Host cell and ultraviolet reactivation of ultraviolet-irradiated Mycoplasmaviruses.

The mycoplasma Acholeplasma laidlawii was shown to have mechanisms for both host cell and ultraviolet (UV) reactivation of UV-irradiated mycoplasmaviruses. Host cell reactivation was examined by comparing the survival abilities of UV-irradiated double-stranded deoxyribonucleic acid mycoplasmavirus plated on both untreated and on acriflavine-treated cells. Acriflavine treatment inhibited cell exision repair. Decreased survival on the acriflavine-treated cells demonstrated host cell reactivation. UV reactivation was studied by comparing the survival of UV-irradiated virus plated on untreated cells with its survival on cells that received a small UV dose before plating. The UV-irradiated cells gave increased virus survival, showing UV reactivation. Similar experiments with a single-stranded deoxyribonucleic acid mycoplasmavirus showed that this virus could be UV reactivated, but not host cell reactivated.     

Resistance of a Bacillus subtilis mutant to a group of temperate bacteriophages

A mutant of Bacillus subtilis 168 was isolated which resists infection by all the group III temperate bacteriophages except SPR, while allowing full infection by phages of the other groups (I, II and IV). The mutation conferring this phenotype, pha-3, shows 52-54% PBS1-mediated cotransduction with the hisAl marker, mapping therefore in the gtaA and gtaB region of the B. subtilis chromosome. Nevertheless, it does not affect the infection by phages sensitive to gta mutations.     

Adsorption of bacteriophages phi 29 and 22a to protoplasts of Bacillus subtilis 168.

Adsorption of bacteriophages phi 29 and 22a to protoplasts of Bacillus subtilis 168 is described. The number of binding sites on bacilli and protoplasts is determined for each phage. Bacilli and protoplasts possess roughly the same number of sites per unit area for phi 29, i.e., approximately 700 sites per bacillus. There are also approximately 700 sites per bacillus for 22a, but only about one-third as many sites per unit area on the protoplast surface. A model for phi 29 adsorption is proposed.     

Genome homology and divergence in the SP beta-related bacteriophages of Bacillus subtilis

Chromosomal organization in related temperate Bacillus subtilis bacteriophages SP beta, phi 3T, rho 11, Z, and E was compared. DNA-DNA hybridization studies done in conjunction with available restriction fragment maps of SP beta, phi 3T, and rho 11 demonstrated that DNA homology between these three phages extended over most of their respective genomes, although each contained unique chromosomal segments, phi 3T, rho 11, Z, and E, but not SP beta, possessed apparently homologous structural genes (thyP) for thymidylate synthetase. DNA from all thyP-containing phages transformed thymine auxotrophs of B. subtilis SP beta lysogens to prototrophy. This transformation commonly involved incorporation of the thyP gene into SP beta prophage within a region corresponding to the middle of the viral chromosome. Chimeric plasmids containing the thyP gene from phi 3T or cloned fragments of SP beta DNA were used in DNA-DNA hybridization studies to locate the thymidylate synthetase gene near the center of the phi 3T chromosome, and to demonstrate that the organization of this region resembled the analogous portion of the SP beta genome. Profiles of virion structural proteins from the five phages were also very similar, further suggesting functional homology between these viruses. However, despite these evidences of relatedness, populations of fragments generated by digesting SP beta, phi 3T, rho 11, Z, and E DNA with restriction enzymes were quite dissimilar.     

Early-blocked asporogenous mutants of Bacillus subtilis are lysogenized at reduced frequency by temperate bacteriophages.

The establishment of lysogeny in early-blocked asporogenous (Spo-) mutants of Bacillus subtilis 168, which were also defective in the production of antibiotics (Abs-), by temperate phage phi105 or SPO2 was studied. It was found that the frequency of lysogenization of Spo-Abs-mutants was 10 to 20% that of the wild-type bacteria. There was no difference in the efficiency of plating and the burst size of phi105 between wild-type and mutant strains. Phi105 lysogens of mutant strains were as stable as those of the wild type. Several rifampin-resistant mutants defective in the production of antibiotics were isolated. They were also defective in spore formation and lysogenized by phi105 at reduced frequency.     

