The ABC toxin complex from Yersinia entomophaga can package three different cytotoxic components expressed from distinct genetic loci in an unfolded state: the structures of both shell and cargo.

Bacterial ABC toxin complexes (Tcs) comprise three core proteins: TcA, TcB and TcC. The TcA protein forms a pentameric assembly that attaches to the surface of target cells and penetrates the cell membrane. The TcB and TcC proteins assemble as a heterodimeric TcB-TcC subcomplex that makes a hollow shell. This TcB-TcC subcomplex self-cleaves and encapsulates within the shell a cytotoxic `cargo' encoded by the C-terminal region of the TcC protein. Here, we describe the structure of a previously uncharacterized TcC protein from Yersinia entomophaga, encoded by a gene at a distant genomic location from the genes encoding the rest of the toxin complex, in complex with the TcB protein. When encapsulated within the TcB-TcC shell, the C-terminal toxin adopts an unfolded and disordered state, with limited areas of local order stabilized by the chaperone-like inner surface of the shell. We also determined the structure of the toxin cargo alone and show that when not encapsulated within the shell, it adopts an ADP-ribosyltransferase fold most similar to the catalytic domain of the SpvB toxin from Salmonella typhimurium. Our structural analysis points to a likely mechanism whereby the toxin acts directly on actin, modifying it in a way that prevents normal polymerization.

Linocin M18 protein from the insect pathogenic bacterium Brevibacillus laterosporus isolates.

Brevibacillus laterosporus (Bl) is a Gram-positive and spore-forming bacterium. Insect pathogenic strains have been characterised in New Zealand, and two isolates, Bl 1821L and Bl 1951, are under development for use in biopesticides. However, growth in culture is sometimes disrupted, affecting mass production. Based on previous work, it was hypothesised that Tectiviridae phages might be implicated. While investigating the cause of the disrupted growth, electron micrographs of crude lysates showed structural components of putative phages including capsid and tail-like structures. Sucrose density gradient purification yielded a putative self-killing protein of ~30 kDa. N-terminal sequencing of the ~30 kDa protein identified matches to a predicted 25 kDa hypothetical and a 31.4 kDa putative encapsulating protein homologs, with the genes encoding each protein adjacent in the genomes. BLASTp analysis of the homologs of 31.4 kDa amino acid sequences shared 98.6% amino acid identity to the Linocin M18 bacteriocin family protein of Brevibacterium sp. JNUCC-42. Bioinformatic tools including AMPA and CellPPD defined that the bactericidal potential originated from a putative encapsulating protein. Antagonistic activity of the ~30 kDa encapsulating protein of Bl 1821L and Bl 1951during growth in broth exhibited bacterial autolytic activity. LIVE/DEAD staining of Bl 1821L cells after treatment with the ~30 kDa encapsulating protein of Bl 1821L substantiated the findings by showing 58.8% cells with the compromised cell membranes as compared to 37.5% cells in the control. Furthermore, antibacterial activity of the identified proteins of Bl 1821L was validated through gene expression in a Gram-positive bacterium Bacillus subtilis WB800N. KEY POINTS: • Gene encoding the 31.4 kDa antibacterial Linocin M18 protein was identified • It defined the autocidal activity of Linocin M18 (encapsulating) protein • Identified the possible killing mechanism of the encapsulins.

Lysis Cassette-Mediated Exoprotein Release in Yersinia entomophaga Is Controlled by a PhoB-Like Regulator.

