Cloning of a foreign gene coding for alpha-amylase in Bacillus subtilis.
Explore the source record for details and available documents.
Amylases↗
Biomedical subjects
F E Young
Publications and source records attributed to F E Young.
Restriction-fragment map of the temperate Bacillus subtilis bacteriophage SPO2.
The endonucleases BglI, BglII, EcoRI, SalI, SmaI, and XbaI were used to fragment the phage SPO2 DNA. Electrophoretic analysis using ethidiumbromide agarose gels showed the phage to have nine BglI sites, one BglII site, four EcoRI sites, one SalI site, one SmaI site, and six XbaI sites. Using partial digestions, multiple endonuclease digestion, and autoradiography the fragments were sized and ordered into a circular map of 23 Md. Such an analysis locates the endonuclease sites, indicates which endonucleases are potentially useful in cloning with SPO2, and allows insertions and/or deletions in the SPO2 DNA to be characterized.
Bacillus subtilis↗
Isolation and characterization of viable deletion mutants of Bacillus subtilis bacteriophage SPO2.
Spontaneous deletion mutants of the bacteriophage SPO2, which are viable and retain their temperate character, were isolated using a heat-EDTA enrichment step. They were identified by endonuclease digestion and agarose-gel electrophoresis of phage DNA. Two of the nine mutants were characterized in detail. Both mutants have a 2.3 Md deletion removing the single BglII site and two of the XbaI fragments. The deletion extends 1.0 Md to one side of the former BglII site and 1.3 Md on the other side. This region of the SPO2 genome is non-essential for either lysogeny or viable phage production and thus is a suitable region for the insertion of exogenous DNA fragments.
Bacillus subtilis↗
Cell envelope of Neisseria gonorrhoeae CS7: peptidoglycan protein complex.
Treatment of cells grown to exponential phase with 4% sodium dodecyl sulfate for 3 h at 100 degrees C resulted in solubilization of all cellular components except for peptidoglycan. In most strains, cells cultured in liquid gonococcal broth at pH 7.2 yielded a peptidoglycan composed primarily of N-acetylmuramic acid N-acetylglucosamine, alanine, glutamic acid, and diaminopimelic acid in a molar ratio of 1:1:2:1:1. The peptidoglycan in these cells accounted for 1 to 2% (dry weight) of the cells. However, in cells cultured at pH 6.0, the dry weight of peptidoglycan increased to 4 to 13%. Preliminary investigations indicated that the apparent increase in weight is strain dependent and is due in part to associated protein(s). Neisseria gonorrhoeae strain CS7 had elevated amounts of protein associated with the peptidoglycan regardless of growth pH. The peptidoglycan-protein complex could not be dissociated by additional extraction with sodium dodecyl sulfate, 10 M LiCl2, or ethylenediaminetetraacetate or by 7.5% polyacrylamide gel electrophoresis. The complex could be degraded by lysozyme, trypsin, chymotrypsin, Pronase B, and Chalaropsis sp. muramidase.
Amino Acids↗
Selective association of the chromosome with membrane in a stable L-form of Bacillus subtilis.
A stalbe L-form (Sal-1) of Bacillus subtilis was found to have retained a markedly modified chromosome-membrane association when compared to intact cells. The membrane-deoxyribonucleic acid complex of the L-form was similar to that of its parental strain in quantity and stability. Genetic analysis of the L-form membrane-deoxyribonucleic acid complex revealed enrichment for markers close to the replication origin, but not for internal markers, indicating preferential attachment of the origin of chromosomal replication to the membrane. These results are in close agreement with those found for the parental bacterial form. In contrast, the replication termius region was not preferentially attached to the membrane of the L-form, even though it is enriched in the bacterial form. The association of the chromosome with the membrane at the replication terminus does not appear to be necessary for cell growth and separation, but because the L-form divides aberrantly, it may be one of the factors required for normal deoxyribonucleic acid segregation and septation.
Bacillus subtilis↗
Evidence for peptidoglycan-associated protein(s) in Neisseria gonorrhoeae.
