Uptake and catabolism of D-xylose in Salmonella typhimurium LT2.

Salmonella typhimurium LT2 grows on D-xylose as sole carbon source with a generation time of 105 to 110 min. The following activities are induced at the indicated time after the addition of the inducer, D-xylose: D-xylulokinase (5 min), D-xylose isomerase (7 to 8 min), and D-xylose transport (10 min). All other pentoses and pentitols tested failed to induce isomerase or kinase. Synthesis of D-xylose isomerase was subject to catabolite repression, which was reversed by the addition of cyclic adenosine monophosphate. Most of the radioactive counts from D-[14C]xylose were initially accumulated in the cell in the form of D-xylose or D-xylulose. D-Xylose uptake in a mutant which was deficient in D-xylose isomerase was equal to that of the wild type. The apparent Km for D-xylose uptake was 0.41 mM. Some L-arabinose was accumulated in D-xylose-induced cells, and some D-xylose was accumulated in L-arabinose-induced cells. D-Xylitol and L-arabinose competed against C-xylose uptake, but D-arabinose, D-lyxose, and L-lyxose did not. Osmotic shock reduced the uptake of D-xylose by about 50%; by equilibrium dialysis, a D-xylose-binding protein was detected in the supernatant fluid after spheroplasts were formed from D-xylose-induced cells.     

Xylose Transport by the Anaerobic Thermophile Thermoanaerobacter ethanolicus and the Characterization of a D-Xylose-Binding Protein

Thermoanaerobacter ethanolicus is a xylose-utilizing thermophilic anaerobe that produces considerable amounts of ethanol. A protein in xylose-growing cells was solubilized from cell membranes by extraction with octyl-β-glucoside. Internal peptide sequencing revealed that the protein was the product of a gene, xylF, encoding a putative D-xylose-binding protein. Metabolic labeling with ¹⁴C palmitic acid suggested that this is a lipoprotein that is anchored to the cell membrane via a cysteine residue. Binding was highly specific for xylose as evident by the lack of competition by sugars with structures similar to xylose. The apparent K _d of the protein for xylose was approximately 1.5 μM, and this value was very similar to the affinity constant determined for xylose transport by whole cells at low substrate concentrations. Uptake experiments with cells also suggested the presence of a separate low-affinity system. Binding activity varied less than 20% over a pH range of 4–8, and the level of activity was virtually unaffected when temperature was varied between 40°C and 80°C. This is the first biochemical characterization of a D-xylose-binding protein from a thermophilic organism. Received: 22 April 1998 / Accepted: 21 May 1998     

A Nonconserved Carboxy-Terminal Segment of GroEL Contributes to Reaction Temperature

The role of the C-terminal segment of the GroEL equatorial domain was analyzed. To understand the molecular basis for the different active temperatures of GroEL from three bacteria, we constructed a series of chimeric GroELs combining the C-terminal segment of the equatorial domain from one species with the remainder of GroEL from another. In each case, the foreign C-terminal segment substantially altered the active temperature range of the chimera. Substitution of L524 of Escherichia coli GroEL with the corresponding residue (isoleucine) from psychrophilic GroEL resulted in a GroE with approximately wild-type activity at 25 °C, but also at 10 °C, a temperature at which wild-type E. coli GroE is inactive. In a detailed look at the temperature dependence of the GroELs, normal E. coli GroEL and the L524I mutant became highly active above 14 °C and 12 °C respectively. Similar temperature dependences were observed in a surface plasmon resonance assay of GroES binding. These results suggested that the C-terminal segment of the GroEL equatorial domain has an important role in the temperature dependence of GroEL. Moreover, E. coli acquired the ability to grow at low temperature through the introduction of cold-adapted chimeric or L524I mutant groEL genes.     

