The dnaA region of Wolbachia, an intracellular bacterial parasite of insects, is unique. A glnA cognate was found upstream of the dnaA gene, while neither of the two open reading frames detected downstream of dnaA has any homologue in the database. This unusual gene arrangement may reflect requirements associated with the unique ecological niche this agent occupies

Babaratsas, Alekos

Bourtzis, Kostas

O’Neill, Scott L.

Savakis, Charalambos

Sun, Ling V.

PubMed

O'Neill, Scott L.

University of Queensland eSpace

JOURNAL OF BACTERIOLOGY,
0021-9193/99/$04.0010
Aug. 1999, p. 4708–4710 Vol. 181, No. 15
Copyright © 1999, American Society for Microbiology. All Rights Reserved.
Gene Organization of the dnaA Region of Wolbachia
LING V. SUN,1 ALEKOS BABARATSAS,2 CHARALAMBOS SAVAKIS,2,3 SCOTT L. O’NEILL,1
AND KOSTAS BOURTZIS1,2*
Department of Epidemiology & Public Health, Yale University School of Medicine, New Haven, Connecticut 06520,1
and Insect Molecular Genetics Group, Institute of Molecular Biology and Biotechnology, FORTH,2 and Division of
Medical Sciences, Medical School, University of Crete,3 Heraklion, Crete, Greece
Received 4 November 1998/Accepted 13 May 1999
The dnaA region of Wolbachia, an intracellular bacterial parasite of insects, is unique. A glnA cognate was
found upstream of the dnaA gene, while neither of the two open reading frames detected downstream of dnaA
has any homologue in the database. This unusual gene arrangement may reflect requirements associated with
the unique ecological niche this agent occupies.
Wolbachia is an obligatory intracellular and maternally in-
herited bacterium. It is widespread in insects (23) and is also
known to infect other invertebrates (13). It is responsible for
various reproductive alterations in the hosts it infects, such as
cytoplasmic incompatibility, feminization of genetic males, and
parthenogenesis (4, 13, 22). Although there is an appreciable
and increasing amount of knowledge about the distribution,
phylogeny, and population genetics of Wolbachia, little is
known about its genomic organization.
In Escherichia coli, the DnaA protein is essential for initia-
tion of bidirectional replication at the chromosomal origin of
replication (8, 10, 21). The dnaA gene has been cloned from a
number of eubacterial species, and the sequences are all highly
conserved (12). Moreover, the gene arrangement of the dnaA
region is also conserved, including (together with the dnaA
gene) the rpmH, rnpA, dnaN, recF, and gyrB genes or a subset
of these genes in close proximity (24). In this paper, we report
the cloning and characterization of the dnaA chromosomal
region of Wolbachia and show that this bacterium has a unique
gene arrangement in this region.
The Wolbachia-infected Drosophila simulans Riverside
(DSR) strain was used as a source of Wolbachia (9). A tetra-
cycline-treated derivative strain of DSR was used as the Wol-
bachia-free control strain. Both strains were routinely grown
on cornflour-sugar-yeast medium at 25°C. Unless otherwise
mentioned, standard molecular methods were used (17). In
previous work we have shown that at least part of the Wolba-
chia dnaA gene is located in a 3.3-kb EcoRI genomic fragment
(3). After digestion with EcoRI, DNA from the DSR strain was
size fractionated by electrophoresis, and DNA fragments of 3
to 4 kb in size were recovered and cloned into EcoRI-cut
lZAP (Stratagene, La Jolla, Calif.). The previously described
PCR-derived dnaA fragment (2, 3) was used as a probe for the
detection of recombinants by plaque hybridization. The insert
containing the partial dnaA fragment was excised from lZAP
phagemids and was subcloned into a pBluescript vector. Both
strands of the insert (3,324 bp) were sequenced by using the gd
transposon-facilitated DNA sequencing method (19). This
EcoRI fragment did not contain the 59 coding region of the
dnaA gene which was cloned by the following method. Total
DNA of DSR strain flies was digested with XbaI and ligated to
similarly digested pBluescript. PCR was performed with the
primer dnaA p1 (59-GCT ATA GCA TGC ATT AGA TGT
G-39) and either the T3 or T7 primer, both of which recognize
pBluescript. This was followed by nested PCR with the internal
primer dnaA p2 (59-GAA CCT TGG ATC CAG CGG CG-39).
The resulting PCR product of about 1.6 kb was cloned into
pBluescript. Pfu Taq polymerase (Stratagene, Inc.) was used in