Secretion of exoproteins is a key component of bacterial virulence, and is tightly regulated in response to environmental stimuli and host-dependent signals. The entomopathogenic bacterium Yersinia entomophaga MH96 produces a wide range of exoproteins including its main virulence factor, the 2.46 MDa insecticidal Yen-Tc toxin complex. Previously, a high-throughput transposon-based screening assay identified the region of exoprotein release (YeRER) as essential to exoprotein release in MH96. This study defines the role of the YeRER associated ambiguous holin/endolysin-based lysis cluster (ALC) and the novel RoeA regulator in the regulation and release of exoproteins in MH96. A mutation in the ambiguous lysis cassette (ALC) region abolished exoprotein release and caused cell elongation, a phenotype able to be restored through trans-complementation with an intact ALC region. Endogenous ALC did not impact cell growth of the wild type, while artificial expression of an optimized ALC caused cell lysis. Using HolA-sfGFP and Rz1-sfGFP reporters, Rz1 expression was observed in all cells while HolA expression was limited to a small proportion of cells, which increased over time. Transcriptomic assessments found expression of the genes encoding the prominent exoproteins, including the Yen-Tc, was reduced in the roeA mutant and identified a 220 ncRNA of the YeRER intergenic region that, when trans complemented in the wildtype, abolished exoprotein release. A model for Y. entomophaga mediated exoprotein regulation and release is proposed. IMPORTANCE While theoretical models exist, there is not yet any empirical data that links ALC phage-like lysis cassettes with the release of large macro-molecular toxin complexes, such as Yen-Tc in Gram-negative bacteria. In this study, we demonstrate that the novel Y. entomophaga RoeA activates the production of exoproteins (including Yen-Tc) and the ALC at the transcriptional level. The translation of the ALC holin is confined to a subpopulation of cells that then lyse over time, indicative of a complex hierarchical regulatory network. The presence of an orthologous RoeA and a HolA like holin 5' of an eCIS Afp element in Pseudomonas chlororaphis, combined with the presented data, suggests a shared mechanism is required for the release of some large macromolecular protein assemblies, such as the Yen-Tc, and further supports classification of phage-like lysis clusters as type 10 secretion systems.

Genome sequence of the entomopathogenic Serratia entomophila isolate 626 and characterisation of the species specific itaconate degradation pathway.

BACKGROUND: Isolates of Serratia entomophila and S. proteamaculans (Yersiniaceae) cause disease specific to the endemic New Zealand pasture pest, Costelytra giveni (Coleoptera: Scarabaeidae). Previous genomic profiling has shown that S. entomophila isolates appear to have conserved genomes and, where present, conserved plasmids. In the absence of C. giveni larvae, S. entomophila prevalence reduces in the soil over time, suggesting that S. entomophila has formed a host-specific relationship with C. giveni. To help define potential genetic mechanisms driving retention of the chronic disease of S. entomophila, the genome of the isolate 626 was sequenced, enabling the identification of unique chromosomal properties, and defining the gain/loss of accessory virulence factors relevant to pathogenicity to C. giveni larvae. RESULTS: We report the complete sequence of S. entomophila isolate 626, a causal agent of amber disease in C. giveni larvae. The genome of S. entomophila 626 is 5,046,461 bp, with 59.1% G + C content and encoding 4,695 predicted CDS. Comparative analysis with five previously sequenced Serratia species, S. proteamaculans 336X, S. marcescens Db11, S. nematodiphila DH-S01, S. grimesii BXF1, and S. ficaria NBRC 102596, revealed a core of 1,165 genes shared. Further comparisons between S. entomophila 626 and S. proteamaculans 336X revealed fewer predicted phage-like regions and genomic islands in 626, suggesting less horizontally acquired genetic material. Genomic analyses revealed the presence of a four-gene itaconate operon, sharing a similar gene order as the Yersinia pestis ripABC complex. Assessment of a constructed 626::RipC mutant revealed that the operon confer a possible metabolic advantage to S. entomophila in the initial stages of C. giveni infection. CONCLUSIONS: Evidence is presented where, relative to S. proteamaculans 336X, S. entomophila 626 encodes fewer genomic islands and phages, alluding to limited horizontal gene transfer in S. entomophila. Bioassay assessments of a S. entomophila-mutant with a targeted mutation of the itaconate degradation region unique to this species, found the mutant to have a reduced capacity to replicate post challenge of the C. giveni larval host, implicating the itaconate operon in establishment within the host.

Isolation, Purification, and Characterisation of a Phage Tail-Like Bacteriocin from the Insect Pathogenic Bacterium Brevibacillus laterosporus.

