Erythromycin resistant mutations in Bacillus subtilis cause temperature sensitive sporulation.

Summary All of several hundred erythromycin resistant (ery^R) single site mutants ofBacillus subtilis W168 are temperature sensitive for sporulation (spo^ts). The mutants and wild type cells grow vegetatively at essentially the same rates at both permissive (30° C) and nonpermissive (47° C) temperatures. In addition, cellular protein synthesis, cell mass increases and cell viabilities are similar in mutant and wild type strains for several hours after the end of vegetative growth (47° C). In the mutants examined, the temperature sensitive periods begin when the sporulation process is approximately 40% completed, and end when the process is 90% complete. At nonpermissive temperatures, the mutants produce serine and metal proteases at 50% of the wild type rate, accumulate serine esterase at 16% of the wild type rate, and do not demonstrate a sporulation related increase in alkaline phosphatase activity.The ery^R and spo^ts phenotypes cotransform 100%, and cotransduce 100% using phage PBS1. Revertants selected for ability to sporulate normally at 47° C (spo⁺), simultaneously regain parental sensitivity to erythromycin. No second site revertants are found.Ribosomes from ery^R spo^ts strains bind erythromycin at less than 1% of the wild type rate. A single 50S protein (L17) from mutant ribosomes shows an altered electrophoretic mobility. Ribosomes from spo⁺ revertants bind erythromycin like parental ribosomes and their proteins are electrophoretically identical to wild type. These data indicate that the L17 protein of the 50S ribosomal subunit fromBacillus subtilis may participate specifically in the sporulation process.     

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.     

Evaluation of host and pathogen responses for screening soil amendments to control Sclerotium rolfsii

Three strains of Bradyrhizobium, 280A, 2209A and 32H1, that nodulated peanuts (Arachis hypogaea L.), were tested for their ability to grow and survive at elevated temperatures of up to 42°C in laboratory culture. Strain 32H1 was unable to grow at 37°C and was more sensitive to elevated temperatures than the other two strains. All three produced heat-shock proteins of molecular weights 17 kDa and 18 kDa. Two greenhouse experiments were conducted to determine the effect of high root temperature on nodulation, growth and nitrogen fixation of peanut. Two peanut varieties (Virginia cv NC7 and Spanish cv Pronto) were inoculated and exposed to root temperatures of 30°, 37° and 40°C. Nodulation and nitrogen fixation were strongly affected by root temperature but there was no variety × temperature interaction. At a constant 40°C root temperature no nodules were formed. Nodules were formed when roots were exposed to this temperature with diurnal cycling but no nitrogen fixation occurred. Highest plant dry weight, shoot nitrogen content and total nitrogen were observed at a constant root temperature of 30°C. Increasing root temperature to 37°C reduced average nitrogen content by 37% and total nitrogen by 49% but did not reduce nodulation. The symbiotic performance of the strains corresponded to their abilities to grow and survive at high temperature in culture.     

Mutants of Agrobacterium tumefaciens with temperature sensitivity in respect to their tumor inducing ability

Summary The isolation of six mutants of Agrobacterium tumefaciens which can induce tumors at low temperatures (22°C) but fail to do so at 28°C is described. At the nonpermissive temperature the following characteristics of the mutants are the same as those of the wild type: growth rates in vitro, growth rates in planta, and sensitivity towards agrocin 84, a marker for the presence of the virulence-plasmid. The tumors induced by the mutants at low temperature grow without addition of hormones at both 22°C and 28°C. The induction of the tumors but not the maintenance of the tumorous phenotypes are affected in the mutants isolated.     

Sequential cold-sensitive mutations in Aspergillus fumigatus.

