Whole genome sequence of Bacillus thuringiensis ATCC 10792 and improved discrimination of Bacillus thuringiensis from Bacillus cereus group based on novel biomarkers.

BACKGROUND: Among the Bacillus cereus group, B. thuringiensis, is one of the most extensively used biological control agent. The present study reports the complete genome and four novel plasmid analysis of the type strain B. thuringiensis ATCC 10792. METHODS: Complete genome sequencing of Bacillus thuringiensis ATCC 10792, assembled using de-novo (v.3.2.0, assembly name MIRA3), Pac-Bio sequencers and Hierarchical Genome Assembly Process software (version 4.1) and real-time polymerase chain reaction (qPCR) is a consistent technique for quantifying gene expression based on specific biomarkers, in addition the efficiency of the primers were analysed based on artificially spiked food samples on lettuce, kimbab and spinach with B. thuringiensis ATCC 10792. RESULTS: Complete genome annotation was performed, and a total of 6269 proteins with 5427594 bps were identified and four novel plasmid (poh2, poh3, poh4, poh5) a total of 134, 131, 96, 21 proteins with 113294; 92,949; 86488; 11332 bps were identified. Six selective genes (lipoprotein-lipo, methyltransferase-MT, S-layer homology domain protein-BC, flagellar motor protein-motB, transcriptional regulator-XRE, crystal protein-cry2) and associated four novel plasmids were investigated along with the characteristics and expression profiles of two housekeeping genes (chaperonin protein-GroEL and topoisomerase enzyme-gyrB). Although from the assessment of 120 strains, both GroEL and gyrB showed 100% specificity towards detection of both B. thuringiensis in artificially spiked vegetable samples. All the eight genes revealed no specificity towards any of the 9 non- Bacillus strains. CONCLUSION: In our study based on the complete genome and plasmid sequence of B. thuringiensis ATCC 10792, among the six discriminating genes, specifically GroEL, gyrB and XRE showed promising results in identifying B. thuringiensis ATCC 10792, and there detection limit was 3.0-9.6 log CFU/g in the food samples respectfully. The key role in control of the predatory biological agent.

New perspectives on Mega plasmid sequence (poh1) in Bacillus thuringiensis ATCC 10792 harbouring antimicrobial, insecticidal and antibiotic resistance genes.

Bacillus thuringiensis promotes the growth of numerous economically important crops. The present study presents the complete genome sequence for a mega plasmid present in the type strain of B. thuringiensis ATCC 10792, a typical spore-forming Gram-positive bacterium with insecticidal activity, and investigates its genetic characteristics. The genome was sequenced and assembled de novo using Pac-Bio sequencers and the Hierarchical Genome Assembly Process, respectively. Further genome annotation was performed, and a total of 489 proteins and a novel mega-plasmid (poh1) with 584,623 bps were identified. The organization of poh1 revealed the genes involved in the insecticidal toxin pathway. The genes responsible for antimicrobial, insecticidal and antibiotic activities were well conserved in poh1, indicating an intimate association with plant hosts. The poh1 plasmid contains the gene encoding a novel crystal protein kinase responsible for production of zeta toxin, which poisons insects and other Gram-negative bacteria through the global inhibition of peptidoglycan synthesis. Lantibiotics are a group of bacteriocins that include the biologically active antimicrobial peptide Paenibacillin. Further, poh1 also contains the genes that encode the gramicidin S prototypical antibiotic peptide and tetracycline resistance protein. In conclusion, the strain-specific genes of B. thuringiensis strain ATCC 10792 were identified through complete genome sequencing and bioinformatics data based on major pathogenic factors that contribute to further studies of the pathogenic mechanism and phenotype analyses.

Unique biomarkers as a potential predictive tool for differentiation of Bacillus cereus group based on real-time PCR.

