Isolation and Characterization of a Composite Plasmid Rms201 Mutant Temperature Sensitive for Replication

A mutant temperature-sensitive for R-plasmid replication, Rms201ts14, was isolated from composite plasmid Rms201 after mutagenesis of P1 transducing lysate with 100 mM hydroxylamine for 40 h at 37°C. When Escherichia coli ML1410(Rms201ts14)⁺ was grown at temperatures between 40 and 42°C in L broth, antibiotic-sensitive cells were segregated. When the incubation temperature of ML1410(Rms201ts14)⁺ in L-broth was shifted to 42 from 30°C, the increase in the number of antibiotic-resistant cells ceased 90 min after the temperature shift. However, the total number of cells continuously increased, and only 3% of the cells retained the plasmid at 5 h after the temperature shift to 42°C. At 30°C the amounts of covalently closed circular deoxyribonucleic acid per chromosome of Rms201ts14 and Rms201 were 3.8 and 6.3%, respectively. Incorporation of radioactive thymidine into the covalently closed circular deoxyribonucleic acid of Rms201ts14 did not take place at 42°C, whereas radioactive thymidine was incorporated into the covalently closed circular deoxyribonucleic acid of Rms201 at a rate of 4%/chromosome even at 42°C. The synthesis of plasmid covalently closed circular deoxyribonucleic acid in a cell harboring Rms201ts14 was almost completely blocked at 42°C. These results indicated that the gene(s) responsible for plasmid deoxyribonucleic acid replication was affected in the mutant Rms201ts14. Temperature-sensitive miniplasmid pMSts214, which has a molecular weight of 5.3 × 10⁶ and encodes ampicillin resistance, was isolated from Rms201ts14. Similarly, miniplasmid pMS201, which encodes single ampicillin resistance, was isolated from its parent, Rms201, and its molecular weight was 4.7 × 10⁶. These results indicate that the gene(s) causing temperature sensitivity for replication of Rms201 resides on the miniplasmid.     

SOS induction by thermosensitive replication mutants of miniF plasmid

Summary MiniF, a 9.3 kb fragment of the dispensable F plasmid, carries genes necessary for its replication and partition as well as for the expression of an SOS signal. The arrest of replication of a thermo-sensitive miniFts at 42°C induced SOS functions such as prophage , sfiA expression, W-reactivation of UV-irradiated phage . Two miniF ts9 and ts17 mutations were located within the KpnI fragment (43.6–46.9) in the minimal oriS replicon. Blocking miniF replication by incBC ⁺ incompatibility genes situated in trans on a second plasmid also induced SOS functions. In contrast, if miniFts17 plasmid escaped the replication block at 42°C by being inserted into pR325, there was no SOS induction. SOS induction by the arrest of miniF replication required the miniF lynA ⁺ locus in cis, the host recA ⁺ and lexA ⁺ genes. We found that SOS induction was increased greatly near the stationary phase and that cell viability declined. During host cell exponential growth, miniFts9 and miniFts17 plasmids were lost rapidly, although SOS induction persisted for several cell generations. We postulate that lynA expresses a persistent product that may lead to the unwinding of chromosomal DNA.     

Isolation and characterization of an operator-constitutive mutation in the recA gene of E. coli K-12

Summary The recA gene of E. coli is regulated by a specific repressor, the lexA protein, which binds to an operator in the recA regulatory region. We describe in this paper the isolation and characterization of a mutant thought to carry an operator-constitutive mutation in the recA gene. This mutation has the following properties: 1) It partially supresses the UV sensitivity of lexA ^– strains. 20 It maps near the recA gene. 3) It allows constitutive high-level synthesis of recA protein in both lexA ^– and lexA ⁺ backgrounds. 4) It allows constitutive synthesis of the recA messenger RNA. 5) It is cis–acting. The mutation does not restore induced cellular mutagenesis in a lexA ^– background. The expression of induced repair and mutagenesis of UV irradiated phage lambda or the regulation of the lexA gene is not affected by the presence of the mutation in either a lexA ⁺ or lexA ^– strain. These observations confirm other findings that high levels of recA protein synthesis per se is not sufficient for the expression of UV inducible functions and that the lexA protein represses other genes besides the recA gene.Abbreviations UV ultraviolet - Kd kilodalton - PAGE polyacrylamide gel electrophoresis     

