Imparired DNA synthesis and envelope defect in a mutant of Salmonella typhimurium

Summary S. typhimurium mutants with temperature-sensitive synthesis of DNA have been isolated. One of these mutants,dna-26, has been studied in detail. DNA synthesis is stopped indna-26 without any residual replication after shift to 42° though increase in cell mass is not inhibited. Mutantdna-26 shows increased sensitivity to deoxycholate, to nalidixic acid and rifampicin. This suggests a cell envelope defect. Inhibition of DNA synthesis at 42° can be phenotypically cured indna-26 by 0.25 M NaCl and KCl and 0.44 M sucrose but not by 0.44 M glycerol. This DNA synthesis induced by hypertonic medium proceeds at a slower rate than increase in cell mass but is predominantly due to normal sequential chromosome replication. The position of mutationdna-26 has been approximately mapped in thepurD region of the chromosome.     

Temperature-sensitive,lipopolysaccharide-deficient mutants of Salmonella typhimurium

Two mutants of Salmonella typhimurium LT2, which were temperature-sensitive for lipopolysaccharide (LPS) synthesis, were isolated from a galE ^- strain based on their resistance to phage C21 and sensitivity to sodium deoxycholate at 42°C. They produced LPS of chemotype Rc at 30°C and deep-rough LPS at 42°C. P22-mediated transductional analysis showed that the mutations responsible for temperature sensitivity are located in the rfa cluster where several genes involved in the synthesis of the LPS core are mapped. A plasmid, carrying rfaC, D and F genes of Escherichia coli K-12, complemented these mutations. These genes are responsible for the synthesis of the inner-core region of the LPS molecule. This indicates that genetic defects in these temperature-sensitive mutants affect the inner-core region of LPS.     

DNA synthesis in P1 infected E. coli mutants temperature-sensitive in DNA replication

Summary P1 DNA is synthesized in the E. coli ts dna mutants 165/70 (elongation defect) and 252 (initiation defect) at elevated temperatures. In strain 165/70, P1 infection at 41°C leads to phage production accompanied by a transient recovery of bacterial DNA synthesis. No phages are produced byt P1 DNA is still synthesized in strain 252 if infected after host DNA replication has come to a halt at 42°C.     

Temperature-sensitive initiation of DNA replication in a mutant of Escherichia coli K12

Summary A mutant of E. coli K12 appears to be temperature-sensitive in the process of initiation of DNA replication. After a temperature shift from 33 to 42°C, the amount of residual DNA synthesis (Fig. 1) and the number of residual cell divisions (Figs. 2,4) indicate that rounds of DNA replication in process are completed, but new rounds cannot be initiated. Following the alignment of chromosomal DNA by amino acid starvation at 33° C no residual DNA synthesis at 42°C takes place (Fig. 5). When the temperature is lowered to 33°C after a period of inhibition at 42°C, the following observations are made: 1. DNA replication resumes and proceeds synchroneously, (Figs. 7, 8a), 2. cells start to divide again only after a lag period of about 1 hour 3. a temporary increase in cell volume is correlated with the frequency of initiation of DNA synthesis (Fig. 8a, b). In a lysogenic mutant strain prophage is inducible; with all bacteriophages tested, replication of phage DNA is not inhibited at 42°C.     

Nuclear mutants of Neurospora crassa temperature-sensitive for the synthesis of cytochrome aa3. I. Isolation and preliminary characterization.

Summary Three nuclear mutants of Neurospora crassa, temperature-sensitive for the synthesis of cytochrome aa ₃ have been isolated. When grown at 41°C the mutants have large amounts of KCN-insensitive respiration, reduced amounts of cytochrome aa ₃ and cytochrome c oxidase activity, and grow more slowly than wild-type cultures grown at the same temperature. When the mutants are grown at 23°C, they are virtually indistinguishable from wild-type strains.The mutants were selected on the basis of their slow growth at 41°C in medium containing salicylhydroxamic acid, and by their inability to reduce 2,3,5-triphenyltetrazolium chloride at 41°C. The selection technique was designed to eliminate mutants that did not carry thermolabile electron transport chain components. However, studies on the thermolability of the cytochrome oxidase activity in isolated mitochondria indicate that the enzyme of the mutants is no more susceptible to heat denaturation than is the enzyme in wild-type mitochondria. This suggests that the synthesis or assembly of cytochrome aa ₃ may be altered in the mutants at the restrictive temperature.Supported by National Research Council of Canada Grant Number A-6351Recipient of a National Research Council of Canada Postgraduate Scholarship     

