Frank E. Young

Isolation of a sequence-specific endonuclease (BamI) from Bacillus amyloliquefaciens H

A restriction endonuclease has been isolated from Bacillus amyloliquefaciens H (strain RUB500). The enzyme, Bam I, cleaves adenovirus-2 DNA at three sites, phase λ DNA at five sites, λ p lac DNA at four sites, 80 pt DNA at 14 sites, and 3T + DNA at four sites. However, it does not cleave DNA from bacteriophage SPO2, 105 or 29.

New shuttle vectors for Bacillus subtilis and Escherichia coli which allow rapid detection of inserted fragments

Two new shuttle vectors have been constructed by fusing the Escherichia coli plasmid pUC9 with the Staphylococcus aureus plasmids pU110 and pC194. The resulting hybrids replicate in both E. coli and Bacillus subtilis and contain seven restriction sites within a part of the lacZ gene. Insertion of foreign DNA into those sites can be easily detected in E. coli and hybrid plasmids can subsequently be transformed into B. subtilis.

Tunicamycin, an inhibitor of Bacillus peptidoglycan synthesis: A new site of inhibition

Abstract Bacillus subtilis membranes can transfer either N-acetylmuramyl-pentapeptide phosphate or N-acetylglucosaminyl phosphate from UMP directly onto undecaprenyl phosphate. Tunicamycin blocks only the latter transfer and inhibits peptidoglycan synthesis by toluenized cells of Bacillus megaterium utilizing added nucleotide sugar precursors or cell wall synthesis by intact cells of B. subtilis . Tunicamycin prevents formation of the cell wall disaccharide lipid intermediate by blocking transfer of N-acetylglucosamine onto undecaprenyl muramyl pentapeptidyl pyrophosphate.

Transformation of Bacillus subtilis and Escherichia coli by a hybrid plasmid pCD1.

The gene thyP3 from Bacillus subtilis bacteriophage phi 3T was cloned in the plasmid pMB9. The resulting chimeric plasmid, pCD1, is effective in transforming both Escherichia coli and Bacillus subtilis to thymine prototrophy. The activity of the thyP3 gene product, thymidylate synthetase, was assayed and found to be 9 times greater in a transformed strain of Escherichia coli than in a phi 3T lysogen of Bacillus subtilis. The physical location of restriction sites has been determined for two related plasmids pCD1 and pCD2. Hybridization studies clearly indicate that the plasmid gene responsible for Thy+ transformation is the gene from the bacteriophage phi 3T. The lack of restriction in this transformation process is consistent with our previous studies using bacterial DNA in heterospecific exchanges indicating that the nucleotide sequence surrounding the gene is the dominant factor in determining interspecific transformation.

Identification of three homology classes of small, cryptic plasmids in intestinal Bacteroides species

A systematic analysis of the plasmid content of intestinal Bacteroides spp. was made. Eight of fifteen clinical isolates and seven of nineteen normal rectal flora isolates examined contained small plasmids (less than 5 Mda). The majority of these small plasmids could be assigned to three homology classes by Southern hybridization. Further investigation demonstrated the presence of subclasses within one of the homologous classes. Class I plasmids were 1.8 Mda, Class III were 3.7 Mda, and Class II plasmids were of three different molecular weights: 2.6 Mda (IIA), 3.2 Mda (IIB), and 4.0 Mda (IIC). Among representative plasmids from each class there was remarkable sequence similarity based upon digestion patterns obtained using the restriction endonucleases, AluI and DdeI. In addition, similar polypeptide products were observed using purified Class I plasmids as template in an Escherichia coli in vitro coupled transcription-translation system. Small plasmids were found in seven of the ten recognized species of intestinal bacteroides, indicating that no species barrier exists for these plasmids. In addition, since plasmids from all three classes were found together in a single isolate, it was concluded that these plasmids are not incompatible.

