Prophage PBSX Research Articles

Characterization of Heterogeneity and Dynamics of Lysis of Single Bacillus subtilis Cells upon Prophage Induction During Spore Germination, Outgrowth, and Vegetative Growth Using Raman Tweezers and Live-Cell Phase-Contrast Microscopy.

A prophage comprises a bacteriophage genome that has integrated into a host bacterium's DNA, which generally permits the cell to grow and divide normally. However, the prophage can be induced by various stresses, or induction can occur spontaneously. After prophage induction, viral replication and production of endolysins begin until the cell lyses and phage particles are released. However, the heterogeneity of prophage induction and lysis of individual cells in a population and the dynamics of a cell undergoing lysis by prophage induction have not been fully characterized. Here, we used Raman tweezers and live-cell phase-contrast microscopy to characterize the Raman spectral and cell length changes that occur during the lysis of individual Bacillus subtilis cells from spores that carry PBSX prophage during spores' germination, outgrowth, and then vegetative growth. Major findings of this work are as follows: (i) After addition of xylose to trigger prophage induction, the intensities of Raman spectral bands associated with nucleic acids of single cells in induced cultures gradually fell to zero, in contrast to the much smaller changes in protein band intensities and no changes in nucleic acid bands in uninduced cultures; (ii) the nucleic acid band intensities from an individual induced cell exhibited a rapid decrease, following a long lag period; (iii) after the addition of nutrient-rich medium with xylose, single spores underwent a long period (228 ± 41.4 min) for germination, outgrowth, and vegetative growth, followed by a short period of cell burst in 1.5 ± 0.8 min at a cell length of 8.2 ± 5.5 μm; (iv) the latent time (Tlatent) between the addition of xylose and the start of cell burst was heterogeneous in cell populations; however, the period (ΔTburst) from the latent time to the completion of cell lysis was quite small; (v) in a poor medium with l-alanine alone, addition of xylose caused prophage induction following spore germination but with longer Tlatent and ΔTburst times and without cell elongation; (vi) spontaneous prophage induction and lysis of individual cells from spores in a minimal nutrient medium were observed without xylose addition, and cell length prior to cell lysis was ∼4.1 μm, but spontaneous prophage induction was not observed in a rich medium; (vii) in a rich medium, addition of xylose at a time well after spore germination and outgrowth significantly shortened the average Tlatent time. The results of this study provide new insights into the heterogeneity and dynamics of lysis of individual B. subtilis cells derived from spores upon prophage induction.

Crystal structures of the Bacillus subtilis prophage lytic cassette proteins XepA and YomS.

As part of the Virus-X Consortium that aims to identify and characterize novel proteins and enzymes from bacteriophages and archaeal viruses, the genes of the putative lytic proteins XepA from Bacillus subtilis prophage PBSX and YomS from prophage SPβ were cloned and the proteins were subsequently produced and functionally characterized. In order to elucidate the role and the molecular mechanism of XepA and YomS, the crystal structures of these proteins were solved at resolutions of 1.9 and 1.3 Å, respectively. XepA consists of two antiparallel β-sandwich domains connected by a 30-amino-acid linker region. A pentamer of this protein adopts a unique dumbbell-shaped architecture consisting of two discs and a central tunnel. YomS (12.9 kDa per monomer), which is less than half the size of XepA (30.3 kDa), shows homology to the C-terminal part of XepA and exhibits a similar pentameric disc arrangement. Each β-sandwich entity resembles the fold of typical cytoplasmic membrane-binding C2 domains. Only XepA exhibits distinct cytotoxic activity in vivo, suggesting that the N-terminal pentameric domain is essential for this biological activity. The biological and structural data presented here suggest that XepA disrupts the proton motive force of the cytoplasmatic membrane, thus supporting cell lysis.

A system for the expression and release of heterologous proteins from the core ofBacillus subtilisspores

Methodologies that exploit the durability of Bacillus subtilis spores by displaying heterologous proteins or antigenic molecules on the spore surface for mucosal vaccine delivery and other applications are well established. Here we extend the concept by engineering spores intended as oral delivery vehicles for therapeutic proteins. The method is exemplified by the expression and deposition of human growth hormone in the developing spore core, where the protein is shielded from physicochemical and biological degradation by the protective spore structure. Lysates from physically disrupted spores are shown to stimulate differentiation of a pre-adipocyte cell line to mature adipocyte cells, indicating that the spore-core located human growth hormone is folded correctly and functional. We also introduce a methodology for controlled release of heterologous proteins from the spore core, which utilises components of the PBSX prophage to lyse spores during germination and outgrowth. With further development, spore core expression, coupled with an engineered autolytic germination mechanism, may permit the use of spores as oral delivery carriers of therapeutic proteins.

