THE INFLUENCE OF ACIDITY ON THE MULTIPLICATION OF LACTIC STREPTOCOCCAL BACTERIOPHAGE IN LIQUID MEDIA

SUMMARY: Mass lysis of lactic streptococci infected with baeteriophage at 30° was prevented at pH 5·10. At lower pH values no multiplication of phage followed infection, and prolonged incubation at 30° resulted in loss of phage particles from unlysed samples. Adsorption of phage particles on host cells was unaffected by acidity, but no phage penetration of host cells took place. Host cell properties were apparently unchanged by adsorption of phage particles in acid whey.     

Transfection of Escherichia coli and Salmonella typhimurium Spheroplasts: Host-Controlled Restriction of Infective Bacteriophage P22 Deoxyribonucleic Acid

Under proper conditions, one infective center was obtained for 3 x 10(8) molecules of P22 phage deoxyribonucleic acid (DNA) when lysozyme-ethylenediaminetetraacetic acid spheroplasts of Escherichia coli were transfected in the presence of 25 mug of protamine sulfate per ml. A 3- to 50-fold B-specific and K-specific E. coli restriction of the incoming P22 DNA was observed. When P22 DNA-infected E. coli spheroplasts were plated with infertile r(LT) (+)m(LT) (+)Salmonella typhimurium indicator, an additional 70-fold restriction was observed. In the presence of protamine sulfate, penicillin spheroplasts of S. typhimurium SB1330 could be transfected b P22 DNA with efficiencies sometimes approaching those obtained with the E. coli spheroplasts; thus, facilitation of transfection by protamine sulfate is not limited to E. coli or to lysozyme-ethylenediaminetetraacetic acid spheroplasts. The application of these results to studies of transfection among other genuses and to studies of in vitro host-controlled restriction and modification for the two loci in S. typhimurium and the one locus in E. coli is discussed.     

Frequency of infection and efficiency of transfection of Rhizobium meliloti cells and spheroplasts.

Competent cultures of Rhizobium meliloti cells and spheroplasts obtained by various methods were infected with DNA of phage 1A. The Frequency of infection among the cells and spheroplasts was 2 X 10(-8)-5 X 10(-10). The efficiency of transfection calculated from the ratio of plaque forming units to infective DNA molecule of phage 1A was 5 X 10(-8) to 10(-10). Frequency of infection and efficiency of transfection among the competent cells were by one order of magnitude higher in the case of the spheroplasts. The use of various media did not noticeably alter the efficiency of transfection.     

Absence of Immunity to Superinfection in Spheroplasts of a Lysogen

Penicillin-induced spheroplasts of Salmonella typhimurium strain LT2 lysogenic for bacteriophage P22, appear to lose immunity to superinfection by homologous phage.     

Intercellular Transfer by Phage Receptor Site Lipopolysaccharide

TREATMENT of Escherichia coli cells with lysozyme and EDTA partially removes the outer layer of the cell wall containing lipopolysaccharide (LPS), leaving osmotically unstable spheroplasts¹. These can be infected with phage nucleic acid² and can produce viable phage particles. Removal of LPS-containing phage receptor sites^3–5, however, leaves spheroplasts resistant to infection by intact phages¹. We now show that LPS, obtained from phage-sensitive cells by aqueous phenol extraction, can provide functional phage receptor sites to spheroplasts prepared from cells lacking receptor sites.     

Osmotic Properties of Spheroplasts from Saccharomyces cerevisiae Grown at Different Temperatures

Spheroplasts were prepared from cells of Saccharomyces cerevisiae NCYC 366, grown at 30 or 15 C, by incubating cells with snail-gut juice after pretreatment with 2-mercaptoethanol. Walls of cells grown batchwise or in continuous culture at 15 C were more resistant to digestion with snail juice than walls on cells grown under the same conditions as 30 C. Spheroplasts lysed when suspended in hypotonic solutions of mannitol. The resistance of spheroplasts to osmotic lysis tended to increase when the test temperature was lowered below 30 C. The increased resistance was greater with spheroplasts from cells grown at 15 C. Cations, especially Ca²⁺, protected spheroplasts against osmotic lysis. In general, the protective effects, measured at 30 C, were smaller with spheroplasts from cells grown at 15 C compared with 30 C. Citrate and ethylenediaminetetraacetate (EDTA) decreased the resistance of spheroplasts to osmotic lysis. On the whole, the decrease was greater with spheroplasts from cells grown at 30 C rather than 15 C. In the presence of EDTA, spheroplasts from cells grown at 30 C were less resistant to osmotic lysis at 5 C than at 30 C; when spheroplasts from cells grown at 15 C were similarly examined, they were more resistant to lysis at 5 C than at 30 C. Spheroplast membranes from cells grown at 15 C had slightly but significantly greater contents of Mg²⁺, Ca²⁺, K⁺, and Na⁺ compared with spheroplast membranes from cells grown at 15 C. Mg²⁺ and Ca²⁺ were more easily extracted with EDTA from membranes of 30 C-grown cells than from 15 C-grown cells.     

