Studies on the metabolic role of peptidyl-tRNA hydrolase

Summary A mutant strain of Eschrichia coli that is temperature-sensitive for growth stopped protein biosynthesis at 43° C after a brief lag (Fig. 1). Cell-free extracts from the strain showed no specific defect in aminoacyl-tRNA synthetases, binding initiator tRNA to ribosomes (Table 1), protein chain elongation (Tables 2, 5) or protein chain termination (Tables 3, 4) at high temperature.The partially purified enzyme peptidyl-tRNA hydrolase, however, was temperature-sensitive (Table 6); the mutant hydrolase was inactivated rapidly at 43° C (Table 7). Mixing experiments ruled out the presence, in the mutant enzyme preparation, of an inhibitor and also demonstrated, on the mutant enzyme, a protective effect by wild type enzyme that was not shown by general coli proteins (Tables 8, 9).Interrupted mating allowed the temperature-sensitive growth phenotype to be mapped near to and before trp (Figs. 4, 5). Co-transsduction, mediated by bacteriophage P1, with trp ⁺ (frequency 7.5%) located the marker at 24 min on the coli map. All transductants for temperature-sensitive growth also had temperature-sensitive peptidyl-tRNA hydrolase activity in crude sonicates (Table 10). We provisionally conclude that the temperature-sensitive protein synthesis and growth are caused by a single genetic change in the structural gene (pth) for peptidyl-tRNA hydrolase.After shift to 43° C the polysomes of the mutant cells broke down into 70S particles (Figs. 2, 3). A defect in protein biosynthesis thus appeared to be located after termination and before reformation of new polysomes.The metabolic role of peptidyl-tRNA hydrolase is discussed in the light of these experiments.Journal paper No. J-7465 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa, project no. 1747.     

Plasmid pKM101-mediated mutagenesis in Escherichia coli is inducible

Summary A tif-1 umuC36 double mutant of Escherichia coli was constructed. It has been found that the umuC36 mutation prevents both increased spontaneous mutagenesis and enhanced reactivation of UV-irradiated , phenomena normally observed in the tif-1 strain grown at 42°C. When the plasmid pKM101 was introduced into tif-1 umuC36, an elevated spontaneous reversion rate of the his-4 mutation observed at 30°C was further increased 6-fold at 42°C. This was accompanied by a 10-fold increase in the ability of tif-1umuC36 containing pKM101 and grown before infection at 42°C to reactivate UV-irradiated .     

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.     

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.     

Amber mutations in the structural gene for RNA polymerase sigma factor of Escherichia coli

Summary Amber mutants of Escherichia coli K-12 affected in the structural gene (rpoD) for th subunit of RNA polymerase have been obtained from a strain harboring a temperature-sensitive amber suppressor (supF-Ts6) which is active only at low temperatures. These mutants grow normally at low temperature (30°C) but do not grow at high temperature (42°C) due to the inability to synthesize factor. In one mutant studied in detail (rpoD40), the rate of -factor synthesis at 30°C is about half that of the wild type and is decreased to 10%–15% within 1 h of incubation at 42°C. The synthesis of core polymerase subunits or bulk protein is virtually unaffected at least for 2 h. The defect of the mutant in synthesis and growth at high temperature can be suppressed by any of the amber suppressors tested (supD, supE or supF). RNA-polymerase holoenzymes prepared from the mutant cells carrying each of the suppressors (grown at 42°C) exhibit different thermostabilities attributable to alterations in the factor. The reduced synthesis in the mutant is accompanied by the synthesis of polypeptide tentatively identified as amber fragment. These results as well as the genetic mapping data indicate that the amber mutation (rpoD40) resides within the structural gene for the factor and directly affects synthesis upon inactivation of the suppressor at high temperature.     

