1887

Abstract

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.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.000626
2018-03-02
2019-10-23
Loading full text...

Full text loading...

/deliver/fulltext/micro/164/4/495.html?itemId=/content/journal/micro/10.1099/mic.0.000626&mimeType=html&fmt=ahah

References

  1. Poinar GO. Biology and taxonomy of Steinernematidae and Heterorhabditidae. In Entomopathogenic Nematodes in Biological Control Boca Raton, FL: CRC Press; 1979
    [Google Scholar]
  2. Akhurst RJ, Boemare NE. Biology and taxonomy of Xenorhabdus. In Entomopathogenic Nematodes in Biological Control Boca Raton, FL: CRC Press; 1990
    [Google Scholar]
  3. Forst S, Dowds B, Boemare N, Stackebrandt E. Xenorhabdus and Photorhabdus spp.: bugs that kill bugs. Annu Rev Microbiol 1997; 51: 47– 72 [CrossRef] [PubMed]
    [Google Scholar]
  4. Boemare NE. Biology, taxonomy and systematics of Photorhabdus and Xenorhabdus. In Entomopathogenic Nematology Wallingford, UK: CABI Publishing; 2002
    [Google Scholar]
  5. Forst S, Clarke D. Bacteria-Nematode Symbiosis, Entomopathogenic Nematology Wallingford, UK: CABI Publishing; 2002
    [Google Scholar]
  6. Herbert EE, Goodrich-Blair H. Friend and foe: the two faces of Xenorhabdus nematophila. Nat Rev Microbiol 2007; 5: 634– 646 [CrossRef] [PubMed]
    [Google Scholar]
  7. Nielsen-Leroux C, Gaudriault S, Ramarao N, Lereclus D, Givaudan A. How the insect pathogen bacteria Bacillus thuringiensis and Xenorhabdus/Photorhabdus occupy their hosts. Curr Opin Microbiol 2012; 15: 220– 231 [CrossRef] [PubMed]
    [Google Scholar]
  8. Bird AF, Akhurst RJ. The nature of the intestinal vesicle in nematodes of the family Steinernematidae. Int J Parasitol 1983; 13: 599– 606 [CrossRef]
    [Google Scholar]
  9. Snyder H, Stock SP, Kim SK, Flores-Lara Y, Forst S. 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. Appl Environ Microbiol 2007; 73: 5338– 5346 [CrossRef] [PubMed]
    [Google Scholar]
  10. Kim SK, Flores-Lara Y, Patricia Stock S. Morphology and ultrastructure of the bacterial receptacle in Steinernema nematodes (Nematoda: Steinernematidae). J Invertebr Pathol 2012; 110: 366– 374 [CrossRef] [PubMed]
    [Google Scholar]
  11. Singh S, Reese JM, Casanova-Torres AM, Goodrich-Blair H, Forst S. Microbial population dynamics in the hemolymph of Manduca sexta infected with Xenorhabdus nematophila and the entomopathogenic nematode Steinernema carpocapsae. Appl Environ Microbiol 2014; 80: 4277– 4285 [CrossRef] [PubMed]
    [Google Scholar]
  12. Lee MM, Stock SP. A multigene approach for assessing evolutionary relationships of Xenorhabdus spp. (gamma-Proteobacteria), the bacterial symbionts of entomopathogenic Steinernema nematodes. J Invertebr Pathol 2010; 104: 67– 74 [CrossRef] [PubMed]
    [Google Scholar]
  13. Murfin KE, Lee MM, Klassen JL, McDonald BR, Larget B et al. Xenorhabdus bovienii strain diversity impacts coevolution and symbiotic maintenance with Steinernema spp. nematode hosts. MBio 2015; 6: e00076-15 [CrossRef] [PubMed]
    [Google Scholar]
  14. McMullen JG, McQuade R, Ogier JC, Pagès S, Gaudriault S et al. Variable virulence phenotype of Xenorhabdus bovienii (γ-Proteobacteria: Enterobacteriaceae) in the absence of their vector hosts. Microbiology 2017; 163: 510– 522 [CrossRef] [PubMed]
    [Google Scholar]