Explore the source record for details and available documents.
Amino Acids↗
Mechanism of integrating foreign DNA during transformation of Bacillus subtilis.
Genes encoding thymidylate synthetase from Bacillus subtilis bacteriophages were cloned in Escherichia coli. Chimeric plasmids pCD1 and pCD3 were constructed from site-specific endonuclease digests of bacteriophage phi3T DNA cloned in pMB9 in E. coli. Similar cloning techniques with bacteriophage beta22 DNA yielded chimeric plasmids pCD4, pCD5, and pCD6. Endonuclease digests of DNA from pCD1 and pCD3 propagated in E. coli or from DNA isolated from bacteriophage phi3T propagated in B. subtilis transformed B. subtilis from Thy- to Thy+. Intact DNA from bacteriophage beta22, endonuclease digests of beta22 DNA, and a chimeric plasmid (pCD5) composed only of the thybeta22 gene and pMB9 did not transform B. subtilis from Thy- to Thy+ even though pCD5 could transform Thy- E. coli to Thy+. However, if the thybeta22 fragment from pCD5 was introduced into another chimeric plasmid, pCD2, that contains a region of homology to the chromosome of B. subtilis in addition to pMB9, transformation of Thy- clones of B. subtilis was possible. Furthermore, Southern hybridization analyses of the digests of chromosomal DNA from the Thy+ transformants established that the entire chimeric plasmid was incorporated into the chromosome of B. subtilis. Treatment of these plasmids with site-specific endonucleases abolished transformation. These results indicated that the entire chimeric plasmid can be incorporated into the chromosome of B. subtilis by a Campbell-like model. Therefore, an additional mechanism for transformation exists whereby plasmids can be integrated if sufficient chromosomal homology is maintained.
Bacillus subtilis↗
D-alanine incorporation into macromolecules and effects of D-alanine deprivation on active transport in Bacillus subtilis.
An auxotroph of Bacillus subtilis 168 unable to synthesize D-alanine loses the ability to support endogenously energized transport when deprived of D-alanine. Revertants of the mutant retain transport activity. The loss of transport is specific for substrates taken up by active transport; substrates taken up by group translocation are transported at normal rates. The loss of transport can be retarded by pretreatment of the cells with inhibitors of protein synthesis. Since the loss of transport could be due to an alteration in a D-alanine-containing polymer, we investigated the incorporation of D-[14C]alanine into macromolecules. The major D-alanine-containing polymers in B. subtilis are peptidoglycan and teichoic acid, with 4 to 6% of the D-[14C]alanine label found in trypsin-soluble material. Whereas the peptidoglycan and teichoic acid undergo turnover, the trypsin-soluble material does not. Treatment of the trypsin-soluble material with Pronase releases free D-alanine. Analysis of acid-hydrolyzed trypsin-soluble material indicated that approximately 75% of the radioactivity is present as D-alanine, with the remainder present as L-alanine. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of partially purified D-[14C]alanine-labeled membranes indicated the presence of two peaks of radioactivity (molecular weights, 230,000 and 80,000) that could be digested by trypsin. The results suggest that D-alanine may be covalently bound to cellular proteins.
Alanine↗
Biochemical and genetic properties of site-specific restriction endonucleases in Bacillus globigii.
Bacillus globigii contains two site-specific endonucleases, BPGLI AND BglI. A rapid technique for selection of mutants deficient in each of these enzymes was developed using sensitivity to infection by bacteriophage SP50 as an indication of the levels of enzyme. Mutants defective in BglI, BglII, and both BglI and BglII retained the wild-type modification phenotype. Genetic and biochemical studies have established that these enzymes are involved in restriction in vivo. Simplified purification procedures for BglI and BglII using these mutants are described.
Bacillus↗
Regulation of bacterial cell walls: correlation between autolytic activity and cell wall turnover in Staphylococcus aureus.
