Stress responses and metal tolerance of <Emphasis Type="Italic">Chlamydomonas acidophila</Emphasis> in metal-enriched lake water and artificial medium

Chlamydomonas acidophila faces high heavy-metal concentrations in acidic mining lakes, where it is a dominant phytoplankton species. To investigate the importance of metals to C. acidophila in these lakes, we examined the response of growth, photosynthesis, cell structure, heat-shock protein (Hsp) accumulation, and metal adsorption after incubation in metal-rich lake water and artificial growth medium enriched with metals (Fe, Zn). Incubation in both metal-rich lake water and medium caused large decreases in photosystem II function (though no differences among lakes), but no decrease in growth rate (except for medium + Fe). Concentrations of small Hsps were higher in algae incubated in metal-rich lake-water than in metal-enriched medium, whereas Hsp60 and Hsp70A were either less or equally expressed. Cellular Zn and Fe contents were lower, and metals adsorbed to the cell surface were higher, in lake-water-incubated algae than in medium-grown cells. The results indicate that high Zn or Fe levels are likely not the main or only contributor to the low primary production in mining lakes, and multiple adaptations of C. acidophila (e.g., high Hsp levels, decreased metal accumulation) increase its tolerance to metals and permit survival under such adverse environmental conditions. Supposedly, the main stress factor present in the lake water is an interaction between low P and high Fe concentrations.     

Thermotolerance and gene expression following heat stress in the whitefly Bemisia tabaci B and Q biotypes

The whitefly Bemisia tabaci (Gennadius) causes tremendous losses to agriculture by direct feeding on plants and by vectoring several families of plant viruses. The B. tabaci species complex comprises over 10 genetic groups (biotypes) that are well defined by DNA markers and biological characteristics. B and Q are amongst the most dominant and damaging biotypes, differing considerably in fecundity, host range, insecticide resistance, virus vectoriality, and the symbiotic bacteria they harbor. We used a spotted B. tabaci cDNA microarray to compare the expression patterns of 6000 ESTs of B and Q biotypes under standard 25 °C regime and heat stress at 40 °C. Overall, the number of genes affected by increasing temperature in the two biotypes was similar. Gene expression under 25 °C normal rearing temperature showed clear differences between the two biotypes: B exhibited higher expression of mitochondrial genes, and lower cytoskeleton, heat-shock and stress-related genes, compared to Q. Exposing B biotype whiteflies to heat stress was accompanied by rapid alteration of gene expression. For the first time, the results here present differences in gene expression between very closely related and sympatric B. tabaci biotypes, and suggest that these clear-cut differences are due to better adaptation of one biotype over another and might eventually lead to changes in the local and global distribution of both biotypes.     

Effects of dissolved oxygen tension and agitation rate on the production of heat-shock protein glycoprotein 96 by MethA tumor cell suspension culture in stirred-tank bioreactors

Heat-shock protein glycoprotein (gp96) serves as a natural adjuvant for chaperoning antigenic peptide into the immune surveillance pathway. In our laboratory, MethA tumor cell suspension culture process has been recently developed for gp96 production in spinner flask. In this work, effects of dissolved oxygen tension (DOT) and agitation rate on this process were studied in stirred-tank bioreactor. The optimal conditions for gp96 production were different with those for MethA tumor cell growth. MethA tumor cell growth pattern was not much changed by various levels of DOT and agitation rate, while gp96 biosynthesis was more sensitive to DOT and agitation rate. Compared with 50% of DOT, the production and specific productivity of gp96 was increased by 27 and 66% at 10% of DOT, respectively. Compared with the agitation rate of 100 rpm, the production and volumetric productivity of gp96 was increased by 48 and 144% at the agitation rate of 200 rpm, respectively. Low DOT (i.e., 10% of air saturation) and high agitation rate (i.e., 200 rpm) were identified to be favorable for gp96 biosynthesis. The results of this work might be useful to scale-up the bioprocess into the pilot scale.     

表达载体pHsh对大肠杆菌热休克系统中负调控机制的影响

pHsh是一种由σ32识别和启动外源基因表达的新型高效的大肠杆菌表达载体。正常E.coli细胞在热激诱导条件下,σ32的浓度在5 min内到达高峰,随后被3个负调控蛋白Dnak、DnaJ、GrpE结合导致失活或降解,整个热休克反应持续约12min。在携带有外源基因的高拷贝pHsh 的E.coli细胞中,外源基因却能持续高效表达4～10 h,这一现象表明了此时细胞中的σ32比没有携带质粒的细胞内σ32的浓度要高。σ32浓度的增高有可能是由于3个负调控蛋白Dnak、DnaJ、GrpE在细胞内的含量比正常情况下降低的结果。为了验证这一推测,从E.coli中克隆了Dnak、DnaJ、GrpE的编码基因,表达并初步纯化了其重组蛋白以作分子标记,采用双向电泳技术,分析携带质粒（pHsh+）和不携带质粒的E.coli(pHsh-)细胞在热休克后胞内蛋白质组的差异。该项实验通过与检索到的标准的E.coli蛋白质组图谱进行比较鉴别出的两个蛋白Dnak、GrpE,并通过对比目标点的大小和深浅发现pHsh+中的Dnak均少于pHsh─中的目标蛋白,所得结果与上述假设一致。     

The Small Heat-shock Protein HspL Is a VirB8 Chaperone Promoting Type IV Secretion-mediated DNA Transfer

