De novo engineering and metabolic flux analysis of inosine biosynthesis in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Wild-type B. subtilis strain W168 was de novo engineered for inosine biosynthesis. Inactivation of deoD and purA led to 0.15 ± 0.04 and 6.44 ± 0.39 g inosine/l yields, respectively. The deoD purA double mutant accumulated 7.6 ± 0.34 g inosine/l, with a 4.7% (w/w) conversion ratio from glucose to inosine. Comparative metabolic flux analysis revealed that the fluxes from inosine to hypoxanthine and from inosine monophosphate to adenosine monophosphate in the double mutant decreased to 14.0 and 0.61% of those in the wild-type strain. The major role of purA was demonstrated when inactivation of deoD and purA were found to contribute additively to inosine accumulation. This work is expected to contribute to the improvement of the fermentative production of purine nucleosides in the microbial industry.     

Cloning and characterization of the groE locus from Actinobacillus pleuropneumoniae

A 4.4-kb DNA fragment was cloned from Actinobacillus pleuropneumoniae (strain 4074, serotype 1) by genetic complementation with Escherichia coli groES-groEL mutant strains. Sequence analysis of this fragment revealed a purine nucleoside phosphorylase (DeoD)-encoding gene homolog (deoD), heat-shock response-encoding genes for the small (groES) and large subunits (groEL) and a partial open reading frame encoding an alcohol dehydrogenase homolog (adhE). The predicted amino-acid sequence of groES and groEL genes showed extensive sequence identity (80–95%) with other Pasteurellaceae. The gene organization surrounding the groE locus was different from that of Haemophilus infuenzae. When expressed in E. coli, groES-groEL genes were capable of complementing the growth of a λ lytic phage, indicating a structural as well as functional conservation.     

Regulation of the synthesis of nucleoside catabolic enzymes in Escherichia coli: Further analysis of a deo OC mutant strain

Summary Four genes, deoA, deoB, deoC, and deoD, involved in the synthesis of nucleoside and deoxynucleoside catabolic enzymes, are located contiguously in the order C-A-B-D on the linkage map of E. coli. They constitute two overlapping operons, one transcribing all the four genes and the other deoB and deoD. To the left of deoC are located two promoter-operator regions in the order deoPO-cytPO. They are involved in controlling the expression of the tetracistronic mRNA. For efficient binding of RNA polymerase at the cytPO site the cAMP+CRP complex is required, whereas binding of RNA polymerase at the deoPO site is independent of this complex. Evidence is available for the existence of yet another controlling site, PO-3, located between deoA and deoB; this controls the expression of deoB and deoD. Both the operons are transcribed in a clockwise direction. An operator constitutive (O ^c) type mutant affecting the synthesis of all four deo enzymes has been analysed. Because of this mutation the strain has become insensitive to catabolite repression. The results confirm the order of the gene in the controlling region to be deoPO-cytPO and the mutation, previously analysed as a deletion, appears to have deleted cytPO deoC region of the chromosome.     

Regulation of the deo operon in Escherichia coli: the double negative control of the deo operon by the cytR and deoR repressors in a DNA directed in vitro system.

