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.

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.

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.

The BC component of ABC toxins is an RHS-repeat-containing protein encapsulation device.

The ABC toxin complexes produced by certain bacteria are of interest owing to their potent insecticidal activity and potential role in human disease. These complexes comprise at least three proteins (A, B and C), which must assemble to be fully toxic. The carboxy-terminal region of the C protein is the main cytotoxic component, and is poorly conserved between different toxin complexes. A general model of action has been proposed, in which the toxin complex binds to the cell surface via the A protein, is endocytosed, and subsequently forms a pH-triggered channel, allowing the translocation of C into the cytoplasm, where it can cause cytoskeletal disruption in both insect and mammalian cells. Toxin complexes have been visualized using single-particle electron microscopy, but no high-resolution structures of the components are available, and the role of the B protein in the mechanism of toxicity remains unknown. Here we report the three-dimensional structure of the complex formed between the B and C proteins, determined to 2.5 Å by X-ray crystallography. These proteins assemble to form an unprecedented, large hollow structure that encapsulates and sequesters the cytotoxic, C-terminal region of the C protein like the shell of an egg. The shell is decorated on one end by a ß-propeller domain, which mediates attachment of the B-C heterodimer to the A protein in the native complex. The structure reveals how C auto-proteolyses when folded in complex with B. The C protein is the first example, to our knowledge, of a structure that contains rearrangement hotspot (RHS) repeats, and illustrates a marked structural architecture that is probably conserved across both this widely distributed bacterial protein family and the related eukaryotic tyrosine-aspartate (YD)-repeat-containing protein family, which includes the teneurins. The structure provides the first clues about the function of these protein repeat families, and suggests a generic mechanism for protein encapsulation and delivery.

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.

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.

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.

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.

Three-dimensional structure of the toxin-delivery particle antifeeding prophage of Serratia entomophila.

The Serratia entomophila antifeeding prophage (Afp) is a bullet-shaped toxin-delivery apparatus similar to the R-pyocins of Pseudomonas aeruginosa. Morphologically it resembles the sheathed tail of bacteriophages such as T4, including a baseplate at one end. It also shares features with the type VI secretion systems. Cryo-electron micrographs of tilted Afp specimens (up to 60 degrees) were analyzed to determine the correct cyclic symmetry to overcome the limitation imposed by exclusively side views in nominally untilted specimens. An asymmetric reconstruction shows clear 6-fold cyclic symmetry contrary to a previous conclusion of 4-fold symmetry based on analysis of only the preferred side views (Sen, A., Rybakova, D., Hurst, M. R., and Mitra, A. K. (2010) J. Bacteriol. 192, 4522-4525). Electron tomography of negatively stained Afp revealed right-handed helical striations in many of the particles, establishing the correct hand. Higher quality micrographs of untilted specimens were processed to produce a reconstruction at 2.0-nm resolution with imposed 6-fold symmetry. The helical parameters of the sheath were determined to be 8.14 nm for the subunit rise along and 40.5° for the rotation angle around the helix. The sheath is similar to that of the T4 phage tail but with a different arrangement of the subdomain of the polymerizing sheath protein(s). The central tube is similar to the diameter and axial width of the Hcp1 hexamer of P. aeruginosa type VI secretion system. The tube extends through the baseplate into a needle resembling the "puncture device" of the T4 tail. The tube contains density that may be the toxin and/or a length-determining protein.

Role of antifeeding prophage (Afp) protein Afp16 in terminating the length of the Afp tailocin and stabilizing its sheath.

