A mutation in the gene for trigger factor suppresses the defect in cell division in the divE42 mutant of Escherichia coli K12

A total of sixteen spontaneously generated, independent suppressor mutants was isolated from a mutant (divE42) of Escherichia coli K12 that is defective in cell division. One of the suppressor mutants, designated TR4, had a novel phenotype: it was able to grow at 42° C but not at 32° C. The Kohara genomic library was screened for complementing clones. Clone 148 was able to complement the mutation responsible for the cold-sensitive phenotype, and the gene for trigger factor (tig), which encodes a ribosome-associated peptidyl-prolyl cis/trans isomerase, was identified as the mutated gene by deletion analysis with the insert DNA from clone 148. DNA sequencing revealed that the mutation in the tig gene of the TR4 suppressor mutant was a single nucleotide insertion (+A) at a distance of 834 nucleotides from the initiation codon for this enzyme. When the wild-type tig gene was introduced into the TR4 suppressor mutant, the bacteria were able to grow at 32° C but not at 42° C, an indication that the intergenic suppressor mutation was recessive to the wild-type allele. A model is proposed that accounts for the phenotypes of the divE42 mutant and the TR4 suppressor mutant. Received: 3 March 1998 / Accepted: 14 July 1998     

Studies on temperature-dependent ultraviolet light-sensitive mutants of bacteriophage T4: the structural gene for T4 endonuclease V.

Mutants of bacteriophage T4 which exhibit increased sensitivity to ultraviolet radiation specifically at high temperature were isolated after mutagenesis with hydroxylamine. At 42 °C the mutants are twice as sensitive to ultraviolet light as T4D, whereas at 30 °C they exhibit survival curves almost identical to that of the wild-type strain. Complementation tests revealed that the mutants possess temperature-sensitive mutations in the v gene.Evidence is presented to show that T4 endonuclease V produced by the mutants is more thermolabile than the enzyme of the wild-type. (1) Extracts of cells infected with the mutants were capable of excising pyrimidine dimers from ultraviolet irradiated T4 DNA at 30 °C, but no selective release of dimers was induced at 42 °C. (2) Endonuclease V produced by the mutant was inactivated more rapidly than was the enzyme from T4D-infected cells when the purified enzymes were incubated in a buffer at 42 °C. From these results it is evident that the v gene is the structural gene for T4 endonuclease V, which plays an essential role in the excision-repair of ultraviolet light-damaged DNA.The time of action of the repair endonuclease was determined by using the mutant. Survival of a temperature-sensitive v mutant, exposed to ultraviolet light, increased when infected cells were incubated at 30 °C for at least ten minutes and then transferred to 42 °C. It appears that repair of DNA proceeds during an early stage of phage development.     

Genetic interaction of DED1encoding a putative ATP-dependent RNA helicase with SRM1 encoding a mammalian RCC1 homolog in Saccharomyces cerevisiae

The Saccharomyces cerevisiae temperature-sensitive mutants srm1-1, mtr1-2 and prp20-1 carry alleles of a gene encoding a homolog of mammalian RCC1. In order to identify a protein interacting with RCC1, a series of suppressors of the srm1-1 mutation were isolated as cold-sensitive mutants and one of the mutants, designated ded1-21, was found to be defective in the DED1 gene. The double mutant, srm1-1 ded1-21, could grow at 35°?C, but not at 37°?C. A revertant of srm1-1 ded1-21 that became able to grow at 37°?C acquired another mutation in the SRM1 gene, indicating the tight relationship between SRM1 and DED1. In all the rcc1 ^- strains examined, the amount of mutated SRM1 proteins was reduced or not detectable at the nonpermissive temperature. While mutated SRM1 protein was stabilized in all of the rcc1 ^- strains by the ded1-21 mutation, the ded1-21 mutation suppressed both srm1-1 and mtr1-2, but not the prp20-1 mutation, contrary to the previous finding that overproduction of the S. cerevisiae Ran homolog GSP1 suppresses prp20-1, but not srm1-1 or mtr1-2.     

Ribosomal protein modification in Escherichia coli. I. A mutant lacking the N-terminal acetylation of protein S5 exhibits thermosensitivity.