The aim of the study was to develop unique biomarkers for qPCR detection of Bacillus cereus group. Clinical and soil isolates were identified by specifically designed biomarkers - Lipoprotein (OPL-114-lipo), Methyltransferase (MT-17) and S-layer homology domain protein (151-1BC). In order to design biomarkers, we used 120 bacterial strains grouped into B. cereus and non-Bacillus group. The B. cereus group was confirmed by 108 strains of B. cereus and B. thuringiensis (30 reference and 78 wild), along with 3 strains of B. mycoides, B. pseudomycoides, and B. weihenstephanensis; while the non-Bacillus group was composed of 9 Gram-positive and Gram-negative strains. Direct analysis of samples revealed specificity towards identification and characterization of B. cereus group. The newly developed markers OPL-114-lipo and MT-17 showed specificity of 95% and 81%, respectively in identification of B. cereus. They are efficient tools to identify contaminated sources and the degree of bacterial contamination. Environmental and food samples do not require band isolation, re-amplification, sequencing or sequence identification. Thus, reducing the time and cost of analysis. Hence, it will be an alternative approach to traditional culture methods. Commercial food processing industries will be able to employ these biomarkers specific for B. cereus group as a detection tool to reduce economic loss due to B. cereus contamination.

Molecular discrimination of Bacillus cereus group species in foods (lettuce, spinach, and kimbap) using quantitative real-time PCR targeting groEL and gyrB.

The aim of the study was to identify and evaluate specific biomarkers to differentiate within Bacillus cereus group species from contaminated food samples with the use of real-time PCR. A total of 120 strains, comprising of 28 reference, 2 type, 78 wild strains of B. cereus and B. thuringiensis along with 12 strains representing 2 bacterial groups - B. mycoides, B. pseudomycoides, B. weihenstephanensis (B. cereus group); B. amyloliquefaciens, B. subtilis, Enterococcus faecalis, Escherichia coli, Listeria monocytogenes, Micrococcus luteus, Salmonella enterica, Staphylococcus aureus, Streptococcus pyogenes (non-Bacillus sp.) were identified by applying valid biomarkers (groEL and gyrB). In addition, the presence of B. cereus group was determined in three different artificially contaminated vegetable samples (lettuce, spinach, and kimbap), using prominent biomarkers targeting on chaperonin protein (GroEL) and topoisomerase enzyme protein (gyrB). Direct analysis of samples revealed the specificity towards identification and characterization of the B. cereus group among wild, reference and type strains and the type strain inoculated in vegetables. Our results demonstrated two existing biomarkers groEL and gyrB with a high specificity of 98% and 96% respectively to analyze the total B. cereus group. Further, we also reported the detection limit of groEL and gyrB in food samples was 3.5 and 3.7 log CFU/g respectively. Thus, the developed real-time PCR approach can be a reliable and effective tool for the identification of B. cereus group strains present in environment and food samples. This does not require band isolation, re-amplification, sequencing or sequence identification, thus reducing the time and cost of analysis.

Novel motB as a potential predictive tool for identification of B. cereus, B. thuringiensis and differentiation from other Bacillus species by triplex real-time PCR.

Quantitative triplex real-time PCR (qPCR) offers an alternative method for detection of bacterial contamination. It provides quantitation of the number of gene copies. In our study, we established a qPCR assay to detect and quantify the specificity towards Bacillus cereus and B. thuringiensis. The assay was designed to detect a 280 bp fragment of motB gene encoding the flagellar motor protein, specific for detection of B. cereus and B. thuringiensis, excluding other group species B. pseudomycoides, B. mycoides and B. weihenstephanensis. Specificity of the assay was confirmed with 111 strains belonging to Bacillus cereus group and performed against 58 B. cereus, 50 B. thuringiensis, 3 other Bacillus bacteria and 9 non-Bacillus bacteria. Detection limit was determined for each assay. Direct analysis of samples revealed the specificity towards identification and characterization of B. cereus group cultured in nutrient media. Based on results, it was observed that motB showed 97% specificity towards B. cereus strains, 98% for B. thuringiensis but other B. cereus group showed less sensitivity (0%), thus, provides an efficient tool to identify B. cereus and B. thuringiensis. Further, environmental and food samples do not require band isolation, re-amplification or sequence identification. Thus, reducing the time and cost of analysis.

Continuous exposure to 60 Hz magnetic fields induces duration- and dose-dependent apoptosis of testicular germ cells.