Agrobacterium tumefaciens is a plant pathogen that utilizes a type IV secretion system (T4SS) to transfer DNA and effector proteins into host cells. In this study we discovered that an α-crystallin type small heat-shock protein (α-Hsp), HspL, is a molecular chaperone for VirB8, a T4SS assembly factor. HspL is a typical α-Hsp capable of protecting the heat-labile model substrate citrate synthase from thermal aggregation. It forms oligomers in a concentration-dependent manner in vitro. Biochemical fractionation revealed that HspL is mainly localized in the inner membrane and formed large complexes with certain VirB protein subassemblies. Protein-protein interaction studies indicated that HspL interacts with VirB8, a bitopic integral inner membrane protein that is essential for T4SS assembly. Most importantly, HspL is able to prevent the aggregation of VirB8 fused with glutathione S-transferase in vitro, suggesting that it plays a role as VirB8 chaperone. The chaperone activity of two HspL variants with amino acid substitutions (F98A and G118A) for both citrate synthase and glutathione S-transferase-VirB8 was reduced and correlated with HspL functions in T4SS-mediated DNA transfer and virulence. This study directly links in vitro and in vivo functions of an α-Hsp and reveals a novel α-Hsp function in T4SS stability and bacterial virulence.     

Phylogeny of the α-crystallin-related heat-shock proteins

Summary Phylogenetic relationships were examined among 35 -crystallin-related heat-shock proteins from animals, plants, and fungi. Approximately one-third of the aligned amino acids in these proteins were conserved in 74% of the proteins, and three blocks of consensus sequence were identified. Relationships were established by maximum parsimony and distance matrix analyses of the aligned amino acid sequences. The inferred phylogeny trees show the plant proteins clearly divided into three major groups that are unrelated to taxonomy: the chloroplast-localized proteins and two groups that originate from a common ancestral plant protein. The animal proteins, in contrast, branch in accordance with taxonomy, the only clear exception being the -crystallin subgrouping of vertebrates. This analysis indicates that the small heat-shock proteins of animals have diverged more widely than have the plant proteins, one group of which is especially stable.Offprint requests to: N. Plesofsky-Vig     

Quantitative mRNA expression profiling of heat-shock protein families in rainbow trout cells

We isolated multiple HSPs from rainbow trout Oncorhynchus mykiss RTG-2 cells and quantitatively compared their mRNA levels between unstressed and heat-shocked cells using real-time RT-PCR analysis. Consequently, we isolated nine cDNAs encoding HSPs from heat-shocked RTG-2 cells, namely, Hsp90betaa, Hsp90betab, Grp78, Hsp70a, Hsc70a, Hsc70b, Cct8, Hsp47, and DnaJ homolog. Quantitative RT-PCR analyses, in which Hsp70b isolated previously was included, showed that the mRNA accumulation levels of Hsp70a, Hsp70b, Hsc70a, Hsc70b, and Hsp47 were significantly increased after heat shock, and the increased levels of two Hsp70s, Hsp70a, and Hsp70b, were most conspicuous. In the case of Hsc70s, the increased level of Hsc70b was more remarkable than that of Hsc70a. These results demonstrate the importance of a comprehensive expression analysis of HSPs for better understanding of the cellular stress response in fish, especially in tetraploid species such as rainbow trout.     

SUMO1 attenuates stress-induced ROS generation by inhibiting NADPH oxidase 2

Small ubiquitin-like modifier 1 (SUMO1) is a member of the superfamily of ubiquitin-like proteins. Despite its structural similarity with ubiquitin, SUMO1 does not seem to play any role in protein degradation and its precise biological function is poorly understood. During our studies on heat-shock responses, we found that heat-shock stress increased SUMO1 conjugation in a dose-dependent manner. Intriguingly, SUMO1 conjugation resulted in decrease of intracellular ROS generation and protection cells from death under heat-shock stress. We showed that NADPH oxidase 2 (NOX2) is a target protein of sumoylation by SUMO1 using immunoprecipitation and is colocalized with SUMO1 at plasma membrane. Additionally, we demonstrated that the attenuation in intracellular ROS generation resulted from inhibition of NADPH oxidase complex (NOX) activity. These results suggested that SUMO1 plays an important role in modulation of NOX activity required for ROS generation.     

Predicting relatedness of bacterial genomes using the chaperonin-60 universal target (cpn60 UT): application to Thermoanaerobacter species

D.R. Zeigler determined that the sequence identity of bacterial genomes can be predicted accurately using the sequence identities of a corresponding set of genes that meet certain criteria [32]. This three-gene model for comparing bacterial genome pairs requires the determination of the sequence identities for recN, thdF, and rpoA. This involves the generation of approximately 4.2 kb of genomic DNA sequence from each organism to be compared, and also normally requires that oligonucleotide primers be designed for amplification and sequencing based on the sequences of closely related organisms. However, we have developed an analogous mathematical model for predicting the sequence identity of whole genomes based on the sequence identity of the 542-567 base pair chaperonin-60 universal target (cpn60 UT). The cpn60 UT is accessible in nearly all bacterial genomes with a single set of universal primers, and its length is such that it can be completely sequenced in one pair of overlapping sequencing reads via di-deoxy sequencing. These mathematical models were applied to a set of Thermoanaerobacter isolates from a wood chip compost pile and it was shown that both the one-gene cpn60 UT-based model and the three-gene model based on recN, rpoA, and thdF predicted that these isolates could be classified as Thermoanaerobacter thermohydrosulfuricus. Furthermore, it was found that the genomic prediction model using cpn60 UT gave similar results to whole-genome sequence alignments over a broad range of taxa, suggesting that this method may have general utility for screening isolates and predicting their taxonomic affiliations.