Molecular Studies on the Role of a Root Surface Agglutinin in Adherence and Colonization by Pseudomonas putida.

Pseudomonas putida aggressively colonizes root surfaces and is agglutinated by a root surface glycoprotein. Mutants of P. putida derived chemically or by Tn5 insertion demonstrated enhanced or decreased agglutinability. Two nonagglutinable Tn5 mutants (Agg) and two mutants with enhanced agglutinability (Agg) possessed Tn5 in unique restriction sites. Agg mutants colonized root surfaces of seedlings grown from inoculated seeds, but at levels lower than those observed with the Agg parent. In short-term binding studies, Agg cells adhered at levels that were 20- to 30-fold less than those for Agg parental cells. These data suggest that the agglutination interaction plays a role in the attachment of P. putida to root surfaces.

Studies of esterase 6 in Drosophila melanogaster. XIII. Purification and characterization of the two major isozymes.

Esterase-6 (EST 6; carboxylic-ester hydrolase; EC 3.1.1.1) from Drosophila melanogaster was purified to homogenity. Purified enzyme occurs as two closely moving isozymes, slow (EST 6S) and fast (EST 6F), on native polyacrylamide gel electrophoresis. Except for slight differences in their mobility, the two isozymes share similar molecular and catalytic properties. Both isozymes are glycoproteins and have an apparent molecular weight of 62,000 to 65,000 as judged by analytical gel filtration and sodium dodecyl sulfate (SDS) electrophoresis. They have identical mobility on SDS-polyacrylamide gels and an isoelectric point of 4.5. Each isozyme has a single active catalytic site as confirmed by titration with 0,0-diethyl-p-nitrophenyl phosphate (Paraoxon). We conclude that EST 6 is a monomeric enzyme. The amino acid composition of the two isozymes is very similar and both variants lack half-cystine residues. The low pI of the enzyme is due in part to a relatively high proportion of glutamic and aspartic amino acid residues. Characterization of the kinetic parameters of the isozymes using beta-naphthyl and p-nitrophenyl esters revealed no statistically significant differences in catalytic efficiency. There is, however, a suggestion that the two isozymes may differ in their substrate specificity.

Genetic and hormonal regulation of esterase 6 activity in male Drosophila melanogaster.

The esterase 6 locus in Drosophila melanogaster is the structural gene for a carboxylesterase (E.C. 3.1.1.1) and is polymorphic for two major electrophoretic variants (slow and fast). Isogenic lines containing X chromosomes extracted from natural populations and substituted into a common genetic background were used to detect unlinked factors that affect the activity of the Est 6 locus. Twofold activity differences of esterase 6 were found among males from these derived lines, which differ only in their X chromosome. These unlinked activity modifiers identify possible regulatory elements. Immunoelectrophoresis was used to estimate quantitatively the levels of specific cross-reacting material in the derived lines. The results show that the variation in activity is due to differences in the amount of EST 6 present. Physiological studies of the control of EST 6 levels reveal that both juvenile hormone and 20 hydroxyecdysone stimulate the production of EST 6 activity in adult males. These results suggest that the effects of X chromosomes on EST 6 activity may be effected by modulating the level of adult hormone titers in Drosophila.