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.     

Plasmid (pKM101)-mediated enhancement of repair and mutagenesis: Dependence on chromosomal genes in Escherichia coli K-12

Summary The drug resistance plasmid pKM101 plays a major role in the Ames Salmonella/microsome carcinogen detecting system by enhancing chemical mutagenesis. It is shown that in Escherichia coli K-12 the plasmid pKM101 enhances both spontaneous and methyl methanesulfonate-caused reversion of an ochre mutation, bacterial survival after ultraviolet irradiation, and reactivation of ultraviolet-irradiated in unirradiated cells. All these effects are shown to be dependent on the recA ⁺ lexA⁺ genotype but not on the recB ⁺ recC⁺ or recF ⁺ genotypes. The recA lexA-dependence of the plasmid-mediated repair and mutagenesis suggests an interaction with the cell's inducible error-prone repair system. The presence of pKM101 is shown to cause an additional increase in methyl methanesulfonate mutagenesis in a tif mutant beyond that caused by growth at 42°. The presence of the plasmid raises the level of the Weigle-reactivation curve for the reactivation of ultraviolet-irradiated in E. coli and causes a shift of the maximum to a higher UV fluence. These observations suggest that pKM101 does not exert its effects by altering the regulation of the cell's error-prone repair system but rather by supplying a mechanistic component or components.     

The novel gene(s) ARD of plasmid pKM101: alleviation of EcoK restriction

Summary The host-controlled K restriction of unmodified phage was 10-100-fold alleviated in the wild-type strain E. coli K12, carrying plasmid pKM101 of incompability group N. pKM101-mediated release of K restriction was also observed in lexA ^-, recA ^-, and recB ^- strains of E. coli K12. By restriction mapping Tn5 insertions in pKM101, which reduced pKM101-mediated alleviation of restriction, were shown to be located within the BglIIB fragment approximately 11 kb anticlockwise from the RI site of pKM101. We have termed the gene(s) promoting the alleviation of K restriction of phage ard (alleviation of restriction of DNA). It was shown (1) that ard function affected only the EcoK restriction system and not the EcoB, EcoRI, EcoRIII, or EcoPI system, (2) ard gene(s) did not mediate EcoK type modification of DNA and did not increase the modification activity of the EcoK system in a way similar to that observed with gene ral of bacteriophage .     

The fate of newly made DNA in Escherichia coli mutants thermosensitive in DNA synthesis

Summary E. coli mutants exist in which DNA synthesis is thermosensitive. In one class of these mutants DNA synthesis stops immediately if a critical temperature (42°C) is reached. When DNA replication in such mutants is followed by ³H thymidine incorporation at 33°C, it is found that 1. only the newly made DNA is degraded at 42°C, 2. the discontinuously replicated DNA is lost predominantly at 42°C, 3. 1–3% of the chromosomal DNA is rendered acid soluble at 42°C without concomitant loss of viability of the cells at 33°C.Replication of phage DNA is inhibited in the same mutant at 42°C. However, when DNA synthesis is followed in infected cells at 33°C it is found that 1. no degradation of specific DNA seems to occur at 42°C in the early phase of infection, 2. replicating DNA molecules in the late phase of infection are completed at 42°C before DNA synthesis comes to a halt.     

A new bacterial gene (groPC) which affects lambda DNA replication.

Summary A bacterial mutation affecting DNA replication, called groPC756, has been mapped between the thr and leu bacterial loci. Most of the parental DNA does not undergo even one round of replication in this host. Lambda mutants, called , which map in the P gene are able to overcome the inhibitory effect of the groPC756 mutation. It is shown that the mutation at the groPC locus also interferes with bacterial growth at 42°C. A -transducing phage, carrying the groPC⁺ allele, was isolated as a plaqueformer on groPC756 bacteria. Upon lysogenization, it restores both the gro ⁺ and temperature resistant phenotypes.     

Effect of the plasmid ColIb-P9 on cellular processes related to DNA repair

Summary The plasmid ColIb-P9 introduced into Escherichia coli K12 umuC mutant cells suppresses the deficiencies in mutagenesis and repair of mutants after UV-irradiation. These data suggest that ColIb-P9 encodes a product with a function similar to that of the chromosomal gene umuC. Tn5 insertion mutants of ColIb-P9 were isolated with an altered ability to restore UV-mutagenesis in the umuC mutant. The same plasmid mutations were shown to eliminate the effects of ColIb-P9 on UV-mutagenesis, survival after UV and mitomycin C treatment, reactivation of UV-irradiated in unirradiated cells, Weigle-reactivation, induction of colicin E1 synthesis. The ColIb-P9 genes responsible for the enhancement of UV-mutagenesis were cloned within a 14 Md SalI fragment. Their location was established by restriction analysis of the mutant plasmid ColIb 6-13::Tn5.While the action of the plasmids ColIb-P9 and pKM101 is similar, these plasmids were shown to have opposite effects on cell survival and colicin E1 synthesis after mitomycin C treatment. A study of the mutant plasmids ColIb::Tn5 and pGW12 (muc ^- mutant of pKM101) has shown the difference in the effects of ColIb-P9 and pKM101 to be associated with the plasmid genes responsible for the protective and mutagenesis-enhancing effects of these plasmids in UV-irradiated cells.Abbreviations MC mitomycin C - ICS induction of colicin synthesis     

