Distribution and evolution of bacteriophage WO in Wolbachia, the endosymbiont causing sexual alterations in arthropods.

Wolbachia are obligatory intracellular and maternally inherited bacteria, known to infect many species of arthropod. In this study, we discovered a bacteriophage-like genetic element in Wolbachia, which was tentatively named bacteriophage WO. The phylogenetic tree based on phage WO genes of several Wolbachia strains was not congruent with that based on chromosomal genes of the same strains, suggesting that phage WO was active and horizontally transmitted among various Wolbachia strains. All the strains of Wolbachia used in this study were infected with phage WO. Although the phage genome contained genes of diverse origins, the average G+C content and codon usage of these genes were quite similar to those of a chromosomal gene of Wolbachia. These results raised the possibility that phage WO has been associated with Wolbachia for a very long time, conferring some benefit to its hosts. The evolution and possible roles of phage WO in various reproductive alterations of insects caused by Wolbachia are discussed.

Wolbachia infection and cytoplasmic incompatibility in the cricket Teleogryllus taiwanemma.

Wolbachia are cytoplasmically inherited bacteria found in many arthropods. They induce various reproductive alterations in their hosts, including cytoplasmic incompatibility, thelytokous parthenogenesis, feminization and male-killing. In this study, we examined Wolbachia infection and its effects on the host cricket Teleogryllus taiwanemma. In a phylogenetic study based on the wsp gene coding for a Wolbachia surface protein, the Wolbachia strain harboured by T. taiwanemma was clustered together with those harboured by Laodelphax striatellus, Tribolium confusum, Acraea encedon, Trichogramma deion and Adalia bipunctata. Crossing experiments using the Wolbachia-infected and uninfected strains of cricket showed that the infection is associated with the expression of unidirectional cytoplasmic incompatibility: the egg hatch rate in the incompatible cross between the infected males and uninfected females was 20.3 %. We also examined the distribution of Wolbachia within the host using polymerase chain reaction assays; they were detected in the antennae, heads, forewings, hindwings, testes, ovaries, Malpighian tubules, foot muscles and fat bodies. Quantitative polymerase chain reaction assays showed that the bacterial density was highest in the fat bodies, followed by the ovaries and testes. Wolbachia were not detected in the haemolymph or in mature spermatozoa. The spermatozoa of the infected male may be modified by the presence of Wolbachia during its development. To examine this possibility, we compared the profiles of sperm proteins between the infected and uninfected males using two-dimensional gel electrophoresis. However, no differences in the protein profiles were observed.

The first detection of the insertion sequence ISW1 in the intracellular reproductive parasite Wolbachia.

Wolbachia are maternally inherited intracellular rickettsia-like bacteria known to infect a wide range of arthropods. They are associated with a number of different reproductive phenotypes in their hosts, such as cytoplasmic incompatibility, parthenogenesis, and feminization. We report on a novel insertion sequence (IS), ISW1, which was identified in the region downstream of groEL of a Wolbachia strain, wTai. The 573-bp-long ISW1 sequence is the first IS element observed in this organism, displays significant similarity to IS200, and lacks terminal inverted repeats. There were more than 20 copies of ISW1 on the chromosome of wTai. Sequence analysis of nine distinct ISW1 copies and their flanking regions showed that the copies were identical and suggested that ISW1 has no preference for its insertion sites. Possible roles of ISW1 in the adaptation of Wolbachia to intracellular environments and in various reproductive alterations caused by this bacterium are discussed.

Purification and some properties of lignostilbene-alpha, beta-dioxygenase isozyme IV from Pseudomonas paucimobilis TMY1009.

