SelectingRhizobium phaseoli strains for use with beans (Phaseolus vulgaris L.) in Kenya: Tolerance of high temperature and antibiotic resistance

Forty one strains ofRhizobium phaseoli were screened for the ability to multiply at high temperatures on yeast extract-mannitol agar. Most strains were tolerant of 30°C, eight strains were tolerant of 45°C and two of 47°C although the rate of multiplication was reduced at 45–47°C. The high temperature-tolerant strains were isolated from Kenyan soils and were fast-growing. Seven of the eight strains tolerant of 45–47°C lost their infectiveness after incubation at high temperature but four strains tolerant of 40°C remained infective after incubation at that temperature.Thirty six strains were resistant to 200 g ml^–1 streptomycin sulphate and 29 strains to 200 g ml^–1 spectinomycin dihydrochloride. Eight strains were resistant to both antibiotics each at 200 g ml^–1. Two of the double-labelled antibiotic-resistant mutants lost their infectiveness onPhaseolus vulgaris. The response to acidity was unaltered and two of the mutants showed a decrease in temperature tolerance. The doublelabelled mutants were recoverable from two Kenyan soils.     

Isolation and Primary Identification of Methylotrophic Yeast Hansenula polymorpha Mutants for Peroxisome Biogenesis

After exposure of cells of the methylotrophic yeast Hansenula polymorphaHF246leu1-1 to N-nitro-N-nitrosoguanidine, a collection of 227 mutants unable to grow on methanol at elevated temperature (45°C) was obtained. Ninety four ts mutants (35% of the total number of mutants), which were unable to grow on methanol only at 45°C but could grow at optimal temperature (37°C), were isolated. Complementation analysis of mutants using 12 deletion mutants for genes of peroxisome biogenesis (PEX) (available in this yeast species by the beginning of our work) allowed to assign 51 mutants (including 16 ts) to the separate group of mutants unable to complement deletion mutants with defects in eight PEX genes. These mutants were classified into three groups: group 1 contained 10pex10 mutants (4ts mutants among them); group 2 included 19 mutants that failed to complement otherpex testers: 1 pex1; 2 pex4(1ts); 6 pex5(5ts); 3 pex8; 1 pex13; 6 (3ts) pex19; group 3 contained 22 multiple mutants. In mutants of group 3, hybrids with several testers do not grow on methanol. All mutants (51) carried recessive mutations, except for mutant 108, in which the mutation was dominant only at 30°C, which suggests that it is ts-dominant. Recombination analysis of mutants belonging to group 2 revealed that only five mutants (two pex5 and three pex8) carried mutations for the corresponding PEX genes. Analysis of the spore population from the hybrids of remaining 14 mutants with the pex tester demonstrated the presence of methanol-utilizing segregants, which indicates mutation localization in other genes. In 19 mutants, random analysis of ascospores from hybrids obtained upon crossing mutants of group 3 with a strain lacking peroxisomal disorders (ade11) revealed a single mutation causing the appearance of a multiple phenotype. A more detailed study of two mutants from this group allowed us to localize this mutation in the only PEX gene (PEX1 or PEX2). The revealed disorder of complementation interactions between nonallelic genes is under debate.     

A Charged Second-Site Mutation in the Fusion Peptide Rescues Replication of a Mutant Avian Sarcoma and Leukosis Virus Lacking Critical Cysteine Residues Flanking the Internal Fusion Domain

