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The Escherichia coli argU10 (Ts) mutation in the argU gene, encoding the minor tRNA Arg species for the rare codons AGA and AGG, causes pleiotropic defects, including growth inhibition at high temperatures, as well as the Pin phenotype at 30°C. In the present study, we first showed that the codon selectivity and the arginine-accepting activity of the argU tRNA are both essential for complementing the temperature-sensitive growth, indicating that this defect is caused at the level of translation. An in vitro analysis of the effects of the argU10 (Ts) mutation on tRNA functions revealed that the affinity with elongation factor Tu-GTP of the argU10 (Ts) mutant tRNA is impaired at 30 and 43°C, and this defect is more serious at the higher temperature. The arginine acceptance is also impaired significantly but to similar extents at the two temperatures. An in vivo analysis of aminoacylation levels showed that 30% of the argU10 (Ts) tRNA molecules in the mutant cells are actually deacylated at 30°C, while most of the argU tRNA molecules in the wild-type cells are aminoacylated. Furthermore, the cellular level of this mutant tRNA is one-tenth that of the wild-type argU tRNA. At 43°C, the cellular level of the argU10 (Ts) tRNA is further reduced to a trace amount, while neither the cellular abundance nor the aminoacylation level of the wild-type argU tRNA changes. We concluded that the phenotypic properties of the argU10 (Ts) mutant result from these reduced intracellular levels of the tRNA, which are probably caused by the defective interactions with elongation factor Tu and arginyl-tRNA synthetase.

Mutations in the Escherichia coli gene for initiation of DNA replication, dnaA, which suppress the polymerization defect and growth inhibition caused by temperature-sensitive (Ts) mutations in the polymerization gene, dnaX, are called Cs,Sx. We show here that these mutations, on their own, also cause defects in initiation, including inhibition of initiation at both low (20 degrees C) and high (44 degrees C) temperatures and asynchronous initiation at both the permissive (34 degrees C) and suppression (39 degrees C) temperatures. These findings suggests a relationship between partially defective initiation and suppression of the polymerization defect, both of which occur at 39 degrees C.

The Escherichia coli RecR protein participates in a recombinational DNA repair process. Its gene is located in a region of chromosome that extends from 502 to 509 kilobases on the physical map and that contains apt, dnaX, orf12-recR, htpG, and adk. Most, if not all, of these are involved in nucleic acid metabolism. The orf12-recR reading frames consist of 935 base pairs and overlap by one nucleotide, with the 3' A of the orf12 termination codon forming the 5' nucleotide of the recR initiation codon. The orf12-recR promoter was located upstream of orf12 by sequence analysis, promoter cloning, and S1 nuclease protection analysis. The start point of transcription was determined by primer extension. The transcript 5' end contained a long, apparently untranslated region of 199 nucleotides. Absence of a detectable promoter specific for recR and the overlap of the orf12 and recR reading frames suggest that translation of recR is coupled to that of orf12. By maxicell analysis, it was determined that both orf12 and recR are translated.

Arginine is coded for by CGN (N = G, A, U, C), AGA and AGG. In Escherichia coli there is little tRNA for AGA and AGG and the use of these codons is strongly avoided in virtually all genes. Recently, we demonstrated that the presence of tandem AGA or AGG codons in mRNA causes frameshifts with high frequency. Here, we show that phaseshifts can be suppressed when cells are transformed with the gene for tRNA(T4Arg) or E. coli tRNA(argU,Arg) demonstrating that such errors are the result of tRNA depletion. Bacteriophage T4 encoded tRNA(Arg) (anticodon UCU) corrects shifts at AGA-AGA but not at AGG-AGG, suggesting that this tRNA can only read AGA. Similarly, comparison of the translational efficiencies in an argU (Ts) mutant and in its isogenic wild type parent indicates that argU tRNA (anticodon UCU) reads AGA but not AGG. An argU (Ts) mutant barely reads through AGA-AGA at 42 degrees C but translation of AGG-AGG is hardly, if at all, affected. Overexpression of argU+ relaxes the codon specificity. The thermosensitive mutant in argU, previously called dnaY because it is defective in DNA replication, can be complemented for growth by the gene for tRNA(T4Arg). This implies that the sole function of the argU gene product is to sustain protein synthesis and that its role in replication is probably indirect.