Mapping of genes determining nonpermissiveness and host-specific restriction to bacteriophages in Bacillus subtilis Marburg.

Summary Bacillus subtilis Marburg is nonpermissive for the multiplication of bacteriophages SP10 and NR2. A permissive mutant was derived from the Marburg strain, and the genetic determinants of nonpermissiveness were analyzed by PBS1 transduction. The simultaneous presence of two genes as mutant alleles, nonA and nonB, was necessary for permissiveness. The gene nonA is linked very closely to rfm (cotransfer: 95%); nonB is located between dal and purB (cotransfer of nonB and purB6: 48%). The genetic determinant of host-specific restriction intrinsic to the Marburg strain (hsrM) was found to be identical or very closely linked to nonB. The segregation of nonB and hsrM has never been observed in the course of transduction analysis. The mutation, hsrM1, diminishes the restriction activity, but not the host-controlled modification.     

Early development of bacteriophages SP01 and SP82G in minicells of Bacillus subtilis.

SP01- and SP82G-infected Bacillus subtilis CU403 divIVBI minicells synthesize 13 easily detectable early RNA species with molecular weights ranging from 60 × 10³ to 430 × 10³. Comparison of in vivo and in vitro translation of early messenger RNA indicates that five early mRNAs of SP01 are synthesized but not translated unless protein synthesis has been permitted in the infected minicell, providing evidence for a translation control mechanism. A sequential appearance of 48 polypeptides has been determined in SP01-infected minicells. The polypeptides have been grouped into two classes of early polypeptides, i.e. those encoded by early mRNA and three subsequent classes as demonstrated by the analysis of polypeptides synthesized in minicells infected with the SP01 mutants, susF21, susF4 and susF14. Phage capsid proteins are not synthesized in minicells. RNA synthesized in infected minicells is subject to turnover. The individual mRNA species have differing functional stabilities ranging from a loss of only 50% functional activity, in 20 minutes at 37 °C, to loss of over 99% activity.Infection of anucleate minicells has been shown to be a very simple method for comparison of closely related phages (slight differences are detected between SP01- and SP82G-encoded mRNA and polypeptides), detection of polypeptides affected by amber mutations and the analysis of early events in phage development in the absence of host syntheses.     

Sporulation-converting bacteriophages for Bacillus pumilus.

Thirty-three sporulation-converting bacteriophages for Bacillus pumilus NRS576 were assigned to two apparently unrelated groups on the basis of morphology and antiserum neutralization. Bacterial sporulation mutants responded similarly (conversion or nonconversion) to representatives of both phage groups. Evidence is presented indicating that PMB1 and related phages specify a restriction and/or modification system.     

Bacillus subtilis bacteriophages SP beta c1 is a deletion mutant of SP beta

Summary The restriction fragment patterns of two mutant forms of the temperate Bacillus subtilis bacteriophage SP have been examined. The DNA of a heat-inducible mutant, SPc2, which has a molecular size of 128 kilobases (kb), yields the same restriction pattern as the wild type SPc⁺ DNA. The DNA of a clear-plaque mutant, SPc1, has a molecular size of 117 kb, and is deleted for an 11 kb region of phage DNA. Neither SPc1 nor SPc2 DNA is cleaved by the endonuclease HaeIII.     

Detergent-resistant variants of Bacillus subtilis with reduced cell diameter.

Variants of Bacillus subtilis resistant to the detergent Triton X-100 may exhibit: (i) normal cell morphology, (ii) reduced cell diameter, or (iii) helical cell shape. One variant of type ii was studied in some detail. Triton resistance, cell diameter reduction, and poor sporulation all may have resulted from a single mutation. High concentrations of Triton caused rapid lysis of wild-type cells. B. subtilis adapted to low Triton concentrations such that, upon subsequent exposure to higher concentrations, growth continued, although it bacame inhibited at very high concentrations. The variant studied retained its sensitivity to Triton-induced lysis but, after adaptation, grew at very high Triton levels. In this strain, cell diameter and cross-sectional area were reduced to about 73 and 50%, respectively, of those of wild type, yet the cells grew at normal rates, and DNA/protein/RNA ratios were largely unaltered. Peptidoglycan content per unit of cell surface area was higher in the variant than in the wild type under at least certain growth conditions.     