Summary The synthesis of the four enzymes of the deo operon in Escherichia coli is known from in vivo experiments to be subject to a double negative control, exerted by the products of the cytR and deoR genes.A DNA-directed in vitro protein synthesizing system makes the deo enzymes (exemplified by thymidine phosphorylase) in agreement with in vivo results. Enzyme synthesis is stimulated by cyclic AMP and repressed by the cytR and deoR gene products. Repression by the cytR repressor is reversed by cytidine or adenosine in the presence of cyclic AMP, while repression by the deoR repressor is reversed by deoxyribose-5-phosphate.Assays for the presence of the cytR and deoR repressors were established by use of S-30 extracts prepared from the regulatory mutants.Dissociation constants for repressor-operator binding as well as for repressor-inducer interactions have been estimated from the results.Abbreviations and Symbols deoA (previously designated tpp) Genes coding for: thymidine, phosphorylase - deoB (previously designated drm) deoxyribomutase - deoC (previously designated dra) deoxyriboaldolase - deoD (previously designated pup) purine nucleoside phosphorylase - udp uridine phosphorylase - cytR regulatory gene for cdd, udp, deoC, deoA, deoB, and deoD - deoR (previously designated nucR) regulatory gene for deoC, deoA, deoB, and deoD Enzymes (EC 2.4.2.1) Purine nucleoside phosphorylase or purine nucleoside: orthophosphate(deoxy)ribosyltansferase - (EC 2.4.2.4) thymidine phosphorylase or thymidine: orthophosphate deoxyribosyltransferase - (EC 2.4.2.3) uridine phosphorylase or uridine: orthophosphate ribosyltransferase - (EC 4.1.2.4) deoxyriboaldolase or 2-deoxy-D-ribose-5-phosphate: acetaldehydelyase - (EC 2.7.5.6) phosphodeoxyribomutase The deo operon is defined as the gene cluster consisting of deoC deoA deoB deoD. The deo enzymes are the four enzymes encoded by the four genes of the deo operon. cAMP: cyclic adenosine 3,5-monophosphate. CRP: cyclic AMP receptor protein. dRib-5P: deoxyribose-5-phosphate. THUR: 3,4,5,6-tetrahydrouridine; EDTA: ethylene-diamine-tetra-acetate.     

Expression of the Heat Shock Response in a Tomato Interspecific Hybrid Is Not Intermediate between the Two Parental Responses

While it is apparent that the heat shock response is ubiquitous, variabilities in the nature of the heat shock response between closely related species have not been well characterized. The heat shock response of three genotypes of tomato, Lycopersicon esculentum, Lycopersicon pennellii, and the interspecific sexual hybrid was characterized. The two parental genotypes differed in the nature of the heat shock proteins synthesized; the speciesspecific heat shock proteins were identified following in vivo labeling of leaf tissue with [³⁵S]methionine and cysteine. The duration of, and recovery from, heat shock varied between the two species: L. esculentum tissue recovered more rapidly and protein synthesis persisted longer during a heat shock than in the wild species, L. pennellii. Both species induced heat shock protein synthesis at 35°C and synthesis was maximal at 37°C. The response of the F1 to heat shock was intermediate to the parental responses for duration of, and recovery from, heat shock. In other aspects, the response of the F1 to heat shock was not intermediate to the parental responses: the F1 induced only half of the L. esculentum specific heat shock proteins, and all of the L. pennellii specific heat shock proteins. A discussion of the inheritance of the regulation of the heat shock response is presented.     

The effect of abrupt changes in sodium chloride concentration upon the viability of gram-negative bacteria at 2 and 40 degrees C

“Osmotic” shock between 0.14 m and 4.5 m NaCl is highly temperature dependent for postlog populations of E. coli, A. aerogenes, and P. aeruginosa, suggesting that killing is not due solely to simple physical osmotic action. Thermal shock in postlog E. coli and A. aerogenes does not occur, although in P. aeruginosa populations when temperature fluctuations are repetitious appreciable loss of viability does occur. Thermal shock concomitant with osmotic shock, on the other hand, is lethal to postlog populations of all 3 species. Thus, during freezing-thawing concentrated solute is a prime candidate for causing death in postlog E. coli, A. aerogenes, and P. aeruginosa populations.     

Destabilization and recovery of a yeast prion after mild heat shock

Yeast prion [PSI⁺] is a self-perpetuating amyloid of the translational termination factor Sup35. Although [PSI⁺] propagation is modulated by heat shock proteins (Hsps), high temperature was previously reported to have little or no effect on [PSI⁺]. Our results show that short-term exposure of exponentially growing yeast culture to mild heat shock, followed by immediate resumption of growth, leads to [PSI⁺] destabilization, sometimes persisting for several cell divisions after heat shock. Prion loss occurring in the first division after heat shock is preferentially detected in a daughter cell, indicating the impairment of prion segregation that results in asymmetric prion distribution between a mother cell and a bud. Longer heat shock or prolonged incubation in the absence of nutrients after heat shock led to [PSI⁺] recovery. Both prion destabilization and recovery during heat shock depend on protein synthesis. Maximal prion destabilization coincides with maximal imbalance between Hsp104 and other Hsps such as Hsp70-Ssa. Deletions of individual SSA genes increase prion destabilization and/or counteract recovery. The dynamics of prion aggregation during destabilization and recovery are consistent with the notion that efficient prion fragmentation and segregation require a proper balance between Hsp104 and other (e.g., Hsp70-Ssa) chaperones. In contrast to heat shock, [PSI⁺] destabilization by osmotic stressors does not always depend on cell proliferation and/or protein synthesis, indicating that different stresses may impact the prion via different mechanisms. Our data demonstrate that heat stress causes asymmetric prion distribution in a cell division and confirm that the effects of Hsps on prions are physiologically relevant.     