The Escherichia coli dnaA73, dnaA721, and dnaA71 alleles, which encode A213D, R432L, T435K substitutions, respectively, were originally isolated as extragenic suppressors of a temperature-sensitive dnaX mutant. As the A213D substitution resides in a domain that functions in ATP binding and the R432L and T435K substitutions affect residues that recognize the DnaA box motif, they might be expected to reduce ATP and specific DNA binding, respectively. Therefore, a major objective was to quantify the biochemical defects of the mutant DnaAs to understand how the altered proteins suppress the temperature-sensitive phenotype of a dnaX mutant. A second purpose was to address the paradox that mutant proteins with substitutions of amino acids essential for recognition of the DnaA box motifs within the E. coli replication origin (oriC) may well be inactive in initiation, yet chromosomal dnaA mutants expressing DnaA proteins with the R432L and T435K substitutions are viable at temperatures from 30 to 39 degrees C. We show biochemically that mutant DnaAs carrying R432L and T435K substitutions fail to bind to the DnaA box sequence. The A213D mutant is sevenfold reduced in its affinity for ATP compared to wild-type DnaA, and its affinity for the DnaA box sequence is also reduced. However, the reduced activity of the A213D mutant in oriC plasmid replication appears to arise from a defect in DnaA oligomerization. Although the T435K mutant fails to bind to the DnaA box sequence, other results suggest that DnaA oligomerization stabilizes the binding of the mutant DnaA to oriC to support its partial activity in initiation in vitro. These results support a model that suppression of dnaX occurs by reducing the frequency of initiation to a manageable level for the mutant DnaX so that viability is maintained.

Temperature sensitivity of DNA polymerization and growth of a dnaX (Ts) mutant is suppressible at 39 to 40°C by mutations in the initiator gene, dnaA . These suppressor mutations concomitantly cause initiation inhibition at 20°C and have been designated Cs,Sx to indicate both phenotypic characteristics of cold-sensitive initiation and suppression of dnaX (Ts) . One dnaA (Cs,Sx) mutant, A213D, has reduced affinity for ATP, and two mutants, R432L and T435K, have eliminated detectable DnaA box binding in vitro. Two models have explained dnaA (Cs,Sx) suppression of dnaX , which codes for both the τ and γ subunits of DNA polymerase III. The initiation deficiency model assumes that reducing initiation efficiency allows survival of the dnaX (Ts) mutant at the somewhat intermediate temperature of 39 to 40°C by reducing chromosome content per cell, thus allowing partially active DNA polymerase III to complete replication of enough chromosomes for the organism to survive. The stabilization model is based on the idea that DnaA interacts, directly or indirectly, with polymerization factors during replication. We present five lines of evidence consistent with the initiation deficiency model. First, a dnaA (Cs,Sx) mutation reduced initiation frequency and chromosome content (measured by flow cytometry) and origin/terminus ratios (measured by real-time PCR) in both wild-type and dnaX (Ts) strains growing at 39 and 34°C. These effects were shown to result specifically from the Cs,Sx mutations, because the dnaX (Ts) mutant is not defective in initiation. Second, reduction of the number of origins and chromosome content per cell was common to all three known suppressor mutations. Third, growing the dnaA (Cs,Sx) dnaX (Ts) strain on glycerol-containing medium reduced its chromosome content to one per cell and eliminated suppression at 39°C, as would be expected if the combination of poor carbon source, the Cs,Sx mutation, the Ts mutation, and the 39°C incubation reduced replication to the point that growth (and, therefore, suppression) was not possible. However, suppression was possible on glycerol medium at 38°C. Fourth, the dnaX (Ts) mutation can be suppressed also by introduction of oriC mutations, which reduced initiation efficiency and chromosome number per cell, and the degree of suppression was proportional to the level of initiation defect. Fifth, introducing a dnaA (Cos) allele, which causes overinitiation, into the dnaX (Ts) mutant exacerbated its temperature sensitivity.