Structural differentiation of the Bacillus subtilis 168 cell wall.

Exponential-growth-phase cultures of Bacillus subtilis 168 were probed with polycationized ferritin (PCF) or concanavalin A (localized by the addition of horseradish peroxidase conjugated to colloidal gold) to distinguish surface anionic sites and teichoic acid polymers, respectively. Isolated cell walls, lysozyme-digested cell walls, and cell walls treated with mild alkali to remove teichoic acid were also treated with PCF. After labelling, whole cells and walls were processed for electron microscopy by freeze-substitution. Thin sections of untreated cells showed a triphasic, fibrous wall extending more than 30 nm beyond the cytoplasmic membrane. Measurements of wall thickness indicated that the wall was thicker at locations adjacent to septa and at pole-cylinder junctions (P < 0.001). Labelling studies showed that at saturating concentrations the PCF probe labelled the outermost limit of the cell wall, completely surrounding individual cells. However, at limiting PCF concentrations, labelling was observed at only discrete cell surface locations adjacent to or overlying septa and at the junction between pole and cylinder. Labelling was rarely observed along the cell cylinder or directly over the poles. Cells did not label along the cylindrical wall until there was visible evidence of a developing septum. Identical labelling patterns were observed by using concanavalin A-horseradish peroxidase-colloidal gold. Neither probe appeared to penetrate between the fibers of the wall. We suggest that the fibrous appearance of the wall seen in freeze-substituted cells reflects turnover of the wall matrix, that the specificity of labelling to discrete sites on the cell surface is indicative of regions of extreme hydrolytic activity in which alpha-glucose residues of the wall teichoic acids and electronegative sites (contributed by phosphate and carboxyl groups of the teichoic acids and carboxyl groups of the peptidoglycan polymers) are more readily accessible to our probes, and that the wall of exponentially growing B. subtilis cells contains regions of structural differentiation.     

Cosegregation of cell wall and DNA in Bacillus subtilis.

Cosegregation of cell wall and DNA of a lysis-negative mutant of Bacillus subtilis was examined by continuously labeling (i) cell wall, (ii) DNA, and (iii) both cell wall and DNA. After four to five generations of chase in liquid media it was found by light microscope autoradiography that the numbers of wall segregation units per cell are 29 and 9 in rich and minimal medium, respectively. Under the same conditions the numbers of segregation units of DNA were almost 50% lower: 15 and 5, respectively. Simultaneous labeling of cell wall and DNA (iii) provided figures almost identical to those obtained for cell wall alone, (i), implying cosegregation of the two components. Statistical analysis ruled out their random distribution into daughter cells. Measurements of the positions of grain clusters at the end of the chase period along chains of cells, each derived from a single cell at the beginning of chase, show that cell wall units are localized according to a symmetrical pattern, whereas those of DNA are distributed in an asymmetrical but highly regular way. It appears that of two cell wall units of the same age one only has a strand of DNA attached to it. We present a simple diagrammatic model of cell wall organization and DNA-cell wall association which is compatible with our observations. Finally, we discuss previous experiments pertinent to cosegregation of cell wall and DNA obtained with cells grown on solid media as well as with germinating spores; an explanation for the independent segregation of cell wall and DNA observed in the latter case is advanced.     

Relation between cell wall turnover and cell growth in Bacillus subtilis.

The kinetics of cell wall turnover in Bacillus subtilis have been examined in detail. After pulse labeling of the peptidoglycan with N-acetylglucosamine, the newly formed peptidoglycan is stable for approximately three-quarters of a generation and is then degraded by a process that follows first-order kinetics. Deprivation of an auxotroph of amino acids required for protein synthesis results in a cessation of turnover. If a period of amino acid starvation occurs during the lag phase of turnover, then the initiation of turnover is delayed for a period of time equivalent to the starvation period. During amino acid starvation, new cell wall peptidoglycan is synthesized and added to preexisting cell wall. This peptidoglycan after resumption of growth is also subject to degradation (turnover). It is suggested that cell wall turnover is dependent on cell growth and elongation. Several possible control mechanisms for cell wall autolytic enzymes are discussed in light of these observations.     