A functional analysis of the repeated methionine initiator tRNA genes (IMT) in yeast

Summary Standard laboratory yeast strains have from four to five genes encoding the methionine initiator tRNA (IMT). Strain S288C has four IMT genes with identical coding sequences that are colinear with the RNA sequence of tRNA _I ^Met . Each of the four IMT genes from strain S288C is located on a different chromosome. A fifth IMT gene with the same coding sequence is present in strain A364A but not in S288C. By making combinations of null alleles in strain S288C, we show that each of the four IMT genes is functional and that tRNA _I ^Met is not limiting in yeast strains with three or more intact genes. Strains containing a single IMT2, 3 or 4 gene grow only after amplification of the remaining IMT gene. Strains with only the IMT1 gene intact are viable but grow extremely slowly; normal growth is restored by the addition of another IMT gene by transformation, providing a direct test for IMT function.Abbreviations IMT and imt (imt=initiator methionine tRNA), designate the genotype of the wild-type and the mutant alleles respectively, of the initiator methionine transfer RNA gene - met-tRNA _I ^Met methionylated initiator methionine transfer RNA - eIF-2 eukaryotic initiation factor two - GTP guanosine 5-triphosphate The calculation of Td values (the temperature at which half of the duplex is dissociated) for oligonucleotides used as probes in hybridizations was based on the assumption that the increase in Td value was 4° C for each G:C base pair and 2° C for each A:T base pair (Wallace et al. 1981)     

Nuclear and mitochondrial revertants of a mitochondrial mutant with a defect in the ATP synthetase complex

Summary Yeast strain 990 carries a mutation mapping to the oli1 locus of the mitochondrial genome, the gene encoding ATPase subunit 9. DNA sequence analysis indicated a substitution of valine for alanine at residue 22 of the protein. The strain failed to grow on nonfermentable carbon sources such as glycerol at low temperature (20°C). At 28°C the strain grew on nonfermentable carbon sources and was resistant to the antibiotic oligomycin. ATPase activity in mitochondria isolated from 990 was reduced relative to the wild-type strain from which it was derived, but the residual activity was oligomycin resistant. Subunit 9 (the DCCD-binding proteolipid) from the mutant strain exhibited reduced mobility in SDS-polyacrylamide gels relative to the wild-type proteolipid. Ten revertant strains of 990 were analyzed. All restored the ability to grow on glycerol at 20°C. Mitotic segregation data showed that eight of the ten revertants were attributable to mitochondrial genetic events and two were caused by nuclear events since they appeared to be recessive nuclear suppressors. These nuclear mutations retained partial resistance to oligomycin and did not alter the electrophoretic behavior of subunit 9 or any other ATPase subunit. When mitochondrial DNA from each of the revertant strains was hybridized with an oligonucleotide probe covering the oli1 mutation, seven of the mitochondrial revertants were found to be true revertants and one a second mutation at the site of the original 990 mutation. The oli1 gene from this strain contained a substitution of glycine for valine at residue 22. The proteolipid isolated from this strain had increased electrophoretic mobility relative to the wild-type proteolipid.Abbreviations DCCD dicyclohexylcarbodiimide - SDS sodium dodecyl sulfate - PMSF phenylmethylsulfonyl fluoride - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonate - SMP submitochondrial particles - mit^- mitochondrial point mutant     

Radiation sensitivity of a mutant of Escherichia coli K-12 associated with DNA replication: Evidence for a new repair function

Summary The isolation and properties of a new radiation sensitive mutant of Escherichia coli K-12 are described which shows a correlation between radiation sensitivity and replication of irradiated DNA. The mutation, called rer, is located between argB and purD loci. The mutant, when grown in tryptone broth after irradiation, is sensitive to UV and -rays and incorporates little or no ³H-thymidine but in minimal glucose-salts medium both the radiation sensitivity and incorporation of ³H-thymidine remain identical to that of the parent strain. Studies with a temperature sensitive double mutant rer dnaC show that 1 hr incubation of irradiated cells at 42° C before their transfer to 30° C results in higher survival as compared to their incubation at 30° C only. It is suggested that rer controls the replication of irradiated DNA and thus regulates the coordination between replication and repair of DNA.     

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.     

