Transcriptional analysis of a Photorhabdus sp. variant reveals transcriptional control of phenotypic variation and multifactorial pathogenicity in insects.

Photorhabdus luminescens lives in a mutualistic association with entomopathogenic nematodes and is pathogenic for insects. Variants of Photorhabdus frequently arise irreversibly and are studied because they have altered phenotypic traits that are potentially important for the host interaction. VAR* is a colonial and phenotypic variant displaying delayed pathogenicity when directly injected into the insect, Spodoptera littoralis. In this study, we evaluated the role of transcriptomic modulation in determining the phenotypic variation and delayed pathogenicity of VAR* with respect to the corresponding wild-type form, TT01α. A P. luminescens microarray identified 148 genes as differentially transcribed between VAR* and TT01α. The net regulator status of VAR* was found to be significantly modified. We also observed in VAR* a decrease in the transcription of genes supporting certain phenotypic traits, such as pigmentation, crystalline inclusion, antibiosis, and protease and lipase activities. Three genes encoding insecticidal toxins (pit and pirB) or putative insecticidal toxins (xnp2) were less transcribed in VAR* than in the TT01α. The overexpression of these genes was not sufficient to restore the virulence of VAR* to the levels of ΤΤ01α, which suggests that the lower virulence of VAR* does not result from impaired toxemia in insects. Three loci involved in oxidative stress responses (sodA, katE, and the hca operon) were found to be downregulated in VAR*. This is consistent with the greater sensitivity of VAR* to H(2)O(2) and may account for the impaired bacteremia in the hemolymph of S. littoralis larvae observed with VAR*. In conclusion, we demonstrate here that some phenotypic traits of VAR* are regulated transcriptionally and highlight the multifactorial nature of pathogenicity in insects.

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.

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.

flhDC, the flagellar master operon of Xenorhabdus nematophilus: requirement for motility, lipolysis, extracellular hemolysis, and full virulence in insects.

Xenorhabdus is a major insect pathogen symbiotically associated with nematodes of the family Steinernematidae. This motile bacterium displays swarming behavior on suitable media, but a spontaneous loss of motility is observed as part of a phenomenon designated phase variation which involves the loss of stationary-phase products active as antibiotics and potential virulence factors. To investigate the role of one of the transcriptional activators of flagellar genes, FlhDC, in motility and virulence, the Xenorhabdus nematophilus flhDC locus was identified by functional complementation of an Escherichia coli flhD null mutant and DNA sequencing. Construction of X. nematophilus flhD null mutants confirmed that the flhDC operon controls flagellin expression but also revealed that lipolytic and extracellular hemolysin activity is flhDC dependent. We also showed that the flhD null mutant displayed a slightly attenuated virulence phenotype in Spodoptera littoralis compared to that of the wild-type strain. Thus, these data indicated that motility, lipase, hemolysin, or unknown functions controlled by the flhDC operon are involved in the infectious process in insects. Our investigation expands the view of the flagellar regulon as a checkpoint coupled to a major network involving bacterial physiological aspects as well as motility.

Swarming and Swimming Changes Concomitant with Phase Variation in Xenorhabdus nematophilus.

Xenorhabdus spp., entomopathogenic bacteria symbiotically associated with nematodes of the family Steinernematidae, occur spontaneously in two phases. Phase I, the variant naturally isolated from the infective-stage nematode, provides better conditions than the phase II variant for nematode reproduction. This study has shown that Xenorhabdus phase I variants displayed a swarming motility when they were grown on a suitable solid medium (0.6 to 1.2% agar). Whereas most of the phase I variants from different Xenorhabdus spp. were able to undergo cycle of rapid and coordinately population migration over the surface, phase II variants were unable to swarm and even to swim in semisolid agar, particularly in X. nematophilus. Optical and electron microscopic observations showed nonmotile cells with phase II variants of X. nematophilus F1 which lost their flagella. Flagellar filaments from strain F1 phase I variants were purified, and the molecular mass of the flagellar structural subunit was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 36.5 kDa. Flagellin from cellular extracts or culture medium of phase II was undetectable with antiserum against the denatured flagellin by immunoblotting analysis. This suggests that the lack of flagella in phase II cells is due to a defect during flagellin synthesis. The importance of such a difference of motility between both phases is discussed in regard to adaptation of these bacteria to the insect prey and the nematode host.

Fast and accurate identification of Xenorhabdus and Photorhabdus species by restriction analysis of PCR-amplified 16S rRNA genes.

Thirteen bacterial strains of Xenorhabdus and 14 strains of Photorhabdus originating from a wide range of geographical and nematode host sources were typed by analyzing 16S rRNA gene (rDNA) restriction patterns obtained after digestion of PCR-amplified 16S rDNAs. Eight tetrameric restriction endonucleases were examined. A total of 17 genotypes were identified, forming two heterogeneous main clusters after analysis by the unweighted pair-group method using arithmetic averages: group I included all Xenorhabdus species and strains, symbionts of Steinernema, whereas group II encompassed the Photorhabdus strains, symbionts of Heterorhabditis. To identify the four valid species of Xenorhabdus and unclassified strains and all the genotypes of Photorhabdus luminescens, three restriction enzymes are required: CfoI, AluI, and HaeIII. Our results, in substantial agreement with DNA-DNA pairing and 16S rDNA sequence data, indicate that amplified 16S rDNA restriction analysis is a simple and accurate tool for identifying entomopathogenic nematode bacterial symbionts.