  15. Emelianoff V, Sicard M, Le Brun N, Moulia C, Ferdy JB. Effect of bacterial symbionts Xenorhabdus on mortality of infective juveniles of two Steinernema species. Parasitol Res 2007; 100: 657– 659 [CrossRef] [PubMed]
    [Google Scholar]
  16. Ogier JC, Pagès S, Bisch G, Chiapello H, Médigue C et al. Attenuated virulence and genomic reductive evolution in the entomopathogenic bacterial symbiont species, Xenorhabdus poinarii. Genome Biol Evol 2014; 6: 1495– 1513 [CrossRef] [PubMed]
    [Google Scholar]
  17. Bisch G, Pagès S, McMullen JG, Stock SP, Duvic B et al. Xenorhabdus bovienii CS03, the bacterial symbiont of the entomopathogenic nematode Steinernema weiseri, is a non-virulent strain against lepidopteran insects. J Invertebr Pathol 2015; 124: 15– 22 [CrossRef] [PubMed]
    [Google Scholar]
  18. Bisch G, Ogier JC, Médigue C, Rouy Z, Vincent S et al. Comparative genomics between two Xenorhabdus bovienii strains highlights differential evolutionary scenarios within an entomopathogenic bacterial species. Genome Biol Evol 2016; 8: 148– 160 [CrossRef] [PubMed]
    [Google Scholar]
  19. Shrestha S, Kim Y. An entomopathogenic bacterium, Xenorhabdus nematophila, inhibits hemocyte phagocytosis of Spodoptera exigua by inhibiting phospholipase A2. J Invertebr Pathol 2007; 96: 64– 70 [CrossRef] [PubMed]
    [Google Scholar]
  20. Vigneux F, Zumbihl R, Jubelin G, Ribeiro C, Poncet J et al. The xaxAB genes encoding a new apoptotic toxin from the insect pathogen Xenorhabdus nematophila are present in plant and human pathogens. J Biol Chem 2007; 282: 9571– 9580 [CrossRef] [PubMed]
    [Google Scholar]
  21. Ffrench-Constant R, Waterfield N. An ABC guide to the bacterial toxin complexes. Adv Appl Microbiol 2006; 58: 169– 183 [PubMed] [Crossref]
    [Google Scholar]
  22. Sheets JJ, Hey TD, Fencil KJ, Burton SL, Ni W et al. Insecticidal toxin complex proteins from Xenorhabdus nematophilus: structure and pore formation. J Biol Chem 2011; 286: 22742– 22749 [CrossRef] [PubMed]
    [Google Scholar]
  23. Singh S, Orr D, Divinagracia E, McGraw J, Dorff K et al. Role of secondary metabolites in establishment of the mutualistic partnership between Xenorhabdus nematophila and the entomopathogenic nematode Steinernema carpocapsae. Appl Environ Microbiol 2015; 81: 754– 764 [CrossRef] [PubMed]
    [Google Scholar]
  24. Bode HB. Entomopathogenic bacteria as a source of secondary metabolites. Curr Opin Chem Biol 2009; 13: 224– 230 [CrossRef] [PubMed]
    [Google Scholar]
  25. Morales-Soto N, Snyder H, Forst S. Interspecies competition in a bacteria-nematode mutualism. In Defensive Mutualism in Microbial Symbiosis Boca Raton, FL: CRC Press; 2009
    [Google Scholar]
  26. Couche GA, Gregson RP. Protein inclusions produced by the entomopathogenic bacterium Xenorhabdus nematophilus subsp. nematophilus. J Bacteriol 1987; 169: 5279– 5288 [CrossRef] [PubMed]
    [Google Scholar]
  27. Goetsch M, Owen H, Goldman B, Forst S. Analysis of the PixA inclusion body protein of Xenorhabdus nematophila. J Bacteriol 2006; 188: 2706– 2710 [CrossRef] [PubMed]
    [Google Scholar]
  28. Xu J, Hurlbert RE. Toxicity of irradiated media for Xenorhabdus spp. Appl Environ Microbiol 1990; 56: 815– 818 [PubMed]
    [Google Scholar]
  29. Holdorf MM, Owen HA, Lieber SR, Yuan L, Adams N et al. Arabidopsis ETHE1 encodes a sulfur dioxygenase that is essential for embryo and endosperm development. Plant Physiol 2012; 160: 226– 236 [CrossRef] [PubMed]