all PCRs to maximize the sequence fidelity of the PCR prod-
uct. Sequencing of both strands of this fragment was done at
the Keck Sequencing Facility, Yale University. DNA and pro-
tein sequences were analyzed with the University of Wisconsin
Genetics Computer Group programs (7).
The coding map of the Wolbachia dnaA region, which is
present as a single copy in the genome (data not shown), is
presented in Fig. 1. Three complete open reading frames
(ORFs) and the start of a fourth one were found and analyzed
by the BLAST program. The first ORF (Fig. 1) was identified
as the Wolbachia glnA cognate, a gene which encodes the
enzyme glutamine synthetase (GS), an enzyme responsible for
ammonia assimilation and glutamine biosynthesis. There are
two major families of GS: GS I (440 to 470 amino acids), which
is present in most prokaryotes; and GS II (340 to 370 amino
acids), which is present in eukaryotes and some prokaryotes
(14). The Wolbachia glnA cognate is only 735 bp long. The
deduced protein is much shorter than most of its homologues,
FIG. 1. Coding map of the Wolbachia dnaA region. The boxes represent ORFs. The thin vertical lines indicate the presence of putative DnaA boxes. The entire
region (4,838 bp) was sequenced (for details see text).
* Corresponding author. Mailing address: Insect Molecular Genet-
ics Group, Institute of Molecular Biology and Biotechnology, FORTH,
Heraklion, Crete, Greece. Phone: 30 81 39 45 41. Fax: 30 81 39 11 01.
E-mail: bourtzis@imbb.forth.gr.
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with a molecular weight (MW) of 30 and predicted isoelectric
point of approximately 6.7. Sequence analysis indicated that
the putative Wolbachia GlnA protein is a member of the GS I
family (data not shown) and is closely related to a hypothetical
protein of Rickettsia prowazekii and two E. coli GlnA cognates
as determined based on analysis made by the MegAlign pro-
gram of the DNASTAR software (Table 1).
The next ORF encodes the Wolbachia dnaA homologue,
which contains 454 amino acids with an MW of 52 and a
calculated isoelectric point of 8.6. It is very similar to other
bacterial dnaA genes, being most closely related to the R.
prowazekii homologue (Table 1). This similarity is most pro-
nounced in the ATP-binding domain and the carboxy-terminal
region, which includes the DNA binding domain (12). Neither
of the two ORFs (orf1 and orf2) found downstream of the
dnaA gene has any obvious homologue in the databases. In
addition, the entire dnaA region contains 15 potential DnaA
boxes with 100% homology to the degenerate consensus se-
quence (YYHTMCRHM) (18), approximately three times the
number of boxes expected by chance.
The gene arrangement in the Wolbachia dnaA region, glnA-
dnaA-orf1-orf2, has not been observed in any other known
bacterial genome (1, 6, 11, 15, 16, 20, 25). In most of the
various bacteria studied so far, the dnaA gene is present in a
quite conserved gene cluster while exceptions can be explained
by small chromosomal rearrangements in this region. It has
been suggested that this conserved gene arrangement is of
ancestral origin and evolved more than one billion years ago
(24). However, several diverse bacteria do not follow this rule.
The exceptions identified so far are as follows: (i) the hyper-
thermophilic bacterium Aquifex aeolicus, which belongs to the
family Aquificaceae, the most deeply branching family of bac-
teria (5); (ii) two marine cyanobacteria, Prochlorococcus mari-
nus and Synechocystis sp., which have a light-dependent cell
cycle (15, 16); (iii) the gastric pathogen Helicobacter pylori (20);
and (iv) four members of the a subdivision of the Proteobac-
teria, namely, Caulobacter crescentus, which divides asymmetri-
cally (25), Rhizobium meliloti, which can differentiate from a
free-living to a symbiotic nitrogen-fixing bacterium within the
root nodules of alfalfa, a change which is accompanied by a
cessation of DNA replication and cell division (11), and R.
prowazekii (1) and Wolbachia, both intracellular bacteria. It is
likely that the unique arrangements in each of these bacteria
may reflect adaptive changes associated with a unique mode of
regulation of the dnaA gene and DNA replication, which in
turn might reflect their unique physiology and the environmen-
tal niches they occupy. It is notable that the dnaA regions of
Wolbachia and its closest known relative, Rickettsia, have dif-
ferent organizations, suggesting that such adaptive changes
