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.     

L-lysine production at 65°C by auxotrophic-regulatory mutants ofBacillus stearothermophilus

Summary The amino acid L-lysine was produced from auxotrophic-regulatory mutants ofBacillus stearothermophilus at a temperature of 60–65°C. One of the mutants (AEC 12 A5, S-(2-aminoethyl)-cysteine^r, homoserine^–), produced L-lysine at the concentration of 7.5 g/l in shaken flasks in minimal medium containing 5% glucose. Culture conditions for optimizing L-lysine production were not investigated. The aspartokinase activity of the wild strainB. stearothermophilus Zu 183 was inhibited by lysine alone and by threonine plus lysine. AEC resistant mutants showed an aspartokinase activity genetically desensitized to the feedback inhibition. Optimal temperature and pH of aspartokinase were 45°C and 9.5, respectively. The data provide significant evidence that mutants of the speciesB. stearothermophilus have a potential value for amino acid production.     

Isolation and initial characterization of a Schizosaccharomyces pombe mutant exhibiting temperature-dependent radiation sensitivity due to a mutation in a previously unidentified rad locus

Summary We have isolated a mutant of the yeast Schizosaccharomyces pombe which exhibits sensitivity to UV light when grown at either 30° or 37°C, as compared to the parental wild-type strain. This increased sensitivity is more pronounced when cells are grown at 37°C. The mutant is also sensitive to 18 MeV electrons at the high temperature. Tetrad analysis of spores generated by crossing the mutant and a Rad⁺ strain revealed that sensitivity to both types of radiation cosegregate 2:2, relative to wild-type resistance, indicating that a single altered chromosomal locus is responsible for the radiation sensitivities observed. In addition, analysis of spores resulting from crosses between the mutant and all other known S. pombe rad mutants indicates that the temperature-dependent sensitivity described in this report is mediated by a mutation in a previously unidentified rad locus.     

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.     

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.     

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     

The induction of sensitivity to gibberellin in aleurone tissue of developing wheat grains

Aleurone tissue from undried immature developing wheat grains (Triticum aestivum L. cv. Sappo), normally insensitive to gibberellic acid, can be made to respond to the hormone by a series of temperature treatments. Incubation of the de-embryoed grains at temperatures above 27° C for at least 8 h causes the tissue to become sensitive. Prolonged incubation at temperatures below 27° C does not effect a change in sensitivity. In addition to the requirement for exposure to an elevated temperature for a period of several hours the tissue must also subsequently be subjected to a period at a lower temperature for just a few seconds for the response to be observed. Once sensitized, the tissue remains responsive to gibberellic acid for substantial periods of time. Exposure of the tissue to temperatures which induce sensitivity to gibberellic acid also results in an increased leakage of amino acids. It is suggested that the increase in sensitivity to gibberellin requires two separate processes to take place. One could be a homeoviscous adaptation of the cell membranes in response to elevated temperature, the other a subsequent, permanent change in conformation of membrane components.     

Response characteristics of cold cell on the antenna ofLocusta migratoria L.

Summary The dendritic outer segment of the cell which is most likely the cold unit in the poreless coeloconic sensilla onLocusta migratoria antennae, has finger-like projections up to 1.5 m long and 0.13 m thick (Fig. 1). This unit responds to constant temperature, to slowly changing temperature and to step changes. Under stationary conditions impulse frequency attained 35 imp/s. Between 14 °C and 41 °C the higher frequencies were associated with the higher temperatures (Fig. 5). In this range the differential sensitivity is positive but not large: + 0.8 (imp/s)/°C. Its resolving power for steady temperature is 4.7 °C.Downward step changes produced by shifting between airstreams at different temperatures yield far higher frequencies (Figs. 2, 3). Step amplitudes were between –0.1 °C and –12 °C; the conditioning temperature from which the steps were initiated, was between 16 °C and 33 °C. Frequency peaked during the first 50 ms after stimulus onset (Fig. 2) and reached its highest values (310–340 imp/s) at initial temperatures above 30 °C and steps larger than –10 °C (Fig. 4). The mean differential sensitivity from 23 curves was –19 (imp/s)/°C and the resolving power 0.6 °C.During slowly changing temperature the impulse frequency was governed by two parameters simultaneously: ambient temperature and its rate of change. Rates were between 0.001 °C/s or less, and 0.03 °C/s in either direction. Frequency was higher during slow cooling at a given temperature than during slow warming (Fig. 6). The average differential sensitivity to the rate of change was –210 (imp/s)/(°C/s). Further, the larger responses to cooling developed at lower ambient temperatures (differential sensitivity: –1.0 (imp/s)/°C). It is to be noted that this sign is negative, in contrast to the sign for differential sensitivity to constant temperature and also for the influence of initial temperature on the response to downward step changes.Abbreviations b Slope of characteristic curve, differential sensitivity - F impulse frequency in imp/s - imp/s impulses/s - P _w partial pressure of water vapor in torr - r correlation coefficient - T temperature in °C - T T-step - x resolving power in °C     

