Yuan Zhiminggot his PhD on microbiology and biotechnology at Sun Yat-sen University, and then trained and worked in France, Denmark, and United States for several years. He has been working as a Professor in Wuhan Institute of Virology, the President of Wuhan Branch, Chinese Academy of Sciences since year 2000. As a principal investigator, his research group works on the diagnosis and drug discovery of aborviruses, genomics, insecticidal proteins and their mode of action, genetically modification of entomopathogenic bacteria (Bacillus spp), as well as the production, standardization and application of bio-pesticide and other microbial agents. Since 2003, he was appointed the Chairman of Institutional Biosafety Committee, member of National Laboratory Biosafety Committee, advisor of different national technical committees on biosafety management, general manager of National High-Level Biosafety Laboratory in Wuhan. He also works as a group leader on Sino-French biosafety legislation cooperation. In addition, he was elected as President of Hubei Society for Microbiology in 2008. Along providing expertise and consultative services to both private companies and government institutions on matters of biotechnology and biosafety, he has given many lectures to national and international audiences. He published more than 150 scientific papers on Bacillus spp and aborviruses during his career
The Biological Control of Arbovirus Vectors research group is mainly engaged in researches on the detection of aborvirus and the biological control of mosquito vectors. The research interests of this group include: aborvirus diagnosis technology development and the interaction of aborvirus and mosquitoes, entomopathogenic bacteria and insecticidal gene resources, microbial genomics and comparative genomics, insecticidal proteins and their mode of action, construction of engineering strains with higher toxicity and wider active spectrum, production, standardization and the application of bio-pesticide and other microbial.
The research on agricultural microbiology and its application was strategically determined to be one of the four main aspects when the institute was founded in 1956. Leaded by Prof. Shaobing Luo and Yongmei Zhang, the research on entomopathogenic bacteria B. thuringiensis and B. sphaericus and their application for agricultural pest and medical-importance vector control started in mid-60s and late 70s in last century, respectively. The great achievements on active strain isolation, insect pathogenesis, fermentation, formulation processing, standardization and application were well acknowledged in national and international levels.Succeeded by Prof. Zhiming Yuan in 1998, the group is continuously working on theproduction, standardization and application of bio-pesticides based on the two bacteria, and meanwhile, great effort is focused on mosquitocidal bacterial resources and their diversity, insecticidal proteins and their mode of action, comparative genomics and the functional genes, genetic improvement of the bacteria and their risk assessment.
A formulation (JianbaoÒ) based on a high toxic B. sphaericuslocal isolateC3-41which was developed and has been successfully used for mosquito control for 20 years in more than 80 cities in China. Four types of binary toxins have been identified, and their function/structure and the toxin determinants have been elucidated. Besides, a series of genetically modified bacteria with broader active spectrum and higher toxicities have been constructed through the co-expression and domain swapping of binary toxin and other crystal toxins from B. thuringiensis. The resistance mechanism and genetic basis of target mosquitoes to B. sphaericus was explored and the synergism of different mosquitocidal toxins was used for resistance management. Furthermore, the whole genome of B. sphaericus C3-41 was compared with closely related Bacillus spp. and a new cluster (lysinibacillus) has been suggested, which was further supported by a newly developed multilocus sequence typing (MLST). In addition, the genetic mechanism that B. sphaericus fall to use sugar as the sole carbon resource has been explored by molecular biology technology, genome comparison and metabonomics, anovel large plasmid (178-kb) pBsph was identified and the unique plasmid replication/segregation system of pBsph were analyzed, four protein associated with spore germination, a collagen-like glycoprotein required for the formation of filamentous structure of the internal exosporium, a gene for S-layer protein production were identified, the data is of importance for further understanding the elementary biology of this unique bacterium.
Since 2003, the group has extended its researches to the monitoring of arboviruses in mosquitoes and their transmission risk assessment in integrated mosquito control program.
The main objectives of our 10 ongoing projects are to study the resistance and cross resistance in mosquitoes against chemical pesticides and B. sphaericus, to elucidate the sugar metabolism and regulation system, to identify the genes responsible for spore germination and exosporium formation, with the aim to genetic modification of bacteria for Aedes and resistant Culex control, and to develop the reliable techniques for arboviruses and other biorisk 4 viral pathogens detection, to monitor the occurrence of arboviruses in mosquitoes for transmission risk assessment and mosquito control strategy.