Requirement of protein and RNA synthesis for lambda repressor inactivation by tif-1: effects of chloramphenicol, neomycin and rifampicin.

Summary The inactivation of repressor was followed by the specific DNA binding assay during the course of lysogenic induction provoked by incubation at 42°C of an E. coli tif-1 lysogenic strain. The presence of up to 400 g/ml chloramphenicol during the inducing treatment did not impair the loss of repressor binding activity, whilst concentrations of 200 g/ml neomycin and 100 g/ml rifampicin effectively inhibited the inactivation of repressor.Residual protein synthesis in the presence of chloramphenicol, neomycin and rifampicin was 5%, 5% and 27% respectively of that observed in the drug-free control. This residual synthesis did not appear to involve amplification of the X-protein. These results suggest that tif-mediated inactivation of the repressor requires the activation of some specific gene(s), the translation of which appears to be resistant to chloramphenicol.     

DNA repair properties of Escherichia coli tif-1, recAo281 and lexA1 strains deficient in single-strand DNA binding protein

Summary Mutations affecting single-strand DNA binding protein (SSB) impair induction of mutagenic (SOS) repair. To further investigate the role of SSB in SOS induction and DNA repair, isogenic strains were constructed combining the ssb ⁺, ssb-1 or ssb-113 alleles with one or more mutations known to alter regulation of damage inducible functions. As is true in ssb ⁺ strains tif-1 (recA441) was found to allow thermal induction of prophage ⁺ and Weigle reactivation in ssb-1 and ssb-113 strains. Furthermore, tif-1 decreased the UV sensitivity of the ssb-113 strain slightly and permitted UV induction of prophage ⁺ at 30°C. Strains carrying the recAo281 allele were also constructed. This mutation causes high constitutive levels of RecA protein synthesis and relieves much of the UV sensitivity conferred by lexA ^– alleles without restoring SOS (error-prone) repair. In contrast, the recAo281 allele failed to alleviate the UV sensitivity associated with either ssb ^– mutation. In a lexA1 recAo281 background the ssb-1 mutation increased the extent of postirradiation DNA degradation and concommitantly increased UV sensitivity 20-fold to the level exhibited by a recA1 strain. The ssb-113 mutation also increased UV sensitivity markedly in this background but did so without greatly increasing postirradiation DNA degradation. These results suggest a direct role for SSB in recombinational repair apart from and in addition to its role in facilitating induction of the recA-lexA regulon.     

Phenotypic instability in a tif-1 mutant of Escherichia coli

Summary The mutant T44() of Escherichia coli K12, grown in the presence of adenine, develops an increased tolerance to streptomycin. In cultures grown on streptomycin, the ts character (tif) may temporarily be suppressed but, on further transfer, both the temperature-sensitive phenotype and streptomycin tolerance disappear. In a cell-free system, the relative efficiency of translation of MS2 and poly U messenger RNAs was, respectively, 75 and 50% lower in extracts from cultures grown at 37° with adenine than in extracts from 30° cultures. Similar results were obtained when adenine was added in vitro to an extract from a culture grown at 37° in the absence of adenine, using MS2 RNA as messenger. Moreover, the 37° extracts showed a much lower misincorporation of isoleucine into polyphenylalanine in the poly U system. In addition, the Mg⁺⁺ concentration required for optimal translational activity was higher for the 37° than for the 30° extracts. Extracts from a culture grown in L medium at 37° or from a tif ^-/F tif ⁺ merodiploid grown at 37° with adenine behaved similarly to that from the 30° culture when poly U was used as messenger RNA. It is suggested that the tif ⁺ gene product may play a regulatory role in ribosomal function and the pleiotropic nature of the tif-1 mutation could be due to impairment of translational activity augmented by elevated temperature or by adenine.     

