Cloning and characterization of a thermostable superoxide dismutase from the thermophilic bacterium Rhodothermus sp XMH10

A superoxide dismutase (SOD) gene was cloned from the thermophilic bacterium Rhodothermus sp. XMH10 for the first time and highly expressed in Escherichia coli. The Rhodothermus sp. XMH10 SOD (RhSOD) gene encodes 209 amino acids with a putative molecular weight of 23.6 kDa and a pI value of 5.53. The recombinant RhSOD was detected to be an iron type SOD and existed as a dimer on its natural status. Experiments revealed that this RhSOD showed high activity at 50-70 degrees C and pH 5.0. Compared to SODs from other thermophiles, it was highly thermostable, maintaining more than 90% of its activity after incubation at 70 degrees C for 12 h, only totally inactivated after more than 4-h incubation at 80 degrees C. It also showed much higher resistance to KCN, NaN3 and H2O2 as compared to other SODs

Sequence analysis and characterizations of two novel plasmids isolated from Thermus sp 4C

Two novel plasmids, named pS4C and pL4C, were isolated from the thermophilic bacterium Thermus sp. 4C. The pS4C with a length of 5015 by and 58.25% of G+C content, contains 9 putative open reading frames (ORFs). The larger plasmid, pL4C, consisting of 21,248 bp, has a G+C content of 68.60% and 34 putative ORFs. Both plasmids encode their own replication protein. The ORF 22 of pL4C and the ORF 4 of pS4C encode proteins with high sequence similarities to integrase (97%) and transposase (97%), respectively, which are both involved in DNA rearrangement and exchange. Furthermore, sequence analysis of pL4C also showed that several plasmid-encoded genes may be involved in DNA modification and repair, such as DNA G:T-mismatch repair endonuclease and micrococcal nuclease-like protein. These proteins may be involved in raising the repair efficiency and other minor editing needs. Interestingly, the elimination of plasmids significantly lowered the growth temperature of Thermos sp. 4C. Few reports dealing with the DNA repair enzymes on the plasmid from Thermus strains were published so far. (c) 2007 Elsevier Inc. All rights reserved

A novel JNK from Litopenaeus vannamei involved in white spot syndrome virus infection

National Basic Research Program of China (973 Program) [2012CB114403]; National High Technology Research and Development Program of China (863 Program) [2012AA092205]; China Agriculture Research System-47; National Natural Science Foundation of China [30830084]; Scientific Research Foundation of Third Institute of Oceanography, SOA [2011018]The c-Jun N-terminal kinase (JNK), a member of MAP kinases, is a serine/threonine-specific protein kinase which responds to extracellular stimuli and regulate various cellular activities. It is well documented in innate immune responses and reported to be involved in various viral infections of mammals. In present study, we cloned INK homolog in a crustacean, Litopenaeus vannamei (designated as LvJNK) and studied its role in white spot syndrome virus (WSSV) infection. Sequence analysis displayed that LvJNK shared high similarity with other members of the JNK subfamily, including the conserved TPY motif and serine/threonine protein kinase (S_TKc) domain. Western blot analysis showed that the activation of LvJNK took place in WSSV infection. Lvjnk silencing mediated by specific dsRNA in shrimps could significantly inhibit the proliferation of the virus. Moreover, inhibition of shrimp JNK signaling pathway by specific inhibitor resulted in the reduction of WSSV replication and the delay of WSSV gene transcription. These results indicate for the first time that shrimp JNK is activated in response to WSSV infection and WSSV could benefit from JNK activation. It may facilitate our understanding of the molecular mechanism of virus infection and provided a potential target for preventing the WSSV infection. (c) 2012 Elsevier Ltd. All rights reserved

Identification of differentially expressed genes from Rhodothermus sp XMH10 in response to low temperature using random arbitrarily primed PCR