The Escherichia coli DNA polymerase III τ and γ subunits are single-strand DNA-dependent ATPases (the latter requires the δ and δ′ subunits for significant ATPase activity) involved in loading processivity clamp β. They are homologous to clamp-loading proteins of many organisms from phages to humans. Alignment of 27 prokaryotic τ/γ homologs and 1 eukaryotic τ/γ homolog has refined the sequences of nine previously defined identity and functional motifs. Mutational analysis has defined highly conserved residues required for activity in vivo and in vitro. Specifically, mutations introduced into highly conserved residues within three of those motifs, the P loop, the DExx region, and the SRC region, inactivated complementing activity in vivo and clamp loading in vitro and reduced ATPase catalytic efficiency in vitro. Mutation of a highly conserved residue within a fourth motif, VIc, inactivated clamp-loading activity and reduced ATPase activity in vitro, but the mutant gene, on a multicopy plasmid, retained complementing activity in vivo and the mutant gene also supported apparently normal replication and growth as a haploid, chromosomal allele.

Temperature sensitivity of DNA polymerization and growth, resulting from mutation of the tau and gamma subunits of Escherichia coli DNA polymerase III, are suppressed by Cs,Sx mutations of the initiator gene, dnaA. These mutations simultaneously cause defective initiation at 20 degrees C. Efficient suppression, defined as restoration of normal growth rate at 39 degrees C to essentially all the cells, depends on functional oriC. Increasing DnaA activity in a strain capable of suppression, by introducing a copy of the wild-type allele, increasing the suppressor gene dosage or introducing a seqA mutation, reversed the suppression. This suggests that the suppression mechanism depends on reduced activity of DnaACs, Sx. Models that assume that suppression results from an initiation defect or from DnaACs,Sx interaction with polymerization proteins during nascent strand synthesis are proposed.

Extragenic suppressor mutations which had the ability to suppress a dnaX2016(Ts) DNA polymerization defect and which concomitantly caused cold sensitivity have been characterized within the dnaA initiation gene. When these alleles (designated Cs, Sx) were moved into dnaX+ strains, the new mutants became cold sensitive and phenotypically were initiation defective at 20 degrees C (J.R. Walker, J.A. Ramsey, and W.G. Haldenwang, Proc. Natl. Acad. Sci. USA 79:3340-3344, 1982). Detailed localization by marker rescue and DNA sequencing are reported here. One mutation changed codon 213 from Ala to Asp, the second changed Arg-432 to Leu, and the third changed codon 435 from Thr to Lys. It is striking that two of the three spontaneous mutations occurred in codons 432 and 435; these codons are within a very highly conserved, 12-residue region (K. Skarstad and E. Boye, Biochim. Biophys. Acta 1217:111-130, 1994; W. Messer and C. Weigel, submitted for publication) which must be critical for one of the DnaA activities. The dominance of wild-type and mutant alleles in both initiation and suppression activities was studied. First, in initiation function, the wild-type allele was dominant over the Cs, Sx alleles, and this dominance was independent of location. That is, the dnaA+ allele restored growth to dnaA (Cs, Sx) strains at 20 degrees C independently of which allele was present on the plasmid. The dnaA (Cs, Sx) alleles provided initiator function at 39 degrees C and were dominant in a dnaA(Ts) host at that temperature. On the other hand, suppression was dominant when the suppressor allele was chromosomal but recessive when it was plasmid borne. Furthermore, suppression was not observed when the suppressor allele was present on a plasmid and the chromosomal dnaA was a null allele. These data suggest that the suppressor allele must be integrated into the chromosome, perhaps at the normal dnaA location. Suppression by dnaA (Cs, Sx) did not require initiation at oriC; it was observed in strains deleted of oriC and which initiated at an integrated plasmid origin.

