Structure of the Lysinibacillus sphaericus Tpp49Aa1 pesticidal protein elucidated from natural crystals using MHz-SFX.

The Lysinibacillus sphaericus proteins Tpp49Aa1 and Cry48Aa1 can together act as a toxin toward the mosquito Culex quinquefasciatus and have potential use in biocontrol. Given that proteins with sequence homology to the individual proteins can have activity alone against other insect species, the structure of Tpp49Aa1 was solved in order to understand this protein more fully and inform the design of improved biopesticides. Tpp49Aa1 is naturally expressed as a crystalline inclusion within the host bacterium, and MHz serial femtosecond crystallography using the novel nanofocus option at an X-ray free electron laser allowed rapid and high-quality data collection to determine the structure of Tpp49Aa1 at 1.62 Å resolution. This revealed the packing of Tpp49Aa1 within these natural nanocrystals as a homodimer with a large intermolecular interface. Complementary experiments conducted at varied pH also enabled investigation of the early structural events leading up to the dissolution of natural Tpp49Aa1 crystals-a crucial step in its mechanism of action. To better understand the cooperation between the two proteins, assays were performed on a range of different mosquito cell lines using both individual proteins and mixtures of the two. Finally, bioassays demonstrated Tpp49Aa1/Cry48Aa1 susceptibility of Anopheles stephensi, Aedes albopictus, and Culex tarsalis larvae-substantially increasing the potential use of this binary toxin in mosquito control.

Evaluation of inhibitory compounds produced by bacteria isolated from a hydrogen-producing bioreactor during the self-fermentation of wheat straw.

AIMS: The objective of this study was to evaluate the inhibitory activity of compounds secreted by bacteria isolated from a hydrogen-producing bioreactor to understand how these microorganisms interact in this community. METHODS AND RESULTS: In vitro inhibitory assays were performed using samples secreted by bacteria subject to different treatments to determine if their inhibitory effect was due to organic acids, non-proteinaceous compounds or bacteriocin-like inhibitory substances (BLIS). Bacterial isolated were suppressed 43%, 30% and 27% by neutralized, precipitated and non-neutralized cell-free supernatants, respectively. Non-hydrogen producers (non-H2 P) lactic acid bacteria (LAB) (Lactobacillus plantarum LB1, Lactobacillus pentosus LB7, Pediococcus acidilactici LB4) and hydrogen producers (H2 P) LAB (Enterococcus faecium F) were inhibited by the production of organic acids, non-proteinaceous compounds and BLIS. Meanwhile, the obligate anaerobe H2 P (Clostridium beijerinckii B) inhibited by the production of non-proteinaceous compounds and BLIS. The presence of BLIS was confirmed when proteolytic enzymes affected the inhibitory activity of secreted proteins in values ranging from 20% to 42%. The BLIS produced by L. plantarum LB1, P. acidilactici LB4, L. pentosus LB7 and E. faecium F showed molecular masses of ~11, 25, 20 and 11 kDa, respectively. CONCLUSIONS: It was demonstrated antagonistic interactions between Lactobacillus-Enterococcus and Pediococcus-Enterococcus species, generated by the secretion of organic acids, non-proteinaceous compounds and BLIS. SIGNIFICANCE AND IMPACT OF THE STUDY: We report the interactions between LAB isolated from hydrogen-producing bioreactors. These interactions might impact the dynamics of the microbial population during hydrogen generation. Our work lays a foundation for strategies that allow controlling bacteria that can affect hydrogen production.

De novo phasing with X-ray laser reveals mosquito larvicide BinAB structure.

BinAB is a naturally occurring paracrystalline larvicide distributed worldwide to combat the devastating diseases borne by mosquitoes. These crystals are composed of homologous molecules, BinA and BinB, which play distinct roles in the multi-step intoxication process, transforming from harmless, robust crystals, to soluble protoxin heterodimers, to internalized mature toxin, and finally to toxic oligomeric pores. The small size of the crystals-50 unit cells per edge, on average-has impeded structural characterization by conventional means. Here we report the structure of Lysinibacillus sphaericus BinAB solved de novo by serial-femtosecond crystallography at an X-ray free-electron laser. The structure reveals tyrosine- and carboxylate-mediated contacts acting as pH switches to release soluble protoxin in the alkaline larval midgut. An enormous heterodimeric interface appears to be responsible for anchoring BinA to receptor-bound BinB for co-internalization. Remarkably, this interface is largely composed of propeptides, suggesting that proteolytic maturation would trigger dissociation of the heterodimer and progression to pore formation.