The Gram-positive and spore-forming bacterium Brevibacillus laterosporus (Bl) belongs to the Brevibacillus brevis phylogenetic cluster. Isolates of the species have demonstrated pesticidal potency against a wide range of invertebrate pests and plant diseases. Two New Zealand isolates, Bl 1821L and Bl 1951, are under development as biopesticides for control of diamondback moth and other pests. However, due to the often-restricted growth of these endemic isolates, production can be an issue. Based on the previous work, it was hypothesised that the putative phages might be involved. During investigations of the cause of the disrupted growth, electron micrographs of crude lysate of Bl 1821L showed the presence of phages' tail-like structures. A soft agar overlay method with PEG 8000 precipitation was used to differentiate between the antagonistic activity of the putative phage and phage tail-like structures (bacteriocins). Assay tests authenticated the absence of putative phage activity. Using the same method, broad-spectrum antibacterial activity of Bl 1821L lysate against several Gram-positive bacteria was found. SDS-PAGE of sucrose density gradient purified and 10 kD MWCO concentrated lysate showed a prominent protein band of ~48 kD, and transmission electron microscopy revealed the presence of polysheath-like structures. N-terminal sequencing of the ~48 kD protein mapped to a gene with weak predicted amino acid homology to a Bacillus PBSX phage-like element xkdK, the translated product of which shared >90% amino acid similarity to the phage tail-sheath protein of another Bl published genome, LMG15441. Bioinformatic analysis also identified an xkdK homolog in the Bl 1951 genome. However, genome comparison of the region around the xkdK gene between Bl 1821L and Bl 1951 found differences including two glycine rich protein encoding genes which contain imperfect repeats (1700 bp) in Bl 1951, while a putative phage region resides in the analogous Bl 1821L region. Although comparative analysis of the genomic organisation of Bl 1821L and Bl 1951 PBSX-like region with the defective phages PBSX, PBSZ, and PBP 180 of Bacillus subtilis isolates 168 and W23, and Bacillus phage PBP180 revealed low amino acids similarity, the genes encode similar functional proteins in similar arrangements, including phage tail-sheath (XkdK), tail (XkdO), holin (XhlB), and N-acetylmuramoyl-l-alanine (XlyA). AMPA analysis identified a bactericidal stretch of 13 amino acids in the ~48 kD sequenced protein of Bl 1821L. Antagonistic activity of the purified ~48 kD phage tail-like protein in the assays differed remarkably from the crude lysate by causing a decrease of 34.2% in the number of viable cells of Bl 1951, 18 h after treatment as compared to the control. Overall, the identified inducible phage tail-like particle is likely to have implications for the in vitro growth of the insect pathogenic isolate Bl 1821L.

Identification of genes involved in exoprotein release using a high-throughput exoproteome screening assay in Yersinia entomophaga.

Bacterial protein secretion is crucial to the maintenance of viability and pathogenicity. Although many bacterial secretion systems have been identified, the underlying mechanisms regulating their expression are less well explored. Yersinia entomophaga MH96, an entomopathogenic bacterium, releases an abundance of proteins including the Yen-Tc into the growth medium when cultured in Luria Bertani broth at ≤ 25°C. Through the development of a high-throughput exoproteome screening assay (HESA), genes involved in MH96 exoprotein production were identified. Of 4,080 screened transposon mutants, 34 mutants exhibited a decreased exoprotein release, and one mutation located in the intergenic region of the Yen-Tc operon displayed an elevated exoprotein release relative to the wild-type strain MH96. DNA sequencing revealed several transposon insertions clustered in gene regions associated with lipopolysaccharide (LPSI and LPSII), and N-acyl-homoserine lactone synthesis (quorum sensing). Twelve transposon insertions were located within transcriptional regulators or intergenic regions. The HESA will have broad applicability for identifying genes associated with exoproteome production in a range of microorganisms.

Identification of Diverse Toxin Complex Clusters and an eCIS Variant in Serratia proteamaculans Pathovars of the New Zealand Grass Grub (Costelytra Giveni) and Manuka Beetle (Pyronota Spp.) Larvae.

