INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Oct. 1991, p. 563-565 Vol. 41, No. 4 0020-77~3~9~~040563-03$02.0010 Copyright 0 1991, International Union of Microbiological Societies

NOTES

Arsenophonus nasoniae gen. nov., sp. nov. the Causative Agent of the Son-Killer Trait in the Parasitic vitripennis ROBERT L. GHERNA,l* JOHN H. WERREN,, WILLIAM WEISBURG,3t ROSE COTE,l CARL R. WOESE,3 LINDA MANDELC0,3 AND DONALD J. BRENNER4 Department of Bacteriology, American Type Culture Collection, Rockville, Maryland 20852'; Department of Biology, University of Rochester, Rochester, New York 14627,; Department of Genetics and Development, University of Illinois, Urbana, Illinois 618013; and Meningitis and Special Pathogens Branch, Divisian of Bacterial Diseases, Center for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia 303334

A bacterial strain was previously isolated from a parasitic wasp, , and shown to cause the son-killer trait in . The 16s rRNA sequence, DNA probes, and whole-cell fatty acid profiles suggest that it belongs to the family . The strain's properties indicate a closer relationship to the than to the genus , Citrobacter, or , We propose the name nasoniae gen. nov., sp. nov., for this bacterium. Strain SKI4 (ATCC 49151) is the type strain.

A variety of cytoplasmically inherited microorganisms of members of the family Enterobacteriaceae. Supplemen- that distort the sex ratio of their host species are known. tation of these formulations with 1% proteose peptone (Difco Some of these organisms, such as microsporidia and the no. 0120) improved growth; however, results were negative "sex ratio" spiroplasma, distort the sex ratio by causing the for most tests. The API 20E system (Analytab Products, death of male offspring of their host species (1, 20). Re- Plainview, N.Y.) was used to confirm negative results ob- cently, a gram-negative bacterium was isolated and shown to tained from media used in plates and tubes. Carbon utiliza- be the cause of male egg mortality in the wasp tion was determined in broth and on agar by using a basal Nasonia vitripennis; N. vitripennis is a parasite of the pupae medium with the following composition (in grams per liter): of various fly species (19). proteose peptone, 10.0; Na,S04, 2.0; K,HP04, 1.5; KH, The son-killer trait (17) occurs in approximately 5% of PO,, 0.5; MgSO,. 7H20, 0.1; phenol red, 0.015; ferric female wasps from natural populations thus far surveyed ammonium citrate, 0.02; and CaC1, . 2H,O, 0.03. All carbon (16). The bacterium which causes this trait is transmitted sources were filter sterilized and added to a final concentra- both maternally and by infection, and it appears to act by tion of 1% (wthol). Nitrogen utilization was determined with preventing the development of unfertilized eggs from in- the same liquid and agar basal medium by using 1% (wthol) fected females. On the basis of the cytopathological study by glucose as the carbon source and omitting phenol red and Huger et al. (8) and other findings, the bacterium is believed proteose peptone. Results of the biochemical tests are pre- to be transmitted from infected female wasps to the he- sented under the species description, below. molymph of the fly pupa it parasitizes via stinging and then DNA was isolated and purified by the Marmur method (12) perorally to the feeding wasp larvae. In this paper, we as modified by Brenner et al. (3). The total DNA was present genomic, phenotypic, and chemotaxonomic evi- hybridized with probes (provided by David E. Kohne, dence that this isolate constitutes a new genus and species Gen-Probe, Inc., San Diego, Calif.) consisting of a tritiated within the family Enterobacteriaceae, for which we propose rRNA sequence specific for members of the the name . family Enterobacteriaceae or a tritiated E. coli rRNA se- The bacterial strain used in this study, SKI4, was isolated quence enriched for, but not exclusive for, members of the in 1983 from a parasitic wasp (N.vitripennis) strain collected family Enterobacteriaceae at 60, 70, and 75°C by the hy- in Utah (19). The culture was grown at 26°C on GC medium droxyapatite method (3). Table 1 depicts the level of DNA- base (Difco no, 0289) supplemented with Kellogg's additive rRNA among members of the family Enterobac- (10). All biochemical tests and carbon and nitrogen utiliza- teriaceae and strain SKI4 by using the enriched probe. tion tests were conducted at 26 and 30°C. Test media were Salmonella serotype typhimurium LT2 shows 74% related- inoculated with cell suspensions prepared from 3-day-old ness to E. coli K-12 probes at 60°C and 61% relatedness at cultures grown on brain heart infusion broth (Difco no. 0037) 7WC, whereas Proteus mirabilis Pr14 shows 60 and 42% at 3WC, and were harvested by centrifugation and relatedness at 60 and 70"C, respectively. Strain SKI4 was 62 washed three times with sterile physiological saline. All test and 42% related to the probe. media were incubated for 10 days before evaluation of the The rRNA sequencing was performed on unfractionated results, unless noted otherwise. RNA by using primer extension by avian reverse tran- SKI4 grows poorly or not at all on conventional biochem- scriptase with dideoxynucleotide termination. The primers ical test media used in the identification and characterization consisted of a set specific for 16s rRNA. Sequences were aligned by methods previously described (ll), and pairwise evolutionary distances (expressed as estimated changes per * Corresponding author. 100 nucleotides) were computed from the percent similarities t Present address: Gene-Trak Systems, Framingham, MA 01701. with the correction of Jukes and Cantor (9), as modified by