Mitochondrial and Innate Immunity Transcriptomes from Spodoptera frugiperda Larvae Infected with the Spodoptera frugiperda Ascovirus.

Ascoviruses are large, enveloped DNA viruses that induce remarkable changes in cellular architecture during which the cell is partitioned into numerous vesicles for viral replication. Previous studies have shown that these vesicles arise from a process resembling apoptosis yet which differs after nuclear lysis in that mitochondria are not degraded but are modified by the virus, changing in size, shape, and motility. Moreover, infection does not provoke an obvious innate immune response. Thus, we used in vivo RNA sequencing to determine whether infection by the Spodoptera frugiperda ascovirus 1a (SfAV-1a) modified expression of host mitochondrial, cytoskeletal, and innate immunity genes. We show that transcripts from many mitochondrial genes were similar to those from uninfected controls, whereas others increased slightly during vesicle formation, including those for ATP6, ATP8 synthase, and NADH dehydrogenase subunits, supporting electron microscopy (EM) data that these organelles were conserved for virus replication. Transcripts from 58 of 106 cytoskeletal genes studied increased or decreased more than 2-fold postinfection. More than half coded for mitochondrial motor proteins. Similar increases occurred for innate immunity transcripts and their negative regulators, including those for Toll, melanization, and phagocytosis pathways. However, those for many antimicrobial peptides, such as moricin, increased more than 20-fold. In addition, transcripts for gloverin-3, spod_x_tox, Hdd23, and lebocin, also antimicrobial, increased more than 20-fold. Interestingly, a phenoloxidase inhibitor transcript increased 12-fold, apparently to interfere with melanization. SfAV-1a destroys most fat body cells by 7 days postinfection, so innate immunity gene transcripts apparently occur in remaining cells in this tissue and possibly other major tissues, namely, epidermis and tracheal matrix.IMPORTANCE Ascoviruses are large DNA viruses that infect insects, inducing a cellular pathology that resembles apoptosis but which differs by causing enormous cellular hypertrophy followed by cleavage of the cell into numerous viral vesicles for replication. Previous EM studies suggest that mitochondria are important for vesicle formation. Transcriptome analyses of Spodoptera frugiperda larvae infected with SfAV-1a showed that mitochondrial transcripts were similar to those from uninfected controls or increased slightly during vesicle formation, especially for ATP6, ATP8 synthase, and NADH dehydrogenase subunits. This pattern resembles that for chronic disease-inducing viruses, which conserve mitochondria, differing markedly from viruses causing short-term viral diseases, which degrade mitochondrial DNA. Though mitochondrial transcript increases were low, our results demonstrate that SfAV-1a alters host mitochondrial expression more than any other virus. Regarding innate immunity, although SfAV-1a destroys most fat body cells, certain immunity genes were highly upregulated (greater than 20-fold), suggesting that these transcripts may originate from other tissues.

Agave: a natural renewable resource with multiple applications.

Agaves are a group of succulent plants that thrive in arid or semiarid environments. Indeed, genes associated with their resilience are a potential resource for genetic engineering of other agronomically important crops grown in adverse climates. Agave is mainly used for the production of distilled (spirits) and non-distilled alcoholic beverages, including tequila, mezcal, bacanora, raicilla, and pulque, all of which have special connections to Mexican history and culture, and contribute to the Mexican economy. In recent years, there has been growing interest to maximize the use of agave plant materials for other purposes, as the bulk of their biomass pre- and post-production is wasted. In traditional practice, during the passage from fields to factories, only agave cores are used, and the leaves and bagasse are not always harnessed. To place this in perspective, during the period from 2010 to 2019, 2674.7 × 106 L of tequila was produced in Mexico, which required 9 607 400 tons of agave cores. This generated approximately the same amount of leaves and 3 842 960 tons of bagasse. The economic base of agave plants can be expanded if expended biomass could be transformed into products that are useful for applications in food, forage, ensilage, agriculture, medicine, energy, environment, textiles, cosmetics, and esthetics. This review focuses on the current utility of agave plants, as well as our perspective for future studies and uses of this formidable plant. © 2020 Society of Chemical Industry.

