d-Tartrate utilization correlates with phylogenetic subclade in Pseudomonas cichorii.

Pseudomonas cichorii is divided into two subclades based on the 16S ribosomal RNA gene sequence and core genome multilocus sequence typing. It was shown that subclade 2 strains utilize d-tartrate as a sole carbon source, whereas subclade 1 strains do not. Draft genome sequencing was performed with P. cichorii strains to identify d-tartrate utilization genes. By genome comparative and homology search studies, an ∼7.1-kb region was identified to be involved in d-tartrate utilization. The region is subclade 2 specific, and contains tarD and dctA genes, which encode a putative enzyme and transporter of d-tartrate, respectively. When the region was introduced into subclade 1 strains, the transformants were able to utilize d-tartrate. Partial fragments of tarD and dctA were amplified from all subclade 2 strains tested in this study by PCR using gene-specific primers, but not from subclade 1 strains. This is the first report on the genetic analysis of biochemical characteristics corresponding to a specific phylogenetic group in P. cichorii.

Complete Genome Sequences of Ralstonia solanacearum Strains Isolated from Zingiberaceae Plants in Japan.

Here, we report the complete genome sequences of three Ralstonia solanacearum strains isolated from Zingiberaceae plants in Japan. The total genome sizes of these strains ranged from 5.87 to 6.05 Mb. Strains MAFF 211472, MAFF 211479, and MAFF 311693 each carried one chromosome and one megaplasmid. MAFF 311693 contained an additional 71.9-kb plasmid.

Identification and characterization of three microsporidian genera concurrently infecting a silkworm, Bombyx mori, in Brazil.

Microsporidia are important entomopathogens known for infecting insects such as the silkworm (Bombyx mori) thus impairing global silk production. This study aimed to identify and characterize the microsporidia isolated from a diseased larva of silkworm, collected from a sericulture farm in southern Brazil. Identification was performed by phylogenetic analysis of the nucleotide sequences of the SSU rRNA genes. Characterization was performed by analyzing spore sizes, tissue tropism, internal and external symptoms, and pathogenicity against B. mori. Microsporidia belonging to three different genera were identified, namely, Endoreticulatus, Nosema and Tubulinosema. After inoculation of the mixed spores of the microsporidian isolates into B. mori larvae, a high prevalence of Tubulinosema spp. was observed. This isolate showed high prevalence on the silk glands and a late mortality, initially of around 10% until the 20th day post-inoculation but reaching 91.5% upon pupation. Therefore, we demonstrated that Tubulinosema spp. causes chronic infection with slow pathogenicity. We identified for the first time three different microsporidians concurrently infecting B. mori in Brazil. Tubulinosema is of particular interest because of its potential threat to silk production; it affects the formation of silk glands in B. mori while not presenting distinguishable external symptoms or causing the immediate death of these insects. Further studies focusing on this species, mainly regarding its life cycle within the host and the sublethal effects of surviving individuals, demonstrate the importance of describing it as a new species and improving the characterization of the disease in order to prevent its spread.

Characterization and Genome Structure of Virulent Phage EspM4VN to Control Enterobacter sp. M4 Isolated From Plant Soft Rot.

Enterobacter sp. M4 and other bacterial strains were isolated from plant soft rot disease. Virulent phages such as EspM4VN isolated from soil are trending biological controls for plant disease. This phage has an icosahedral head (100 nm in diameter), a neck, and a contractile sheath (100 nm long and 18 nm wide). It belongs to the Ackermannviridae family and resembles Shigella phage Ag3 and Dickeya phages JA15 and XF4. We report herein that EspM4VN was stable from 10°C to 50°C and pH 4 to 10 but deactivated at 70°C and pH 3 and 12. This phage formed clear plaques only on Enterobacter sp. M4 among tested bacterial strains. A one-step growth curve showed that the latent phase was 20 min, rise period was 10 min, and an average of 122 phage particles were released from each absorbed cell. We found the phage's genome size was 160,766 bp and that it annotated 219 open reading frames. The genome organization of EspM4VN has high similarity with the Salmonella phage SKML-39; Dickeya phages Coodle, PP35, JA15, and Limestone; and Shigella phage Ag3. The phage EspM4VN has five tRNA species, four tail-spike proteins, and a thymidylate synthase. Phylogenetic analysis based on structural proteins and enzymes indicated that EspM4VN was identified as a member of the genus Agtrevirus, subfamily Aglimvirinae, family Ackermannviridae.

