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Mutations in prophage phi11 that impair the transducibility of their Staphylococcus aureus lysogens for methicillin resistance.

Methicillin resistance (mec) is not transduced into Staphylococcus aureus 8325-4, but is transduced into this host after it has been lysogenized with phage phi11 and has acquired the penicillinase plasmid pI524 by a separate transduction (Cohen and Sweeney, 1970, 1973). Strain 8325-4 is competent for transformation of typical plasmid or chromosomal markers and for mec only if it is lysogenic for phi11 or a related prophage (Sjöström et al., 1974, 1975). A mutant strain of phi11 that was temperature sensitive (Ts) for vegetative multiplication did not mediate competence for transformation of its 8325-4 lysogen if the lysogen had been grown at a nonpermissive temperature (Sjöström and Philipson, 1974). We isolated four Ts mutants of phi11 that did not mediate transducibility of their 8325-4(pI524) lysogens for mec after growth at nonpermissive temperatures (40 to 42 degrees C). Transduction of typical plasmid or chromosomal markers was not affected. These phi11-Ts mutants mediated normal competence of their lysogens for transformation of a tetracycline resistance plasmid. Similarly, phi11-Ts mutants that rendered their lysogens temperature sensitive for transformation did not depress the frequency of transduction of mec. These two types of phi11-Ts mutants fell into two different genetic complementation groups that differed in the physiology of deoxyribonucleic acid synthesis and in the time of expression of the mutations during a single-burst growth cycle at a nonpermissive temperature. A virulent mutant of phi11, which plaqued with 100% efficiency on 8325(phi11), also failed to condition strain 8325-4 for transducibility of mec but retained the ability to confer competence for transformation of a tetracycline resistance plasmid. Different genetic loci and physiological functions are involved in phi11 mutations that affect transducibility of mec and those that affect competence for transformation of markers generally in S. aureus 8325-4.

DNA, Viral

Analysis of the phase variation in lambda reduced immunity lysogens.

Two distinct phases characterized by different levels of immunity that appear in some E. coli strains lysogenic for reduced immunity mutants of bacteriophage lambda are identified as single and tandem double lysogens respectively on the basis of DNA-DNA hybridization experiments and the requirement of the phage xis function for the transition from a single to a double, and of the host recA function for the transition from a double to a single lysogen (in a xis- condition). Rim lysogens with a further increase in immunity, containing some 5 copies of the lambda genome per host genome, have also been observed. It is argued that the different levels of immunity are a direct reflection of the CI gene dosage effect. An unexplained finding is that rim single lysogens yield double lysogens with a frequency of near 1% per generation, whereas cured cells fail to appear even at a frequency 100 times lower.

Cell Division

Lysogenic conversion for multiple characters in a strain of Staphylococcus aureus.

Lysogenization of nonlysogenic strains of Staphylococcus aureus was performed with two different bacteriophages, LS1 and LS2, that were unable to plaque on any of the strains of S. aureus tested. Infection of recipient strains was achieved when protoplasts were inoculated with LS1 or LS2 or when bacterial cultures were simultaneously inoculated with a virulent phage together with LS1 or LS2. Lysogenization was demonstrated by changes in phenotypic characters of the host strain and by liberation of bacteriophages from the modified strains as shown by electron microscopic examination. The lysogenic strains differed from the host strains by the following characters: they were coagulase, deoxyribonuclease, and lipase negative; they were untypable by the basic set of phages; they did not ferment mannitol under anaerobic conditions; and they produced only l-(+)-lactic acid by glucose fermentation. Their cell walls contained less glycine and concomitantly more serine than those of the host strains. Furthermore, they were devoid of protein A. Conversely, some antigenic factors as well as the presence of ribitol in the cell wall teichoic acid, indicated a parental relationship between the host strains and the derived lysogenic ones. Phages LS1 and LS2 could be excluded from the lysogenic strains by invading phages, and the revertant nonlysogenic strains recovered all of the characteristics of the initial host strains. It was thus concluded that the phenomenon described was due to lysogenic conversion. The origin of phages LS1 and LS2 is discussed.

Cell Wall

Increased reproductive fitness of Escherichia coli lambda lysogens.

