Noël Boemare

The genome sequence of the entomopathogenic bacterium Photorhabdus luminescens.

Photorhabdus luminescens is a symbiont of nematodes and a broad-spectrum insect pathogen. The complete genome sequence of strain TT01 is 5,688,987 base pairs (bp) long and contains 4,839 predicted protein-coding genes. Strikingly, it encodes a large number of adhesins, toxins, hemolysins, proteases and lipases, and contains a wide array of antibiotic synthesizing genes. These proteins are likely to play a role in the elimination of competitors, host colonization, invasion and bioconversion of the insect cadaver, making P. luminescens a promising model for the study of symbiosis and host-pathogen interactions. Comparison with the genomes of related bacteria reveals the acquisition of virulence factors by extensive horizontal transfer and provides clues about the evolution of an insect pathogen. Moreover, newly identified insecticidal proteins may be effective alternatives for the control of insect pests.

Stages of Infection during the Tripartite Interaction between Xenorhabdus nematophila, Its Nematode Vector, and Insect Hosts

ABSTRACT Bacteria of the genus Xenorhabdus are mutually associated with entomopathogenic nematodes of the genus Steinernema and are pathogenic to a broad spectrum of insects. The nematodes act as vectors, transmitting the bacteria to insect larvae, which die within a few days of infection. We characterized the early stages of bacterial infection in the insects by constructing a constitutive green fluorescent protein (GFP)-labeled Xenorhabdus nematophila strain. We injected the GFP-labeled bacteria into insects and monitored infection. We found that the bacteria had an extracellular life cycle in the hemolymph and rapidly colonized the anterior midgut region in Spodoptera littoralis larvae. Electron microscopy showed that the bacteria occupied the extracellular matrix of connective tissues within the muscle layers of the Spodoptera midgut. We confirmed the existence of such a specific infection site in the natural route of infection by infesting Spodoptera littoralis larvae with nematodes harboring GFP-labeled Xenorhabdus. When the infective juvenile (IJ) nematodes reached the insect gut, the bacterial cells were rapidly released from the intestinal vesicle into the nematode intestine. Xenorhabdus began to escape from the anus of the nematodes when IJs were wedged in the insect intestinal wall toward the insect hemolymph. Following their release into the insect hemocoel, GFP-labeled bacteria were found only in the anterior midgut region and hemolymph of Spodoptera larvae. Comparative infection assays conducted with another insect, Locusta migratoria, also showed early bacterial colonization of connective tissues. This work shows that the extracellular matrix acts as a particular colonization site for X. nematophila within insects.

The PhlA Hemolysin from the Entomopathogenic Bacterium Photorhabdus luminescens Belongs to the Two-Partner Secretion Family of Hemolysins

Photorhabdus is an entomopathogenic bacterium symbiotically associated with nematodes of the family Heterorhabditidae. Bacterial hemolysins found in numerous pathogenic bacteria are often virulence factors. We describe here the nucleotide sequence and the molecular characterization of the Photorhabdus luminescens phlBA operon, a locus encoding a hemolysin which shows similarities to the Serratia type of hemolysins. It belongs to the two-partner secretion (TPS) family of proteins. In low-iron conditions, a transcriptional induction of the phlBA operon was observed by using the chloramphenicol acetyltransferase reporter gene, causing an increase in PhlA hemolytic activity compared to iron-rich media. A spontaneous phase variant of P. luminescens was deregulated in phlBA transcription. The phlA mutant constructed by allelic exchange remained highly pathogenic after injection in the lepidopteran Spodoptera littoralis, indicating that PhlA hemolysin is not a major virulence determinant. Using the gene encoding green fluorescent protein as a reporter, phlBA transcription was observed in hemolymph before insect death. We therefore discuss the possible role of PhlA hemolytic activity in the bacterium-nematode-insect interactions.

Phenotypic and DNA Relatedness between Nematode Symbionts and Clinical Strains of the Genus Photorhabdus (Enterobacteriaceae)

Bacterial strains isolated from wide ranges of nematode hosts and geographic sources and strains isolated from human clinical specimens were used to assess the taxonomic structure of the genus Photorhabdus. The following two methods were used: DNA relatedness and phenotypic characterization. Analysis of the DNA relatedness data revealed that all of the strains studied were congeneric and that the genus Photorhabdus is, on the basis of DNA relatedness data, more homogeneous than the other genus of nematode-symbiotic bacteria, the genus Xenorhabdus. In contrast to previous reports, only two DNA relatedness groups were identified in the genus Photorhabdus. These groups corresponded to the symbiotic strains and the clinical strains. There appeared to be some subgroups within the symbiotic strain group on the basis of the interactions of the strains with nematodes, which corresponded to some extent with the DNA relatedness data. However, there were significant ambiguities in the DNA relatedness data, and this group could not be subdivided on the basis of DNA relatedness data or phenotypic data. The distinct functional differences within and between the DNA relatedness groups of symbiotic Photorhabdus strains indicated that there are biologically significant sub-groups within the genus Photorhabdus that cannot be defined at this time. Further investigation of the taxonomy of Photorhabdus by using different approaches and a suitably wide range of strains is recommended. However, it is clear that the clinical strains form a recognizable subgroup within the genus even though no formal subtaxon can be defined at this time.