Lignostilbene-alpha, beta-dioxygenase isozyme IV was purified from ultrasonic extracts of Pseudomonas paucimobilis TMY1009 through four steps of column chromatography. The fraction obtained gave a single band on SDS-PAGE and a single peak on reversed-phase HPLC and DEAE-HPLC. The specific activity and Km of purified isozyme IV were 110 mu kat/g and 4.2 microM for 4.4'-dihydroxy-3,3'-dimethoxystilbene, 150 mu kat/g and 3.3 microM for 4,2'-dihydroxy-3,3'-dimethoxy-5'-(2"-carboxyvinyl)stilbene. The molecular mass of intact isozyme IV was estimated to be 94 kDa by gel permeation chromatography, and that of its subunits was 52 kDa by SDS-PAGE under denaturing conditions. The N-terminal amino acid sequence of isozyme IV differed slightly from that of other isozymes. Isozyme IV seemed to be composed of two identical subunits, gamma gamma.

Cloning of a lignostilbene-alpha,beta-dioxygenase isozyme gene from Pseudomonas paucimobilis TMY1009.

A genomic DNA library of Pseudomonas paucimobilis TMY1009 was constructed using a cosmid vector pWE15. Screening of the library for lignostilbene-alpha,beta-dioxygenase (LSD) isozyme genes was done with a common probe for the alpha, beta, and gamma subunits, which composed LSD isozymes. The positive clones obtained by colony hybridization were further confirmed by Southern hybridization. A 4.2-kb BamHI-HindIII fragment hybridized with the probe was subcloned into pUC118 to yield the plasmid pKHE1700. Escherichia coli MV1184 carrying pKHE1700 produced one of the LSD isozymes. The cloned LSD was purified and compared with LSD isozymes from P. paucimobilis TMY1009. The behavior on column chromatographies through all purification steps accorded with that of LSD-III. Furthermore, the mobility on polyacrylamide gel electrophoresis, the elution profile on reversed-phase HPLC, and the partial amino acid sequence were common to the cloned LSD and the native LSD-III. Thus the cloned LSD could be identified as LSD-III. It was found that the gene of LSD-III (lsdB) was composed of 489 codons.

Cloning, expression, and sequence analysis of a lignostilbene-alpha,beta-dioxygenase gene from Pseudomonas paucimobilis TMY1009.

From the genomic library of Pseudomonas paucimobilis TMY1009 constructed with the cosmid pHC79, an 8-kb BamHI-KpnI fragment encoding lignostilbene-alpha,beta-dioxygenase (LSD) was cloned into pUC19 (designated pKSN2510). E. coli JM109 having pKSN2510 produced a small amount of LSD only when the lac promoter was induced by isopropyl-beta-D-thio-galactopyranoside (IPTG). The 1.9-kb SalI fragment containing the LSD gene on pKSN2510 was subcloned into pUC119 in opposite directions (designated pKHN2560 and pKHN2590). The LSD gene on pKHN2590 was expressed in E. coli MV1184 using the lac promoter. LSD produced by E. coli MV1184 transformed with pKHN2590 (cloned LSD) was purified and found to be identical with LSD-I, which was the major isozyme produced by P. paucimobilis TMY1009. Cloned 1.9-kb nucleotide was sequenced and one open reading frame composed to 486 codons was found.

Structural and enzymatical comparison of lignostilbene-alpha,beta-dioxygenase isozymes, I, II, and III, from Pseudomonas paucimobilis TMY1009.

Three isozymes of lignostilbene-alpha,beta-dioxygenase (LSD) from Pseudomonas paucimobilis TMY1009 were separated on QAE-Toyopearl chromatography. All active fractions were further chromatographed on DEAE-Toyopearl, Butyl-Toyopearl, and Sephacryl S-300 columns. Then the isozymes I, II, and III were purified homogeneously. All three isozymes consisted of two subunits with the same mol. mass. According to the N-terminal amino acid sequences up to 25 residues of these three isozymes and the reversed-phase HPLC patterns of peptidase-digested them, it was found that LSD-I, II, and III consisted of alpha alpha, alpha beta, and beta beta subunits, respectively. They showed different specificities for several substrates that are stilbene and styrene derivatives.