Suppressor mutations located within dnaA can suppress the temperature sensitivity of a dnaZ polymerization mutant, indicating in vivo interaction of the products of these genes. The suppressor allele of dnaA [designated dnaA(SUZ, Cs)] could not be introduced, even at the permissive temperature, by transduction into temperature-sensitive (Ts) dnaC or dnaG recipients; it was transduced into dnaB(Ts) and dnaE(Ts) strains but at very low frequency. Recipient cells which were dnaA+ dnaE(Ts) were killed by the incoming dnaA(SUZ, Cs) allele, and it is presumed that combinations of dnaA(SUZ, Cs) with dnaB(Ts), dnaC(Ts), or dnaG(Ts) are lethal also. In one specific case, the lethality required the presence of three alleles: the incoming dnaA suppressor mutation, the resident dnaA+ gene, and the dnaB(Ts) gene. This was shown by the fact that dnaB(Ts) could readily be introduced into a dnaA(SUZ, Cs) dnaB+ recipient. That is, in the absence of dnaA+, the dnaA suppressor and dnaB(Ts) double mutant was stable. One model to explain these results proposes that the dnaA protein functions not only in initiation but also in the replication complex which contains multiple copies of dnaA and other replication factors.

Three mutants of Escherichia coli K-12 which form filaments during 42 C incubation have been characterized. The mutant strains AX621, AX629, and AX655 continued to grow and to synthesize deoxyribonucleic acid at 42 C for 150 to 180 min, after which time growth ceased. When cultures of the mutants were transferred from 42 to 28 C, septation of the filaments began after a 25- to 30-min period and continued at a greater than normal rate until no filaments remained. Addition of chloramphenicol at the time of transfer from 42 to 28 C prevented cell division in strain AX655 and caused lysis of strains AX621 and AX629. The temperature sensitivity mutation in each strain mapped near leu . For strain AX621, the mutation was specifically located between leu and nadC by P1 transduction. Properties of these strains are compared with those of other cell division mutants.

The physiological effects of incubation at nonpermissive temperatures of Escherichia coli mutants that carry a temperature-sensitive dnaZ allele [ dnaZ (Ts) 2016 ] were examined. The temperature at which the dnaZ (Ts) protein becomes inactivated in vivo was investigated by measurements of deoxyribonucleic acid (DNA) synthesis at temperatures intermediate between permissive and nonpermissive. DNA synthesis inhibition was reversible by reducing the temperature of cultures from 42 to 30°C; DNA synthesis resumed immediately after temperature reduction and occurred even in the presence of chloramphenicol. Inasmuch as DNA synthesis could be resumed in the absence of protein synthesis, we concluded that the protein product of the dnaZ allele (Ts) 2016 is renaturable. Cell division, also inhibited by 42°C incubation, resumed after temperature reduction, but the length of time required for resumption depended on the duration of the period at 42°C. Replicative synthesis of cellular DNA, examined in vitro in toluene-permeabilized cells, was temperature sensitive. Excision repair of ultraviolet light-induced DNA lesions was partially inhibited in dnaZ (Ts) cells at 42°C. The dnaZ + product participated in the synthesis of both Okazaki piece (8–12 S ) and high-molecular-weight DNA. During incubation of dnaZ (Ts)(λ) lysogens at 42°C, prophage induction occurred, and progeny phage were produced during subsequent incubation at 30°C. The temperature sensitivity of both DNA synthesis and cell division in the dnaZ (Ts) 2016 mutant was suppressed by high concentrations of sucrose, lactose, or NaCl. Incubation at 42°C was neither mutagenic nor antimutagenic for the dnaZ (Ts) mutant.

Escherichia coli lon-cells were subjected to treatments which produced a decrease in the DNA/mass ratio of the cell. Thymine starvation, a shift-up from minimal medium to rich medium, and exposure to BUdR each caused greater inhibition of cell division in lon-cells than in lon+cells. DNA metabolism was found to be the same in both lon+and lon-cells during these treatments. The results are consistent with the hypothesis that the lon-defect leads to inhibition of cell division under conditions which produce a decreased DNA/mass ratio.

