Arsenophonus nasoniae gen. nov., sp. nov. the Causative Agent of

Arsenophonus nasoniae gen. nov., sp. nov. the Causative Agent of

INTERNATIONAL
JOURNALOF SYSTEMATIC
BACTERIOLOGY,
Oct. 1991, p. 563-565
0020-77~3~9~~040563-03$02.0010
Copyright 0 1991, International Union of Microbiological Societies
Vol. 41, No. 4
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 (ATCC49151) is the type strain.
A variety of cytoplasmically inherited microorganisms
that distort the sex ratio of their host species are known.
Some of these organisms, such as microsporidia and the
"sex ratio" spiroplasma, distort the sex ratio by causing the
death of male offspring of their host species (1, 20). Recently, a gram-negative bacterium was isolated and shown to
be the cause of male egg mortality in the parasitoid wasp
Nasonia vitripennis; N . vitripennis is a parasite of the pupae
of various fly species (19).
The son-killer trait (17) occurs in approximately 5% of
female wasps from natural populations thus far surveyed
(16). The bacterium which causes this trait is transmitted
both maternally and by infection, and it appears to act by
preventing the development of unfertilized eggs from infected females. On the basis of the cytopathological study by
Huger et al. (8) and other findings, the bacterium is believed
to be transmitted from infected female wasps to the hemolymph of the fly pupa it parasitizes via stinging and then
perorally to the feeding wasp larvae. In this paper, we
present genomic, phenotypic, and chemotaxonomic evidence that this isolate constitutes a new genus and species
within the family Enterobacteriaceae, for which we propose
the name Arsenophonus nasoniae.
The bacterial strain used in this study, SKI4, was isolated
in 1983 from a parasitic wasp ( N . vitripennis) strain collected
in Utah (19). The culture was grown at 26°C on GC medium
base (Difco no, 0289) supplemented with Kellogg's additive
(10). All biochemical tests and carbon and nitrogen utilization tests were conducted at 26 and 30°C. Test media were
inoculated with cell suspensions prepared from 3-day-old
cultures grown on brain heart infusion broth (Difco no. 0037)
at 3WC, and bacteria were harvested by centrifugation and
washed three times with sterile physiological saline. All test
media were incubated for 10 days before evaluation of the
results, unless noted otherwise.
SKI4 grows poorly or not at all on conventional biochemical test media used in the identification and characterization
of members of the family Enterobacteriaceae. Supplementation of these formulations with 1% proteose peptone (Difco
no. 0120) improved growth; however, results were negative
for most tests. The API 20E system (Analytab Products,
Plainview, N.Y.) was used to confirm negative results obtained from media used in plates and tubes. Carbon utilization was determined in broth and on agar by using a basal
medium with the following composition (in grams per liter):
proteose peptone, 10.0; Na,S04, 2.0; K,HP04, 1.5; KH,
PO,, 0.5; MgSO,. 7H20, 0.1; phenol red, 0.015; ferric
ammonium citrate, 0.02; and CaC1, . 2H,O, 0.03. All carbon
sources were filter sterilized and added to a final concentration of 1% (wthol). Nitrogen utilization was determined with
the same liquid and agar basal medium by using 1%(wthol)
glucose as the carbon source and omitting phenol red and
proteose peptone. Results of the biochemical tests are presented under the species description, below.
DNA was isolated and purified by the Marmur method (12)
as modified by Brenner et al. (3). The total DNA was
hybridized with probes (provided by David E. Kohne,
Gen-Probe, Inc., San Diego, Calif.) consisting of a tritiated
Escherichia coli rRNA sequence specific for members of the
family Enterobacteriaceae or a tritiated E . coli rRNA sequence enriched for, but not exclusive for, members of the
family Enterobacteriaceae at 60, 70, and 75°C by the hydroxyapatite method (3). Table 1 depicts the level of DNArRNA homology among members of the family Enterobacteriaceae and strain SKI4 by using the enriched probe.
Salmonella serotype typhimurium LT2 shows 74% relatedness to E. coli K-12 probes at 60°C and 61% relatedness at
7WC, whereas Proteus mirabilis Pr14 shows 60 and 42%
relatedness at 60 and 70"C, respectively. Strain SKI4 was 62
and 42% related to the probe.