DNA-replication of spi - mutants of bacteriophage lambda in recombination-deficient bacteria

Summary Phage imm ²¹ c spi ^– infecting recA ^– cells gives a burst of 6 progeny phages compared to 120 in rec ⁺ cells. Parental spi ^– DNA is not degraded in recA ^– cells. The synthesis of early replication products is enhanced by a factor of 2 yielding 30 closed circular progeny DNA molecules per cell compared to 15 in the control. These DNA supercoils include 9% of dimer molecules under red ^– recA ^– and red ^– rec ⁺ conditions. On the other hand, the formation of linear phage DNA molecules in recA ^– cells is reduced by a factor of 5 to 6, if compared to spi ^– DNA in rec ⁺ and spi ⁺ DNA in recA ^– cells, respectively. The specific biological activity of these linear molecules in the helper phage assay system is unimpaired. Intermediates of late spi ^– replication under recA ^– conditions are supposed to be the unprotected targets of the action of the recB ⁺ recC ⁺ nuclease.     

Induction of recA+-protein synthesis in Escherichia coli.

Summary Escherichia coli was infected with precA ⁺to determine the genetic and physiological factors controlling recA ⁺gene expression. When precA ⁺replication was prevented by superinfection immunity, recA ⁺protein synthesis was induced by UV radiation. The recA ⁺gene is negatively controlled by the lexA ⁺gene product because i) a dominant lexA mutation, lexA3, prevented induction of recA ⁺protein synthesis ii) a recessive lexA mutation, tsl-1, caused induction of recA ⁺protein synthesis. Conversely positive control of recA ⁺gene expression requires recA ⁺protein because i) a co-dominant tif-1 mutation (a recA mutation) caused induction of recA ⁺protein synthesis ii) a recessive mutation, recA1, prevented cis-induction of recA protein synthesis. recA ⁺protein and Protein X of UV irradiated bacteria co-migrated and were subject to the same physiological and genetic controls. It is concluded that Protein X is recA ⁺protein. lysogenic induction was prevented by TPCK, a protease inhibitor. However TPCK did not prevent induction of recA ⁺protein synthesis, indicating that induction of the two processes occurs in different ways. It is suggested that the lexA ⁺and recA ⁺proteins normally combine to repress the recA ⁺gene. Derepression might occur after DNA damaging treatments because the amount of this complex would be reduced by recA ⁺protein i) binding to single-stranded DNA and/or ii) being activated to function proteolytically towards regulatory molecules such as repressor.     

P. mirabilis RecA protein catalyses cleavage of E. coli LexA protein and the lambda repressor in vitro

Summary The cloned recA ⁺ gene of Proteus mirabilis substitutes for a defective RecA protein in Escherichia coli recA ^– mutants, and restores recombination, repair and phage induction functions to near normal levels. In a previous report, we described the purification and charactrisation of the recombination activities of the P. mirabilis RecA protein (West et al. 1983b). In this paper, we show that the purified protein catalyses the cleavage of both the Escherichia coli LexA protein and the bacteriophage lambda repressor in vitro. These results provide a direct biochemical basis for the interspecies complementation observed in vivo and suggest that P. mirabilis has an SOS regulatory network similar to that of E. coli.     

Repressor and rex product of coliphage lambda: lack of collaboration and joint controls

Summary Exposure to 41° C for 10 to 100 minutes rapidly inactivates repressor bearing several ts mutations in the A or B region of gene cI, but does not result in simultaneous rapid loss of the rex function, which restricts phage T4 rII. One may conclude that the rex product does not directly collaborate with the repressor protein. Immediate loss of the rex activity at 47.5° C, observed with most of the cIts mutants and even cI⁺, appears to be unrelated to the repressor inactivation. In tof ⁺ lysogens carrying nonlethal cIts prophage mutants, the prolongation of induction at 41° C ultimately results in irreversible loss of the rex function, but only after about six cell generations. In similar experiments with tof deficient lysogens, loss of the rex activity requires about eleven cell generations and the rex function is regained in less than 30 minutes after return of the lysogen to 30° C. Two methods of rex assay, the more sensitive phage yield method and the infective center method, were employed.     