The grass grub endemic to New Zealand, Costelytra giveni (Coleoptera: Scarabaeidae), and the manuka beetle, Pyronota festiva and P. setosa (Coleoptera: Scarabaeidae), are prevalent pest species. Through assessment of bacterial strains isolated from diseased cadavers of these insect species, 19 insect-active Serratia proteamaculans variants and a single Serratia entomophila strain were isolated. When independently bioassayed, these isolates differed in host range, the rate of disease progression, and 12-day mortality rates, which ranged from 60 to 100% of the challenged larvae. A Pyronota spp.-derived S. proteamaculans isolate caused a transient disease phenotype in challenged C. giveni larvae, whereby larvae appeared diseased before recovering to a healthy state. Genome sequence analysis revealed that all but two of the sequenced isolates contained a variant of the S. entomophila amber-disease-associated plasmid, pADAP. Each isolate also encoded one of seven distinct members of the toxin complex (Tc) family of insect-active toxins, five of which are newly described, or a member of the extracellular contractile injection (eCIS) machine family, with a new AfpX variant designated SpF. Targeted mutagenesis of each of the predicted Tc- or eCIS-encoding regions abolished or attenuated pathogenicity. Host-range testing showed that several of the S. proteamaculans Tc-encoding isolates affected both Pyronota and C. giveni species, with other isolates specific for either Pyronota spp. or C. giveni. The isolation of several distinct host-specific pathotypes of Serratia spp. may reflect pathogen-host speciation. IMPORTANCE New pathotypes of the insect pathogen Serratia, each with differing virulence attributes and host specificity toward larvae of the New Zealand manuka beetle and grass grub, have been identified. All of the Serratia proteamaculans isolates contained one of seven different insect-active toxin clusters or one of three eCIS variants. The diversity of these Serratia-encoded virulence clusters, resulting in differences in larval disease progression and host specificity in endemic scarab larvae, suggests speciation of these pathogens with their insect hosts. The differing virulence properties of these Serratia species may affect their potential infectivity and distribution among the insect populations. Based on their differing geographic isolation and pathotypes, several of these Serratia isolates, including the manuka beetle-active isolates, are likely to be more effective biopesticides in specific environments or could be used in combination for greater effect.

Investigating the Process of Sheath Maturation in Antifeeding Prophage: a Phage Tail-Like Protein Translocation Structure.

The antifeeding prophage (Afp) produced by the bacterium Serratia entomophila is the archetypical external contractile injection system (eCIS). Afp and its orthologues are characterized by three sheath proteins, while contractile bacteriophages and pyocins encode only one. Using targeted mutagenesis, transmission electron microscopy (TEM), and pulldown studies, we interrogated the roles of the three sheath proteins (Afp2, Afp3, and Afp4) in Afp assembly, in particular the interaction between the two sequence-related helical-sheath-forming proteins Afp2 and Afp3 and their cross talk with the tail termination sheath capping protein (TrP) Afp16 in the sheath maturation process. The expressed assemblies for the afp2-deficient mutant were mostly a mixture of isolated tail fibers, detached baseplates without tail fibers, and sheathless inner tube baseplate complexes (TBCs) with a length similar to that of mature Afp, which were surrounded in many cases by fibrillar polymerized material. In the afp3-deficient mutant, variable-length TBCs with similar but shorter fibrillar polymerized material, largely bereft of tail fibers, were observed, while only detached baseplate assemblies were seen for the afp4-deficient mutant. Furthermore, we found that (i) only trans complementation of afp2 with its mutated counterpart restored mature Afp particles with full biological activity, (ii) purified Afp3 pulled down Afp2 by forming a sodium dodecyl sulfate (SDS)-resistant complex but not vice versa, (iii) Afp16 had a higher affinity for binding Afp2 or Afp3 than Afp4, and (iv) Afp4 is required for the association of the polymerized sheath on the baseplate via Afp2. A proposed model for sheath maturation and assembly in Afp is presented. IMPORTANCE Members of the contractile bacteriophage-related but evolutionarily divergent eCIS contain not one but three sheath proteins, two of which, namely, Afp2 and Afp3 in the Afp, arranged as alternate hexameric stacks constitute the helical sheath. We revealed that Afp2 and Afp3, even though they are highly similar, possess markedly distinct, crucial roles in Afp assembly. We find that Afp3, by virtue of its interaction with the tail-terminating protein Afp16, regulates tube and sheath length, while Afp2 is critical for proper sheath polymerization and the assembly of the baseplate. The resulting model for the Afp assembly will further guide the manipulation of Afp and its related eCISs as nanodelivery vehicles for pest control and phage therapy.