The Serratia entomophila antifeeding prophage Afp, forms a phage-tail-like particle that acts on the New Zealand grass grub, Costelytra zealandica with a 3-day LD50 of approximately 500 Afp particles per larva. Genes (afp1-18) encoding components of Afp were expressed and their products purified allowing morphological assessment of the products by transmission electron microscopy (TEM). Expression of afp1-15 resulted in the formation of a non-sheathed structure termed the tube-baseplate complex or TBC, composed of an irregular-length tube attached to a baseplate with associated tail fibres. Expression of afp1-16 produced mature, normal-length Afp particles, whereas coexpression of afp16 with afp1-15 in trans resulted in the formation of aberrant Afp particles of variable lengths. A C-terminally truncated Afp16 mutant resulted in a phenotype intermediate between mature Afp and TBC. The addition of purified Afp16 to Afp unravelled by acidic treatment resulted in the formation of shorter tubes when specimen pH was adjusted to 7 than those formed in the absence of Afp16. Analysis of TEM images of purified Afp16 revealed a hexameric ring-like structure similar to that formed by gp3 of phage T4 and gpU of phage λ. Our results suggest that Afp16 terminates tube elongation and is involved in sheath formation.

3D structure of the Yersinia entomophaga toxin complex and implications for insecticidal activity.

Toxin complex (Tc) proteins are a class of bacterial protein toxins that form large, multisubunit complexes. Comprising TcA, B, and C components, they are of great interest because many exhibit potent insecticidal activity. Here we report the structure of a novel Tc, Yen-Tc, isolated from the bacterium Yersinia entomophaga MH96, which differs from the majority of bacterially derived Tcs in that it exhibits oral activity toward a broad range of insect pests, including the diamondback moth (Plutella xylostella). We have determined the structure of the Yen-Tc using single particle electron microscopy and studied its mechanism of toxicity by comparative analyses of two variants of the complex exhibiting different toxicity profiles. We show that the A subunits form the basis of a fivefold symmetric assembly that differs substantially in structure and subunit arrangement from its most well characterized homologue, the Xenorhabdus nematophila toxin XptA1. Histopathological and quantitative dose response analyses identify the B and C subunits, which map to a single, surface-accessible region of the structure, as the sole determinants of toxicity. Finally, we show that the assembled Yen-Tc has endochitinase activity and attribute this to putative chitinase subunits that decorate the surface of the TcA scaffold, an observation that may explain the oral toxicity associated with the complex.

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.

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.

Histopathological effects of the Yen-Tc toxin complex from Yersinia entomophaga MH96 (Enterobacteriaceae) on the Costelytra zealandica (Coleoptera: Scarabaeidae) larval midgut.

Yersinia entomophaga MH96, which was originally isolated from the New Zealand grass grub, Costelytra zealandica, produces an orally active proteinaceous toxin complex (Yen-Tc), and this toxin is responsible for mortality in a range of insect species, mainly within the Coleoptera and Lepidoptera. The genes encoding Yen-Tc are members of the toxin complex (Tc) family, with orthologs identified in several other bacterial species. As the mechanism of Yen-Tc activity remains unknown, a histopathological examination of C. zealandica larvae was undertaken in conjunction with cultured cells to identify the effects of Yen-Tc and to distinguish the contributions that its individual subunit components make upon intoxication. A progressive series of events that led to the deterioration of the midgut epithelium was observed. Additionally, experiments using a cell culture assay system were carried out to determine the cellular effects of intoxication on cells after topical application and the transient expression of Yen-Tc and its individual components. While observations were broadly consistent with those previously reported for other Tc family members, some differences were noted. In particular, the distinct stepwise disintegration of the midgut shared features associated with both apoptosis and necrotic programmed cell death pathways. Second, we observed, for the first time, a contribution of toxicity from two chitinases associated with the Yen-Tc complex. Our findings were suggestive of the activities encoded within the subunit components of Yen-Tc targeting different sites along putative programmed cell death pathways. Given the observed broad host range for Yen-Tc, these targeted loci are likely to be widely shared among insects.

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.

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.

The main virulence determinant of Yersinia entomophaga MH96 is a broad-host-range toxin complex active against insects.