Mutants of thermotolerant fungus Aspergillus fumigatus I-21 (ATCC 32722) unable to grow at 37 degrees C were sought. Cold-sensitive mutants were enriched from progeny spores of gamma-irradiated conidia by two or more incubations at various nonpermissive temperatures alternating with filtrations through chessecloth. The approximate minimum, optimum, and maximum growth temperatures of the parent were 12, 40, and 50 degrees C, respectively. Mutants unable to grow at 37 degrees C were not successfully isolated directly from the wild type. A mutant unable to grow at 25 degrees C was isolated and mutations further increasing the cold sensitivity by increments of 3-5 degrees C were found to occur. Mutants completely unable to grow at 37 degrees C were obtained by five sequential mutations. All mutants grew as fast as the wild-type parent at 45 degrees C and higher. Each mutant produced revertants able to grow not only at the nonpermissive temperature used for its isolation but also at lower temperatures.     

Protein synthesis kinetics with ribosomes from temperature-sensitive mutants of Escherichia coli.

The kinetics of MS2 ribonucleic acid (RNA) directed protein synthesis have been investigated at seven temperatures between 30 and 47 degrees C by using ribosomes isolated from a wild type strain and seven temperature-sensitive mutants of Escherichia coli. The amount of MS2 coat protein formed at each temperature was determined by gel electrophoresis of the products formed with control ribosomes. With ribosomes from each of the mutant strains, the activation energy required to drive protein synthesis below the maximum temperature (up to 40 degrees C) was increased relative to the control (wild type) activity. Preincubation of the ribosomes at 44 degrees C revealed the kinetics of thermal inactivation, with ribosomes from each of the mutants having a half-life for inactivation less than that of the control ribosomes. A good correlation was observed between the relative activity of the different ribosomes at 44 degrees C and their relative rate of thermal inactivation. Mixing assays allowed the identification of a temperature-sensitive ribosomal subunit for each of the mutants. Defects in one or more of three specific steps in protein synthesis (messenger RNA binding, transfer RNA binding, transfer RNA binding, and subunit reassociation) were identified for the ribosomes from each mutant. The relationship between temperature sensitivity and protein synthesis in these strains is discussed.     

Evaluation of different temperature-sensitive mutant phenotypes ofSalmonella enteritidis as vaccine potentials

Temperature-sensitive (ts) mutants ofSalmonella enteritidis were isolated after mutagenesis with UV light and enrichment with antibiotic. Mutants were characterized according to their growth profile at the permissive (28°C) and the nonpermissive (37°C) temperatures, persistence of surface antigens, reversion frequencies, and potentials for inducing humoral immunity and protection against challenge with the parental wild-type (wt) in mice. We obtained 32 strains ofS. enteritidis able to grow well at 28°C, but capable of only limited or no replication at 37°C. The ts mutants were positive for factor 9 in an agglutination assay and were susceptible to infection with phage P22. Three mutants of different phenotypes were selected for protection studies. A single intraperitoneal (i.p.) immunization with any of the mutants studied induced significant protection from i.p. challenge with 100 LD₅₀ of the wt strain.     

Thermotolerance and ethanol production are at variance in mutants of Schizosaccharomyces pombe

Summary Genetically similar mutants of Schizosaccharomyces pombe that failed to grow but survived at 44 °C for 72 h, exhibited a significantly delayed thermal death. These thermotolerant mutants registered a decrease in ethanol production by 25–50% at low as well as high temperatures. The data on tetrad products derived from crosses of mutants with the wild type parent, corroborated the constant co-segregation of thermotolerance and low ethanol production and most probably under the influence of a single gene, tetl. Reduced ethanol tolerance of mutant strains seemed to have been a probable reason for low ethanol production.     

Growth and macromolecular synthesis phenotypes of a heat-sensitive mutant strain, rip-1, of Neurospora crassa