The rRNA sequencing was performed on unfractionated
RNA by using primer extension by avian reverse transcriptase with dideoxynucleotide termination. The primers
consisted of a set specific for 16s rRNA. Sequences were
aligned by methods previously described ( l l ) , and pairwise
evolutionary distances (expressed as estimated changes per
100 nucleotides) were computed from the percent similarities
with the correction of Jukes and Cantor (9), as modified by
* Corresponding author.
t Present address: Gene-Trak Systems, Framingham, MA 01701.
563
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564
INT. J . SYST.BACTERIOL.
NOTES
TABLE 1. Levels of DNA-rRNA homology among members of
the family Enterobacteriaceae and A . nasoniae
% Relatedness to E . coli
K-12 probe
Source of unlabeled DNA
Escherichia coli K-12
I?scherichia coli DO32
Escherichia coli 3914-70
Salmonella serotype typhimurium LT2
iDrovidencia ulcalifaciens 3370-67
Yersinia enterocolitica 497-70
Proteus mirabilis Pr14
Arsenophonus nasoniae SKI4
Xenorhabdus nemutophilus 9012-80
,4eromonas hydrophila 9176-76
Legionella pneumophila Philadelphia 1
Legionella rubrilucens WA-270A-C2
’‘Vibrio neocistes” 9076-79
60°C
70°C
75°C
100
100
97
74
75
71
60
62
60
39
27
20
14
100
100
63
89
57
34
43
24
30
25
21
10
16
4
97
61
44
48
42
42
40
31
17
12
12
G. J. Olsen (15) to accommodate the actual nucleotide ratios.
The dendrogram was constructed from the evolutionary
distance matrix by using the algorithm of De Soete (4). Of
the genera tested, the phylogenetic tree depicted in Fig. 1
shows Proteus to be the closest to strain SKI4. The sequence of Oceanospirillum linum served as an outgroup,
establishing the root of the tree. Although the phylogenetic
tree does not include members of the genus Xenorhabdus, an
examination of the sequence data on the genus Xenorhabdus
(5) showed that the two groups are different and distinct.
These data are consistent with the DNA-rRNA probe data.
Table 2 presents the distance matrix used to compile the
dendrogram (Fig. 1).
Whole-cell fatty acids were analyzed by extracting the
‘
5‘i
FIG. 1. Detailed phylogenetic tree for members of the family
Enterobacteriaceae and A . nasoniae, derived from the evolutionary
distance matrix of Table 2. Oceanospirillum linum served as the
outgroup.
TABLE 2. Evolutionary distances among members
of the family Enterobacteriaceae
Genus or
species
(1) E. coli
(2) Citrobacter
(3) Serratia
(4)Proteus
( 5 ) SKI4
(6) 0. linum
Evolutionary distanceu
~
1
2
3
4
5
3.0
3.9
6.7
8.8
15.8
7.2
8.6
14.5
6.2
8.3
14.9
6.7
15.6
16.5
The distances were calculated as described in the text. Only positions in
the alignment represented by a nucleotide of known composition in all
sequences being considered were used in the analysis. Oceanospirillum linum
served as the outgroup.
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
chromatograph (model 5898A) equipped with a 5% phenylmethyl silicone capillary column (0.2 mm by 25 m) and a
flame ionization detector. Peaks were automatically integrated, and fatty acid identities and percentages were calculated by using computer software from Microbial ID, Inc.
(Newark, Del.).
Strain SKI4 is characterized by having a large amount of
C16:o(41%) and 16:l cis 9 (27%) fatty acids and a smaller
amount of 14:O fatty acids (9.7%). This fatty acid profile
appears closer to that of the genus Providencia and supports
the DNA probe and 16s rRNA data, suggesting that strain
SKI4 is a member of the family Enterobacteriaceae.
The DNA-rRNA probe and 16s rRNA sequence data,
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 Providencia (39 to 42%) (7).
