Temperature-Sensitive Mutants of Bacteriophage Mu

Temperature-sensitive mutants of bacteriophage Mu, which grow at 32 C but not at 42 C, have been isolated. These mutants fall into two groups. Group 1 mutants fail to lyse host cells at nonpermissive temperatures, whereas lysis occurs normally with the group 2 mutants. All of the group 1 mutants apparently belong to the cistrons mapping to the left of gene C, whereas the group 2 mutants have lesions in various genes between D and S.     

Sporulation and Antibiotic Production by Bacillus subtilis Mutants Deficient in Intracellular Proteases

Erythropoietin (Ep) was isolated from the urine of patients with aplastic anemia [Yanagawa et al., J. Biol. Chem., 259, 2707 (1984)] and burst-promoting activity (BPA) was extensively purified from the residue obtained after removal of Ep. These erythropoietic factors were studied for their effcects on erythroid burst-colony formation of human peripheral blood mononuclear cells in methylcellulose cultures. Reddish bursts were formed with the addition of Ep alone. Addition of BPA not only elevated the number of bursts but also greatly reduced the amount of Ep required for burst formation. The presence of BPA alone in cultures did not permit bursts to form but did permit the growth of small colonies that did not contain hemoglobin (Hb). Addition of Ep to these small colonies led to the formation of erythroid bursts. Administration of Ep to the cultures could be delayed for 6 days without decreasing the number of bursts if the cultures were initiated in the presence of BPA; in the absence of BPA, the erythroid precursors (BUF-E) were rapidly lost if Ep was not provided at the start of the cultures. BPA produced larger bursts than those formed in the presence of Ep alone. Microassays of Hb in the bursts indicated that BPA increased the amonut of Hb per burst. This increase could not be entirely explained by the augumentation in cell number per burst but was partly ascribable to the increased amount of Hb per cell.     

Transducing Bacteriophage for Bacillus cereus

A phage, designated CP-51, that carries out generalized transduction in Bacillus cereus 569 was isolated from soil. All auxotrophic mutants tested, those requiring tryptophan, histidine, leucine, isoleucine, methionine, or phenylalanine, were transduced to prototrophy. The phage was extremely unstable when stored at 2 to 4 C, but stability was enhanced by storage at higher temperatures. The optimal temperature of those tested for maintenance of plaque-forming units was 15 C.     

Temperature-Sensitive Induction of Bacteriophage in Bacillus subtilis 168

In a temperature-sensitive mutant of Bacillus subtilis 168, induction of the defective phage PBSX occurred at 48 C. Cell lysis began after 90 min of growth at 48 C, and cell viability began to decrease after 10 to 30 min. The loss in viability at the nonpermissive temperature was prevented by azide or cyanide. Deoxyribonucleic acid (DNA), ribonucleic acid, and protein synthesis were not inhibited at 48 C. Temperature induction of the temperate phage SPO2 also occurred in this mutant. The temperature-sensitive mutation, designated tsi-23, was linked by transduction to purB6 and pig, the order being purB6 pig tsi-23. Mutation tsi-23 was transformable to wild type by B. subtilis 168 DNA but not by DNA from the closely related strains W23 or S31. DNA from the latter two strains transformed auxotrophic markers of strain 168 at frequencies close to those found with 168 donor DNA. Upon temperature induction, cellular DNA was broken to a size of 22S, characteristic of DNA in PBSX particles. The DNA isolated from temperature-induced PBSX did not give an increased Ade(+)/Met(+) transformant ratio relative to cellular DNA nor contain preferential break points as determined by transformation of four closely linked markers.     

Mutagenic Effect of Temperature-Sensitive Mutants of Gene 42 (dCMP Hydroxymethylase) of Bacteriophage T4

Certain temperature-sensitive mutants of gene 42 of bacteriophage T4 increase the reversion rates of some rII mutants in the same genome by about 4 to 10 times. This effect was usually found at 34 C, an intermediate permissive temperature, but not at 28 C.     

Intragenic Complementation Between Temperature-Sensitive Mutants of Gene 42 (dCMP Hydroxymethylase) of Bacteriophage T4

Interallelic complementation between certain temperature-sensitive mutants of gene 42 of bacteriophage T4 was demonstrated by measuring the incorporation of labeled thymine into DNA.     