The lon − mutants of Escherichia coli grow apparently normally except that, after temporary periods of inhibition of deoxyribonucleic acid synthesis, septum formation is specifically inhibited. Under these conditions, long, multinucleate, nonseptate filaments result. The lon − mutation also creates a defect such that wild-type bacteriophage λ fails to lysogenize lon − mutants efficiently and consequently forms clear plaques on a lon − host. Two lines of evidence suggest that this failure probably results from interference with expression of the λ cI gene, which codes for repressor, or with repressor action:-(i) when a lon − mutant was infected with a λ cII, cIII , or c Y mutant, there was an additive effect between the lon − mutation and the λ c mutations upon reduction of lysogenization frequency; and (ii) lon − mutants permitted the growth of the λ cro − mutant under conditions in which the repressor was active. The isolation of λ mutants (λtp) which gained the ability to form turbid plaques on lon − cells is also reported.

The ras − mutant of Escherichia coli K-12 is sensitive to ultraviolet (UV) light but only slightly sensitive to X-irradiation (1.5-fold increase). Other phenotypic properties include normal recombination ability and normal host cell reactivation ability but an abnormally high frequency of UV-induced mutation. The response of the ras − mutant to UV has been studied biochemically. After low doses of UV, the ras − mutant degraded excessive amounts of deoxyribonucleic acid, and long delays in resumption of deoxyribonucleic acid synthesis occurred. Pyrimidine dimers were excised at the normal rate. Although the mutant had the capability of initiating repair replication, the process was not completed after the high UV dose required to allow detection of repair replication. The ras − mutant, after low UV doses, left three to four times as many single-strand breaks not rejoined as did the wild-type strain.

Thermosensitive mutants of E. coli which form filaments at 42 C were islated. These mutants were divided into 3 classes according to optical density and cell number patterns during growth at 42 C and after being shifted from 42 C to 30 C. Nuclear regions in 2 classes were positioned along the filament with the same number per unit length of cell as were those of wild-type cells. However, one class appeared to be defective in nuclear region positioning in that the nuclear bodies of its filaments were separated by large areas of cytoplasm.

Septum formation is a key step in bacterial division, but the mechanism which controls periodic septum formation is unknown. In an attempt to understand this mechanism, lon − mutants, in which septum formation is blocked by very doses of ultraviolet light (UV), were investigated. UV must act on some part of the apparatus of cytokinesis; thus, identification of the UV target would identify part of this apparatus. As likely possibilities, UV might damage the septum-forming site or it might damage deoxyribonucleic acid (DNA), since DNA replication is normally coordinated with septum formation. To distinguish between these possibilities, DNA was specifically sensitized by incorporating bromodeoxyuridine into lon − bacteria. These bacteria were strongly sensitized to longer wavelength UV (2,900 to 3,100 A) so that they failed to form septa, grew into filaments which lysed, and did not form colonies. Various control experiments supported the conclusion that UV inhibits septum formation as a result of alterations in DNA metabolism. A relationship thus exists between DNA metabolism and septum formation.

Thelon−mutation is responsible for a defect in cell division ofEscherichia colilightly irradiated with ultraviolet light (UV). Theselon−mutants can be isolated readily by a procedure described here. Physiological studies were performed with the objective of determining the role of thelongene. Unirradiatedlon−mutants grow normally, except that a correlation of this mutation with capsule formation has been noted previously. These two properties can be separated, however. After irradiation,lon−grows as long filaments because septum formation is prevented. The filaments eventually lyse. Mass increase and deoxyribonucleic acid and enzyme synthesis appear to proceed normally. Thus, the lesion produced by UV appears to be highly specific. In bacteria that carry both genes (merozygotes),lon+is dominant tolon−. Septum formation is restored to irradiatedlon−bacteria by introduction oflon+by conjugation. Also, normal growth can be restored by nutritional variations. It is concluded thatlon+is able to nullify the effects of the UV lesion under conditions wherelon−cannot. Possibly, capsule precursors that can accumulate in the latter are responsible for the difference because they interfere with repair of the UV lesion.

To define in vivo interactions of Escherichia coli DNA replication components, extragenic suppressors of a dnaZ(TS) mutant were isolated. A temperature-sensitive dnaZ mutant, which is defective in polymerization, was placed at 39 degrees C to select temperature-insensitive revertants. Some of these revertants also were cold sensitive, a phenotypic property that facilitated study of the suppressor. Mapping of the cold sensitivity indicated that some of the suppressor mutations are intragenic but others are located within the initiation gene, dnaA. The dnaA mutations that suppress the dnaZ(TS) defect are designated dnaA(SUZ, CS). The dnaA(SUZ, CS) strains have a defect in DNA synthesis at low temperature that is typical of an initiation defect. These data suggest that the dnaA product, an initiation factor, interacts in vivo with the dnaZ protein, a polymerization factor.