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.     

Suppression of the lexC (ssbA) mutation of Escherichia coli by a mutant of bacteriophage P1

Summary A new mutant of bacteriophage P1 designated lxc that suppresses the phenotype of lexC and ssbA mutants of Escherichia coli was isolated and characterized. The properties of lexC mutants suppressed by the lxc mutation include temperature sensitive growth at 42° C, sensitivity to ultraviolet light and alkylating agents, and a nonmutagenic response following exposure to ultraviolet irradiation. A bac mutant of bacteriophage P1 that suppresses the temperature sensitivity of dnaB mutants does not affect the phenotype of lexC or ssbA mutants. Neither the lxc or bac mutations affect the ultraviolet light sensitivity of strains with the mutations uvrA155, lexA102, or recA56.     

Thermophilic mutants of Pseudomonas fluorescens

Summary A series of heat tolerant mutants of Pseudomonas fluorescens were obtained which can grow at temperatures up to 54°C, in contrast to a maximum growth temperature of 37°C for the wild type. The minimum temperatures allowing growth of the mutant strains increased to the same extent as their maximum temperatures. Antibiotic sensitivity patterns suggested the mutants had altered ribosomes, but the purified mutant ribosomes showed no significant increase in thermostability. The virulence of the wild and mutant strains for mice correlated with their relative abilities to grow at the mouse body temperature of approximately 37°C.     

Temperature sensitivity as a general phenomenon in a collection of chlorophyll-deficient mutants of sweetclover (Melilotus alba)

A collection of chlorophyll (Chl)-deficient mutants of sweetclover (Melilotus alba) with defects in eight nuclear loci were grown at 17 or 26° C. Plants grown at either temperature were examined for Chl content, Chla/b ratio, expression of the light-harvesting complex II (LHC-II) apoproteins, and protochlorophyllide (Pchlide) biosynthetic capacity. Except for thech4 mutant, the parental strain and all mutants accumulate more Chl when grown at 26° C than at 17° C. Thech5 mutants, lacking Chl b under any growth condition, and thech12 mutant showed little temperature-dependent phenotypic plasticity, whereas this was a marked phenomenon in the other mutants. Thech10 andch11 mutants demonstrated extreme temperature sensitivity with regard to the production of Chlb and the Chlb-binding LHC-II apoproteins. When excised trifoliolates were supplemented with exogenously supplied -aminolevulinic acid, only thech4 mutant was markedly impaired in the ability to produce Pchlide. These data indicate that temperature-sensitive phenotypic plasticity is a common phenomenon of chlorophyll-deficient mutants and substantiate that only a minority of Chl-deficient mutants is impaired in the biosynthesis of Chl.This research was supported by Grants GM84-CRCR-1-1479 (J.C.O.) and 89-00641 (J.M.) of the United States Department of Agriculture and by National Science Foundation Grant DMB87-03100 (J.M.). This is paper No. 8971, Nebraska Agricultural Research Division.     

Natural premature protein synthesis termination can be reduced in Escherichia coli by decreased translation rates.

