Isolation of a Nucleoprotein Complex from Bacillus Subtilis Chromatin

To gain insight into the nature of bacterial chromatin, my thesis work was aimed at developing, adapting and applying methodology to stabilize proteins interacting with DNA in vivo. I examined the crosslinked chromatin for the possible existence of subunits involved in chromosome function. Putative chromatin subunits were isolated and then further examined for the presence of DNA and protein components. DNA and protein components were then isolated and characterized. A method was developed to stabilize the bacterial chromosome in its condensed, organized native form. A reversible crosslinker, formaldehyde, was used to stabilize nucleoids. The degree of nucleoid stabilization was examined by using computer-aided image analysis of 4,$\sp\prime$6-diamidino-2-phenylindole (DAPI) stained nucleoids. Putative chromatin subunits were isolated from the crosslinked chromatin by digestion with micrococcal nuclease (MN). The rationale is that MN will digest all regions of bacterial DNA, not complexed protein. DNA regions complexes with protein are resistant to nuclease digestion and are therefore enriched. Agarose gel electrophoresis, nondenaturing polyacrylamide electrophoresis, SDS polyacrylamide gel electrophoresis and two-dimensional polyacrylamide electrophoresis were used to examine crosslinked chromatin. Putative digests for discrete resistant fragments were isolated by nondenaturing polyacrylamide gel electrophoresis and by two-dimensional polyacrylamide gel electrophoresis. Three major chromatin subunits have been found. Each chromatin subunit was found to be associated with different proteins when isolated on nondenaturing polyacrylamide gels. Proteins associated with each nuclease resistant chromatin subunit were resolved on SDS polyacrylamide gels. Proteins were further characterized by immunoblotting with antibodies against DNA binding proteins, protein HU, RNA polymerase subunits $\beta/\beta\sp\prime,$ gyrase subunit $\beta$ and a replication site protein, 64 kD protein. A protein recognized by HU antibodies was found associated with one of the nuclease digest products. A second nuclease resistant fragment resolved from digests on nondenaturing polyacrylamide gels was recognized by antibodies against the 64 kD protein. Using in vivo labeling of DNA with tritiated thymidine, a nuclease resistant structure was shown to be associated with DNA. End labeling was used to show that a DNA associated with the chromatin fragment migrated as a single-stranded DNA of approximately 38 bases in denaturing gels following reversal of crosslinks