A dnaW mutant, isolated on the basis of inability to effect conjugal DNA transfer at high temperature, has been shown by complementation and enzyme assay to be defective in the adk (adenylate kinase; EC 2.7.4.3) locus. The adk mutant, known to have reduced ATP concentration at the nonpermissive temperature (Cousin and Belaich 1966), was used to demonstrate a donor energy requirement for stable aggregate formation and for chromosome transfer in conjugation.

Mutations ts2158 and ts1882, which confer temperature sensitivity of septum formation, map near leu in the region of min 2.0 to 2.1 on the Escherichia coli chromosome. These mutants stop division abruptly and grow as filaments at 42 C; when returned to 28 C, division resumes after about 30 min to produce short cells. The product of the gene defined by these mutations probably is required during all stages of septum formation rather than specifically for initiation of septation. Filaments that formed at 42 C contained incomplete constrictiions (septa). When actively dividing filaments (i.e., those incubated at 28 C until division resumed) were shifted to 42 C a second time, division again stopped abruptly and incomplete constrictions persisted during the incubation at 42 C. Filaments that were subjected to 28 C incubation for a brief time (10, 20, or 30 min) before being shifted again to 42 C did not resume division as would be expected of a strain defective in initiating septation. Mutations ts 1882 and ts2158 and recessive to the ts+ allele, which is consistent with the interpretation that these mutations cause the loss of a function. They did not complement each other and presumably represent one cistron. Mutants carrying ts1882 and ts2158 mutations were compared with a mutant defective in the ftA allele, also known to map near leu.

Escherichia coli mutants with temperature-sensitive (ts) mutations in dnaX and dnaY genes have been isolated. Based on transduction by phage PI, dnaX and Y have been mapped at minutes 10.4-10.5 and 12.1, respectively, in the sequence dnaX purE dnaY. Both dnaXts36 and YtslO are recessive to wild-type alleles present on episomes. F13 carries both dnaX + and Y +; the shorter F210 carries dnaY +, but not X +. Lambda transducing phages that carry dnaX + or Y + have been isolated, and hybrid plasmids of Col E1 and E. coli DNA from the CLARKE and CARBON (1976) collection also carry portions of the dnaX purE dnaY region. Results obtained with the λ transducing phages and the hybrid plasmids suggest that dnaX is a different gene from the previously characterized dnaZ gene, which is also near minute 10.5.—The dnaXts36 mutant, after a shift to 42°, stopped DNA synthesis gradually, and the total amount of DNA increased two-fold. When this mutant was shifted to M°, the rate of DNA synthesis dropped immediately and the final increment of DNA was only 10% of the initial amount. Replicative DNA synthesis in toluene-treated cells was completely inhibited at 42° and was partially in-hibited even at 30°.—When the dnaYtslO mutant was shifted to 42°, DNA synthesis gradually stopped, and the amount of DNA increased 3.6-fold. At 44°, residual DNA synthesis amounted to a two-fold increase. Replicative DNA synthesis in vitro in toluene-treated cells was inactivated after 20 minutes at 42° or by "preincubation" of cells at 42° before toluene treatment.— The dnaX and dnaY products probably function in polymerization of DNA, although participation also in initiation cannot be excluded.

Lambda tp mutants, selected for their ability to form turbid plaques on lon hosts, overproduce repressor. The tp1 and tp2 mutations have been located within (or adjacent to) the cIII gene. The tp1 mutation reduced late gene expression, as measured by endolysin synthesis (in the absence of functional cI repressor) and progeny phage yield. The tp4 mutation was mapped in the cY-cII region, and complementation tests indicated that tp4 affects the diffusible product of the cII gene. The tp4 mutation also reduced progeny production, but did not markedly affect endolysin synthesis.