Most research on the adaptation of thermophiles is focused on their adaptation to heat stress; only a few studies are focused on their cold adaptation. In this report, the thermophilic bacterium Rhodothermus sp. XMH10 was examined to gain a better understanding of gene expression in response to low temperature. Random arbitrarily primed polymerase chain reaction (RAP-PCR) was used to isolate and identify differentially expressed genes of bacteria grown at 45 degrees C (lowest) compared to those at 75 degrees C (optimal). Fifty-three differential cDNA fragments in total were isolated. Among them, 35 different cDNAs were analyzed by Northern blot, and 17 were confirmed to be differentially expressed at the transcriptional levels. These genes reflected a profile of differential expression and were involved in many physiological processes such as metabolism, cell membrane alterations, and regulatory adaptive response; most of them have never been previously reported. This study provides some new information on the adaptation of thermophilic bacteria to environmental temperature stress

Molecular cloning and characterization of a cDNA encoding extracellular signal-regulated kinase from Litopenaeus vannamei

Extracellular signal-regulated kinase (ERR) is a serine/threonine-specific kinase, which is activated by downstream signaling molecules of cellular activation, cytokine and chemokine stimulation and various other stimuli. Here we cloned an ERK gene from Litopenaeus vannamei and designated it as lverk. The lverk cDNA contained an open reading frame of 1098 bp encoding 365 amino acids. LVERK had a conserved TEY motif and serine/threonine protein kinase (S_TKc) domain, and close phylogenetic relationship to Penaeus monodon and Marsupenaeus japonicus ERR. Immunofluorescence staining analysis showed that following serum stimulation LVERK was located in cytoplasm and nucleus, but phospho-LVERK was prominently in nucleus, suggesting conserved ERR signaling module occurred in shrimp cells. Then during the white spot syndrome virus (WSSV) infection, LVERK and phospho-LVERK increased at the early stage of infection. Once silencing of lverk in vivo, the replication of WSSV was obviously inhibited. Moreover, treatment of mitogen-activated protein kinase kinase inhibitor in vitro could result in reduction of WSSV proliferation and delay of viral early gene transcription. Our results indicated a role of LVERK involved in WSSV infection. Understanding how WSSV influences ERR signaling pathway to dismantle an effective immune response may lead to insight into pathogenic progression and possible disease control. (C) 2012 Elsevier Ltd. All rights reserved.NSFC/HK-RGC Joint Research Scheme [N_HKUST609/09]; NSFC [20931160426

Characterization of two novel ADP ribosylation factors from the shrimp Marsupenaeus japonicus

National 501 Natural Science Foundation of China [30830084]; National Basic Science Research Program of China [2006CB101804]; Earmarked fund for Modern Agro-industry Technology Research SystemADP-ribosylation factors (Arfs) that play an essential role in intracellular trafficking and organelle structure are small GTP-binding proteins, which have been identified recently to be involved in virus infection. However, little is known about the Arfs and their relationships with viral infection in the economically important crustaceans to date. In the present study, two novel members of the Arf family, designated as MjArf1 and MjArfn respectively, were cloned from the shrimp Marsupenaeus japonicus. Sequence and phylogenetic analysis showed that MjArf1 belongs to Class I Arf, which has very high homology in sequence to the known Arf 79F of insects and Arf1 of other animals (96-99%), whereas MjArfn is an unidentified Arf, which has only 62-66% identity to other known Arfs. In High Five cells, the distribution of MjArf1 was dependent on its GDP/GTP binding state but the distribution of MjArfn was not affected by that. Both Arfs were ubiquitously expressed in examined tissues. Further investigation with real-time quantitative PCR revealed that MjArf1 and MjArfn were significantly up-regulated after WSSV challenge. In virus-resistant shrimps, however, no distinct fluctuation of MjArf1 expression was found and MjArfn was even found to be notably repressed. These results suggested that MjArf1 and MjArfn might be involved in the shrimp innate immune response in WSSV infection and MjArfn might play a role in WSSV invasion. These studies may contribute to a better understanding of host defense and/or virus invasion interaction and for the control of marine crustacean diseases. (C) 2010 Elsevier Ltd. All rights reserved

Molecular characteristics of the tubeworm, Ridgeia piscesae, from the deep-sea hydrothermal vent

