Chaomin Sun

Two RNA-binding motifs in eIF3 direct HCV IRES-dependent translation

The initiation of protein synthesis plays an essential regulatory role in human biology. At the center of the initiation pathway, the 13-subunit eukaryotic translation initiation factor 3 (eIF3) controls access of other initiation factors and mRNA to the ribosome by unknown mechanisms. Using electron microscopy (EM), bioinformatics and biochemical experiments, we identify two highly conserved RNA-binding motifs in eIF3 that direct translation initiation from the hepatitis C virus internal ribosome entry site (HCV IRES) RNA. Mutations in the RNA-binding motif of subunit eIF3a weaken eIF3 binding to the HCV IRES and the 40S ribosomal subunit, thereby suppressing eIF2-dependent recognition of the start codon. Mutations in the eIF3c RNA-binding motif also reduce 40S ribosomal subunit binding to eIF3, and inhibit eIF5B-dependent steps downstream of start codon recognition. These results provide the first connection between the structure of the central translation initiation factor eIF3 and recognition of the HCV genomic RNA start codon, molecular interactions that likely extend to the human transcriptome.

A single gene directs both production and immunity of halocin C8 in a haloarchaeal strain AS7092

Halocin C8 (HalC8) is an extremely stable and hydrophobic microhalocin with 76 amino acids, and has a wide inhibitory spectrum against the haloarchaea. It is derived from the C‐terminus of a 283‐amino‐acid prepro‐protein (ProC8), which was demonstrated by molecular cloning of the halC8 gene, and verified by the N‐terminal amino acid sequencing as well as MALDI‐TOF‐MS analysis of the purified HalC8. The production of this halocin is controlled through both transcription regulation and protein processing: the halC8 transcripts and HalC8 activity rapidly increased to maximal levels upon transition from exponential to stationary phase. However, while halC8 transcripts remained abundant, the HalC8 processing was inhibited during stationary phase. Remarkably, agar‐diffusion test revealed the unprocessed ProC8 and its 207‐amino‐acid N‐terminal peptide (HalI), with or without the putative Tat signal sequence, were capable to block the halocin activity of HalC8 in vitro. In addition, heterologous expression of HalI in Haloarcula hispanica rendered this sensitive strain remarkable resistance to HalC8, indicating that HalI encodes the immunity property of the producer. In accordance with this immunity function, HalI and ProC8 were both found localized on the cellular membrane. Protein interaction assay revealed that HalI likely sequestrated the HalC8 activity by specific binding. To our knowledge, this is the first report on halocin immunity, and our results that a single gene encodes both peptide antibiotic and immunity protein also provide a novel immune mechanism for peptide antibiotics.

Expression of SARS-coronavirus nucleocapsid protein in Escherichia coli and Lactococcus lactis for serodiagnosis and mucosal vaccination

The nucleocapsid (N) protein of the severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) is an important antigen for the early diagnosis of SARS and the development of vaccines. It was expressed in Escherichia coli as a fusion with human glutathione S-transferase (hGST) and was confirmed by Western blotting analysis. This recombinant N protein (hGST-N) was purified and used to measure the SARS-CoV N-specific antibody in the sera of eight SARS patients by enzyme-linked immunosorbent assay. Specific antibody response to this purified recombinant N protein was 100% positive in the SARS patients’ sera, while none of the control sera from 30 healthy people gave a positive reaction in the same assay. The SARS-CoV N protein was also expressed in Lactococcus lactis in the cytoplasm or secreted into the medium. The N-producing strain MG1363/pSECN and the purified hGST-N protein were respectively administered to mice, either orally or intranasally. Results indicated that orally delivered MG1363/pSECN induced significant N-specific IgG in the sera. In conclusion, our work provides a novel strategy to produce the SARS-CoV N protein for serodiagnosis and for L. lactis-based mucosal vaccines.

A novel polysaccharide from Sargassum integerrimum induces apoptosis in A549 cells and prevents angiogensis in vitro and in vivo.