The dnaH locus is the fourth gene to be identified as required for deoxyribonucleic acid polymerization in Escherichia coli . A temperature-sensitive mutant defective in this gene exhibited an abrupt decrease in rate of deoxyribonucleic acid synthesis when shifted to 42 C. The locus mapped in the proC-purE region of the chromosome by conjugation and was co-transducible with purE. dnaH + is carried on the F′ 13 episome and is dominant over the dnaH − mutation.

Suppressors of a temperature-sensitive dnaZ polymerization mutant of Escherichia coli have been identified by selecting temperature-insensitive revertants. Those suppressed strains which concomitantly became cold sensitive were chosen for further study. Intragenic suppressor mutations, which caused cold-sensitive defects in DNA polymerization, were located in dnaZ by transduction with lambda dnaZ+ phages. Extragenic suppressor mutations were mapped within the initiation gene dnaA. These suppressor-containing strains were defective in initiation at low temperature as determined by measurements of DNA synthesis in vivo and in toluene-treated cells. The occurrence of suppressor mutations of dnaZ(Ts) within the dnaA gene is considered evidence that the dnaA and dnaZ products interact in vivo. A second indication of a dnaA-dnaZ protein-protein interaction was provided by the observation that the introduction of additional copies of the dnaZ+ gene into a strain carrying the dnaA suppressor mutation was lethal [whether the strain was dnaZ+ or dnaZ(Ts)].

The Escherichia coli DNA replication genes dnaZ and dnaX have previously been localized very near each other at 10.4 to 10.5 min on the chromosome map. These genes were cloned from a dnaZ+X+ plasmid of the Clarke and Carbon collection by identifying complementing fragments and both were located on a 2.1 kilobase pair (kb) fragment. The organization of the Z and X genes was investigated by Tn5 mutagenesis of a Z+X+ plasmid. Insertions which abolished Z or X complementing activity were mapped by restriction enzyme analysis within the 2.1 kb fragment. With the exception of one atypical insertion, all the insertions inactivated both Z and X complementation. The protein products of the dnaZ-dnaX region were labelled in minicells containing dnaZ+X+ and dnaZX::Tn5 plasmids. The 2.1 kb ZX region (which has a maximum coding capacity of 77,000 daltons of protein in a single reading frame) directed the synthesis of two proteins, one of 75,000 daltons, designated dnaX, and another of 56,500 daltons, designated dnaZ. Tn5 insertion into the ZX region interrupted the synthesis of these proteins; the detection of truncated fragments of dnaX determined the direction of transcription. In vitro, using a coupled transcription-translation system dependent on plasmid DNA, synthesis of the 75,000 dalton dnaX protein was demonstrated, but there was no detectable synthesis of the smaller dnaZ protein. Probably, therefore, the 75,000 dalton dnaX protein is cleaved in vivo to generate the dnaZ protein. It is possible that the 75,000 dalton product is the tau subunit of DNA polymerase III because they migrated similarly in electrophoresis.

The Escherichia coli lon cell division mutant, which is lysogenized with reduced frequency by λ and other lambdoid phages, produced only one-half the λ repressor activity found in lon+ cells after λ infection. A similar reduction of repressor level was observed in λcro-infected lon cells, compared to λcro-infected lon+ cells, indicating that the lon effect on repressor activity is not the result of an altered interaction with the λcro+ gene product. λ late gene functions were not efficiently shut off in λ+-infected lon cells, as demonstrated by elevated endolysin levels and phage yields. Although lon cells are lysogenized inefficiently, lon lysogens can be isolated and are stable. Mono-lysogens of lon+ and lon strains contained similar repressor levels. Thus, the lon defect results in reduced repressor activity during the establishment of repression, but not during maintenance of lysogeny. λ mutants (λtp) have been selected for the ability to form turbid plaques on lon hosts. After infection by λtp, repressor levels in both lon+ and lon cells were twofold higher than the levels in λ+-infected cells. Established lon+ and lon mono-lysogens of the λtp mutants contained repressor activity levels similar to those in λ+ mono-lysogens.