Genome-based identification of active prophage regions by next generation sequencing in Bacillus licheniformis DSM13.

Prophages are viruses, which have integrated their genomes into the genome of a bacterial host. The status of the prophage genome can vary from fully intact with the potential to form infective particles to a remnant state where only a few phage genes persist. Prophages have impact on the properties of their host and are therefore of great interest for genomic research and strain design. Here we present a genome- and next generation sequencing (NGS)-based approach for identification and activity evaluation of prophage regions. Seven prophage or prophage-like regions were identified in the genome of Bacillus licheniformis DSM13. Six of these regions show similarity to members of the Siphoviridae phage family. The remaining region encodes the B. licheniformis orthologue of the PBSX prophage from Bacillus subtilis. Analysis of isolated phage particles (induced by mitomycin C) from the wild-type strain and prophage deletion mutant strains revealed activity of the prophage regions BLi_Pp2 (PBSX-like), BLi_Pp3 and BLi_Pp6. In contrast to BLi_Pp2 and BLi_Pp3, neither phage DNA nor phage particles of BLi_Pp6 could be visualized. However, the ability of prophage BLi_Pp6 to generate particles could be confirmed by sequencing of particle-protected DNA mapping to prophage locus BLi_Pp6. The introduced NGS-based approach allows the investigation of prophage regions and their ability to form particles. Our results show that this approach increases the sensitivity of prophage activity analysis and can complement more conventional approaches such as transmission electron microscopy (TEM).

Comparison of Responses to Double-Strand Breaks betweenEscherichia coliandBacillus subtilisReveals Different Requirements for SOS Induction

DNA double-strand breaks are particularly deleterious lesions that can lead to genomic instability and cell death. We investigated the SOS response to double-strand breaks in both Escherichia coli and Bacillus subtilis. In E. coli, double-strand breaks induced by ionizing radiation resulted in SOS induction in virtually every cell. E. coli strains incapable of SOS induction were sensitive to ionizing radiation. In striking contrast, we found that in B. subtilis both ionizing radiation and a site-specific double-strand break causes induction of prophage PBSX and SOS gene expression in only a small subpopulation of cells. These results show that double-strand breaks provoke global SOS induction in E. coli but not in B. subtilis. Remarkably, RecA-GFP focus formation was nearly identical following ionizing radiation challenge in both E. coli and B. subtilis, demonstrating that formation of RecA-GFP foci occurs in response to double-strand breaks but does not require or result in SOS induction in B. subtilis. Furthermore, we found that B. subtilis cells incapable of inducing SOS had near wild-type levels of survival in response to ionizing radiation. Moreover, B. subtilis RecN contributes to maintaining low levels of SOS induction during double-strand break repair. Thus, we found that the contribution of SOS induction to double-strand break repair differs substantially between E. coli and B. subtilis.

SOS Induction in a Subpopulation of Structural Maintenance of Chromosome (Smc) Mutant Cells in Bacillus subtilis

The structural maintenance of chromosome (Smc) protein is highly conserved and involved in chromosome compaction, cohesion, and other DNA-related processes. In Bacillus subtilis, smc null mutations cause defects in DNA supercoiling, chromosome compaction, and chromosome partitioning. We investigated the effects of smc mutations on global gene expression in B. subtilis using DNA microarrays. We found that an smc null mutation caused partial induction of the SOS response, including induction of the defective prophage PBSX. Analysis of SOS and phage gene expression in single cells indicated that approximately 1% of smc mutants have fully induced SOS and PBSX gene expression while the other 99% of cells appear to have little or no expression. We found that induction of PBSX was not responsible for the chromosome partitioning or compaction defects of smc mutants. Similar inductions of the SOS response and PBSX were observed in cells depleted of topoisomerase I, an enzyme that relaxes negatively supercoiled DNA.

Chromosome DNA fragmentation and excretion caused by defective prophage gene expression in the early-exponential-phase culture ofBacillus subtilis

Bacillus subtilis 168 and its major autolysin mutant, AN8, were shown to excrete two size classes of DNA when cultured in Luria-Bertani medium. Pulsed-field gel electrophoresis of DNA harvested from the cell surface demonstrated the presence of 13-kb-long and circa 50-kb-long strands. Restriction digestion of both sizes of DNA resulted in a smearing pattern, as observed by agarose gel electrophoresis. Shotgun sequencing of DNase I partial digests of 50-kb DNA fragments revealed that the strands originate from various sites on the chromosome. SDS-PAGE analysis of cell surface fractions and culture supernatants demonstrated the presence of several proteins that were thought to be associated with the DNA. Of these, three major proteins were identified, i.e., XkdG, XkdK, and XkdM, by tandem mass spectrometry, all of which were proteins of a defective prophage PBSX residing in the Bacillus subtilis chromosome. Disruption of these PBSX genes resulted in a reduction of 13-kb fragment generation and excretion and also a great reduction of 50-kb fragment excretion. Electron microscopy showed that a few mature phages and numerous membrane vesicle-like particles existed in the cell surface fractions of strain 168. The present findings suggest that the spontaneous generation and excretion of chromosome DNA fragments in Bacillus subtilis are both closely related to the expression of defective prophage genes.

