Transcription initiation of the copy-number control and better-thanrandom segregation genes of the broad-host-range and low-copynumber plasmid pSM19035 are subjected to repression by the autoregulated pSM19035-encoded product in Bacillus subtilis cells. The promoters of the copS (Pcop1 and Pcop2), ␦ (P␦), and (P) genes have been mapped. These promoters are embedded in a set of either seven copies of a 7-bp direct repeat or in a block consisting of two 7-bp direct repeats and one 7-bp inverted repeat; the blocks are present either two or three times. The cooperative binding of protein to the repeats on the Pcop1, Pcop2, P␦, and P promoters represses transcription initiation by a mechanism that does not exclude A RNAP from the promoters. These results indicate that protein regulates plasmid maintenance by controlling the copy number on the one hand and by regulating the amount of proteins required for better-than-random segregation on the other hand.plasmid replication ͉ Gram-positive bacteria ͉ protein-DNA interactions ͉ transcriptional repressor

Prototype strain MG409 (arg11-1) is a severe arginine bradytroph with greatly reduced ornithine and arginine pools, although all known enzymes required for arginine biosynthesis are functional. To identify the function required for normal arginine production impaired in MG409, we have cloned, sequenced, and performed a first molecular characterization of ARG11.We show that the ARG11 open reading frame encodes a putative 292-residue protein with a predicted molecular mass of 31.5 kDa. Sequence similarities, a tripartite organization, and six potential hydrophobic transmembrane spans suggest that Arg11p belongs to the mitochondrial integral inner membrane carrier family. We have used immuno-Western blotting and hemagglutinin epitope-tagged derivatives of Arg11p, Arg8p (a mitochondrial matrix marker), and Arg3p (a cytosolic marker) to demonstrate that Arg11p is confined to the mitochondria and behaves like an integral membrane protein.A deletion created in ARG11 causes the same arginineleaky behavior as the original arg11-1 mutation, which yields a premature stop codon at residue 266. Arg11p thus appears to fulfill a partially redundant function requiring its 27 carboxyl-terminal amino acids. As a working hypothesis, we propose that Arg11p participates in the export of matrix-made ornithine into the cytosol.

Reversal of the silencing of a tetracycline-regulated minimal promoter requires a chromatin-remodeling activity for subsequent promoter activation by the Tet-VP16 fusion protein. Thus, distinct regulatory elements may be combined to obtain long-term regulation and persistent expression of exogenous genes in eukaryotic cells.