Characterization of a thermostable glucose dehydrogenase with strict substrate specificity from a hyperthermophilic archaeon Thermoproteus sp. GDH-1

A hyperthermophilic archaeon was isolated from a terrestrial hot spring on Kodakara Island, Japan and designated as Thermoproteus sp. glucose dehydrogenase (GDH-1). Cell extracts from cells grown in medium supplemented with glucose exhibited NAD(P)-dependent glucose dehydrogenase activity. The enzyme (TgGDH) was purified and found to display a strict preference for d-glucose. The gene was cloned and expressed in Escherichia coli, resulting in the production of a soluble and active protein. Recombinant TgGDH displayed extremely high thermostability and an optimal temperature higher than 85 °C, in addition to its strict specificity for d-glucose. Despite its thermophilic nature, TgGDH still exhibited activity at 25 °C. We confirmed that the enzyme could be applied for glucose measurements at ambient temperatures, suggesting a potential of the enzyme for use in measurements in blood samples.     

Synergistic effect of high-light and low temperature on cell growth of the Δ12 fatty acid desaturase mutant in Synechococcus sp. PCC 7002

The effect of polyunsaturated fatty acids on photosynthesis and the growth of the marine cyanobacterium Synechococcus sp. PCC 7002 was examined using wild-type and Δ12 fatty acid desaturase mutant strains. Under a light intensity of 250 μmol m⁻² s⁻¹, wild-type cells could grow exponentially in a temperature range of 20–38 °C, but growth was non-exponential below 20 °C and ceased at 12 °C. The Δ12 desaturase mutant cells lacking polyunsaturated fatty acids had the same growth rate as wild-type cells in a temperature range of 25–38 °C but grew slowly at 22 °C, and no cell growth took place below 18 °C. Under a very high-light intensity of 2.5 mmol m⁻² s⁻¹, wild-type cells could grow exponentially in a temperature range of 30–38 °C, although the high-light grown cells became chlorotic because of nitrogen limitation. The temperature sensitive phenotype in the Δ12 desaturase mutant was enhanced in cells grown under high-light illumination; the mutant cells could grow at 38 °C, but were killed at 30 °C. The decrease of oxygen evolution and nitrate consumption by whole cells as a function of temperature was similar in both wild type and the Δ12 desaturase mutant. No differences were observed in either light-induced damage of oxygen evolution or recovery from this damage. No inactivation of oxygen evolution took place at 22 °C under the normal light intensity of 250 μmol m⁻² s⁻¹. These results suggest that growth of the Δ12 desaturase mutant at low temperature is not directly limited by the inactivation of photosynthesis, and raise new questions about the functions of polyunsaturated membrane lipids on low temperature acclimation in cyanobacteria. This revised version was published online in June 2006 with corrections to the Cover Date.     

Secreted β-galactosidase from a Flavobacterium sp. isolated from a low-temperature environment

The bacterial strain Flavobacterium sp. 4214 isolated from Greenland was found to express β-galactosidase (EC 3.2.1.23) at temperatures below 25°C. A chromosomal library of Flavobacterium sp. 4214 was constructed in Escherichia coli, and the gene gal4214-1 encoding a β-galactosidase of 1,046 amino acids (114.3 kDa) belonging to glycosyl hydrolase family 2 was isolated. This was the only gene encoding β-galactosidase activity that was identified in the chromosomal library. Expression levels in both Flavobacterium sp. 4214 and in initial recombinant E. coli strains were insufficient for biochemical characterization. However, a combination of T7 promoter expression and introduction of an E. coli host that complemented rare transfer RNA genes yielded 15 mg of β-galactosidase per liter of culture. Gal4214-1-His protein was found to be active in monomeric conformation. The protein was secreted from the cytoplasm, probably through an N-terminal signaling sequence. The Gal4214-1-His protein was found to have optimum activity at a temperature of 42°C, but with short-term stability at temperatures above 25°C.     