Sporulation and Enterotoxin Production by Mutants of Clostridium perfringens

The ability of Clostridium perfringens type A to produce an enterotoxin active in human food poisoning has been shown to be directly related to the ability of the organism to sporulate. Enterotoxin was produced only in a sporulation medium and not in a growth medium in which sporulation was repressed. Mutants with an altered ability to sporulate were isolated from an sp(+) ent(+) strain either as spontaneous mutants or after mutagenesis with acridine orange or nitrosoguanidine. All sp(0) (-) mutants were ent(-). Except for one isolate, these mutants were not disturbed in other toxic functions characteristic of the wild type and unrelated to sporulation. A total of four of seven osp(0) mutants retained the ability to produce detectable levels of enterotoxin. None of the ent(-) mutants produced gene products serologically homologous to enterotoxin. A total of three sp(-) mutants, blocked at intermediate stages of sporulation, produced enterotoxin. Of these mutants, one was blocked at stage III, one probably at late stage IV, and one probably at stage V. A total of three sp(+) revertants isolated from an sp(-) ent(-) mutant regained not only the ability to sporulate but also the ability to produce enterotoxin. The enterotoxin appears to be a sporulation-specific gene product; however, the function of the enterotoxin in sporulation is unknown.     

Isolation and Properties of a Temperature-Sensitive Sporulation Mutant of Bacillus subtilis

A thermosensitive sporulation mutant (t(s)-4) of Bacillus subtilis was isolated, and its morphological, physiological, and enzymatic properties were investigated. This mutant is able to grow equally well at 30 and 42 C, but is unable to sporulate at the higher temperature. Electron microscope studies have shown that the t(s)-4 mutant is blocked at stage zero of spore development. This was further confirmed by its inability to produce antibiotic when grown at the restrictive temperature and by the relatively low ribonucleic acid (RNA) and protein turnover during the stationary growth phase, characteristic for stage zero asporogenic mutants. At the permissive temperature, however, antibiotic production and RNA and protein turnover took place at the rate normally found in sporogenic strains of B. subtilis. The above properties were not altered in the parent strain when grown at either 30 or 42 C. By shifting cultures of the t(s)-4 mutant from 30 to 42 C and from 42 to 30 C at different stages of growth, we have been further able to show that the event affected at the high temperature takes place at a very early stage of spore development. As a consequence of this early block in the sporulation process, the t(s)-4 mutant grown at 42 C became defective in the late spore-specific enzymes involved in the biosynthesis of dipicolinic acid. This study suggests that the sporulation process is mediated by a regulatory protein which is altered in the thermosensitive mutant when grown at the restrictive temperature. As a result of this alteration, a pleiotropic phenotype is produced which has lost the ability to catalyze the late biochemical reactions required for spore formation.     

Regulation of Extracellular Protease Production in Bacillus cereus T: Characterization of Mutants Producing Altered Amounts of Protease

Twenty-nine mutants of Bacillus cereus T were selected on casein agar for their inability to produce large amounts of extracellular protease. They all formed spores, and 27 were also auxotrophs for purines or pyrimidines. Upon reversion to prototrophy, a large fraction regained the capacity to produce protease. Conversely, reversion to normal protease production resulted in loss of the purine or pyrimidine requirement in a large fraction of the revertants. One spontaneous low-protease-producing pyrimidine auxotroph studied in detail grew as well as the wild type and produced spores which were identical to those produced by the wild type on the basis of heat resistance, dipicolinic acid content, density, and appearance in the electron microscope. The rate of protein turnover in the mutant was the same as the wild type. The mutant did grow poorly, however, when casein was the principal carbon source. A mutant excreting 5 to 10 times as much protease as the wild type was isolated as a secondary mutation from the hypoproducer discussed above. Loss of the pyrimidine requirement in this case did not alter the regulation of protease production. Although the secondary mutant grew somewhat faster in most media than the wild type, the final cell yield was lower. The spores of this mutant appeared to have excess coat on the basis of both electron microscopic and chemical studies. There appear to be closely related but distinct catabolic controls for both extracellular protease and spore formation. These controls can be dissociated as for the hypoproducers but can also appear integrated as for the hyperprotease producer.     

