Methylation of DNA in prokaryotes.
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Amino Acid Sequence↗
Biomedical subjects
T A Trautner
Publications and source records attributed to T A Trautner.
BsuBI--an isospecific restriction and modification system of PstI: characterization of the BsuBI genes and enzymes.
The enzymes of the Bacillus subtilis BsuBI restriction/modification (R/M) system recognize the target sequence 5'CTGCAG. The genes of the BsuBI R/M system have been cloned and sequenced and their products have been characterized following overexpression and purification. The gene of the BsuBI DNA methyltransferase (M.BsuBI) consists of 1503 bp, encoding a protein of 501 amino acids with a calculated M(r) of 57.2 kD. The gene of the restriction endonuclease (R.BsuBI), comprising 948 bp, codes for a protein of 316 amino acids with a predicted M(r) of 36.2 kD. M.BsuBI modifies the adenine (A) residue of the BsuBI target site, thus representing the first A-N6-DNA methyltransferase identified in B. subtilis. Like R.PstI, R.BsuBI cleaves between the A residue and the 3' terminal G of the target site. Both enzymes of the BsuBI R/M system are, therefore, functionally identical with those of the PstI R/M system, encoded by the Gram negative species Providencia stuartii. This functional equivalence coincides with a pronounced similarity of the BsuBI/PstI DNA methyltransferases (41% amino acid identity) and restriction endonucleases (46% amino acid identity). Since the genes are also very similar (58% nucleotide identity), the BsuBI and PstI R/M systems apparently have a common evolutionary origin. In spite of the sequence conservation the gene organization is strikingly different in the two R/M systems. While the genes of the PstI R/M system are separated and transcribed divergently, the genes of the BsuBI R/M system are transcribed in the same direction, with the 3' end of the M gene overlapping the 5' end of the R gene by 17 bp.
Amino Acid Sequence↗
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.
Amino Acid 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.
Amino Acid Sequence↗
High plasticity of multispecific DNA methyltransferases in the region carrying DNA target recognizing enzyme modules.
Multispecific cytosine C5 DNA methyltransferases (MTases) methylate more than one specific DNA target. This is due to the presence of several target recognizing domains (TRDs) in these enzymes. Such TRDs form part of a variable centre in the MTase primary sequence, which separates conserved enzyme core sequences responsible for general steps in the methylation reaction. By deleting, rearranging and exchanging several TRDs of multispecific MTases, we demonstrate their modular character; they mediate target recognition independent of a particular TRD or core sequence context. We show also that multispecific MTases can accommodate inert material of non-MTase origin within their variable region without losing their activity. The remarkable plasticity with respect to the material that can be integrated into this region suggests that the enzyme core sequences preceding or following it form separable functional domains. In spite of the documented flexibility multispecific MTases could not be endowed with novel specificities by integration of putative TRDs of monospecific MTases, pointing to differences between multi- and monospecific MTases in the way their core and TRD sequences interact.
Base Sequence↗
Intramolecular homologous recombination in Bacillus subtilis 168.
Plasmid resolution from a phage::plasmid chimera was used to measure directly intramolecular recombination in Bacillus subtilis. The system is based on a sigma-replicating plasmid (pC194) cloned into a dispensable region of the lytic bacteriophage SPP1. The plasmid, which confers chloramphenicol resistance, is resolved when SPP1::pC194 phages infect B. subtilis cells, provided the chimera carries a functional, intact copy of the plasmid repH gene. Intramolecular homologous recombination was independent of the RecA and RecL-RecR functions, but dependent on RecF, RecB, RecG, RecP, RecH and AddAB functions. These results are consistent with the hypothesis that B. subtilis has multiple pathways for genetic recombination and allow us to tentatively place the recB and recG genes into a new epistatic group epsilon.
Bacillus subtilis↗
Cloning, characterization and evolution of the BsuFI restriction endonuclease gene of Bacillus subtilis and purification of the enzyme.