Reversion of Q beta RNA phage mutants by homologous RNA recombination.

Q beta phage RNAs with inactivating insertion (8-base) or deletion (17-base) mutations within their replicase genes were prepared from modified Q beta cDNAs and transfected into Escherichia coli spheroplasts containing Q beta replicase provided in trans by a resident plasmid. Replicase-defective (Rep-) Q beta phage produced by these spheroplasts were detected as normal-sized plaques on lawns of cells containing plasmid-derived Q beta replicase, but were unable to form plaques on cells lacking this plasmid. When individual Rep- phage were isolated and grown to high titer in cells containing plasmid-derived Q beta replicase, revertant (Rep+) Q beta phage were obtained at a frequency of ca. 10(-8). To investigate the mechanism of this reversion, a point mutation was placed into the plasmid-derived Q beta replicase gene by site-directed mutagenesis. Q beta mutants amplified on cells containing the resultant plasmid also yielded Rep+ revertants. Genomic RNA was isolated from several of the latter phage revertants and sequenced. Results showed that the original mutation (insertion or deletion) was no longer present in the phage revertants but that the marker mutation placed into the plasmid was now present in the genomic RNAs, indicating that recombination was one mechanism involved in the reversion of the Q beta mutants. Further experiments demonstrated that the 3' noncoding region of the plasmid-derived replicase gene was necessary for the reversion-recombination of the deletion mutant, whereas this region was not required for reversion or recombination of the insertion mutant. Results are discussed in terms of a template-switching model of RNA recombination involving Q beta replicase, the mutant phage genome, and plasmid-derived replicase mRNA.     

Transfection of Escherichia coli Spheroplasts IV. Transfection of rec+ and rec? Spheroplasts by Native,Denatured, and Renatured T5 Bacteriophage DNA After Repair of Single-Strand Breaks by Polynucleotide Ligase

Transfection of Escherichia coli spheroplasts by native T5 phage DNA was not affected by treatment with polynucleotide ligase. Denatured T5 phage DNA infectivity, only 0.1% of the native DNA level, was increased slightly by polynucleotide ligase treatment. Renatured T5 phage DNA infectivity was also increased slightly by polynucleotide ligase treatment. To form an infective center with rec(+) spheroplasts, 1.6 to 2.1 native T5 phage DNA molecules were required; however, 1.4 T5 phage DNA molecules were required to form an infective center with recA(-)B(-) spheroplasts, and one molecule was sometimes sufficient for rec B(-) spheroplasts. Polynucleotide ligase treatment of T5 phage DNA had no effect on these parameters. Thus, the single-strand interruptions of T5 phage DNA are probably not essential to the survival of the parental T5 phage DNA, and T5 phage DNA, especially the denatured form, is highly sensitive to some nucleases in E. coli spheroplasts.     

UV-sensitivity and UV-mutability of infectious lambdaDNA: reactivation, protection and mutability in various assay systems

Summary The UV-sensitivity of phage and its infectious DNA have been compared in experiments involving infection of normal cells by phage and transfection of lysozyme-EDTA spheroplasts or Ca⁺⁺-treated cells by phage DNA. It is shown that UV-irradiated DNA undergoes extensive HCR. Since intact phage and free phage DNA have the same survival after UV-irradiation in Hcr^- spheroplasts and cells, resp., and since survival is also identical in Ca⁺⁺-treated Hcr⁺ cells it is concluded that DNA in solution or packaged in the phage head provides the same target for the induction of lethal UV lesions. This conclusion is supported by the observation that cysteamine provides a similar radioprotection to the intact phage and its free DNA. Spheroplasts of Hcr⁺ cells, however, have an HCR capacity reduced by about 20% when compared with normal or Ca⁺⁺-treated cells. Moreover, UV-reactivation of irradiated DNA, which is absent in spheroplasts, occurs efficiently in Ca⁺⁺-treated cells. Possible reasons for the physiological difference between spheroplasts and normal cells are discussed. c-mutations, which are readily induced by UV in phage assayed with E. coli mul ^-, could not be induced in DNA when assayed with spheroplasts or Ca⁺⁺-treated cells of this strain. No mutants were also found with DNA extracted from UV-irradiated phage. The significance of the mode of entry of UV-irradiated DNA into a cell for the production of mutations is discussed.     