Evolution of virulence in a novel family of transmissible mega-plasmids.

Some Serratia entomophila isolates have been successfully exploited in biopesticides due to their ability to cause amber disease in larvae of the Aotearoa (New Zealand) endemic pasture pest, Costelytra giveni. Anti-feeding prophage and ABC toxin complex virulence determinants are encoded by a 153-kb single-copy conjugative plasmid (pADAP; amber disease-associated plasmid). Despite growing understanding of the S. entomophila pADAP model plasmid, little is known about the wider plasmid family. Here, we sequence and analyse mega-plasmids from 50 Serratia isolates that induce variable disease phenotypes in the C. giveni insect host. Mega-plasmids are highly conserved within S. entomophila, but show considerable divergence in Serratia proteamaculans with other variants in S. liquefaciens and S. marcescens, likely reflecting niche adaption. In this study to reconstruct ancestral relationships for a complex mega-plasmid system, strong co-evolution between Serratia species and their plasmids were found. We identify 12 distinct mega-plasmid genotypes, all sharing a conserved gene backbone, but encoding highly variable accessory regions including virulence factors, secondary metabolite biosynthesis, Nitrogen fixation genes and toxin-antitoxin systems. We show that the variable pathogenicity of Serratia isolates is largely caused by presence/absence of virulence clusters on the mega-plasmids, but notably, is augmented by external chromosomally encoded factors.

In vivo transcriptome analysis provides insights into host-dependent expression of virulence factors by Yersinia entomophaga MH96, during infection of Galleria mellonella.

The function of microbes can be inferred from knowledge of genes specifically expressed in natural environments. Here, we report the in vivo transcriptome of the entomopathogenic bacterium Yersinia entomophaga MH96, captured during initial, septicemic, and pre-cadaveric stages of intrahemocoelic infection in Galleria mellonella. A total of 1285 genes were significantly upregulated by MH96 during infection; 829 genes responded to in vivo conditions during at least one stage of infection, 289 responded during two stages of infection, and 167 transcripts responded throughout all three stages of infection compared to in vitro conditions at equivalent cell densities. Genes upregulated during the earliest infection stage included components of the insecticidal toxin complex Yen-TC (chi1, chi2, and yenC1), genes for rearrangement hotspot element containing protein yenC3, cytolethal distending toxin cdtAB, and vegetative insecticidal toxin vip2. Genes more highly expressed throughout the infection cycle included the putative heat-stable enterotoxin yenT and three adhesins (usher-chaperone fimbria, filamentous hemagglutinin, and an AidA-like secreted adhesin). Clustering and functional enrichment of gene expression data also revealed expression of genes encoding type III and VI secretion system-associated effectors. Together these data provide insight into the pathobiology of MH96 and serve as an important resource supporting efforts to identify novel insecticidal agents.

Atomic structures of an entire contractile injection system in both the extended and contracted states.

Contractile injection systems are sophisticated multiprotein nanomachines that puncture target cell membranes. Although the number of atomic-resolution insights into contractile bacteriophage tails, bacterial type six secretion systems and R-pyocins is rapidly increasing, structural information on the contraction of bacterial phage-like protein-translocation structures directed towards eukaryotic hosts is scarce. Here, we characterize the antifeeding prophage AFP from Serratia entomophila by cryo-electron microscopy. We present the high-resolution structure of the entire AFP particle in the extended state, trace 11 protein chains de novo from the apical cap to the needle tip, describe localization variants and perform specific structural comparisons with related systems. We analyse inter-subunit interactions and highlight their universal conservation within contractile injection systems while revealing the specificities of AFP. Furthermore, we provide the structure of the AFP sheath-baseplate complex in a contracted state. This study reveals atomic details of interaction networks that accompany and define the contraction mechanism of toxin-delivery tailocins, offering a comprehensive framework for understanding their mode of action and for their possible adaptation as biocontrol agents.

Cryo-EM structures of the pore-forming A subunit from the Yersinia entomophaga ABC toxin.