Extracellular proteases modify cell wall turnover in Bacillus subtilis.

The rate of turnover of peptidoglycan in exponentially growing cultures of Bacillus subtilis was observed to be sensitive to extracellular protease. In protease-deficient mutants the rates of cell wall turnover were greater than that of wild-type strain 168, whereas hyperprotease-producing strains exhibited decreased rates of peptidoglycan turnover. The rate of peptidogylcan turnover in a protease-deficient strain was decreased when the mutant was grown in the presence of a hyperprotease-producing strain. The addition of phenylmethylsulfonyl fluoride, a serine protease inhibitor, to cultures of hyperprotease-producing strains increased their rates of cell wall turnover. Isolated cell walls of all protease mutants contained autolysin levels equal to or greater than that of wild-type strain 168. The presence of filaments, or cells with incomplete septa, was observed in hyperprotease-producing strains or when a protease-deficient strain was grown in the presence of subtilisin. The results suggest that the turnover of cell walls in B. subtilis may be regulated by extracellular proteases.     

Distribution of bacteriophage phi 3T homologous deoxyribonucleic acid sequences in Bacillus subtilis 168, related bacteriophages, and other Bacillus species.

The Bacillus subtilis 168 chromosome was found to share extensive homology with the genome of bacteriophage phi 3T. At least three different regions of the bacterial genome hydridized to ribonucleic acid complementary to phi 3T deoxyribonucleic acid (DNA). The thymidylate synthetase gene, thyA, of B. subtilis and the sequences adjacent to it were shown to be homologous to the region in the phi 3T DNA containing the phage-encoded thymidylate synthetase gene, thyP3. SP beta, a temperate bacteriophage known to be integrated into the B. subtilis 168 chromosome, was demonstrated to be closely related to phi 3T. Other regions of the bacterial genome were also found to hybridize to the phi 3T probe. The nature and location of these sequences in the bacterial and phage chromosomes were not identified. It was shown however, that they were not homologous to either the thyP3 gene or the DNA surrounding the thyP3 gene. The chromosomes of other Bacillus species were also screened for the presence of phi 3T homologous sequences, and the thyP3 gene was localized in the linear genomes of phages phi 3T and rho 11 by heteroduplex mapping. It is suggested that the presence of sequences of phage origin in the B. subtilis 168 chromosome might contribute to the restructuring and evolution of the viral and bacterial DNAs.     

Host cell reactivation by excision repair is error-free in human cells

Do host cell repair processes affect the mutagenesis of UV-irradiated virus in human cells? The answer was obtained by investigating the mutagenesis of UV-irradiated herpes simplex virus after the irradiated virus was grown in human cells that possess normal repair capacity (normal) or lack excision repair (XPA) or post-replication repair (XP var). Evidence is presented which indicate that XPA cells express no host cell reactivation, while XP var cells express the normal level. Viral mutagenesis was measured as the fraction of the progeny of the surviving virus capable of plaque formation in the presence of iododeoxycytidine. In the normal and XPA cells mutagenesis of the irradiated virus increased linearly with UV exposure. The UV exposure needed to yield a given mutagenesis level for virus grown in XPA cells was much lower than that for virus grown in normal cells. However, when the mutation frequencies were compared at similar virus survival levels, the data from virus grown in normal cells and in XPA cells were indistinguishable. Mutagenesis in XP var cells increased as dose squared and was similar in magnitude to that in normal cells. Thus the excision repair of normal cells which provided host cell reactivation by removing lethal UV damage also removed mutagenic lesions from the virus with the same efficiency, while the repair deficiency of XP var cells had a minor role in host cell reactivation and in mutagenesis. This demonstrates that in human cells host cell reactivation by excision repair is primarily an error-free process.