may have arisen independently in the corresponding lineages.
Nucleotide sequence accession number. The nucleotide se-
quence (4,838 bp) reported in the present study has been
deposited in the EMBL database under the accession no.
AJ012073.
We thank Androniki Nirgianiki for excellent technical assistance
and Georgia Houlaki for secretarial assistance.
This work was supported by grants from the Greek Secretariat for
Research and Technology (PENED 15774), the MacArthur Founda-
tion, the McKnight Foundation, and the National Institutes of Health
(AI 40620).
REFERENCES
1. Andersson, S. G., A. Zomorodipour, J. O. Andersson, T. Sicheritz-Ponten,
U. C. Alsmark, R. M. Podowski, A. K. Naslund, A. S. Eriksson, H. H.
Winkler, and C. G. Kurland. 1998. The genome sequence of Rickettsia
prowazekii and the origin of mitochondria. Nature 396:133–140.
2. Bourtzis, K., A. Nirgianaki, G. Markakis, and C. Savakis. 1996. Wolbachia
infection and cytoplasmic incompatibility in Drosophila species. Genetics
144:1063–1073.
3. Bourtzis, K., A. Nirgianaki, P. Onyango, and C. Savakis. 1994. A prokaryotic
dnaA sequence in Drosophila melanogaster: Wolbachia infection and cyto-
plasmic incompatibility among laboratory strains. Insect Mol. Biol. 3:131–
142.
4. Bourtzis, K., and S. L. O’Neill. 1998. Wolbachia infections and their influ-
ence on arthropod reproduction. BioScience 48:287–293.
5. Burggraf, S., G. J. Olsen, K. O. Stetter, and C. R. Woese. 1992. A phyloge-
netic analysis of Aquifex pyrophilus. Syst. Appl. Microbiol. 15:353–356.
6. Deckert, G., P. V. Warren, T. Gaasterland, W. G. Young, A. L. Lenox, D. E.
Graham, R. Overbeek, M. A. Snead, M. Keller, M. Aujay, R. Huber, R. A.
Feldman, J. M. Short, G. J. Olsen, and R. V. Swanson. 1998. The complete
genome of the hyperthermophilic bacterium Aquifex aeolicus. Nature 392:
353–358.
7. Devereux, J., M. Haebert, and O. Smithies. 1984. A comprehensive set of
sequence analysis programs for the VAX. Nucleic Acids Res. 12:387–395.
8. Fuller, R. S., J. M. Kaguni, and A. Kornberg. 1981. Enzymatic replication of
the origin of the Escherichia coli chromosome. Proc. Natl. Acad. Sci. USA
78:7370–7374.
9. Hoffmann, A. A., M. Turelli, and G. M. Simmons. 1986. Unidirectional
incompatibility between populations of Drosophila simulans. Evolution 40:
692–701.
10. Kaguni, J. M., R. S. Fuller, and A. Kornberg. 1982. Enzymatic replication of
E. coli chromosomal origin is bidirectional. Nature 296:623–627.
11. Margolin, W., D. Bramhill, and S. R. Long. 1995. The dnaA gene of Rhizo-
bium meliloti lies within an unusual gene arrangement. J. Bacteriol. 177:
2892–2900.
12. Messer, W., and C. Weigel. 1996. Initiation of chromosome replication, p.
1579–1601. In F. C. Neidhardt, R. Curtis III, J. L. Ingraham, E. C. C. Lin,
K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and
H. E. Umbarger (ed.), Escherichia coli and Salmonella: cellular and molec-
ular biology. American Society for Microbiology, Washington, D.C.
13. O’Neill, S. L., A. A. Hoffmann, and J. H. Werren (ed.). 1997. Influential
passengers: inherited microorganisms and arthropod reproduction. Oxford
University Press, Oxford, United Kingdom.
14. Reitzer, L. J. 1996. Ammonia assimilation and the biosynthesis of glutamine,
glutamate, aspartate, asparagine, L-alanine, and D-alanine, p. 391–407. In
F. C. Neidhardt, R. Curtis III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B.
Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger
(ed.), Escherichia coli and Salmonella: cellular and molecular biology. Amer-
ican Society for Microbiology, Washington, D.C.
15. Richter, S., W. R. Hess, M. Krause, and W. Messer. 1998. Unique organi-
zation of the dnaA region from Prochlorococcus marinus Ccmp1375, a ma-
rine cyanobacterium. Mol. Gen. Genet. 257:534–541.
16. Richter, S., and W. Messer. 1995. Genetic structure of the dnaA region of the
TABLE 1. The products of the dnaA region of Wolbachia and the identities to their closest relatives
Wolbachia gene Product Nearest relative(s) Accession no. % Identity
glnA Putative GS R. prowazekii hypothetical protein AJ235272 38.2
E. coli putative GS AE000228 34.9
E. coli putative GS D90767 36.0
E. coli GS (GlnA) AE000462 26.9
dnaA DnaA protein R. prowazekii DnaA protein Q59758 58.7
orf1 None
orf2 None
VOL. 181, 1999 NOTES 4709
 o
n
 O
ctober 12, 2015 by University of Queensland Library
http://jb.asm.org/
D
ow
nloaded from
 
