Thomas A. Trautner

The relationship between molecular structure and transformation efficiency of some S. aureus plasmids isolated from B. subtilis.

SummaryDNA preparations of the chloramphenicol resistance determining S. aureas plasmids pC194, pC223, and PUB112 can be fractionated by gel electrophoresis into various bands. Electromicroscopic investigations of these various molecular species obtained with pC194 indicated that, depending on the preparations, 70 to 80% of the molecules were monomers, while the rest consisted of various classes of concatemeric and/or interlocked multimers. Measurements of the specific transforming activity of the various molecular classes indicated that the monomers had less than one thousandth the activity of the multimeric plasmid DNA. pC194 DNa of high specific transforming activity could also be obtained by ligation of HindIII generated monomers into concatemeric DNA.

A Gene Essential for De Novo Methylation and Development in Ascobolus Reveals a Novel Type of Eukaryotic DNA Methyltransferase Structure

Molecular mechanisms determining methylation patterns in eukaryotic genomes still remain unresolved. We have characterized, in Ascobolus, a gene for de novo methylation. This novel eukaryotic gene, masc1, encodes a protein that has all motifs of the catalytic domain of eukaryotic C5-DNA-methyltransferases but is unique in that it lacks a regulatory N-terminal domain. The disruption of masc1 has no effect on viability or methylation maintenance but prevents the de novo methylation of DNA repeats, which takes place after fertilization, through the methylation induced premeiotically (MIP) process. Crosses between parents harboring the masc1 disruption are arrested at an early stage of sexual reproduction, indicating that the activity of Masc1, the product of the gene, is crucial in this developmental process.

Methylation of DNA in Prokaryotes

A much wider variety of biological functions of postreplicative DNA methylation is observed in prokaryotes than in eukaryotes. In eukaryotes DNA methylation is primarily a means of the control of gene expression. Many chapters of this book are devoted to various aspects of this function. In prokaryotes, DNA methylation affects such diverse phenomena as determination of accessibility of DNA to digestion by endonucleases, control of initiation of DNA replication, and the definition of origins of packaging in the maturation of phage DNA, which will be dealt with in this article. We shall also be concerned with the enzymes, which facilitate methylation, the DNA methyltransferases. In the eukaryotes, as far as we know at this time, the various DNA methyltranferases encountered represent a rather homogeneous group, whereas in prokaryotes, we find a very diverse set of DNA methyltransferases. Beyond their biological significance, DNA methyltransferases represent a remarkable class of enzymes in their own right. Not only are they paradigms for sequence specific DNA binding proteins, but they also show specificity in their catalytic interaction with defined DNA sequences. Furthermore, their universal distribution, the multitude of enzymes with different or identical specificities observed among prokaryotes and the obligatory coexistence of isospecific restriction and methylating enzymes in restriction/modification systems make DNA methyltransferases choice candidates for evolutionary studies.

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.

One way to do experiments on gene conversion

SummaryCompetent cells of B. subtilis were transfected with heteroduplex SPP1 DNA, made by annealing complementary strands of wild type and 21 plaque type mutant DNAs. The frequencies of cells yielding mutant and wild type, only wild type, and only mutant phages were determined by single burst analyses of transfected cells. The data obtained reveal that an effective mechanism is operating in B. subtilis, which converts heterozygous to homozygous molecules prior to their replication. This “correction” mechanism is asymmetric with regard to the strand which is preferentially corrected in a given heteroduplex pair. The direction of asymmetry thus defined depends on the marker introduced into a particular heteroduplex. The efficiency of correction varies with the markers used and is correlated to the position of markers in the genetic map. From this correlation, the direction of replication of the SPP1 genome is deduced. The frequency distribution of wild type and mutant phages in cells yielding both genotypes indicates that both strands of the input DNA contribute equally to the production of progeny, i.e. DNA replication is symmetric.

The role of recombination in transfection of B. subtilis.

SummaryA comparative study of transfection with four different phage DNAs is being presented. Two types of transfection systems are distinguished, one with nearly linear dependence of the number of infective centers produced on the concentration of the phage DNA, the other type displaying multihit dose response. Studies of genetic recombination in transfection show that in systems of the latter type two (SPP 1) or three (SP 50) input genomes have to cooperate in a recombination event prior to replication. This obligatory process, termed primary recombination, is exclusively mediated by the host recombination system and cannot be effected by the phage recombination system.

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.

Different specific activities of the monomeric and oligomeric forms of plasmid DNA in transformation of B. subtilis and E. coli

Summary(1) The low residual transforming activity in preparations of monomeric, supercoiled, circular (CCC) forms of the plasmids pC194 and pHV14 could be attributed to the presence in such isolates of a small number of contaminating multimeric molecules. (2) E. coli derived preparations of pHV14, an in vitro recombinant plasmid capable of replication in both E. coli and B. subtilis, contain oligomeric forms of plasmid DNA in addition to the prevalent monomeric CCC form. The specific transforming activity of pHV14 DNA for E. coli is independent of the degree of oligomerization, whereas in transformation of B. subtilis the specific activity of the purified monomeric CCC molecules is at least four orders of magnitude less than that of the unfractionated preparation. (3) Oligomerization of linearized pHV14 DNA by T4 ligase results in a substantial increase of specific transforming activity when assayed with B. subtilis and causes a decrease when used to transform E. coli.

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.

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.