To evaluate duration- and dose-dependent effects of continuous exposure to a 60 Hz magnetic field (MF) on the testes in mice, BALB/c male mice were exposed to a 60 Hz MF at 100 µT for 24 h a day for 2, 4, 6, or 8 weeks, and at 2, 20, or 200 µT for 24 h a day for 8 weeks. Any exposures to MF did not significantly affect body or testicular masses. However, the apoptotic cells among testicular germ cells were increased duration-dependent at exposures of 100 µT for 6 and 8 weeks and dose-dependent at exposures of 20 and 200 µT for 8 weeks. The number of sperm in epididymis and the diameter of seminiferous tubule decreased in mice exposed to 100 and 200 µT for 8 weeks, respectively. To induce the apoptosis of testicular germ cell in mice, the minimum dose is 20 µT at continuous exposure to a 60 Hz MF for 8 weeks and the minimum duration is 6 weeks at continuous exposure of 100 µT. Taken together, these results suggest that continuous exposure to a 60 Hz MF might affect, duration- and dose-dependent biological processes including apoptotic cell death and spermatogenesis in the male reproductive system of mice.

Transduced Tat-glyoxalase protein attenuates streptozotocin-induced diabetes in a mouse model.

Diabetes mellitus (DM) is characterized by hyperglycemia. Glyoxalase 1 (GLO) has considerable potential as a possible therapeutic agent for DM. However, the precise action of GLO remains unclear in DM. In this study, we examined the protective effects of GLO protein in a streptozotocin (STZ)-induced diabetes animal model using cell-permeable Tat-GLO protein. Purified Tat-GLO protein was efficiently transduced into RINm5F cells in a time- and dose-dependent manner and protected cells against sodium nitroprusside (SNP)-induced cell death and DNA fragmentation. Furthermore, Tat-GLO protein significantly inhibited blood glucose levels and altered the serum biochemical parameters in STZ-induced diabetic mice. These results demonstrate that transduced Tat-GLO protein protects pancreatic cells by the inhibition of STZ-mediated toxicity. Therefore, Tat-GLO protein could be useful as a therapeutic agent against DM.

Improvement of the diagnostic sensitivity of scrub typhus using a mixture of recombinant antigens derived from Orientia tsutsugamushi serotypes.

Diagnosis of scrub typhus is difficult because its symptoms are very similar to other acute febrile illnesses, such as leptospirosis, murine typhus, and other viral hemorrhagic fevers. To differentiate scrub typhus from other acute febrile diseases, a rapid and reliable serological diagnosis is important. We have developed a chimeric recombinant antigen cr56 and two other recombinant antigens, r21 and kr56, from various serotypes of Orientia tsutsugamushi. They were tested for the detection of antibodies against O. tsutsugamushi in the patient's serum samples using enzyme-linked immunosorbent assay (ELISA) and dot-blot analyses. As of conventional immunofluorescence assay (IFA), when the mixture of these three recombinant antigens was used, both sensitivity and specificity of the recombinant antigens were increased up to 98% in IgM and IgG at ELISA and dot blotting. Additionally, both sensitivity and specificity by detection of IgM and IgG antibodies at rapid diagnostic test (RDT), using the mixture of three antigens and gold conjugated antibodies, were 99%. Our results suggest the use of mixture of these recombinant antigen proteins in ELISA or RDT is suitable as a diagnostic test for scrub typhus.

Functional assessment of Nramp-like metal transporters and manganese in Caenorhabditis elegans.

Nramp1 (natural resistance-associated macrophage protein-1) is a functionally conserved iron-manganese transporter in macrophages. Manganese (Mn), a superoxide scavenger, is required in trace amounts and functions as a cofactor for most antioxidants. Three Nramp homologs, smf-1, smf-2, and smf-3, have been identified thus far in the nematode Caenorhabditis elegans. A GFP promoter assay revealed largely intestinal expression of the smf genes from early embryonic through adult stages. In addition, smf deletion mutants showed increased sensitivity to excess Mn and mild sensitivity to EDTA. Interestingly, these smf deletion mutants demonstrated hypersensitivity to the pathogen Staphylococcus aureus, an effect that was rescued by Mn feeding or knockdown of the Golgi calcium/manganese ATPase, pmr-1, indicating that Mn uptake is essential for the innate immune system. This reversal of pathogen sensitivity by Mn feeding suggests a protective and therapeutic role of Mn in pathogen evasion systems. We propose that the C. elegans intestinal lumen may mimic the mammalian macrophage phagosome and thus could be a simple model for studying Mn-mediated innate immunity.

Differentiation of Bacillus thuringiensis From Bacillus cereus Group Using a Unique Marker Based on Real-Time PCR.

