Restriction of hemimethylated DNA by the Bacillus subtilis R system

Summary The effects of restriction by the BsuR system on hemimethylated SPP1 DNA were investigated. In vitro, single-stranded nicks were introduced in the nonmodified strand of the hemimethylated DNA at the same sites as recognized in nonmodified homoduplex DNA. Transfection with BsuR-treated hemimethylated DNA was severely reduced.In vivo, transfection with hemimethylated DNA was also severely reduced in competent B. subtilis R cells. In contrast, transfection of protoplasts of the R strain with this DNA was not affected. The apparent restriction by competent cells was attributed to the special mode of processing of transfecting DNA.     

Restriction and modification in B. subtilis

Summary 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 strands¹ 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.Abbreviations EGTA ethyleneglycolbis-(aminoethylether)tetraacetic acid - m⁺ modified - m^- non-modified - moi multiplicity of infection - r⁺ m⁺ restricting and modifying - r^- m^- mon-restricting and non-modifying - SSC 0.15M NaCl+0.015 M trisodium citrate - SDS sodium dodecyl sulphate     

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

Summary 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.     

Genetic studies on site-specific endodeoxyribonucleases in Bacillus subtilis: multiple modification and restriction systems in transformants of Bacillus subtilis 168

Summary We transformed B. subtilis 168 with DNA from B. subtilis IAM1231, IAM1192 and ATCC6633. When we examined the restriction activities of the transformants in vivo and in vitro using phage 105C we found the following: (1) Cells of either IAM1231 or IAM1192 have two modification and restriction systems (Bsu1231(1)-system and Bsu1231(II)-system in IAM1231, and Bsu1192(I)-system and Bsu1192(II)-systems in IAM1192), and cells of ATCC6633 have only one system (Bsu6633-system). (2) The restriction enzymes of all of these five systems are site-specific endonucleases. (3) The nucleotide sequence specificities of the enzymes involved in Bsu1231(I)-system, Bsu1192(I)-system and Bsu6633-system are the same; and those of Bsu1231(II)-system and Bsu1192(II)-system are the same. The sequence specificities of these two groups are different from each other and also different from those of the Bsu168-system of B. subtilis 168, the BsuR-system of B. subtilis R and the Bsu1247(I)-and Bsu1247(II)-systems which are systems of B. subtilis IAM1247. (4) Transformants possessing four different modification and restriction systems (Bsu1231(I)-, Bsu1247(I)-, BsuR- and Bsu168-systems) were constructed. (5) Transformation of two derivatives of 168 that were m _R ⁺ r _R ⁺ by DNA from IAM1231 produced 16 transformants that had the Bsu1231(II) restriction system, but had lost the BsuR system. Transformation of a derivative of 168 that was m _1247(II) ⁺ r _1247(II) ⁺ by DNA from m _1231(II) ⁺ r _1231(II) ⁺ -or m _R ⁺ r _R ⁺ -derivative of 168 produced about 100 each of transformants that had the Bsu1231(II)-restriction system or the BsuR-restriction system. But all these transformants lost the Bsu1247(II)-system.     

Restriction and modification in B. subtilis. Purification and general properties of a restriction endonuclease from strain R.

Summary All Bacillus subtilis R-type strains showing the phenomena of restriction and modification contain an endonuclease that inactivates in vitro the biological activity of a variety of DNAs lacking R-specific modification, such as transfecting SPP1, SPO2 and 105 DNA, and transforming B. subtilis 168-type DNA. The corresponding DNAs carrying R-specific modification are resistant to the enzyme. The enzyme has been purified approximately 400-fold and is essentially free from contaminating double strand-directed unspecific exo-or endonuclease activity. Only Mg²⁺ is required as cofactor. The substrate DNAs are cleaved at specific sites. The double-stranded fragments produced from SPP1 DNA (molecular weight 2.5×10⁷) have an average molecular weight of about 3×10⁵.     

Host-controlled modification and restriction in Bacillus subtilis: Bsu 168-system and BsuR-system in B. subtilis 168.

