Capsular polysaccharide synthesis, cell division and radiation sensitivity are controlled by the capR(a synonym for Ion) gene, as well as other genes in E. coli K-12. The overall objectives of the proposed research are: (a) to understand the mechanism of control of cell division at a molecular level as related to mutations in E. coli K12 designated capR, sulA and sulB. The capR mutation causes sensitivity to UV, X-rays, ozone and furadantin (a radiomimetic drug). After treatment with UV, capR mutants form a nonseptate filament that does not divide and will not produce a clone. Either mutation in sulA or sulB reverses the sensitivity of capR mutants to UV and furadantin. capR sulA, and sulB map in different regions of the E. coli K12 chromosome. (b) To understand how capR mutations also control at the molecular level, synthesis of the enzymes of polysaccharide synthesis and overproduction of the capsular polysaccharide (with emphasis on the galETK operon). (c) To understand how our recently cloned genes specifying a major outer membrane protein and two other polypeptides (none of which are the capR gene product) control capsular polysaccharide synthesis in capR mutants (see Progress Report). We have cloned the capR ion gene on a tetracylcine resistance plasmid and are identifying the polypeptide(s) that it specifies in minicell producing bacteria. We will purify the capR polypeptide(s) and determine the function(s) in vitro (repressor, protease, nuclease, etc.). Similar techniques, including lambda bacteriophage cloning vectors, are being used to isolate the sulA ion and sulB ion genes and the proteins they specify. We have cloned a 2 times 10 to the 6th power dalton DNA fragment that controls capsular polysaccharide synthesis and three polypeptides that it specifies, including the normal major outer membrane polypeptide a (or 3b), are needed to shut off capsule synthesis. We will purify them and study whether one is a precursor of a and will determine their function(s) in vitro (repressor, protease, nuclease, etc.).