cyanobacterium Synechocystis sp. strain PCC6803. J. Bacteriol. 177:4245–
4251.
17. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a
laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Har-
bor, N.Y.
18. Schaefer, C., and W. Messer. 1991. DnaA protein/DNA interaction. Modu-
lation of the recognition sequence. Mol. Gen. Genet. 226:34–40.
19. Strathmann, M., B. A. Hamilton, C. A. Mayeda, M. I. Simon, E. M. Meyer-
owitz, and M. J. Palazzolo. 1991. Transposon-facilitated DNA sequencing.
Proc. Natl. Acad. Sci. USA 88:1247–1250.
20. Tomb, J.-F., O. White, A. R. Kerlavage, R. A. Clayton, G. G. Sutton, R. D.
Fleischman, K. A. Ketchum, H. P. Klenk, S. Gill, B. A. Dougherty, K. Nelson,
J. Quackenbush, L. Zhou, E. F. Kirkness, S. Peterson, B. Loftus, D. Rich-
ardson, R. Dodson, H. G. Khalak, A. Glodek, K. McKenney, L. M. Fitzeger-
ald, N. Lee, M. A. Adams, E. K. Hickey, D. E. Berg, J. D. Gocayne, T. R.
Utterback, J. D. Peterson, J. M. Kelley, M. D. Cotton, J. M. Weidman, C.
Fujii, C. Bowman, L. Watthey, E. Wallin, W. S. Hayes, M. Borodovsky, P. D.
Karp, H. O. Smith, C. M. Fraser, and J. C. Venter. 1997. The complete
genome sequence of the gastric pathogen Helicobacter pylori. Nature 388:
539–547.
21. Tomizawa, J., and G. Selzer. 1979. Initiation of DNA synthesis in Escherichia
coli. Annu. Rev. Biochem. 48:999–1034.
22. Werren, J. H. 1997. Biology of Wolbachia. Annu. Rev. Entomol. 42:587–609.
23. Werren, J. H., D. Windsor, and L. R. Guo. 1995. Distribution of Wolbachia
among neotropical arthropods. Proc. R. Soc. Lond. Ser. B Biol. Sci. 262:
197–204.
24. Yoshikawa, H., and N. Ogasawara. 1991. Structure and function of DnaA
and the DnaA-box in eubacteria: evolutionary relationships of bacterial
replication origins. Mol. Microbiol. 5:2589–2597.
25. Zweiger, G., and L. Shapiro. 1994. Expression of Caulobacter dnaA as a
function of the cell cycle. J. Bacteriol. 176:401–408.
4710 NOTES J. BACTERIOL.
 o
n
 O
ctober 12, 2015 by University of Queensland Library
http://jb.asm.org/
D
ow
nloaded from
 


Gene Organization of the dnaA Region of Wolbachia

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