563 564 NOTES INT. J. SYST.BACTERIOL.

TABLE 1. Levels of DNA-rRNA homology among members of TABLE 2. Evolutionary distances among members the family Enterobacteriaceae and A. nasoniae of the family Enterobacteriaceae

% Relatedness to E. coli Genus or Evolutionary distanceu K-12 probe ~ Source of unlabeled DNA species 1 2 3 4 5 60°C 70°C 75°C (1) E. coli Escherichia coli K-12 100 100 100 (2) Citrobacter 3.0 I?scherichia coli DO32 100 63 (3) Serratia 3.9 Escherichia coli 3914-70 97 97 89 (4)Proteus 6.7 7.2 6.2 Salmonella serotype typhimurium LT2 74 61 57 (5) SKI4 8.8 8.6 8.3 6.7 iDrovidencia ulcalifaciens 3370-67 75 44 34 (6) 0. linum 15.8 14.5 14.9 15.6 16.5 enterocolitica 497-70 71 48 43 Proteus mirabilis Pr14 60 42 24 The distances were calculated as described in the text. Only positions in Arsenophonus nasoniae SKI4 62 42 30 the alignment represented by a nucleotide of known composition in all Xenorhabdus nemutophilus sequences being considered were used in the analysis. Oceanospirillum linum 9012-80 60 40 25 served as the outgroup. ,4eromonas hydrophila 9176-76 39 31 21 Legionella pneumophila Philadelphia 1 27 17 10 Legionella rubrilucens WA-270A-C2 20 12 16 ’‘Vibrio neocistes” 9076-79 14 12 4 fatty acids from cells grown on brain heart infusion agar slants grown at 30°C for 3 days. Methyl esters were prepared by the method of Moss and Dees (14). The fatty acid analyses were performed by using a Hewlett-Packard gas G. J. Olsen (15) to accommodate the actual nucleotide ratios. chromatograph (model 5898A) equipped with a 5% phenyl- The dendrogram was constructed from the evolutionary methyl silicone capillary column (0.2 mm by 25 m) and a distance matrix by using the algorithm of De Soete (4). Of flame ionization detector. Peaks were automatically inte- the genera tested, the phylogenetic tree depicted in Fig. 1 grated, and fatty acid identities and percentages were calcu- shows Proteus to be the closest to strain SKI4. The se- lated by using computer software from Microbial ID, Inc. quence of Oceanospirillum linum served as an outgroup, (Newark, Del.). establishing the root of the tree. Although the phylogenetic Strain SKI4 is characterized by having a large amount of tree does not include members of the genus Xenorhabdus, an C16:o(41%) and 16:l cis 9 (27%) fatty acids and a smaller examination of the sequence data on the genus Xenorhabdus amount of 14:O fatty acids (9.7%). This fatty acid profile (5) showed that the two groups are different and distinct. appears closer to that of the genus Providencia and supports These data are consistent with the DNA-rRNA probe data. the DNA probe and 16s rRNA data, suggesting that strain Table 2 presents the distance matrix used to compile the SKI4 is a member of the family Enterobacteriaceae. dendrogram (Fig. 1). The DNA-rRNA probe and 16s rRNA sequence data, Whole-cell fatty acids were analyzed by extracting the along with the fatty acid profiles and phenotypic properties, indicate that strain SKI4 is a new species and that it is closely related to the genus Proteus and a member of the family Enterohacteriaceae. The G+C content (13), 39%, ‘ agrees closely with those of the genera Proteus and Provi- dencia (39 to 42%) (7). Although most genera constituting the family Enterobac- teriaceae have been discovered as a result of their direct or incidental human association, few insects have been system- atically screened for such bacteria. In fact, Proteus strains have been isolated from blowflies, and large populations of Proteus strains have been found in the guts of blowfly larvae (6). Xenorhabdus species isolated from nematodes and as- signed to the family Enterobacteriaceae (18) are similar to strain SKI4 in the inability to reduce nitrate to nitrite. Unlike strain SKI4, however, Xenorhabdus species are motile by means of peritrichous flagellation. On the basis of the distinctions described above, we propose a new genus and new species for the son-killer strain, Arsenophonus naso- niae. Characteristics useful in distinguishing A. nasoniae from other members of the family Enterobacteriaceae are summarized in Table 3. Description of Arsenophonus gen. nov. Arsenophonus (Ar. se. no. pho.’ nus. Gr. n. amen, a male; Gr. suffix phonus, slayer; N. L. masc. n. Arsenophonus, male killer). The type species is Arsenophonus nasoniae. The essential character- istics of the genus are given in the description of the single 5‘i species, A. nasoniae. FIG. 1. Detailed phylogenetic tree for members of the family Description of Arsenophonus nasoniae sp. nov. Arsenopho- Enterobacteriaceae and A. nasoniae, derived from the evolutionary nus nasoniae (na. so.’ ni. ae. N. L. Nasonia, genus of a fly; distance matrix of Table 2. Oceanospirillum linum served as the nasoniae, of the genus Nasonia.) The cells are nonmotile, outgroup. non--forming, long rods, occasionally filamentous in VOL. 41, 1991 NOTES 565