The respiratory chain of the facultative thermophile,Bacillus coagulans

Two oxidases were found to be present in membranes from the facultative thermophile Bacillus coagulans grown at 55°C, compared to one in cells grown at 37°C. Cytochrome spectra and inhibitors of the respiratory chain identified them as cytochrome oxidases aa ₃ and d. Both were present in membranes from 55°C grown cells, but only cytochrome oxidase aa ₃ was found in membranes from 37°C grown cells. The presence of cytochrome d in 55°C grown cultures was found to be due to decreased oxygen tension and not to the high growth temperature. This was confirmed by (a) induction of cytochrome d at 37°C under conditions of oxygen limitation and (b) its repression at 55°C under conditions of high aeration and its subsequent induction on lowering the dissolved oxygen concentration in chemostat cultures. Two cytochromes b (_max 558 and _max 562) were present in both 37°C and 55°C grown cells. Results from the inhibition of substrate oxidation by membranes suggested different pathways of electron transport by the respiratory chain.     

Mutations affecting growth of the Escherichia coli cell under a condition of DNA polymerase I-deficiency

Summary We constructed a strain of E. coli K12 carrying polA1 (an amber mutation of the DNA polymerase I gene; De Lucia and Cairns, 1969), and sup-126 (a temperature-sensitive amber suppressor; Nagata and Horiuchi, 1973). DNA polymerizing activity of the enzyme in this strain is virtually undetectable if the cells are grown at 42°C, but if grown at 30°C it is sufficiently present. By mutagenizing this strain, and after appropriate screening, we obtained mutants no longer able to grow at 42°, but able to do so when the normally functioning polA gene is present. One of them, called TS41, was most extensively studied. It acquired a mutation named pdeB41 which was found to be located between ilv and metE on the E. coli linkage map. Its phenotype is pleiotropic. The mutation by itself, i.e., if present in a polA ⁺ cell, does not kill the cell at 42°, but does so as in TS41 when it is reconstructed into a pdeB41 polA1 sup-126 triple mutant by P1 transduction. The mutation by itself renders the cell sensitive to UV, and tolerant to phage deficient in recombination. It is also a mutator.     

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.     

Identification of the C. coli dnaK (groPC756) gene product.

Summary The E. coli dnaK (groPC756) gene product is essential for bacteriophage DNA replication. Bacterial DNA segments carrying this gene have been cloned onto a bacteriophage vector. The product of the dnaK gene has been identified on SDS polyacrylamide gels after infection of UV-irradiated E. coli cells. The dnaK gene codes for a polypeptide with an apparent molecular weight of 93,000-Mr. Transducing phages carrying amber mutations in the dnaK gene fail to induce the synthesis of the 93,000-Mr polypeptide chain upon infection of sup ⁺ bacteria, but do so upon infection of supF bacteria. E. coli carrying the dnaK756 mutation are, in addition, temperature sensitive for growth at 43° C. It is shown that the dnaK756 mutation results in an overproduction of the dnaK gene product at that temperature.     

A point mutation in the Nul gene of bacteriophage lambda facilitates phage growth in Escherichia coli with himA and gyrB mutations

Summary A mutant of was isolated that grows in the Escherichia coli himA/gyrB-him320(Ts) double mutant at 42°C; conditions which are non-permissive for wild-type growth. The responsible mutation, ohm1, alters the 40th codon of the Nul reading frame. The Nul and A gene products comprise the terminase protein which cleaves concatameric DNA into unit-length phage genomes during DNA packaging. The Nul-ohm1 gene product acts in trans to support growth in the double himA/gyrB mutant, and cos154 growth in the single himA mutant. The observation that an alteration in Nul suppresses the inhibition of growth in the double himA/gyrB mutant implicates DNA gyrase, as well as integration host factor, in the DNA: protein interactions that occur at the initiation of packaging.     

Reduced expression of a distal gene of the prn gene cluster in deletion mutants of Aspergillus nidulans: genetic evidence for a dicistronic messenger in an eukaryote.

Summary Temperature sensitive dnaAts46 mutants, in which initiation of chromosome replication is blocked at 42° C, are unable to maintain a dv plasmid at the permissive temperature unless the plasmid carries a mutation in gene P of the type permitting phage to grow in groP (dnaB) bacteria. The growth rate of dnaAts46 mutants seems to be impaired by the presence of the dvP mutant plasmid.Cold sensitive dnaAcos mutants which overinitiate replication at low temperature and grow normally only at 40° and above, can maintain efficiently dvP ⁺ plasmids as well as dvP mutants. Cold sensitivity of dnaAcos mutants is suppressed by the presence of the plasmid dvP ⁺ and by certain dvP mutants, but not by others.The gene P product seems to act by reducing the initiation potential of both types of dnaA mutants, aggravating the initiation defect in dnaAts46 and correcting the overinitiation of dnaAcos.     