Lambda lysogens of Escherichia coli reproduce more rapidly than nonlysogens during aerobic growth in glucose-limited chemostats. If the environment is changed to anaerobic growth, the situation is reversed, and the lysogen reproduces more slowly than the nonlysogen. Based on a tetrazolium dye assay, the increased fitness of the lambda lysogen during aerobic growth seems to result from a continued high metabolic rate as glucose becomes limiting, whereas the metabolic rate of the nonlysogen declines. The lambda rex gene is required for the growth advantage of lysogens since lack of rex function causes lambda lysogens to lose their reproductive advantage over nonlysogens.

Aerobiosis

Reproductive fitness of P1, P2, and Mu lysogens of Escherichia coli.

P1, P2, and Mu lysogens of Escherichia coli reproduce more rapidly than nonlysogens during aerobic growth in glucose-limited chemostats. Thus, prophage-containing stains of E. coli are reproductively more fit than the corresponding nonlysogens. If mixed populations are grown by serial dilution under conditions in which growth is not limited, both the lysogen and nonlysogen manifest identical growth rates. The increased fitness of the lysogens in glucose-limited chemostats correlates with a higher metabolic activity of the lysogen as compared with the nonlysogen during glucose exhaustion. We propose that P1, P2, Mu, and lambda prophage all confer an evolutionarily significant reproductive growth advantage to E. coli lysogenic strains.

Aerobiosis

X-ray sensitivity of Escherichia coli lysogenic for bacteriophage P2.

Strains of Escherichia coli C or K lysogenic for the non-inducible phage P2 show a lower survival following X-ray irradiation as compared to nonlysogenic strains. This difference in X-ray sensitivity is not accompanied by a significant difference in X-ray induced mutability. The capacity of X-irradiated P2 lysogens to multiply any of a number of unirradiated infecting phages is severely impaired. These effects of X-ray treatment can be most simply explained as a consequence of the fact that protein and RNA syntheses are strongly inhibited in P2 lysogens after X-irradiation. All the above events specifically occurring in X-rayed P2 lysogens are dependent on the P2 gene old.

Bacterial Proteins

Conditions for induction of bacteriophage from lysogenic Bacillus megaterium with aflatoxin B1.

The present study was conducted to determine whether or not aflatoxin B1 was an effective inducing agent for lysogenic bacteria and to characterize some of the parameters involved in induction. A lysogenic strain of Bacillus megaterium (NRRL-B-3695) and an indicator strain of this species (NRRL-B-3694) were used. Cultures of the lysogenic strain were incubated for various periods of time in the presence of aflatoxin B1. Plaque-forming units as well as colony-forming units were then determined. Results of the present study indicated that bacteriophage lysogenizing B. megaterium could be induced with aflatoxin B1. The optimum concentration for induction was 25 micrograms of toxin per ml of early-log-phase culture. Evidence suggested that: (i) higher concentrations of aflatoxin B1 formed hydrophobic complexes which would not efficiently induce B. megaterium; (ii) the toxic effect of aflatoxin B1 severely limited the number of cells which could be induced prior to killing action of the toxin; and (iii) concentrations less than 25 micrograms of aflatoxin B1 per ml were not efficient inducers of bacteriophage production nor did they demonstrate the toxic effect observed at higher concentrations.

Aflatoxins

Lysogenic conversion in Klebsiella pneumoniae: system which requires active immunity regulation for expression of the conversion phenomenon.

We have previously described Klebsiella pneumoniae MirM7b, which, although stably lysogenic for the inducible and nondefective phages FR2 and AP3, is not immune to superinfection by these same viruses. MirA12b, a strain which is lysogenic for FR2 and AP3 and immune to superinfection, has been derived from MirM7b. The sensitivity of this strain and that of the nonimmune parent to several bacteriophages have been compared in this work. It has been found that, whereas MirM7b is sensitive to coliphages P1, T3, T7, and phiI, MirA12b is fully resistant to all of them. It is shown that phages FR2 and AP3 convert Klebsiella strains to resistance to coliphage P1 and coliphages T3, T7, and phiI, respectively, and cause loss of surface antigens in lysogenic cells. To determine such a conversion, both FR2 and AP3 require expression of immunity to superinfection. This explains the differences that exist between MirM7b and MirA12b in both phage sensitivity and surface antigens. Hypotheses are presented to explain the peculiar need for an active superinfection repressor to express lysogenic conversion.