L-asparaginase synthesis by Escherichia coli B

We have studied the influence of strain of organism, temperature, and medium on the production of the antileukemic intracellular enzyme L-asparaginase by E. coli B grown in shaken flasks. Five strains of E. coli B exhibited wide differences in their capacities to synthesize the EC-2 form of L-asparaginase active against leukemia. For the most productive strain, when grown in a casein hydrolysate medium, maximal production of L-asparaginase occurred at 25°C. At this temperature, the organism required glycerol, glucose, or other mono-saccharides to synthesize L-asparaginase. Synthesis was stimulated when glycerol was used in place of glucose, but not in its presence. The effect of glycerol on L-asparaginase synthesis was most evident when the cells were grown at 37°C, rather than at 25°C. With 0.25% glucose, cells had a specific activity of 409 I.U./g; with glycerol cells had a specific activity of 553 I.U./g. At 25°C, both cell and L-asparaginase synthesis were increased by the use of 0.25% glycerol resulting in only a slight increase in specific activity of the cells. The addition of zinc, copper, manganese, iron, L-asparagine, L-glutamine, or L-aspartic acid had no effect on L-asparaginase synthesis in the casein hydrolysate medium. L-aspartic acid (10^?2 M) enhanced L-asparaginase synthesis in a synthetic medium that lacked these metals or L-asparagine, L-glutamine, or L-aspartic acid; cells grown under these conditions had a specific activity of 90 I.U./g. In the casein hydrolysate medium, cell morphology was correlated with temperature of incubation.     

Temperature-sensitivity of D1 protein metabolism in isolated Zea mays chloroplasts

The effects of low temperature on the synthesis and stability of the 32 kDa D1 protein of photosystem II were investigated in chloroplasts isolated from maize (Zea mays cv. LG11) leaves. The synthesis of D1 by intact chloroplasts in vitro was strongly dependent on temperature; the Q₁₀ for the initial rate of incorporation of [³⁵S]-methionine into D1 was ca. 2.6 over the range 13–25°C. The synthesis of other thylakoid polypeptides exhibited a similar temperature dependence, whilst synthesis of stromal proteins was considerably less temperature-dependent, with the exception of two polypeptides of ca. 56 and 59.5 kDa. The stability of newly-synthesized D1 in the thylakoid membranes was dependent both on the temperature at which the plants were grown and on the temperature during the pulse-labelling period when the protein was synthesized. In chloroplasts isolated from maize leaves grown at 25°C, D1 that was synthesized and assembled at 25 °C in vitro was rapidly degraded during the chase period. At lower chase temperatures the protein was more stable. When chloroplasts from 25°C-grown leaves were pulse-labelled at 13°C, the stability of D1 was markedly enhanced at all temperatures during the chase period. This effect was even more pronounced in chloroplasts isolated from plants grown at 14°C. The implications of these results are discussed with regard to the ability of maize to recover from photoinhibitory damage at low temperatures.     

Regulation of Synthesis of Chlorophyll, Carotene, Ribulosel,5-diP Carboxylase and Phosphoribulokinase in a Temperature-sensitive Chlorophyll Mutant of Medicago sativa

The activities of several enzymes were studied in a temperature-sensitive chlorophyll mutant of alfalfa (Medicago saliva). In leaves grown at 10°C photosynthetic capacity was essentially nil with ribulose-1,5-diP carboxylase, chlorophyll, and carolene present in greatly limiting concentrations. The activity of phosphoribulokinase was 3.5 times lower at 10°C than at 27°C, but was still sufficiently high at 10°C to not limit the rate of CO₂ fixation. Activities of phosphoriboisomerase, phosphoenolpyruvate carboxylase, glucose-6-P dehydrogenase and malate dehydrogenase were not different at 10°C and 27°C. The low fraction I protein content (which also accounts for the ribulose-1,5-dip carboxylase activity in alfalfa) indicated that synthesis of the carboxylase was effectively blocked at 10°C. A large, comparable increase in carboxylase activity and in concentration of fraction I protein in alfalfa leaves grown at 27°C indicated that the carboxylase was synthesized de novo. The initial induction of the carboxylase, chlorophyll, and carotene may be related, but after induction the carboxylase was not linearly correlated with the other two and had a different temperature optima. Nevertheless, the synthesis of each appeared to be regulated by the temperature-sensitive gene of this mutant.     