A thermosensitive mutant (JE386) of Escherichia coli which harbours an alteration in protein S5 of the smaller ribosomal subunit has been isolated. Genetic studies have shown that the lesion causing thermosensitivity also causes the alteration in protein S5, and that this mutation is not in the structural gene for S5 (rpsE). Hence the mutation has been termed rimJ (ribosomal modification). Protein-chemical studies of protein S5 purified from JE386 and its wild-type parent indicated an alteration in the N-terminal tryptic peptide. Amino acid sequence analysis of the N-terminal peptides showed complete homology between wild-type and mutant, suggesting that the N-terminal modification (acetylation) of the parent was absent in the mutant. Gradient transmission mapping has located the rimJ mutation at 31 minutes on the current E. coli genetic map. By constructing a derivative of the mutant heterozygous for rimJ, it has been found that the wild-type allele is dominant over the mutant one. Ts⁺ revertants of JE386 have been isolated which show either a wild-type ribosomal protein electrophoresis pattern, or an additional alteration in either protein S4 or S5. The mutations in S4 and S5 may compensate the lesion caused by the rimJ mutation of JE386, that is even though the N-terminus of S5 remains unacetylated, bacteria can grow at 42 °C. Furthermore, a mutation near or at strA carried by JE386 has been found to be involved in the phenotypic expression of the rimJ mutation. This mutation was also found to be present in four other strA mutants. Possible implications of the modification of ribosomal proteins in vivo are discussed.     

ParD: a new gene coding for a protein required for chromosome partitioning and septum localization in Escherichia coli

A new gene, parD, has been located at 88.5 min on the genetic map of E. coli. Cells carrying an amber mutation in this gene, together with a temperature-sensitive suppressor tRNA, are able to grow, synthesize DNA and divide at both 30°C and 42°C. At 42°C, however, they are defective both in the separation of replicated chromosomes and in the placement of septa. Both the amount of DNA and the number of septa per cell mass are normal in cells growing at 42°C: only the localization of the chromosomes and septa are altered. As a result, cells of random sizes are produced at 42°C and the smallest of these contain no DNA.     

Eight UCA codons differentially affect the expression of the lacZ gene in the divE42 mutant of Escherichia coli

In the divE mutant, which has a temperature-sensitive mutation in the tRNA1(Ser) gene, the synthesis of beta-galactosidase is dramatically decreased at the non-permissive temperature. In Escherichia coli, the UCA codon is only recognized by tRNA1(Ser). Several genes containing UCA codons are normally expressed at 42 degrees C in the divE mutant. Therefore, it is unlikely that the defect is due to the general translational deficiency of the mutant tRNA1(Ser). In this study, we constructed mutant lacZ genes, in which one or several UCA codons at eight positions were replaced with other serine codons such as UCU or UCC, and we examined the expression of these mutant genes in the divE mutant. We found that a single UCA codon at position 6 or 462 was sufficient to cause the same level of reduced beta-galactosidase synthesis as that of the wild-type lacZ gene, and that the defect in beta-galactosidase synthesis was accompanied by a low level of lacZ mRNA. It was also found that introduction of an rne-1 pnp-7 double mutation restored the expression of mutant lacZ genes with only UCA codons at position 6 or 462. A polarity suppressor mutation in the rho gene had no effect on the defect in lacZ gene expression in the divE mutant. We propose a model to explain these results.     

The S / M checkpoint at 37°C and the recovery of viability of the mutant polδts3 require the crb2 + /rhp9 + gene in fission yeast

We have isolated a mutant in fission yeast, in which mitosis is uncoupled from completion of DNA replication when DNA synthesis is impaired by a thermosensitive mutation in the gene encoding the catalytic subunit of DNA polymerase δ. By functional complementation, we cloned the wild-type gene and identified it as the recently cloned checkpoint gene crb2 ⁺ /rhp9 ⁺ . This gene has been implicated in the DNA damage checkpoint and acts in the Chk1 pathway. Unlike the deleted strain dcrb2, cells bearing the crb2-1 allele were not affected in the DNA repair checkpoint after UV or MMS treatment at 30°?C, but were defective in this checkpoint function when treated with MMS at 37°?C. We analysed the involvement of Crb2 in the S/M checkpoint by blocking DNA replication with hydroxyurea, by using S phase cdc mutants, or by overexpression of the mutant PCNA L68S. Both crb2 mutants were unable to maintain the S/M checkpoint at 37°?C. Furthermore, the crb2 ⁺ gene was required, together with the cds1 ⁺ gene, for the S/M checkpoint at 30°?C. Finally, both the crb2 deletion and the crb2-1 allele induced a rapid death phenotype in the polδts3 background at both 30°?C and 37°?C. The rapid death phenotype was independent of the checkpoint functions.     