Class I defensins (BraDef) from broccoli (Brassica oleracea var. italica) seeds and their antimicrobial activity.

The objective of this study was to determine whether seeds of Brassica oleracea var. italica (i.e. broccoli, an edible plant) produce defensins that inhibit phytopathogenic fungi and pathogenic bacteria of clinical significance. Crude extracts obtained from broccoli seeds were fractioned by molecular exclusion techniques and analyzed by liquid chromatography-high-resolution mass spectrometry. Two peptides were identified, BraDef1 (10.68 kDa) and BraDef2 (9.9 kDa), which were categorized as Class I defensins based on (a) their primary structure, (b) the presence of four putative cysteine disulfide bridges, and (c) molecular modeling predictions. BraDef1 and BraDef2 show identities of, respectively, 98 and 71%, and 67 and 85%, with defensins from Brassica napus and Arabidopsis thaliana. BraDef (BraDef1 + BraDef2) disrupted membranes of Colletotrichum gloeosporioides and Alternaria alternata and also reduced hyphal growth of C. gloeosporioides by ~ 56% after 120 h of incubation. Pathogenic bacteria (Bacillus cereus 183, Listeria monocytogenes, Salmonella typhimurium, Pseudomonas aeruginosa, and Vibrio parahaemolitycus) were susceptible to BraDef, but probiotic bacteria such as Bifidobacterium animalis, Lactobacillus acidophilus, and Lactobacillus casei were not inhibited. To our knowledge, this is the first report of defensins present in seeds of B. oleracea var. italica (i.e. edible broccoli). Our findings suggest an applied value for BraDef1/BraDef2 in controlling phytopathogenic fungi and pathogenic bacteria of clinical significance.

Regulator ThnR and the ThnDE ABC transporter proteins confer autoimmunity to thurincin H in Bacillus thuringiensis.

The structural gene that encodes thurincin H, a bacteriocin produced by Bacillus thuringiensis, is harboured in a genetic cluster (thnP, E, D, R, A1, A2, A3, B, T, I) that controls its synthesis, modification, secretion and autoimmunity. The specific genes in the cassette that confer immunity in B. thuringiensis to thurincin H are unknown. To identify these immunity determinants, we generated constructs that were used to transform a natural thurincin H-sensitive B. thuringiensis strain (i.e. Btk 404), and resistance or susceptibility to the bacteriocin in resultant recombinants was evaluated. When Btk 404/pHT3101-ThnARDEP and Btk 404/pHT3101-ThnABTI were exposed to thurincin H, immunity was demonstrated by the former only, indicating that ThnI does not play a role in resistance to the bacteriocin as previously proposed. Furthermore, we generated different sub-cassettes under the control of divergent promoters pThnR and pThur of the thurincin H locus, and pChi, and using the green fluorescent protein gene as reporter, which demonstrated that all promoters were recognised by ThnR, except pChi. We show for the first time that the small operon composed of thnR, thnD and thnE is required for immunity of B. thuringiensis to thurincin H, and thnI is not necessary for this response.

Co-synthesis of kenyacin 404 and heterologous thurincin H enhances the antibacterial activity of Bacillus thuringiensis.