Acclimation of photosystem II to high temperature in a suspension culture of soybean (Glycine max) cells requires proteins that are associated with the thylakoid membrane

In a study of the responses of photosystem II (PSII) to high temperature in suspension-cultured cells of soybean (Glycine max L. Merr.), we found that high temperatures inactivated PSII via two distinct pathways. Inactivation of PSII by moderately high temperatures, such as 41°C, was reversed upon transfer of cells to 25°C. The recovery of PSII required light, but not the synthesis of proteins de novo. By contrast, temperatures higher than 45°C inactivated PSII irreversibly. An increase in the growth temperature from 25 to 35°C resulted in an upward shift of 3°C in the profile of the heat-induced inactivation of PSII, which indicated that the thermal stability of PSII had been enhanced. This acclimative response was reflected by the properties of isolated thylakoid membranes: PSII in thylakoid membranes from cells that had been grown at 35°C exhibited greater thermal stability than that from cells grown at 25°C. Disruption of the vesicular structure of thylakoid membranes with 0.05% Triton X-100 decreased the thermal stability of PSII to a similar level in both types of thylakoid membrane. Proteins released by Triton X-100 from thylakoid membranes from cells grown at 35°C were able to increase the thermal stability of Triton-treated thylakoid membranes. These observations suggest that proteins that are associated with thylakoid membranes might be involved in the enhancement of the thermal stability of PSII.     

Cloning of the d-Xylose Uptake Gene Linked to the xyl A Gene in Escherichia coli

An Escherichia coli mutant (MX-5) deficient in d-xylose utilization was isolated. The d-xylose uptake and d-xylose isomerase activities of the mutant were much lower than those of the parental strain (C600). The genes responsible for the d-xylose uptake by E. coli were cloned onto vector plasmid pBR322, and the resultant hybrid plasmid was designated as pXP5. Hybrid plasmid pXP5 improved the growth rate of the mutant (MX-5) on d-xylose, and also both the d-xylose uptake and d-xylose isomerase activities of the mutant were recovered when pXP5 was introduced into the mutant cells. Based on these results, it was suggested that one (xyl T) of the d-xylose transport genes could be closely linked to the d-xylose isomerase gene (xylA) known to be present at 80 min on E. coli chromosomal DNA.     

Production of infectious particles at the nonpermissive temperature by a temperature-sensitive mutant of bacteriophage SH-133 specific forPseudomonas facilis

The preliminary characterization of a unique temperature-sensitive (ts) mutant of bacteriophage SH-133, designatedts18, is reported. The mutant showed a substantial reduction in the ability to form plaques at the nonpermissive temperature (32°C) when compared with its plaqueforming ability at the permissive temperature (27°C). However, the supernatant fromts18-infected cells grown at 32°C exhibited significant infectivity when assayed at 27°C, which indicates that the reduced titer ofts18 at 32°C is not due to its inability to form phage particles at that temperature. Phage particles produced at 32°C, but not at 27°C, were thermolabile when tested at 32°C. The thermolability of phage yields from cells mixedly infected at 32°C with increasing wild-type/ts18 input ratios was independent of the quantity of wild-type gene product per cell. Thermostable phage particles were yielded byts18-infected cells that received short pulses of permissive temperature during the latter part of the latent period. These data indicate that the defect of the mutant is due to the production of a nonstructural assembly protein that misfunctions when viral maturation proceeds at the nonpermissive temperature.     