The dnaY gene of E. coli, thought to be involved in the polymerization phase of DNA replication, was localized on a fragment 118 base pairs in length. This fragment, cloned in two different vectors and tested in a dnaY (Ts) recA host, has dnaY + complementing activity. The nucleotide sequence of the 118 base pairs and flanking bases was determined. The dnaY complementing activity was inactivated by transposon insertion and by localized chemical mutagenesis. Three independent insertions of Tn5 into the 118 base pair region eliminated dnaY activity. Eight single-base-change mutations that resulted in loss of dnaY activity also were located within the 118 base pair region. Analysis of the nucleotide sequence reveals a potential promoter but reveals no open reading frames likely to be translated into polypeptides. However, an RNA transcript of the dnaY region is synthesized in vivo. Perhaps the active product of dnaY is a small RNA or perhaps the dnaY region functions as a site.

There are several classes of Escherichia coli mutants defective in radiation repair. These include strains defective in pyrimidine dimer excision, in photoreactivation, in recombination, in repair of X-ray damage, and ultraviolet (UV)-conditional mutants which do not divide after UV. Another mutant ( ras − ) has been isolated. The ras − has increased UV sensitivity, but only slightly increased X-ray sensitivity (1.5-fold increase). Ability to effect genetic recombination, to reactivate irradiated bacteriophage T1, and to be photoreactivated is normal. UV-induced mutation frequency is greatly increased in the mutant. The ras − apparently lacks the ability to repair some UV damage in the bacterial cell but can repair UV damage to bacteriophage DNA. The ras locus is located between lac and purE on the chromosome map.

The Escherichia coli lon − mutants apparently are defective in the ability to recommence cell division after temporary periods of deoxyribonucleic acid (DNA) synthesis inhibition. They are also more susceptible to cell division inhibition by the basic dye, crystal violet (CV), than are lon + strains. In enriched broth, the lon + strain continued to grow and divide in the presence of CV, but lon − cell division was inhibited and filamentous growth resulted. In a supplemented minimal medium containing CV, lon − cell division was only temporarily inhibited. There was no detectable specific effect on DNA synthesis, although CV slowed the rate of mass increase in both media. Trichloroacetic acid-insoluble lipid synthesis was preferentially inhibited in both lon + and lon − strains. In CV-containing enriched broth, diaminopimelic acid incorporation into trichloroacetic acid-insoluble compounds occurred at a rate greater than the rate of mass increase in both lon + and lon − strains. In a CV-containing supplemented minimal medium, diaminopimelic acid was incorporated to a greater extent by lon − cells than by lon + cells.

The Escherichia coli ras- mutant was sensitized to UV by bromodeoxyuridine. The extent of sensitization indicates that the ras- mutation probably increases UV-sensitivity by an effect on excision repair. This effect probably is uncontrolled degradation without concomitant resynthesis in a small proportion of the repair events.

The replicative polymerase of Escherichia coli, DNA polymerase III, consists of a three-subunit core polymerase plus seven accessory subunits. Of these seven, tau and gamma are products of one replication gene, dnaX. The shorter gamma is created from within the tau reading frame by a programmed ribosomal -1 frameshift over codons 428 and 429 followed by a stop codon in the new frame. Two temperature-sensitive mutations are available in dnaX. The 2016(Ts) mutation altered both tau and gamma by changing codon 118 from glycine to aspartate; the 36(Ts) mutation affected the activity only of tau because it altered codon 601 (from glutamate to lysine). Evidence which indicates that, of these two proteins, only the longer tau is essential includes the following. (i) The 36(Ts) mutation is a temperature-sensitive lethal allele, and overproduction of wild-type gamma cannot restore its growth. (ii) An allele which produced tau only could be substituted for the wild-type chromosomal gene, but a gamma-only allele could not substitute for the wild-type dnaX in the haploid state. Thus, the shorter subunit gamma is not essential, suggesting that tau can be substitute for the usual function(s) of gamma. Consistent with these results, we found that a functional polymerase was assembled from nine pure subunits in the absence of the gamma subunit. However, the possibility that, in cells growing without gamma, proteolysis of tau to form a gamma-like product in amounts below the Western blot (immunoblot) sensitivity level cannot be excluded.