Lipid Composition of Bacillus cereus During Growth and Sporulation

The lipid composition of Bacillus cereus during growth and sporulation was examined. The total lipid extract accounted for 2 to 3% of the dry weight of the cells and consisted of neutral lipids (30 to 40%) and phospholipids (60 to 70%). Phospholipids were separated by thin-layer chromatography into eight components; phosphatidyl ethanolamine, phosphatidyl glycerol, and diphosphatidyl glycerol were the major phospholipids and accounted for over 90% of the total. Also identified was a diglycosyl diglyceride and an alanine ester of phosphatidyl glycerol. Diphosphatidyl glycerol was more difficult to extract than the other components in vegetative and stationary-phase cells, but became increasingly easy to extract during spore maturation, and during sporulation cellular levels increased. Phosphatidyl glycerol had a high turnover rate; it accounted for about 70% of the phospholipid synthesis throughout sporulation but only represented between 30 and 40% of the total phospholipid at any time. Phosphatidyl ethanolamine, on the other hand, accounted for about 20% of the synthesis but was the major phospholipid (50 to 60% of the total).     

Sporulation of Tricarboxylic Acid Cycle Mutants of Bacillus subtilis

A mutant of Bacillus subtilis 168 lacking aconitase (EC 4.2.1.3) was found to be blocked at stage 0 or I of sporulation. Although adenosine triphosphate levels, which normally decrease in tricarboxylic acid cycle mutants at the completion of exponential growth, could be maintained at higher levels by feeding metabolizable carbon sources, this did not permit the cells to progress further into the sporulation sequence. When post-exponential-phase cells of mutants blocked in the first half of the tricarboxylic acid cycle were resuspended with an energy source in culture fluid from post-exponential-phase wild-type B. subtilis or Escherichia coli, good sporulation occurred. The spores produced retained the mutant genotype and were heat stable but lost refractility and heat stability several hours after their production.     

Cold Lability of Bacillus cereus Bacteriophage CP-51

Phage CP-51 was rapidly inactivated when stored at the usual refrigerator temperatures (2 to 4 C) and even more rapidly when exposed to 0 C. The loss in viability resulting from exposure to cold appeared to correlate with the increase in number of phage particles having contracted tails. High concentrations (0.01 M) of Mg²⁺, Ca²⁺, or Mn²⁺ stabilized the phage considerably, but even in the presence of these divalent cations, it was much less stable at 0 C than at 15 C.     

The DNA-Delay Mutants of Bacteriophage T4

Mutants of phage T4 defective in genes 39, 52, 58-61, and 60 (the DNA delay or DD genes) are characterized by a delay in phage DNA synthesis during infection of a nonpermissive Escherichia coli host. Amber (am) mutants defective in these genes yield burst sizes varying from 30 to 110 at 37 C in E. coli lacking an am suppressor. It was found that when DD am mutants are grown on a non-permissive host at 25 C, rather than at 37 C, phage yield is reduced on the average 61-fold. At 25 C incorporation of labeled thymidine into phage DNA is also reduced to 3 to 10% of wild-type levels. Mutants defective in the DD genes were found to promote increased recombination as well as increased base substitution and addition-deletion mutation. These observations indicate that the products of the DD genes are necessary for normal DNA synthesis. The multiplication of the DD am mutants on an Su⁻ host at 37 C is about 50-fold inhibited if prior to infection the host cells were grown at 25 C. This suggests that a compensating host function allows multiplication of DD am mutants at 37 C in the Su⁻ host, and that this function is active in cells grown at 37 C prior to infection, but is inactive when the prior growth is at 25 C. Further results are described which suggest that the products of genes 52, 60, and 39 as well as a host product interact with each other.     

Genetic Analysis of Pleiotropic Negative Sporulation Mutants in Bacillus subtilis

Genetic studies were undertaken on 14 pleiotropic negative sporulation mutants. These mutants (spoA) which are blocked early in the sporulation process were found to map near the terminus of the Bacillus subtilis chromosome in a region enriched in genes involved in spore formation. Two- and three-factor crosses by transduction and transformation led to the conclusion that the pleiotropic spoA mutations formed a linked cluster. The genetic distance across the cluster calculated from transformation data was compatible with the mutant sites defining a single gene. Suppressor studies revealed that either a nonsense or missense mutation in the spoA locus generated a pleiotropic negative phenotype. It was concluded that the locus codes for a protein, and the absence of this protein is responsible for the pleiotropic phenotype.     

Biochemical Studies of Bacterial Sporulation and Germination XXI. Temperature-Sensitive Mutants for Initiation of Germination

Mutants of Bacillus megaterium have been isolated that are temperature-sensitive for the initiation of germination.