Short‐term high temperature treatment reduces viability and inhibits respiration and DNA repair enzymes in Araucaria angustifolia cells

We evaluated the effect of global warming on Araucaria angustifolia (Bert.) O. Kuntze, a critically endangered native tree of Southern Brazil, by studying the effects of short‐term high temperature treatment on cell viability, respiration and DNA repair of embryogenic cells. Compared with control cells grown at 25°C, cell viability was reduced by 40% after incubation at 30 and 37°C for 24 and 6 h, respectively, while 2 h at 40 and 42°C killed 95% of the cells. Cell respiration was unaffected at 30–37°C, but dramatically reduced after 2 h at 42°C. The in vitro activity of enzymes of the base excision repair (BER) pathway was determined. Apurinic/apyrimidine endonuclease, measured in extracts from cells incubated for 2 h at 42°C, was completely inactivated while lower temperatures had no effect. The activities of three enzymes of the mitochondrial BER pathway were measured after 30‐min preincubation of isolated mitochondria at 25–40°C and one of them, uracil glycosylase, was completely inhibited at 40°C. We conclude that cell viability, respiration and DNA repair have different temperature sensitivities between 25 and 37°C, and that they are all very sensitive to 40 or 42°C. Thus, A. angustifolia will likely be vulnerable to the short‐term high temperature events associated with global warming.     

The deletion of Ffh in Escherichia coli induces adverse physiological and morphological changes

The Escherichia coli Ffh protein is homologous to the SRP54 subunit of the eukaryotic signal recognition particle (SRP) that is involved in targeting and translocation of membrane proteins. The functions of Ffh in E. coli were investigated using the mutant with the Ffh deficiency. The mutant showed lower growth rate at 30°C and rapidly lost viability at the non-permissive temperature of 42°C. In addition, the amount of the total membrane proteins decreased sharply in the mutant. The mutant cells cultured at either 30 or 42°C appeared to have an elongated shape as compared to the wild type cells. Transmission electron microscopy revealed that the membrane layer of the mutant cells was thinner than that of the wild type cells. The article is published in the original.     

Peracetic acid activity on biofilm formed by Escherichia coli isolated from an industrial water system

One of the major problems in industrial water systems is the generation of biofilm, which is resistant to antimicrobial agents and causes failure of sanitization policy. This work aimed to study the anti-biofilm activity of peracetic acid (PAA) at contact times and temperatures combinations. To this end, a 96-well microtiter-based calorimetric method was applied in in vitro biofilm production using Escherichia coli, isolated from the water supply system of a pharmaceutical plant. The phenotypic and phylogenetic tests confirmed that the isolated bacteria belong to strains of Escherichia coli. The anti-biofilm activity of peracetic acid on formed biofilm was investigated at concentrations of 0·15–0·5% for a contact time of 5–15 min at 20–60°C. The maximum biofilm formation by MTP method using an Escherichia coli isolate was achieved in 96-h incubation in TSB containing wells at 37°C. Biofilm formation rate shown to be high by the environmental isolate compared with that of standard strain. PAA at concentrations above 0·25%, the temperature of 40°C and a minimum of 10 min of contact time was effective in the eradication of biofilm in an MTP-based system.     

FLOURY ENDOSPERM11‐2 encodes plastid HSP70‐2 involved with the temperature‐dependent chalkiness of rice (Oryza sativa L.) grains