Ribosomal assembly deficiency in an Escherichia coli thermosensitive mutant having an altered L24 ribosomal protein.

Localized P1 mutagenesis was used to screen for conditionally lethal mutations in ribosomal protein genes. One such mutation, 2859mis, has been mapped inside the ribosomal protein gene cluster at 72 minutes on the Escherichia coli chromosome and cotransduces at 98% with rpsE (S5). The 2869mis mutation leads to thermosensitivity and impaired assembly in vivo of 50 S ribosomal particles at 42 °C. The strain carrying the mutation has an altered L24 ribosomal protein which at 42 °C shows weaker affinity for 23 S RNA than the wild-type protein. The mutational alteration involves a replacement of glycine by aspartic acid in protein L24 from the mutant. We conclude therefore that the 2859mis mutation affects the structural gene for protein L24 (rplX).     

A mutant of Escherichia coli blocked in peptide elongation: altered elongation factor Ts

Among our transfer RNA-dependent growth mutants, one, HAK88, was found that carries an altered elongation factor Ts. The activity of mutant EFTs to bind GDP to EFTu, or to form the ternary complex (aminoacyl-tRNA-EFTu-GTP) is thermolabile. The effect of magnesium on the formation of EFTu-GDP from the EFTu-EFTs complex of HAK8 shows that a four to fivefold increase of the duplex formation occurs when the magnesium concentration is increased from 10^?6m to 10^?2m at 0 °C and at 41 °C. However, at higher temperatures, formation of the binary EFTu-GDP from the EFTu-EFTs complex of HAK88 is depressed, even at 10^?3m to 10^?2m-magnesium. The binding of GDP to the wild-type or mutant EFTu-EFTs complex at 0 °C and 42 °C indicates that the formation of EFTu-GDP is inhibited at 42 °C only when mutant complex is used for the assay. Binding of GTP to complete bacteriophage Qβ replicase (which is known to contain EFTs) formed in phage-infected HAK88 is also inhibited at 42 °C.     

A conditional lethal mutation in an Escherichia coli strain with a longer chemical lifetime of messenger RNA.

We have analysed an Escherichia coli temperature-sensitive mutant with altered messenger RNA stability, and it was found that: (1) the unstable fraction of pulse-labelled RNAs decayed with a half-life at 42 °C of about two minutes in the parent strain PA3092; the half-life was 11 to 12 minutes in the mutant HAK75. Puromycin enhanced the decay rate about twofold in both PA3092 and HAK75; the addition of chloramphenicol inhibited the degradation significantly in both strains. The rate of ribosomal RNA accumulation in the mutant cells at 42 °C did not differ from that in the wild-type cells. (2) Sedimentation analysis by sodium dodecyl sulphate/sucrose density-gradient centrifugation of bulk mRNA as well as tryptophan mRNA of the wild-type strain showed the expected rapid reduction in the size and level of those mRNA molecules at three minutes and five minutes respectively, after addition of rifampicin at 42 °C. In contrast, the cells of HAK75 retained almost full-length trp mRNA and bulk mRNA at 5 to 12 minutes after the addition of rifampicin at 42 °C, even though the total level of radioactivity in the mRNA fraction had decreased to about 60 to 75% of the initial activity. (3) Even though mRNA molecules were chemically protected at the non-permissive temperature in the mutant, the functional decay of both β-galactosidase and tryptophan synthetase occurred at a rate comparable to that in the parental strain. (4) We isolated temperature-resistant revertants from the mutant at a frequency of 5 × 10^?8, and these revertants (TR1 and TR2) had the normal decay rate of unstable RNA.     