Many polysaccharides isolated from plants have exhibited promising antitumor activities. The aim of this study is to investigate the antitumor activity of the novel polysaccharide named SPS from Sargassum integerrimum, elucidate the underlying anticancer mechanism in a human lung cancer cell line A549, and evaluate its anti-angiogenic activity both in vitro and in vivo. The results show that SPS significantly reduces A549 cells viability in a dose- and time-dependent manner via MTT method. Flow cytometry analysis indicates that SPS could induce cell apoptosis, the loss of mitochondrial membrane potential (MMP), generation of reactive oxygen species (ROS) and G2/M phase cell cycle arrest of A549 cells. Up-regulation of the expressions of P53 and Bax, down-regulation of the expression of Bcl-2, and activation of cleaved caspase-3, caspase-9 and PARP are also detected by western blotting after the treatment of SPS. In addition, SPS inhibits the proliferation, migration and cord formation of human umbilical vein endothelial cells (HUVECs) in vitro, and prevents the vascular development of zebrafish embryos in vivo. Altogether, our data prove the anticancer and anti-angiogenesis properties of SPS, and provide further insights into the potential pharmacological application of SPS as antitumor and anti-angiogenic agent against lung cancer.

Antibiofilm and Anti-Infection of a Marine Bacterial Exopolysaccharide Against Pseudomonas aeruginosa

Pseudomonas aeruginosa is a well-known pathogenic bacterium that forms biofilms and produces virulence factors, thus leading to major problems in many fields, such as clinical infection, food contamination, and marine biofouling. In this study, we report the purification and characterization of an exopolysaccharide EPS273 from the culture supernatant of marine bacterium P. stutzeri 273. The exopolysaccharide EPS273 not only effectively inhibits biofilm formation but also disperses preformed biofilm of P. aeruginosa PAO1. High performance liquid chromatography traces of the hydrolyzed polysaccharides shows that EPS273 primarily consists of glucosamine, rhamnose, glucose and mannose. Further investigation demonstrates that EPS273 reduces the production of the virulence factors pyocyanin, exoprotease, and rhamnolipid, and the virulence of P. aeruginosa PAO1 to human lung cells A549 and zebrafish embryos is also obviously attenuated by EPS273. In addition, EPS273 also greatly reduces the production of hydrogen peroxide (H2O2) and extracellular DNA (eDNA), which are important factors for biofilm formation. Furthermore, EPS273 exhibits strong antioxidant potential by quenching hydroxyl and superoxide anion radicals. Notably, the antibiofouling activity of EPS273 is observed in the marine environment up to 2 weeks according to the amounts of bacteria and diatoms in the glass slides submerged in the ocean. Taken together, the properties of EPS273 indicate that it has a promising prospect in combating bacterial biofilm-associated infection, food-processing contamination and marine biofouling.

Molecular Characterization of the Minimal Replicon and the Unidirectional Theta Replication of pSCM201 in Extremely Halophilic Archaea

A 3,463-bp plasmid, pSCM201, was isolated from a halophilic archaeon, Haloarcula sp. strain AS7094. The minimal replicon that is essential and sufficient for autonomous replication and stable maintenance in Haloarcula hispanica was determined by deletion analysis of the plasmid. This minimal replicon ( approximately 1.8 kb) consisted of only two functionally related segments: (i) a putative origin (ori201) containing an AT-rich region and sets of repeats and (ii) an adjacent gene encoding a putative replication initiation protein (Rep201). Electron microscopic observation and Southern blotting analysis demonstrated that pSCM201 replicates via a theta mechanism. Precise mapping of the putative origin suggested that the replication initiated from a fixed site close to the AT-rich region and proceeded unidirectionally toward the downstream rep201 gene, which was further confirmed by electron microscopic analysis of the ClaI-digested replication intermediates. To our knowledge, this is the first unidirectional theta replication plasmid experimentally identified in the domain of archaea. It provides a novel plasmid system to conduct research on archaeal DNA replication.

Genomic and Transcriptomic Insights into Calcium Carbonate Biomineralization by Marine Actinobacterium Brevibacterium linens BS258

Calcium carbonate (CaCO3) biomineralization has been investigated due to its wide range of scientific and technological implications, however, the molecular mechanisms of this important geomicrobiological process are largely unknown. Here, a urease-positive marine actinobacterium Brevibacterium linens BS258 was demonstrated to effectively form CaCO3 precipitates. Surprisingly, this bacterium could also dissolve the formed CaCO3 with the increase of the Ca2+ concentration. To disclose the mechanisms of biomineralization, the genome of B. linens BS258 was further completely sequenced. Interestingly, the expression of three carbonic anhydrases was significantly up-regulated along with the increase of Ca2+ concentration and the extent of calcite dissolution. Moreover, transcriptome analyses revealed that increasing concentration of Ca2+ induced KEGG pathways including quorum sensing (QS) in B. linens BS258. Notably, most up-regulated genes related to QS were found to encode peptide/nickel ABC transporters, which suggested that nickel uptake and its associated urease stimulation were essential to boost CaCO3 biomineralization. Within the genome of B. linens BS258, there are both cadmium and lead resistance gene clusters. Therefore, the sequestration abilities of Cd2+ and Pb2+ by B. linens BS258 were checked. Consistently, Pb2+ and Cd2+ could be effectively sequestered with the precipitation of calcite by B. linens BS258. To our knowledge, this is the first study investigating the microbial CaCO3 biomineralization from both genomic and transcriptomic insights, which paves the way to disclose the relationships among bacterial metabolisms and the biomineralization.