Cloning of a foreign gene coding for α-amylase in Bacillussubtilis

Abstract Foreign DNA has been introduced into the genome of bacteriophage O3T, producing a specialized transducing bacteriophage containing the genetic information encoding α-amylase from Bacillus amyloliquefaciens H. Genetic and physical studies demonstrated that the gene(s) is inserted into the bacteriophage genome. These bacteriophage carrying the gene(s) encoding α-amylase lysogenized and replicated in Bacillus subtilis with normal efficiency. In these lysogens, the gene(s) encoding α-amylase appears to map near the bacteriophage attachment site rather than the chromosomal amy E locus. This method of construction of specialized bacteriophage should be applicable to the cloning of other genes for which no primary selection exists.

The effect of carbohydrate depletion on procoagulant activity and in vivo survival of highly purified human factor VIII

Human factor VIII procoagulant protein (factor VIII) was purified using a modification of our previously described method, in which Sephacryl S-400 elution, rather than QAE-cellulose chromatography, served as the final purification step. The protein had a specific activity of more than 2500 U/mg and consisted of a single polypeptide (Mr 100 000) when analyzed by SDS-polyacrylamide gel electrophoresis. Factor VIII was shown to be a glycoprotein by staining with periodic acid-Schiffs reagent following electrophoresis. Treatment of factor VIII with a mixture of exo- and endoglycosidases caused a reduction by about 50% in the intensity of periodic acid-Schiff staining, as determined by scanning densitometry, and an increase in electrophoretic mobility (equivalent to a new Mr 95 000). Removal of this portion of the total carbohydrate had no significant effect on factor VIII clotting activity or on thrombin potentiation of clotting activity. The in vivo survival curves of a native and sugar-depleted 125I-labeled factor VIII both showed similar patterns of initial rapid decay to 60 and 40% activity, respectively, followed by a one-half decay time of 4 h for both. These results suggest that the carbohydrate portion of human factor VIII does not contribute significantly to either clotting function in vitro or to biological turnover in vivo.

Epidemiology of Antibiotic and Heavy Metal Resistance in Bacteria: Resistance Patterns in Staphylococci Isolated from Populations Not Known to be Exposed to Heavy Metals

Staphylococci were isolated from clinical specimens obtained from patients not known to be exposed to abnormal levels of heavy metals. The antibiotic and heavy metal resistance patterns of these strains were determined by using a disk diffusion test and computer sorting. Though not absolute, an association of resistance to mercury and tetracycline in coagulase-negative strains was found, in contrast to resistance to copper and penicillin in coagulase-producing strains. A high degree of correlation was observed between the resistance to phenyl mercury and inorganic mercury, but no correlation was obtained between resistance to methylmercury and other metals. In general, strains resistant to many agents were usually coagulase negative. A possible mechanism and implications of these associations are considered.

Expression of thymidylate synthetase activity in Bacillus subtilis upon integration of a cloned gene from Escherichia coli

The gene from Escherichia coli encoding thymidylate synthetase was cloned in the plasmid pBR322. The resulting chimeric plasmid, pER2, was effective in transforming both E. coli and Bacillus subtilis to thymine prototrophy. Uncloned linear E. coli chromosomal DNA was unable to transform thymine-requiring strains of B. subtilis to thymine independence. Linearization of the chimeric plasmid, pER2, with restriction enzymes markedly diminished its ability to transform B. subtilis auxotrophs. The Thy+ transformants derived from the transformation of B. subtilis with pER2 DNA did not contain detectable extrachromosomal DNA as demonstrated by Southern hybridization patterns and centrifugation in CsCl gradients of DNA isolated from B. subtilis colonies transformed with the chimeric plasmid. We conclude that the DNA from the chimeric plasmid was integrated into the chromosome of B. subtilis, demonstrating that extensive homology is not required for the integration of foreign DNA. This is the first reported case of a gene from a Gram-negative bacterium functioning in a Gram-positive organism.

Transformation of Bacillussubtilis using gently lysed L-forms; a new mapping technique

Summary Transformation of competent Bacillus subtilis 168 strains using gently lysed L-forms of the same genospecies resulted in efficiencies between 3–10 percent for single marker transformtion. Transformation of multiple auxotrophs to prototrophy occurred at probabilities above those predicted by congression, indicating linkage between distant markers. L-form DNA was also able to transform for non-selected aberrant colony morphologies at high frequencies which apparently are division mutants.