Strategy and implementation of a system for protein engineering.

This paper describes an overall view of an industrial protein engineering project from conception to successful completion. The choice of rational design was determined by the availability of an excellent three-dimensional crystal structure and the availability of information in the literature to define a strategy. The design strategy was refined extensively during the course of the project. The development of methods for mutagenesis, expression, verification, purification, and characterization of mutant enzymes is dictated in part by the enzyme property one chooses to modify and must be rapid yet accurate. Such an approach would be applicable to improve the stability of any other protein or enzyme. Using this approach, we successfully increased the stability of subtilisin BL over 10-fold at 50 degrees C with an overall success rate greater than 60%.

Five unique temperate phages from a polylysogenic strain of Bacillus thuringiensis subsp. aizawai.

Five temperate phages were isolated from strain 4042B of Bacillus thuringiensis subsp. aizawai. The phages, which were heteroimmune, could also be distinguished by their host ranges, plaque and particle morphologies, serological specificities, and locations of restriction endonuclease cleavage sites on their chromosomes. Besides maintaining a stable lysogenic relationship with the 4042B host strain, each phage formed a stable lysogen with Bacillus cereus.

In vitro activity of the spermicide nonoxynol-9 against Chlamydia trachomatis.

The in vitro activity of nonoxynol-9 against four serotypes (C, D, H, and K) of Chlamydia trachomatis was investigated. A constant inoculum of each serotype was exposed to serial twofold dilutions (1:100 to 1:800) of Koromex, Conceptrol, or reference preparations (not containing nonoxynol-9) for 4 and 24 h at 37 degrees C. The mixtures of nonoxynal-9 or nonnonoxynol preparations and control inocula were dispensed into triplicate wells containing McCoy cell monolayers. After incubation at 37 degrees C, the monolayers were fixed and stained with iodine and examined for evidence of infection with C. trachomatis. All nonoxynol-9-containing preparations showed marked antichlamydial activity as judged by percent reduction of glycogen-containing intracytoplasmic inclusions. The reference preparations, which did not contain nonoxynol-9, were markedly less active when tested in this in vitro system.

Restriction fragments that exert promoter activity during postexponential growth of Bacillus subtilis.

Two restriction fragments of Bacillus subtilis DNA were identified which caused the cat-86 gene present on the promoter cloning plasmid pPL703 to be activated predominantly during postexponential growth of host cells. The postexponential increase was observed in both sporulation-positive strains and in a spoOA mutant of B. subtilis. However, the postexponential increase in the cat-86 gene product, chloramphenicol acetyltransferase, was diminished or not observed when the plasmid-containing cells were grown in the presence of excess glucose. The promoter-containing fragment, designated as 33, was mapped to a site on the B. subtilis chromosome adjacent to hisA. The other fragment, 14, mapped to a site adjacent to ctrA. When present on a high-copy vector, both fragments caused a reduction in the sporulation frequency of host cells. Fragment 33 in high copy number conferred on B. subtilis cells three additional phenotypic changes: brown colony color, intracellular inclusions, and, in a protease-deficient mutant, the production of extracellular protease activity. These activities were observed only in postexponential-phase cultures.

Use of restriction enzymes to investigate the source of a primary cytomegalovirus infection in a pediatric nurse.

The risk of transmission of cytomegalovirus (CMV) infection from congenitally infected infants to nonimmune medical attendants is unknown. The case of a CMV-seronegative, pregnant nurse who seroconverted after caring for an infant with symptomatic CMV infection is reported. She elected to be aborted and the fetal tissue contained CMV. Isolates from the nurse, the fetal tissue, and the infant to whom the nurse was exposed were examined for genetic relatedness by restriction enzyme analysis. As expected, the isolates from the nurse and the fetal tissue were identical. However, the virus isolated from the symptomatic infant was different from the strain infecting the nurse. These data indicate that the nurse acquired her infection from a source other than the index infant, either within the hospital or within the community.