Our group has been working on mosquito control with biological agents and on B. sphaericus and B. thuringiensis sp. israelensis for 30 years. The mosquitocidal agents based on these two bacteria have been successfully used for mosquito control in China. However, the appearance of resistance in targets and narrow active spectrum of B. sphaericus hinder its further effective application in mosquito control program. Therefore, to isolate and genetically construct the better mosquito killer, to develop new applicable formulation and application technology will be our long-tern research goals, with an aim to integrate our mosquito control program in national infectious disease monitoring and control program. Besides, we are interested in the genetic diversity of B. sphaericus and evolution of mosquitocidal toxins, unique energy and nitrogen metabolism and exosporium formation, with the aim to well understand this bacterium, to answer the questions: where do the toxin genes come from? Why the toxic strains have high genetic homology, while non-toxic strains not? Why this bacterium could not utilize sugar for growth, how it evolved?
WP1. Application and genetic engineering of bio-pesticide
The research group isolated and collected a large amount of mosquitocidal B. sphaericusstrains from worldwide, of which the high toxicC3-41 isolated from a mosquito breeding-site in China was developed as liquid formulation (JianbaoÒ) for commercial application. This local pesticide was used for mosquito breeding-site control in about 80 cities all over China and about 10,000 hectares of mosquito breeding-sites were treated each year. In certain southern cities, such as Shengzhen, Dongguan and Fushan, C3-41 formulation has been continually used for more than 20 years and the mosquito population has been successfully controlled.
The resistances of target C. quinquefasciatus to different B. sphaericus strains under long-continuous selection pressures were investigated in field and in laboratory. It was found that Mtx1 has a different mode of action from the binary toxin, and that it could be an alternative toxin to delay or overcome resistance development to binary toxin in C. quinquefasciatus. In addition, it was foundthatchitinase ChiAc can synergize the toxicity of the binary toxin against resistant mosquitoes and thus may be useful in managing mosquito resistance to B. sphaericus (Cai et al., 2007). Moreover, previous works showed that the resistance to B. sphaericus in Culex colonies was associated with the absence of the 60-kDa binary toxin receptor (Cpm1/Cqm1), an alpha-glucosidase in the larval midgut microvilli. Recently, we found that single nucleotide deletion of cqm1 gene results in the development of resistance to B.sphaericus in C. quinquefasciatus (Guo et al., 2013).
The mosquitocidal genes, including bin1-4, mtx1，mtx2、cry, cyt and vip and some other genes related with toxicity (e.g. chiAc), were cloned separately or in different combinations, the expression and synergism of these genes were analyzed. Furthermore, through conjugation, DNA shuffling, toxin domain exchange and normal molecular biology technology, different engineering mosquitocidal strains were constructed. It was observed that Cry4A，Cry11A, Cry11B, Cyt1A, or ChiAa could synergized with Bin toxins, as the activity against mosquito of the recombinant B. sphaericus strains carrying the corresponding toxin gene combinations have been improved, and the insecticidal spectrum has been broadened (Zhang et al, 2006, Cai et al., 2007）.
WP2. Genetic population and diversity of B. sphaericus
For the genetic population and diversity studies of B. sphaericus,the whole genome of B. sphaericus C3-41 was sequenced and compared with other closely related Bacillus species, the results indicated that B. sphaericus is most similar to that of Bacillus sp. strain NRRL B-14905, a marine species that, like B. sphaericus, is unable to metabolize polysaccharides. It was found that there are two copies of binary toxin gene (binA and binB), both in chromosome and in a newly identified 178 kb plasmid pBsph, in B. sphaericus C3-41 (Hu et al., 2008).
Bacteria Plasmids are mainly associated with important physiological functions. However, there is few research on large plasmid of B. sphaericus. We found that there is a 178 kb plasmid, named pBsph, in B. sphaericus C3-41, which carries binary toxin gene (binA and binB) and is associated with the mosquitocidal toxicity. The minimal replicon of pBsph has been identified by molecular cloning which comprises of TubR-Bs, TubZ-Bs, 167 bp and 267 bp up- and down-stream DNA fragment,the three corresponding typical components NTPase, DNA binding protein and cis element, and their interplay mode were characterized, and the polymerizaiton activity of TubZ-Bs in vivo and in vitro was visualized by electron and fluorescence microscopy (Ge, submitted). The results will have a general value for the classification, evolution and biological function of bacteria plasmids.
In order to characterize the isolates and explore the population structure of B. sphaericus, based on the complete genome sequence,the genetic diversity of 35 B. sphaericus strains was analyzed by a newly developed multilocus sequence typing (MLST) scheme, toxin gene pool, and the potential insecticidal activity among different B. sphaericuswas surveyed as well. The results demonstrated that strains assigned to the same sequence type (ST) had the same occurrence of toxin genes. Further sequence analysis revealed that toxic strains presented a nearly clonal population structure, whereas nontoxic strains had a high level of heterogeneity and were significantly distinct from toxic strains. This study will have theoretical and practical value for further study of the diversity and phylogenic relationship of B. sphaericus toxins and for further application of B. sphaericus as biopesticide (Ge et al., 2011).