Cell wall turnover was examined in parent and mutant strains of Staphylococcus aureus. Peptidoglycan and teichoic acid were observed to undergo turnover in the wild-type strain during exponential growth; however, the rate of turnover did not decrease when the growth rate slowed, as the culture entered stationary phase. Isolated native cell walls and crude soluble autolytic enzyme were prepared from cells harvested during exponential and postexponential phases of growth. Native cell walls from both phases of growth autolyzed in buffer at identical rates; similarily, crude soluble enzyme from both preparations degraded radioactive cell walls at the same rate. Therefore, the activity of the autolysin in both exponential and postexponential cells was similar. The autolysis of whole cells of a mutant tar-1 was enhanced by 1.0 M NaCl. When 1.0 M NaCl was present under growing conditions, the rate of cell wall turnover was greatly increased. The presence of chloramphenicol, which inhibits whole-cell autolysis, also inhibited turnover. Analysis of the cell wall material recovered from spent medium revealed products consistent with the known mode of action of the endogenous autolysin. It is concluded that cell wall turnover in S. aureus is independent of the stage of culture growth but is dependent instead on the activity of the autolysin.
Bacteriolysis↗
Incorporation of N-acetyl-D-glucosamine from UDP-N-acetyl-D-glucosamine by isolated membranes of Bacillus subtilis. Identification of undecaprenyl poly(N-acetylglucosaminyl pyrophosphate).
Membrane isolated from Bacillus subtilis strain 168 incorporated GlcNAc from UDP-GlcNAc directly onto undecaprenyl phosphate via transphosphorylation and subsequent transglucosylations. Chain lengths of 6, 4, and 1 units of GlcNAc were found. Approximately 80% of the isotope incorporated was extracted into chloroform:methanol (2:1 v/v), and could be distinguished from the undecaprenyl disaccharide cell wall intermediate by a different elution pattern on DEAE-cellulose (acetate form). The GlcNAc-lipid(s) were eluted from a similar column in chloroform:methanol:water (10:10:3, v/v) with 6 mM NH4COOH indicating a pyrophosphate linkage between the lipid and the GlcNAc. The GlcNAc-lipid(s) were not degraded by conditions which completely deacylated [32P]glyceryl phospholipids, but were rapidly hydrolyzed by mild acid treatment (0.005 N HCl, 90 degrees) with the release of oligosaccharide phosphate (typical of sugars linked to undecaprenyl pyrophosphate). Catalytic hydrogenation of the GlcNAc-lipid(s) resulted in the release of water-soluble sugar phosphate. Under these same conditions, undecaprenyl pyrophosphate and undecaprenyl disaccharide cell wall intermediate were similarly effected while [32P]glyceryl phospholipids remained intact. The formation of GlcNAc-lipid(s) in vitro was inhibited if membranes were prepared from cells previously treated with bacitracin. Thus, the GlcNAc-lipid(s) has the properties of undecaprenyl poly(N-acetylglucosaminyl pyrophosphate) and may represent a new synthetic role of the polyisoprenyl lipid in B. subtilis.
Bacillus subtilis↗
Recognition sequence of specific endonuclease BamH.I from Bacillus amyloliquefaciens H.
Explore the source record for details and available documents.
Adenoviridae↗
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.
Bacillus subtilis↗
Effect of site-specific endonuclease digestion on the thyP3 gene of bacteriophage phi 3T and the thyP11 gene of bacteriophage rho11.
phi 3T and rho11 are closely related bacteriophages of Bacillus subtilis which can "convent" thymine auxotrophs to thymine prototrophs upon infection or transfection. The effect of endonuclease digestion on the ability of both bacteriophage and prophage DNA from phi eT and rho11 to transform for thymine prototrophy was determined. All of the endonucleases tested: BamHI, Bg/II, BsuRI, EcoRI, HindII+ III, and HpaII reduced the efficiency of thyP transformation to an equal extent in prophage and bacteriophage DNA. Only HpaII completely abolished thyP transformation. The reduction in transformation with BamHI, Bg/II, BsuRI, EcoRI, and HpaII fragments is size related. The thyP transforming fragments generated by these endonucleases are potentially clonable.
Bacillus subtilis↗
Inducible resistance to D-cycloserine in Bacillus subtilis 168.