Gnotobiological study of infective juveniles and symbionts of Steinernema scapterisci: A model to clarify the concept of the natural occurrence of monoxenic associations in entomopathogenic nematodes.

Gnotobiology of Steinernema scapterisci and bacteriological study of its symbiont confirmed that this nematode harbors a symbiotic species of Xenorhabdus, as do other Steinermena species. Based on phenotypic and 16S rDNA data, this Xenorhabdus strain UY61 could be distinguished from other Xenorhabdus species. Bacteria reported previously as being associated with this nematode and belonging to several other genera were probably contaminating bacteria located in the intercuticular space of the infective juveniles (IJs). These bacteria were detrimental to nematode reproduction in Galleria mellonella. Axenic S. scapterisci and its symbiont Xenorhabdus strain UY61 alone were not pathogenic to G. mellonella. The combination of both partners reestablished the pathogenicity of the complex toward G. mellonella. This combination also gave the best yields of IJs when produced in this insect and in vitro production on artificial diet.

Cobalamin (vitamin B12) biosynthesis: identification and characterization of a Bacillus megaterium cobI operon.

A 16 kb DNA fragment has been isolated from a Bacillus megaterium genomic library and fully sequenced. The fragment contains 15 open reading frames, 14 of which are thought to constitute a B. megaterium cobalamin biosynthetic (cob) operon. Within the operon, 11 genes display similarity to previously identified Salmonella typhimurium cobalamin biosynthetic genes (cbiH60, -J, -C, -D, -ET, -L, -F, -G, -A, cysGA and btuR), whereas three do not (cbiW, -X and -Y). The genes of the B. megaterium cob operon were compared with the cobalamin biosynthetic genes of Pseudomonas denitrificans, Methanococcus jannaschii and Synechocystis sp. Taking into account the presence of cbiD and cbiG, the absence of a cobF, cobG and cobN, -S and -T, it was concluded that B. megaterium, M. jannaschii and Synechocystis sp., like S. typhimurium, synthesize cobalamin by an anaerobic pathway, in which cobalt is added at an early stage and molecular oxygen is not required.

Cobalamin (vitamin B12) biosynthesis: functional characterization of the Bacillus megaterium cbi genes required to convert uroporphyrinogen III into cobyrinic acid a,c-diamide.

The function of individual genes of the Bacillus megaterium cobI operon genes in cobalamin (vitamin B12) biosynthesis was investigated by their ability to complement defined Salmonella typhimurium cob mutants. This strategy confirmed the role of cbiA, -D, -F, -J, -L and cysGA. Furthermore the operon as a whole was used to restore corrin biosynthesis in Escherichia coli, which, although closely related to S. typhimurium, does not possess the CobI pathway. When the B. megaterium cob operon was cloned into a plasmid and transformed into an E. coli strain containing the S. typhimurium cbiP, it conferred upon the host strain the ability to make the cobyric acid de novo. However, cobyric acid synthesis was observed only when the strain was grown anaerobically. Derivatives of the corrin-producing E. coli strain were constructed in which genes of the B. megaterium cob operon had been inactivated. These strains were used to demonstrate that, whereas B. megaterium cbiD, -G and -X are essential for cobyric acid synthesis, the cbiW and -Y genes could be deleted without detriment to cobyric acid production in E. coli.

Salmonella typhimurium cobalamin (vitamin B12) biosynthetic genes: functional studies in S. typhimurium and Escherichia coli.

In order to study the Salmonella typhimurium cobalamin biosynthetic pathway, the S. typhimurium cob operon was isolated and cloned into Escherichia coli. This approach has given the new host of the cob operon the ability to make cobalamins de novo, an ability that had probably been lost by this organism. In total, 20 genes of the S. typhimurium cob operon have been transferred into E. coli, and the resulting recombinant strains have been shown to produce up to 100 times more corrin than the parent S. typhimurium strain. These measurements have been performed with a quantitative cobalamin microbiological assay which is detailed in this work. As with S. typhimurium, cobalamin synthesis is only observed in the E. coli cobalamin-producing strains when they are grown under anaerobic conditions. Derivatives of the cobalamin-producing E. coli strains were constructed in which genes of the cob operon were inactivated. These strains, together with S. typhimurium cob mutants, have permitted the determination of the genes necessary for cobalamin production and classification of cbiD and cbiG as cobl genes. When grown in the absence of endogenous cobalt, the oxidized forms of precorrin-2 and precorrin-3, factor II and factor III, respectively, were found to accumulate in the cytosol of the corrin-producing E. coli. Together with the finding that S. typhimurium cbiL mutants are not complemented with the homologous Pseudomonas denitrificans gene, these results lend further credence to the theory that cobalt is required at an early stage in the biosynthesis of cobalamins in S. typhimurium.