Identification of an insecticidal toxin produced by Enterobacter sp. strain 532 isolated from diseased Bombyx mori silkworms.

Although Enterobacter sp. 532 shows pathogenicity in Bombyx mori, the insecticidal mechanisms are unclear. Here, we identified and characterised an insecticidal protein from Enterobacter. The insecticidal protein was purified from the strain and inoculated into B. mori larvae. Intracellular proteins were prepared, purified and separated by preparative native polyacrylamide gel electrophoresis (PAGE); one protein band had insecticidal activity. Sodium dodecyl sulfate-PAGE showed the presence of several bands, indicating that the insecticidal protein formed a complex. Peptide mass fingerprinting of a prominent 255.3-kDa band revealed 64 peptides that matched one protein with 33.0% sequence coverage. This protein was a homologue of the A component of the toxin complex (Tc), and the VRP1 domain was conserved; thus, the gene was named itcA (insecticidal toxin complex A). In the itcA downstream region, B and C component gene homologues were found, and these genes were located on an 86.2-kb contig sequence. Two repA genes and 27 genes related to conjugation transfer of plasmids were located on the contig, suggesting that the contig originated from a mobilisable plasmid. Therefore, these findings suggested that the strain may have acquired the Tc genes by horizontal transfer. This is the first description of Tc produced by the genus Enterobacter.

Expression, Purification, and Characterization of Recombinant Human α1-Antitrypsin Produced Using Silkworm-Baculovirus Expression System.

Human α1-antitrypsin (AAT) is the most abundant serine proteinase inhibitor (serpin) in the human plasma. Commercially available AAT for the medications of deficiency of α1-antitrypsin is mainly purified from human plasma. There is a high demand for a stable and low-cost supply of recombinant AAT (rAAT). In this study, the baculovirus expression vector system using silkworm larvae as host was employed and a large amount of highly active AAT was recovered from the silkworm serum (~ 15 mg/10 ml) with high purity. Both the enzymatic activity and stability of purified rAAT were comparable with those of commercial product. Our results provide an alternative method for mass production of the active rAAT in pharmaceutical use.

Lipidation of BmAtg8 is required for autophagic degradation of p62 bodies containing ubiquitinated proteins in the silkworm, Bombyx mori.

p62/Sequestosome-1 (p62/SQSTM1, hereafter referred to as p62) is a major adaptor that allows ubiquitinated proteins to be degraded by autophagy, and Atg8 homologs are required for p62-mediated autophagic degradation, but their relationship is still not understood in Lepidopteran insects. Here it is clearly demonstrated that the silkworm homolog of mammalian p62, Bombyx mori p62 (Bmp62), forms p62 bodies depending on its Phox and Bem1p (PB1) and ubiquitin-associated (UBA) domains. These two domains are associated with Bmp62 binding to ubiquitinated proteins to form the p62 bodies, and the UBA domain is essential for the binding, but Bmp62 still self-associates without the PB1 or UBA domain. The p62 bodies in Bombyx cells are enclosed by BmAtg9-containing membranes and degraded via autophagy. It is revealed that the interaction between the Bmp62 AIM motif and BmAtg8 is critical for the autophagic degradation of the p62 bodies. Intriguingly, we further demonstrate that lipidation of BmAtg8 is required for the Bmp62-mediated complete degradation of p62 bodies by autophagy. Our results should be useful in future studies of the autophagic mechanism in Lepidopteran insects.

Expression of a Vibrio parahaemolyticus toxin in Escherichia coli results in chromosomal DNA degradation.

A toxin-antitoxin system, vp1842/vp1843, locates within a superintegron on the Vibrio parahaemolyticus genome chromosome I whose toxin gene vp1843 encodes a DNA nicking endonuclease. We found that the vp1843 expression in Escherichia coli cells strongly induced chromosomal DNA degradation. On the basis of these observations, we discuss a possible physiological role of vp1842/vp1843 in V. parahaemolyticus.

Unique clade of alphaproteobacterial endosymbionts induces complete cytoplasmic incompatibility in the coconut beetle.