The efficiency of a novel biomarker (the transcriptional regulator, XRE) was tested and evaluated in differentiating Bacillus thuringiensis from Bacillus cereus group species in environmental and spiked samples based on PCR and real-time PCR. Totally 120 strains, representing two bacterial groups, B. cereus group and non-Bacillus sp., were used to evaluate the performance of XRE and crystal protein (cry2, an existing biomarker). Further, three diverse samples (kimbap, lettuce, and spinach) were inoculated with B. thuringiensis and prominent biomarkers XRE and cry2 were used as targets. Direct analysis of the detection results for the pure cultures of B. cereus group wild-types, references and type strains revealed an accuracy rate of 97.5% targeting XRE, and 83.3% targeting cry2. The real-time PCR was constructed with a R 2-value of 0.993. For the artificially contaminated samples, a concentration of 103 CFU/g of B. thuringiensis in spiked food samples could be detected using real-time PCR targeting XRE. A good performance was obtained with XRE in discriminating B. thuringiensis from B. cereus groups, as well as detecting B. thuringiensis in spiked food samples with PCR or real-time PCR. Therefore, this real-time PCR targeting XRE can be used as a dependable and promising tool to identify B. thuringiensis in foods.

Differential requirement of unfolded protein response pathway for calreticulin expression in Caenorhabditis elegans.

Accumulation of unfolded proteins in the endoplasmic reticulum triggers the unfolded protein response (UPR) pathway, which increases the expression of chaperones to maintain the homeostasis. Calreticulin is a calcium-binding chaperone located in the lumen of endoplasmic reticulum (ER). Here we show that in response to a UPR inducing reagent, tunicamycin, the expression of calreticulin (crt-1) is specifically up-regulated in Caenorhabditis elegans. Tunicamycin (TM) induced expression of the crt-1 requires IRE-1 and XBP-1 but is ATF-6 and PEK-1 independent. Analysis of the crt-1 promoter reveals a putative XBP-1 binding site at the -284 to -278 bp region, which was shown to be necessary for TM-mediated induction. Genetic analysis of crt-1 mutants and mutants of UPR pathway genes show various degrees of developmental arrest upon TM treatment. Our results suggest that the TM-induced UPR pathway culminates in the up-regulation of crt-1, which protects the worm from deleterious accumulation of unfolded proteins in the ER. Knockdown of the crt-1, pdi-2, or pdi-3 increased the crt-1 expression, whereas knockdown of the hsp-3 or hsp-4 did not have any effect on crt-1 expression, indicating the existence of complex compensatory networks to cope up with ER stress.

In vivo screening platform for shiga toxin-producing Escherichia coli (STEC) using Caenorhabditis elegans as a model.

Shiga toxin-producing Escherichia coli (STEC) strains are the main cause of bacillary dysentery, although STEC strains generally induce milder disease symptoms compared to Shigella species. This study aimed to determine the virulence of STEC using the nematode Caenorhabditis elegans as a model host. Worm killing, fertility and bacterial colonisation assays were performed to examine the potential difference in the virulence of STEC strains compared to that of the control E. coli OP50 strains on which worms were fed. A statistically significant difference in the survival rates of C. elegans was observed in that the STEC strains caused death in 8-10 days and the E. coli OP50 strains caused death in 15 days. STEC strains severely reduced the fertility of the worms. The intestinal load of bacteria in the adult stage nematodes harbouring the E. coli OP50 strains was found to be 3.5 log CFU mL-1. In contrast, the STEC strains E15, E18 and E22 harboured 4.1, 4.2 and 4.7 log CFU ml-1 per nematode, respectively. The heat-killed STEC strains significantly increased the longevity of the worms compared to the non-heated STEC strains. In addition, PCR-based genomic profiling of shiga toxin genes, viz., stx1 and stx2, identified in selected STEC strains revealed that these toxins may be associated with the virulence of the STEC strains. This study demonstrated that C. elegans is an effective model to examine and compare the pathogenicity and virulence variation of STEC strains to that of E. coli OP50 strains.

In vitro and in vivo defensive effect of probiotic LAB against Pseudomonas aeruginosa using Caenorhabditis elegans model.