Antigenic structure of Brucella suis spheroplasts

Baughn, Robert E. (University of Tennessee, Memphis), and Bob A. Freeman. Antigenic structure of Brucella suis spheroplasts. J. Bacteriol. 92:1298-1303. 1966.-Immunoelectrophoresis was used to differentiate between the antigenic mosaics of normal cells of Brucella suis and of spheroplasts prepared by treatment with penicillin, glycine, and a combination of these agents. Smooth cells possessed at least 13 antigens, 10 of which were precipitated with homologous antiserum. Three additional antigens were visualized by reaction with spheroplast antisera. Spheroplasts induced with glycine were the least complex, with only six antigens. Penicillin-glycine spheroplasts were similar, but possessed one additional antigen. Penicillin spheroplasts were the most complex, with eight antigens. Although there appeared to be quantitative differences between the antigens of spheroplasts and normal cells, no completely new antigens were detected in spheroplasts. Serum absorption studies indicated that four antigens were associated with the surface of normal B. suis, none of which occurred in spheroplasts.     

Effect of Polymyxin on the Bacteriophage Receptors of the Cell Walls of Gram-Negative Bacteria

Treatment of gram-negative bacteria with lethal doses of polymyxin B and colistin resulted in the formation of projections of the outer layer of the cell wall. Phages T3, T4, and T7, which use wall lipopolysaccharide as receptors, were specifically prevented from adsorbing to Escherichia coli B cells treated with polymyxin, whereas phages T1, T2, T5, and T6 were not. In the systems of phage P22C-Salmonella typhimurium LT2 and phage C21-S. typhimurium variant SL1069, the phage were prevented from adsorbing to the host cell treated with the antibiotics. Electron microscopic observations show that phage T2 adsorbed irreversibly to the normal smooth surface between the projections on the outer layer caused by the drug treatment. These results indicate that lipopolysaccharide is affected by polymyxin functionally and morphologically, but lipoprotein is not. The purified lipopolysaccharide showed a ribbon-like structure when viewed face on and showed trilamellar structure when viewed edge on. The lipopolysaccharide from E. coli B was irreversibly adsorbed by phages T3, T4, and T7, but not phage T2. Often, phage T4 adsorbed to both sides of the lipopolysaccharide strand at comparable distances. Phage P22C adsorbed through the spikes of the tail-plates to the lipopolysaccharide from S. typhimurium LT2. Lipopolysaccharide which was treated with low doses of the drug (2.5 to 6.25 mug of polymyxin B per ml to 100 mug of lipopolysaccharide per ml) turned into the coiled form and was partially broken down into short segments with coiled form. The loosely coiled lipopolysaccharide retains both its function as the receptor and its trilamellar structure. Treatment with high doses of the drug (12.5 to 25 mug of polymyxin B per ml to 100 mug of lipopolysaccharide per ml) caused the collapse of the trilamellar structure of the strand. These collapsed lipopolysaccharides became flat and fused with each other, making an amorphous mass, and finally they were broken into small collapsed fragments.     

Bacteriophage conversion of heat-labile enterotoxin in Escherichia coli.

A temperate phage designated obeta1 (omicron beta) was mitomycin C induced and isolated from heat-labile enterotoxin (LT)-producing Escherichia coli E2631-C2. Phage obeta1 infected the nonlysogenic, nontoxigenic, mitomycin C-sensitive strain of E. coli K-12 (CSH38) and converted it to lysogeny and enterotoxigenicity. After the establishment of lysogeny, E. coli CSH38(obeta1) produced produced LT and phage particles at maximal levels following mitomycin C induction. The LT Tox+ character is carried by the temperate phage obeta1.     

In vitro reconstitution of intercompartmental protein transport to the yeast vacuole