Proteins released during high toxin production in Clostridium difficile.

The mechanism by which toxins A and B are released by Clostridium difficile is unknown and information about the other extracellular proteins of this bacterium is limited. The authors identified exported proteins from C. difficile strain VPI 10463 during conditions promoting high toxin production. Toxins A and B were released in a 1:1 ratio and the proportion of toxin in the extracellular fraction reached 50% during the stationary phase as compared to a proportion of <1% for typical cytoplasmic proteins, showing that toxin export was not due to bacterial lysis. A 47 kDa protein, released with similar kinetics to the toxins, was processed and showed weak similarity to the channel-forming protein TolC. Another protein released during high toxin production was unprocessed and showed similarity to XkdK encoded by the prophage PBSX in Bacillus subtilis, a protein supposedly exported via phage-specific holins. The two most abundant extracellular C. difficile proteins, found during both high and low toxin production, were processed and identified as shed S-layer proteins. As shown by N-terminal sequencing and PCR-based methods, there was a considerable sequence variation of the S-layer gene slpA in different serogroup reference strains. To conclude, C. difficile uses the classical Sec-dependent and probably also holin-like pathways to secrete a comparatively small repertoire of proteins.

Overproduction, isolation, and DNA-binding characteristics of Xre, the repressor protein from the Bacillus subtilis defective prophage PBSX.

PBSX is a phage-like bacteriocin (phibacin) of Bacillus subtilis 168. Lysogeny is maintained by the PBSX-encoded repressor, Xre. The Xre protein was overproduced in Escherichia coli and isolated by affinity chromatography. Gel retardation and DNase I footprinting studies indicated that Xre binds to four sites close to its own gene. These sites overlap putative promoters for xre and a divergent transcriptional unit, containing the middle genes.

Lytic enzymes associated with defective prophages of Bacillus subtilis: Sequencing and characterization of the region comprising the N-acetylmuramoyl-L-alanine amidase gene of prophage PBSX

Prophage induction in Bacillus subtilis strains 168, S31 and W23 is accompanied by synthesis of two endolysins. The synthesis of those of strain 168, with molecular masses of 32 and 34 kDa, was shown to be controlled by the repressor of the defective phage PBSX. The 32 kDa protein corresponds to an N-acetylmuramoyl-L-alanine amidase, and plays the major role in PBSX-mediated lysis. Its structural gene, xlyA, is the last in the PBSX late operon, whose four most distal open reading frames have been cloned and sequenced. Analysis of the nucleotide sequence suggests that the two open reading frames preceding xlyA, designated xhlA and xhlB, encode polypeptides whose combined action could play the role of a holin. The open reading frame upstream of xhlA, designated xepA, encodes an exoprotein. The phage amidase, although endowed with a signal peptide, is apparently, like Xep, exported by a holin-like mechanism which does not involve the cleavage of the signal peptide. The presence on the B. subtilis chromosome of other, similar, genes, and their possible widespread occurrence, is discussed.

Analysis of Bacillus subtilis 168 prophage-associated lytic enzymes; identification and characterization of CWLA-related prophage proteins.

The CWLA lytic amidase of Bacillus subtilis 168 was purified and antisera raised against the purified protein. No expression of cwlA could be demonstrated under any conditions by the use of the antisera and cwlA::lacZ fusion analysis. Two lytic enzymes of apparent molecular masses 34 and 30 kDa (as measured by renaturing SDS-PAGE) were found to be mitomycin C-inducible, the larger of which corresponds to a protein immunologically related to CWLA. Both of these inducible lysins were found to be encoded by prophage PBSX. Prophage SP beta was shown by renaturing SDS-PAGE to produce a 43 kDa lytic enzyme unrelated immunologically to CWLA. The smaller of the two PBSX enzymes was purified and found to be an N-acetylmuramyl-L-alanine amidase of 32 kDa (as measured by SDS-PAGE and Coomassie blue staining) which cross-reacts only weakly with the anti-CWLA sera. The potential origin of cwlA and its possible relationship to the other phage lytic enzymes are discussed.

