Characterization of the pixB gene in Xenorhabdus nematophila and discovery of a new gene family.

Xenorhabdus nematophila are Gram-negative bacteria that engage in mutualistic associations with entomopathogenic nematodes. To reproduce, the nematodes invade insects and release X. nematophila into the haemolymph where it functions as an insect pathogen. In complex medium, X. nematophila cells produce two distinct types of intracellular crystalline inclusions, one composed of the methionine-rich PixA protein and the other composed of the PixB protein. Here we show that PixB crystalline inclusions were neither apparent in X. nematophila cells grown in medium that mimics insect haemolymph (Grace's medium) nor in cells grown directly in the insect haemocoel. The identified pixB gene was regulated by a conserved σ70 promoter while the pixA promoter was less well conserved. Expression of pixA and pixB under biological conditions was analysed using GFP promoter reporters. Microplate fluorescence detection and flow cytometry analyses revealed that pixB was expressed at high levels in Grace's medium and in insect haemolymph and at lower levels in complex medium, while pixA was expressed at lower levels under all conditions. Although pixB was highly expressed in Grace's medium, PixB crystalline inclusions were not present, suggesting that under biological conditions PixB production may be controlled post-transcriptionally. Although a pixB-minus strain was constructed, the function of PixB remains unresolved. The pixB gene was present in few Xenorhabdus species and pixB-type genes were identified in some Proteobacteria and Gram-positive species, while pixA was only present in Xenorhabdus species. Two conserved sequences were identified in PixB-type proteins that characterize this previously unrecognized gene family.

Method for fluorescent marker swapping and its application in Steinernema nematode colonization studies.

An allelic exchange vector was constructed to replace gfp by mCherry in bacteria previously tagged with mini-Tn5 derivatives. The method was successfully applied to a gfp-labeled Yersinia pseudotuberculosis strain and the re-engineered bacterium was used to study the colonization of Steinernema nematodes hosting their Xenorhabdus symbiont using dual-color confocal microscopy.

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).

New insights into the colonization and release processes of Xenorhabdus nematophila and the morphology and ultrastructure of the bacterial receptacle of its nematode host, Steinernema carpocapsae.

We present results from epifluorescence, differential interference contrast, and transmission electron microscopy showing that Xenorhabdus nematophila colonizes a receptacle in the anterior intestine of the infective juvenile (IJ) stage of Steinernema carpocapsae. This region is connected to the esophagus at the esophagointestinal junction. The process by which X. nematophila leaves this bacterial receptacle had not been analyzed previously. In this study we monitored the movement of green fluorescent protein-labeled bacteria during the release process. Our observations revealed that Xenorhabdus colonizes the distal region of the receptacle and that exposure to insect hemolymph stimulated forward movement of the bacteria to the esophagointestinal junction. Continued exposure to hemolymph caused a narrow passage in the distal receptacle to widen, allowing movement of Xenorhabdus down the intestine and out the anus. Efficient release of both the wild type and a nonmotile strain was evident in most of the IJs incubated in hemolymph, whereas only a few IJs incubated in nutrient-rich broth released bacterial cells. Incubation of IJs in hemolymph treated with agents that induce nematode paralysis dramatically inhibited the release process. These results suggest that bacterial motility is not required for movement out of the distal region of the receptacle and that hemolymph-induced esophageal pumping provides a force for the release of X. nematophila out of the receptacle and into the intestinal lumen.

Inactivation of ompR promotes precocious swarming and flhDC expression in Xenorhabdus nematophila.

The response regulator OmpR is involved in numerous adaptive responses to environmental challenges. The role that OmpR plays in swarming behavior and swarm-cell differentiation in the symbiotic-pathogenic bacterium Xenorhabdus nematophila was examined in this study. Swarming began 4 h sooner in an ompR mutant strain than in wild-type cells. Precocious swarming was correlated with elevated expression of fliC, early flagellation, and cell elongation. The level of flhDC mRNA was elevated during the early period of swarming in the ompR strain relative to the level in the wild type. These findings show that OmpR is involved in the temporal regulation of flhDC expression and flagellum production and demonstrate that this response regulator plays a role in the swarming behavior of X. nematophila.