Effect of the dnaN mutation on the growth of small DNA phages

Summary The effect of the dnaN mutation on the growth of single-stranded DNA phages was studied by burst experiments. In HC138 dnaN cells exposed to 42.5° C at 5 min before infection, growth of spherical (microvirid or isometric) phages such as 3, Kh-1 and X174 was partially reduced at the nonpermissive temperature. When infection was performed at 30 min after temperature shift-up, viral replication was completely inhibited at 42.5° C in the dnaN strain but not in a dna ⁺ revertant. At 41° C, multiplication of filamentous (inovirid) phages M13 and fd was restricted specifically in HC138 F⁺ dnaN bacteria. When dnaN cells lysogenic for i²¹ were grown at 42.5° C for 60 min and then shifted down to 33° C, a burst of i²¹ occurred with concomitant cellular lysis, manifesting induction of the prophage development.     

Studies on the E. coli groNB (nusB) gene which affects bacteriophage lambda N gene function

Summary Escherichia coli mutants, called groNB, which block the growth of bacteriophage at the level of action of the gene N product, have been isolated as survivors at 42°C of bacteria carrying a) the defective prophage bio1 1 i cI857 H1 or b) the pcR1 plasmid containing the EcoRI immunity fragment of phage cI857. In addition, groNB bacterial mutants have been isolated at 37° C, as large colony formers in the presence of i cI h ⁴³⁴, i cI h , and i cI h ⁸⁰ phage. The groNB locus is located at 9 minute of the E. coli genetic map with the order of the neighboring loci being proC tsx groNB purE. Most groNB mutations isolated at 42° C were found to interfere in addition with bacterial growth at low temperatures, since (a) the GroNB phenotypes of growth inhibition and bacterial cold sensitivity cannot be separated by P1 transduction, and (b) some cold resistant revertants simultaneously become Gro⁺ for growth. Lambda transducing phages carrying the groNB ⁺ bacterial gene have been isolated. GroNB mutant bacterial lysogenized by the transducing phage acquire the Gro⁺ phenotype and simultaneously the cold resistant phenotype, suggesting that the groNB mutations are recessive to the wild-type gene.     

Repair of ultraviolet-light damaged ColE1 factor carrying Escherichia coli genes for guanine synthesis.

Summary Hybrid ColE1 plasmids called ColE1-cos-guaA or ColE1-cos-gal can be efficiently transduced into various E. coli K-12 cells through packaging into phage particles. Using these plasmids, repair of ultraviolet-light (UV) damaged ColE1 DNAs was studied in various UV sensitive E. coli K-12 mutants. (1) The host mutations uvrA and uvrB markedly reduced host-cell reactivation of UV-irradiated ColE1-cos-guaA. (2) Pre-existing hybrid ColE1 plasmids had no effect on the frequency of phage-mediated transduction of another differentially marked hybrid ColE1 DNAs. (3) ColE1-cos-guaA and ColE1-cos-gal DNAs could temporarily but not stably co-exist in E. coli K-12 recA cells. (4) The presence of ColE1-cos-gal in uvrB cells promoted the repair of super-infected UV-irradiated ColE1-cos-guaA about 7-fold. (5) The same ColE1-cos-gal plasmid in a uvrB recA double mutant did not have this promoting effect. These results indicate that the effect of resident hybrid ColE1 plasmids is manifested by the host recA ⁺ gen function(s) and suggest that ColE1 plasmid itself provides no recA ⁺-like functions.     

Lambda phage mutants insensitive to temperature-sensitive repressor

Summary The isolation of recombinant carrying virC mutation from newly isolated virulent carrying virL virC virR, (Horiuchi et al., 1969) was succeeded and the genetic character of virC mutation producing clear plaque was studied. virL virC shows weak-virulent character and produces clear plaque on CIts lysogen but not on wild type lysogen. virC shows avirulent character and no plaque is produced on these lysogen. The virC mutation is located very closely to and on the left side of the virR region (Fig. 1) which is presumed to be the operator of the right-side operon including O and P cistrons. The genetic characters of virL, virR and virC, were compared with v ₁, v ₂, v ₃ mutations of classical vir (Jacob and Wollman, 1954) and c ₁₇ mutation of another type of virulent (Da Silva and Jacob, 1968). The results indicate that virL, virC or virR mutation is similar to v ₂, v ₁ or v ₃ mutation, respectively, and an effect of virC mutation on producing virulent character was somewhat similar to that of c ₁₇ mutation and was stronger than that of virR mutation. The length of virR regions was suggested to be smaller than one tenth of that of the CI cistron.     

Prophage inactivation in recB-proficient Escherichia coli K12 (lambda) lysogens after ultraviolet irradiation

Summary If ultraviolet irradiated, -lysogenic Escherichia coli K12 bacteria are incubated for 4 to 6 h at 30° C, prophage becomes inactive in the non-surviving cells. This was demonstrated by the use of cIts857ind1 prophage which can be induced by heat but not by ultraviolet light. An analysis with various bacterial mutants showed that RecBC recombination enzyme is required in conjunction with RecA protein for prophage inactivation.