Toward a detailed understanding of protein sorting in the late secretory pathway, we have reconstituted intercompartmental transfer and proteolytic maturation of a yeast vacuolar protease, carboxypeptidase Y (CPY). This in vitro reconstitution uses permeabilized yeast spheroplasts that are first radiolabeled in vivo under conditions that kinetically trap ER and Golgi apparatus-modified precursor forms of CPY (p1 and p2, respectively). After incubation at 25 degrees C, up to 45% of the p2CPY that is retained in the perforated cells can be proteolytically converted to mature CPY (mCPY). This maturation is specific for p2CPY, requires exogenously added ATP, an ATP regeneration system, and is stimulated by cytosolic protein extracts. The p2CPY processing shows a 5-min lag period and is then linear for 15-60 min, with a sharp temperature optimum of 25-30 degrees C. After hypotonic extraction, the compartments that contain p2 and mCPY show different osmotic stability characteristics as p2 and mCPY can be separated with centrifugation into a pellet and supernatant, respectively. Like CPY maturation in vivo, the observed in vitro reaction is dependent on the PEP4 gene product, proteinase A, which is the principle processing enzyme. After incubation with ATP and cytosol, mCPY was recovered in a vacuole-enriched fraction from perforated spheroplasts using Ficoll step-gradient centrifugation. The p2CPY precursor was not recovered in this fraction indicating that intercompartmental transport to the vacuole takes place. In addition, intracompartmental processing of p2CPY with autoactivated, prevacuolar zymogen pools of proteinase A cannot account for this reconstitution. Stimulation of in vitro processing with energy and cytosol took place efficiently when the expression of PEP4, under control of the GAL1 promoter, was induced then completely repressed before radiolabeling spheroplasts. Finally, reconstitution of p2CPY maturation was not possible with vps mutant perforated cells suggesting that VPS gene product function is necessary for intercompartmental transport to the vacuole in vitro.     

Kinetics of synthesis, processing, and membrane transport of heat-labile enterotoxin, a periplasmic protein in Escherichia coli

We report the detection in vivo of precursors to the A and the B subunits of the heat-labile enterotoxin (LT) in Escherichia coli. Both pre-LT A (Mr = 29,500) and pre-LT B (Mr = 13,500) are present in the spheroplast fraction of the bacteria after separation of the cells in spheroplasts and periplasm. Two smaller LT A related polypeptides (17 and 23 kDa) were also detected in the spheroplast fraction. Both were degraded with a half-time of about 40 s. Mature subunits (Mr = 27,500 for LT A, and 11,500 for LT B) are released from the spheroplasts soon after processing and occur freely in the periplasm not associated with the cytoplasmic or the outer membranes. Processing occurs mainly post-translationally for both the A and the B subunits. However, they show different kinetics of processing and subsequent segregation into the periplasm. Whereas pre-LT B is processed and released within seconds after chain termination, pre-LT A is processed and released more slowly, and a subfraction of mature LT A may reside in the cytoplasmic membrane for several minutes.     

Defective Bacteriophage PBSH in Bacillus subtilis: II. Intracellular Development of the Induced Prophage

Treatment of Bacillus subtilis strain 168 with mitomycin C caused induction of a defective prophage, PBSH. During induction, extensive deoxyribonucleic acid (DNA) synthesis took place. Concurrently, a change in marker frequency of the bacterial DNA was noticed. The frequency of only one marker, ade-16, the marker closest to the origin of the bacterial chromosome, was enhanced manyfold. DNA from whole phage particles transformed all bacterial markers at a frequency equal to that of DNA in the noninduced culture, except ade-16, the frequency of which was enhanced 30 to 100 times. Analysis of a double isotope experiment demonstrated that 14% of the phage DNA was derived from preinduction bacterial DNA. The other 86% of DNA in phage particles was DNA replicated after induction. Density label experiments with 5-bromodeoxyuridine showed that postinduction DNA synthesis took place preferentially at the origin region of the bacterial chromosome. Measurement of the molecular weight of DNA replicated after induction clearly showed that postinduction DNA replication is chromosomal. No evidence for prophage detachment and autonomous phage DNA replication was found. The data indicated that, after mitomycin C action, the bacterial chromosome under-went multiple reinitiation at the origin, while normal sequential DNA replication was stopped. The pool of replicated bacterial DNA was fragmented randomly. This DNA was packaged into PBSH particles which were released after cell lysis.     

Purification of Streptococcus group C bacteriophage lysin.

A simple procedure for the purification of Streptococcus group C phage lysin to apparent homogeneity is described. The electrophoretically pure, enzymatically stable polypeptide of 98,000 molecular weight converted Streptococcus (groups A, F, and H) cells into spheroplasts within 5 min at 0 degrees C or within less than a minute at 37 degrees C.     