TABLE 3. Characteristics for differentiating A. nasoniae from other members of the family Enterobacteriaceae

Characteristic" of Parameter ~ ~~ ~~~~ A. nusoniue E. coli K. pneumoniae P. mirabilis P. stuartii S. typhi S. sonnei Indole - + - Methyl red - + - Voges-Proskauer - - + Hydrogen sulfide on triple sugar iron agar - - - Gelatin liquefaction + - - Sucrose + [-I + ~ ~~ ~~ ~ ~ ~ ~ ~~ ~ ~ ~ ~ +, 90% or more of the strains are positive; d, strains are 26 to 75% positive; [-I, 11 to 25% negative. Data for Enterobacteriaceae strains are from Bergey's Manual of Systematic Bacteriology, vol. 1 (2), and are reprinted with permission of the publisher. young cultures (0.40 to 0.57 pm wide by 6.9 to 10.0 pm long). Enterobacteriaceae associated with human wounds. J. Clin. Colonies are mucoid, grey-white, round, and convex with Microbiol. 15: 1133-1 140. entire edges. Does not utilize (NH,),SO,, KNO,, complete 4. De Soete, G. 1983. A least squares algorithm for fitting additive defined amino acid mixtures, or acid-hydrolyzed peptones trees to proximity data. Psychometrika 48:621-626. 5. Ehlers, R.-U., U. Wyss, and E. Stackebrandt. 1988. 16s rRNA (Casamino Acids) as nitrogen sources. Enzymatically di- cataloguing and the phylogenetic position of the genus Xe- gested proteins best serve as nitrogen sources. Utilizes norhabdus. Syst. Appl. Microbiol. 10:121-125. glucose, fructose, and sucrose as primary carbon sources; 6. Erdmann, G. R. 1987. Antibacterial action of myiasis-causing weak growth is obtained with cellobiose, maltose, trehalose, flies. Parasitol. Today 317:214-216. and D-xylose. Acid is produced from D-glucose, fructose, 7. Falkow, S., I. R. Ryman, and 0. Washington. 1962. Deoxyribo- and sucrose. Growth is negative with adonitol, L-arabinose, nucleic acid base composition of Proteus and Providencia dulcitol, glycerol, i-inositol, lactose, D-mannitol, and raffi- organisms. J. Bacteriol. 83:1318-1321. nose. Positive for gelatin liquefaction and catalase. Negative 8. Huger, A., S. W. Skinner, and J. H. Werren. 1985. Bacterial for nitrate reduction, Voges-Proskauer, methyl red, indole, infections associated with the son-killer trait in the parasitoid wasp, Nasonia (= Mormoniella) vitripennis. J. Invertebr. hydrogen sulfide, oxidase, o-nitrophenyl-P-D-galactopyrano- Pathol. 46:272-280. side, dehydrolase, lysine and ornithine decarboxy- 9. Jukes, T. H., and C. R. Cantor. 1969. Evolution of protein lases, and urease. Minimum, optimum, and maximum tem- molecules, p. 21-132. In H. N. Munro (ed.), mammalian protein peratures are 15, 30, and 35"C, respectively. Cells grow at a . Academic Press, New York. pH of 6.2 to 8.7, with an optimum pH range of 7.4 to 8.0. The 10. Kellogg, D. S., Jr., W. L. Peacock Jr., W. E. Deacon, L. Brown, G+C content of the DNA is 39.5 mol%. The type strain is and C. L. Pirkle. 