The restriction endonuclease (R.BsuFI) of Bacillus subtilis recognizes the target DNA sequence 5' CCGG. The R.BsuFI gene was found in close proximity to the cognate M.BsuFI gene, which had previously been characterized (1). Cloning of the R.BsuFI gene in E.coli was only possible with the M.BsuFI Mtase gene present on a compatible plasmid. The cloned R.BsuFI gene was expressed in E. coli and restriction activity was observed in vivo and in vitro. The R.BsuFI gene consists of 1185 bp, coding for a protein of 395 amino acids with a calculated molecular weight of 45.6 kD. The R.BsuFI enzyme was purified to homogeneity following overexpression. It presumably works as a dimer and cleaves the 5' CCGG target sequence between the two cytosines to produce sticky ends with 5' CG overhangs, like the isoschizomers R.MspI and R.HpaII. The relatedness between R.BsuFI and R.MspI is reflected by significant similarities of the amino acid sequences of both enzymes. This is the first case where such similarities have been observed between isoschizomeric restriction endonucleases which belong to 5mC specific R/M systems. This observation suggests that R.BsuFI and R.MspI genes derive from a common ancestor. In spite of such functional and evolutionary relatedness, the R/M systems differ in the arrangement of their R and M genes. In the BsuFI system transcription of the two genes is convergent, whereas divergent transcription occurs in the MspI system.
Amino Acid Sequence↗
'Pseudo' domains in phage-encoded DNA methyltransferases.
5-Cytosine-DNA-methyltransferases, which are found in many organisms ranging from bacteriophages to mammals, transfer a methyl group from S-adenosylmethionine to the carbon-5 of a cytosine residue in specific DNA target sequences. Some phage-encoded methyltransferases methylate more than one sequence: these enzymes contain several independent target-recognizing domains each responsible for recognizing a different site. The amino-acid sequences of these multispecific methyltransferases reveal that some enzymes in addition carry domains that do not contribute to the enzymes' methylation potential, but strongly resemble previously identified target-recognizing domains. Here we show that introducing defined amino-acid alterations into these inactive domains endows these enzymes with additional methylation specificities. Gel retardation analysis demonstrates that these novel methylation specificities correlate with the acquisition of additional DNA-binding potential of the proteins.
Amino Acid Sequence↗
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.
Amino Acid Sequence↗
Functional analysis of the Bacillus subtilis bacteriophage SPP1 pac site.
Encapsidation of the DNA of the virulent Bacillus subtilis phage SPP1 follows a processive unidirectional headful-mechanism and initiates at a unique genomic location (pac). We have cloned a fragment of SPP1 DNA containing the pac site flanked by reporter genes into the chromosome of B. subtilis. Infection of such cells with SPP1 led to highly efficient packaging, initiated at the inserted pac site, of chromosomal DNA. The directionality in the packaging of this DNA was the same as observed with vegetative phage DNA. Mutagenizing the chromosomal pac insert defined an 83 base pair segment containing the pac cleavage site which is sufficient to direct phage specific DNA encapsidation. The packaging recognition signal as defined can also be utilized by the SPP1 related phages 41c, SF6 and rho 15.
Bacillus subtilis↗
The amino acid sequence of the CCGG recognizing DNA methyltransferase M.BsuFI: implications for the analysis of sequence recognition by cytosine DNA methyltransferases.
The Bacillus subtilis FI DNA methyltransferase (M.BsuFI) modifies the outer cytosine of the DNA sequence CCGG, causing resistance against R.BsuFI and R.MspI restriction. The M.BsuFI gene was cloned and expressed in B.subtilis and Escherichia coli. As derived from the nucleotide sequence, the M.BsuFI protein has 409 amino acids, corresponding to a molecular mass of 46,918 daltons. Including these data we have compared the nucleotide and amino acid sequences of different CCGG recognizing enzymes. These analyses showed that M.BsuFI is highly related to two other CCGG specific methyltransferases, M.MspI and M.HpaII, which were isolated from Gram-negative bacteria. Between M.BsuFI and M.MspI the sequence similarity is particularly significant in a region, which has been postulated to contain the target recognition domains (TRDs) of cytosine-specific DNA methyltransferases. Apparently M.BsuFI and M.MspI, derived from phylogenetic distant organisms, use highly conserved structural elements for the recognition of the CCGG target sequence. In contrast the very same region of M.HpaII is quite different from those of M.BsuFI and M.MspI. We attribute this difference to the different targeting of methylation within the sequence CCGG, where M.HpaII methylates the inner, M.BsuFI/M.MspI the outer cytosine. Also the CCGG recognizing TRD of the multispecific B.subtilis phage SPR Mtase is distinct from that of the host enzyme, possibly indicating different requirements for TRDs operative in mono- and multispecific enzymes.
