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 Wasp Nasonia 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, Nasonia vitripennis, and shown to cause the son-killer trait in wasps. The 16s rRNA sequence, DNA probes, and whole-cell fatty acid profiles suggest that it belongs to the family Enterobacteriaceae. The strain's properties indicate a closer relationship to the genus Proteus than to the genus Escherichia, Citrobacter, or Salmonella, We propose the name Arsenophonus 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 parasitoid 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 Escherichia coli rRNA sequence specific for members of the the name Arsenophonus nasoniae. 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 homology 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 bacteria 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 Yersinia 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.