Suppression of a +1 T mutation by a nearby substitution in the mitochondrial cox1 gene of Chlamydomonas reinhardtii : a new type of frameshift suppression in an organelle genome

In Chlamydomonas reinhardtii, mutants defective in the cytochrome pathway of respiration lack the capacity to grow under heterotrophic conditions (in darkness on acetate). In the dark^? strain dum18, a +1?T addition in a run of four Ts, located at codon 145 of the mitochondrial cox1 gene encoding subunit I of cytochrome c oxidase, is responsible for the mutant phenotype. A leaky revertant (su11) that grows heterotrophically at a lower rate than wild-type cells was isolated from dum18. Its respiration sensitivity to cyanide was low and its cytochrome c oxidase activity was only 4% of that of the wild-type enzyme. Meiotic progeny obtained from crosses between revertant and wild-type cells inherited the phenotype of the mt ^? parent, showing that the suppressor mutation, like dum18 itself, is located in the mitochondrial genome. In order to map the su11 mutation relative to dum18, a recombinational analysis was performed on the diploid progeny. It demonstrated that su11 was very closely linked to the dum18 mutation – less than 20–30?bp away. The cox1 gene of the su11 revertant was then sequenced. In addition to the +1?T frameshift mutation still present at codon 145, an A?→?C substitution was found at codon 146, leading to the replacement of a glutamic acid by an alanine in the polypeptide chain. No other mutations were detected in the cox1 coding sequence. As the new GCG codon (Ala) created at position 146 is very seldom used in the mitochondrial genome of C. reinhardtii, we suggest that the partial frameshift suppression by the nearby substitution is due to an occasional abnormal translocation of the ribosome (+1 base shift) facilitated both by the run of Ts and the low level of weak interaction of alanyl-tRNA.     

A New Fungal Metabolite and Root Growth-stimulating Activity of Its Methyl Ester

Escherichia coli rodA mutant AOS151 grows as round cells at 30 and 42°C (H. Matsuzawa, K. Hayakawa, T. Sato, and K. Imahori, J. Bacteriol., 115, 436–442 (1973)). The mutant was found to be resistant to mecillinam at both temperatures. lip⁺ transductants were prepared by Pl phage transduction via strain AOS151, the cotransduction frequency of round morphology (Rod^?) at 42°C with the lip gene being about 90%. At 42°C all 54 Rod^? transductants tested were resistant to mecillinam. At 30°C all but two of these Rod^? (at 42°C)-type transductants were rod-shaped, and all were sensitive to mecillinam; the two strains grew as ovoid cells. The original rodA mutant AOS151 probably involves an additional mutation(s), that expresses the round cell shape at lower temperature, whereas the rodA51 mutation alone seems to result in temperature-sensitive expression of round cell morphology and mecillinam resistance. rodA mutant cells cultured at either 30 or 42°C had wild-type penicillin-binding protein 2, judging from penicillin-binding activity, electrophoretic mobility, and thermosensitivity.     

Genetic and enzymatic characterization of conditional lethal mutants of the yeast Schizosaccharomyces pombe with a temperature-sensitive DNA ligase.

The DNA ligase activities of wild type and temperature-sensitive lethal cdc 17 mutants of Schizosaccharomyces pombe have been studied by measuring effects on the conversion of relaxed DNA circles containing a single nick to a closed circular form. Such assays have revealed that all cdc 17 mutants have a thermosensitive DNA ligase deficiency, that this deficiency cosegregates 2:2 with their temperature-sensitive cdc-lethality in three tetrads derived from a cross against wild type, and that genetic reversion of the temperature-sensitive cdc^? phenotype is accompanied by a restoration of DNA ligase activity; all of which implies that the temperature-sensitive cdc^? phenotype of cdc 17 mutants is due to a single nuclear mutation causing a DNA ligase deficiency. Both wild type and mutant enzymes have been partially purified by chromatography in heparin/agarose columns. The wild-type enzyme is completely stable in vitro at both permissive (25 °C) and restrictive (35 °C) temperatures, whereas that of two different mutants, though completely stable at 25 °C, is rapidly inactivated at 35 °C, implying that their mutations are located in the structural gene for DNA ligase.     