The entry process of the avian sarcoma and leukosis virus (ASLV) family of retroviruses requires first a specific interaction between the viral surface (SU) glycoproteins and a receptor on the cell surface at a neutral pH, triggering conformational changes in the viral SU and transmembrane (TM) glycoproteins, followed by exposure to low pH to complete fusion. The ASLV TM glycoprotein has been proposed to adopt a structure similar to that of the Ebola virus GP2 protein: each contains an internal fusion peptide flanked by cysteine residues predicted to be in a disulfide bond. In a previous study, we concluded that the cysteines flanking the internal fusion peptide in ASLV TM are critical for efficient function of the ASLV viral glycoproteins in mediating entry. In this study, replication-competent ASLV mutant subgroup A [ASLV(A)] variants with these cysteine residues mutated were constructed and genetically selected for improved replication capacity in chicken fibroblasts. Viruses with single cysteine-to-serine mutations reverted to the wild-type sequence. However, viruses with both C9S and C45S (C9,45S) mutations retained both mutations and acquired a second-site mutation that significantly improved the infectivity of the genetically selected virus population. A charged-amino-acid second-site substitution in the TM internal fusion peptide at position 30 is preferred to rescue the C9,45S mutant ASLV(A). ASLV(A) envelope glycoproteins that contain the C9,45S and G30R mutations bind the Tva receptor at wild-type levels and have improved abilities to trigger conformational changes and to form stable TM oligomers compared to those of the C9,45S mutant glycoprotein.All retroviruses have envelope glycoproteins that interact with a receptor protein on the cell surface to initiate entry (18, 36). The viral glycoprotein is synthesized as a precursor polyprotein consisting of the surface (SU) glycoprotein, which contains the domains that bind with the cellular receptor, and the transmembrane (TM) glycoprotein, which tethers the protein to the viral surface and contains the domains responsible for fusion of the viral and cellular membranes (32). After synthesis, the precursor viral glycoproteins form trimers through the interaction of the TM domains. The SU and TM domains are then cleaved by a cellular protease, forming a mature, metastable complex capable of mediating viral entry. A specific receptor protein interaction with the SU domain of the mature Env is required to initiate a conformational change in the trimer, separating the globular SU domains to allow the TM glycoproteins to form a structure that projects the fusion peptide toward the target membrane. Two domains in TM, the N-terminal heptad repeat and the C-terminal heptad repeat, are critical for the formation of the extended structure (13, 31)}. The fusion peptide is thought to interact with a target membrane irreversibly, forming an extended prehairpin TM oligomer structure anchored in both the viral and target membranes (35). The cooperation of several of these extended prehairpin TM oligomer structures is most likely required to complete fusion. The viral and target membranes are brought into close proximity when the C-terminal heptad repeats fold back into grooves formed by the N-terminal heptad repeats, forming presumably the most stable TM structure, the six-helix bundle (6HB). Fusion of the membranes proceeds through the initial mixing of the outer lipid leaflets, hemifusion, followed by initial fusion pore formation, pore widening, and the completion of fusion. The 6HB may undergo some additional structural rearrangement in order to bring the fusion peptide and membrane-spanning domain of TM into close proximity to form the final trimeric hairpin structure (22, 24, 33).Until recently, the triggering of class I virus fusion proteins was thought to occur by one of two mechanisms (13, 35, 36). In one mechanism, the viral glycoproteins interact with receptors on the cell surface, resulting in the trafficking of the virion into an endocytic compartment, followed by the triggering of structural rearrangements in the viral glycoproteins to initiate fusion by exposure to low pH (e.g., influenza virus hemagglutinin [HA]). In a second entry mechanism, the interaction of the viral glycoproteins with receptors on the cell surface in a neutral pH environment triggers the structural rearrangements in the viral glycoproteins directly, initiating viral entry. Retroviruses predominately employ the second entry mechanism, although two cellular protein receptors may be required to complete the