    [Google Scholar]
  30. Alexeyev MF. The pKNOCK series of broad-host-range mobilizable suicide vectors for gene knockout and targeted DNA insertion into the chromosome of gram-negative bacteria. Biotechniques 1999; 26: 824– 828 [PubMed]
    [Google Scholar]
  31. Jubelin G, Pagès S, Lanois A, Boyer MH, Gaudriault S et al. Studies of the dynamic expression of the Xenorhabdus FliAZ regulon reveal atypical iron-dependent regulation of the flagellin and haemolysin genes during insect infection. Environ Microbiol 2011; 13: 1271– 1284 [CrossRef] [PubMed]
    [Google Scholar]
  32. Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 1997; 25: 3389– 3402 [CrossRef] [PubMed]
    [Google Scholar]
  33. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA et al. Clustal W and Clustal X version 2.0. Bioinformatics 2007; 23: 2947– 2948 [CrossRef] [PubMed]
    [Google Scholar]
  34. Kumar S, Stecher G, Tamura K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 2016; 33: 1870– 1874 [CrossRef] [PubMed]
    [Google Scholar]
  35. Zuckerkandl E, Pauling L. Evolutionary divergence and convergence in proteins. In Evolving Genes and Proteins 1965
    [Google Scholar]
  36. Cowles KN, Cowles CE, Richards GR, Martens EC, Goodrich-Blair H et al. The global regulator Lrp contributes to mutualism, pathogenesis and phenotypic variation in the bacterium Xenorhabdus nematophila. Cell Microbiol 2007; 9: 1311– 1323 [CrossRef] [PubMed]
    [Google Scholar]
  37. Huber B, Feldmann F, Köthe M, Vandamme P, Wopperer J et al. Identification of a novel virulence factor in Burkholderia cenocepacia H111 required for efficient slow killing of Caenorhabditis elegans. Infect Immun 2004; 72: 7220– 7230 [CrossRef] [PubMed]
    [Google Scholar]
  38. Park D, Ciezki K, van der Hoeven R, Singh S, Reimer D et al. Genetic analysis of xenocoumacin antibiotic production in the mutualistic bacterium Xenorhabdus nematophila. Mol Microbiol 2009; 73: 938– 949 [CrossRef] [PubMed]
    [Google Scholar]
  39. Volgyi A, Fodor A, Szentirmai A, Forst S. Phase variation in Xenorhabdus nematophilus. Appl Environ Microbiol 1988; 64: 1188– 1193
    [Google Scholar]
  40. Alatorre-Rosas R, Kaya HK. Interspecific competition between entomopathogenic nematodes in the genera Heterorhabditis and Steinernema for an insect host in sand. J Invertebr Pathol 1990; 55: 179– 188 [CrossRef]
    [Google Scholar]
  41. Koppenhöfer AM, Kaya HK. Coexistence of two steinernematid nematode species (Rhabditida: Steinernematidae) in the presence of two host species. Applied Soil Ecology 1996; 4: 221– 230 [CrossRef]
    [Google Scholar]
  42. Půza V, Mrácek Z. Mixed infection of Galleria mellonella with two entomopathogenic nematode (Nematoda: Rhabditida) species: Steinernema affine benefits from the presence of Steinernema kraussei. J Invertebr Pathol 2009; 102: 40– 43 [CrossRef] [PubMed]
    [Google Scholar]
  43. Battesti A, Hoskins JR, Tong S, Milanesio P, Mann JM et al. Anti-adaptors provide multiple modes for regulation of the RssB adaptor protein. Genes Dev 2013; 27: 2722– 2735 [CrossRef] [PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.000626
Loading
/content/journal/micro/10.1099/mic.0.000626
Loading

Data & Media loading...

Supplements

Supplementary File 1

PDF
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error