This study aimed to investigate in vitro and in vivo the probiotic characteristics of lactic acid bacteria (LAB) isolated from Korean traditional fermented foods. Caenorhabditis elegans  (C. elegans) was used for analytical assays of fertility, chemotaxis, life-span, worm-killing and bacterial colonization in the intestinal lumen of the worm. All 35 strains of LAB reduced fertility and slowed development in the worms. The worm-killing assay showed that LAB significantly increased the lifespan (P < 0.05) and reduced the susceptibility to virulent PA14; however, the heat-killed LAB did not. The bacterial colonization assay revealed that LAB proliferated and protected the gut of the worm against infection by Pseudomonas aeruginosa PA14. In addition, specific LAB Pediococcus acidilactici(P. acidilactici DM-9), Pediococcus brevis (L. brevis SDL1411), and Pediococcus pentosaceus (P. pentosaceus SDL1409) strains showed acid resistance (66-91%), resistance to pepsin (64-67%) and viability in simulated intestinal fluid (67-73%) based on in vitro probiotic analyses. Taken together, these results suggest that C. elegans may be a tractable model for screening efficient probiotics.

Prevalence and Toxin Characteristics of Bacillus thuringiensis Isolated from Organic Vegetables.

The prevalence and toxin characteristics of Bacillus thuringiensis isolated from 39 organic vegetables were investigated. B. thuringiensis was detected in 30 out of the 39 organic vegetables (76.9%) with a mean value of 2.60 log CFU/g. Twenty-five out of the 30 B. thuringiensis isolates (83.3%) showed insecticidal toxicity against Spodoptera exigua. The hblCDA, nheABC, and entFM genes were found to be the major toxin genes, but the ces gene was not detected in any of the tested B. thuringiensis isolates. The hemolysin BL enterotoxin was detected in all 30 B. thuringiensis isolates (100%). The non-hemolytic enterotoxin complex was found in 27 out of 30 B. thuringiensis isolates (90.0%). The B. thuringiensis tested in this study had similar toxin gene characteristics to B. cereus, which possessed more than one toxin gene. B. thuringiensis could have the potential risk of foodborne illness based on the toxin genes and toxin-producing ability.

VHA-8, the E subunit of V-ATPase, is essential for pH homeostasis and larval development in C. elegans.

Vacuolar H+-ATPase (V-ATPase) is an ATP-dependent proton pump, which transports protons across the membrane. It is a multi-protein complex which is composed of at least 13 subunits. The Caenorhabditis elegans vha-8 encodes the E subunit of V-ATPase which is expressed in the hypodermis, intestine and H-shaped excretory cells. VHA-8 is necessary for proper intestinal function likely through its role in cellular acidification of intestinal cells. The null mutants of vha-8 show a larval lethal phenotype indicating that vha-8 is an essential gene for larval development in C. elegans. Interestingly, characteristics of necrotic cell death were observed in the hypodermis and intestine of the arrested larvae suggesting that pH homeostasis via the E subunit of V-ATPase is required for the cell survival in C. elegans.

Highly efficient and specific separation of Staphylococcus aureus from lettuce and milk using Dynabeads protein G conjugates.

An immunomagnetic separation method using antibody-coated Dynabeads® Protein G was developed for specific and efficient separation of Staphylococcus aureus in lettuce and whole milk. The amount of immunomagnetic beads (IMBs) and conjugation conditions were optimized. A high capture efficiency was obtained with 0.4 mg of IMBs, an immunoreaction time of 20 min, and a separation time of 1 min without wash. Under optimal conditions, the capture efficiency (CE) for 100-105 CFU/mL of S. aureus was higher than 91.46%. The IMBs showed high specificity even with a high constant number (107 CFU/mL) of Bacillus cereus, Micrococcus luteus, and Lactobacillus plantarum. The CE of IMBs against S. aureus at concentrations from 102 to 105 CFU/mL ranged from 78.70 to 94.77% for lettuce and 60.0 to 73.27% for milk samples. This IMS can be an appropriate selection for combining with bacterial detection method or efficient isolation procedure for S. aureus from foods.

Dynabeads protein G antibody conjugates combined with modified brain heart infusion broth for the enrichment and separation of Bacillus cereus in artificially contaminated vegetables.