conformational changes in the viral glycoproteins necessary to complete entry (e.g., human immunodeficiency virus type 1). However, the entry process of the avian sarcoma and leukosis virus (ASLV) family of retroviruses demonstrates a third entry mechanism for the action of class I virus fusion proteins (25). ASLV entry requires both a specific interaction between the viral glycoproteins and receptors at the cell surface at neutral pH, triggering initial conformational changes in the viral glycoproteins, and a subsequent exposure to low pH to complete fusion (2, 3, 22-24).The fusion peptides of ASLVs are not at the N terminus of the cleaved TM, as in all other retroviral TM proteins, but in a proposed internal loop (TM residues 22 to 37) flanked by two cysteine residues (residues C9 and C45) (Fig. ​(Fig.1).1). The ASLV TM glycoprotein has been proposed to adopt a structure similar to that of the Ebola virus GP2 protein: both contain an internal fusion peptide flanked by cysteine residues predicted to be in a disulfide bond (10). Other viruses contain internal fusion peptides also predicted to be in looped structures (35). In a study to determine if the cysteines that flank the ASLV fusion peptide are required for function, mutant ASLV Env proteins were constructed with one or both of these cysteines changed to serine (C9S, C45S, or C9S C45S [C9,45S]) (8). The mutant subgroup A ASLV [ASLV(A)] Env proteins were expressed, processed, and incorporated into virions at levels similar to those of wild-type (WT) ASLV(A) Env. The mutant and WT ASLV(A) Env proteins bound the Tva receptor with similar affinities. However, murine leukemia virus (MLV) virions pseudotyped with the mutant Envs were ∼500-fold less infectious (titer, ∼2 × 10³ inclusion-forming units [IFU]/ml) than MLV virions pseudotyped with WT ASLV(A) Env (titer, ∼1 × 10⁶ IFU/ml). The ability of the mutant Envs to mediate cell fusion was also greatly impaired compared to that of WT ASLV(A) Env in a cell-cell fusion assay. We concluded that the cysteines flanking the internal fusion peptide in ASLV TM are critical for efficient function of the ASLV viral glycoproteins in mediating entry. In a recent study, the cysteines flanking the fusion peptide region were shown to be critical for the lipid mixing stage of fusion (6).Open in a separate windowFIG. 1.Schematic representations of the ASLV-based RCASBP retroviral vector and the major domains of the envelope glycoproteins. The RCASBP(A)AP replication-competent vector contains a subgroup A env and a reporter gene coding for heat-stable AP. The hypervariable domains (vr1, vr2, hr1, hr2, and vr3) of the SU glycoprotein, the proteolytic cleavage site, the putative fusion peptide region (shaded box), and the membrane-spanning domain (MSD) of the TM glycoprotein are shown schematically. The first 45 residues of the TM glycoprotein are shown for wild-type subgroup A Env (WT) and for the three mutants tested in this study, with either a substitution of serine for the cysteine at position 9 in TM (C9S), a substitution of serine for the cysteine at position 45 in TM (C45S), or both substitutions (C9,45S). The complete sequence of the ASLV(A) WT TM glycoprotein is shown, with the fusion peptide region, N-terminal and C-terminal heptad repeat regions (N-alpha-helix; C-alpha helix), and membrane-spanning domain indicated.Very little is known about the structures of fusion peptides in the context of full-length, trimeric, viral glycoproteins upon interaction with target membranes. Also, natural membrane targets contain a variety of lipid and protein compositions in an asymmetrical organization that is difficult to reproduce experimentally (27). In addition, little is known about how fusion proteins with internal fusion peptide regions interact with target membranes or the possible conformational changes that might be required to complete the fusion process (19, 20). In this study, replication-competent ASLV(A) viruses containing the C9S, C45S, or C9,45S mutations were constructed and genetically selected for improved replication in chicken fibroblasts in order to further explore the importance of these cysteines for proper TM function. Viruses with single cysteine-to-serine mutations reverted to the WT sequence. However, viruses with both the C9S and the C45S mutation retained both mutations and acquired a second-site mutation that significantly enhanced the infectivity of the genetically selected virus population. Unexpectedly, the selected second-site mutation was a charged residue located in the middle of the hydrophobic fusion peptide within TM.     