Maternally inherited bacterial endosymbionts in arthropods manipulate host reproduction to increase the fitness of infected females. Cytoplasmic incompatibility (CI) is one such manipulation, in which uninfected females produce few or no offspring when they mate with infected males. To date, two bacterial endosymbionts, Wolbachia and Cardinium, have been reported as CI inducers. Only Wolbachia induces complete CI, which causes 100% offspring mortality in incompatible crosses. Here we report a third CI inducer that belongs to a unique clade of Alphaproteobacteria detected within the coconut beetle, Brontispa longissima This beetle comprises two cryptic species, the Asian clade and the Pacific clade, which show incompatibility in hybrid crosses. Different bacterial endosymbionts, a unique clade of Alphaproteobacteria in the Pacific clade and Wolbachia in the Asian clade, induced bidirectional CI between hosts. The former induced complete CI (100% mortality), whereas the latter induced partial CI (70% mortality). Illumina MiSeq sequencing and denaturing gradient gel electrophoresis patterns showed that the predominant bacterium detected in the Pacific clade of B. longissima was this unique clade of Alphaproteobacteria alone, indicating that this endosymbiont was responsible for the complete CI. Sex distortion did not occur in any of the tested crosses. The 1,160 bp of 16S rRNA gene sequence obtained for this endosymbiont had only 89.3% identity with that of Wolbachia, indicating that it can be recognized as a distinct species. We discuss the potential use of this bacterium as a biological control agent.

Characterization of Recombinant Thermococcus kodakaraensis (KOD) DNA Polymerases Produced Using Silkworm-Baculovirus Expression Vector System.

The KOD DNA polymerase from Thermococcus kodakarensis (Tkod-Pol) has been preferred for PCR due to its rapid elongation rate, extreme thermostability and outstanding fidelity. Here in this study, we utilized silkworm-baculovirus expression vector system (silkworm-BEVS) to express the recombinant Tkod-Pol (rKOD) with N-terminal (rKOD-N) or C-terminal (rKOD-C) tandem fusion tags. By using BEVS, we produced functional rKODs with satisfactory yields, about 1.1 mg/larva for rKOD-N and 0.25 mg/larva for rKOD-C, respectively. Interestingly, we found that rKOD-C shows higher thermostability at 95 °C than that of rKOD-N, while that rKOD-N is significantly unstable after exposing to long period of heat-shock. We also assessed the polymerase activity as well as the fidelity of purified rKODs under various conditions. Compared with commercially available rKOD, which is expressed in E. coli expression system, rKOD-C exhibited almost the same PCR performance as the commercial rKOD did, while rKOD-N did lower performance. Taken together, our results suggested that silkworm-BEVS can be used to express and purify efficient rKOD in a commercial way.

Alkaline protease contributes to pyocyanin production in Pseudomonas aeruginosa.

The role of the alkaline protease (AprA) in pyocyanin production in Pseudomonas aeruginosa was investigated. AprA was overproduced when a plasmid carrying the aprA gene was introduced to an aprA-deletion mutant strain, EG03; thus, aprA-complemented EG03 was used as an overproducing strain. The complemented strain produced higher pyocyanin than the mutant strain in all commercially available media evaluated. Particularly, pyocyanin production was higher in the complemented than in the parental strain in brain-heart infusion and tryptic soy broths. These results suggested that protein degradation products by AprA were utilized for pyocyanin production. Protein-rich media were used in subsequent validation studies. Similar results were obtained when the basal medium was supplemented with casein or skim milk as the sole organic nitrogen source. However, gelatin failed to induce abundant pyocyanin production in the complemented strain, despite the presence of protein degradation products by AprA as assessed by SDS-PAGE. Thus, gelatin degradation products may not be suitable for pyocyanin synthesis. In conclusion, AprA could contribute to pyocyanin production in the presence of several proteins or peptides.

Species associations and distributions of soil entomopathogenic fungi Metarhizium spp. in Japan.

Metarhizium Sorokin (Hypocreales: Clavicipitaceae) is a genus of facultative parasites of insects found in soils from various environments and is used for pest management. Understanding the habitat selection of Metarhizium spp. is critical to improve the efficacy and persistence of these fungi as microbial insecticides. This study sought to determine the habitat preferences of Metarhizium spp. in Japan. We identified 302 isolates of Metarhizium spp. as eight species by a combination of PCR-RFLP and phylogenetic analysis of DNA sequences. M. pingshaense was the predominant species in Japan and was most frequently isolated from both forest and agricultural environments. On the other hand, M. brunneum and M. pemphigi were comparatively restricted to forest environments. A similar species association was detected in a small area that was intensively investigated, where 7 species including 14 genotypes were isolated from soil. The results of this study have revealed different habitat preferences among Metarhizium spp. in Japan.

