Characterization of a cold-sensitive hisW mutant of Salmonella typhimurium

Previous studies of hisW mutants of Salmonella typhimurium have led to the suggestion that such strains are defective in tRNA maturation. (J. E. Brenchley and J. Ingraham, J. Bacteriol. 114:528-536, 1973). In this study, we report that one hisW strain is defective in the accumulation of all stable RNA species. Polyacrylamide gel electrophoresis of radiolabeled RNA indicated tha at the nonpermissive temperature (20 degrees C) all stable RNa species in the cold-sensitive hisW3333 mutant were synthesized and rapidly degraded. We propose that the cold sensitivity of this strain is caused by such a restriction in stable RNA accumulation at low temperature. In vitro and in vivo studies demonstrated that the RNA degraded in this strain was synthesized de novo and was not preexisting RNA. Furthermore, physiological and genetic recovery from the cold-sensitive hisW phenotype resulted in relatively normal RNA synthesis and accumulation. Thus, the RNA alterations observed in this strain were not explained by defects in a tRNA modification enzyme. Rather, these findings suggest the existence of defective RNA processing and that a control mechanism for the overall synthesis or accumulation of stable RNA species is altered in the hisW3333 mutant.     

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.     

Poliovirus mutant that contains a cold-sensitive defect in viral RNA synthesis.

By manipulating an infectious cDNA clone of poliovirus, we have introduced a single-codon insertion into the 3A region of the viral genome which has been proposed to encode a functional precursor of the virion-linked protein VPg. The resulting mutant was cold sensitive in monkey kidney cells. Viral RNA synthesis was poor at 32.5 degrees C, although no other function of the virus was obviously affected. The synthesis of both positive and negative strands was severely depressed. Temperature shift experiments suggest that a normal level of production of the affected function was required only during the early (exponential) phase of RNA synthesis. Analysis of viral polyprotein processing at the nonpermissive temperature revealed that some of the normal cleavages were not made, most likely as a consequence of the defect in RNA synthesis or as a result of the concomitant reduction in the level of virally encoded proteases.     

Correlation of long-range membrane order with temperature-dependent growth characteristics of parent and a cold-sensitive, branched-chain-fatty-acid-deficient mutant of Listeria monocytogenes

Listeria monocytogenes is a food-borne, pathogenic, psychrotolerant bacterium that grows at refrigeration temperatures. Long-range membrane order of the parent (10403S) and of a cold-sensitive mutant ( cld-1) deficient in odd-numbered, branched-chain fatty acids was measured using the width of the central line of spectra of an electron paramagnetic resonance probe, 4,4-dimethyl-2-heptyl-2-hexyloxazolidine- N-oxyl (7N14), that locates deep in the hydrocarbon region of the membranes. The line width decreased from 0.9 to 0.5 milliTesla (mT) over the temperature range of 0-10 degrees for strain 10403S and -5 to 32 degrees C for strain cld-1 independent of protein state (heat denatured or intact). This provided new evidence for phase transitions in the membranes. When strain cld-1 was grown in medium supplemented with 2-methylbutyric acid, which restores anteiso fatty acids and the ability to grow at low temperature, the change in central line width as a function of temperature resembled that of strain 10403S. The temperatures at which the central line width became 0.8 mT corresponded to those at which growth became very slow in both strains (3-5 degrees C for 10403S, 15 degrees C for cld-1) as determined by Arrhenius plots. These data underscore the critical role of odd-numbered anteiso fatty acids in influencing the lower temperature limits of growth through their effects on long-range membrane fluidity.     

Mutant of Escherichia coli exhibiting a cold-sensitive phenotype for growth on lactose

As part of a study on the effect of low temperature on cellular regulatory processes, a class of lactose-negative mutants of Escherichia coli K-12 was isolated which could use lactose as a sole carbon and energy source at 37 C, but which could not use this sugar at 20 C. The lactose operon of the mutants functioned normally at 20 C. Galactose exhibited a strong inhibitory effect on growth, especially at 20 C. Growth of the mutants on glycerol was stopped at 20 C and slowed considerably at 37 C if galactose was added to the medium. Making the mutants galactose-positive eliminated the cold sensitivity of lactose utilization. One mutant was shown to be galactose-1-phosphate uridyl transferase-negative, galactose-kinase heat-sensitive, and uridine diphosphate-galactose-4-epimerase-positive. It is postulated that the mutant is able to phosphorylate galactose at 20 C (if only at a very low rate), but lacking transferase it is poisoned by the accumulation of galactose-1-phosphate. At 37 C, galactokinase is nonfunctional and the mutant grows on the glucose moiety of lactose.     