Summary A heat-sensitive mutant of Neurospora crassa, strain 4M(t), was isolated using ultraviolet-light mutagenesis followed by the inositol-less death enrichment technique. The heat-sensitivity is the result of a single gene mutation which maps to the distal end of the right arm of linkage group II. The mutation defines the rip-1 gene locus. Both conidial germination and mycelial extension are inhibited in the mutant at 35°C and above (the nonpermissive temperature) but prolonged incubation at that temperature is not lethal to either cell type. Analysis of the lateral mycelial growth rates of wild type and of the rip-1 mutant at a variety of temperatures between 10 and 40°C indicated that the maximal growth rate occurs at 35°C in the wild type, and at 25°C in the rip-1 strain. The rip-1 mutant grows 239-times slower at 35°C than at 25°C, whereas the wild type grows 1.4-times faster. Temperature shift-up experiments showed that even 3 h at 20°C is not sufficient to allow germination at 37°C, thereby showing that the mutant cannot accumulate enough heat-sensitive product at the permissive temperature to contribute to germination at 37°C. The reciprocal temperature shift-down experiments showed that the molecular events at 37°C may be qualitatively useful for germination after shifting to 20°C. Studies of macromolecular synthesis showed that the biochemical defect in the heat-sensitive strain appears to affect RNA synthesis before protein synthesis, although there were differences in the relative effects depending on the age of the germinating conidia and the inhibition of the two processes was never complete. Messenger RNA synthesis is normal in the mutant at 37°C. Previous work has shown that the rip-1 mutant strain has a conditional defect in the accumulation of 25S rRNA and, hence, in 60S ribosomal subunit production (Loo et al. 1981). There are also indications from those studies that the 60S ribosomal subunit may be functionally impaired at the higher temperature. Thus, the growth and macromolecular synthesis phenotypes may result as a consequence of a conditional, ribosome function defect and leads to the hypothesis that the mutation in the rip-1 strain may be in a gene for a 60S ribosomal subunit component, perhaps a ribosomal protein.     

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     

A slow-growing, streptomycin resistant mutant of Escherichia coli affected in protein synthesis and ribosomal assembly

Summary The Escherichia coli K12 strain derivative MX 109, carries the strA938 mutant allele. It was isolated as a spontaneous high-level streptomycin resistant mutant, having the additional characteristic of growing slowly at a wide range of temperatures. Thus far, these two phenotypic characteristics have not been genetically segregated and, probably, are the result of a single mutational event at the strA locus.A number of pleiotropic alterations are also observed in cells of MX109 when compared two wild type cells, very likely as a consequence of the expression of the strA938 mutant allele. These are, i) decrease in their polysome content, ii) increase in their RNA to protein ratio, iii) increase in the density of the cells, iv) accumulation during growth of 30S and 50S ribosomal precursors, and v) low efficiency of their ribosomes to support polypeptide synthesis in vitro. Most of these effects are observed at 37° C but are more marked if cells or extracts are incubated at 20° C. These observations point to a reduced efficiency for protein synthesis of the ribosomes of MX109, as the primary effect exerted by the 30S-protein product of the strA938 mutant allele.     

Temperature sensitive mutant of Trichoderma reesei defective in secretion of cellulase

Summary Temperature sensitive mutants of Trichoderma reesei derived from hypersecretory strain RL-P37 were isolated and characterized. Compared to the parent strain, one mutant (LU-ts 1) grew well in the mycelial phase at both permissive (25°C) and non-permissive (37°C) temperatures. However, the secretion of overall protein and active cellulases was significantly reduced in the mutant at the higher temperature. No accumulation of active cellulases or intracellular proteins was observed in the mycelia of LU-ts 1 at 37°C. The inhibitory effects of temperature on cellulase secretion in LU-ts 1 were reversible. Isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoretic analyses confirmed that the secretion of the major cellulases was greatly reduced in LU-ts 1 at 37°C. Molecular characterization of the various temperature sensitive secretion mutants of T. reesei should help elucidate the crucial aspects of the secretory pathway of this cellulolytic fungus.     

The growth of Epidermophyton floccosum and E. stockdaleae at different temperatures

The ability of 17 strains of genus Epidermophyton (15 strains belonging to Epidermophyton floccosum, one to E. floccosum var. nigricans and one to E. stockdaleae) to grow at different temperatures (4 °C, 25 °C, 28 °C, 31 °C, 34 °C, 37 °C and 40 °C) was stated.The strains were inoculated on Sabouraud Dextrose Agar and regularly controled over a period of 14 days when the plates were incubated at 25 °C, 28 °C, 31 °C, 34 °C, 37 °C and 40 °C, and over a period of 70 days when the temperature was 4 °C. The optimal growth of E. floccosum was observed at 28 °C and 31 °C, and no signs of growth were recorded neither at 4 °C nor at 40 °C. The optimal development of E. stockdaleae was observed at 25 °C and 28 °C. This species grew from 4 °C to 31 °C.     