WP3. Metabolism and gene regulation
Except N-acetylglucosamine, all B. sphaericus could not metabolize any sugars as carbon resource, thus hindering the further application of B. sphaericus as larvicidal agent because of high production cost of formulations. Unfortunately, little was known on this special sugar metabolism in B. sphaericus. The comparison between B. sphaericus and Bacillus sp. strain NRRL B-14905 confirmed both strains hold glucokinase (glk) and phosphofructokinase (pfk), butlack of phosphate glucose isomerase (pgi) genes, which encode key enzymesin the EMP pathway (Hu et al., 2008). The expression and activity of Glk and Pfkwas observed in B. sphaericus and heterogenous Escherichia coli (Han et al., 2007).In addition, CcpA, a pleiotropic transcriptional regulator that acts as the key factor in the regulation of carbon and nitrogen metabolism, was knocked out by homologous recombinant. By comparing the metabonome of the wild and the ccpA mutant, the NMR and the rebuilt network map suggests CcpA regulates and promotes sugar and amino acid metabolism of B. sphaericus (Li et al., 2013). Therefore, a pgi gene fromB. cereus ATCC 33018 was cloned and expressed in B. sphaericus 2297 and 2362 under the control of binary toxin promoter and cry3A promoter, separately. However, the EMP pathway of B. sphaericus could not be totally restored (Han unpublished). Ortholog analysis indicated that some phosphotransferase system (PTS)-catalyzed transports and ABC sugar transporters functioning in sugar binding and transport are present in the other six compared species but absent from B. sphaericus C3-41 and Bacillus sp. strain NRRL B-14905, both of which may, therefore, be defective in the transport of sugars (Hu et al., 2008). Preliminary nuclear magnetic resonance (NMR) results indicated that the cell extracts of the recombinant B. sphaericus strains which express heterogenous pgi gene could convert glucose into acid in vitro (Han et al., unpublished). Thus, the lack of key enzyme(s) and sugar transport systems in the two bacteria may be the main reasons for the inability of polysaccharide utilization.Moreover, the abundance of proteolytic enzymes and transport systems was observed in B. sphaericus C3-41 and Bacillus sp. strain NRRL B-14905, which may endow these bacteria with exclusive metabolic pathways for a wide variety of organic compounds and amino acids. Our findings provide additional data to further elucidate the specific metabolic pathway and for genetic-improvement of B. sphaericus for further mosquito control.
WP4. Sporulation and germination
Suffering nutrient limitation, B. sphaericus undergoes sporulation via a series of well defined morphological stages. However, only a small number of genes involved in sporulation have been identified. To identify genes associated with sporulation, and to understand the relationship between sporulation and crystal protein synthesis, a random mariner-based transposon insertion mutant library of B. sphaericus strain 2297 was constructed and seven sporulation-defective mutants were selected. Sequencing of the DNA flanking of the transposon insertion identified several genes involved in sporulation. The morphologies of mutants were determined by electron microscopy and synthesis of crystal proteins was analyzed by SDS-PAGE and Western blot. Four mutants blocked at early stages of sporulation failed to produce crystal proteins and had lower larvicidal activity. However, the other three mutants were blocked at later stages and could form crystal proteins, and the larvicidal activity was similar to wild type. These results indicated that crystal protein synthesis in B. sphaericus is dependent on sporulation initiation. Furthermore, the organization and composition of bacterial exosporium have been analyzed. Bioinformatic analyses revealed that Bsph_0411, encoding a protein of 1,142 amino acid residues, shared 52% amino acid identity with BclA from Bacillus cereus and contained 327 triplet repeats including 117 GAT and 85 GVT that is typical of a collagen-like region. Immunogold labeling assays showed that Bsph_0411 was a glycoprotein and located exclusively on the surface of the exosporium. Thin-section electron microscopy revealed that the null mutation of Bsph_0411 resulted in the absence of the filamentous structure in the internal exosporium, indicating an involvement of Bsph_0411 in the formation of exosporium. In vivo visualization of the Bsph_0411-GFP fusion protein in B. sphaericus revealed a dynamic pattern of fluorescence that follows the formation sites of the exosporium around the forespore, and the fluorescence was mainly localized on the mother cell proximal pole of the mature spores. Further studies revealed that the deletion of Bsph_0411 led to significant decreases in heat resistance of the spores, suggested a role in the protection of the spores. The results demonstrated that the glycoprotein Bsph_0411 participated in the formation of the B. sphaericus exosporium and contributed to the heat resistance of the spores, and the information is of importance for further understanding the mechanism of the protection of the exosporium for the Bin toxins and spores.