Antigens, Bacterial

Induction of lambda prophage and of mutations to streptomycin resistance in separate small fractions of a lysogenic derivative of Escherichia coli B/r by very low doses of ultraviolet light.

The number of induced mutations to streptomycin resistance is compared at doses of ultraviolet (UV) light between 0.2 and 6.4 J/m2 in a Uvr- (excision-deficient) derivative of E. coli B/r, strain WU, and in its lambda lysogen, strain WU(lambda). At UV doses up to about 1 J/m2, which converts about 5% of the lysogenic population into enfective centers, no difference is observed in the number of mutations to streptomycin resistance produced by the two strains. It is concluded that the capacity to produce UV-induced mutations is not coupled with lysis due to the induction of lambda prophage at low doses of UV radiation. At UV doses above 1 J/m2, the number of mutatants detected in the lysogenic strain decreases appreciably compared to the number detected in the nonlysogen, and is only about 10% as high at UV doses of 3 J/m2 and higher, doses which cause maximal induction of prophage. The results are compatible with the operation of a common "all-or-none" induction signal resulting in expression of UV-inducible functions at high UV doses, but not at low doses.

Coliphages

Mechanism of defective lysogenization by phage P1 in a lon-mutant of Escherichia coli K-12.

Phage P1 cannot lysogenize a lon- mutant of Escherichia coli K-12, which is defective in the regulation of cellular division cycle to result in snake formation (14). P1 mutants, called P1pla, can lysogenize the lon- host. These mutations have been classified into two complementation groups: one is cis-dominant; the other is trans-dominant. A temperature-sensitive lon- mutant was isolated, which exhibited the lon- phenotype at 42 C but not at 33 C. A temperature-shift experiment of the P1-lysogenic derivative of the lon- ts mutant showed lysis of the culture and induction of the phage production. It is proposed that P1 plasmid may be under a certain regulatory circuit of the division cycle of the host bacterium by indirectly regulating the production of P1 immune repressor, or alternatively by directly derepressing the functions of P1 prophage.

Cell Division

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.

Anti-Bacterial Agents

Close association between shape alteration and loss of immunity to superinfection in a wild-type Klebsiella pneumoniae stable lysogen which can be both immune and nonimmune to superinfection.

Klebsiella pneumoniae MirM7 is a wild-type strain which grows as cocci at pH 7 and above and as rods at pH 6.5 and below. Cultures of this strain and an auxotrophic derivative, MirM7b, have been found to undergo spontaneous lysis after purification from possible contaminating viruses. Lysates always contained two phages, FR2 and AP3, most often at high titers. FR2 and AP3 plated with the same efficiency on both MirM7b and K59 (another K. pneumoniae strain sensitive to FR2 and AP3) and lysogenized 45 and 54% of the K59-infected cells, respectively. These findings raise the possibility that MirM7b is lysogenic for FR2 and AP3, although nonimmune to their superinfection. The fact that mitomycin C and N-methyl-N'-nitro-N-nitrosoguanidine can induce phages FR2 and AP3 from MirM7b confirmed this possibility. When MirM7b was infected with FR2 several strains immune to FR2 and AP3, which were all rod shaped, were obtained. Furthermore, 19 derivatives, rod shaped at all pH's have been isolated from MirM7b. They were all immune to both FR2 and AP3. From mating experiments between the MirM7b donor derivative, strain M720, and either K59 or MirCV5, a rod-shaped MirM7b derivative cured from the prophages, cysteine recombinants were obtained which were most often (80%) immune to FR2 and AP3. Nonimmune and still lysogenic recombinants were obtained by mating M720 with a rod-shaped immune MirM7b derivative; the majority of the non-immune strains maintained the rod shape. Five coccus-shaped recombinants were also isolated; they were nonimmune to superinfection. Several physiological properties of strain MirM7b and the other nonimmune coccal recombinants have been studied in comparison with those of the rod-shaped immune derivatives. All of the coccal strains have shown several alterations with respect to the rods. The role of possible derepressed prophage genes in the various physiological alterations of MirM7 is discussed, and the analogies between this system and those of vertebrate cells transformed by proviruses are stressed.

Bacteriophages

Characteristics of Staphylococcus aureus associated with lysogenic conversion to loss of beta-hemolysin production.