OBJECTIVES: To develop a recombinant strain of Bacillus thuringiensis that synthesizes two bacteriocins that enhance the antibacterial potency of the strain and that could have applied clinical and industrial value. RESULTS: We cloned the thurincin H cluster into the pHT3101 vector by assembling two genetic cassettes harboring genes for the synthesis, modification, immunity and transport of thurincin H. This construct was used to transform a thurincin H-sensitive strain of B. thuringiensis that synthesizes the kenyacin 404 to generate the recombinant Btk 404/pThurH which was immune to thurincin H and produces bacteriocins of approximately 3 kDa. A significant increase in the inhibitory activity, respectively, ~ 40 and 300%, was observed when compared with parental Btm 269 and Btk 404. Btk 404/pThurH showed increased activity against ten Gram-positive bacteria, including B. cereus, Listeria monocytogenes and B. pseudomycoides, and the Gram-negative bacterium, Sphingobacterium cabi. However, an antagonistic effect against Vibrio parahaemolyticus, relative to native strains, was observed. CONCLUSIONS: We have generated a recombinant strain of B. thuringiensis that co-synthesizes two bacteriocins (kenyacin 404, thurincin H) with improved inhibitory activity, when compared with parental strains. To our knowledge, this is the first study that shows that B. thuringiensis could be manipulated to produce two bacteriocins, one being of heterologous origin, that enhance the antibacterial activity of the recombinant strain.

ICTV Virus Taxonomy Profile: Ascoviridae.

The family Ascoviridae includes viruses with circular dsDNA genomes of 100-200 kbp characterized by oblong enveloped virions of 200-400 nm in length. Ascoviruses mainly infect lepidopteran larvae and are mechanically transmitted by parasitoid wasps in which they may also replicate. Most known members belong to the genus Ascovirus, except one virus, that of the genus Toursvirus, which replicates in both its lepidopteran and parasitoid vector hosts. Ascoviruses cause high mortality among economically important insect pests, thereby controlling insect populations. This is a summary of the current International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Ascoviridae, which is available at www.ictv.global/report/ascoviridae.

The thnR gene is a negative transcription regulator of the thurincin H genetic cassette in Bacillus thuringiensis subsp. morrisoni.

Thurincin H is a bacteriocin synthesized by some strains of Bacillus thuringiensis. In this study, the thurincin H genetic cassette, which contains ten genes, from a Mexican strain of B. thuringiensis subsp. morrisoni (Btm) was cloned and sequenced. To study the function of the thnR gene component in the cassette, we generated various constructs with or without thnR for expression in Btm. All transformants harboring thnR in recombinant plasmids showed a decrease of ~15 to ~90 % in inhibitory activity against the target strain, Bacillus cereus 183. Importantly, a ~90 % reduction in inhibition occurred with Btm harboring a construct containing thnR alone, suggesting that ThnR, indeed, functions as a negative transcription regulator of the thurincin H cassette. Based on sequence homology, ThnR was classified as a member of the YtrA subfamily of the GntR superfamily of transcriptional regulators.

Recombinant Bacillus thuringiensis subsp. kurstaki HD73 strain that synthesizes Cry1Ac and chimeric ChiA74∆sp chitinase inclusions.

In this study, the endochitinase chiA74 gene lacking its secretion signal peptide sequence (chiA74∆sp) was fused in frame with the sequence coding for the C-terminal crystallization domain and transcription terminator of cry1Ac. The chimeric gene was expressed under the strong pcytA-p/STAB-SD promoter system in an acrystalliferous Cry-B strain of Bacillus thuringiensis and B. thuringiensis subsp. kurstaki HD73. We showed that the chimeric ChiA74∆sp produced amorphous inclusions in both Cry-B and HD73. In addition to the amorphous inclusions putatively composed of the chimera, bipyramidal Cry1Ac crystals, smaller than the wild-type crystal, were observed in recombinant HD73, and chitinase activity was remarkably higher (75-fold) in this strain when compared with parental HD73. Moreover, we observed that lyophilized samples of a mixture containing Cry1Ac, amorphous inclusions, and spores maintained chitinase activity. Amorphous inclusions could not be separated from Cry1Ac crystals by sucrose gradient centrifugation. Interestingly, the chitinase activity of purified Cry1Ac/amorphous inclusions was 51-fold higher compared to purified Cry1Ac inclusions of parental HD73, indicating that the increased enzymatic activity was due primarily to the presence of the atypical amorphous component. The possibility that the chimera is occluded with the Cry1Ac crystal, thereby contributing to the increased endochitinolytic activity, cannot be excluded. Finally, bioassays against larvae of Spodoptera frugiperda with spore/crystals of HD73 or spore-crystal ChiA74∆sp chimeric inclusions of recombinant HD73 strain showed LC50s of 396.86 and 290.25 ng/cm2, respectively. Our study suggests a possible practical application of the chimera in formulations of B. thuringiensis-based lepidopteran larvicides.