Thermal stress responses in Antarctic yeast, Glaciozyma antarctica PI12, characterized by real-time quantitative PCR

Living organisms have some common and unique strategies to response to thermal stress. However, the amount of data on thermal stress response of certain organism is still lacking, especially psychrophilic yeast from the extreme habitat. Therefore, it is not known whether psychrophilic yeast shares the common responses of other organisms when exposed to thermal stresses. In this work, the cold shock and heat shock responses in Antarctic psychrophilic yeast Glaciozyma antarctica PI12 which had an optimal growth temperature of 12 °C were determined. The expression levels of 14 thermal stress-related genes were measured using real-time quantitative PCR (qPCR) when the yeast cells were exposed to cold shock (0 °C), mild cold shock (5 °C), and heat shock (22 °C) conditions. The expression profiles of the 14 genes at these three temperatures varied indicating that these genes had their specific roles to ensure the survival of the yeast. Under cold shock condition, the afp4 and fad genes were over-expressed possibly as a way for the G. antarctica PI12 to avoid ice crystallization in the cell and to maintain the membrane fluidity. Under the heat shock condition, hsp70 was significantly up-regulated possibly to ensure the proteins fold properly. Among the six oxidative stress-related genes, MnSOD and prx were up-regulated under cold shock and heat shock, respectively, possibly to reduce the negative effects caused by oxidative stress. Interestingly, it was found that the trehalase gene, nth1 that plays a role in degrading excess trehalose, was down-regulated under the heat shock condition possibly as an alternative way to accumulate trehalose in the cells to protecting them from being damaged.     

The natural alkaloid sanguinarine promotes the expression of heat shock protein genes in Arabidopsis

Small-molecule heat shock response inducers are known to enhance heat tolerance in plants. In this paper, we report that a plant alkaloid enhances the heat tolerance of Arabidopsis. We investigated 12 commercially available alkaloids to determine whether they enhance the heat tolerance of Arabidopsis seedlings using an in vitro assay system with geldanamycin, which is a known heat shock response inducer, as a positive control. Accordingly we found that the isoquinoline alkaloid sanguinarine can enhance heat tolerance in Arabidopsis. No such effect was shown for the other 11 alkaloids. The sanguinarine treatment increased the expression of heat shock protein genes such as HSP17.6C-CI, HSP70, and HSP90.1, which were up-regulated by geldanamycin. Treatments with other isoquinoline alkaloids (berberine and papaverine), which showed few heat tolerance-enhancing effects, did not promote the expression of the heat shock protein genes. These results suggest that sanguinarine influenced the heat tolerance of Arabidopsis by enhancing the expression of heat shock protein genes.     

