Search PubMed⌕ Search

Biomedical subjects

T A Trautner

Publications and source records attributed to T A Trautner.

At least 73 records · Page 4Linked to original sources
PubMed1981

Restriction and modification in Bacillus subtilis: two DNA methyltransferases with BsuRI specificity. I. Purification and physical properties.

Two S-adenosyl-L-methionine:DNA (cytosine 5)-methyltransferases, termed M.BsuRIa and M.BsuRIb, were purified 3,000- and 4,000-fold, respectively, from Bacillus subtilis strain OG3R (r+m+) by successive column chromatography. The molecular weights determined by gel filtration were 37,000 for M.BsuRIa and 40,000 for M.BsuRIb. The sedimentation coefficients s20,w were 3.55 for both enzymes as determined by glycerol gradient centrifugation, corresponding to molecular weights of 43,000. Analysis of the two methyltransferases by agarose gel electrophoresis under native conditions, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showed correspondence of the M.BsuRIa activity with one protein band at a molecular weight of 41,000, whereas M.BsuRIb activity was associated with two protein bands with molecular weights of 42,000 and 39,000, respectively.

Bacillus subtilis↗
PubMed1981

DNA methyltransferases affecting the sequence 5'CCGG.

B. subtilis phage SPbeta and Moraxella sp. code for DNA methyltransferases which methylate both cytosines of the sequence 5'CCGG. Experiments using a B. subtilis strain whose DNA is sensitive to HpaII and resistant to MspI degradation, indicated that methylation of the outer C of this sequence provides protection against the restriction enzyme MspI.

Bacillus subtilis↗
PubMed1981

Cloning and expression of the Bacillus subtilis phage SPP1 in E. coli. I. Construction and characterization of lambda/SPP1 hybrids.

We have constructed lambda/SPP1 hybrid phages by in vitro ligation of EcoRI fragments of the Bacillus subtilis phage SPP1 DNA to a lambdoid bacteriophage vector. EcoRI digestion of SPP1 generated 15 DNA fragments of which 13 could be cloned. The SPP1 DNA of such hybrids was stably maintained and replicated in Escherichia coli, as indicated by marker rescue experiments in B. subtilis. EcoRI fragment 1 of SPP1 could not be cloned although subfragments of fragment 1 resulting from spontaneous deletions which occurred during the cloning regime were consistently obtained. A region within EcoRI fragment 1 responsible for its incompatibility with replication in E. coli was defined by these experiments.

Bacillus subtilis↗
PubMed1981

Plasmid transformation in Bacillus subtilis. The significance of partial homology between plasmid and recipient cell DNAs.

A series of hybrid plasmids consisting of pC194 or pUB112 and B. subtilis DNA were constructed. In contrast to plasmid pC194, purified monomeric forms of such plasmids were active in transformation, provided the recipient cells were recombination proficient. Similarly the monomers of pC194 derived plasmids, containing bacteriophage phi 105 DNA were able to transform phi 105 lysogenic but not nonlysogenic cells. From the results it is concluded that the presence of DNA/DNA homology between chromosomal DNA of the recipient cell and part of the hybrid plasmids used is a sufficient condition to endow monomeric plasmids with transforming activity.

Bacillus subtilis↗
PubMed1981

Plasmid transformation in Bacillus subtilis. Alterations introduced into the recipient-homologous DNA of hybrid plasmids can be corrected in transformation.

Various alterations (deletions, additions, inversions) were introduced into portions of pC194/B. subtilis or pC194/phi 105 hybrid plasmid molecules which are homologous to the DNA of recipients in transformation. These plasmids are stably maintained in transformations of recombination deficient cells. In transformations of recombination proficient cells, they can be corrected to those plasmid forms into which the alterations were originally introduced. This correction is most pronounced when transformations are performed with monomeric ccc forms of hybrid plasmids. It is suggested that correction is a consequence of mismatch repair occurring in the synapsis of homologous portions of plasmid and resident DNAs.

