Cloning and characterization of a gene encoding the major surface protein of the bacterial endosymbiont Wolbachia pipientis

Abstract

The maternally inherited intracellular symbiont Wolbachia pipientis is well known for inducing a variety of reproductive abnormalities in the diverse arthropod hosts it infects. It has been implicated in causing cytoplasmic incompatibility, parthenogenesis, and the feminization of genetic males in different hosts. The molecular mechanisms by which this fastidious intracellular bacterium causes these reproductive and developmental abnormalities have not yet been determined. In this paper, we report on (i) the purification of one of the most abundantly expressed Wolbachia proteins from infected Drosophila eggs and (ii) the subsequent cloning and characterization of the gene (wsp) that encodes it. The functionality of the wsp promoter region was also successfully tested in Escherichia coli. Comparison of sequences of this gene from different strains of Wolbachia revealed a high level of variability. This sequence variation correlated with the ability of certain Wolbachia strains to induce or rescue the cytoplasmic incompatibility phenotype in infected insects. As such, this gene will be a very useful tool for Wolbachia strain typing and phylogenetic analysis, as well as understanding the molecular basis of the interaction of Wolbachia with its host.

FIG. 1

WSP is the dominant protein in silver-stained SDS-polyacrylamide gels of Wolbachia fractions from DSR eggs; DSRT, uninfected control; left lane, marker proteins.

FIG. 2

wsp gene sequence and deduced amino acid sequence from Wolbachia harbored by DSR. Several putative regulatory regions are indicated.

FIG. 3

Southern blot showing a single hybridizing fragment in total fly DNA digested with EcoRI and probed with 610 bp of wsp from DSR, indicating a single-copy gene. Lanes: 1, DSR; 2, DSH; 3, DSW/Mau.

FIG. 4

E. coli strains transformed with different plasmid constructs were used to assay the wsp upstream sequence for its ability to drive expression of a CAT gene. Shown is a reverse negative of an autofluorogram of a thin-layer chromatogram of CAT activity revealed by the cleavage of a CAT substrate. Presence of CAT is demonstrated by cleavage of the fluorescently labeled 1-deoxyCAM substrate (lower band) to generate the higher band. Lanes 5 to 8 show 10-fold dilutions of lanes 1 to 4. The unmodified pKK232-8 vector (lanes 1 and 5) shows no CAT activity. The insertion of the wsp upstream sequence at the 5′ end of the CAT gene induced its expression (lanes 2 and 6). Similarly, the insertion of the lac promoter induced expression of the CAT gene in the presence (lanes 3 and 7) and in the absence (lanes 4 and 8) of IPTG.

FIG. 5

Alignment of the deduced amino acid sequence of WSP from DSR with sequences of homologous outer surface proteins: MSP4 from A. marginale (L01987), MAP1 from Cowdria ruminantium (U50832), and the tia invasion (inv.) determinant from E. coli (U20318). Only the second putative transmembrane domain shown in Fig. 2 is shared by all homologs. Amino acids identical or similar (3 distance units out of 22, using the PAM250 table of the MegAlign software program, version 3.11 [DNASTAR Inc., Madison, Wis.]) to those in the WSP sequence are in boldface.

FIG. 6

The amino acid sequence alignment of a segment of WSP of three Wolbachia strains capable of expressing CI (DSR, DSH, and C. cautella) compared to four strains incapable of expressing the phenotype (DSW/Mau, DS Coffs, DM Harwich, and DM CantonS). Differences are in boldface.