Type 6 secretion system components hcp and vgrG support mutualistic partnership between Xenorhabdus bovienii symbiont and Steinernema jollieti host.

Xenorhabdus, like other Gram-negative bacteria, possesses a Type 6 Secretion System (T6SS) which acts as a contact-dependent molecular syringe, delivering diverse proteins (effectors) directly into other cells. The number of T6SS loci encoded in Xenorhabdus genomes are variable both at the inter and intraspecific level. Some environmental isolates of Xenorhabdus bovienii, encode at least one T6SS locus while others possess two loci. Previous work conducted by our team demonstrated that X. bovienii [Jollieti strain SS-2004], which has two T6SSs (T6SS-1 and T6SS-2), hcp genes are required for biofilm formation. Additionally, while T6SS-1 hcp gene plays a role in the antibacterial competition, T6SS-2 hcp does not. In this study, we tested the hypothesis that vgrG genes are also involved in mutualistic and pathogenic interactions. For this purpose, targeted mutagenesis together with wet lab experiments including colonization, competition, biofilm, and virulence experiments, were carried out to assess the role of vgrG in the mutualistic and antagonistic interactions in the life cycle of XBJ. Our results revealed that vgrG genes are not required for biofilm formation but play a role in outcompeting other Xenorhabdus bacteria. Additionally, both vgrG and hcp genes are required to fully colonize the nematode host. We also demonstrated that hcp and vgrG genes in both T6SS clusters are needed to support the reproductive fitness of the nematodes. Overall, results from this study revealed that in X. bovieni jollieti strain, the twoT6SS clusters play an important role in the fitness of the nematodes in relation to colonization and reproduction. These results lay a foundation for further investigations on the functional significance of T6SSs in the mutualistic and pathogenic lifecycle of Xenorhabdus spp.

Variable virulence phenotype of Xenorhabdus bovienii (γ-Proteobacteria: Enterobacteriaceae) in the absence of their vector hosts.

Xenorhabdus bovienii bacteria have a dual lifestyle: they are mutualistic symbionts to many species of Steinernema nematodes and are pathogens to a wide array of insects. Previous studies have shown that virulence of X.bovienii-Steinernema spp. pairs decreases when the nematodes associate with non-cognate bacterial strains. However, the virulence of the X. bovienii strains alone has not been fully investigated. In this study, we characterized the virulence of nine X. bovienii strains in Galleria mellonella and Spodoptera littoralis and performed a comparative genomic analysis to correlate observed phenotypes with strain genotypes. Two X. bovienii strains were found to be highly virulent against the tested insect hosts, while three strains displayed attenuated insect virulence. Comparative genomic analyses revealed the presence of several clusters present only in virulent strains, including a predicted type VI secretion system (T6SS). We performed intra-species-competition assays, and showed that the virulent T6SS+ strains generally outcompeted the less virulent T6SS- strains. Thus, we speculate that the T6SS in X. bovienii may be another addition to the arsenal of antibacterial mechanisms expressed by these bacteria in an insect, where it could potentially play three key roles: (1) competition against the insect host microbiota; (2) protection of the insect cadaver from necrotrophic microbial competitors; and (3) outcompeting other Xenorhabdus species and/or strains when co-infections occur.

Morphology and ultrastructure of the bacterial receptacle in Steinernema nematodes (Nematoda: Steinernematidae).

Infective juveniles of entomopathogenic nematodes in the genus Steinernema harbor symbiotic bacteria, Xenorhabdus spp., in a discrete structure located in the anterior portion of the intestine known as the 'bacterial receptacle' (formerly known as the bacterial or intestinal vesicle). The receptacle itself is a structured environment in which the bacteria are spatially restricted. Inside this receptacle, bacterial symbionts are protected from the environment and grow to fill the receptacle. Until now, no comparative study across different Steinernema spp. has been undertaken to investigate if morphological variation in this structure exists at the interspecific level. In this study, we examined the bacterial receptacles of 25 Steinernema spp. representatives of the currently accepted five evolutionary clades. Our observations confirmed the bacterial receptacle is a modification of the two most anterior cells of the ventricular portion of the intestine. Size of the bacterial receptacle varied across the examined species. Steinernema monticolum (clade II) had the largest receptacle of all examined species (average: 46×17 µm) and S. rarum (no clade affiliation) was noted as the species with the smallest observed receptacle (average: 8×5 µm). At the morphological level, species can be grouped into two categories based on the presence or absence of vesicle within the receptacle. The receptacles of all examined species harbored an intravesicular structure (IVS) with variable morphology. All examined taxa members of the 'feltiae' (clade III) and 'intermedium' (clade II) clades were characterized by having a vesicle. This structure was also observed in S. diaprepesi (clade V), S. riobrave (clade IV) and S. monticolum (clade I).

The rectal glands of Heterorhabditis bacteriophora (Rhabditida: Heterorhabditidae) hermaphrodites and their role in symbiont transmission.