Okadaic acid causes breakdown of self-incompatibility in Brassica oleracea: evidence for the involvement of protein phosphatases in the incompatible response

Summary Detached pistils from inbred lines of Brassica oleracea L. var alboglabra were fed with okadaic acid (OA), an inhibitor of serine/threonine protein phosphatases, via the transpiration stream. Following self-pollination, pollen tubes were observed to have grown into or through the styles of pistils treated with OA, but not those of untreated controls. Treatment with 1 M OA was sufficient to completely overcome self-incompatibility (SI) in an inbred line homozygous for the S63 allele, though an OA concentration of 5 M was required to cause breakdown of SI in an inbred line homozygous for the S29 allele. At the higher concentration used, pollen tube growth was arrested before the pollen tubes reached the ovary, but this effect was also noted in cross-pollinated styles treated in the same manner. These data provide evidence for the involvement of type 1 and/or type 2A protein phosphatases in the Brassica SI signal transduction mechanism. Present address after November 1993: Department of Biology, Colorado State University, Fort Collins, Colorado, USA     

Cold-sensitive mutations in the Z gene of prophage P2 that result in increased sensitivity of the lysogens to a low molecular weight product of the host bacteria

Summary Z mutants of bacteriophage P2 form clear plaques and are unable to give rise to stable lysogens in Escherichia coli C. To study the function of the Z gene in lysogenization by P2, temperature-sensitive mutants were isolated. Those that were classified as Z mutants by complementation were all cold-sensitive (cs); they were unable to form lysogens at 30° C, but had wild type phenotype at 42° C. When lysogens carrying such mutants, prepared at 42° C, were shifted to the lower temperature, the bacteria continued to multiply at the normal rate until they reached concentrations of about 5 × 10⁷ per ml, at which point the viable titer began to decrease. Inactivation of the bacteria at even lower concentrations occurred if they were transferred to medium taken from overnight cultures of the same strain, suggesting that they were sensitive to some material that had accumulated in the culture medium.The lethal material was produced not only by csZ lysogens, but by all derivatives of Escherichia coli C tested, including non-lysogens, and at both 30° C and 42° C. Only csZ lysogens were sensitive to it, however, and only at the lower temperature. A preliminary characterization of the material indicates that it is heat-stable, of low molecular weight and does not adsorb to activated charcoal.This work was supported by Research Grant 72 from the Swedish Medical Research Council     

Structure and dynamic properties of the single disulfide-deficient α-amylase inhibitor [C45A/C73A]tendamistat: An NMR study

Covalent linkages such as disulfide bonds are important for the stabilization of proteins. In the present NMR study we compare the structure and the dynamics of the single disulfide-deficient variant C45A/C73A of the α-amylase inhibitor tendamistat and the wild-type protein, which contains two disulfide bonds (C11-C27 and C45-C73). Complete proton assignment was achieved by standard homonuclear 2D techniques for the variant. Chemical shift differences, intra-strand NOE effects and protected amide proton were used to compare the connectivity of the secondary structure elements of variant and wild-type. Dynamic properties of the wild-type protein were studied by ¹³C_α heteronuclear NOE experiments with carbon in natural abundance. ¹⁵N isotope labeling was necessary to obtain the relaxation parameters of the variant, because of sample degradation. The ¹⁵N resonance assignment was achieved by a ¹⁵N 3D-NOESY-HMQC. Removal of the C45-C73 bond by the C45A/C73A mutation has no influence upon the β-barrel structure of tendamistat beside very local changes at the mutation site. The relaxation data revealed only subtle differences between variant and wild-type on a subnanosecond time scale. Only the N-terminus and G62 in the connecting loop between the anti-parallel β-sheets showed an increased mobility. The results are discussed in respect to thermodynamic stability and the secretion efficiency of tendamistat. Proteins 33:285–294, 1998. © 1998 Wiley-Liss, Inc.     

Photoperiodism and thermoperiodism in the predatory mite Amblyseius potentillae are probably based on the same mechanism

Summary A comparative study was made of the photoperiodic and thermoperiodic induction of diapause in the phytoseiid mite Amblyseius potentillae. Sensitivity to thermoperiod was found to be highest during the protonymphal and deutonymphal stages, with some sensitivity still being present in the young adult. Summation of both photoperiodic and thermoperiodic cycles was shown to take place, which demonstrated the presence of a photoperiodic counter as well as a thermoperiodic counter in these mites. Vitamin A appeared to be necessary for some early step in the physiological mechanism of diapause induction and not just for the expression of the diapause response. The light sensitivity threshold for photoperiodic induction of diapause was found to be extremely low, viz. less than 0.02 W/cm². Moreover, the light sensitivity threshold appeared to be strongly temperature dependent in A. potentillae. Experiments in which the mites experienced various sequences of short-day photoperiods and short-day thermoperiods, applied either concurrently or in succession, showed that the information collected by the photoperiodic counter and the thermoperiodic counter is integrated into one induction sum. These results strongly suggest that photoperiodic and thermoperiodic induction of diapause in these mites is based on the same physiological mechanism.Abbreviations DD continuous darkness - LL continuous light - LD light-dark cycle (e.g. LD 16:8 is a cycle of 16 h of light and 8 h of darkness) - TC thermoperiodic cycle (e.g. TC 16:8 (27°: 15°) is a thermoperiod with a 16 h thermophase of 27 °C and an 18 h cryophase of 15°C)     

The effect of tunicamycin on Leishmania brasiliensis. Glycosylation and the cell surface components