1963. Neisseria gonorrhoeae. I. Virulence SKI4 (= ATCC 49151), isolated from an N. vitripennis genetically linked to clonal variation. J. Bacteriol. 851274- son-killer strain collected near Salt Lake City, Utah. A. 1279. 11. Lane, D., B. Pace, G. J. Olsen, D. A. Stahl, M. L. Sogin, and nasoniae is the causative agent of the son-killer trait in the N. R. Pace. 1985. Rapid determination of 16s ribosomal RNA parasitic wasp N. vitripennis. sequences for phylogenetic analysis. Proc. Natl. Acad. Sci USA 82:6955-6959. We thank Charles Mills, American Type Culture Collection, 12. Marmur, J. 1961. A procedure for the isolation of deoxyribo- Rockville, Maryland, for the fatty acid analyses and Thomas MacA- nucleic acid from micro-organisms. J. Mol. Biol. 3:208-218. doo, Virginia Polytechnic Institute and State University, Blacks- 13. Marmur, J., and P. Doty. 1962. Determination of the base burg, for valuable advice in selecting the orthography of the genus composition of deoxyribonucleic acid from its thermal denatur- and species epithet. ation temperature. J. Mol. Biol. 5109-118. The efforts of W.G.W., C.R.W., and L.M. were supported by a 14. Moss, C. W., and S. B. Dees. 1976. Cellular fatty acids and grant from the National Science Foundation, Systematic Biology metabolic products of Pseudomonas species obtained from Division, to C.R.W. R.L.G. and R.C. were supported by NSF grant clinical specimens. J. Clin. Microbiol. 4:492-502. BSR-8415014. 15. Olsen, G. J. Personal communication. 16. Skinner, S. W. 1983. Ph.D. thesis. University of Utah, Salt Lake REFERENCES City. Andreadis, T. G., and D. W. Hall. 1979. Significance of transo- 17. Skinner, S. W. 1985. Son-killer: a third extrachromosomal varial infections of Amblyospora sp. (Microsporidia: factor affecting sex ratio in the parasitoid wasp, Nasonia Thelohaniidae) in relation to parasite maintenance in the mos- (= Mormoniella) vitripennis. Genetics 109:745-754. quito Culex salinarius. J. Invertebr. Pathol. 34:152-157. 18. Thomas, G. M., and G. 0. Poinar. 1979. Xenorhahdus gen. Brenner, D. J. 1984. Family 1. Enterobacteriaceae Rahn 1937, nov., a genus of entomopathogenic nematophilic bacteria of the nom. fam. cons. Opin. 15, Jud. Comm. 1958, 73; Ewing, family Enterobacteriaceae. Int. J. Syst. Bacteriol. 29:352-360. Farmer, and Brenner 1980, 674; Judicial Commission 1981, 104, 19. Werren, J. H., S. W. Skinner, and A. Huger. 1986. Male-killing p. 408-420. In N. R. Krieg and J. G. Holt (ed.), Bergey's manual bacteria in a parasitic wasp. Science 231:990-992. of systematic bacteriology, vol. 1. The Williams & Wilkins Co., 20. Williamson, D. L., and D. F. Poulson. 1979. Sex ratio organisms Baltimore. (spiroplasmas) of Drosophila, p. 175-208. In R. F. Whitcomb Brenner, D. J., A. C. McWhorter, J. K. Leete Knutson, and and J. G. Tully (ed.), The mycoplasmas, vol. 3. Academic A. G. Steigerwalt. 1982. : a new species of Press, New York.