Contributions of 5'-UTR and 3'-UTR cis elements to Cyt1Aa synthesis in Bacillus thuringiensis subsp. israelensis.

The biopesticide used most effectively to control mosquito and blackfly vectors of human diseases worldwide is Bacillus thuringiensis subsp. israelensis. The high efficacy of this bacterium is due to synergistic interactions among four protein entomotoxins assembled individually into a single parasporal body (PB) during sporulation. Cyt1Aa, the primary synergist, is the most abundant toxin, comprising approximately 55% of the PB's mass. The other proteins are Cry11Aa at ∼35%, and Cry4Aa and Cry4Ba, which together account for the remaining ∼10%. The molecular genetic basis for the comparatively large amount of Cyt1Aa synthesized is unknown. Here, in addition to the known strong BtI (σE) and BtII (σK) promoters, we demonstrate a third promoter (BtIII) that has high identity to the σE promoter of Bacillus subtilis, contributes to the large amount of Cyt1Aa synthesized. We also show that a cyt1Aa-BtIII construct was not functional in a σE-deficient strain of B. subtilis. Comparison of transcription levels and protein profiles for recombinant strains containing different combinations of BtI, BtII and BtIII, or each promoter alone, showed that BtIII is active throughout sporulation. We further demonstrate that a stable stem-loop in the 3'-untranslated region (3'-UTR, predicted ΔG=-27.6) contributes to the high level of Cyt1Aa synthesized.

Bacteriocinogenic Bacteria Isolated from Raw Goat Milk and Goat Cheese Produced in the Center of México.

Currently, there are few reports on the isolation of microorganisms from goat milk and goat cheese that have antibacterial activity. In particular, there are no reports on the isolation of microorganisms with antibacterial activity from these products in central Mexico. Our objective was to isolate bacteria, from goat products, that synthesized antimicrobial peptides with activity against a variety of clinically significant bacteria. We isolated and identified Lactobacillus rhamnosus, L. plantarum, L. pentosus, L. helveticus and Enterococcus faecium from goat cheese, and Aquabacterium fontiphilum, Methylibium petroleiphilum, Piscinobacter aquaticus and Staphylococcus xylosus from goat milk. These bacteria isolated from goat cheese were able to inhibit Staphylococcus aureus, Bacillus cereus, Escherichia coli, Listeria monocytogenes, L. inoccua, Pseudomona aeruginosa, Shigella flexneri, Serratia marcescens, Enterobacter cloacae and Klebsiella pneumoniae. In addition, bacteria from goat milk showed inhibitory activity against B. cereus, L. lactis, E. coli, S. flexneri, E. cloacae and K. pneumonia; S. aureus, L. innocua, S. agalactiae and S. marcescens. The bacteriocins produced by these isolates were shown to be acid stable (pH 2-6) and thermotolerant (up to 100 °C), but were susceptible to proteinases. When screened by PCR for the presence of nisin, pediocin and enterocin A genes, none was found in isolates recovered from goat milk, and only the enterocin A gene was found in isolates from goat cheese.

Heterologous expression, purification and biochemical characterization of endochitinase ChiA74 from Bacillus thuringiensis.

ChiA74 is a secreted endochitinase produced by Bacillus thuringiensis. Previously we have partially characterized the physical parameters that affect enzymatic activity of ChiA74 in crude preparations of bacterial secretomes. In the present study, we cloned the chiA74 open reading frame (ORF) lacking the 5' sequence coding for its secretion signal peptide (chiA74Δsp) into a cold shock expression vector (pColdI) for production of the enzyme in Escherichia coli BL21-Rosetta 2. As a result, the N-terminal end of ChiA74Δsp ORF was fused to an artificial sequence of 28 amino acid, including a 6× histidine tag for purification of recombinant 6×His tagged-ChiA74Δsp (rChiA74, ∼74kDa). Along with a protein of ∼74kDa, we co-purified its ∼55kDa processed form which was confirmed by Western blot analysis. Optimal endochitinase activity of purified rChiA74 occurred at pH 7 and 40°C. Most divalent cations (e.g. Ba(+2), Ca(+2), Mn(+2), Mg(+2), Zn(+2) and Cu(+2)) at concentration of 10mM reduced chitinase activity by ∼30%, and Hg(+2) (10mM) drastically inhibited ChiA74 activity by ∼75-100%. The Vmax, Km and kcat for rChiA74 were 0.11±0.01nmol/min, 2.15µM±0.45 and 3.81s(-1), respectively, using 4-MU-GlcNAc3 as substrate. Using purified rChiA74 and colloidal chitin as substrate, chitin-derived oligosaccharides with degree of polymerization of 2 and 1 were detected.