Culture conditions of Leishmania cells were developed to allow the study of the effect of tunicamycin (TM) on glycosylation and on the cell surface components. Leishmania incorporate [¹⁴C]-mannose and [³⁵S]-methionine in vitro. The incorporation of [¹⁴C]-mannose is linear for 150 min and is inhibited by TM (2 g/ml) in a time dependent effect which reaches a plateau of 45% inhibition at 36 h. Under the same experimental conditions [³⁵S]-methionine incorporation into protein is slightly affected. This is reflected by an almost identical polypeptide pattern for TM treated and non-treated cells when analyzed on SDS-PAGE. On the contrary, strong differences were detected on the labeled compounds analyzed on SDS-PAGE followed by autoradiography when the precursor used was [¹⁴C]-mannose. A shift in the electrophoretic mobility of most of the glycopeptides synthesized in the presence of TM was observed, which is also reflected in the structure of the main Leishmania cell surface components.The findings are discussed in the light of biological implications.     

The nature of ompA mutants of Escherichia coli K12 exhibiting temperature-sensitive bacteriophage resistance

Summary A class of ompA mutants of Escherichia coli, exhibiting temperature-sensitive resistance towards phages using the OmpA protein as receptor, was analysed. The mutants produce detectable levels of the protein at 42°C but not at 30°C (Manning and Reeves 1976). They were found to have a deletion (one isolate) or insertions (three isolates) upstream of the coding part of the ompA gene. Several previously characterized mutants possessing insertions or a deletion in the non-translated 5 area of the gene also exhibited a similar temperature-sensitive phage resistance. This cold-sensitive phenotype is explained in terms of the recent discovery that the stability of ompA mRNA is regulated by the rate of cell growth (Nilsson et al. 1984).     

Abscisic acid and mefluidide in the regulation of cold hardiness development in Solanum tuberosum L. potato

Plants of Solanum tuberosum L. potato do not cold acclimate when exposed to low temperature such as 5°C, day/night. When ABA (45 M) was added to the culture medium, stem-cultured plantlets of S. tuberosum, cv. Red Pontiac, either grown at 20°C/15°C, day/night, or at 5°C, increased in cold hardiness from –2°C (killing temperature) to –4.5°C. The increase in cold hardiness could be inhibited in both temperature regimes if cycloheximide (70 M) was added to the culture medium at the inception of ABA treatment. Cycloheximide did not inhibit cold hardiness development, however, when it was added to the culture medium 3 days after ABA treatment.When pot-grown plants were foliar sprayed with mefluidide (50 M), ABA content increased from 10 nmol to 30 nmol g^–1 dry weight and plants increased in cold hardiness from –2°C to about –3.5°C. The increases in free ABA and cold hardiness occurred only in plants grown at 20°C/15°C; neither ABA nor cold hardiness increased in plants grown at 5°C.The results suggest that an increase in ABA and a subsequent de novo synthesis of proteins are required for the development of cold hardiness in S. tuberosum regardless of temperature regime, and that the inability to synthesize ABA at low temperature, rather than protein synthesis, appears to be the reason why S. tuberosum does not cold acclimate.     

Thermus kawarayensis sp. nov., a new member of the genus Thermus, isolated from Japanese hot springs

A long-rod-shaped thermophilic microorganism, strain KW11, was isolated from a hot springs located in the Kawarayu, Gunma, Japan. Cloning and preliminary sequence analysis of 16S rDNA showed that this isolate belongs to the genus Thermus. The cells were 10–20 m long, about 0.8 m in diameter, and produced no pigment in contrast with most of the Thermus species previously reported. KW11 was an aerobic heterotroph and grew at temperatures ranging from 40–73°C, with optimal growth occurring at 68°C. The pH range for growth was from 5.8–8.9, with optimal growth around pH 7. KW11 was sensitive to ampicillin, penicillin G, kanamycin, and streptomycin. The G+C content of DNA was 69 mol%. The main fatty acids were 16:0 (52.9%), iso-15:0 (22.1%), and iso-17:0 (15.6%). The 16S rDNA sequence of KW11 showed 96.0, 95.8, and 95.4% similarity with the sequences of T. aquaticus, T. igniterrae, and T. thermophilus, respectively, and less than 95% with other Thermus species. The physiological differences and phylogenetic evidence indicated that strain KW11 represents T. kawarayensis, a novel species of the genus Thermus. The type strain is isolate KW11^T (JCM12314, DSM16200).     