ABC toxins are pore-forming virulence factors produced by pathogenic bacteria. YenTcA is the pore-forming and membrane binding A subunit of the ABC toxin YenTc, produced by the insect pathogen Yersinia entomophaga. Here we present cryo-EM structures of YenTcA, purified from the native source. The soluble pre-pore structure, determined at an average resolution of 4.4 Å, reveals a pentameric assembly that in contrast to other characterised ABC toxins is formed by two TcA-like proteins (YenA1 and YenA2) and decorated by two endochitinases (Chi1 and Chi2). We also identify conformational changes that accompany membrane pore formation by visualising YenTcA inserted into liposomes. A clear outward rotation of the Chi1 subunits allows for access of the protruding translocation pore to the membrane. Our results highlight structural and functional diversity within the ABC toxin subfamily, explaining how different ABC toxins are capable of recognising diverse hosts.

Serratia proteamaculans Strain AGR96X Encodes an Antifeeding Prophage (Tailocin) with Activity against Grass Grub (Costelytra giveni) and Manuka Beetle (Pyronota Species) Larvae.

A highly virulent Serratia proteamaculans strain, AGR96X, exhibiting specific pathogenicity against larvae of the New Zealand grass grub (Costelytra giveni; Coleoptera: Scarabaeidae) and the New Zealand manuka beetle (Pyronota festiva and P. setosa; Coleoptera: Scarabaeidae), was isolated from a diseased grass grub larva. A 12-day median lethal dose of 4.89 × 103 ± 0.92 × 103 cells per grass grub larva was defined for AGR96X, and death occurred within 5 to 12 days following the ingestion of a high bacterial dose. During the infection period, the bacterium rapidly multiplied within the insect host and invaded the hemocoel, leading to a mean bacterial load of 8.2 × 109 cells per larva at 6 days postingestion. Genome sequencing of strain AGR96X revealed the presence of a variant of the Serratia entomophila antifeeding prophage (Afp), a tailocin designated AfpX. Unlike Afp, AfpX contains two Afp16 tail-length termination protein orthologs and two putative toxin components. A 37-kb DNA fragment encoding the AfpX-associated region was cloned, transformed into Escherichia coli, and fed to C. giveni and Pyronota larvae, causing mortality. In addition, the deletion of the afpX15 putative chaperone component abolished the virulence of AGR96X. Unlike S. entomophila Afp, the AfpX tailocin could be induced by mitomycin C. Transmission electron microscopy analysis revealed the presence of Afp-like particles of various lengths, and when the purified AfpX tailocin was fed to grass grub or manuka beetle larvae, they underwent phenotypic changes similar to those of larvae fed AGR96X.IMPORTANCESerratia proteamaculans strain AGR96X shows dual activity against larvae of endemic New Zealand pasture pests, the grass grub (Costelytra giveni) and the manuka beetle (Pyronota spp.). Unlike Serratia entomophila, the causal agent of amber disease, which takes 3 to 4 months to kill grass grub larvae, AGR96X causes mortality within 5 to 12 days of ingestion and invades the insect hemocoel. AGR96X produces a unique variant of the S. entomophila antifeeding prophage (Afp), a cell-free phage-like entity that is proposed to deliver protein toxins to the grass grub target site, causing a cessation of feeding activity. Unlike other Afp variants, AGR96X Afp, named AfpX, contains two tail-length termination proteins, resulting in greater variability in the AfpX length. AfpX shows dual activity against both grass grub and manuka beetle larvae. AGR96X is a viable alternative to S. entomophila for pest control in New Zealand pasture systems.

Dispersal of the Invasive Pasture Pest Heteronychus arator into Areas of Low Population Density: Effects of Sex and Season, and Implications for Pest Management.