Cloning and nucleotide sequence of the Bacillus subtilis ansR gene, which encodes a repressor of the ans operon coding for L-asparaginase and L-aspartase

Previous work has shown that expression of the Bacillus subtilis ans operon which codes for L-asparaginase and L-aspartase, is both increased and made insensitive to repression by NH4+ by the aspH1 mutation. In current work, the gene in which the aspH1 mutation resides has been identified and sequenced; this gene, termed ansR, is immediately upstream of, but transcribed in the opposite direction from, the ans operon. The promoter region of ansR contains -10 and -35 sequences similar to those recognized by RNA polymerase containing the major vegetative-cell sigma factor sigma A, and ansR appears to be monocistronic. The ansR gene codes for a 116-residue protein, but the aspH1 mutant allele has an additional guanine residue at codon 55, resulting in generation of a truncated polypeptide of only 58 residues. Insertional inactivation of ansR resulted in a phenotype identical to that of the aspH1 mutant. The predicted amino acid sequence of the ansR gene product (AnsR) was homologous to that of the repressor of B. subtilis prophage PBSX, and a helix-turn-helix motif, characteristic of many DNA-binding proteins, was present in the AnsR amino-terminal region. These results suggest that ansR codes for a repressor of the ans operon.

Cloning and characterization of genes induced by hydrogen peroxide in Bacillus subtilis.

Transcriptional fusions of Bacillus subtilis DNA to the lacZ gene were screened for induction, initially by ethanol and then by hydrogen peroxide (H2O2). Two fusions were identified which were induced late following treatment with sublethal concentrations of H2O2 (100 microM). The oxy-1 promoter was induced 4-5-fold and mapped to 11 degrees while the oxy-2 promoter was induced 20-fold and mapped close to the right of the defective prophage PBSX, at about 120 degrees. The oxy-2 fusion was induced by mitomycin C as well as H2O2, which correlated with the induction of PBSX by these agents. This was probably not a transcriptional induction, but rather a consequence of the induction of PBSX replication extending into adjacent regions of the chromosome.

Prophage induction in thermosensitive DNA mutants of Bacillus subtilis.

Incubation of thermosensitive dna mutants of Bacillus subtilis at the non-permissive temperature leads in some instances to induction of defective prophage PBSX and cell lysis. A clear distinction can be made between mutants affected in DNA replication at the growing point (extension mutants) and those unable to initiate new rounds of replication (initiation mutants). The former promote PBSX induction to a variable and mutation-specific extent, whereas the latter do not exhibit any signs of induction. Analysis of mutants carrying two dna mutations suggests that products of some dna genes involved in initiation and in extension are not essential for induction but can substantially amplify its extent. However, mitomycin C treatment of dna mutants which have completed their residual DNA synthesis leads to a PBSX induction essentially identical to that obtained by mitomycin C treatment of the wild-type strain, which precludes an essential role for any of the mutated proteins in this induction process. On the basis of our observations we propose that the induction signal is related to the number of blocked replication forks: the larger that number, the higher the proportion of induced cells within the population.

Bacteriophage PBSX-induced deletion mutants of Bacillus subtilis 168 constitutive for alkaline phosphatase.

Mutants of Bacillus subtilis with deletions extending from the PBSX prophage, and in some cases removing pro(AB) and metC, have been found to be constitutive for vegetatively synthesized alkaline phosphatase. Such deletions were isolated by selecting for heat-resistant derivatives of a strain carrying a xhi-1479 mutation causing heat-inducibility of the defective phage PBSX. These deletions remove the phoS gene, a regulatory gene for alkaline phosphatase; it is concluded that the phoS gene product exerts negative control on alkaline phosphatase synthesis. Deletion mapping, combined with previously published linkage data, indicates a gene order of PBSX-phoS-pro(AB)-metC.

Properties of Bacillus subtilis 168 derivatives freed of their natural prophages

An isogenic set of Bacillus subtilis 168 strains which are non-inducible for prophage PBSX and are cured of prophage SPβ has been constructed. By utilizing these strains, prophage SPβ has been shown to control the inducible DNA modification system which exists in this bacterium. However, neither the PBSX nor the SPβ prophages alter the ability of the bacterium to undergo genetic recombination, to repair damaged DNA or to sporulate. Prophageless B. subtilis would be a useful host for the φ3T cloning vector, because of the absence of vector—prophage interactions.

Selection of Bacillus subtilis 168 mutants with deletions of the PBSX prophage.