Val133 and Cys137 in transmembrane segment 2 are close to Arg935 and Gly939 in transmembrane segment 11 of human P-glycoprotein

P-glycoprotein (P-gp; ABCB1) transports a wide variety of structurally diverse compounds out of the cell. The protein has two homologous halves joined by a linker region. Each half consists of a transmembrane (TM) domain with six TM segments and a nucleotide-binding domain. The drug substrate-binding pocket is at the interface between the TM segments in each half of the protein. Preliminary studies suggested that the arrangement of the two halves of P-gp shows rotational symmetry (i.e. "head-to-tail" arrangement). Here, we tested this model by determining whether the cytoplasmic ends of TM2 and TM3 in the N-terminal half are in close contact with TM11 in the C-terminal half. Mutants containing a pair of cysteines in TM2/TM11 or TM3/TM11 were subjected to oxidative cross-linking with copper phenanthroline. Two of the 110 TM2/TM11 mutants, V133C(TM2)/G939C(TM11) and C137C(TM2)/A935C (TM11), were cross-linked at 4 degrees C, when thermal motion is reduced. Cross-linking was specific since no cross-linked product was detected in the 100 double Cys TM3/TM11 mutants. Vanadate trapping of nucleotide or the presence of some drug substrates inhibited cross-linking of mutants V133C(TM2)/G939C(TM11) and C137C(TM2)/A935C(TM11). Cross-linking of TM2 and TM11 also blocked drug-stimulated ATPase activity. The close proximity of TM2/TM11 and TM5/TM8 (Loo, T. W., Bartlett, M. C., and Clarke, D. M. (2004) J. Biol. Chem. 279, 7692-7697) indicates that these regions between the two halves must enclose the drug-binding pocket at the cytoplasmic side of P-gp. They may form the "hinges" required for conformational changes during the transport cycle.     

Temperature-sensitive mutant rho-115 rho-RNA binary complexes, and stabilization by substrates and analogues

Summary To determine the molecular basis for the temperature-sensitivity of pure rho RNA-dependent ATPase from Escherichia coli mutant rho-115 cells, we investigated mutant rho binding to [³H] polyC as measured by retention on nitrocellulose filters. Complexes of wild-type rho and polyC incubated at 37°C and 45°C were similarly stable. At 37°C mutant rho-polyC binary complexes were inactivated at a slightly faster rate than complexes with wild-type rho. Upon shift to 45°C the quantity of rho-115 bound to polyC declined immediately, resulting in one-fifth of the quantity of complexes observed at 37°C. Shift back to 37°C restored the level of observed complexes by two-fold. The inclusion of ATP or the analogue - methylene ATP during 45°C incubation resulted in stable mutant rho-polyC complexes. The hydrolysis product ADP was also effective in stabilizing binary complexes at 45°C but this effect was observed with an order of magnitude more ADP than ATP. Adenine, adenosine, AMP or P_i had no stabilizing effect. We conclude that the mutant rho-115 protein exhibits a structural instability as a result of binding RNA. Furthermore ATP confers a wild-type phenotype upon rho-115 protein, probably as a result of conformational change due to binding of this compound. The effect of ATP on the stability of mutant rho-polyC binary complexes supports the model of ATP modulation of rho-RNA interaction proposed by Galluppi and Richardson (1980).     

Synergistic effects of ethanol and temperature on yeast mitochondria

At temperatures lower than 37°C, the ethanol inhibition constant (Ki) for growth or fermentation inrho ⁺ cells of theSaccharomyces cerevisiae strain S288C was always higher (1.1M) than inrho ^– mutants (0.7M). At 37°C these differences disappeared, and both strains were equally inhibited by ethanol (Ki=0.7m). Mitochondrial activity can be inhibited by high ethanol concentration and temperature. In fact, the stronger inhibition by ethanol of therho ⁺ strain at 37°C was due to the fact that, under these conditions, this strain loses the advantage conferred by mitochondrial activity since the induction ofrho ^– cells in the population is very high. This does not result in an increase in the frequency ofrho ^– mutants because of the poor viability of these mutants in conditions of high temperature and ethanol. In consequence, S288C strain becomes as strongly inhibited by ethanol as therho ^– mutant strains. Differences in viability were not related to the fatty acids and ergosterol composition of the strain. In the presence of ethanol, bothrho ⁺ andrho ^– strains modified their lipids in the same way, but these changes did not improve their ethanol tolerance. They were not due to differences in adaptation to ethanol either, since after successive transfers in ethanol, growth () and fermentation () rates in therho ^– mutants were increasingly inhibited with time, whereas in the S288C strain inhibition of and by ethanol remained unaltered. Rather,rho ^– mutants are less viable thanrho ⁺ cells because of the inability of the former to respire. At 37°C the Ki increased to 0.9M ethanol either when mitochondrial from highly ethanol-tolerant wine yeasts were transferred torho ^– mutants of the strain S288C or when the mitochondria of strain S288C were preadapted by growing the strain in glycerol instead of glucose before it was cultivated in ethanol.     