Participation of the host protein(s) in the morphogenesis of bacteriophage P22

Summary Spontaneous mutants of S. typhimurium resistant to thiolutin are conditionally non-permissive for phage P22 development (Joshi and Chakravorty 1979). At 40° C non-infective phage particles are produced. Phage development in two nonpermissive hosts (18/MC4 and 153/MC4) has been studied in detail. The steps at which the phage morphogenesis is interfered with differ in the two mutants. The electron micrograph of the particles produced in the mutant 18/MC4 reveals the presence of normal-looking particles; these particles contain phage DNA, adsorb to the permissive host but fail to inject their DNA. The particles produced in the mutant 153/MC4 which fail to adsorb to the host are found to be tail fibre-less. These observations indicate the involvement of host protein(s) in phage P22 morphogenesis.     

Inhibition of Bacteriophage φX174 DNA Replication in dnaB Mutants of Escherichia coli C

Bacteriophage phiX174 DNA replication was examined in temperature-sensitive dnaB mutants of Escherichia coli C to determine which stages require the participation of the product of this host gene. The conversion of the infecting phage single-stranded DNA to the double-stranded replicative form (parental RF synthesis) is completely inhibited at the nonpermissive temperature (41 C) in two of the three dnaB mutants tested. The efficiency of phage eclipse and of phage DNA penetration of these mutant host cells at 41 C is the same as that of the parent host strain. The defect is most likely in the synthesis of the complementary strand DNA. The semiconservative replication of the double-stranded replicative form DNA (RF replication) is inhibited in all three host mutants after shifting from 30 to 41 C. Late in infection, the rate of progeny single-stranded phage DNA synthesis increases following shifts from 30 to 41 C. Approximately the same amounts of phage DNA and of infectious phage particles are made following the shift to 41 C as in the control left at 30 C. The simplest interpretation of our data is that the product of the host dnaB gene is required for phiX174 parental RF synthesis and RF replication, but is not directly involved in phage single-stranded DNA synthesis once it has begun. The possible significance of the synthesis of parental RF DNA at 41 C in one of the three mutants is discussed.     

Regulation of polar surface structures in Caulobacter crescentus: Pleiotropic mutations affect the coordinate morphogenesis of flagella, pili and phage receptors

Summary A large number of Caulobacter mutants resistant to DNA or RNA phages were isolated. These phage-resistant mutants exhibited phenotypic variations with respect to cell motility and sensitivity to other phages.The majority of the mutants was resistant to both DNA and RNA phages tested. In addition, these mutants were either motile or non-motile. The analysis of spontaneous revertants from these mutants indicated that a single mutation is involved in these phenotypic variations. Other mutants were resistant to RNA phages and only to a certain DNA phage tested, and were also motile or non-motile.Several temperature-sensitive phage-resistant mutants were also isolated. One of them, CB13 ple-801, exhibited the wild type phenotype when grown at 25°C. However, at a higher temperature (35°C), the mutant cells became non-motile and resistant to both DNA and RNA phages. These phenotypes seem to be attributed to the concommitant loss of flagella, pili and phage receptors. In other respects (cell growth and morphology, and asymmetric stalk formation), CB13 ple-801 was normal at 35°C. The spontaneous revertants from CB13 ple-801 simultaneously regained the wild type phenotypes in all respects.It is suggested that a single mutation pleiotropically affects the formation of flagella, pili and phage receptors.     

DNA synthesis in an Escherichia coli dna B dnaC mutant.

Summary An Escherichia coli K12 dnaB dnaC mutant was constructed by P1 transduction of the dnaC allele into a dnaB recipient strain dnaB dnaC transductants were discriminated from dnaB mutants by their inability to grow at 40° C after lysogenization with phage P1bac. The dnaB dnaC mutant character was verified by 1. P1 transduction, and 2. by in vitro complementation with dnaB and dnaC wild type protein fractions.DNA synthesis was studied in strains containing dnaB, dnaC, or dnaB dnaC alleles in an otherwise uniform genetic background with the dnaB character either unsuppressed or suppressed by P1bac prophage. Degradation at 42° C of [³H]-thymidine pulselabeled DNA in dnaB and dnaB dnaC mutants is suppressed by P1bac. However, unlike the dnaC mutant, the P1bac lysogen of the dnaB dnaC mutant exhibits an abrupt cessation of DNA synthesis and less residual cell divisions at 42° C indicating an inhibition of DNA chain elongation rather than a defect in DNA initiation. It is suggested that denaturation of the dnaB protein affects the dnaC function.     