Through transposon mutagenesis and DNA sequence analysis, the main disease determinant of the entomopathogenic bacterium Yersinia entomophaga MH96 was localized to an ~32-kb pathogenicity island (PAI) designated PAI(Ye96). Residing within PAI(Ye96) are seven open reading frames that encode an insecticidal toxin complex (TC), comprising not only the readily recognized toxin complex A (TCA), TCB, and TCC components but also two chitinase proteins that form a composite TC molecule. The central TC gene-associated region (~19 kb) of PAI(Ye96) was deleted from the Y. entomophaga MH96 genome, and a subsequent bioassay of the ΔTC derivative toward Costelytra zealandica larvae showed it to be innocuous. Virulence of the ΔTC mutant strain could be restored by the introduction of a clone containing the entire PAI(Ye96) TC gene region. As much as 0.5 mg of the TC is released per 100 ml of Luria-Bertani broth at 25°C, while at 30 or 37°C, no TC could be detected in the culture supernatant. Filter-sterilized culture supernatants derived from Y. entomophaga MH96, but not from the ΔTC strain grown at temperatures of 25°C or less, were able to cause mortality. The 50% lethal doses (LD50s) of the TC toward diamondback moth Plutella xylostella and C. zealandica larvae were defined as 30 ng and 50 ng, respectively, at 5 days after ingestion. Histological analysis of the effect of the TC toward P. xylostella larva showed that within 48 h after ingestion of the TC, there was a general dissolution of the larval midgut.

Nucleotide sequence of the Serratia entomophila plasmid pADAP and the Serratia proteamaculans pU143 plasmid virulence associated region.

Some strains of Serratia entomophila and S. proteamaculans cause amber disease of the New Zealand grass grub Costelytra zealandica (Coleoptera: Scarabaeidae), an important pasture pest in New Zealand. The disease determinants of S. entomophila, are encoded on a 153,404-bp plasmid, termed pADAP for amber disease associated plasmid. The S. proteamaculans strain 143 (Sp143) exhibits an unusual pathotype, where only 60-70% of C. zealandica larvae infected with the bacterium succumb to disease. DNA sequence analysis of the Sp143 pU143 virulence associated region identified high DNA similarity to the pADAP sep virulence associated region, with DNA sequence variation in the sepA gene and the variable region of the sepC component. No pADAP anti-feeding prophage orthologue was detected in the Sp143 genome. The region of pADAP replication was cloned and found to replicate in S. entomophila but not in Escherichia coli. DNA sequence analysis of the plasmid pSG348 repA gene from the French isolate of Serratia grimesii, identified 93% DNA identity to the pADAP repA gene. A comparison of the pU143 virulence associated region with the completed pADAP nucleotide sequence is given.

Yersinia entomophaga sp. nov., isolated from the New Zealand grass grub Costelytra zealandica.

A Gram-negative, rod-shaped, non-spore-forming bacterium (MH96(T)) was isolated from diseased larvae of the New Zealand grass grub, Costelytra zealandica (Coleoptera: Scarabaeidae). On the basis of 16S rRNA gene sequence similarity, strain MH96(T) is a member of the genus Yersinia, which is a member of the class Gammaproteobacteria. The most similar 16S rRNA gene sequence to that of MH96(T) is that of the type strain of Yersinia mollaretii (98.5 % similarity) followed by those of the type strains of Yersinia aldovae, Y. frederiksenii and Y. rohdei (all 98.4 % similarity). Multilocus sequence typing of five housekeeping genes (dnaJ, glnA, gyrB, groEL and recA) identified Yersinia ruckeri (81-92 % similarity) as the closest relative. The results of DNA-DNA hybridization and physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain MH96(T) from the four most closely related Yersinia species with validly published names, including a Y. ruckeri isolate. Strain MH96(T) therefore represents a novel species, for which the name Yersinia entomophaga sp. nov. is proposed, with the type strain MH96(T) ( = DSM 22339(T)  = ATCC BAA-1678(T)).