SPP1 DNA replicative forms: growth of phage SPP1 in Bacillus subtilis mutants temperature-sensitive in DNA synthesis.

Summary The development of bacteriophages SPP1, and 29 has been studied in several B. subtilis mutants defective in host DNA replication, under non permissive conditions.Several gene products, involved in the synthesis of host DNA, are required for 29 replication, while SPP1 seems to require obly the host DNA polymerase III. In addition both phages are unable to grow in a dna A mutant (ribonucleotide reductase). Taking advantage of the fact that SPP1 DNA is actively replicated in several dna mutants at non-permissive temperature, we have studied the structure of the replicative intermediates of this phage in the absence of interfering host DNA synthesis.Fast sedimenting forms of SPP1 DNA can be isolated from phage infected cells and evidence of covalently joined concatemers has been obtained, suggesting the presence of terminally repeated sequences.     

Genetic studies on temperature-sensitive mutants ofBacillus subtilis bacteriophage SPP 1

A total of 30ts mutants ofBacillus subtilis bacteriophage SPP1 were isolated and subjected to complementation test. On the basis of this test 21 mutants were classified into 4 functional groups; the classification of the remaining 9 mutants was unclear. The frequency of recombination by mutual crossing was determined in representatives of individual groups; this made it possible to place these mutants on a linear map comprising a total length of 7.62 recombination units.     

Characterization of Bacillus subtilis mutants temperature-sensitive in the synthesis of ribonucleic acid

Two mutants of Bacillus subtilis temperature-sensitive in RNA synthesis were isolated. One mutation (rna-20) was demonstrated to be an allele of a previously identified gene (Riva et al., 1976). The other mutation (rna-16) identified a different gene and was mapped near aroI. The rna-16 mutation at the permissive temperature affected the spore outgrowth process. Purified RNA polymerase from rna-16 did not show any temperature sensitivity or structural defect.     

Bacillus subtilis tau subunit of DNA polymerase III interacts with bacteriophage SPP1 replicative DNA helicase G40P

Genetic evidence suggests that the Bacillus subtilis dnaX gene only encodes for the τ subunit of both DNA polymerases III (Pol IIIs). The B.subtilis full-length protein and their mutant derivatives τ(373– 563) (lacking the N-terminal, domains I–III or amino acid residues 1–372) and τ(1–372) (lacking the C-terminal region or amino acids 373–563) have been purified. The τ protein forms tetramers, τ(373– 563) forms dimers, whereas τ(1–372), depending on the ionic strength, forms trimers or tetramers in solution. In the absence of single-stranded (ss) DNA and a nucleotide cofactor, τ interacts with the SPP1 hexameric replicative G40P DNA helicase in solution or with G40P-ATP bound to ssDNA, with a 1:1 stoichiometry. G40P(109–442), lacking the N-terminal amino acid residues 1–108, interacts with the C-terminal moiety of τ. The data indicate that the interaction of G40P with the τ subunit of Pol III, is relevant for the loading of the Pol IIIs into the SPP1 G38P-promoted open complex.     

Membrane attachment of the chromosome in Bacillus subtilis mutants temperature-sensitive in DNA replication

We have examined three mutants of Bacillussubtilis temperature sensitive in DNA initiation and one temperature sensitive in DNA elongation, in order to investigate whether these lesions can cause or can result in a detachment of the membrane-bound chromosomal region.Our results argue against any effect of the mutations examined on the association between the chromosome and the membrane.     

The genome of Bacillus subtilis phage SPP1: structure of an early promoter

The strongest of five 'early' promoters of Bacillus subtilis phage SPP1 was localized in a DNA restriction fragment by analysis of RNA polymerase binding and R-loop formation. The nucleotide sequence of the promoter region was established. The signal structures identified were similar to those recognized by the sigma 55 RNA polymerase of B. subtilis. The promoter precedes an open reading frame with 51 codons. A protein with the Mr predicted from the nucleotide sequence was identified in minicells.     

Crystal structure of Bacillus subtilis SPP1 phage gp23.1, a putative chaperone

SPP1 is a siphophage infecting the gram‐positive bacterium Bacillus subtilis. The SPP1 tail electron microscopy (EM) reconstruction revealed that it is mainly constituted by conserved structural proteins such as the major tail proteins (gp17.1), the tape measure protein (gp18), the Distal tail protein (Dit, gp19.1), and the Tail associated lysin (gp21). A group of five small genes (22–24.1) follows in the genome but it remains to be elucidated whether their protein products belong or not to the tail. Noteworthy, an unassigned EM density accounting for ～245 kDa is present at the distal end of the SPP1 tail‐tip. We report here the gp23.1 crystal structure at 1.6 Å resolution, a protein that lacks sequence identity to any known protein. We found that gp23.1 forms a hexamer both in the crystal lattice and in solution as revealed by light scattering measurements. The gp23.1 hexamer does not fit well in the unassigned SPP1 tail‐tip EM density and we hypothesize that this protein might act as a chaperone.     