Identification of ten genes that control ribosome formation in yeast

Summary Twenty-three temperature-sensitive mutants of Saccharomyces cerevisiae, all of which undergo a rapid cessation of net RNA accumulation following a shift from the permissive (23°) to the restrictive temperature (36°), have been characterized. Genetic studies demonstrate that these mutants belong to ten different complementation groups and that, in most cases, their properties are the result of a single, recessive mutation in a nuclear gene. Although the mutants were isolated for heat sensitivity, mutants from 2 of the complementation groups are cold sensitive (at 13°) as well. The mutants continue to synthesize protein, including an enzyme, alkaline phosphatase, for two to four hours following a shift from 23° to 36°, suggesting that they are capable of messenger RNA synthesis and the translation of messenger RNA with fidelity at the restrictive temperature. The small amount of RNA that is synthesized in these mutants at the restrictive temperature has been examined on sucrose gradients and by acrylmide gel electrophoresis; in addition, the RNA components in polyribosomes have been fractionated by a new technique that separates messenger RNA from ribosomal RNA. As a result of these analyses we conclude that these mutants are strongly inhibited in the accumulation of 5S, 7S, 17S, and 25S RNA components but are only slight if at all inhibited in the synthesis of messenger RNA and 4S RNA. The results reported here define ten genes, designated rna 2 through rna 11, that play an essential role in the formation or maturation of ribosomes in yeast.     

Physiological and genetical studies on a mutant of Salmonella typhimurium which is temperature-sensitive for DNA synthesis

Summary The following evidence supports the view that a temperature-sensitive mutant of Salmonella typhimurium (11 G) is defective in DNA synthesis initiation: a) the increment in DNA synthesis at 38° is abolished by prior completion of rounds of replication at 25°. b) The extent of the increment at 38° is greatly increased by prior growth in the presence of a DNA synthesis inhibitor. c) Density gradient centrifugation demonstrates that the terminal region of the chromosomes is preferentially replicated at 38°. d) Preferential replication of the chromosome origins occurs at 25° after a period at 38°. The parental strain in the presence of a DNA synthesis inhibitor or the mutant at 38° (without inhibitor) show increased sensitivity to the detergent sodium deoxycholate, possibly due to a secondary effect of DNA synthesis inhibition on membrane composition. Strains of 11 G carrying episomes transfer the episomes very poorly at 38° suggesting a rôle for the chromosomal initiation apparatus in episome transfer. Continued cell division of 11 G with the production of DNA-less cells at 38° is not due to the presence of a rec mutation and no secondary mutation responsible for such division has been found. The lesion maps close to leu on the Salmonella chromosome.     

Isolation and genetic analysis of temperature-sensitive mutants of B. subtilis defective in DNA synthesis

Summary Fifty temperature-sensitive mutants defective in DNA synthesis at high temperature have been identified among 655 temperature-sensitive mutants isolated at random from a mutagenised population of B. subtilis. They are distributed in a non-random fashion in 9 genetic linkage groups, located in different regions of the B. subtilis genome. It is suggested that at least 14 genes are involved in B. subtilis DNA replication.     

Replication of bacteriophages in Escherichia coli mutants thermosensitive in DNA synthesis

Summary In E. coli mutants thermosensitive in DNA synthesis the capacity for replication of bacteriophages , P1 and T4 was studied in order to obtain more information about the biochemical lesions in such strains. Two mutant types were used. In one of them DNA synthesis stops immediately at the restrictive temperature (mutant 165/70). In the other type DNA synthesis continues at the elevated temperature for a residual time period before it comes to a halt (mutant 252). The thermolabile synthetic steps involved in both mutant types are presently still unknown.The temperate phages and P1 differ in their ability to replicate in the mutant types at temperatures non-permissive for host cell DNA synthesis. Replication of phage is blocked in 165/70 but can still take place in 252 after host DNA synthesis has come to a halt. Phage P1 shows the opposite behaviour. It grows in the mutant 165/70 but its ability to replicate in 252 at 42° C is restricted to the period of residual host cell DNA synthesis observed in uninfected cells. Replication of phage T4 on the other hand is unimpeded in both mutants at restrictive temperatures.     

