Andras Fodor

Prepupal larval mosaics in Drosophila melanogaster

THE metamorphosis of insects provides a model system for the study of hormone action. In Drosophila, metamorphosis is initiated by the formation of a puparium with a rigid cuticle that becomes tanned early in the prepupal period. Subsequently many larval tissues degenerate and the imaginal tissues develop to produce the adult insect. These processes are initiated in Drosophila by the steroid moulting hormone, β-ecdysone1. We are interested in the mechanisms by which β-ecdysone elicits adult development. The recovery of mutants which cannot respond to the hormone would facilitate investigation into the nature of the action of β-ecdysone. We have isolated systematically a series of late larval and prepupal X-linked lethals (ref. 2 and unpublished results of I.K. et al.), about 15% of which are non-pupariating (npr) lethals in which metamorphosis is not initiated. We have investigated whether the npr condition is a result of the autonomous inability of the mutant target tissues to respond normally to β-ecdysone or rather is a stage-specific failure in the production of the hormone. Our results indicate that in most npr lethals the tissue cannot respond to the hormone. Futhermore, we estimate that there are 100–200 such lethals in the genome of D. melanogaster.

Phylogenetic evidence for the taxonomic heterogeneity of Photorhabdus luminescens.

The sequences of the 16S rRNA gene of 40 strains of bacterial symbionts isolated from the nematodes Heterorhabditis spp. and seven bacterial symbionts of the nematodes Steinernema spp. which were isolated from different geographical areas, as well as the type strain of Xenorhabdus japonicus, were determined and compared to each other and to the sequences of several reference strains of members of the Enterobacteriaceae. The data confirmed the separate status of the two genera of symbionts of entomopathogenic rhabditid nematodes. The symbionts of Heterorhabditis spp. clustered with the type strain of Photorhabdus luminescens, while the symbionts of Steinernema spp. grouped with Xenorhabdus species. X. japonicus clustered with the other Xenorhabdus species. Phylogenetic analysis of 15 almost complete 16S ribosomal DNA (rDNA) sequences of the Heterorhabditis symbionts indicated that there were several subclusters. The properties correlated with these subclusters are not yet apparent, although there may be some geographical and ecological correlations. For example, among the nematode-symbiotic bacteria, the members of subclusters I and III are from southeastern and midwestern North America, respectively, while the members of subclusters II and IV are primarily from Europe and Australia, respectively. The nonsymbiotic strains of P. luminescens form a highly homologous subcluster by themselves. The results of DNA-DNA hybridization studies performed with a few selected strains of five of the 16S rDNA subclusters support the existence of several genospecies within P. luminescens.

Comparison of proteolytic activities produced by entomopathogenic Photorhabdus bacteria: strain- and phase-dependent heterogeneity in composition and activity of four enzymes.

ABSTRACT Twenty strains (including eight phase variant pairs) of nematode-symbiotic and insect-pathogenic Photorhabdus bacteria were examined for the production of proteolytic enzymes by using a combination of several methods, including gelatin liquefaction, zymography coupled to native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and activity measurement with two chromogen substrate types. Four protease activities (∼74, ∼55, ∼54, and ∼37 kDa) could be separated. The N-terminal sequences of three of the proteases were determined, and a comparison with sequences in databases allowed identification of these proteases as HEXXH metallopeptidases. Thus, the 74-kDa protease (described formerly as Php-B [J. Marokházi, G. Kóczán, F. Hudecz, L. Gráf, A. Fodor, and I. Venekei, Biochem. J. 379:633-640, 2004) is an ortholog of OpdA, a member the thimet oligopeptidase family, and the 55-kDa protease is an ortholog of PrtA, a HEXXH+H peptidase in clan MB (metzincins), while the 37-kDa protease (Php-C) belongs to the HEXXH+E peptidases in clan MA. The 54-kDa protease (Php-D) is a nonmetalloenzyme. PrtA and Php-C were zymographically detected, and they occurred in several smaller forms as well. OpdA could not be detected by zymography. PrtA, Php-C, and Php-D were secreted proteases; OpdA, in contrast, was an intracellular enzyme. OpdA activity was found in every strain tested, while Php-D was detected only in the Brecon/1 strain. There was significant strain variation in the secretion of PrtA and Php-C activities, but reduced activity or a lack of activity was not specific to secondary-phase variants. The presence of PrtA, OpdA, and Php-C activities could be detected in the hemolymph of Galleria melonella larvae 20 to 40 h postinfection. These proteases appear not to be directly involved in the pathogenicity of Photorhabdus, since strains or phase variants lacking any of these proteases do not show reduced virulence when they are injected into G. melonella larvae.

Using a DNA Microarray To Investigate the Distribution of Insect Virulence Factors in Strains of Photorhabdus Bacteria

Photorhabdus is an insect-pathogenic bacterium in which oral toxicity to insects is found in two distinct taxonomic groups. Using a DNA microarray and comparative genomics, we show that oral toxicity is associated with toxin complex genes tcaABC and that this locus can be mobilized or deleted within different strains.