Penicillin-Binding Component of Bacillus cereus

(14)C-penicillin is irreversibly bound by Bacillus cereus 569. Incubation of penicillin-treated cells in a cell wall digestion medium results in solubilization of approximately 60% of the irreversibly bound lable. The extent of the solubilization is the same when cells are prepared by either a cold or 37 C treatment procedure. However, spheroplasts prepared by the cold treatment are leaky. When the resulting spheroplasts are incubated in supplemented medium, reduced rates and levels of penicillinase synthesis, relative to induced whole-cell controls, are observed. Spheroplasts from both cold and 37 C prepared cells exhibit this phenomena, although the spheroplasts from 37 C prepared cells synthesized approximately sixfold higher levels of penicillinase. The size distribution of the label solubilized during the preparation of spheroplasts was examined by using Bio Gel P-150 columns. Although no label appeared in the exclusion volume fractions when the cell wall digest of the 37 C treated cells was chromatographed, approximately 10% of the label from cold-treated cells did appear. These results suggest that the presence of irreversibly bound penicillin is required for the synthesis of induced levels of penicillinase and that the irreversibly bound penicillin can be solubilized as a labile complex with material which is excluded from BioGel P-150. It may be concluded that the penicillin-binding lipoprotein complex which has been previously observed is the penicillin-specific binding site. However, the location of this complex in relation to the cell membrane could not be determined.     

Evidence for the Direct Action of Colicin K on Aerobic 32Pi Uptake in Escherichia coli In Vivo and In Vitro

Pentachlorophenol (PCP)-sensitive incorporation of (32)P-labeled orthophosphate ((32)P(i)) into nucleotides and nucleic acids by disrupted spheroplasts of Escherichia coli was inhibited by addition of colicin K. Incorporation by intact cells was also inhibited by a similar concentration of colicin K. Various colicin K-resistant mutants were isolated, and their ability to incorporate (32)P(i) was tested. When T6(r)-colK(r) mutants (T6 phage-resistant) and tol I mutants (T6-sensitive, colicin E-sensitive) were converted to disrupted spheroplasts, their (32)P(i)-incorporation became sensitive to colicin K. On the contrary, incorporation by disrupted spheroplasts from tol II mutants (T6-sensitive, colicin E-resistant) was fairly resistant to colicin K like that of intact cells. A modification of the cell surface of T6(r)-colK(r) mutants, caused by mutation to novobiocin-permeable, T4 phage-resistant cells, restored the sensitivity of the cells to colicin K. The modified T6(r)-colK(r) cells did not adsorb T6 phage or colicin K, indicating that the receptors for T6 phage or colicin K are not reactivated by this modification. Similar treatment of tol I mutants did not have this effect. These observations strongly suggest that colicin K can act on its target on the cell membrane if it can penetrate the cell surface to reach this target. The receptor for colicin K on the cell surface, which may be part of the T6 phage-receptor, may have some unknown function in relation to the action of colicin K in normal cells, but tends to become dispensable if the cells become permeable to colicin K.     

Study of the stimulating action of sturin, its fractions, and the internal protein of T2 phage in transfection of E. coli spheroplasts by Lambda phage DNA

The stimulating action of sturin and its fractions in the transfection of E. coli spheroplasts with DNA of phage lambda has been shown, and the optimum conditions for the infection of sturin-treated spheroplasts with phage DNa have been established. The biological effect was most pronounced in arginine-enriched low-molecular sturin fraction B. The treatment of spheroplasts with the internal protein of phage T2 did not stimulate transfection. These findings suggest that the specificity of the stimulating action of protamine depends on the peculiarities of its amino acid composition, viz. a high content of arginine which is present in protein molecules in the form of blocks made up of 2-6 amino acid residues.     

Further studies on the biosynthesis of gramicidin S and proteins in a cell-free system from Bacillus brevis

1. An improved 11000g cell-free system for the incorporation of [(14)C]valine into gramicidin S has been obtained. The cell-free extract used was the supernatant obtained by treating Bacillus brevis with ultrasonics for 1min. followed by centrifugation at 11000g. The optimum pH for the incorporation was 8.2-8.4 and the optimum Mg(2+) concentration 0.05m. The presence of ammonium sulphate (0.1m) and K(+) (0.01m) increased the incorporation. 2. Cell-free extracts prepared from cells harvested in the early phase of growth (extinction value 0.1) incorporated negligible amounts of [(14)C]valine into gramicidin S compared with that incorporated by cell-free extracts prepared from cells harvested in the late phase of growth (extinction value 0.5). This was not due to the presence of inhibitors in the cell-free extracts prepared from cells harvested early, since there was no marked decrease in gramicidin S synthesis in a mixture of extracts prepared from cells harvested early and late in the growth phase. 3. The small incorporation of [(14)C]valine into protein, which took place in cell-free extracts from cells harvested in the late growth phase, was not inhibited by puromycin, chloramphenicol and ribonuclease. However, the substantial incorporation that took place in cell-free extracts prepared from cells harvested in the early phase of growth was completely inhibited by puromycin, chloramphenicol and ribonuclease. On mixing cell-free extracts prepared from cells harvested early and late in the growth phase, it appeared that the small incorporation that occurs in extracts from cells harvested in the late phase of growth was not due to cellular inhibitors.