Expression and Characterization of Recombinant Serratia liquefaciens Nucleases Produced with Baculovirus-mediated Silkworm Expression System.

Baculovirus-Bombyx mori protein expression system has mainly been used for translation of eukaryotic proteins. In contrast, information pertaining to bacterial protein expression using this system is not sufficient. Therefore, recombinant nucleases from Serratia liquefaciens (rSlNucAs) were expressed in a Baculovirus-B. mori protein expression system. rSlNucAs containing the native signal peptide (rSlNucA-NSP) or silkworm 30-K signal peptide (rSlNucA-30K) at the NH2-terminus were constructed to enable secretion into the extracellular fraction. Both rSlNucA-30K and rSlNucA-NSP were successfully secreted into hemolymph of B. mori larvae. Affinity-purified rSlNucAs showed high nuclease activity. Optimum pH was 7.5 and half of maximum activity was maintained between pH 7.0 and 9.5. Optimum temperature was 35 °C. rSlNucAs showed sufficient activity in twofold-diluted radioimmunoprecipitation assay buffer and undiluted, mild lysis buffer. Genomic DNA of Escherichia coli was efficiently digested by rSlNucAs in the bacterial lysate. The results in this study suggest that rSlNucAs expressed by the Baculovirus-B. mori protein expression system will be a useful tool in molecular biology. Functional recombinant protein of bacteria was produced by Baculovirus-B. mori protein expression system. This system may be highly suitable for bacterial extracellular protein secreted via Sec pathway.

Effect of antibiotics on extracellular protein level in Pseudomonas aeruginosa.

Pseudomonas aeruginosa PAO1 organisms harbouring different plasmids were cultured in broths containing appropriate antibiotic(s). Extracellular proteins were more abundant in the presence of tetracycline or kanamycin than in the presence of other antibiotics. Zymography revealed that alkaline protease (AprA) production was interfered by these antibiotics. Extracellular proteins were not observed at the same level when AprA-deficient EG03 strains were cultured in the presence of different antibiotics. The extracellular protein levels were dependent on the antibiotics and plasmid derivative groups. Levels of extracellular protein were not significantly different between PAO1 (pBBR1MCS-5) and EG03 (pAprcomp-MCS5), and profiles of the extracellular proteome were comparable. In contrast, the level of EG03 (pBBR1MCS-MCS5) extracellular protein was higher than those observed in the other two strains. These results suggested that although AprA partially contributes to the alteration of extracellular protein level, the effect is limited.

Comparative proteomic analysis of hemolymph proteins from Autographa californica multiple nucleopolyhedrovirus (AcMNPV)-sensitive or -resistant silkworm strains during infections.

We reported previously that baculovirus AcMNPV host-ranges in silkworm strains are controlled by a novel third chromosomal locus. To further isolate the potential host factor and uncover the functional pathway involved, in this study we analyzed hemolymph proteins from AcMNPV-resistant or -sensitive silkworm strains infected with baculoviruses. All the protein spots from 2D electrophoresis were characterized by MALDI-TOF MS and further systematically assessed for differentially regulated proteins at different stages of infection. Subsequently, six candidates were selected for functional analysis using Bm5 cells, where the candidates were knocked-down or overexpressed. We observed that mRNA expression levels of beta-N-acetylglucosaminidase and prophenoloxidase subunit 2 are significantly upregulated during AcMNPV infections in Bm5 cells. Ultimately, we found that RNA interference of ribosomal protein RpL34 causes serious damages to cell viability as well as abortive infection, indicating that ribosomal components are essential for productive baculovirus infection.

Characterization of KfrA proteins encoded by a plasmid of Paenibacillus popilliae ATCC 14706(T).