Effects of pressure stress on the fission yeast Schizosaccharomyces pombe cold-sensitive mutant nda3

To investigate the influence of pressure stress on the cell cycle of Schizosaccharomyces pombe, we used a cold-sensitive nda3-KM311 mutant which arrests cell division at a step similar to the mitotic prophase, proposed by Hiraoka and colleagues (Cell 39 (1984) 349-358), under the restrictive temperature, 20 degrees C. The nda3-KM311 cells were first aerobically grown at 30 degrees C, transferred to 20 degrees C for 4 h and shifted to a permissive temperature of 36 degrees C for 15 min. The cells were treated with 100-200 MPa pressure and studied by electron and fluorescence microscopy. At 100 MPa, the nuclear membrane was damaged and the matrix of mitochondria had an electron-dense area. At 150 MPa, the nuclear membrane was broken over broad areas; numerous small vacuoles had fused into large pieces. Actin patches were concentrated in the central region and actin rings were seen in the 20 degrees C-grown cells. Even at 100 MPa, specific actin distribution was lost. Although at 100 MPa, long and fine actin cables were seen all over the cells, large actin patches and the actin rings remained in the center of the cell. They changed into thick and short cables at 150 MPa and above 200 MPa they decomposed but the actin ring was visible even with faint fluorescence. Immunoelectron microscopic observation confirmed this phenomenon.     

Physiological properties of cold-sensitive suppressor mutations of a temperature-sensitive dnaZ mutant of Escherichia coli

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)].     

Neurospora crassa cytoplasmic ribosomes: cold-sensitive mutant defective in ribosomal ribonucleic acid synthesis.

Experiments were conducted to characterize further the biochemical defects of crib-1 (PJ30201), a coldsensitive mutant strain of Neurospora crassa with a defect in ribosome biosynthesis. The results are as follows. (i) The critical temperature for the expression of the mutant growth and ribosome phenotypes is in the range of 18 to 20 C. (ii) No preferential breakdown of 37S cytoplasmic ribosomal subunits synthesized by crib-1 at 25 C occurs after a shift to 10 C. (iii) Ribosomal subunits synthesized by crib-1 at 25 C function normally in in vivo protein synthesis at 10 C. (iv) Whereas wild type synthesizes both ribosomal subunits in a coordinate manner after either a temperature shift-down (25 to 10 C) of a shift-up (10 to 25 C), noncoordinate synthesis of ribosomal subunits owing to underproduction of 37S subunits occurs in the crib-1 strain immediately after a temperature shift-down. (v) After a shift from 10 to 25 C crib-1 exhibits a 12-h lag before the growth rate and the rate of synthesis of 37S subunits begin to increase significantly. (vi) At 10 C crib-1 synthesizes unequal amounts of 25S and 17S ribosomal ribonucleic acid (rRNA) molecules, resulting from a greatly reduced accumulation of stable 17S rRNA. The mutant phenotypes of crib-1 are proposed to be the result of a defect in rRNA processing.     

A cold-sensitive mutant ofNeurospora crassa obtained using tritium-suicide enrichment that is conditionally defective in the biosynthesis of cytoplasmic ribosomes

The purpose of this investigation was to use tritium-suicide enrichment with a mutagenized population of wild-typeNeurospora crassa to isolate cold-sensitive mutants with conditional defects in the production of cytoplasmic ribosomes. Eighty-six cold-sensitive mutant strains were obtained following tritium-suicide enrichment using [5-³H]uridine. Zone sedimentation analysis of cytoplasmic ribosomes produced by the strains at 10°C (the nonpermissive temperature) indicated that one strain,PJ31562, is defective in the accumulation of ribosomal subunits at that temperature. The properties of strainPJ31562 are: (1) At 10°C the growth rate is 28 times slower than at 25°C, whereas the factor for the wild type is 5.1. At 25°C the mutant's growth rate is 90% that of the wild type. (2) At 10°C the mutant accumulates the two ribosomal subunits, 60 and 37 S, in markedly disproportionate amounts apparently as a result of the underproduction of, or an instability of, the 17 S ribosomal RNA component of the small ribosomal subunit. At 25°C the mutant strain still exhibits a disproportionality in ribosomal subunit accumulation but to a much lesser degree than at 10°C. (3) Genetic studies have shown that a single nuclear gene is responsible for both the cold sensitivity and ribosome biosynthesis defect of strainPJ31562. The mutation involved is located in linkage group IV and appears to be closely linked to, and not allelic with, the cold-sensitive mutation carried by strainPJ30201 which has been shown previously to exhibit a similar phenotype with respect to ribosomal subunit accumulation, and which defines thecrib-1 locus. Thus tritium-suicide enrichment can be used to isolate cold-sensitive mutants ofNeurospora among which a relatively low frequency have conditional defects in ribosome production.     