Amino Acid Sequence↗
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.
Amino Acid Sequence↗
Analysis of structural and biological parameters affecting plasmid deletion formation in Bacillus subtilis.
Deletions generated following stimulation by the deletion hot spot of plasmid pHV15-1 were studied in Bacillus subtilis. Nucleotide sequencing showed that deletions extend between short direct repeat sequences. Such direct repeat sequences may have homology to the sequence of the hot spot. Deletion formation is recE-independent, but requires an active exonuclease V (AddAB) enzyme. Other structural parameters like plasmid size and structure influence deletion formation.
Bacillus subtilis↗
Organization of target-recognizing domains in the multispecific DNA (cytosine-5)methyltransferases of Bacillus subtilis phages SPR and phi 3T.
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Bacillus subtilis↗
Chimeric multispecific DNA methyltransferases with novel combinations of target recognition.
DNA target recognizing domains of different multispecific DNA-cytosine-methyltransferases can be rearranged through engineering of the corresponding genes to generate enzymes with novel combinations of target recognition.
DNA (Cytosine-5-)-Methyltransferases↗
Sequential order of target-recognizing domains in multispecific DNA-methyltransferases.
In the multispecific DNA(cytosine-5)-methyltransferases (Mtases) of Bacillus subtilis phages SPR and phi 3T the domains responsible for recognition of DNA methylation targets CCA/TGG, CCGG, GGCC (SPR) and GCNGC, GGCC (phi 3T) represent contiguous sequences of approximately 50 amino acids each. These domains are tandemly arranged and do not overlap. They are part of a 'variable' segment within the enzymes which is flanked by 'conserved' amino acids, which are very similar amongst bacterial monospecific and the multispecific Mtases studied here. These results follow from a mutational analysis of the SPR and phi 3T Mtase genes. They further support our concept of a modular enzyme organization, according to which variability of type II Mtases with respect to target recognition is achieved by a combination of the same enzyme core with a variety of target-recognizing domains.
Amino Acid Sequence↗
Construction and use of chimeric SPR/phi 3T DNA methyltransferases in the definition of sequence recognizing enzyme regions.
Multispecific DNA methyltransferases (Mtases) of temperate Bacillus subtilis phages SPR and phi 3T methylate the internal cytosine of the sequence GGCC. They differ in their capacity to methylate additional sequences. These are CCGG and CC(A/T)GG in SPR and GCNGC in phi 3T. Introducing unique restriction sites at equivalent locations within the two genes facilitated the construction of chimeric genes. These expressed Mtase activity at a level comparable to that of the parental genes. The methylation specificity of chimeric enzymes was correlated with the location of chimeric fusions. This analysis, which also included the use of mutant genes, showed that domains involved in the recognition of target sequences unique to each enzyme [CCGG, CC(A/T)GG or GCNGC] are represented by the central non-conserved parts of the proteins, whilst recognition of the sequence (GGCC), which is a target for both enzymes, is determined by an adjacent conserved region.
Bacillus subtilis↗
Organization of multispecific DNA methyltransferases encoded by temperate Bacillus subtilis phages.
B. subtilis phage rho 11s codes for a multispecific DNA methyltransferase (Mtase) which methylates cytosine within the sequences GGCC and GAGCTC. The Mtase gene of rho 11s was isolated and sequenced. It has 1509 bp, corresponding to 503 amino acids (aa). The enzyme's Mr of 57.2 kd predicted from the nucleotide sequence was verified by direct Mr determinations of the Mtase. A comparison of the aa sequence of the rho 11s Mtase with those of related phages SPR and phi 3%, which differ in their methylation potential, revealed generalities in the building plan of such enzymes. At least 70% of the aa of each enzyme are contained in two regions of 243 and 109 aa at the N and C termini respectively, which are highly conserved among the three enzymes. In each enzyme, variable sequences separate the conserved regions. Variability is generated through the single or multiple use of related and unrelated sequence motifs. We propose that the recognition of those DNA target sequences, which are unique for each of the three enzymes, is determined by these variable regions. Evolutionary relationships between the three enzymes are discussed.
Amino Acid Sequence↗