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Journal of Bacteriology, August 1999, p. 4708-4710, Vol. 181, No. 15
0021-9193/99/$04.00+0
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
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ABSTRACT |
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.
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TEXT |
Wolbachia is an
obligatory intracellular and maternally inherited 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
initiation 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 tetracycline-treated derivative strain of DSR was
used as the Wolbachia-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 Wolbachia 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 
ZAP (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 ZAP phagemids and was
subcloned into a pBluescript vector. Both strands of the insert (3,324 bp) were sequenced by using the 
transposon-facilitated DNA
sequencing method (19). This EcoRI fragment did
not contain the 5' 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
(5'-GCT ATA GCA TGC ATT AGA TGT G-3') and either the T3 or T7 primer, both of which recognize pBluescript. This was followed by nested PCR
with the internal primer dnaA p2 (5'-GAA CCT TGG ATC CAG CGG CG-3'). 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 product. Sequencing of both strands of this fragment was done at the Keck Sequencing Facility, Yale University. DNA and protein 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, 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 program of the DNASTAR software (Table
1).
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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).
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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 pronounced 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 sequence
(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 hyperthermophilic
bacterium Aquifex aeolicus, which belongs to the family
Aquificaceae, the most deeply branching family of bacteria
(5); (ii) two marine cyanobacteria, Prochlorococcus
marinus 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 
subdivision of the Proteobacteria,
namely, Caulobacter crescentus, which divides asymmetrically
(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 environmental niches they occupy. It is notable that the
dnaA regions of Wolbachia and its closest known
relative, Rickettsia, have different organizations, suggesting that such adaptive changes may have arisen independently in
the corresponding lineages.
Nucleotide sequence accession number.
The nucleotide sequence
(4,838 bp) reported in the present study has been deposited in the EMBL
database under the accession no. AJ012073.
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ACKNOWLEDGMENTS |
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 Foundation, the
McKnight Foundation, and the National Institutes of Health (AI 40620).
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FOOTNOTES |
*
Corresponding author. Mailing address: Insect Molecular
Genetics 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{at}imbb.forth.gr.
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Journal of Bacteriology, August 1999, p. 4708-4710, Vol. 181, No. 15
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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