Genome sequence of a crustacean iridovirus, IIV31, isolated from the pill bug, Armadillidium vulgare.

Members of the family Iridoviridae are animal viruses that infect only invertebrates and poikilothermic vertebrates. The invertebrate iridovirus 31 (IIV31) was originally isolated from adult pill bugs, Armadillidium vulgare (class Crustacea, order Isopoda, suborder Oniscidea), found in southern California on the campus of the University of California, Riverside, USA. IIV31 virions are icosahedral, have a diameter of about 135 nm, and contain a dsDNA genome 220.222 kbp in length, with 35.09 mol % G+C content and 203 ORFs. Here, we describe the complete genome sequence of this virus and its annotation. This is the eighth genome sequence of an IIV reported.

Bacillus thuringiensis subsp. kurstaki HD1 as a factory to synthesize alkali-labile ChiA74∆sp chitinase inclusions, Cry crystals and spores for applied use.

BACKGROUND: The endochitinase ChiA74 is a soluble secreted enzyme produced by Bacillus thuringiensis that synergizes the entomotoxigenecity of Cry proteins that accumulate as intracellular crystalline inclusion during sporulation. The purpose of this study was to produce alkaline-soluble ChiA74∆sp inclusions in B. thuringiensis, and to determine its effect on Cry crystal production, sporulation and toxicity to an important agronomical insect, Manduca sexta. To this end we deleted the secretion signal peptide-coding sequence of chiA74 (i.e. chiA74∆sp) and expressed it under its native promoter (pEHchiA74∆sp) or strong chimeric sporulation-dependent cytA-p/STAB-SD promoter (pEBchiA74∆sp) in Escherichia coli, acrystalliferous B. thuringiensis (4Q7) and B. thuringiensis HD1. RESULTS: Based on mRNA analyses, up to ~9-fold increase in expression of chiA74∆sp was observed using the cytA-p/STAB-SD promoter. ChiA74∆sp (~70 kDa) formed intracellular inclusions that frequently accumulated at the poles of cells. ChiA74∆sp inclusions were dissolved in alkali and reducing conditions, similar to Cry crystals, and retained its activity in a wide range of pH (5 to 9), but showed a drastic reduction (~70%) at pH 10. Chitinase activity of E. coli-pEHchiA74∆sp was ~150 mU/mL, and in E. coli-pEBchiA74∆sp, 250 mU/mL. 4Q7-pEBchiA74∆sp and 4Q7-pEHchiA74∆sp had activities of ~127 mU/mL and ~41 mU/mL, respectively. The endochitinase activity in HD1-pEBchiA74∆sp increased 42x when compared to parental HD1 strain. HD1-pEBchiA74∆sp and HD1 harbored typical bipyramidal Cry inclusions, but crystals in the recombinant were ~30% smaller. Additionally, a 3x increase in the number of viable spores was observed in cultures of the recombinant strain when compared to HD1. Bioassays against first instar larvae of M. sexta with spore-crystals of HD1 or spore-crystal-ChiA74∆sp inclusions of HD1-pEBchiA74∆sp showed LC50s of 67.30 ng/cm² and 41.45 ng/cm², respectively. CONCLUSIONS: Alkali-labile ChiA74∆sp inclusion bodies can be synthesized in E. coli and B. thuringiensis strains. We demonstrated for the first time the applied utility of synthesis of ChiA74∆sp inclusions, Cry crystals and spores in the same sporangium of HD1, a strain used successfully worldwide to control economically significant lepidopteran pests of agriculture. Our findings will allow to us develop strategies to modify expression of ChiA74∆sp while maximizing Cry crystal synthesis in commercial strains of B. thuringiensis.

