) base-pairs) of the region of the genes required for pac cleavage revealed five presumptive genes. We have assigned gene 1 and gene 2 to two of these open reading frames (orf), giving the gene order gene 1-gene 2-orf 3-orf 4-orf 5. The direction of transcription of the gene 1 to orf 5 operon and the length of the mRNAs was determined. We have identified, upstream from gene 1, the major transcriptional start point (P1). Transcription originating from P1 requires a phage-encoded factor for activity. The organization of gene 1 and gene 2 of SPP1 resembles the organization of genes in the pac/cos region of different Escherichia coli double-stranded DNA phages. We propose that the conserved gene organization is representative of the packaging machinery of a primordial packaging system.

Identification of a gene in Bacillus subtilis bacteriophage SPP1 determining the amount of packaged DNA.

The virulent Bacillus subtilis bacteriophage SPP1 encapsidates its DNA by a headful mechanism. Analyzing phage missense mutants, which package less DNA than SPP1 wild-type but show no other affected properties, we have identified a gene whose product is involved in the sizing of phage DNA during maturation. Characterization of this gene and its product provides an experimental access to the poorly understood mechanism of DNA sizing in packaging. The gene (gene 6 or siz) was cloned and sequenced. An open reading frame (ORF) coding for a 57.3 kDa polypeptide was identified. All the single nucleotide substitutions present in different siz mutants affect the net charge of that protein. The gene was further characterized by assignment of several nonsense mutations (sus) to the ORF. Phages carrying the latter type of mutations could be complemented in trans when gene 6 is provided by a plasmid.

Shape and DNA packaging activity of bacteriophage SPP1 procapsid: protein components and interactions during assembly.

The procapsid of the Bacillus subtilis bacteriophage SPP1 is formed by the major capsid protein gp13, the scaffolding protein gp11, the portal protein gp6, and the accessory protein gp7. The protein stoichiometry suggests a T=7 symmetry for the SPP1 procapsid. Overexpression of SPP1 procapsid proteins in Escherichia coli leads to formation of biologically active procapsids, procapsid-like, and aberrant structures. Co-production of gp11, gp13 and gp6 is essential for assembly of procapsids competent for DNA packaging in vitro. Presence of gp7 in the procapsid increases the yield of viable phages assembled during the reaction in vitro five- to tenfold. Formation of closed procapsid-like structures requires uniquely the presence of the major head protein and the scaffolding protein. The two proteins interact only when co-produced but not when mixed in vitro after separate synthesis. Gp11 controls the polymerization of gp13 into normal (T=7) and small sized (T=4?) procapsids. Predominant formation of T=7 procapsids requires presence of the portal protein. This implies that the portal protein has to be integrated at an initial stage of the capsid assembly process. Its presence, however, does not have a detectable effect on the rate of procapsid assembly during SPP1 infection. A stable interaction between gp6 and the two major procapsid proteins was only detected when the three proteins are co-produced. Efficient incorporation of a single portal protein in the procapsid appears to require a structural context created by gp11 and gp13 early during assembly, rather than strong interactions with any of those proteins. Gp7, which binds directly to gp6 both in vivo and in vitro, is not necessary for incorporation of the portal protein in the procapsid structure.

Head morphogenesis genes of the Bacillus subtilis bacteriophage SPP1.

We have identified and characterized the phage cistrons required for assembly of SPP1 heads. A DNA fragment containing most of the head morphogenesis genes was cloned and sequenced. The 3'-end of a previously identified gene (gene 6) and eight complete open reading frames (7 to 15) were predicted. We have assigned genes 7, 8, 9, 11, 12, 13, 14 and 15 to these orfs by correlating genetic and immunological data with DNA and protein sequence information. G7P was identified as a minor structural component of proheads and heads, G11P as the scaffold protein, G12P and G15P as head minor proteins and G13P as the coat protein. Characterization of intermediates in head assembly, which accumulate during infection with mutants deficient in DNA packaging or in morphogenetic genes, allowed the definition of the head assembly pathway. No proteolytic processing of any of the head components was detected. Removal of G11P by mutation leads to the accumulation of prohead-related structures and aberrant particles which are similar to the assemblies formed by purified G13P in the absence of other phage-encoded proteins. The native molecular masses of G11P and G13P are about 350 kDa and larger than 5000 kDa, respectively (predicted molecular masses 23.4 kDa and 35.3 kDa, respectively). G13P, upon denaturation and renaturation, assembles from protomers into some prohead-related structures. The organization of the DNA packaging and head genes of SPP1 resembles the organization of genes in the analogous regions of phage lambda and P22.