Prophage induction and cell division in E. coli. II. Linked (recA, zab) and unlinked (lex) suppressors of tif-1-mediated induction and filamentation

Summary The pleiotropy of tif-1, a mutation in E. coli K12 causing, among other effects, cellular filamentation at 42° and thermal induction of lysogenic derivatives, can be explained by the participation of the tif ⁺ gene product in more than one reaction pathway. Pathways that involve the tif ⁺ product may be analyzed by selection of secondary mutations that reverse both tif-1-mediated prophage induction and cell filamentation. Among revertants of a tif-1 lysogen among 20% are recombination deficient. These appear to carry a recA mutation. In addition to this class is a rarer (7%) phenotypically distinguishable class of revertants, called zab, first described here. Markers tif-1, recA and zab are closely linked. Mutations lex which are dominant and located near malB also appear (3%) among tif-1 revertants. The lex ⁺ function is needed for normal UV, X-ray and mitomycin C induction of prophage .The zab mutation resembles recA in causing (1) high sensitivity to UV, X-rays and mitomycin C, (2) drastic DNA degradation following UV irradiation but normal capacity to repair UV-damaged infecting phage (Hcr⁺), (3) failure to carry out UV reactivation and UV mutagenesis of UV-irradiated bacteriophage , (4) a markedly reduced level of spontaneous induction of . In contrast, other capacities, strikingly diminished by recA, are affected less, if at all by zab. Thus zab (1) permits 30–60% normal recombination proficiency, (2) shows real, although very low inducibility of by UV or mitomycin C, (3) permits 100% efficiency of plating of red gam, and (4) does not degrade DNA spontaneously.The hypothesis is proposed that the tif-1 mutation is a regulatory mutation controlling the activity, or more likely the synthesis of repair enzyme(s). The level of these repair enzyme(s), rather than DNA lesions, may govern the stability of the prophage repressor and the capacity of the bacteria to form septa.     

Expression of the plasmid pKM101-determined DNA repair system in recA? and lex? strains of Escherichia coli

Summary pKM101, a plasmid R factor of the N compatibility group increases methylmethane sulfonate mutagenesis and diminishes UV-killing in recA ⁺ lex ⁺ and recA ⁺ lex ^– strains, but not in recA ^– lex ⁺ strains. The induction of a reclex dependent colicin is not present in lex ^– strains carrying the pKM101 factor. These facts indicate that pKM101 acts through an error-prone DNA repair system, which is recA ⁺ dependent, but not lex ⁺ dependent.This paper is published on the occasion of Dr. C. Callerio's seventy-fifth birthday     

Lysogenic induction of lambdoid phages in lexA mutants of Escherichia coli

Summary UV irradiation of lexA3 mutants of E. coli caused lysogenic induction of prophage , _i21, _i434 and 80. Maximal induction in lexA3 lysogens needed less UV than in lexA ⁺ bacteria and gave 25–100% of the normal levels of infective centres induced. Assays of gene expression arising from derepression of a defective prophage showed at least 40% of the normal levels of induction by mitomycin C in lexA3 bacteria. The need for post-irradiation protein synthesis for lysogenic induction in lexA3 lysogens was reduced by increasing the basal level of recA protein with a recA ⁺ plasmid. It is concluded that in lexA E. coli some recA protein synthesis, too small to be detected by physical means, is needed for UV induced lysogenic induction.     