Regulation of histidine biosynthetic enzymes in a mutant of Escherichia coli with an altered histidyl-tRNA synthetase

Summary A mutant of Escherichia coli was isolated that grew at a normal rate in minimal medium at 26°C, grew at a normal rate in minimal medium at 37°C only if exogenous histidine was supplied, and grew more slowly than normal at 42°C even in the presence of histidine. In very rich media the growth rate of the mutant was normal at 26°C and 30°C, but not at 37°C or 42°C. It may be described as a temperature-conditional histidine bradytroph with a decreased ceiling to its growth rate.The histidyl-tRNA synthetase of the mutant was found to be abnormal; in crude extracts the enzyme activity was less stable and had approximately a tenfold higher apparent K _Mfor histidine than normal.Under many growth conditions the histidine biosynthetic enzymes in the mutant were derepressed several hundred fold compared to the wild strain, even in the presence of exogenous genous histidine. In general, the degree of derepression in the mutant was proportional to the difference in growth rate between the mutant and normal strains; this relationship, however, did not hold below 30°C or above 37°C.The properties of the mutant could be related to the properties of its histidyl-tRNA synthetase by assuming that the enzyme participates both in protein synthesis and in histidine biosynthetic enzyme regulation and that at low temperature it functions relatively more effectively in protein synthesis than in repression, while at high temperature it functions relatively more effectively in repression.Abbreviations used tRNA transfer RNA - AICAR aminoimidazole carboxamide ribose-5-phosphate     

Cloning of a gene encoding a thermo-stable endo-beta-1,4-glucanase from Thermoascus aurantiacus and its expression in yeast

A gene encoding a thermo-stable endo--1,4-glucanase was isolated from the thermophilic fungus, Thermoascus aurantiacusIFO9748, and designated as eg1. Induction of this gene expression at 50°C was stronger than at 30°C. The deduced amino acid sequence encoded by eg1 showed that it belongs to the glycoside hydrolase family 5. The cloned gene was expressed in Saccharomyces cerevisiae and the gene product was purified and characterized. No significant activity loss was detected over 2 h at 70°C and the product was stable from pH 3–10. The enzyme was optimally active at 70°C over 20 min and the optimal pH was 6.     

Cold-active DnaK of an Antarctic psychrotroph Shewanella sp. Ac10 supporting the growth of dnaK-null mutant of Escherichia coli at cold temperatures

Shewanella sp. Ac10 is a psychrotrophic bacterium isolated from the Antarctica that actively grows at such low temperatures as 0°C. Immunoblot analyses showed that a heat-shock protein DnaK is inducibly formed by the bacterium at 24°C, which is much lower than the temperatures causing heat shock in mesophiles such as Escherichia coli. We found that the Shewanella DnaK (SheDnaK) shows much higher ATPase activity at low temperatures than the DnaK of E. coli (EcoDnaK): a characteristic of a cold-active enzyme. The recombinant SheDnaK gene supported neither the growth of a dnaK-null mutant of E. coli at 43°C nor phage propagation at an even lower temperature, 30°C. However, the recombinant SheDnaK gene enabled the E. coli mutant to grow at 15°C. This is the first report of a DnaK supporting the growth of a dnaK-null mutant at low temperatures.     

Thermotolerant nalidixic acid-resistant mutants ofEscherichia coli

Nalidixic acid-resistant mutants ofEscherichia coli CGSC #6353 capable of growth at 48°C were obtained by mutagenesis withN-methyl-N-nitro-N-nitrosoguanidine. Cotransductional analyses employing phage P1 indicated that the mutation resulting in the phenotype of growth at 48°C is an allele of thegyrA structural gene. Similar thermal inactivation kinetics were observed for ribosomes isolated from a thermotolerant (T/r) mutant grown at both 37°C and 48°C and from the parental strain grown at 37°C. Cell-free extracts prepared from the T/r mutant grown at 48°C exhibited a sharp increase in protein synthesis at 55°C, whereas this effect was not displayed by extracts from the mutant or parental strains grown at 37°C. In addition, preincubation at 55°C enhanced protein synthesis at 37°C up to 15-fold in an extract prepared from the T/r mutant grown at 48°C, whereas comparable values were 2.6- to 3.0-fold for extracts from the mutant and parental strains grown at 37°C.     