Resistance to d-cycloserine could be induced in Bacillus subtilis 168 by sublethal concentrations of d-cycloserine. Sensitivity to the antibiotic could be regained by growth in the absence of d-cycloserine. The bactericidal activity of d-cycloserine apparently was not altered by resistant cells, and peptidoglycan synthesis was still inhibited by d-cycloserine in resistant cells. The d-cycloserine resistance apparently resulted from a decreased uptake of the antibiotic. The decrease in d-cycloserine transport could be prevented by simultaneous treatment of the cells with rifampin and d-cycloserine. d-Cycloserine was transported by the same system as glycine in B. subtilis. d-Cycloserine was able to exchange for intracellular glycine in both sensitive and resistant cells, suggesting that d-cycloserine is not excluded from the cell in resistant cultures.
Bacillus subtilis↗
D-Cycloserine-induced alterations in the transport of D-alanine and glycine in Bacillus subtilis 168.
d-Alanine, l-alanine, and glycine transport was investigated in Bacillus subtilis 168 cells that were phenotypically resistant to d-cycloserine. These cells showed enhanced rates of uptake as compared with that observed in sensitive cells. The usual enhancement in d-alanine and glycine transport resulting from treatment of the cells with d-cycloserine could be prevented by the addition of rifampin. Kinetic analyses of the initial rate of glycine transport indicated an increase in the V(max) for transport in resistant cells, with no alteration in the K(m) for glycine. Investigations of the net transport of glycine revealed that resistant cells maintained a higher gradient of glycine than did sensitive cells. Kinetic analyses of the net transport of glycine suggested that a new system for the accumulation of glycine was present in d-cycloserine-resistant cells.
Alanine↗
Cell wall and morphological changes induced by temperature shift in Bacillus subtilis cell wall mutants.
Bacillus subtilis RUB1012 and RUB1013 have the following phenotype when grown at 45 degrees C: no growth on tryptose blood agar base, growth as clumps of spheres in broth culture, a slow autolysis rate, and a low proportion of teichoic acid to peptidoglycan. Revertants of strain RUB1012 (RUB2032, RUB2012, and RUB2042) that could grow on tryptose blood agar base were isolated. Each revertant had a different proportion of teichoic acid to peptidoglycan. The nanomoles of phosphorus per milligram of cell wall at the nonpermissive temperature were 141, 160, 236, and 541 for strain RUB1012 and revertants RUB2032, 2012, and 2042, respectively, as compared with 1,100 for the parent strain. With most bacteriophage tested, plating efficiency was related to the amount of glucosylated teichoic acid. Scanning electron microscopy was used to study strain RUB2032 during a shift from growth at 30 degrees C to growth at 45 degrees C. The change from rod to sphere began with the thickening of the cylindrical portion of the cell. Caps of the cells appeared to be immune to the thickening process. During growth, the cells became progressively shorter and thicker, and cell separation was inhibited. When cells of strain RUB2032 were shifted from growth at 45 degrees C to growth at 30 degrees C, accumulation of an amorphous material on the outer surfaces of the cells preceded the change from sphere to rod morphology. Cells remained clumped, with rods appearing at the periphery of the clumps. Analysis by DNA-mediated transformation and PBS1-mediated transduction indicated that strains RUB1012 and RUB1013 have multiple mutations mapping in the same region as other cell wall mutations.
Bacillus subtilis↗
Rapid method for auxotyping multiple strains of Neisseria gonorrhoeae.
A rapid method for auxotyping strains was developed that uses microtiter plates. This miniplate technique enables rapid identification of major auxotypes present in clinical strains. Additional growth requirements can be identified by adding individual amino acid supplements to complete gonococcal genetic medium. Analysis of 8 clones from 40 patients revealed that 10 had more than 1 auxotype. Deoxyribonucleic acid-mediated transformation can be used to establish whether the strains with apparently more than one auxotype are defective in the same locus in each of the involved biosynthetic pathways. Selection of more than one clone is required in precise epidemiological studies.
Amino Acids↗