Stanislav Zadražil

Restriction and modification in Bacillus subtilis 168

SummaryGene hsrM (nonB) of Bacillus subtilis 168, causing non-permissiveness to phage SP10 (Saito et al. 1979) and reduced plating efficiency of unmodified phage ϕ105, is responsible for non-permissiveness of B. subtilis 168 for phages π15 and PZA. Upon transformation to sporulation deficiency (allele spoOA) B. subtilis 168 becomes permissive for ϕ15 and PZA and loses the ability to restrict ϕ105. spoOA str-1 double transformants of B. subtilis 168, however, retain the restriction 168 and non-permissiveness for ϕ15 and PZA phages, in spite of their Spo− phenotype. Therefore it appears that a functional product of the spoOA gene is required for expression of gene hsrM in wild-type bacteria, but is not essential in streptomycin-resistant bacteria. Phage genomes (PZA) were trapped in spores of the restriction deficient strain with much higher efficiency than in the wild-type.

Tolerated variations in a genome: the case of closely related Bacillus phages PZA, phi 29 and phi 15--a review.

Restriction-site analysis was used to estimate the relationship of bacteriophages PZA, phi 29 and phi 15. Complete nucleotide sequences of PZA and luminal diameter 29 genomes were compared and tolerated variations were assessed. Most of the base-pair changes are silent nucleotide substitutions in the third position of codons but amino acid changing substitutions are also observed. The terminal portions of the phage genomes diverged faster than their central parts. Gene mutations in phage PZA were induced by hydroxylamine and their frequency was compared with the evolutionary mutability.

Isolation procedure for bacterial DNA based on gel permeation chromatography on a sepharose column.

Abstract Gel permeation chromatography of bacterial lysates, no matter what type of lysis is used, on a Sepharose 4B column equilibrated with buffered 2 M NaCl was demonstrated to be generally useful as a simple method for the quantitative and reproducible isolation of high-molecular-weight DNA (average molecular weight 2.2 × 10 7 daltons). Conventional analyses of this DNA and its re-chromatography on a MAK column and electrophoretic fractionations in polyacrylamide and agarose gels show less heterogeneity and a lower polysaccharide content in comparison with DNA preparations isolated by other complex processes, no RNA contamination and 2–3% of residual proteins. The method is also recommended for bacteria affected by different factors, where classical methods for the quantitative isolation of DNA fail.

NEW MEMBERS OF BACILLUS SUBTILIS PHAGE GROUP CONTAINING A PROTEIN LINK IN THEIR CIRCULAR DNA

ABSTRACT Newly described phages (PZE and PZA) are morphologically similar to o29. Comparison of these three phages has revealed that they have the following characteristics in common: the contour length of DNA molecules corresponds to 11-12 Md, mol.% of G+C amounts to about 40%, as established by Tm, buoyant densities and by enzymatic digestion of 32 P-labelled DNA to mononucleotides. Circular DNA from the particles has transfecting activity and can be linearized by proteases. While o29 and PZE seem to be nearly identical organisms, the phage PZA differs in host range properties, antigenic activity DNA restriction patterns and efficiency of DNA-DNA hybridization.

Fractionation of DNA from Bacillus subtilis and its transforming activity for various markers

The physical, chemical, and functional heterogeneity of tranforming DNA was studied by preparative fractionation techniques providing resolution with respect to differences in molecular weight (gel filtration, sucrose density gradient), base composition (CsCl density gradient), or both these parameters simultaneously (methylalbumin-coated celite 545 MAK column). A comparison of the basic characteristics of the obtained fractions (melting temperature, Tm; density, ρ; sedimentation coefficient, S20,w; and transforming activity for ade, leu, and met markers) showed that the factor decisive for functional activity represents, in addition to the sequential arrangement of nucleotides in the chain, the average base composition. Hence, using the methylalbumin column or CsCl density gradient centrifugation, DNA fractions can be isolated which show a several times higher transforming activity for any of the markers examined. By contrast, the remaining fractionation methods, even though considerably decreasing the heterogeneity of the fractions as regards their molecular weight (such as zonal centrifugation), do not offer a possibility of fractionation of the activity for individual markers. This indicates a statistically random degradation of transforming DNA during its isolation. The order of the investigated markers according to their guanine-cytosine content is ade leu met and corresponds also to the order of their positions on the genetic map of Bacillus subtilis.

Expression and properties of prochymosin derivatives containing extensions of various length in the pro-part

A series of hybrid prochymosin derivatives containing portions of the simian virus 40 small-t antigen in the pro-part was constructed. Portions comprising 93, 63, 47, 12, and 1 amino acid (aa) from the N terminus of the small-t antigen were separately fused via eight polylinker-encoded amino acids to a prochymosin product commencing with the 5th aa of the pro-part. All the DNAs coding for the hybrid proteins were put under pL-promoter control in the expression constructs. Expression revealed that only fusion of the 47-aa or 12-aa stretch of the small-t antigen to prochymosin gave stable protein products and that only the latter one allowed the hybrid prochymosin to be activated to chymosin. The products containing 93 aa and 63 aa of small-t antigen were unstable and degraded. Complete removal of the small-t antigen portion led to mRNA instability, probably owing to inefficient initiation of translation.

Transcriptional and Translational Signals in Phages PZA and ø29

There is a group of small and morphologically similar bacteriophages that lytically infect cells of Bacillus subtilis (1,2). This report is concerned with two of them, namely PZA and o29. They both contain linear, duplex DNA, 18 kb long. Genomes of the two phages were compared by restriction analysis (3) and the nucleotide sequence of selected regions were determined (3–7 and unpublished data). These studies have shown that the genomes of phages PZA and o29 are closely related.

Mechanism of resistance to 5-azacytidine inBacillus Subtilis

Spontaneous mutants resistant to 5-azacytidine (5-AzCyd-r) and to 5-aza-2′-deoxycytidine (5-dAzCyd-r) were isolated inBacillus subtilis. There is no cross resistance between the two markers. Genetic transfer by a transformation process was possible with a low efficiency only with the 5-AzCyd-r marker. Independently isolated 5-AzCyd-r mutants did not show any differences in the level of resistance.