Induced Levels of Heat Shock Proteins in a dnaK Mutant of Lactococcus lactis

The bacterial heat shock response is characterized by the elevated expression of a number of chaperone complexes and proteases, including the DnaK-GrpE-DnaJ and the GroELS chaperone complexes. In order to investigate the importance of the DnaK chaperone complex for growth and heat shock response regulation in Lactococcus lactis, we have constructed two dnaK mutants with C-terminal deletions in dnaK. The minor deletion of 65 amino acids in the dnaKΔ2 mutant resulted in a slight temperature-sensitive phenotype. BK6, containing the larger deletion of 174 amino acids (dnaKΔ1), removing the major part of the inferred substrate binding site of the DnaK protein, exhibited a pronounced temperature-sensitive phenotype and showed altered regulation of the heat shock response. The expression of the heat shock proteins was increased at the normal growth temperature, measured as both protein synthesis rates and mRNA levels, indicating that DnaK could be involved in the regulation of the heat shock response in L. lactis. For Bacillus subtilis, it has been found (A. Mogk, G. Homuth, C. Scholz, L. Kim, F. X. Schmid, and W. Schumann, EMBO J. 16:4579–4590, 1997) that the activity of the heat shock repressor HrcA is dependent on the chaperone function of the GroELS complex and that a dnaK insertion mutant has no effect on the expression of the heat shock proteins. The present data from L. lactis suggest that the DnaK protein could be involved in the maturation of the homologous HrcA protein in this bacterium.     

Heat Shock Modulates Neutrophil Motility in Zebrafish

Heat shock is a routine method used for inducible gene expression in animal models including zebrafish. Environmental temperature plays an important role in the immune system and infection progression of ectotherms. In this study, we analyzed the impact of short-term heat shock on neutrophil function using zebrafish (Danio rerio) as an animal model. Short-term heat shock decreased neutrophil recruitment to localized Streptococcus iniae infection and tail fin wounding. Heat shock also increased random neutrophil motility transiently and increased the number of circulating neutrophils. With the use of the translating ribosome affinity purification (TRAP) method for RNA isolation from specific cell types such as neutrophils, macrophages and epithelial cells, we found that heat shock induced the immediate expression of heat shock protein 70 (hsp70) and a prolonged expression of heat shock protein 27 (hsp27). Heat shock also induced cell stress as detected by the splicing of X-box binding protein 1 (xbp1) mRNA, a marker for endoplasmic reticulum (ER) stress. Exogenous expression of Hsp70, Hsp27 and spliced Xbp1 in neutrophils or epithelial cells did not reproduce the heat shock induced effects on neutrophil recruitment. The effect of heat shock on neutrophils is likely due to a combination of complex changes, including, but not limited to changes in gene expression. Our results indicate that routine heat shock can alter neutrophil function in zebrafish. The findings suggest that caution should be taken when employing a heat shock-dependent inducible system to study the innate immune response.     

Involvement of heat shock protein 47 in Schistosoma japonicum-induced hepatic fibrosis in mice

Chronic infection with the blood fluke Schistosoma japonicum is associated with both liver cirrhosis and liver cancer. Previously, heat shock protein 47, a collagen-specific molecular chaperone, was shown to play a critical role in the maturation of procollagen. However, less is known about the role of heat shock protein 47 in S. japonicum-induced hepatic fibrosis. We therefore investigated the expression of heat shock protein 47 in S. japonicum-induced liver fibrosis and attempted to determine whether inhibition of heat shock protein 47 could have beneficial effects on fibrosis in vitro and in vivo. In this study, we found that the expression of heat shock protein 47 was significantly increased in patients with Schistosoma-induced fibrosis, as well as in rodent models. Immunohistochemistry revealed heat shock protein 47-positive cells were found in the periphery of egg granulomas. Administration of heat shock protein 47-targeted short hairpin (sh)RNA remarkably reduced heat shock protein 47 expression and collagen deposition in NIH3T3 cells and liver tissue of S. japonicum-infected mice. Life-table analysis revealed a dose-dependent prolongation of survival rates with the treatment of heat shock protein 47-shRNA in murine fibrosis models. Moreover, serum alanine aminotransferase and aspartate transaminase activity, splenomegaly, spleen weight index and portal hypertension were also measured, which showed improvement with the anti-fibrosis treatment. The fibrosis-related parameters assessed were expressions of Col1a1, Col3a1, TGF-β1, CTGF, IL-13, IL-17, MMP-9, TIMP-1 and PAI-1 in the liver. This study demonstrated that heat shock protein 47-targeted shRNA directly reduced collagen production of mouse liver fibrosis associated with S. japonicum. We conclude that heat shock protein 47 plays an essential role in S. japonicum-induced hepatic fibrosis in mice and may be a potential target for ameliorating the hepatic fibrosis caused by this parasite.