Temperature resistant Nocardia biovarieties in Tucumán,Argentina?

Eighty-six regional strains of the pathogenic Nocardia species isolated from soil and human mycetoma were tested for their response to different incubation temperatures and for their tolerance to different temperatures. The aim was to assess whether growth temperature and tolerance to elevated temperatures are valuable criteria for the differentiation of pathogenic species of local strains based on the results obtained from a large number of strains. The results showed that 75.34% of all N. brasiliensis isolates from both sources grew at a temperature higher than 37 °C. 20% of the mycetoma strains and 11.32% of those from soil grew at 45 °C. 98.1% of N. brasiliensis from soil and 55.0% of the mycetoma strains tolerated 50 °C for 8 h and many isolates from both sources endured this temperature for an even longer time and tolerated yet higher temperatures. Both properties (growth temperature and temperature tolerance) are used to identify N. asteroides complex (N. farcinica) and N. otitidiscaviarum, and according to our results they are not suitable to differentiate regional strains of this species. The N. asteroides strains assayed showed an ability to grow at and tolerate elevated temperatures superior to those belonging to the other species. Although adaptation of local N. asteroides and N. otitidiscaviarum strains to temperature is important, it is more significant for N. brasiliensis, because this species is predominant in the Tucumán soil and responsible for the major number of diseases in the area.This revised version was published online in October 2005 with corrections to the Cover Date.     

Isolation and characterization of a ColE1 plasmid containing the entire bio gene cluster of Escherichia coli K12.

Summary Temperature-sensitive mutants of Tetrahymena pyriformis which had previously been selected for their inability to grow at 38°C but which grew normally (or near normally) at 30°C were characterized with respect to their patterns of RNA and protein accumulation at both the permissive and nonpermissive temperatures. Out of 116 such mutants, the majority (72) acted like wild type for these accumulations during a 3 h labelling period although some of them stopped dividing during this time. The remainder exhibited a variety of altered phenotypes for the rate, extent, and timing of RNA and/or protein accumulation. Those mutants which exhibited selective inhibition of RNA accumulation, and were thus potential ribosomal RNA (rRNA) mutants, were further characterized by examining patterns of protein and RNA synthesis in cells starved at the permissive temperature, but re-fed at the permissive and non-permissive temperatures. At least five different types of mutants as defined by patterns of protein and RNA synthesis in refed cells were identified. Direct analysis of the RNA synthesized in cells from 2 of these types of mutants showed that in 5 out of 6 cases rRNA synthesis and/or processing was inhibited within 30 min after shifting to the non-permissive temperature. The other mutant examined was found to show a delayed inhibition of rRNA synthesis.     

Prolonged environmental stress via a two step process selects mutants of Escherichia,Salmonella and Pseudomonas that grow at 54°C

A prolonged incubation of Escherichia, Salmonella or Pseudomonas at 48°C with nalidixic acid selected mutants (T48) able to grow at 48°C. A prolonged incubation at 54°C of the T48 mutants selected mutants (T54) able to grow at 54°C. These mutants were susceptible to the same bacteriophages as the original mesophilic strains. Auxotrophic phenotypes of Escherichia coli and Salmonella typhimurium mesophilic parents were demonstrated by these mutants if they were cultivated on minimal agar with cellobiose at 48°C or 54°C or on a minimal agar with glucose at 37°C. The T48 alleles mapped in the gyrA region of E. coli or S. typhimurium chromosome. In S. typhimurium the T54 alleles, which permit growth at 54°C, were shown by cotransductional analysis to be linked to gyrA.     

Isolation and characterisation of a strain of Saccharomyces cerevisiae deficient in in vitro RNA polymerase B(II) activity.