African black beetle, Heteronychus arator (Scarabaeidae), is an exotic pest of pastures in northern New Zealand. Both adults and larvae feed on pasture grasses. Adults disperse by walking (short range) or flying (long range). Dispersal flights are triggered by warm night temperatures in spring and autumn. Short range adult dispersal in search of mates, food or oviposition sites is poorly understood. This study investigated walking activity of H. arator adults over three seasons in New Zealand pastures. Adult walking activity was monitored using pitfall traps along fence lines and in pasture plots on a dairy farm in Waikato, New Zealand, in spring 2013, spring 2014, and autumn 2015. Beetle populations were reduced by application of a biopesticide bait to compare walking activity between treated and control plots for up to 26 days post-treatment. Marked beetles were released into the pasture plots to measure the distance traveled by recaptured individuals. Trap catches along the fence lines were correlated with air temperatures in 2013. Trap catches were male biased in spring 2014 compared with autumn 2015. Trap numbers in the control plots were nearly double that of treated plots in both seasons. More beetles were caught in the pitfall traps at the edges of the treated plots than in the center. Trap catches were consistent throughout the control plot in spring 2014, but in autumn 2015 more beetles were caught in the center of the control plot than at the edges. Few marked beetles were recaptured with dispersal rates estimated as <0.5 m per day. Warmer temperatures encouraged short range dispersal in H. arator. Males were more active than females during the spring mating season. Edge effects were strong and should be considered in the design of field experiments.

Purification of the Yersinia entomophaga Yen-TC Toxin Complex Using Size Exclusion Chromatography.

The Yersinia entomophaga toxin complex (Yen-TC) is the bacterium's main virulence determinant. Because of its high insect activity, methods were developed to allow the routine isolation and purification of Yen-TC from an overnight bacterial culture using size exclusion chromatography. Here we outline an overnight purification procedure using a 100-ml culture volume, where approximately 2 mg of Yen-TC, with an approximate purity of 95-98 %, can be routinely obtained.

The Draft Genome Sequence of the Yersinia entomophaga Entomopathogenic Type Strain MH96T.

Here we report the draft genome of Yersinia entomophaga type strain MH96T. The genome shows 93.8% nucleotide sequence identity to that of Yersinia nurmii type strain APN3a-cT, and comprises a single chromosome of approximately 4,275,531 bp. In silico analysis identified that, in addition to the previously documented Y. entomophaga Yen-TC gene cluster, the genome encodes a diverse array of toxins, including two type III secretion systems, and five rhs-associated gene clusters. As well as these multicomponent systems, several orthologs of known insect toxins, such as VIP2 toxin and the binary toxin PirAB, and distant orthologs of some mammalian toxins, including repeats-in-toxin, a cytolethal distending toxin, hemolysin-like genes and an adenylate cyclase were identified. The genome also contains a large number of hypothetical proteins and orthologs of known effector proteins, such as LopT, as well as genes encoding a wide range of proteolytic determinants, including metalloproteases and pathogen fitness determinants, such as genes involved in iron metabolism. The bioinformatic data derived from the current in silico analysis, along with previous information on the pathobiology of Y. entomophaga against its insect hosts, suggests that a number of these virulence systems are required for survival in the hemocoel and incapacitation of the insect host.

The role of gluconate production by Pseudomonas spp. in the mineralization and bioavailability of calcium-phytate to Nicotiana tabacum.

Organic phosphorus (P) is abundant in most soils but is largely unavailable to plants. Pseudomonas spp. can improve the availability of P to plants through the production of phytases and organic anions. Gluconate is a major component of Pseudomonas organic anion production and may therefore play an important role in the mineralization of insoluble organic P forms such as calcium-phytate (CaIHP). Organic anion and phytase production was characterized in 2 Pseudomonas spp. soil isolates (CCAR59, Ha200) and an isogenic mutant of strain Ha200, which lacked a functional glucose dehydrogenase (Gcd) gene (strain Ha200 gcd::Tn5B8). Wild-type and mutant strains of Pseudomonas spp. were evaluated for their ability to solubilize and hydrolyze CaIHP and to promote the growth and assimilation of P by tobacco plants. Gluconate, 2-keto-gluconate, pyruvate, ascorbate, acetate, and formate were detected in Pseudomonas spp. supernatants. Wild-type pseudomonads containing a functional gcd could produce gluconate and mineralize CaIHP, whereas the isogenic mutant could not. Inoculation with Pseudomonas improved the bioavailability of CaIHP to tobacco plants, but there was no difference in plant growth response due to Gcd function. Gcd function is required for the mineralization of CaIHP in vitro; however, further studies will be needed to quantify the relative contribution of specific organic anions such as gluconate to plant growth promotion by soil pseudomonads.