Inactivation of a Novel Gene Produces a Phenotypic Variant Cell and Affects the Symbiotic Behavior of Xenorhabdus nematophilus

ABSTRACT Xenorhabdus nematophilus is an insect pathogen that lives in a symbiotic association with a specific entomopathogenic nematode. During prolonged culturing, variant cells arise that are deficient in numerous properties. To understand the genetic mechanism underlying variant cell formation, a transposon mutagenesis approach was taken. Three phenotypically similar variant strains ofX. nematophilus, each of which contained a single transposon insertion, were isolated. The insertions occurred at different locations in the chromosome. The variant strain, ANV2, was further characterized. It was deficient in several properties, including the ability to produce antibiotics and the stationary-phase-induced outer membrane protein, OpnB. Unlike wild-type cells, ANV2 produced lecithinase. The emergence of ANV2 from the nematode host was delayed relative to the emergence of the parental strain. The transposon in ANV2 had inserted in a gene designatedvar1, which encodes a novel protein composed of 121 amino acid residues. Complementation analysis confirmed that the pleiotropic phenotype of the ANV2 strain was produced by inactivation of var1. Other variant strains were not complemented by var1. These results indicate that inactivation of a single gene was sufficient to promote variant cell formation in X. nematophilus and that disruption of genetic loci other than var1 can result in the same pleiotropic phenotype.

Cleavage site analysis of a serralysin-like protease, PrtA, from an insect pathogen Photorhabdus luminescens and development of a highly sensitive and specific substrate.

The aim of this study was the development of a sensitive and specific substrate for protease A (PrtA), a serralysin‐like metzincin from the entomopathogenic microorganism, Photorhabdus. First, cleavage of three biological peptides, the A and B chains of insulin and β‐lipotropin, and of 15 synthetic peptides, was investigated. In the biological peptides, a preference for the hydrophobic residues Ala, Leu and Val was observed at three substrate positions, P2, P1′ and P2′. At these positions in the synthetic peptides the preferred residues were Val, Ala and Val, respectively. They contributed to the efficiency of hydrolysis in the order P1′ > P2 > P2′. Six amino acids of the synthetic peptides were sufficient to reach the maximum rate of hydrolysis, in accordance with the ability of PrtA to cleave three amino acids from both the N‐ and the C‐terminus of some fragments of biological peptides. Using the best synthetic peptide, a fluorescence‐quenched substrate, N‐(4‐[4′(dimethylamino)phenylazo]benzoyl–EVYAVES−5‐[(2‐aminoethyl)amino]naphthalene‐1‐sulfonic acid, was prepared. The ∼ 4 × 106 m−1·s−1 specificity constant of PrtA (at Km ∼ 5 × 10−5 m and kcat ∼ 2 × 102 s−1) on this substrate was the highest activity for a serralysin‐type enzyme, allowing precise measurement of the effects of several inhibitors and pH on PrtA activity. These showed the characteristics of a metalloenzyme and a wide range of optimum pH, similar to other serralysins. PrtA activity could be measured in biological samples (Photorhabdus‐infected insect larvae) without interference from other enzymes, which indicates that substrate selectivity is high towards PrtA. The substrate sensitivity allowed early (14 h post infection) detection of PrtA, which might indicate PrtAs participation in the establishment of infection and not only, as it has been supposed, in bioconversion.

Proteolytic Enzyme Production by Strains of the Insect Pathogen Xenorhabdus and Characterization of an Early-Log-Phase-Secreted Protease as a Potential Virulence Factor

ABSTRACT As a comparison to a similar study on Photorhabdus strains, 15 Xenorhabdus bacterial strains and secondary phenotypic variants of two strains were screened for proteolytic activity by five detection methods. Although the number and intensity of proteolytic activities were different, every strain was positive for proteolytic activity by several tests. Zymography following native PAGE detected two groups of activities with different substrate affinities and a higher and lower electrophoretic mobility that were distinguished as activity 1 and 2, respectively. Zymography following SDS-PAGE resolved three activities, which were provisionally named proteases A, B, and C. Only protease B, an ∼55-kDa enzyme, was produced by every strain. This enzyme exhibited higher affinity to the gelatin substrate than to the casein substrate. Of the chromogenic substrates used, three were hydrolyzed: furylacryloyl-Ala-Leu-Val-Tyr (Fua-ALVY), Fua-LGPA (LGPA is Leu-Gly-Pro-Ala) (a substrate for collagen peptidases), and succinyl-Ala-Ala-Pro-Phe-thiobenzyl (Succ-AAPF-SBzl). All but the Fua-LGPA-ase activity seemed to be from secreted enzymes. According to their substrate preference profiles and inhibitor sensitivities, at least six such proteolytic enzymes could be distinguished in the culture medium of Xenorhabdus strains. The proteolytic enzyme that was secreted the earliest, protease B and the Succ-AAPF-SBzl-hydrolyzing enzyme, appeared from the early logarithmic phase of growth. Protease B could also be detected in the hemolymph of Xenorhabdus-infected Galleria mellonella larvae from 15 h postinfection. The purified protease B hydrolyzed in vitro seven proteins in the hemolymph of Manduca sexta that were also cleaved by PrtA peptidase from Photorhabdus. The N-terminal sequence of protease B showed similarity to a 55-kDa serralysin type metalloprotease in Xenorhabdus nematophila, which had been identified as an orthologue of Photorhabdus PrtA peptidase.