Summary Peptidyl tRNA hydrolase is an essential enzyme for normal growth inasmuch as a mutant strain of Escherichia coli with a temperature-sensitive hydrolase cannot continue protein synthesis at the non-permissive temperature. In the absence of hydrolase peptidyl tRNA rapidly accumulates. Why peptidyl tRNA should be formed is the subject of this report. The rapid rate of protein synthesis is likely one mechanism of formation of peptidyl tRNA. A strA mutant of the hydrolase (pth-1) mutant strain that has a 40% reduction in amino acid polymerization rate can grow at 42° C. StrA mutants with normal polymerization rates, however, cannot grow at 42° C when pth-1 is present. Furthermore, addition of low levels of chloramphenicol (2–4 g/ml) but not several other tested drugs, phenotypically suppressed pth-1 at 42° C. Chloramphenicol, at these concentrations, was found to reduce the amino acid polymerization rate 30–40%. On the other hand, no evidence could be found that amino acyl tRNA selection errors are incorporated into pseudo revertants of the pth-1 strain.This investigation was supported by NSF grant No. PCM 76-11012. Journal Paper No. J-9502 of the Iowa Agriculture and Home Economics Experiment Station. Project No. 2299     

Mechanisms involved in the increased sensitivity ofEscherichia coli to microcin 15m at 42°C

Escherichia coli cells show a markedly increased sensitivity to the antibiotic microcin 15m when briefly treated at 42°C as compared to the effect at 37°C. Furthermore, mutants resistant to the microcin at 37°C become sensitive at 42°C at microcin concentrations that are inactive at 37°C. This effect can be overcome byl-methionine. The mechanism involved seems to be based on an apparent inactivation of the homoserine-O-transsuccinylase activity. As previously established, this enzyme suffers a reversible partial inactivation when the cells are shifted to 42°C and the action of the microcin at this temperature seems to bring this process to a virtually irreversible stage. In mixed cultures of the microcin-producing strain and oneE. coli strain sensitive to the antibiotic, a much stronger growth inhibition of the latter strain has been observed at 42°C than at 37°C.     

Effect of Elevated Temperature on Nitrite and Nitrate Reduction in Leaves and Intact Chloroplasts

Barley (Hordeum vulgare L.) leaves and intact spinach (Spinacia oleracea L.) chloroplasts were exposed to short-term heating, and the aftereffects of heat treatment on in vitro andin vivo activities of nitrate reductase and noncyclic electron transport associated with nitrite reduction were studied. Heating of leaves at temperatures above 40°C led to a monotonic decrease in nitrate reductase in vitro activity. On the contrary, the in vivo enzyme activity, assayed in intact leaf tissues after 5-min heat treatment, increased 1.5 times upon elevating the pretreatment temperature from 37 to 40°C and gradually decreased at higher temperatures. Noncyclic electron transport related to CO₂ fixation in intact chloroplasts decreased gradually after heat exposures above 39°C, unlike the electron transport to nitrite as a terminal acceptor, which was stimulated by heating of intact chloroplast suspensions in the temperature range from 33 to 40°C. The heating at higher temperatures inhibited nitrite photoreduction. It is concluded that the heating of phototrophic cells at sublethal temperatures stimulates the mobilization of inorganic nitrogen and thereby facilitates the repair of thermally induced injuries of proteinaceous cell structures. The stimulation of nitrate reductase activity in vivo at the temperature range 37–40°C provides an evidence for the increase in the availability of reductants in the cytosolic compartment of the leaf cell.     

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.     

The recovery, penetrance, and pleiotropy of X-linked, cold sensitive mutants in Drosophila

Summary The viability at 16°, 22°, and 30°C, loci, visible phenotypes if any, possible effective lethal phase, and female fertility of seven X-linked, recessive cold sensitive mutations are reported. Five of the seven are female sterile at the restrictive temperature of 16°C; two of these five are also female sterile at the permissive temperature of 25°C. For two of the five mutations which are female sterile, escapers at the restrictive temperature exhibit visible phenotypes characteristic of mutants which affect protein synthesis. The possibility that some of the mutants affect ribosomes is considered. One of the mutants, l(1)TW-6 ^cs , is probably a cold sensitive meiotic mutant as well as a cold sensitive zygotic lethal. One of the mutants is a non-conditional visible allele of lozenge.Supported by NSF Grants GB 7707 and GB 20910.     