China Ocean Mineral Resources RD Association [DYXM-115-02-2-16]; Third Institute of Oceanograpy; State Oceanic Administration, ChinaRidgeia piscesae, living around the extremely harsh hydrothermal vent in deep sea, is an ideal model for studying the adaptative mechanism to extreme environment. For insights of its molecular characteristics, a cDNA library of R. piscesae was constructed. A total of 879 expressed sequence tags (ESTs) were sequenced and 199 genes were identified for the first time. They were found to be involved in basal metabolism, adaptation and defense, or signal transduction. Among them, we found 23 various chitin-binding proteins, which are the major component of the chitinous tube that prevents the tubeworms from predators and surrounding extreme environment. Additionally, high polymorphism also exists in other genes, such as myohemerythrin, lysozyme. The gene-expression profile might help to further understand the molecular basis of tubeworm physiology. It will also lay a good foundation for functional studies on the adaptation to extreme environments

Crystal structure of thermostable alkylsulfatase SdsAP from Pseudomonas sp. S9

Correspondence: Yunkun Wu ([email protected]) A novel alkylsulfatase from bacterium Pseudomonas sp. S9 (SdsAP) was identified as a thermostable alkylsulfatases (type III), which could hydrolyze the primary alkyl sulfate such as sodium dodecyl sulfate (SDS). Thus, it has a potential application of SDS biodegradation. The crystal structure of SdsAP has been solved to a resolution of 1.76Å and reveals that SdsAP contains the characteristic metallo-β-lactamase-like fold domain, dimerization domain, and C-terminal sterol carrier protein type 2 (SCP-2)-like fold domain. Kinetic characterization of SdsAP to SDS by isothermal titration calorimetry (ITC) and enzymatic activity assays of constructed mutants demonstrate that Y246 and G263 are important residues for its preference for the hydrolysis of 'primary alkyl' chains, confirming that SdsAP is a primary alkylsulfatase

The role of Litopenaeus vannamei p38 in white spot syndrome virus infection

Major State Basic Research Development Program [2012CB114403]; National High Technology Research and Development Program of China [2012AA092205]; National Natural Science Foundation of China [31001125]; China Agriculture Research System-47; Fujian Science and Technology Project [2011J05079]; Scientific Research Foundation of Third Institute of Oceanography, SOA [2011018]p38 as a member of MAPK family, has been conversed from yeast to mammals. It has been implicated in numerous biological processes, including the responses to stress and inflammation. In this study, three closely related p38 MAPK homologs, Ivp38a, Ivp38b and Ivp38c, which differ only in an internal 25-amino acid sequence, have been cloned from Litopenaeus vannamei. Three isoforms shared p38 conserved TGY motif and catalytic center, as well as had maximum identities to human p38a and Drosophila p38b. Tissue distribution revealed that Ivp38a and Ivp38b were ubiquitously expressed in most tissues, while Ivp38c showed at relatively low levels and in a tissue-specific manner. Western blotting analysis showed that Ivp38 was activated by phosphorylation during WSSV infection. Furthermore, silencing Ivp38 mediated by specific dsRNA in shrimps promoted white spot syndrome virus (WSSV) replication and viral gene transcription at the early stage. These results demonstrated that Ivp38 was involved in WSSV infection and might participate in host defense at the early stage. (C) 2013 Elsevier Ltd. All rights reserved

Isolation and identification of novel microRNAs from Marsupenaeus japonicus

National Natural Science Foundation of China [31001125, 30830084, 30800588]; earmarked fund for Modern Agro-industry Technology Research SystemMicroRNAs (miRNAs) are a class of small noncoding RNAs that function as regulators of gene expression. They play essential roles in various biological processes, such as development, differentiation and immune response. In this study, we identified 35 miRNAs from Marsupenaeus japonicus. Among them, fifteen miRNAs exhibited high homology to the known miRNAs from other arthropods, while the rest might represent novel miRNAs. We further showed a correlation of WSSV infection and the expression levels of 22 miRNAs. This is the first report to identify miRNAs from the shrimp. Our results extend the knowledge of the gene regulation of crustacean, providing clues for future researches of shrimp immunity against virus infection. (C) 2011 Elsevier Ltd. All rights reserved