Summary Two hundred strains of Saccharomyces cerevisiae temperature sensitive for RNA synthesis were selected and screened in crude extracts for DNA-dependent RNA polymerase activities. One strain was isolated which had only residual in vitro RNA polymerase B activity. In normal growth conditions total RNA, poly A⁺ RNA and protein synthesis were indistinguishable from those of the wild type strain at 23°C and after shift to 37°C. A temperature sensitive phenotype was detected only when rpoB containing strains were grown in adverse conditions. The mutant character showed mendelian segregation and was coexpressed with the wild type character in heterozygous diploids. Residual enzyme activity was characterised in crude extracts using synthetic polymers and natural templates in different ionic conditions.     

Isolation and physical mapping of temperature-sensitive mutants defective in heat-shock induction of proteins in Escherichia coli

Summary Mutants of Escherichia coli K12 that are partially or totally defective in induction of major heat-shock proteins and cannot grow at high temperature (42° C) were isolated by localized mutagenesis. These mutants carry a single mutation in the gene htpR (formerly hin) located at min 76 on the E. coli genetic map. Some mutants exhibit delayed (partial) induction of heat-shock proteins or require a higher temperature for induction than the wild type, whereas others are not induced under any of these conditions. The maximum temperature that allows growth varies among different mutants and is correlated with the residual induction capacity. Temperature-resistant revertants obtained from each mutant are fully or partially recovered in heat-shock induction. These results indicate that the inability of htpR mutants to grow at high temperature is due to the defect in heat-shock induction. In addition, a couple of mutants was found that produce significantly higher amounts of heat-shock proteins even at 30° C.The htpR gene has been cloned into plasmid pBR322 using the above mutants, and was localized to a DNA segment of 1.6 kilobase pairs. The mutants harboring certain palsmids that carry a part of htpR produce temperature-resistant recombinants at high frequency. This permits further localization of mutations within the htpR gene. Analysis of proteins encoded by each of the recombinant plasmids including the one carrying a previously isolated amber mutation (htpR165) led to the identification of a protein with an apparent molecular weight of about 36,000 daltons as the htpR gene product.     

Changes in regulation of ribosome synthesis during different stages of the life cycle of Saccharomyces cerevisiae.

Summary Diploid strains of Saccharomyces cerevisiae, each homozygous for one of the temperature sensitive mutations rna2, rna4, rna6 or rna8, are temperature sensitive for ribosome synthesis during vegetative growth, but are not inhibited for ribosomal synthesis at the restrictive temperature under sporulation conditions. The continued ribosome biosynthesis at the restrictive temperature (34° C) during sporulation includes de novo synthesis of both ribosomal RNA and ribosomal proteins. This lack of inhibition of ribosome biosynthesis is found even when cells committed to complete sporulation are returned to vegetative growth medium. The ribosomes synthesized at 34° C are apparently functional, as they are found in polyribosomes. Although the rna mutants do not regulate ribosome synthesis during sporulation, all of these diploid strains fail to complete sporulation at 34° C. The cells are arrested after the second meiotic nuclear division but before ascus formation. The failure to complete sporulation at the restrictive temperature and the inhibition of ribosome biosynthesis during growth are caused by the same mutation, because revertants selected for temperature independent growth were also able to sporulate at 34° C.     

Specific enrichment of mutants of Escherichia coli with an altered acyl CoA synthetase by tritium suicide

Summary In a rich growth medium, tritium-labelled oleate is incorporated preferentially into the lipid fraction of E. coli K12. The incorporation of the radioactive oleate after a temperature shift kills the majority of the cells during subsequent storage. Most of the surviving cells can no longer grow with oleate as the sole carbon source at 40°C or 30°C, though some exhibit a thermosensitive behaviour, and are able to grow at 30°C but not at 40°C. It will be shown that mutants of the first group lack the enzyme acyl CoA synthetase, and that this enzyme is thermolabile in mutants of the second group. In addition one further mutant was isolated which possessed a normal acyl CoA synthetase but which was unable to degrade or incorporate oleic acid.