Phylogeny of Greya (Lepidoptera: Prodoxidae), based on nucleotide sequence variation in mitochondrial cytochrome oxidase I and II: congruence with morphological data

The phylogeny of Greya Busck (Lepidoptera: Prodoxidae) was inferred from nucleotide sequence variation across a 765-bp region in the cytochrome oxidase I and II genes of the mitochondrial genome. Most parsimonious relationships of 25 haplotypes from 16 Greya species and two outgroup genera (Tetragma and Prodoxus) showed substantial congruence with the species relationships indicated by morphological variation. Differences between mitochondrial and morphological trees were found primarily in the positions of two species, G. variabilis and G. pectinifera, and in the branching order of the three major species groups in the genus. Conflicts between the data sets were examined by comparing levels of homoplasy in characters supporting alternative hypotheses. The phylogeny of Greya species suggests that host-plant association at the family level and larval feeding mode are conservative characters. Transition/transversion ratios estimated by reconstruction of nucleotide substitutions on the phylogeny had a range of 2.0-9.3, when different subsets of the phylogeny were used. The decline of this ratio with the increase in maximum sequence divergence among taxa indicates that transitions are masked by transversions along deeper internodes or long branches of the phylogeny. Among transitions, substitutions of A-->G and T-->C outnumbered their reciprocal substitutions by 2-6 times, presumably because of the approximately 4:1 (77%) A+T-bias in nucleotide base composition. Of all transversions, 73%-80% were A<-->T substitutions, 85% of which occurred at third positions of codons; these estimates did not decrease with an increase in maximum sequence divergence of taxa included in the analysis. The high frequency of A<-->T substitutions is either a reflection or an explanation of the 92% A+T bias at third codon positions.      

Characterization and environmental regulation of outer membrane proteins in Xenorhabdus nematophilus.

We have examined the production of the outer membrane proteins of the primary and secondary forms of Xenorhabdus nematophilus during exponential- and stationary-phase growth at different temperatures. The most highly expressed outer membrane protein of X. nematophilus was OpnP. The amino acid composition of OpnP was very similar to those of the porin proteins OmpF and OmpC of Escherichia coli. N-terminal amino acid sequence analysis revealed that residues 1 to 27 of the mature OpnP shared 70 and 60% sequence identities with OmpC and OmpF, respectively. These results suggest that OpnP is a major porin protein in X. nematophilus. Three additional proteins, OpnA, OpnB, and OpnS, were induced during stationary-phase growth. OpnB was present at a high level in stationary-phase cells grown at 19 to 30 degrees C and was repressed in cells grown at 34 degrees C. OpnA was optimally produced at 30 degrees C and was not present in cells grown at lower and higher temperatures. The production of OpnS was not dependent on growth temperature. In contrast, another outer membrane protein, OpnT, was strongly induced as the growth temperature was elevated from 19 to 34 degrees C. In addition, we show that the stationary-phase proteins OpnA and OpnB were not produced in secondary-form cells.     

Molecular analysis of the two-component genes, ompR and envZ, in the symbiotic bacterium Xenorhabdus nematophilus

In Escherichia coli the histidine kinase sensor protein, EnvZ, undergoes autophosphorylation and subsequently phosphorylates the regulatory protein, OmpR. Modulation of the levels of OmpR-phosphate controls the differential expression of ompF and ompC . While the phosphotransfer reaction between EnvZ and OmpR has been extensively studied, the domains involved in the sensing function of EnvZ are not well understood. We have used a comparative approach to study the sensing function of EnvZ. During our search of numerous bacteria we found that the symbiotic/pathogenic bacterium Xenorhabdus nematophilus contained the operon encoding both ompR and envZ . Nucleotide sequence analysis revealed that EnvZ of X. nematophilus (EnvZ^X.n.) is composed of 342 amino acid residues, which is 108 residues shorter than EnvZ of E. coli (EnvZ^E.c.). Amino acid sequence comparison showed that the cytoplasmic domains of the EnvZ moleculsshared 57% sequence identity. In contrast, the large hydrophilic periplasmic domain of EnvZ^E.c. was absent in EnvZ^X.n., and was replaced by a shorter hydrophobic region. Although the periplasmic domains had diverged extensively, envZ^X.n. was able to complement a Δ envZ strain of E. coli . OmpF and OmpC were differentially produced in response to changes in medium osmolarity in this strain. Further genetic analysis established that heterologous phosphorylation between EnvZ^X.n. and OmpR of E. coli (OmpR^E.c.) accounted for the complementation of the Δ envZ strain. In addition we show that the OmpR molecules of X. nematophilus and E. coli share 78% amino acid sequence identity. These results indicate that the EnvZ protein of X. nematophilus was able to sense changes in the osmolarity of the growth environment and properly regulate the levels of OmpR-phosphate in E. coli .