Although most genera constituting the family Enterobacteriaceae have been discovered as a result of their direct or
incidental human association, few insects have been systematically 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 assigned 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 nasoniae. 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 characteristics of the genus are given in the description of the single
species, A . nasoniae.
Description of Arsenophonus nasoniae sp. nov. Arsenophonus nasoniae (na. so.’ ni. ae. N. L. Nasonia, genus of a fly;
nasoniae, of the genus Nasonia.) The cells are nonmotile,
non-spore-forming, long rods, occasionally filamentous in
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VOL. 41, 1991
NOTES
565
TABLE 3. Characteristics for differentiating A . nasoniae from other members of the family Enterobacteriaceae
Characteristic" of
Parameter
~
A . nusoniue
Indole
Methyl red
Voges-Proskauer
Hydrogen sulfide on triple sugar iron agar
Gelatin liquefaction
Sucrose
+, 90%
~
~~
~~
-
+
+
~
K . pneumoniae
+
+
-
-
-
~
~~
~~~~
P . stuartii
S . typhi
S . sonnei
+
-
+
[-I
~
P . mirabilis
-
-
-
~
E . coli
~~
~
~
~
~
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.
Enterobacteriaceae associated with human wounds. J. Clin.
young cultures (0.40 to 0.57 pm wide by 6.9 to 10.0 pm long).
Microbiol. 15:1133-1 140.
Colonies are mucoid, grey-white, round, and convex with
4. De Soete, G. 1983. A least squares algorithm for fitting additive
entire edges. Does not utilize (NH,),SO,, KNO,, complete
trees to proximity data. Psychometrika 48:621-626.
defined amino acid mixtures, or acid-hydrolyzed peptones
5 . Ehlers, R.-U., U. Wyss, and E. Stackebrandt. 1988. 16s rRNA
(Casamino Acids) as nitrogen sources. Enzymatically dicataloguing and the phylogenetic position of the genus X e 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.
7. Falkow, S., I. R. Ryman, and 0. Washington. 1962. Deoxyriboand D-xylose. Acid is produced from D-glucose, fructose,
nucleic acid base composition of Proteus and Providencia
and sucrose. Growth is negative with adonitol, L-arabinose,
organisms. J. Bacteriol. 83:1318-1321.
dulcitol, glycerol, i-inositol, lactose, D-mannitol, and raffi8. Huger, A., S. W. Skinner, and J. H. Werren. 1985. Bacterial
nose. Positive for gelatin liquefaction and catalase. Negative
infections associated with the son-killer trait in the parasitoid
for nitrate reduction, Voges-Proskauer, methyl red, indole,
wasp, Nasonia (= Mormoniella) vitripennis. J. Invertebr.
hydrogen sulfide, oxidase, o-nitrophenyl-P-D-galactopyranoPathol. 46:272-280.
side, arginine dehydrolase, lysine and ornithine decarboxy9. Jukes, T. H., and C. R. Cantor. 1969. Evolution of protein
lases, and urease. Minimum, optimum, and maximum temmolecules, p. 21-132. In H. N. Munro (ed.), mammalian protein
peratures are 15, 30, and 35"C, respectively. Cells grow at a
metabolism. Academic Press, New York.
10. Kellogg, D. S., Jr., W. L. Peacock Jr., W. E. Deacon, L. Brown,
pH of 6.2 to 8.7, with an optimum pH range of 7.4 to 8.0. The
and C. L. Pirkle. 1963. Neisseria gonorrhoeae. I. Virulence
G+C content of the DNA is 39.5 mol%. The type strain is
genetically linked to clonal variation. J. Bacteriol. 851274SKI4 (= ATCC 49151), isolated from an N . vitripennis
1279.
son-killer strain collected near Salt Lake City, Utah. A .
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
We thank Charles Mills, American Type Culture Collection,
Rockville, Maryland, for the fatty acid analyses and Thomas MacAdoo, Virginia Polytechnic Institute and State University, Blacksburg, for valuable advice in selecting the orthography of the genus
and species epithet.
The efforts of W.G.W., C.R.W., and L.M. were supported by a
grant from the National Science Foundation, Systematic Biology
Division, to C.R.W. R.L.G. and R.C. were supported by NSF grant
BSR-8415014.
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