Altered translation initiation factor 2 in the cold-sensitive ssyG mutant affects protein export in Escherichia coli.

The Escherichia coli gene secY (pr1A) codes for an integral membrane protein that plays an essential role in protein export. We previously isolated cold-sensitive mutations (ssy) as extragenic suppressors of temperature-sensitive secY24 mutation. Now we show that the ssyG class of mutations are within infB coding for the translation initiation factor IF2. The mutants produce altered forms of IF2 with a cold-sensitive in vitro activity to form a translation initiation complex. The mutation suppresses not only secY24 but also other secretion-defective mutations such as secA51 and rp10215. The beta-galactosidase enzyme activity of the MalE-LacZ 72-47 hybrid protein is strikingly reduced in the ssyG mutant at the permissive high temperature, while the hybrid protein itself is normally synthesized. This effect, which was observed only for the hybrid protein with a functional signal sequence, may result from some alteration in the cellular localization of the protein. These results suggest that IF2 or the translation initiation step can modulate protein export reactions. The isolation of cold-sensitive ssyG mutations in infB provides genetic evidence that IF2 is indeed essential for normal growth of E. coli cells.     

Suppression of a cold-sensitive mutant initiation factor 1 by alterations in the 23S rRNA maturation region

Genetic selection has been used to isolate second-site suppressors of a defective cold-sensitive initiation factor I (IF1) R69L mutant of Escherichia coli. The suppressor mutants specifically map to a single rRNA operon on a plasmid in a strain with all chromosomal rRNA operons deleted. Here, we describe a set of suppressor mutations that are located in the processing stem of precursor 23S rRNA. These mutations interfere with processing of the 23S rRNA termini. A lesion of RNase III also suppresses the cold sensitivity. Our results suggest that the mutant IF1 strain is perturbed at the level of ribosomal subunit association, and the suppressor mutations partially compensate for this defect by disrupting rRNA maturation. These results support the notion that IF1 is an RNA chaperone and that translation initiation is coupled to ribosomal maturation.     

Regulation of thyroidal inducibility of Na,K-ATPase and binding of epidermal growth factor in wild-type and cold-sensitive E1a mutant type 5 adenovirus-transformed CREF cells

We have analyzed the relationship between expression of the transformed phenotype and thyroid hormone (triiodothyronine, T3) inducibility of Na,K-ATPase and binding of 125I-epidermal growth factor (EGF) to cell membrane receptors in wild-type (wt) and mutant type 5 adenovirus (Ad5)-transformed CREF cells displaying a cold-sensitive (cs) expression of the transformed phenotype. CREF cells respond to thyroid hormone treatment with increased Na,K-ATPase activity and bind similar levels of 125I-EGF at 32 degrees C, 37 degrees C and 39.5 degrees C. In contrast, CREF cells transformed by wt Ad5 or the E1a plus E1b-transforming genes of wt Ad5 are refractile to T3 treatment and bind lower levels of 125I-EGF than CREF cells at all three temperatures. By employing a series of cloned CREF cell lines transformed by a host-range cold-sensitive mutant virus, H5hr1 or H5dl101, or the E1a or E1a plus E1b genes from these viruses, we have investigated expression of the transformed state and its relationship with hormone inducibility and EGF binding. When cs virus, cs E1a- or cs E1a plus E1b-transformed CREF clones were grown at 32 degrees C, a nonpermissive transforming temperature in which cs-transformed cells exhibit properties similar to untransformed CREF cells, T3 induced Na,K-ATPase activity and these cells bound similar levels of 125I-EGF as CREF cells. However, when cs virus- and cs Ela plus E1b-transformed CREF clones were incubated at 37 degrees C or 39.5 degrees C, temperatures at which cs-transformed cells exhibit properties similar to wt Ad5-transformed CREF cells, they did not respond to T3 and bound lower levels of 125I-EGF than CREF cells. In the case of cs E1a-transformed CREF clones, thyroid hormone responsiveness was observed at both 32 degrees C and 37 degrees C, but not at 39.5 degrees C. By performing temperature shift experiments--i.e. 32 degrees C to 37 degrees C, 32 degrees C to 39.5 degrees C, 37 degrees C to 32 degrees C, and 39.5 degrees C to 32 degrees C, it was demonstrated that after a shift from lower to higher temperature a 24-hr lag period was required for cs-transformed CREF cells to lose T3 inducibility and exhibit reduced EGF binding, whereas 96 hr after a shift from higher to lower temperature a 96-hr lag period was required for cs-transformed cells to regain T3 inducibility and increased 125I-EGF binding.(ABSTRACT TRUNCATED AT 400 WORDS)