Differential interference contrast, transmission electron and epifluorescence microscopy techniques were employed to examine the ultrastructure of the rectal glands in Heterorhabditis bacteriophora hermaphrodites, with special attention to the location of Photorhabdus bacteria symbionts within these structures. Three rectal glands were clearly visualized in all examined specimens, with two glands positioned sub-ventrally and another gland located dorsally. The dorsal rectal gland in all examined specimens is larger than the subventral ones. Our observations indicate that Photorhabdus bacteria do not colonize the rectal glands of H. bacteriophora hermaphrodites, but rather are present in the most posterior-intestinal cells.

Diversity and distribution of entomopathogenic nematodes (Steinernematidae, Heterorhabditidae) in South Africa.

A total of 1506 soil samples from different habitats in seven geographic regions of South Africa were evaluated for the presence of entomopathogenic nematodes (EPN). Nematodes were isolated from 5% of the samples. Among the steinernematids, four Steinernema sp. were recovered including Steinernema khoisanae and three new undescribed species. Although steinernematids were recovered from both humid subtropical and semiarid regions, this family accounted for 80% of EPN recovered from the semiarid climate zones characterised by sandy, acidic soils. Eight isolates of S. khoisanae were recovered from the Western Cape province. One of the new undescribed steinernematids (Steinernema sp. 1) was recovered only from the Free State and KwaZulu-Natal provinces where humid subtropical conditions prevail and soils are generally less acidic with higher clay content. A high level of adaptation, however, was noted with Steinernema sp. 2, which was recovered from a wide range of soil conditions and habitats ranging from semiarid (Western Cape province) to humid subtropical (KwaZulu-Natal province). A third undescribed steinernematid, Steinernema sp. 3, seemed better adapted to heavier soils with more than 80% of isolates recovered from fruit orchards in the Free State province. Heterorhabditis bacteriophora was the only heterorhabditid recovered during this survey. This species was particularly prevalent in four provinces ranging from humid subtropical to semiarid regions. Isolation of EPN directly from insect cadavers included Steinernema sp. 2 and one H. bacteriophora from an unidentified white grub (Scarabaeidae) cadaver (i.e., dual infection) and H. bacteriophora from the black vine weevil, Otiorhynchus sulcatus.

Biosynthesis of the cyclooligomer depsipeptide bassianolide, an insecticidal virulence factor of Beauveria bassiana.

Beauveria bassiana is a facultative entomopathogen with an extremely broad host range that is used as a commercial biopesticide for the control of insects of agricultural, veterinary and medical significance. B. bassiana produces bassianolide, a cyclooligomer depsipeptide secondary metabolite. We have cloned the bbBsls gene of B. bassiana encoding a nonribosomal peptide synthetase (NRPS). Targeted inactivation of the B. bassiana genomic copy of bbBsls abolished bassianolide production, but did not affect the biosynthesis of beauvericin, another cyclodepsipeptide produced by the strain. Comparative sequence analysis of the BbBSLS bassianolide synthetase revealed enzymatic domains for the iterative synthesis of an enzyme-bound dipeptidol monomer intermediate from d-2-hydroxyisovalerate and l-leucine. Further BbBSLS domains are predicted to catalyze the formation of the cyclic tetrameric ester bassianolide by recursive condensations of this monomer. Comparative infection assays against three selected insect hosts established bassianolide as a highly significant virulence factor of B. bassiana.

Phylogeny of Cephalobina (Nematoda): molecular evidence for recurrent evolution of probolae and incongruence with traditional classifications.

Nematodes of the suborder Cephalobina include an ecologically and morphologically diverse array of species that range from soil-dwelling microbivores to parasites of vertebrates and invertebrates. Despite a long history of study, certain of these microbivores (Cephaloboidea) present some of the most intractable problems in nematode systematics; the lack of an evolutionary framework for these taxa has prevented the identification of natural groups and inhibited understanding of soil biodiversity and nematode ecology. Phylogenetic analyses of ribosomal (LSU) sequence data from 53 taxa revealed strong support for monophyly of taxa representing the Cephaloboidea, but do not support the monophyly of most genera within this superfamily. Historically these genera have primarily been recognized based on variation in labial morphology, but molecular phylogenies show the same general labial (probolae) morphotype often results from recurrent similarity, a result consistent with the phenotypic plasticity of probolae previously observed for some species in ecological time. Phylogenetic analyses of LSU rDNA also recovered strong support for some other groups of cephalobs, including taxa representing most (but not all) Panagrolaimoidea. In addition to revealing homoplasy of probolae, molecular trees also imply other unexpected patterns of character evolution or polarity, including recurrent similarity of offset spermatheca presence, and representation of complex probolae as the ancestral condition within Cephaloboidea. For Cephalobidae, molecular trees do not support traditional genera as natural groups, but it remains untested if deconstructing probolae morphotypes or other structural features into finer component characters may reveal homologies that help delimit evolutionary lineages.