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The baculovirus egt gene encodes an ecdysteroid UDP-glucosyl transferase (EGT) which catalyzes the transfer of sugar from UDP-sugar to ecdysteroid, the insect molting hormone. EGT activity in cells infected with Lymantria dispar nuclear polyhedrosis virus (LdNPV) was demonstrated. Insects infected with LdNPV having EGT activity were inhibited in larval molting and pupation, while infection of insect with a mutant virus lacking EGT activity did not inhibit molting and pupation. Therefore, expression of the egt gene allowed the virus to interfere with normal development of gypsy moth larvae. Insect growth was retarded and slow weight gain was observed in molt-inhibited fourth-instar larvae. LdNPV infection also affected insect hormonal system. The level of hemolymph ecdysteroids in virus-infected larvae was found to be higher than in uninfected controls. Ecdysteroids in these virus-infected insects were composed mostly of ecdysone glucoside, the product of EGT. Little or no free ecdysteroids were detected in hemolymph in late stages of virus infection. Continuous synthesis of ecdysone by the prothoracic gland was observed during virus replication. Gland activity in virus-infected fourth-instar larvae was higher than controls and continued until the late stages of virus infection even after the time controls had ecdysed into fifth instars. During virus replication, the prothoracic gland was observed to maintain morphological and ultrastructural characteristics indicative of ecdysone biosynthetic activities. It was also demonstrated that the ecdysone conjugate present in the hemolymph of virus-infected insects did not serve as a feedback regulator for ecdysone biosynthesis. Although the exact mechanism for the continued activity of the gland is not known, this study suggests that the lack of feedback inhibition by the ecdysone conjugate may play an important role in raising the level of hormone in virus-infected insects. This altered hormonal level, composition, synthesis, and regulation was not observed in insects infected with an egt gene inactivated mutant virus. Therefore, it is apparent that LdNPV modulates the insect hormonal system through the expression of EGT.

The Bacillus anthracis plasmid pXO1 carries genes for the synthesis of anthrax toxin. Wild-type strains produce maximal amounts of the toxin components, protective antigen (PA), lethal factor (LF), and edema factor (EF), only when grown in the presence of bicarbonate and CO$\sb2$. Previous work in this laboratory identified other pXO1-associated phenotypes in B. anthracis Weybridge A. These include improved growth on minimal medium, reduced frequency of sporulation, and reduced sensitivity to bacteriophages. To localize regions of pXO1 that confer these phenotypes, transposon mutagenesis utilizing pTV1 was used to generate Tn917 insertions in pXO1. Insertion mutants were isolated that (i) exhibited an increase in sensitivity to bacteriophage; (ii) produced LF and EF, but were deficient in production of PA; (iii) were deficient in production of all three toxin components; and (iv) overproduced toxin in the presence and absence of added bicarbonate and CO$\sb2$. The locations of Tn917 insertions in plasmids from the latter two types of mutants were outside of the toxin structural genes indicating that regulatory genes may have been interrupted. A gene was identified between pag and cya that encoded a trans-acting positive regulatory factor designated atxA. A region upstream of lef may be involved in negative and/or bicarbonate regulation of toxin synthesis. Restriction analysis of pXO1 from B. anthracis Weybridge A as well as from several other strains revealed restriction profiles which differed from that previously published for pXO1 from Sterne. A series of DNA-DNA hybridizations with pXO1 (Weybridge A) and pXO1 (Sterne) showed that an inversion of approximately 40 kb had occurred in the toxin-encoding region. The junctions encompassing these regions of pXO1 from Weybridge A and Sterne were cloned. Results from DNA-DNA hybridization studies with the cloned junction fragments suggested that inverted repeats of perfect or near perfect match may be present. Thus far, no differences in the plasmid-associated phenotypes have been found that can be attributed to the inversion.