The frequent occurrence of chalky rice (Oryza sativa L.) grains becomes a serious problem as a result of climate change. The molecular mechanism underlying chalkiness is largely unknown, however. In this study, the temperature‐sensitive floury endosperm11‐2 (flo11‐2) mutant was isolated from ion beam‐irradiated rice of 1116 lines. The flo11‐2 mutant showed significantly higher chalkiness than the wild type grown under a mean temperature of 28°C, but similar levels of chalkiness to the wild type grown under a mean temperature of 24°C. Whole‐exome sequencing of the flo11‐2 mutant showed three causal gene candidates, including Os12g0244100, which encodes the plastid‐localized 70‐kDa heat shock protein 2 (cpHSP70‐2). The cpHSP70‐2 of the flo11‐2 mutant has an amino acid substitution on the 259th aspartic acid with valine (D259V) in the conserved Motif 5 of the ATPase domain. Transgenic flo11‐2 mutants that express the wild‐type cpHSP70‐2 showed significantly lower chalkiness than the flo11‐2 mutant. Moreover, the accumulation level of cpHSP70‐2 was negatively correlated with the chalky ratio, indicating that cpHSP70‐2 is a causal gene for the chalkiness of the flo11‐2 mutant. The intrinsic ATPase activity of recombinant cpHSP70‐2 was lower by 23% at V_max for the flo11‐2 mutant than for the wild type. The growth of DnaK‐defective Escherichia coli cells complemented with DnaK with the D201V mutation (equivalent to the D259V mutation) was severely reduced at 37°C, but not in the wild‐type DnaK. The results indicate that the lowered cpHSP70‐2 function is involved with the chalkiness of the flo11‐2 mutant.     

The Effects of Temperature on 86Rb Uptake by Two Species of Chlamydomonas (Chlorophyta, Chlorophyceae)

⁸⁶Rb uptake was examined in two species of unicellular greenalgae, Chlamydomonas nivalis isolated from snow, and a cellwall-less mutant of the temperate freshwater Chlamydomonas reinhardii.In C. reinhardii cells grown at 20°C and cooled rapidlyto 0°C, ⁸⁶Rb uptake was abolished. Cells cooled rapidlyto –5°C in the absence of ice accumulated ⁸⁶Rb veryrapidly but the time course of this uptake suggested non-selectiveaccumulation through a damaged plasmalemma. Cells grown at 8°Cwere viable, able to divide and motile; they showed no signsof cold-shock and ⁸⁶Rb uptake, albeit slow, was measurable at–5°C in the absence of extracellular ice. Cells ofC. nivalis grown at 20°C were damaged at sub-zero temperaturesalthough they did show an enhanced ⁸⁶Rb uptake at 0°C. Cellsgrown at 5°C were able to accumulate ⁸⁶Rb from media undercooledto -5°C in the absence of extracellular ice, and again showedenhanced uptake at 0°C. The process of acclimation to lowtemperature appears to differ in the two species. Key words: Chlamydomonas, temperature, ⁸⁶Rb uptake, membrane     

Production of piscicolin 126 by Carnobacterium maltaromaticum UAL26 is controlled by temperature and induction peptide concentration

Carnobacterium maltaromaticum UAL26 produces the antimicrobial peptides (bacteriocins) piscicolin 126, first isolated from C. maltaromaticum JG126, and carnobacteriocin BM1, first isolated from C. maltaromaticum LV17. C. maltaromaticum UAL26 is especially inhibitory to strains of Listeria monocytogenes. Bacteriocin activity is not observable in the supernatant of cultures of UAL26 grown in liquid media at 25°C, but at temperatures less than 19°C bacteriocin activity can be detected. In contrast to JG126, the piscicolin 126 operon is downregulated in UAL26 at higher temperature, and piscicolin 126 mRNA is not detected when UAL26 is grown at 25°C. Bacteriocin production in UAL26 grown at 15°C can be induced by addition of 10⁻¹⁰ M of chemically synthesized piscicolin 126 induction peptide (PisN). However, induction of bacteriocin production in UAL26 grown at 25°C requires 10⁻⁷ M of PisN. The sequence of the piscicolin 126 operon in UAL26 contains 34 single nucleotide differences compared with the piscicolin 126 operon in JG126, including single nucleotide differences in the immunity, histidine kinase, dedicated ABC-transporter and accessory genes, as well as a single nucleotide deletion in the transport accessory gene. This deletion causes a frameshift, resulting in truncation of the PisE transport accessory protein in UAL26.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .