The regulatory VirA protein of Agrobacterium tumefaciens does not function at elevated temperatures.

Previous studies have shown that Agrobacterium tumefaciens causes tumors on plants only at temperatures below 32 degrees C, and virulence gene expression is specifically inhibited at temperatures above 32 degrees C. We show here that this effect persists even when the virA and virG loci are expressed under the control of a lac promoter whose activity is temperature independent. This finding suggests that one or more steps in the signal transduction process mediated by the VirA and VirG proteins are temperature sensitive. Both the autophosphorylation of VirA and the subsequent transfer of phosphate to VirG are shown to be sensitive to high temperatures (> 32 degrees C), and this correlates with the reduced vir gene expression observed at these temperatures. At temperatures of 32 degrees C and higher, the VirA molecule undergoes a reversible inactivation while the VirG molecule is not affected. vir gene induction is temperature sensitive in an acetosyringone-independent virA mutant background but not in a virG constitutive mutant which is virA and acetosyringone independent. These observations all support the notion that the VirA protein is responsible for the thermosensitivity of vir gene expression. However, an Agrobacterium strain containing a constitutive virG locus still cannot cause tumors on Kalanchoe plants at 32 degrees C. This strain induces normal-size tumors at temperatures up to 30 degrees C, whereas the wild-type Agrobacterium strain produces almost no tumors at 30 degrees C. These results suggest that at temperatures above 32 degrees C, the plant becomes more resistant to infection by A. tumefaciens and/or functions of some other vir gene products are lost in spite of their normal levels of expression.     

Modulation of gene expression by promoter mutants of the lambdacI857/pRM/pR system

Gene expression driven by the p(R) promoter of the lambdacI857/p(RM)/p(R) system results from inactivation of the temperature-sensitive CI857 repressor. The CI857 repressor, whose gene is transcribed by the divergently orientated p(RM) promoter, is destabilised at temperatures above 30 degrees C. In this study, the lambdacI857/p(RM)/p(R) system was modified by the introduction of a single (A-32G) and a double mutation (A-32G and T-41C). The mutated lambdap(R) expression modules, 32G and 32G/41C, tightly repressed the highly lethal phage PhiX174 lysis gene E at temperatures up to 37 and 39 degrees C, respectively. Expression of protein E and subsequent lysis of Escherichia coli was still induced by a temperature up-shift to 42 degrees C. The impact of the mutations on gene expression levels driven by the lambdap(R) and p(RM) promoters was evaluated at various temperatures using the lacZ reporter gene. Results indicate that the A-32G mutation confers a lambdap(R) promoter-down phenotype. The additional increase in the temperature stability of the 32G/41C expression system is due to the T-41C mutation leading to a higher p(RM) activity. The described lambdap(R) expression modules can be used to obtain a defined expression level at a given temperature and to tightly repress in particular highly lethal genes at different bacterial growth temperatures.     

Enhancement of photoheterotrophic biohydrogen production at elevated temperatures by the expression of a thermophilic clostridial hydrogenase

The working temperature of a photobioreactor under sunlight can be elevated above the optimal growth temperature of a microorganism. To improve the biohydrogen productivity of photosynthetic bacteria at higher temperatures, a [FeFe]-hydrogenase gene from the thermophile Clostridium thermocellum was expressed in the mesophile Rhodopseudomonas palustris CGA009 (strain CGA-CThydA) using a log-phase expression promoter P( pckA ) to drive the expression of heterogeneous hydrogenase gene. In contrast, a mesophilic Clostridium acetobutylicum [FeFe]-hydrogenase gene was also constructed and expressed in R. palustris (strain CGA-CAhydA). Both transgenic strains were tested for cell growth, in vivo hydrogen production rate, and in vitro hydrogenase activity at elevated temperatures. Although both CGA-CThydA and CGA-CAhydA strains demonstrated enhanced growth over the vector control at temperatures above 38?°C, CGA-CThydA produced more hydrogen than the other strains. The in vitro hydrogenase activity assay, measured at 40?°C, confirmed that the activity of the CGA-CThydA hydrogenase was higher than the CGA-CAhydA hydrogenase. These results showed that the expression of a thermophilic [FeFe]-hydrogenase in R. palustris increased the growth rate and biohydrogen production at elevated temperatures. This transgenic strategy can be applied to a broad range of purple photosynthetic bacteria used to produce biohydrogen under sunlight.     

腾冲嗜热厌氧菌葡萄糖激酶在不同温度下的表达和催化活性

尽管在腾冲嗜热厌氧菌的基因组注释中缺乏葡萄糖激酶(Glucokinase,GLK)(EC 2.7.1.2),但是该菌的蛋白质表达谱分析表明,TTE0090可能是一种新型的葡萄糖激酶。利用体外克隆表达的方法,表达了重组TTE0090;此蛋白质不仅具备使葡萄糖磷酸化的催化活性,同时在高温下也能参与反应。采用Western blot和阴离子交换层析的方法进一步检验了TTE0090在腾冲嗜热厌氧菌体内的蛋白质表达及其催化活性,发现体内TTE0090蛋白表达量随温度的升高而降低,而酶比活力却与生长温度呈正相关。这可能预示不同温度下腾冲嗜热厌氧菌中的糖酵解途径的催化通量是相对恒定的。实验数据均表明,TTE0090是存在于腾冲嗜热厌氧菌中的一种新型的葡萄糖激酶。TTE0090基因和其蛋白产物的研究工作,将进一步加深人们对嗜热菌的温度适应性以及它们的生存机理的了解。     

Altered temperature induction sensitivity of the lambda pR/cI857 system for controlled gene E expression in Escherichia coli

Cell lysis of Gram-negative bacteria can be efficiently achieved by expression of the cloned lysis gene E of bacteriophage PhiX174. Gene E expression is tightly controlled by the rightward lambda pR promoter and the temperature-sensitive repressor cI857 on lysis plasmid pAW12. The resulting empty bacterial cell envelopes, called bacterial ghosts, are currently under investigation as candidate vaccines. Expression of gene E is stringently repressed at temperatures up to 30 degrees C, whereas gene E expression, and thus cell lysis, is induced at temperatures higher than 30 degrees C due to thermal inactivation of the cI857 repressor. As a consequence, the production of ghosts requires that bacteria have to be grown at 28 degrees C before the lysis process is induced. In order to reflect the growth temperature of pathogenic bacteria in vivo, it seemed favorable to extend the heat stability of the lambda pR promoter/cI857 repressor system, allowing pathogens to grow at 37 degrees C before induction of lysis. In this study we describe a mutation in the lambda pR promoter, which allows stringent repression of gene E expression at temperatures up to 36 degrees C, but still permits induction of cell lysis at 42 degrees C.     

Covariation of larval gene expression and adult body size in natural populations of Drosophila melanogaster

Understanding adaptive phenotypic variation is one of the most fundamental problems in evolutionary biology. Genes involved in adaptation are most likely those that affect traits most intimately connected to fitness: life-history traits. The genetics of quantitative trait variation (including life histories) is still poorly understood, but several studies suggest that (1) quantitative variation might be the result of variation in gene expression, rather than protein evolution, and (2) natural variation in gene expression underlies adaptation. The next step in studying the genetics of adaptive phenotypic variation is therefore an analysis of naturally occuring covariation of global gene expression and a life-history trait. Here, we report a microarray study addressing the covariation in larval gene expression and adult body weight, a life-history trait involved in adaptation. Natural populations of Drosophila melanogaster show adaptive geographic variation in adult body size, with larger animals at higher latitudes. Conditions during larval development also affect adult size with larger flies emerging at lower temperatures. We found statistically significant differences in normalized larval gene expression between geographic populations at one temperature (genetic variation) and within geographic populations between temperatures (developmental plasticity). Moreover, larval gene expression correlated highly with adult weight, explaining 81% of its natural variation. Of the genes that show a correlation of gene expression with adult weight, most are involved in cell growth or cell maintenance or are associated with growth pathways.     

Low-Temperature-Induced DnaA Protein Synthesis Does Not Change Initiation Mass in Escherichia coli K-12

Expression of the dnaA gene continues in the lag phase following a temperature downshift, indicating that DnaA is a cold shock protein. Steady-state DnaA protein concentration increases at low temperatures, being twofold higher at 14 degrees C than at 37 degrees C. DnaA protein was found to be stable at both low and high temperatures. Despite the higher DnaA concentration at low temperatures, the mass per origin, which is proportional to the initiation mass, was the same at all temperatures. Cell size and cellular DNA content decreased moderately below 30 degrees C due to a decrease in the time from termination to division relative to generation time at the lower temperatures. Analysis of dnaA gene expression and initiation of chromosome replication in temperature shifts suggests that a fraction of newly synthesized DnaA protein at low temperatures is irreversibly inactive for initiation and for autorepression or that all DnaA protein synthesized at low temperatures has an irreversible low-activity conformation.     

Xenogeneic silencing relies on temperature-dependent phosphorylation of the host H-NS protein in Shewanella

Lateral gene transfer (LGT) plays a key role in shaping the genome evolution and environmental adaptation of bacteria. Xenogeneic silencing is crucial to ensure the safe acquisition of LGT genes into host pre-existing regulatory networks. We previously found that the host nucleoid structuring protein (H-NS) silences prophage CP4So at warm temperatures yet enables this prophage to excise at cold temperatures in Shewanella oneidensis. However, whether H-NS silences other genes and how bacteria modulate H-NS to regulate the expression of genes have not been fully elucidated. In this study, we discovered that the H-NS silences many LGT genes and the xenogeneic silencing of H-NS relies on a temperature-dependent phosphorylation at warm temperatures in S. oneidensis. Specifically, phosphorylation of H-NS at Ser42 is critical for silencing the cold-inducible genes including the excisionase of CP4So prophage, a cold shock protein, and a stress-related chemosensory system. By contrast, nonphosphorylated H-NS derepresses the promoter activity of these genes/operons to enable their expression at cold temperatures. Taken together, our results reveal that the posttranslational modification of H-NS can function as a regulatory switch to control LGT gene expression in host genomes to enable the host bacterium to react and thrive when environmental temperature changes.     

Induction of gene expression in bacteria at optimal growth temperatures

Traditional temperature-sensitive systems use either heat shock (40–42 °C) or cold shock (15–23 °C) to induce gene expression at temperatures that are not the optimal temperature for host cell growth (37 °C). This impacts the overall productivity and yield by disturbing cell growth and cellular metabolism. Here, we have developed a new system which controls gene expression in Escherichia coli at more permissive temperatures. The temperature-sensitive cI857-P _L system and the classic lacI-P _lacO system were connected in series to control the gene of interest. When the culture temperature was lowered, the thermolabile cI857 repressor was activated and blocked the expression of lacI from P _L. Subsequently, the decrease of LacI derepressed the expression of gene of interest from P _lacO . Using a green fluorescent protein marker, we demonstrated that (1) gene expression was tightly regulated at 42 °C and strongly induced by lowering temperature to 25–37 °C; (2) different levels of gene expression can be induced by varying culture temperature; and (3) gene expression after induction was sustained until the end of the log phase. We then applied this system in the biosynthesis of acetoin and demonstrated that high yield and production could be achieved using temperature induction. The ability to express proteins at optimal growth temperatures without chemical inducers is advantageous for large-scale and industrial fermentations.     

A portable integrated automatic apparatus for the real-time monitoring of bioluminescence in plants

Real-time monitoring of gene expression by a bioluminescence reporter gene is a powerful method for large-scale, detailed analysis of gene expression in living cells and large-scale screening of mutants. We have developed a portable, compact, integrated automatic bioluminescence-monitoring apparatus that can continuously monitor 960 individual plant seedlings or micro-organism colonies under uniform light conditions at temperatures up to 50 °C. The apparatus gave reproducible and reliable results for both bioluminescence photon counts and period length of bioluminescence rhythms of Arabidopsis reporter strain. Using the apparatus, we measured bioluminescence rhythms in the thermophilic cyanobacterium Thermosynechococcus at temperature up to 43 °C. We also monitored the expression of the flowering regulator gene CONSTANS in Arabidopsis as bioluminescence in high time resolution under different photoperiodic conditions. The high-throughput bioluminescence-monitoring apparatus developed here is a powerful tool for real-time monitoring of gene expression and gene function.     

Cold stress regulates lipid metabolism via AMPK signalling in Cherax quadricarinatus

Lipids play an important role in protecting poikilotherms from cold stress, but relatively little is known about the regulation of lipid metabolism under cold stress, especially in crustaceans. In the present study, red-clawed crayfish Cherax quadricarinatus was employed as a model organism. Animals were divided into four temperature groups (25, 20, 15 and 9 °C) and treated for 4 weeks, with the 25 °C group serving as a control. The total lipid content in the hepatopancreas as well as the triglyceride, cholesterol and free fatty acid levels in the hemolymph were determined. Lipids stored in the hepatopancreas and hemolymph decreased with decreasing temperature, with changes in the 9 °C group most pronounced, indicating that lipids are the main energy source for crayfish at low temperatures. Furthermore, enzyme activity of lipase, fatty acid synthase, acetyl-CoA carboxylase, and lipoprotein esterase, and gene expression analysis of fatty acid synthase gene, acetyl-CoA carboxylase gene and carnitine palmitoyltransferase gene showed that the digestion, synthesis and oxidation of lipids in the hepatopancreas were inhibited under low temperature stress, but expression of sphingolipid delta-4 desaturase (DEGS) was increased, indicating an increase in the demand for highly unsaturated fatty acids at low temperatures. Analysis of the expression of genes related to the AMP-activated protein kinase (AMPK) signalling pathway revealed that the adiponectin receptor gene was rapidly upregulated at low temperatures, which may in turn activate the expression of the downstream AMPKα gene, thereby inhibiting lipid anabolism.     

Hypothermia modulates circadian clock gene expression in lizard peripheral tissues

The molecular mechanisms whereby the circadian clock responds to temperature changes are poorly understood. The ruin lizard Podarcis sicula has historically proven to be a valuable vertebrate model for exploring the influence of temperature on circadian physiology. It is an ectotherm that naturally experiences an impressive range of temperatures during the course of the year. However, no tools have been available to dissect the molecular basis of the clock in this organism. Here, we report the cloning of three lizard clock gene homologs (Period2, Cryptochrome1, and Clock) that have a close phylogenetic relationship with avian clock genes. These genes are expressed in many tissues and show a rhythmic expression profile at 29 degrees C in light-dark and constant darkness lighting conditions, with phases comparable to their mammalian and avian counterparts. Interestingly, we show that at low temperatures (6 degrees C), cycling clock gene expression is attenuated in peripheral clocks with a characteristic increase in basal expression levels. We speculate that this represents a conserved vertebrate clock gene response to low temperatures. Furthermore, these results bring new insight into the issue of whether circadian clock function is compatible with hypothermia.     

A hypothermic-temperature-sensitive gene silencing by the mammalian RNAi

RNA interference (RNAi) has been attracting a great deal of attention. This pathway is highly conserved among most eukaryotes and believed to be important for antiviral reactions and epigenetic gene regulation. Because a temperature-sensitive RNAi was reported in both plant and insect systems, suggesting its evolutional conservation, we analyzed the effect of different temperatures on mammalian RNAi, targeting the ectopic gene expression, and detected suppression at hypothermic temperatures. This phenomenon could be critical and useful to control ectopic and internal gene expressions by RNAi.     

Identification of a grapefruit cDNA belonging to a unique class of citrus dehydrins and characterization of its expression patterns under temperature stress conditions

Citrus fruits are sensitive to low temperatures and this often results in the development of chilling injuries during postharvest storage. In order to gain more insight into the molecular mechanisms involved in the acquisition of fruit chilling tolerance, we initiated a grapefruit ( Citrus paradisi, cv. Marsh Seedless) flavedo cDNA sequencing project and used it to identify a cDNA similar to other Poncirus trifoliata and Citrus unshiu dehydrin genes reported to be responsive to low temperatures. The grapefruit dehydrin cDNA, designated cor15 , encodes a predicted polypeptide of 15.1 kDa, that is almost completely identical with other reported citrus dehydrin proteins, except that it contains two large amino acid repeats, whereas P. trifoliata COR11 has only one such repeat and P. trifoliata COR19 and C. unshiu COR19 have three repeats. Together, the various grapefruit, P. trifoliata and C. unshiu dehydrins form a closely related and unique dehydrin gene family that differs from most other plant dehydrins in having an unusual K-segment similar to that of gymnosperms and in having a serine cluster (S-segment) at an unusual position at the carboxy-terminus. The grapefruit cor15 gene is consistently expressed in the fruit peel tissue at harvest, but its message levels dramatically decrease during storage at 2°C. However, a pre-storage hot water treatment, which enhances fruit chilling tolerance, elicited retention of the constant level of cor15 gene expression during cold storage and eliminated its decline. The hot water treatment had no inductive effect on cor15 gene expression when the fruit were held at non-chilling temperatures. The effects of other stresses, such as exposure to ethylene, UV irradiation and wounding, on cor15 gene expression, were temporary and persisted for 1-2 days after the treatments.     

Replicon-specific regulation of small heat shock genes in Agrobacterium tumefaciens

Four genes coding for small heat shock proteins (sHsps) were identified in the genome sequence of Agrobacterium tumefaciens, one on the circular chromosome (hspC), one on the linear chromosome (hspL), and two on the pAT plasmid (hspAT1 and hspAT2). Induction of sHsps at elevated temperatures was revealed by immunoblot analyses. Primer extension experiments and translational lacZ fusions demonstrated that expression of the pAT-derived genes and hspL is controlled by temperature in a regulon-specific manner. While the sHsp gene on the linear chromosome turned out to be regulated by RpoH (sigma32), both copies on pAT were under the control of highly conserved ROSE (named for repression of heat shock gene expression) sequences in their 5' untranslated region. Secondary structure predictions of the corresponding mRNA strongly suggest that it represses translation at low temperatures by masking the Shine-Dalgarno sequence. The hspC gene was barely expressed (if at all) and not temperature responsive.