A scaffold obtained from whole-genome shotgun sequencing of Paenibacillus popilliae ATCC 14706(T) shares partial homology with plasmids found in other strains of P. popilliae. PCR and sequencing for gap enclosure indicated that the scaffold originated from a 15,929-bp circular DNA. The restriction patterns of a plasmid isolated from P. popilliae ATCC 14706(T) were identical to those expected from the sequence; thus, this circular DNA was identified as a plasmid of ATCC 14706(T) and designated pPOP15.9. The plasmid encodes 17 putative open reading frames. Orfs 1, 5, 7, 8, and 9 are homologous to Orfs 11, 12, 15, 16, and 17, respectively. Orf1 and Orf11 are annotated as replication initiation proteins. Orf8 and Orf16 are homologs of KfrA, a plasmid-stabilizing protein in Gram-negative bacteria. Recombinant Orf8 and Orf16 proteins were assessed for the properties of KfrA. Indeed, they formed multimers and bound to inverted repeat sequences in upstream regions of both orf8 and orf16. A phylogenetic tree based on amino acid sequences of Orf8, Orf16 and Kfr proteins did not correlate with species lineage.

Biochemical characterization of maintenance DNA methyltransferase DNMT-1 from silkworm, Bombyx mori.

DNA methylation is an important epigenetic mechanism involved in gene expression of vertebrates and invertebrates. In general, DNA methylation profile is established by de novo DNA methyltransferases (DNMT-3A, -3B) and maintainance DNA methyltransferase (DNMT-1). DNMT-1 has a strong substrate preference for hemimethylated DNA over the unmethylated one. Because the silkworm genome lacks an apparent homologue of de novo DNMT, it is still unclear that how silkworm chromosome establishes and maintains its DNA methylation profile. As the first step to unravel this enigma, we purified recombinant BmDNMT-1 using baculovirus expression system and characterized its DNA-binding and DNA methylation activity. We found that the BmDNMT-1 preferentially methylates hemimethylated DNA despite binding to both unmethylated and hemimethylated DNA. Interestingly, BmDNMT-1 formed a complex with DNA in the presence or absence of methyl group donor, S-Adenosylmethionine (AdoMet) and the AdoMet-dependent complex formation was facilitated by Zn(2+) and Mn(2+). Our results provide clear evidence that BmDNMT-1 retained the function as maintenance DNMT but its sensitivity to metal ions is different from mammalian DNMT-1.

Phylogenetic relationship of Paenibacillus species based on putative replication origin regions and analysis of an yheCD-like sequence found in this region.

To determine the phylogenetic relationship among Paenibacillus species, putative replication origin regions were compared. In the rsmG-gyrA region, gene arrangements in Paenibacillus species were identical to those of Bacillus species, with the exception of an open reading frame (orf14) positioned between gyrB and gyrA, which was observed only in Paenibacillus species. The orf14 product was homologous to the endospore-associated proteins YheC and YheD of Bacillus subtilis. Phylogenetic analysis based on the YheCD proteins suggested that Orf14 could be categorized into the YheC group. In the Paenibacillus genome, DnaA box clusters were found in rpmH-dnaA and dnaA-dnaN intergenic regions, known as box regions C and R, respectively; this localization was similar to that observed in B. halodurans. A phylogenetic tree based on the nucleotide sequences of the whole replication origin regions suggested that P. popilliae, P. thiaminolyticus, and P. dendritiformis are closely related species.

Draft Genome Sequence of Entomopathogenic Serratia liquefaciens Strain FK01.

In the present study, we determined the draft genome sequence of the entomopathogenic bacterium Serratia liquefaciens FK01, which is highly virulent to the silkworm. The draft genome is ~5.28 Mb in size, and the G+C content is 55.8%.

Expression, purification, and characterization of endo-ß-N-acetylglucosaminidase H using baculovirus-mediated silkworm protein expression system.

Endo-ß-N-acetylglucosaminidase (Endo H) from Streptomyces plicatus hydrolyzes the core di-GlcNAc units of asparagine-linked oligosaccharides and is regarded as an important tool for glycobiology research. In the present study, we established a large-scale system to produce secreted Endo H using a silkworm-baculovirus expression system (silkworm-BES). The recombinant Endo H purified from silkworm hemolymph had activity comparable to that from recombinant Escherichia coli. As well as its well-characterized substrate RNase B, the Endo H from silkworm-BES was able to deglycosylate the high-mannose glycoproteins from silkworm hemolymph. Interestingly, the secretion amount of recombinant Endo H was significantly varied among the different silkworm strains, which could provide valuable information for larger-scale protein productions from silkworm-BES.