Deoxyribonucleic Acid Relatedness and Phenotypic Study of the Genus Xenorhabdus

A collection of 30 clones derived from 20 luminous and nonluminous Xenorhabdus strains was studied by using deoxyribonucleic acid (DNA) relatedness and electron microscopy, as well as growth factor requirement, nutritional ability, and other biochemical tests. Nonluminous Xenorhabdus cells each contained a crystal-like structure, whereas the cells of luminous strains each contained an accumulation of amorphous material associated with mesosomes and microtubules of unknown function. Two DNA relatedness groups of nonluminous strains were delineated, one of which corresponded to Xenorhabdus nematophilus, a species associated with the nematode Steinernema feltiae (= Neoplectana carpocapsae). The other group contained yellow-pigmented bacteria associated with Steinernema bibionis and new Steinernema species. No definite phenotypic characteristic (except pigmentation) separated the two DNA relatedness groups of nonluminous Xenorhabdus strains. Four DNA relatedness groups were delineated among the luminous clones studied. These four groups could be separated by biochemical tests. Unfortunately, different clones derived from the same strain belonged to different DNA relatedness groups. This happened with the type strain of Xenorhabdus luminescens.

Variation in the Effectors of the Type III Secretion System among Photorhabdus Species as Revealed by Genomic Analysis

Entomopathogenic bacteria of the genus Photorhabdus harbor a type III secretion system. This system was probably acquired prior to the separation of the species within this genus. Furthermore, the core components of the secretion machinery are highly conserved but the predicted effectors differ between Photorhabdus luminescens and P. asymbiotica, two highly related species with different hosts.

Whole-Genome Comparison between Photorhabdus Strains To Identify Genomic Regions Involved in the Specificity of Nematode Interaction

The bacterium Photorhabdus establishes a highly specific association with Heterorhabditis, its nematode host. Photorhabdus strains associated with Heterorhabditis bacteriophora or Heterorhabditis megidis were compared using a Photorhabdus DNA microarray. We describe 31 regions belonging to the Photorhabdus flexible gene pool. Distribution analysis of regions among the Photorhabdus genus identified loci possibly involved in nematode specificity.

Steinernema xueshanense n. sp. (Rhabditida, Steinernematidae), a new species of entomopathogenic nematode from the province of Yunnan, southeast Tibetan Mts., China

During a random EPN survey in the northern part of the Yunnan province (southeastern Tibet, Dequen district, town of Dequen) in 2005, soil samples containing an unknown EPN species were collected. The new species is described herein as Steinernema xueshannense n. sp. named after the Xue Shan Mts. a mountain range between Yunnan and Tibet where the nematode was collected. The isolate is a new species belonging to the Steinernema feltiae /kraussei group. S. xueshanense n. sp. is characterized by male, female of both generations and infective juveniles (IJ). IJ lateral field with eight ridges, submarginal pair less distinct, formula 2, 7, 8, 7, 6, 4, 2, Hyaline portion occupies approximately one half of tail length. Second-generation males with distinct mucron and moderately curved spicules. Females wuth a characteristic cone on the tail. Infective juveniles of S. xueshanense n. sp. differ from S. akhursti, S. cholashanense S. kraussei, S. oregonense by different number of ridges in lateral fields. Species which have same number of eight lateral ridges, such as S. silvaticum, S. thanhi, S. weiseri differs from S. xueshanense n. sp. by less prominent sublateral pair whereas those species have all ridges equally spaced and prominent. Only S. feltiae possess the same pattern of ridges as S. xueshanense n. sp., but this species differ by shape of spicules with oblongate manubrium. The description of S. sangi gives only a few characters to compare this species with S. xueshanense n. sp. The exception is the excretory pore position of IJs which is at 40% of pharynx length whereas in other species of this group, including S. xueshanense n. sp., it is situated approximately at 50%. Cross-breedings, sequences of ITS and D2/D3 regions of the ribosomal DNA confirmed the new species identity.

Cloning and nucleotide sequence of a flagellin encoding genetic locus from Xenorhabdus nematophilus: phase variation leads to differential transcription of two flagellar genes (fliCD)

The insect-pathogenic bacterium Xenorhabdus undergoes spontaneous phase variation involving a large number of phenotypes. Our previous study indicated that phase I variants were motile, whereas phase II variants of X. nematophilus F1 were nonflagellated cells which did not synthesize flagellin [Givaudan A., Baghdiguian, S., Lanois, A. and Boemare, N. (1995) Appl. Environ. Microbiol. 61, 1408-1413]. In order to approach the study of the flagellar switching, a locus containing two ORFs from X. nematophilus F1 (phase I) was identified by using functional complementation of flagellin-negative E. coli. The sequence analysis revealed that the first ORF corresponds to the fliC gene coding for flagellin, and showed a high degree of homology between the N-terminal and C-terminal of Xenorhabdus FliC and flagellins from other bacteria. The second identified ORF in the opposite orientation encodes a homologue of the enterobacterial hook-associated protein 2, FliD. Both Xenorhabdus fliCD genes were required for the entire restoration of E. coli motility. A sequence highly homologous to the sigma 28 consensus promoter was identified upstream from the coding sequences from both genes. The structure of the fliC gene and its surrounding region was shown to be the same in both phase variants, but Northern blot analysis revealed that fliC and fliD were, respectively, not and weakly transcribed in phase II variants. In addition, complementation experiments showed that motility and flagellin synthesis of phase II cannot be recovered by placing in trans fliCD genes from phase I. These latter results suggest that a gene(s) higher in the transcriptional hierarchy of the flagellar regulon is switched off in Xenorhabdus phase II variants.