Summary A Bsu168-specific restriction deficient (r ₁₆₈ ^- ) mutant of Bacillus subtilis Marburg 168 was transformed to be BsuR-specific restriction proficient (r _R ⁺ ) with B. subtilis R DNA as efficiently as the Bsu168-specific restriction proficient (r ₁₆₈ ⁺ ) parental strain (hsrM ⁺, hsdR ^-).We constructed r _R ⁺ m _R ⁺ r ₁₆₈ ⁺ m ₁₆₈ ⁺ strain (ISMR 4), r _R ⁺ m _R ⁺ r ₁₆₈ ^- m ₁₆₈ ⁺ strain (ISR 11) and r _R ⁺ m _R ⁺ r ₁₆₈ ^- m ₁₆₈ ^- strain (ISR 6) from strain 101 (r ₁₆₈ ⁺ m ₁₆₈ ⁺ ), strain 1012 (r ₁₆₈ ^- m ₁₆₈ ⁺ ) and strain RM125 (r ₁₆₈ ^- m ₁₆₈ ^- ), respectively by transformation with B. subtilis R DNA, and tested their restriction and modification activities on phage 105C. The results show that the sites recognized by Bsu168-specific restriction and modification enzymes and the sites recognized by BsuR-specific ones are not overlapping.We conclude that the Bsu168-modification and restriction system and the BsuR-modification and restriction system are controlled independently by two distinct sets of genes in the r _R ⁺ m _R ⁺ transformant of r ₁₆₈ ⁺ m ₁₆₈ ⁺ strain B. subtilis 168.     

The effect of restriction on shotgun cloning and plasmid stability in Bacillus subtilis Marburg

Summary Using the bifunctional cloning vehicle pHP13, which carries the replication functions of the cryptic Bacillus subtilis plasmid pTA1060, the effects of BsuM restriction on the efficiency of shotgun cloning of heterologous Escherichia coli DNA were studied. In a restriction-deficient but modification-proficient mutant of B. subtilis, clones were obtained at a high frequency, comparable to frequencies normally obtained in E. coli (10⁴ clones per g target DNA). Large inserts were relatively abundant (26% of the clones contained inserts in the range of 6 to 15 kb), which resulted in a high average insert length (3.6 kb). In the restriction-proficient B. subtilis strain, the class of large inserts was underrepresented. Transformation of B. subtilis with E. coli-derived individual recombinant plasmids was affected by BsuM restriction in two ways. First, the transforming activities of recombinant plasmids carrying inserts larger than 4 kb, were, in comparison with the vector pHP13, reduced to varying degrees in the restricting host. The levels of the reduction increased with insert length, resulting in a 7800-fold reduction for the largest plasmid used (pC23; insert length 16 kb). Second, more than 80% of the pC23 transformants in the restricting strain contained a deleted plasmid. In the non-restricting strain, the transforming activities of the plasmids were fairly constant as a function of insert length (in the range of 0–16 kb), and no structural instability was observed. It is concluded that for shotgun cloning in B. subtilis, the use of restriction-deficient strains is highly preferable. Evidence is presented that in addition to XhoI other sequences are involved in BsuM restriction. It is postulated that AsuII sites are additional target sites for BsuM restriction.     

The genome of B. subtilis phage SPP1:

Summary DNA molecules of B. subtilis phage SPP1 exhibit terminal redundancy and are partially circularly permuted. This was established by the hybridization of selected EcoRI restriction fragments to single strands of SPP1 DNA and by an analysis of the distribution of denaturation loops in partially denatured SPP1 DNA molecules. Deletions in SPP1 DNA are not compensated by an increase in terminally repetitious DNA. This finding, which is unique to SPP1, is discussed in terms of a modification of the Streisinger/Botstein model of phage maturation.     

Restriction and modification in Bacillus subtilis Marburg 168: target sites and effects on plasmid transformation

Summary The effects of the restriction system of Bacillus subtilis strain M on plasmid transformation were studied. Plasmid pHV1401 DNA prepared from B. subtilis transformed the restriction-proficient M strain 100 times more efficiently than the DNA prepared from Escherichia coli, while the two DNA preparations transformed restriction-deficient derivatives of that strain with similar efficiencies. This indicates that transformation with pHV1401 is sensitive to the M restriction system. pHV1401 contains three CTCGAG (XhoI sites). Successive removal of these abolished the effect of restriction. This indicates that the XhoI sites are the targets for the M restriction system.Abbreviations used Ap^r resistance to ampicillin - Cm^r resistance to chloramphenicol - R/M restriction and modification - Tc^r resistance to tetracycline     

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

Summary A gene coding for a modifying DNA-methyltransferase which methylates the central C in the BsuR recognition sequence 5GGCC was identified in the genome of the temperate Bacillus subtilis phage SP. 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 DNA as well as in heterologous DNA.     

Restriction and modification in Bacillus subtilis: inducibility of a DNA methylating activity in nonmodifying cells.

The nonrestricting/nonmodifying strain Bacillus subtilis 222 (r-m-) can be induced to synthesize a DNA-modifying activity upon treatment with either mitomycin C (MC) or UV light. This is shown by the following facts. (i) Infection of MC-pretreated 222 cells with unmodified SPP1 phage yields about 3% modified phage that are resistant to restriction in B. subtilis R (r+m+). The induced modifying activity causes the production of a small fraction of fully modified phage in a minority class of MC-treated host cells. (ii) The MC-pretreated host cells contain a DNA cytosine methylating activity: both bacterial and phage DNAs have elevated levels of 5-methylcytosine. (iii) The MC-induced methylation of SPP1 DNA takes place at the recognition nucleotide sequences of restriction endonuclease R from B. subtilis R. (iv) Crude extracts of MC-pretreated 222 cells have enhanced DNA methyltransferase activities, with a substrate specificity similar to that found in modification enzymes present in (constitutively) modifying strains.     

Efficient processing of abasic sites by bacterial nonhomologous end-joining Ku proteins

Intracellular reactive oxygen species as well as the exposure to harsh environmental conditions can cause, in the single chromosome of Bacillus subtilis spores, the formation of apurinic/apyrimidinic (AP) sites and strand breaks whose repair during outgrowth is crucial to guarantee cell viability. Whereas double-stranded breaks are mended by the nonhomologous end joining (NHEJ) system composed of an ATP-dependent DNA Ligase D (LigD) and the DNA-end-binding protein Ku, repair of AP sites would rely on an AP endonuclease or an AP-lyase, a polymerase and a ligase. Here we show that B. subtilis Ku (BsuKu), along with its pivotal role in allowing joining of two broken ends by B. subtilis LigD (BsuLigD), is endowed with an AP/deoxyribose 5′-phosphate (5′-dRP)-lyase activity that can act on ssDNA, nicked molecules and DNA molecules without ends, suggesting a potential role in BER during spore outgrowth. Coordination with BsuLigD makes possible the efficient joining of DNA ends with near terminal abasic sites. The role of this new enzymatic activity of Ku and its potential importance in the NHEJ pathway is discussed. The presence of an AP-lyase activity also in the homolog protein from the distantly related bacterium Pseudomonas aeruginosa allows us to expand our results to other bacterial Ku proteins.     

Sequence-specific endonucleases in strains of Anabaena and Nostoc

The complements of restriction endonucleases of 12 strains of cyanobacteria were determined in cell-free extracts, and were compared with the complements of restriction activities assessed by measuring the relative efficiencies of plating of cyanophages on those cyanobacteria. The hosts which were susceptible to all of the phages contained endo R · AvaI and endo R · AvaII, and in several cases probably endo R · AvaIII, or isoschizomers of these enzymes. Three hosts which were lysed by only a subset (1 or 3) of the phages contained different restriction endonuclease. Anabaena sp. PCC 7120 showed apparent phenotypic restriction of phage An-22 grown in hosts with (isoschizomers of) AvaI, II and III, but no corresponding endonuclease has yet been detected in vitro. Nostoc sp. ATCC 29131 (PCC 6705) was found to contain a restriction enzyme, NspBII, with hitherot unknown specificity, C(A/C)GC(T/G)G.     

DNA methyltransferase of Bacillus subtilis Marburg: purification,properties and further evidence of specificity

Bacillus subtilis Marburg strain displays DNA methyltransferase activity. This enzyme, M·BsuM, methylates cytosine in the sequence 5'-YTCGAR-3′ (Y = pyrimidine; R = purine). M·BsuM was purified from the exponentially growing cells of B. subtilis 168M. This enzyme (45 ± 1kDa) is monomeric and recognizes only double-stranded DNA. It is inhibited partially by Mg²⁺, Mn²⁺ ions and spermidine and almost totally by sodium dodecyl sulfate, urea and agarose. This enzyme methylates specifically the three methylatable sites of the plasmid pBM3. Relaxation of specificity (‘star’ activity) was observed in the presence of organic solvents. A very low amount of M·BsuM was obtained in the standard Marburg strain. To obtain sufficient enzyme attempts are being made to clone the M·BsuM gene in Escherichia coli by using a constructed plasmid (pBM14) vector. Only one transformant containing a 3-kb insert and showing a low level of expression, was obtained.     