Bacillus subtilis↗
PubMed1981

Plasmid transformation in Bacillus subtilis: fate of plasmid DNA.

Only multimeric, and not monomeric forms of B. subtilis plasmids can transform B. subtilis cells (Canosi et al. 1978). This finding prompted us to study the physico-chemical fate of plasmid DNA in transformation. Competent cells of B. subtilis were exposed to either unfractionated preparations or to preparations of multimeric plasmid DNA. Plasmid DNA was re-extracted from such cells and then analyzed by sedimentation and isopycnic centrifugation and also defined by its sensitivity to nuclease S1 degradation. No double-stranded plasmid DNA could be recovered from cells transformed with unfractionated plasmid preparations which contained predominantly monomeric covalently closed circular (CCC) DNA. Re-extracted plasmid DNA was single-stranded, had a molecular weight considerably smaller than monomer length DNA and had been subject to degradation to acid soluble products. However, when transformations were performed with multimeric DNA (constructed by in vitro ligation of linearized pC194 DNA), both double-stranded and partially double-stranded DNA could be recovered in addition to single-stranded DNA. We assume that plasmid DNA is converted to a single-stranded form in transformation, irrespective of its molecular structure. Double-stranded and partially double-stranded DNAs found in transformation with multimeric DNA would be the products of intramolecular annealing.

Bacillus subtilis↗
PubMed1981

Plasmid transformation in Bacillus subtilis: effects of insertion of Bacillus subtilis DNA into plasmid pC194.

We have constructed a hybrid plasmid pBC1, which consists of plasmid pC194 with an insert of B. subtilis DNA as its HindIII restriction site. This plasmid is stably maintained in B. subtilis. In contrast with pC194, monomeric ccc forms of pBC1 are active in transformation. Transformations with these monomeric molecules of pBC1 have a stringent requirement for recombination proficiency, as defined by recE in the recipient cell. The extent of dependence of the transforming activity of oligomeric pBC1 DNA on the recombination proficiency of the recipient cell decreases with increasing oligomer size. A model of DNA processing during plasmid transformation of B. subtilis is presented.

Bacillus subtilis↗
PubMed1981

In vivo transcription of Bacillus subtilis bacteriophage SPP1.

The temporal program of SPP1 transcription was examined by hybridizing RNA extracted from infected B. subtilis cells, pulse-labelled at various times after infection, to restriction fragments of SPP1 DNA. RNA made early after infection hybridises to contiguous fragments in the left part of the SPP1 molecule, whereas hybridization to fragments in the right part of the chromosome is found late in infection. Viral gene transcription proceeds from right to left on the H-strand throughout the lytic cycle. At late times transcription occurs also from left to right using the L-strand as template. These assignments follow from the established 5'--3' polarity of the complementary (H- and L-) strains of SPP1 DNA and the determination of strand specificity in SPP1 transcription. Early and late transcriptions are also defined physiologically: protein synthesis and phage DNA replication must precede late transcription.

Bacillus subtilis↗
PubMed1981

Promoter sites in the genome of B. subtilis phage SPP1.

Transcriptional complexes formed in vitro using DNA of B. subtilis phage SPP1 as template and E. coli and B. subtilis RNA polymerases were analyzed by electron microscopy. Both enzymes recognize the same five strong promoters in the early region of the genome. Strand selection at these sites was identical with both enzymes. These results correlate well with data obtained from in vivo transcription studies. Transcriptional activity in the late region of the genome was very low, not permitting the identification of promoter sites.

Bacillus subtilis↗
PubMed1981

Structure of Bacillus subtilis bacteriophage SPP1 DNA in relation to its transfection activity.

The availability of a detailed restriction map of SPP1 DNA allowed defined manipulations of such molecules. These were performed to investigate structural requirements for SPP1 transfection. (i) The transfection activity of SPP1 DNA was destroyed by degradation with restriction enzymes. Biological activity could be regenerated when transfection was performed with a combination of two different restriction endonuclease digests, provided that such digests generated widely overlapping DNA fragments. (ii) Unique DNA molecules were constructed from the natural population of circularly permuted SPP1 DNA molecules by using genetic engineering techniques. Such molecules had the same specific transfection activity as did the circularly permuted SPP1 DNA. These results are discussed in the context of current models of DNA processing in transfection.