Structural analysis of Bacillus subtilis SPP1 phage helicase loader protein G39P

The Bacillus subtilis SPP1 phage-encoded protein G39P is a loader and inhibitor of the phage G40P replicative helicase involved in the initiation of DNA replication. We have carried out a full x-ray crystallographic and preliminary NMR analysis of G39P and functional studies of the protein, including assays for helicase binding by a number of truncated mutant forms, in an effort to improve our understanding of how it both interacts with the helicase and with the phage replisome organizer, G38P. Our structural analyses reveal that G39P has a completely unexpected bipartite structure comprising a folded N-terminal domain and an essentially unfolded C-terminal domain. Although G39P has been shown to bind its G40P target with a 6:6 stoichiometry, our crystal structure and other biophysical characterization data reveal that the protein probably exists predominantly as a monomer in solution. The G39P protein is proteolytically sensitive, and our binding assays show that the C-terminal domain is essential for helicase interaction and that removal of just the 14 C-terminal residues abolishes interaction with the helicase in vitro. We propose a number of possible scenarios in which the flexibility of the C-terminal domain of G39P and its proteolytic sensitivity may have important roles for the function of G39P in vivo that are consistent with other data on SPP1 phage DNA replication.     

Analysis of autolysins in temperature-sensitive morphological mutants of Bacillus subtilis.

The content and distribution of autolysin were measured in temperature-sensitive morphological mutants of Bacillus subtilis. Strains RUB1000 and RUB1012 grew as rods at 30 C. At 45 C the mutants contained disproportionately less teichoic acid than peptidoglycan and grew as irregular spheres. The amount of enzyme that could be extracted from rods was at least 31 times the amount extracted from spheres. The rate of autolysis of cell walls was 7- to 28-fold greater in rods than in spheres. The low activity found associated with the cell walls of spheres was not compensated for by larger amounts of autolytic activity in the cytoplasm. No activity was found in the growth medium at either temperature. The failure of the mutant cells to autolyze was due to low amidase activity and relatively resistant cell walls. Revertants of RUB1012 were isolated that had 13, 23, and 55% of the normal proportions of teichoic acid when grown at the nonpermissive temperature. Cell walls from the revertants were as sensitive to added amidase as the wild-type strain. None of the revertant strains regained the wild-type ability to produce more amidase at 45 C. However, the deficiency in autolysin observed with RUB1012 was partially restored in revertants containing higher proportions of teichoic acid.     

In vitro packaging of DNA of the Bacillus subtilis bacteriophage SPP1

In vitro packaging of bacteriophage SPP1 DNA into procapsids is described and the requirements of this process were determined. Combination of proheads with an extract supplying terminase, DNA and phage tails yielded up to 10(7 )viable phages per milliliter of in vitro reaction under optimized conditions. The presence of neutral polymers and polyamines had a concentration and type dependent effect in the packaging reaction. The terminase donor extract lost rapidly activity at 30 degrees C in contrast to the stability of the prohead donor extract. Maturation to infective virions was observed using both procapsids assembled in SPP1 infected cells and procapsid-like structures assembled in Escherichia coli that overexpressed the SPP1 prohead gene clusters. Neither a majority of aberrant capsid-related structures present in the latter material nor procapsids lacking the portal protein inhibited DNA packaging. Addition of purified portal protein reduced DNA packaging activity in vitro only at concentrations 20-fold higher than those found in the SPP1 infected cell. The SPP1 DNA packaged in vitro originated exclusively from the terminase donor extract. This packaging selectivity was not observed in vivo during mixed infections. The data are compatible with a model for processive headful DNA packaging in which terminase and DNA co-produced in the same cell are tightly associated and can effectively discriminate the portal vertex of DNA packaging-proficient proheads from aberrant structures, from portal-less procapsids, and from isolated portal protein.     

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.     

The genome of Bacillus subtilis phage SPP1: the arrangement of restriction endonuclease generated fragments.

Summary SPP1 DNA was cleaved by the restriction endonucleases, BglI, BglII, EcoRI, KpnI, SmaI, and SalI. The molecular weights of the DNA fragments obtained by single enzyme digestion or by consecutive digestion with two enzymes were determined by electron microscopic measurements of contour length and by gel electrophoresis. The major fragments from the six digests could be ordered to give a consistent restriction map of SPP1. The electropherograms of several digests indicated that certain fragments occurred in less than stoichiometric amounts or were heterogeneous in size. Such bands carried a major part of radioactivity, when SPP1 DNA was terminally labelled with P³² prior to degradation by restriction enzymes. These results, and studies of the effect of exonuclease III treatment on restriction enzyme patterns define the terminal restriction fragments. All data obaained support the conclusion drawn in the preceding paper (Morelli et al., 1978b) that the SPP1 genome is terminally redundant and partially circularly permuted.Part of this work is from the doctoral dissertations to be submitted to Stanford University¹ and the Freie Universität Berlin²