A modified brain heart infusion (MBHI) broth and a protocol of immunomagnetic separation (IMS) using antibody-coated Dynabeads® protein G were developed for the enrichment and separation of Bacillus cereus in artificially contaminated vegetable samples. The MBHI consisted of BHI and 0.34 g/L magnesium sulfate, 12.08 g/L sodium pyruvate, 1.82 g/L yeast extract, and polymyxin B. The amount of immunomagnetic beads (IMBs) and immunoreaction time were optimized. The capture efficiency was 58.32% with 0.4 mg IMBs when the immunoreaction time was 20 min. Capture of B. cereus by IMBs did not interfere with competing flora. Pre-enrichment IMS was validated with four B. cereus strains in artificially contaminated baby sprouts, bean sprouts, lettuce, and spinach at two levels (∼0.1 and ∼1 CFU/g). We were able to detect and isolate B. cereus in 40/40 samples of vegetables contaminated at 0.1 CFU/g with IMS after 6 h of enrichment in MBHI.

Opposing functions of calcineurin and CaMKII regulate G-protein signaling in egg-laying behavior of C.elegans.

Ca(2+)/calmodulin-dependent calcineurin has been shown to have important roles in various Ca(2+) signaling pathways. We have previously reported that cnb-1(jh103) mutants, null mutants of a regulatory B subunit, displayed pleiotropic defects including uncoordinated movement and delayed egg laying in Caenorhabditis elegans. Interestingly, gain-of-function mutants of a catalytic A subunit showed exactly opposite phenotypes to those of cnb-1(null) mutants providing an excellent genetic model to define calcium-mediated signaling pathway at the organism level. Furthermore, calcineurin is also important for normal cuticle formation, which is required for maintenance of normal body size in C.elegans. Genetic interactions between tax-6 and several mutants including egl-30 and egl-10, which are known to be involved in G-protein signaling pathways suggest that calcineurin indeed regulates locomotion and serotonin-mediated egg laying through goa-1(Goalpha) and egl-30(Gqalpha). Our results indicate that, along with CaMKII, calcineurin regulates G-protein-coupled phosphorylation signaling pathways in C.elegans.

SHN-1, a Shank homologue in C. elegans, affects defecation rhythm via the inositol-1,4,5-trisphosphate receptor.

Protein localization in the postsynaptic density (PSD) of neurons is mediated by scaffolding proteins such as PSD-95 and Shank, which ensure proper function of receptors at the membrane. The Shank family of scaffolding proteins contain PDZ (PSD-95, Dlg, and ZO-1) domains and have been implicated in the localizations of many receptor proteins including glutamate receptors in mammals. We have identified and characterized shn-1, the only homologue of Shank in Caenorhabditis elegans. The shn-1 gene shows approximately 40% identity over 1000 amino acids to rat Shanks. SHN-1 protein is localized in various tissues including neurons, pharynx and intestine. RNAi suppression of SHN-1 did not cause lethality or developmental abnormality. However, suppression of SHN-1 in the itr-1 (sa73) mutant, which has a defective inositol-1,4,5-trisphosphate (IP(3)) receptor, resulted in animals with altered defecation rhythm. Our data suggest a possible role of SHN-1 in affecting function of IP(3) receptors in C. elegans.

Two thioredoxin reductases, trxr-1 and trxr-2, have differential physiological roles in Caenorhabditis elegans.

Thioredoxin reductase (TrxR) is a member of the pyridine nucleotide-disulfide reductase family, which mainly functions in the thioredoxin system. TrxR is found in all living organisms and exists in two major ubiquitous isoenzymes in higher eukaryotic cells; One is cytosolic and the other mitochondrial. Mitochondrial TrxR functions to protect mitochondria from oxidative stress, where reactive oxidative species are mainly generated, while cytosolic TrxR plays a role to maintain optimal oxido-reductive status in cytosol. In this study, we report differential physiological functions of these two TrxRs in C. elegans. trxr-1, the cytosolic TrxR, is highly expressed in pharynx, vulva and intestine, whereas trxr-2, the mitochondrial TrxR, is mainly expressed in pharyngeal and body wall muscles. Deficiency of the non-selenoprotein trxr-2 caused defects in longevity and delayed development under stress conditions, while deletion mutation of the selenoprotein trxr-1 resulted in interference in acidification of lysosomal compartment in intestine. Interestingly, the acidification defect of trxr-1(jh143) deletion mutant was rescued, not only by selenocystein-containing wild type TRXR-1, but also cysteine-substituted mutant TRXR-1. Both trxr-1 and trxr-2 were up-regulated when worms were challenged by environmental stress such as heat shock. These results suggest that trxr-1 and trxr-2 function differently at organismal level presumably by their differential sub-cellular localization in C. elegans.