Cloning, expression, and characterization of a highly thermostable family 18 chitinase from Rhodothermus marinus

A family 18 chitinase gene chiA from the thermophile Rhodothermus marinus was cloned and expressed in Escherichia coli. The gene consisted of an open reading frame of 1,131 nucleotides encoding a protein of 377 amino acids with a calculated molecular weight of 42,341 Da. The deduced ChiA was a non-modular enzyme with one unique glycoside hydrolase family 18 catalytic domain. The catalytic domain exhibited 43% amino acid identity with Bacillus circulans chitinase C. Due to poor expression of ChiA, a signal peptide-lacking mutant, chiAsp, was designed and used subsequently. The optimal temperature and pH for chitinase activity of both ChiA and ChiAsp were 70°C and 4.5–5, respectively. The enzyme maintained 100% activity after 16 h incubation at 70°C, with half-lives of 3 h at 90°C and 45 min at 95°C. Results of activity measurements with chromogenic substrates, thin-layer chromatography, and viscosity measurements demonstrated that the chitinase is an endoacting enzyme releasing chitobiose as a major end product, although it acted as an exochitobiohydrolase with chitin oligomers shorter than five residues. The enzyme was fully inhibited by 5 mM HgCl₂, but excess ethylenediamine tetraacetic acid relieved completely the inhibition. The enzyme hydrolyzed 73% deacetylated chitosan, offering an attractive alternative for enzymatic production of chitooligosaccharides at high temperature and low pH. Our results show that the R. marinus chitinase is the most thermostable family 18 chitinase isolated from Bacteria so far.     

Isolation and characterization of a temperature-sensitive amber suppressor mutant of Escherichia coli K12

Summary By mutagenizing an E. coli strain carrying an amber suppressor supD ^- (or su _I ⁺), we isolated a mutant whose amber suppressor activity was now temperature-sensitive. The mutant suppressor gene was named sup-126, which was found to be cotransduced with the his gene by phage P1vir at the frequency of ca. 20%. At 30° C it suppresses many amber mutations of E. coli, phage T4, and phage . At 42° C, however, it can suppress none of over 30 amber mutations tested so far. The sup-126 mutation is unambiguous and stable enough to be useful for making production of an amber protein temperature-sensitive.     

Affinity purification and characterization of a yeast epoxide hydrolase

Purification of the membrane-associated epoxide hydrolase from the yeast Rhodosporidium toruloides CBS 0349 to electrophoretic homogeneity was achieved in a single chromatographic step employing the affinity ligand adsorbent Mimetic Green. More than 68% of the total epoxide hydrolase activity present in the whole cells was recovered from the membrane fraction. The enzyme was purified 26-fold with respect to the solubilized membrane proteins and was obtained in a 90% yield. The purified epoxide hydrolase has an apparent monomeric molecular weight of 54 kDa, and a pI of 7.3. The enzyme was optimally active at 30–40 °C, and pH 7.3–8.5. The enzyme is highly glycosylated with a carbohydrate content >42%. The specific activity of the purified enzyme for (±)-1,2-epoxyoctane is 172 mol min^–1 mg protein^–1. The amino acid composition of the protein was determined. This is the first report of a yeast epoxide hydrolase purified to homogeneity in milligram amounts.     