Reprint of "Biodiversity and systematics of nematode-bacterium entomopathogens" (Biol. Control 37 (2006) 32-49)

Nematodes are one of the most abundant animals on earth, and bacteria comprise the most biologically and phylogenetically diverse domains of organisms. On at least two separate occasions a soil dwelling nematode and a bacterium have entered into a mutualistic, insecticidal association. From such origins arose two distinct lineages of nematode–bacterium entomopathogens, Steinernema–Xenorhabdus and Heterorhabditis–Photorhabdus. Herein, we present a summary and discussion of the known evolutionary diversity and systematics of these two groups relative to other nematodes and bacteria, and their shared evolutionary history.

A robust phylogenetic framework for the bacterial genus Photorhabdus and its use in studying the evolution and maintenance of bioluminescence: A case for 16S, gyrB, and glnA

Photorhabdus spp., the only known bioluminescent terrestrial bacteria are well known for their symbiotic association with heterorhabditid nematodes. This association, along with their ability to kill insects, has aroused interest in the evolutionary relationships within this bacterial group. Currently, three species are recognized within the genus Photorhabdus; P. temperata and P. luminescens, which are endosymbionts of Heterorhabditis spp., and P. asymbiotica, which has been isolated from human wounds and has recently been shown to also have a heterorhabditid nematode vector. To examine phylogenetic relationships among these taxa, we utilize total evidence Bayesian, likelihood, and parsimony based analyses of three genetic loci (16S rRNA gene, gyrB, and glnA) to construct a robust evolutionary hypothesis for the genus Photorhabdus. Here we use this phylogeny to evaluate existing specific and sub-specific taxonomic statements within the genus, identify previously undescribed Photorhabdus strains, test the utility of 16S rRNA gene, gyrB, and glnA in resolving various levels of relationships within the genus, and, finally, to investigate the evolution of bioluminescence. The genes examined produced the most robust phylogenetic hypothesis to date for the genus Photorhabdus, as indicated by strong bootstrap and posterior probability values at previously unresolved or poorly resolved nodes. We show that glnA is particularly useful in resolving specific and intra-specific relationships poorly resolved in other studies. We conclude that P. asymbiotica is the sister group to P. luminescens and that the new strains HIT and JUN should be given a new group designation within P. asymbiotica. Furthermore, we reveal a pattern of decline in bioluminescent intensity through the evolution of Photorhabdus, suggesting that this may be a trait acquired and maintained under previous ecological (aquatic) selection pressures that is now gradually being lost in its terrestrial environment.

Novel application of PhastSystem polyacrylamide gel electrophoresis using restriction fragment length polymorphism--internal transcribed spacer patterns of individuals for molecular identification of entomopathogenic nematodes.

A relatively rapid and economic way of identifying and assigning nematodes to taxons, which had already been determined either by comparative sequence analysis of nuclear rDNA internal transcribed spacer (ITS) region or by other methods of molecular or conventional taxonomy, is provided. Molecular identification of entomopathogenic nematodes (EPN) can be upgraded by basing it on PhastSystem polyacrylamide gel electrophoresis (PAGE) analysis of restriction fragment length polymorphism (RFLP) patterns of polymerase chain reaction (PCR)‐amplified DNA derived from single nematodes of Steinernema or Heterorhabditis spp. Although analysis from single worms has previously been made on agarose gel, the resolution on PhastSystem PAGE gel is much higher. The DNA sequences selected for analysis were those constituting the internal transcribed spacer region between the 18S and 26S rDNA genes within the rRNA operon. RFLP analysis was carried out by gel electrophoresis on the PhastSystem (Pharmacia) as detailed elsewhere (Triga et al., Electrophoresis 1999, 20, 1272—1277). The downscaling from conventional agarose to PhastSystem gels resulted in pattern of DNA fragments differing from those obtained with agarose gel electrophoresis under conventional conditions by increasing the number of detected fragments. The approach supported previous species identifications and was able to identify several unclassified isolates, such as those from Hungary and Ireland, and provides a method for identification of previously unclassified strains. We confirmed that Heterorhabditis “Irish Type”, represented by two strains of different geographical origin, comprise a species different from H. megidis. We also confirmed that strain IS5 belongs to the species H. indicus rather than to H. bacteriophora, as had been suggested previously.