Isolation and Characterization of a Temperature-sensitive Sporulation Mutant of Bacillus subtilis

Temperature-sensitive sporulation mutants were isolated spontaneously from Bacillus subtilis 168 TT by a sequential transfer method. A representative mutant strain, ts32, was characterized in detail. The mutant grew normally at 30°C and 42°C, but did not sporulate at 42°C. Electron microscopic observation and physiological analysis showed that the mutant was blocked at stage 0-1 of sporulation. Genetic analysis suggested that the mutation was located at the spo0B locus on the B. subtilis chromosome. Temperature-shift experiments clearly showed that the spo0B gene product functions only at the beginning of sporulation.     

The Effect of Growth Temperature on the Pathogenicity of Campylobacter

Control of Campylobacter in the food chain requires a better understanding of the behaviour of the bacteria in relevant environments. Campylobacter species are largely non-pathogenic in poultry, the body temperature of which is 42 °C. However, the bacteria are highly pathogenic in humans whose body temperature is 37 °C. The aim of this study was to examine if switching from commensal to pathogenic behaviour was related to temperature. We examined the growth, motility and invasion of T84 cells by three species of Campylobacter: C. jejuni 81116, C. jejuni M1, C. coli 1669, C. coli RM2228 and C. fetus fetus NC10842 grown at 37 and 42 °C. Our results suggest that C. jejuni isolates grow similarly at both temperatures but some are more motile at 42 °C and some are more invasive at 37 °C, which may account for its rapid spread in poultry flocks and for infection in humans, respectively. C. coli, which are infrequent causes of Campylobacter infections in humans, is less able to grow and move at 37 °C compared to 42 °C but was significantly more invasive at the lower temperature. C. fetus fetus, which is infrequently found in poultry, is less able to grow and invade at 42 °C.     

Studies on the bacteriophage MS2: XLI. Nature of the azure mutation

Azure (or reverse amber) mutants grow normally on wild-type Escherichia coli but not on host strains harbouring a strong UAG suppressor mutation. Three different bacteriophage MS2 azure mutants obtained by treatment with nitrous acid have been characterized at the nucleotide sequence level. The 3′-end fragment of the ³²P-labelled mutant genomes was isolated by DNA:RNA hybridization and treatment with nuclease S₁, and was analyzed by mini-fingerprinting of the RNA. It is known that the wild-type MS2 polymerase gene ends with a UAG codon, followed seven triplets further by an in-phase UAA triplet. All three azure mutants contained an A → G transition in this UAA second stop codon of the polymerase gene, resulting in a second suppressible UAG (amber) codon. Analysis of revertants demonstrated that the azure mutation can be counteracted either by a true site reversion at the second stop or by the creation of a new stop signal for the polymerase gene, either UAA (ochre) or UGA (opal), before or at the first stop, or beyond the second stop. On the basis of these results, a mechanism for the azure mutation is proposed. Silent mutations (one in the coding region, three in the untranslated 3′-terminal sequence) have also been observed in these phage stocks.     

New killing system controlled by two genes located immediately upstream of the mukB gene in Escherichia coli

The nucleotide sequence was determined of the region upstream of the mukB gene of Escherichia coli. Two new genes were found, designated kicA and kicB (killing of cell); the gene order is kicB-kicA-mukB. Promoter activities were detected in the regions immediately upstream of kicB and kicA, but not in front of mukB. Gene disruption experiments revealed that the kicA disruptant was nonviable, but the kicB-disrupted mutant and the mutant lacking both the kicB and kicA genes were able to grow. When kicA disruptant cells bearing a temperature-sensitive replication plasmid carrying the kicA ⁺ gene were grown at 30° C and then transferred to 42° C, the mutant cells gradually lost colony-forming ability, even in the presence of a mukB ⁺ plasmid. Rates of protein synthesis, but not of RNA or DNA synthesis, fell dramatically during incubation at 42° C. These results suggested that the kicB gene encodes a killing factor and the kicA gene codes for a protein that suppresses the killing function of the kicB gene product. It was also demonstrated that KicA and KicB can function as a post-segregational killing system, when the genes are transferred from the E. coli chromosome onto a plasmid.