HemX is required for production of 2-ketogluconate, the predominant organic anion required for inorganic phosphate solubilization by Burkholderia sp. Ha185.

The bacterium Burkholderia sp. Ha185 readily solubilizes inorganic phosphate by releasing the low molecular weight organic anion, 2-ketogluconate. Using random transposon mutagenesis and in silico analysis, a mutation that caused almost complete abolition of phosphate solubilization was located within hemX, which is part of the hem operon. Burkholderia sp. Ha185 HemX is a multidomain protein, predicted to encode a bifunctional uroporphyrinogen-III synthetase/uroporphyrin-III C-methyltransferase, which has not previously been implicated in phosphate solubilization. Complementation of hemX restored the ability of the mutant to solubilize phosphate in both plate and liquid cultures. Based on a combination of organic-anion profiling, quantitative polymerase chain reaction and in silico analyses, hemX was confirmed to be solely responsible for hydroxyapatite solubilization in Burkholderia sp. Ha185. It is proposed that the biosynthesis of a yet to be determined redox cofactor by HemX is the main pathway for generating 2-ketogluconate via a haem-dependent gluconate 2-dehydrogenase in Burkholderia sp. Ha185.

Temperature-Dependent Galleria mellonella Mortality as a Result of Yersinia entomophaga Infection.

The bacterium Yersinia entomophaga is pathogenic to a range of insect species, with death typically occurring within 2 to 5 days of ingestion. Per os challenge of larvae of the greater wax moth (Galleria mellonella) confirmed that Y. entomophaga was virulent when fed to larvae held at 25°C but was avirulent when fed to larvae maintained at 37°C. At 25°C, a dose of ~4 × 10(7) CFU per larva of a Y. entomophaga toxin complex (Yen-TC) deletion derivative, the Y. entomophaga ΔTC variant, resulted in 27% mortality. This low level of activity was restored to near-wild-type levels by augmentation of the diet with a sublethal dose of purified Yen-TC. Intrahemocoelic injection of ~3 Y. entomophaga or Y. entomophaga ΔTC cells per larva gave a 4-day median lethal dose, with similar levels of mortality observed at both 25 and 37°C. Following intrahemocoelic injection of a Yen-TC YenA1 green fluorescent protein fusion strain into larvae maintained at 25°C, the bacteria did not fluoresce until the population density reached 2 × 10(7) CFU ml(-1) of hemolymph. The observed cells also took an irregular form. When the larvae were maintained at 37°C, the cells were small and the observed fluorescence was sporadic and weak, being more consistent at a population density of ~3 × 10(9) CFU ml(-1) of hemolymph. These findings provide further understanding of the pathobiology of Y. entomophaga in insects, showing that the bacterium gains direct access to the hemocoelic cavity, from where it rapidly multiplies to cause disease.

Afp14 is involved in regulating the length of Anti-feeding prophage (Afp).

The anti-feeding prophage (Afp), a phage-tail-like particle that causes cessation of feeding in the New Zealand grass grub, Costelytra zealandica, is encoded by 18 open reading frames (afp1-18). C-terminal truncations of afp14 resulted in shortened Afp particles, suggesting that Afp14 is involved in Afp length determination. We constructed an Afp assembly system (afp1-18), wherein Afp14 was truncated after the N-terminal 88 residues. This construct, when expressed in trans in Escherichia coli expressing a N-terminal 98-amino acid Afp14 construct, yielded fully assembled Afp but no assembled Afp was detected in the case of a N-terminal 96-amino acid Afp14 construct. These results suggested that the 98 N-terminal, amino acid residues of Afp14 is crucial for the initiation of Afp assembly via baseplate formation. Trans-based expression of wild-type afp14 resulted in Afp particles of varying lengths, all of which were shorter than the wild-type Afp particle. On the other hand, similar expression of Afp14 harboring a C-terminal extension (KLLEH(6)) resulted in elongated Afp particles. This information, combined with bioinformatics data, allowed us to propose a model delineating the mechanism and role of Afp14 in the maturation of the Afp particle.