Altered sequence recognition specificity of a C5-DNA methyltransferase carrying a chimeric 'target recognizing domain'.

A MTase with a chimeric TRD with the N-terminal half derived from a TRD recognizing GCNGC, the C-terminal half from one with CCWGG recognition, was constructed. Its target specificity is reported.

M.BssHII: a new multispecific C5-DNA-methyltransferase.

M.BssHII is a new multispecific C5-DNA-methyltransferase recognizing five different targets. As the enzyme has been isolated from a thermophilic Bacillus, the protein should show enhanced intrinsic thermostability and therefore be a promising candidate for crystallizing a multispecific MTase.

BsuCI, a type-I restriction-modification system in Bacillus subtilis.

A type-I R-M system was identified in B. subtilis. The genes comprising the system have striking similarity to type-I R-M systems observed in Enterobacteriaceae.

DNA methyltransferase genes of Bacillus subtilis phages: comparison of their nucleotide sequences.

The phi 3T DNA methyltransferase (Mtase) and most of the SP beta Mtase genes have been sequenced. With the exception of their promoters, no difference was found between the phi 3T and SP beta Mtase genes which code for an enzyme with a Mr of 50 507, consisting of 443 amino acids (aa). Comparison of the deduced aa sequence of the phi 3T/SP beta type Mtase (target specificity: GGCC and GCNGC) with that of the previously established sequence of the SPR Mtase (Buhk et al., 1984) which has the target specificity GGCC and CCGG, reveals strong similarities between these two types of enzymes. There is, however, one striking difference: both the phi 3T/SP beta and the SPR enzymes contain at different positions inserts of 33 aa, which have no homology to each other. We suggest that the methylation specificity unique to each of the two types of Mtases (GCNGC in phi 3T/SP beta; CCGG in SPR) depends on these inserts, while the GGCC-specific modification potential common to all Mtases is determined by structures conserved in both types of enzymes. A DNA fragment of non-modifying phage Z, which shows homology to both flanks of the SPR Mtase gene, was also sequenced. This segment can be described as a derivative of SPR DNA, in which the Mtase gene and sequences at its 5' end have been deleted, with the deletion extending between two direct repeats of 25 bp.

M.H2I, a multispecific 5C-DNA methyltransferase encoded by Bacillus amyloliquefaciens phage H2.

Bacillus amyloliquefaciens phage H2 codes for a multispecific cytosine-5-DNA- methyltransferase (MTase), M.H2I, which methylates GGCC, GCNGC and [sequence: see text] target sequences. The gene coding for M.H2I was cloned in Escherichia coli and its nucleotide (nt) sequence was determined. It consists of 1509 bp, corresponding to a protein of 503 amino acids (aa) with a calculated Mr of 57,166. A comparison of the aa sequence of M.H2I with those of the multispecific MTases encoded by Bacillus subtilis phages SPR, phi 3T and rho 11S, revealed that M.H2I is closely related to these enzymes. A very high degree of homology was observed between M.H2I and M.rho 11SI, with 96.2% aa identity and 97.8% nt identity of the corresponding genes.

DNA methyltransferase genes of Bacillus subtilis phages: structural relatedness and gene expression.