Genetic engineering of Bacillus subtilis for the commercial production of riboflavin

Recombinant DNA engineering was combined with mutant selection and fermentation improvement to develop a strain of Bacillus subtilis that produces commercially attractive levels of riboflavin. The B. subtilis riboflavin production strain contains multiple copies of a modified B. subtilis riboflavin biosynthetic operon (rib operon) integrated at two different sites in the B. subtilis chromosome. The modified rib operons are expressed constitutively from strong phage promoters located at the 5′ end and in an internal region of the operon. The engineered strain also contains purine analog-resistant mutations designed to deregulate the purine pathway (GTP is the precursor for riboflavin), and a riboflavin analog-resistant mutation in ribC that deregulates the riboflavin biosynthetic pathway. Received 22 June 1998/ Accepted in revised form 6 November 1998     

A determination of the efficiency of uptake of SP50 DNA by competent cells of B. subtilis

Summary Phage SP50 excludes phage SPP1 both in infection and in transfection of B. subtilis. The dependence of the efficiency of exclusion on the concentration of SP50 DNA shows that one SP50 DNA molecule within a competent cell is sufficient to exclude SPP1 phage development. The concentration dependence allows a determination of the efficiency of uptake of SP50 DNA by competent cells. Only 1 out of 200 SP50 DNA molecules in the transfection mixture will become biologically active in excluding SPP1 phage development in the competent cell.     

Methylation of f1 DNA by a restriction endonuclease from Escherichia coli B

Bacteriophage f1 duplex DNA containing hybrid SB sites, the genetic sites which confer upon DNA sensitivity to Escherichia coli B-specific restriction and modification, were prepared in vitro. The hybrid SB sites (modified and mutant) were tested for their ability to be methylated in vitro by endonuclease R · EcoB, the enzyme responsible for both B-specific restriction and modification in vivo. DNA containing hybrid (modified) SB sites can be methylated. One methyl group is added to the DNA per hybrid (modified) SB site. On the other hand, DNA containing hybrid (mutant) SB sites is refractory to modification.The nature and the function of the SB site as well as the implications of these observations for f1 recombination are discussed.     

Inversion and deletion mutants in Bacillus subtilis bacteriophage SPP1 as a consequence of cloning

Summary Properties of an inversion and a deletion mutant of B. subtilis phage SPP1 which arose during cloning are described. The results are related to the biology of this bacteriophage.In preceding communications from our laboratories (Heilmann and Reeve 1982, Behrens et al. 1983) we reported the properties of genetically engineered SPP1 bacteriophages, which could be used as cloning vehicles in B. subtilis. These phages contain a unique restriction site within a dispensable region of their genomes. In the course of cloning experiments using these phage vectors, we have occasionally observed the appearance of not only the original vector and desired hybrid phages, but also of SPP1 phages which had undergone extensive genomic rearrangements. Properties of two such phages, SPP1 inv1, which was found to contain a large inversion and of SPP1 delV, a deletion mutant, which defines an additional dispensable region of the SPP1 genome, are described in this communication.     

Mutational analysis of the nucleoid-associated protein HBsu of Bacillus subtilis

The essential nucleoid-associated protein HBsu of Bacillus subtilis comprises 92 residues, 20% of which are basic amino acids. To investigate the role of the residues located within the DNA-binding arm, the arginine residues R58 and R61 were changed to leucine, while lysine residues K80 and K86 were replaced by alanine. All altered proteins exhibited a reduction in DNA binding capacity, ranging from 10% to 30% of HBsu wild type DNA-binding ability. To investigate the physiological effect of these mutations in B. subtilis, the indigenous hbs gene was replaced by the mutated genes. B. subtilis strain PK20, which carries the HBsu mutation R58L which exhibits the lowest DNA binding ability in vitro, showed the strongest retardation of growth compared to the wild type. Furthermore, PK20 cells displayed an increased rate of cell lysis, diminished sporulation efficiency and a reduced level of negatively supercoiled DNA. These observations suggest that the DNA binding ability of HBsu DNA is important for growth and differentiation and influences DNA topology. Received: 27 July 1998 / Accepted: 22 September 1998     

Specific labelling of replicating SPP1 DNA

Summary Specific labelling of replicating bacteriophage SPP1 DNA can be achieved by infection at nonpermissive temperature of a B. subtilis strain carrying the initation mutation dnaB ts134. Under these conditions host DNA synthesis is reduced by 90 to 95%. This technique was used to identify cistrons of SPP1 involved in phage DNA synthesis and to define intermediates in SPP1 replication.Experiments reported were part of the Doctoral Thesis submitted by K. Burger to the Freie Universität Berlin