Constitutive expression of SOS functions and modulation of mutagenesis resulting from resolution of genetic instability at or near the recA locus of Escherichia coli

Summary Cellular activities normally inducible by DNA damage (SOS functions) are expressed, without DNA damage, in recA441 (formerly tif-1) mutants of Escherichia coli at 42° C but not at 30° C. We describe a strain (SC30) that expresses SOS functions (including mutator activity, prophage induction and copious synthesis of recA protein) constitutively at both temperatures. SC30 is one of four stable subclones (SC strains) derived from an unstable recombinant obtained in a conjugation between a recA441 K12 donor and a recA ⁺ B/r-derived recipient. SC30 does not owe its SOS-constitutive phenotype to a mutation in the lexA gene (which codes the repressor of recA and other DNA damage-inducible genes), since it is lexA ⁺. Each of the SC strains expresses SOS functions in a distinctively anomalous way. We show that the genetic basis for the differences in SOS expression among the SC strains is located at or very near the recA locus. We propose that resolution of genetic instability in this region, in the original recombinant, has altered the pattern of expression of SOS functions in the SC strains.     

Repressor cleavage as a prophage induction mechanism: hypersensitivity of a mutant lambda cI protein to recA-mediated proteolysis

λcIind^s prophage is hypersensitive to derepression by ultraviolet-irradiation. We have utilized this mutant to test current models for prophage derepression. We find that cIind^s repressor is cleaved by RecA protein in vivo at lower ultraviolet doses and with more rapid kinetics than cI⁺ repressor, and that induction of the recA8 gene or other LexA-repressed genes is not required for cleavage. Our results support the concept that RecA-directed proteolysis is the primary mechanism for prophage derepression.     

Induction of lambdoid prophages by amino acid deprivation: Differential inducibility; Role of recA

Summary Lambda prophage in auxotrophic lysogens can be induced by omission of one or combinations of the required amino acids from the culture medium. Such amino acid deprivation can result in nearly as effective induction of lambda as thymine deprivation. Prophage 424 is also induced equally effectively under both conditions although to a lesser extent than lambda. By contrast prophage 21 and i²¹ are differentially induced effectively by thymine deprivation and virtually not at all during amino acid deprivation. The same differential induction of 21 and equivalent induction of and 424 occur when all three prophages are present in the same lysogen. Increasing the levels of repressor with a cI carrying-plasmid prevented amino acidless induction of as did the ind ^– mutation. A recA, but not a recB, mutation in the host prevented induction by amino acid deprivation. A recC mutant host showed increased spontaneous induction of and 21 prophages. The findings reported are used as an argument that the recA protease probably is not itself acting as the inducing protease and that a likely source of the observed specificity is an effector molecule. Different effector molecules may be produced in response to different exigent situations, to which the phage repressors may have evolved sensitivity. i⁸⁰ was inducible both by amino acid and thymine deprivation.     

Red-mediated recombination of phage lambda in a recA? recB?host

Summary The lambda Red recombination system works poorly among unreplicated gam ⁺ lambda chromosomes in recA ^- cells compared to recA ⁺ cells. Recombination is not enhanced in recA ^- recB^-cells. Thus, the inability of Red to promote recombination in recA ^- replication-blocked cross is not due to the hypothetical destruction of recombination intermediates by the recB nuclease. This conclusion strengthens previous proposals that the products of the red genes can operate upon recombinational intermediates which require recA activity for their formation.     

Heat-sensitive DNA-binding activity of thecI product of bacteriophage lambda

Summary The binding of genecI product to DNA was studied at temperatures from 0°C to 46° C. Binding activity of the products ofcIts mutants was higher at 22° C than at 0° C, 26° C or 30° C. BothcI⁺ andcIts products lost DNA-binding activity at 46° C, but after subsequent cooling to 22° C, they regained 50–100% of their activity.     