Heat-resistant derivatives of a Bacillus subtilis 168 strain carrying an xhi mutation, which causes heat-sensitive induction of the PBSX prophage, have been isolated and screened for the acquisition of auxotrophy. Two classes of auxotrophs were isolated, namely Pro- and Pro-Met-; they lacked the ability to produce PBSX, as shown by their resistance to mitomycin C-induced lysis. The proline and methionine requirements and the resistance to mitomycin C were shown to segregate together in phage PBS1-mediated transduction crosses and to be linked to thiB, which is known to be co-transducible with the PBSX prophage. It was therefore proposed that these strains had deletions which removed all or part of the PBSX prophage together with adjacent bacterial DNA encoding the pro(AB) and metC genes. The met mutation was shown to be metC in PBS1 transduction crosses; this gene is known to be co-transducible with the PBSX prophage. The proline requirement was probably due to the deletion of a pro gene which was demonstrated to lie between the PBSX prophage and metC and which was 90% co-transducible with metC. These deletions have been transduced into a strain which was cured of phage SP beta, another bacteriophage carried by B. subtilis 168. No phage particles could be seen in mitomycin C-induced cultures of such strains. The PBSX-deletion strains grew with the same generation time as the PBSX+ parent in L-broth (27 min at 35 degrees C) but they were slower in minimal medium (e.g. 72 min as against 51 min in the PBSX+ strain). Besides being resistant to mitomycin C-induced lysis, the deletion strains were also resistant to lysis induced by thymine starvation of thymine auxotrophs and the loss of viability of these strains after thymine starvation was 100-fold less than in the PBSX+ parent. The deletion strains had not, however, lost the bacterial autolytic enzymes, since they were still susceptible to lysis when placed under semi-anaerobic conditions.

Prophage mutation causing heat inducibility of defective Bacillus subtilis bacteriophage PBSX.

A mutant of Bacillus subtilis 168 has been isolated in which the defective phage PBSX was heat inducible, whereas another phage, phi105, was not so induced. A culture of the mutant grown at 30 degrees C, when shifted to 45 degrees C, began to lyse after 45 min; cell viability began to decrease after 10 min. Heat-induced lysis of the mutant was prevented by chloramphenicol. DNA, RNA, protein, and peptidoglycan synthesis were normal at the nonpermissive temperature up to the time of lysis. The site of xhi-1479 mutation causing this phenotype was linked (50%) in phage PBS1-mediated transduction to the host marker metC and to another PBSX marker xtl and was thus thought to map within the PBSX prophage. The order of markers was argC-thiB-metA-xhi-metC. The xhi mutation was thus distinct from another mutation, tsi-23, causing a similar heat inducibility of PBSX (Siegel and Marmur, 1969), which was unlinked to the metC marker. tsi-23 is therefore thought to be a host mutation, and the available evidence for a scattered phage genome being the cause of the defective nature of PBSX is thus less tenable. It was shown that the mutant, besides carrying the xhi mutation, also carried another closely linked mutation, xki-1479, which caused the PBSX produced to have no killing activity on the sensitive strain W23. The xki mutation was separated from xhi by recombination.

Bacteriophage-specific protein synthesis during induction of the defective Bacillus subtilis bacteriophage PBSX

Particles of PBSX, a defective, noninfectious phage which is inducible from strains of Bacillus subtilis 168, contain at least seven structural proteins resolvable by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Five of these proteins are associated with the phage tail and two with the phage head. An eighth protein, which also may be coded for by the PBSX prophage, has been identified in cells derepressed for PBSX replication.

Isolation and characterization of prophage mutants of the defective Bacillus subtilis bacteriophage PBSX.

Bacillus subtilis mutants with lesions in PBSX prophage genes have been isolated. One of these appears to be a regulatory mutant and is defective for mitomycin C-induced derepression of PBSX; the others are defective for phage capsid formation. All of the PBSX structural proteins are synthesized during induction of the capsid defective mutants; however, several of these proteins exhibit abnormal serological reactivity with anti-PBSX antiserum. The two head proteins X4 and X7 are not immunoprecipitable in a mutant which fails to assemble phage head structures. In the tail mutant, proteins X5 and X6 are not immunoprecipitable, tails are not assembled, and a possible tail protein precursor remains uncleaved. The noninducible mutant does not synthesize any PBSX structural proteins after exposure to mitomycin C. The mutation is specific for PBSX since ø105 and SPO2 lysogens of the mutant are inducible. All of the known PBSX-specific mutations were shown to be clustered between argC and metC on the host chromosome. In addition, the metC marker was shown to be present in multiple copies in cells induced for PBSX replication. This suggests that the derepressed prophage replicates while still integrated and that replication extends into the adjacent regions of the host chromosome.