Bacillus subtilis↗
PubMed1980

Unusual behaviour of SPO1 DNA with respect to restriction and modification enzymes recognizing the sequence 5'-G-G-C-C.

SPO1 DNA contains only 5 cleavage sites for restriction enzymes which recognize and cleave the sequence 5'-G-G-C-C (HaeIII or BsuR). Fragments of SPO1 DNA cloned in E. coli to substitute 5'-hydroxymethyluracil (HMU) by thymine (T) remain resistant to HaeIII indicating that this unexpectedly small number of cleavages by HaeIII is not correlated with the presence of HMU in the normal phage DNA. It was previously shown that SPO1 is neither subject to B. subtilis R restriction (Trautner et al., 1974) nor modification in vivo (Günthert et al., 1975). We now show that SPO1 DNA can however be restricted and modified in vitro.

Bacteriophage lambda↗
PubMed1980

Restriction and modification in B. subtilis: effects on transformation and transfection with native and single-stranded DNA.

The effects of restriction in vivo by competent B. subtilis R cells and in vitro by purified endonuclease BsuR on transformation and transfection with native and denatured DNA were investigated. The results show that transformation by either native, or denatured DNA is not affected by restriction, whereas transfection both with native and denatured SPP1 DNA is severely restricted. In contrast to the results obtained in vivo, the biological activity of native and denatured transforming DNA is destroyed by BsuR in vitro, as is the transfecting activity of native and denatured SPP1 DNA. The sensitivity of denatured DNA, either with mixtures of the complementary strands or with separated single strands alone, is significantly lower than that of native DNA. The results are discussed in the context of possible mechanisms underlying the different responses of transforming and transfecting DNA to in vivo restriction by B. subtilis R cells.

Bacillus subtilis↗
PubMed1980

Restriction and modification in B. subtilis: the role of homology between donor and recipient DNA in transformation and transfection.

Non-modified DNAs from phages SPO2 and phi 105, and prophage DNAs extracted from lysogens carrying these phages, were used to transfect isogenic r+m+ B. subtilis recipients which were either non-lysogenic, or had been lysogenized with a homologous or a non-homologous phage. Restriction of transfecting phage and prophage DNA occurred in non-lysogenic recipients and in recipients lysogenic for a non-homologous phage. No effect of restriction was observed when phage or prophage DNA was used to transfect recipients carrying a homologous prophage. This is analogous to the absence of restriction in transformation and indicates that in B. subtilis the distinction between transforming and transforming and transfecting DNA is not made at the initial stages of DNA uptake and processing, but rather at later stages, where recognition of homologous regions in donor and recipient DNA plays an important role.

Bacillus subtilis↗
PubMed1980

Restriction and modification in Bacillus subtilis: identification of a gene in the temperate phage SP beta coding for a BsuR specific modification methyltransferase.

A gene coding for a modifying DNA-methyltransferase which methylates the central C in the BsuR recognition sequence 5'GGCC was identified in the genome of the temperature Bacillus subtilis phage SP beta. This gene is expressed only after induction of the prophage by either mitomycin C or UV. The presence of active methyltransferase in induced cells leads to modification of BsuR recognition sites in SP beta DNA as well as in heterologous DNA.

Bacillus subtilis↗
PubMed1979

The genome of B. subtilis phage SPP1: physical arrangement in phage genes.

41 genes of SPP1 have been delineated by using complementation analyses of 75 conditionally lethal (ts and sus) mutations. The physical locations of these genes on the SPP1 chromosome have been determined by transfection/marker rescue experiments in which restriction endonuclease generated fragments of SPP1 DNA were used as donor DNA. The physical order of these fragments has been previously established (Ratcliff et al., 1979).

Bacillus subtilis↗
PubMed1979

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

(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, as 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.

Bacillus subtilis↗