Persistent Infection of L Cells with Vesicular Stomatitis Virus: Evolution of Virus Populations

A previous report (Youngner et al., J. Virol. 19:90-101, 1976) documented that noncytocidal persistent infection can be established with wild-type vesicular stomatitis virus (VSV) in mouse L cells at 37°C and that a rapid selection of RNA⁻, group I temperature-sensitive (ts) mutants consistently occurs in this system. To assess the selective advantage of the RNA⁻ts phenotype, evolution of the virus population was studied in persistent infections initiated in L cells by use of VSV ts 0 23 and ts 0 45, RNA⁺ mutants belonging to complementation groups III and V. In L cells persistently infected with ts 0 23, the ts RNA⁺ virus population was replaced gradually by viruses which had a ts RNA⁻ phenotype. VSV ts 0 45 (V) has another marker in addition to reduced virus yield at 39.5°C: a defective protein (G) which renders virion infectivity heat labile at 50°C. Persistent infections initiated with this virus (ts, heat labile, RNA⁺) evolved into a virus population which was ts, heat resistant, and RNA⁻. These findings suggest that the ts phenotype itself is not sufficient to stabilize the VSV population in persistently infected L cells and also indicate that the ts RNA⁻ phenotype may have a unique selective advantage in this system. In addition to the selection of ts RNA⁻ mutants, other mechanisms which also might operate in the maintenance of persistent VSV infections of L cells were explored. Whereas defective-interfering particles did not seem to mediate the carrier state, evidence was obtained that interferon may play a role in the regulation of persistent infections of L cells with VSV.     

Genetic evidence that the elevated levels of Escherichia coli helicase II antagonize recombinational DNA repair

Some phages survive irradiation much better upon multiple than upon single infection, a phenomenon known as multiplicity reactivation (MR). Long ago MR of UV-irradiated λ red phage in E. coli cells was shown to be a manifestation of recA-dependent recombinational DNA repair. We used this experimental model to assess the influence of helicase II on the type of recombinational repair responsible for MR. Since helicase II is encoded by the SOS-inducible uvrD gene, SOS-inducing treatments such as irradiating recA⁺ or heating recA441 cells were used. We found: i) that MR was abolished by the SOS-inducing treatments; ii) that in uvrD background these treatments did not affect MR; and iii) that the presence of a high-copy plasmid vector carrying the uvrD⁺ allele together with its natural promoter region was sufficient to block MR. From these results we infer that helicase II is able to antagonize the type of recA-dependent recombinational repair acting on multiple copies of UV-damaged λ DNA and that its antirecombinogenic activity is operative at elevated levels only.     

Suppressor mutations (rin) that specifically suppress the recA+ dependence of stable DNA replication in Escherichia coli K-12

Summary The sdrA102 mutation confers upon cells the ability to replicate DNA in the absence of protein synthesis. This mutation was combined with the recA200 mutation, which renders the recA protein thermolabile, and had little effect on normal replication. However, the sdrA102 recA200 double mutant exhibited temperature-sensitive stable DNA replication: it replicated DNA continuously in the presence of chloramphenicol at 30°C, whereas at 42°C DNA replication ceased after the DNA content increased only 40–45%. Suppressor mutants (rin; recA-independent) capable of stable DNA replication at 42°C were isolated from the double mutant. The suppressor mutant retained all other recA ^– characteristics, i.e., deficient general recombination, severe UV-sensitivity, and incapability of prophage induction in lysogens. This indicates that the rin mutation specifically suppresses the recA ⁺ dependency of stable DNA replication. It is suggested that the recA ⁺ protein stabilizes a specific structure, similar to an intermediate in recombination, which may function in the initiation of stable DNA replication.     

Phage T4 infection restricts rRNA synthesis byE. coli RNA polymerase

Summary Complementation for the maintenance of lysogeny was studied by superinfecting cIts lysogens at 34° C, and then heating to 43° C. With certain exceptions,ts mutants with defects in the left half of the repressor complementedts mutants with defects in the right half to produce a less heat-labile repressor (Fig. 3). AllcIamber mutants failed to complementcIts mutants. ThecI mutantc50 complements allts mutants. Mutations in Pre (cy) or genescII andcIII do not significantly affect the expression ofcI by a superinfecting genome in an immune lysogen. Mutants with very heat-labile repressors failed to complement cy42 for the establishment of lysogeny at elevated temperatures, while those with less heatsensitive repressors apparently did complementcy.According to a suggested model, the left side of thecI product is concerned primarily with subunit aggregation, while operator binding is the function of the right side of the oligomer.