Staphylococcus aureus strains 7-8 and 57 that produce beta-hemolysin but not staphylokinase (beta + K-) were lysogenically converted by certain serological group F bacteriophages to the loss of beta-hemolysin production and the gain in staphylokinase production (beta-K+). Serological group A phage 42E was found to convert S. aureus strains 7-8(beta-K-) and 57 (beta + K-) to beta - K-. Conversion of beta-hemolysin by lysogenization of a serological group A phage has not previously been reported. Phage 42E conversions differed from the group F conversions since staphylokinase was not affected. This indicates that conversion to beta-K+ involves separate loci on the phage chromosome. Several characteristics associated with virulence of staphylococci of human or animal origin other than staju;plomase production (coagulase, DNase, lipase, gelatinase, mannitol fermentation, and phage-sensitivity patterns) were not correlated with lysogenic conversions to loss of beta-hemolysin.

Bacteriophage Typing

[Phage typing and lysogen typing of Staphylococcus aureus].

A comparison was made between the results of phage and lysogenic typing of S. aureus strains isolated during several outbreaks of staphylococcal infection and S. aureus cultures isolated from the same carriers at different periods. The study of the groups of strains having the same origin showed that the differences in the number of reactions were more pronounced in lysogenic typing than in phage typing. For this reason lysogenic typing can be recommended only for the identification of those strains which cannot be identified with the use of the phages of the International Basic Set. The results of the experiments with induced phages proliferating in a restriction-defective strain indicated that restriction and modification were mainly responsible for the specificity of lytic reactions.

Animals

Induced radioresistance in four strains of Escherichia coli, two with lambda lysogens.

Cells of E. coli that are recA+ and lex+ show a phenomenon of induced radioresistance. A preexposure to ultraviolet light, or ionizing radiation followed by incubation to allow protein synthesis, followed by treatment with rifampin to prevent further induction, renders the cells resistant to further doses of radiation. When this is attempted with lambda lysogens of the same strains, no radioresistance is seen, even though the preexposure is too small to induce lambda itself. If the lysogens are ind-, namely lambda C1857, about the normal radioresistance can be developed by pretreatment. These findings suggest that the lambda repressors can bind to single-strand breaks caused by the inducing agent and can modify the course of induction.

Coliphages

A mutant of Escherichia coli showing constitutive expression of the lysogenic induction and error-prone DNA repair pathways.

A mutant of E. coli (designated the STS mutant) has been isolated in which the phage induction and error-prone DNA repair pathways appear to be expressed constitutively without the cells having received an inducing signal. Phage lambda was not able to lysogenize this mutant, whereas a noninducible mutant of lambda, lambdacIind-, known to synthesize a repressor that is insensitive to the induction mechanism, lysogenized it normally. This result suggested that normal phage repressor was synthesized in the STS mutant but was then inactivated by the induction mechanism. The STS strain also had mutator characteristics, and showed spontaneous, error-prone repair of UV-damaged phage lambda. Derived from a lexA tif sfiA parent strain, the STS mutant carried an additional mutation spr at the lexA locus that resulted in a high level of expression of the induction pathways. The properties of this and related strains provide additional evidence that induction of phage and induction of error-prone DNA repair occur by a similar mechanism, and further suggest a model for the regulation of these pathways.

Coliphages

Prophage substitution and prophage loss from superinfected Escherichia coli recA(P1) lysogens.

It is shown that the plasmid prophage P1 can be displaced by a superinfecting P1 phage in Escherichia coli recA(P1) lysogens. Six widely separated phage markers were used to distinguish between residual recombination and total substitution. It is further shown that superinfection of recA lysogens can lead to loss of both phage (curing). These two phenomena, previously reported in Rec+ strains, are thus independent of host recombination and may result from perturbations of some function involved in plasmid maintenance.

Chloramphenicol

A colorimetric assay of lysogenic induction designed for screening potential carcinogenic and carcinostatic agents.

Simple, rapid colorimetric tests for lysogenic induction (the derepression of a latent bacterial virus) are described. A quantitative test and a more rapid semiquantitative test are based on the assay of the beta-galactosidase synthesized from lacZ gene fused to an operon under lambda repressor control. These biochemical "inductests" are suitable for screening programs designed to detect agents that damage DNA and that are of potential interest in carcinogenesis and cancer chemotherapy.

Bacteriophage lambda