The DNA methyltransferase (Mtase) genes of temperate Bacillus subtilis phages phi 3T, rho 11 and SP beta were cloned and expressed in Escherichia coli. Each gene specifies a 47-kDa1 protein, which modifies BsuR (GGCC) and Fnu4HI (GCNGC) target sequences. Transcription is controlled by phage promoters located on the cloned fragments. The direction of transcription and the approximate position of the Mtase genes were determined. DNA/DNA hybridization experiments revealed close structural relatedness of the phi 3T, rho 11 and SP beta genes. A significant degree of homology was also found among these genes and the Mtase gene of related phage SPR, which codes for an enzyme with different modification specificity. These results suggest a common ancestor of the different phage Mtase genes. Phage Z, the only BsuR-sensitive member of this phage group, lacks a modification gene, but contains regions homologous to sequences flanking the SPR, phi 3T, rho 11 and SP beta Mtase genes.

The complete nucleotide sequence and functional organization of Bacillus subtilis bacteriophage SPP1.

The complete nucleotide sequence of the B. subtilis bacteriophage SPP1 is described. The genome is 44,007 bp in size and has a base composition of 43.7% dG + dC. Only 32.2 kb are essential for phage amplification under laboratory conditions. Transcription using only the 'heavy strand' is asymmetric. Eighty-one orfs organized in five early and four late operons were identified. Experiments have shown that 25 orfs are essential. Of the remaining orfs, functions could be predicted for the products of five of the orfs on the basis of comparison of the deduced amino acid sequence to known proteins. Intergenic regions include most of the 5 PE and the 4 PL promoters. Transcripts are polycistronic. Transcription from the PE promoters is mediated by host RP, whereas recognition of the PL promoters requires an additional unidentified phage-encoded product. Translation of mRNA transcribed from most of the orfs seems to be initiated independently, each from its own ribosomal binding and initiation site, although a few cases of coupled translation have been reported. The organization of SPP1 genes involved in the replication, DNA packaging and phage assembly proteins resembles the organization of genes of equivalent regions of different E. coli double-stranded DNA phages. Absence of aa sequence similarity between analogous proteins of different phages suggested that the conserved gene organization is representative of a primordial bacteriophage.

Sequence analysis of the left end of the Bacillus subtilis bacteriophage SPP1 genome.

The left end of the genome of Bacillus subtilis bacteriophage SPP1 is represented by EcoRI DNA fragments 12 and 1 (EcoRI-12 and EcoRI-1). A number of different deletions were identified in EcoRI-1. A detailed physical and genetic map of EcoRI-1 from wild-type (wt) phage and SPP1 deletion mutants was constructed. Genes encoding essential products involved in late and early stages of phage DNA metabolism were mapped at the left and right ends of the 8.5-kb EcoRI-1, respectively. Deletions fell within the internal 5157-bp DNA segment of EcoRI-1. The nucleotide (nt) sequence of this region and of the endpoints of two deletions, delta X and delta L, were determined. The nt sequence of the junctions in SPP1 delta X and SPP1 delta L showed that, in these deletions, a segment of DNA between short directly repeated sequences of 10 and 13 bp, located 3427 and 4562 bp apart in the wt sequence, had been eliminated. In both cases, the copy of the repeated sequence was retained in the deletion mutant, consistent with the hypothesis that the deletions originated by homologous intramolecular recombination. The corresponding region in wt phage had fifteen presumptive open reading frames (orfs) and the previously identified SPP1 early promoters (PE1). The poor growth phenotype associated with the SPP1 deletion mutants was attributed to premature transcriptional read through from promoter(s) of the early region into late operon brought into close vicinity of the late genes due to the deletion event.

Restriction and modification in Bacillus subtilis: nucleotide sequence, functional organization and product of the DNA methyltransferase gene of bacteriophage SPR.

Bacillus subtilis phage SPR codes for a DNA methyltransferase (Mtase) which methylates the 5' cytosine in the sequence GGCC and both cytosines in the sequence CCGG. A 2126-bp fragment of SPR DNA containing the Mtase gene has been sequenced. This fragment has only one significant open reading frame of 1347 bp, which corresponds to the Mtase gene. Within the sequence the Mtase promoter has been defined by S1 mapping. The size of the SPR Mtase predicted from the deduced amino acid composition is 49.9 kDal. This is in agreement with both the Mr of the purified enzyme and with that of the SPR Mtase gene product identified here by minicell technique. Base changes leading to mutants affected in Mtase activity were localized within the Mtase gene.