Enzymatic properties and nucleotide and amino acid sequences of a thermostable β-agarase from a novel species of deep-sea<Emphasis Type="Italic"> Microbulbifer</Emphasis>

An agar-degrading bacterium, strain JAMB-A7, was isolated from the sediment in Sagami Bay, Japan, at a depth of 1,174 m and identified as a novel species of the genus Microbulbifer. The gene for a novel -agarase from the isolate was cloned and sequenced. It encodes a protein of 441 amino acids with a calculated molecular mass of 48,989 Da. The deduced amino acid sequence showed similarity to those of known -agarases in glycoside hydrolase family 16, with only 34–55% identity. A sequence similar to a carbohydrate-binding module was found in the C-terminal region of the enzyme. The recombinant agarase was hyper-produced extracellularly using Bacillus subtilis as the host, and the enzyme purified to homogeneity had a specific activity of 398 U (mg protein)^–1 at pH 7.0 and 50°C. It was thermostable, with a half-life of 502 min at 50°C. The optimal pH and temperature for activity were around 7 and 50°C, respectively. The pattern of agarose hydrolysis showed that the enzyme was an endo-type -agarase, and the final main product was neoagarotetraose. The activity was not inhibited by NaCl, EDTA, and various surfactants at high concentrations. In particular, sodium dodecyl sulfate had no inhibitory effect up to 2%.     

Genetic studies of the ribosomal proteins in Escherichia coli. XI. Mapping of the genes for L21, L27, S15 and S21 by using hybrid bacteria and over-production of these proteins in the merodiploid strains.

Summary Host capacity for growth of single-stranded DNA phages was investigated with several replication mutants of E. coli. In dnaL708, dnaM709 and dnaS707 mutants, multiplication of K was not restricted even at 42°C. In dnaM710 cells, however, growth of K was severely affected at 42°C but not at 33°C. Upon infection of K, parental replicative form was synthesized at the restrictive temperature, whereas subsequent step (replication of progeny replicative form) was blocked in the dnaZ strain. Growth of X174 and 3, as tested by transfection, was also thermosensitive in the dnaM710 mutant but not in the dnaL708, dnaM709 and dnaS707 strains. In contrast with , microvirid phages could grow in E. coli cells bearing the groPC259, groPC756 or seg-2 mutation.This paper is number 15 in the series entitled Sensivity of Escherichia coli to Viral Nucleic Acid     

recA Gene dependence of replication of the Escherichia coli chromosome initiated by plasmid pUC13 integrated at predetermined sites

Summary Plasmid pUC13 was used to clone DNA fragments of known sites from the chromosome of Escherichia coli. Each chimeric plasmid was introduced individually into the same dnaA46 mutant strain LC381 and suppressive integration (Sin) strains were selected. By means of cotransduction the null mutation recA56 was then introduced into each Sin strain and growth of each recA56 derivative at 42° C was scored. Strains that failed to grow at 42° C depended upon the recA gene for replication. Three factors were shown to limit the viability of LC381 harboring different chimeric plasmids and affect the degree of recA gene dependence of chromosome replication in the Sin strains at 42° C. It is suggested that these three constraints are the consequence of the organization of the E. coli chromosome, particularly the unique ability of terC to retard the progression of replication forks. Two classes of hypotheses concerning the function of the recA gene are considered.     

Characterization and gene cloning of a novel β-mannanase from alkaliphilic <Emphasis Type="Italic">Bacillus</Emphasis> sp. N16-5

An alkaline -mannanase was purified to homogeneity from a culture broth of alkaliphilic Bacillus sp. N16-5. The enzyme had optimum activity at pH 9.5 and 70°C. It was composed of a single polypeptide chain with a molecular weight of 55 kDa deduced from SDS-PAGE, and its isoelectric point was around pH 4.3. The enzyme efficiently hydrolyzed galactomannan and glucomannan, producing a series of oligosaccharides and monosaccharides. The -mannanase gene (manA) contained an open reading frame (ORF) of 1,479 bp, encoding a 32-amino acids signal peptide, and a mature protein of 461 amino acids, with a calculated molecular mass of 50,743 Da. Strain N16-5 ManA, deduced from the manA ORF, exhibited relatively high amino acid similarity to the members of the glycosyl hydrolase family 5. The eight conserved active-site amino acids in family 5 glycosyl hydrolase were found in the deduced amino acid sequence of strain N16-5 ManA.