Complete genome sequence of invertebrate iridovirus IIV-25 isolated from a blackfly larva.

Members of the family Iridoviridae are animal viruses that infect only invertebrates and poikilothermic vertebrates. Invertebrate iridovirus 25 (IIV-25) was originally isolated from the larva of a blackfly (Simulium spp., order Diptera) found in the Ystwyth river near Aberystwyth, Wales. IIV-25 virions are icosahedral, have a diameter of ~130 nm, and contain a dsDNA genome of 204.8 kbp, with a G+C content of 30.32 %, that codes for 177 proteins. Here, we describe the complete genome sequence of this virus and its annotation. This is the fifth genome sequence of an invertebrate iridovirus reported.

Complete genome sequence of invertebrate iridovirus IIV30 isolated from the corn earworm, Helicoverpa zea.

Members of the family Iridoviridae are animal viruses that infect only invertebrates and poikilothermic vertebrates. The invertebrate iridovirus 30 (IIV30) was originally isolated from a larva of the corn earworm, Helicoverpa zea (order lepidoptera, Family Noctuidae) in western Australia. The IIV30 virions are icosahedral, have a diameter of about 130nm, and contain a dsDNA genome of 198.5kbp with 28.11% in GC content and 177 coding sequences. Here we describe its complete genome sequence and annotate the genes for which we could assign a putative function. This is the sixth genome sequence of an invertebrate iridovirus reported.

The chitin-binding domain of Bacillus thuringiensis ChiA74 inhibits gram-negative bacterial and fungal pathogens of humans and plants.

The chitinase ChiA74 is synthesized by Bacillus thuringiensis and possesses a modular organization composed of four domains. In the C-terminal of the enzyme is located the chitin-binding domain (CBD), which has not been isolated as a single unit or characterized. Here, we aimed to isolate the ChiA74's CBD as a single unit, determine the binding properties, and evaluate its antimicrobial and hemolytic activities. We cloned the ChiA74's CBD and expressed it in Escherichia coli BL21. The single domain was purified, analyzed by SDS-PAGE, and characterized. The recombinant CBD (rCBD) showed a molecular mass of ∼14 kDa and binds strongly to α-chitin, with Kd and Bmax of ∼4.7 ± 0.9 µM and 1.5 ± 0.1 µmoles/g chitin, respectively. Besides, the binding potential (Bmax/Kd) was stronger for α-chitin (∼0.31) than microcrystalline cellulose (∼0.19). It was also shown that the purified rCBD inhibited the growth of the clinically relevant Gram-negative bacteria (GNB) Vibrio cholerae, and V. parahemolyticus CVP2 with minimum inhibitory concentrations (MICs) of 121 ± 9.9 and 138 ± 3.2 µg/mL, respectively, and of one of the most common GNB plant pathogens, Pseudomonas syringae with a MIC of 230 ± 13.8 µg/mL. In addition, the rCBD possessed antifungal activity inhibiting the conidia germination of Fusarium oxysporum (MIC = 192 ± 37.5 µg/mL) and lacked hemolytic and agglutination activities against human erythrocytes. The significance of this work lies in the fact that data provided here show for the first time that ChiA74's CBD from B. thuringiensis has antimicrobial activity, suggesting its potential use against significant pathogenic microorganisms. Future works will be focused on testing the inhibitory effect against other pathogenic microorganisms and elucidating the mechanism of action.

Complete genome sequence of invertebrate iridescent virus 22 isolated from a blackfly larva.

Members of the family Iridoviridae are animal viruses that infect only invertebrates and poikilothermic vertebrates. Invertebrate iridescent virus 22 (IIV-22) was originally isolated from the larva of a blackfly (Simulium sp., order Diptera) found in the Ystwyth river, near Aberystwyth, Wales, UK. IIV-22 virions are icosahedral, with a diameter of about 130 nm and contain a dsDNA genome that is 197.7 kb in length, has a G+C content of 28.05 mol% and contains 167 coding sequences. Here, we describe the complete genome sequence of this virus and its annotation. This is the fourth genome sequence of an invertebrate iridovirus to be reported.