Irradiance and temperature effects on photosynthesis of tussock tundra Sphagnum mosses from the foothills of the Philip Smith Mountains,Alaska

Summary Photosynthetic characteristics of three species of Sphagnum common in the foothills of the Brooks Range on the North Slope of Alaska were investigated. Generally, light-saturated rates of net photosynthesis decreased in the order S. squarrosum, S. angustifolium, and S. warnstorfii when plants were grown under common growth chamber conditions. For field-grown S. angustifolium, average light compensation point at 10°C was 37 mol m^-2s^-1 photosynthetic photon flux density (PPFD), and light saturation occurred between 250 and 500 mol m^-2 s^-1. At 20°C, compensation point increased to 127 mol m^-2s^-1 and the PPFD required for light saturation increased to approximately 500 mol m^-2s^-1, while maximum rates of CO₂ uptake increased only slightly. Light response curves of chamber-grown plants exhibited substantially lower compensation points and higher light-saturated rates of CO₂ assimilation than field-grown material, due perhaps to a higher percentage of green, photosynthetically competent tissue. All three species exhibited broad responses to temperature, with optima near 20°C, and maintained at least 75% of maximum assimilation between approx. 13° and 30°C. Rates at 5°C were approx. 50% of maximum. Studies of the microclimate of Sphagnum at the field research site suggest that CO₂ uptake should occur at near light-saturated rates during the day in open tussock tundra but that PPFD may often be limiting under Salix and Betula canopies in a water track drainage. Simulations using a simple model provided a seasonal estimate of 0.78 g dry weight (DW) of S. angustifolium produced from each initial g of photosynthetic tissue under willow canopies, assuming no water limitations. Although the simulation model suggests that production would be 66% higher in open tussock tundra, S. angustifolium is rarely found in this potentially more stressful habitat. To explain the relative abundance of Sphagnum in shaded water track areas as compared to open tussock tundra, we postulate that the vascular plant canopies provide protection from adverse effects of high temperatures, excess irradiance and reduced water availability. Under conditions of normal water availability, removal of the vascular plant cover did not affect the tissue water content of S. squarrosum, but resulted in a strong decrease in photosynthetic capacity, accompanied by chlorophyll bleaching. These results suggest that photoinhibition may limit production under certain conditions.     

Adaptive response to cold temperatures and characterization of cspA in Salmonella typhimurium LT2

Salmonella typhimurium is a major foodborne microbial pathogen which primarily contaminates poultry products causing salmonellosis in humans. S. typhimurium LT2 cultures, when transferred from 37 °C to 5 °C or 10 °C, showed an initial lag period in growth with an approximate generation time of 10–25 h. Western blot assay using E. coli CS7.4 antibody and analysis of radiolabeled total cellular proteins from S. typhimurium cultures after exposure to 10 °C or 5 °C showed elevated expression of a major cold shock protein, CS7.4. Identification of a decreased level of CS7.4 at 37 °C suggests that the expression of this protein may require a large temperature downshift. Putative regulatory protein binding segment on the 5-untranslated region referred as Fragment 7 in S. typhimurium exhibited a 90.6% and a 56.25% nucleotide sequence identity when compared with the Fragment 7 of E. coli and S. enteritidis, respectively. The differences in the nucleotide sequence within the Fragment 7 between S. typhimurium and S. enteritidis may explain the differential expression of CspA at 37 °C. The nucleotide sequence of the open reading frame of S. typhimurium cspA gene showed a single base difference at 816 bp position from a G to a C which altered the amino acid residue from a glycine to an alanine. In addition to CspA, an elevated expression of a 105 kDa, and decreased expression of 6 proteins were evidenced when cultures of S. typhimurium were exposed to 10 °C or 5 °C. Differential expression of the CspA and other proteins in S. typhimurium following exposure to cold temperatures suggest that adaptation and continued growth and survival at cold temperatures in this pathogen may be aided by these cold-responsive proteins.     