Purification and biochemical characterization of an alkaline protease from marine bacteria Pseudoalteromonas sp. 129‐1

An extracellular alkaline protease produced by marine bacteria strain Pseudoalteromonas sp. 129‐1 was purified by ammonium sulphate precipitation, anion exchange chromatography, and gel filtration. The purity of the protease was confirmed by SDS–PAGE and molecular mass was estimated to be 35 kDa. The protease maintained considerable activity and stability at a wide temperature range of 10–60 °C and pH range of 6–11, and optimum activity was detected at temperature of 50 °C and pH of 8. Metallo‐protease inhibitor, EDTA, had no inhibitory effect on protease activity even at concentration up to 15 mM, whereas 15 mM PMSF, a common serine protease inhibitor, greatly inactivated the protease. The high stability of the protease in the presence of surfactants (SDS, Tween 80, and Triton X‐100), oxidizing agent H2O2, and commercial detergents was observed. Moreover, the protease was tolerant to most of the tested organic solvents, and saline tolerant up to 30%. Interestingly, biofilm of Pseudomonas aeruginosa PAO1 was greatly reduced by 0.01 mg ml−1 of the protease, and nearly completely abolished with the concentration of 1 mg ml−1. Collectively, the protease showed valuable feathers as an additive in laundry detergent and non‐toxic anti‐biofilm agent.

Amy63, a novel type of marine bacterial multifunctional enzyme possessing amylase, agarase and carrageenase activities

A multifunctional enzyme is one that performs multiple physiological functions, thus benefiting the organism. Characterization of multifunctional enzymes is important for researchers to understand how organisms adapt to different environmental challenges. In the present study, we report the discovery of a novel multifunctional enzyme Amy63 produced by marine bacterium Vibrio alginolyticus 63. Remarkably, Amy63 possesses amylase, agarase and carrageenase activities. Amy63 is a substrate promiscuous α-amylase, with the substrate priority order of starch, carrageenan and agar. Amy63 maintains considerable amylase, carrageenase and agarase activities and stabilities at wide temperature and pH ranges, and optimum activities are detected at temperature of 60 °C and pH of 6.0, respectively. Moreover, the heteroexpression of Amy63 dramatically enhances the ability of E. coli to degrade starch, carrageenan and agar. Motif searching shows three continuous glycosyl hydrolase 70 (GH70) family homologs existed in Amy63 encoding sequence. Combining serial deletions and phylogenetic analysis of Amy63, the GH70 homologs are proposed as the determinants of enzyme promiscuity. Notably, such enzymes exist in all kingdoms of life, thus providing an expanded perspective on studies of multifunctional enzymes. To our knowledge, this is the first report of an amylase having additional agarase and carrageenase activities.

Precise Determination, Cross-Recognition, and Functional Analysis of the Double-Strand Origins of the Rolling-Circle Replication Plasmids in Haloarchaea

The precise nick site in the double-strand origin (DSO) of pZMX201, a 1,668-bp rolling-circle replication (RCR) plasmid from the haloarchaeon Natrinema sp. CX2021, was determined by electron microscopy and DSO mapping. In this plasmid, DSO nicking occurred between residues C404 and G405 within a heptanucleotide sequence (TCTC/GGC) located in the stem region of an imperfect hairpin structure. This nick site sequence was conserved among the haloarchaeal RCR plasmids, including pNB101, suggesting that the DSO nick site might be the same for all members of this plasmid family. Interestingly, the DSOs of pZMX201 and pNB101 were found to be cross-recognized in RCR initiation and termination in a hybrid plasmid system. Mutation analysis of the DSO from pZMX201 (DSO(Z)) in this hybrid plasmid system revealed that: (i) the nucleotides in the middle of the conserved TCTCGGC sequence play more-important roles in the initiation and termination process; (ii) the left half of the hairpin structure is required for initiation but not for termination; and (iii) a 36-bp sequence containing TCTCGGC and the downstream sequence is essential and sufficient for termination. In conclusion, these haloarchaeal plasmids, with novel features that are different from the characteristics of both single-stranded DNA phages and bacterial RCR plasmids, might serve as a good model for studying the evolution of RCR replicons.