Synthesis of tryptophanase in Escherichia coli: Isolation and characterization of a structural-gene mutant and two regulatory mutants

Summary A mutant of E. coli has been isolated that is temperature-sensitive in respect of tryptophanase. When incubated at 60°C, cell-free extracts of the mutant suffer inactivation of enzyme activity much more rapidly than similar extracts of the wild type. After lysogeny with a specialized transducing phage carrying the wild-type tryptophanase gene, the mutant is able to synthesize tryptophanase that is wild-type in its response to treatment at 60°C. It is concluded that the mutation lies in the structural gene for the enzyme.Two further mutants have been isolated that synthesize tryptophanase constitutively. One mutation renders synthesis of the enzyme indifferent to the presence of inducer; the other mutation allows synthesis of the enzyme in the absence of inducer at about 35% of the fully induced wild-type rate. Neither mutation alleviates catabolite repression. Genetic mapping shows that the constitutive mutations lie very close to the structural-gene mutation, on the side of the structural gene distant from bglR.     

Amino Acid Control over Deoxyribonucleic Acid Synthesis in Escherichia coli Infected With T-Even Bacteriophage

Starvation for a required amino acid of normal or RC(str)Escherichia coli infected with T-even phages arrests further synthesis of phage deoxyribonucleic acid (DNA). This amino acid control over phage DNA synthesis does not occur in RC(rel)E. coli mutants. Heat inactivation of a temperature-sensitive aminoacyl-transfer ribonucleic acid (RNA) synthetase similarly causes an arrest of phage DNA synthesis in infected cells of RC(str) phenotype but not in cells of RC(rel) phenotype. Inhibition of phage DNA synthesis in amino acid-starved RC(str) host cells can be reversed by addition of chloramphenicol to the culture. Thus, the general features of amino acid control over T-even phage DNA synthesis are entirely analogous to those known for amino acid control over net RNA synthesis of uninfected bacteria. This analogy shows that the bacterial rel locus controls a wider range of macromolecular syntheses than had been previously thought.     

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.     

Genetic studies on Rhizobiophage 16-3

Summary A number of temperature-sensitive (ts) and lysis-deficient mutants were isolated from a bacteriophage of Rhizobium meliloti. The ts mutants were grouped by complementation. Functions were classified in relation to the eclipse and latent period. A genetic map of about 40 units was derived from crosses. The genes on the chromosome of the phage are arranged in the order of their presumed functions during the phage growth. They are located on the chromosome in sequence: immunity-early-late-lysis genes.Abbreviations Ant altered antigene - C immunity gene - h host range - K velocity constant of antiphage serum - L lysis gene - m.o.i. multiplicity of infection - NTG N-methyl-N-nitro-Nnitrosoguanidine - P.F.U. plaque forming unit - Rh.41 Rhizobium meliloti strain 41 - ti thermo-inducible - ts temperature-sensitive     

Altered mitochondrial ribosomes in a cold-sensitive mutant of Saccharomyces cerevisiae

A mutation at a new locus denotedtsr1 which lies very close to theery1 locus and 21S rRNA gene in mitochondrial DNA ofSaccharomyces cerevisiae, confers conditional respiratory deficiency on cells grown at low temperature, namely 18°. Studies on mitochondria isolated from a strain carrying the mutatedtsr1 locus demonstrate that the rate of mitochondrial protein synthesis is cold-sensitive at 18°. The large subunit of the mitochondrial ribosomes isolated from the mutant strain is unstable during extraction and the isolated ribosomes are shown to be defective in catalyzing the poly U-directed synthesis of polyphenylalanine. It is concluded that thetsr1 locus is involved in the determination of the properties of the large subunit of the mitochondrial ribosome.     

A mutant ofLactobacillus acidophilus with temperature-sensitive regulation of DNA synthesis

Among other temperature-sensitive mutants ofLactobacillus acidophilus the mutant “ts 9” with temperature-sensitive initiation of DNA synthesis was isolated. In this mutant, the course of DNA synthesis under non-permissive conditions proceeds in two phases. During the first 90–120 min, a slight increase (20–50%) of DNA content takes place. Then during further incubation at 40°C, the capacity for initiation of further DNA synthesis increases and a second round of DNA synthesis starts after 3–4h of incubation. The initiation of DNA synthesis is prevented by chloramphenicol and the preceding lag is temperature-dependent. It is concluded that an accumulation of an initiation factor is required for the onset of a new cycle of DNA synthesis and that in thets 9 mutant this accumulation is inhibited at non-permissive temperature.