Conformational change of bovine serum albumin by heat treatment

The thermal denaturation of bovine serum albumin (BSA) was studied at pH 2.8 and 7.0 in the range of 2–65°C. The relative proportions of -helix, -structure, and disordered structure in the protein conformation were determined as a function of temperature, by the curve-fitting method of circular dichroism spectra. With the rise of temperature at pH 7.0, the proportion of -helix decreased above 30°C and those of -structure and disordered structure increased in the same temperature range. The structural change was reversible in the temperature range below 45°C. However, the structural change was partially reversible upon cooling to room temperature subsequent to heating at 65°C. On the other hand, the structural change of BSA at pH 2.3 was completely reversible in the temperature range of 2–65°C, probably because the interactions between domains and between subdomains might disappear due to the acid expansion. The secondary structure of disulfide bridges-cleaved BSA remained unchanged during the heat treatment up to 65°C at pH 2.8 and 7.0.     

Induction of microspore embryogenesis in Brassica napus L. is accompanied by specific changes in protein synthesis

Culture temperature determines the developmental fate of isolated microspores from Brassica napus L. At 18°C, tricellular pollen develops, whereas culture at 32°C for 8 h leads to the quantitative and synchronous induction of embryogenesis, and ultimately to the formation of embryos. We investigated the changes in protein synthesis that are associated with this 8-h inductive period by using in-situ [³⁵S]methionine labeling, followed by two-dimensional (2-D) gel electrophoretic analysis of the radiolabeled proteins. Qualitative and quantitative computer analyses of 2-D [³⁵S]methionine protein patterns showed six polypeptides specifically labeled under embryogenic culture conditions. Eighteen polypeptides incorporated [³⁵S]methionine at a statistically significant higher rate under embryogenic culture conditions (32°C) than in the controls (18°C), whereas one protein was preferentially labeled under non-embryogenic culture conditions (18°C). These results indicate that only a limited number of proteins detectable in the 2-D gels of microspore extracts are associated with the early induction of embryogenesis. The reproducible identification of the differentially radiolabeled proteins in the 2-D gels allow the sequencing of representative peptides and the isolation of the corresponding cDNAs. This may lead to the identification and characterization of proteins associated with the very first stages of plant embryogenesis.Abbreviations 2-D two-dimensional We would like to thank Dr. H. Van Steeg (Rijks Instituut voor Milieubeheer (RIVM), Bilthoven, The Netherlands) for use of the PhosphorImager apparatus. This research was carried out as part of the EC-Bridge project Regulation of the inductive phase of microspore embryogenesis and EC-Science project The role of mitotic and cytoskeletal genes in the induction of plant cell division.     

Phenology of stranded kelp degradation by the kelp fly Paractora dreuxi mirabilis (Helcomyzidae) at Marion island

Summary During heavy seas, bull-kelp Durvillaea antarctica (Cham.) Har. plants are stranded either singly or in wrack beds on sub-Antarctic Marion Island's (46°54S, 37°45E) few pebbled beaches. In the supralittoral zone, Durvillaea wrack is consumed chiefly by kelp fly larvae (the endemic Paractora dreuxi mirabilis Séguy), whilst two marine amphipods attack wrack in the eulittoral and sometimes in the supralittoral zone. During the summer of 1984 on Marion Island, artificial Durvillaea deposits were established in the supralittoral zone of a beach close to the meteorological station and were closely monitored for six weeks thereafter. Eight days after deposition, temperatures inside the protected wrack bed reached 10°C above ambient, and P. dreuxi larvae attained a maximum biomass of 27 g/kg (dry mass) of kelp in the bed. After 30 days, wrack strings and a protected wrack bed had lost 80 and 65%, respectively, of their original dry mass. A bed left exposed to trampling by seals had disappeared. Over 30 days, P. dreuxi larvae accounted for 35% of the dry mass loss in the wrack bed. P. dreuxi has an opportunistic and flexible ecological strategy, facilitated by a stable, interstitial biotope of particulate kelp detritus from which it colonizes fresh wrack.