Conditions for mutagenesis by N-methyl-N′-nitro-N-nitrosoguanidine and relationships of A. tumefaciens mutants to crown-gall tumor induction

Optimum mutagenesis of Agrobacterium tumefaciens by N-methyl-N′-nitro-N-nitrosoguanidine occurred at pH 6.5 using 250 μg/ml of the mutagen for 3 h at 30°. Antibiotic-resistant mutants and amino acid auxotrophs were selected and scored for crown-gall tumor-inducing ability on Helianthus annuus (sunflower). Mutants resistant to neomycin, kanamycin or rifampicin were not directly affected in their tumor-inducing ability. Mutants that were resistant to neomycin were also resistant to kanamycin and vice versa. Various amino acid auxotrophs varied in virulence. Some of the auxotrophs that required histidine, leucine or tryptophan had simultaneously lost their virulence. The alteration of virulence of the organism is not dependent on its growth since the avirulent auxotrophs when supplemented with the amino acid requirement grew in vivo almost as well as the prototrophic strains and yet remained avirulent.     

The role ofSaccharomyces cerevisiae Cdc40p in DNA replication and mitotic spindle formation and/or maintenance

Successful progression through the cell cycle requires the coupling of mitotic spindle formation to DNA replication. In this report we present evidence suggesting that, inSaccharomyces cerevisiae, theCDC40 gene product is required to regulate both DNA replication and mitotic spindle formation. The deduced amino acid sequence ofCDC40 (455 amino acids) contains four copies of a -transducin-like repeat. Cdc40p is essential only at elevated temperatures, as a complete deletion or a truncated protein (deletion of the C-terminal 217 amino acids in thecdc40-1 allele) results in normal vegetative growth at 23°C, and cell cycle arrest at 36°C. In the mitotic cell cycle Cdc40p is apparently required for at least two steps: (1) for entry into S phase (neither DNA synthesis, nor mitotic spindle formation occurs at 36°C and (2) for completion of S-phase (cdc40::LEU2 cells cannot complete the cell cycle when returned to the permissive temperature in the presence of hydroxyurea). The role of Cdc40p as a regulatory protein linking DNA synthesis, spindle assembly/maintenance, and maturation promoting factor (MPF) activity is discussed.     

Chemotactic behavior of Campylobacter spp. in function of different temperatures (37 °C and 42 °C)

The chemotactic behaviour of Campylobacter strains was determined in the presence of different amino acids at two temperatures (37 °C and 42 °C). Two strains of catalase positive (Campylobacter jejuni) and negative (Campylobacter sputurum) Campylobacter were isolated from river water in Tonekabon, Iran and identified by phenotyping and 16srRNA Gene sequencing methods. Chemotactic responses of the isolates were assessed toward a variety of amino acids viz., L-cystine, L-asparagine, L-histidine, L-aspartic acid, L-serine, L-phenylalanine, L-leucine and L-tryptophan by disc and capillary methods at two temperatures: 37 °C and 42 °C. C. jejuni showed positive chemotactic response towards L-cystine,L-tryptophan, L-phenylalanine, - L-leucine, L-asparagine and L-Serine at both, 37 °C and 42 °C however, it was greater at 37 °C. C. sputurum showed negative or weak response towards all of the amino acids. In addition, C. jejuni illustrated strong chemotactic response to L-asparagine follow by L-serine and weak chemotaxis response to L-phenylalanine and L-cysteine at 37 °C. Overall, C. jejuni showed relatively strong chemotactic response to some amino acids, likewise it was greater at 37 °C. Hence, the human body temperature (37 °C) in compared to avian body temperature (42 °C) probably promotes chemotactic response of C. jejuni, which it might be a reason for causing disease in human being compared to avian.