SummaryThe conjugative ability of the F plasmid of Escherichia coli is highly growth phase dependent, with plasmid transfer efficiency dropping rapidly as donor cells progress through the growth cycle towards stationary phase. Transfer is dependent on the expression of the plasmid transfer ( tra ) genes, which are controlled by three plasmid-encoded regulatory proteins: TraJ, TraY and TraM. Here, we show that the nucleoid-associated host protein, H-NS, acts to repress the expression of traM and traJ as cells enter stationary phase, thereby decreasing mating ability to barely detectable levels. Sequence analysis identified regions of predicted intrinsic curvature, to which H-NS preferentially binds, at the promoters of both traM and traJ . H-NS binding at these regions was then confirmed by electrophoretic mobility shift and DNase I protection footprinting assays. Immunoblot assays displayed a significant increase in TraJ and TraM levels in an hns mutant strain. These findings were further supported by Northern and primer extension analyses which showed that whereas both genes were only expressed in early exponential phase in wild-type cells, hns mutant cells exhibited drastic derepression throughout the growth cycle. Transcriptional fusion studies of the individual promoters demonstrated that H-NS-mediated repression was observed when the promoters of both traM and traJ were present in cis to each other. This suggests that H-NS may bind to an extended region of the F plasmid, acting as a regional silencer of promoters for traJ and traM .

Ino80 is an ATP-dependent nucleosome-remodeling enzyme involved in transcription, replication, and the DNA damage response. Here, we characterize the fission yeast Ino80 and find that it is essential for cell viability. We show that the Ino80 complex from fission yeast mediates ATP-dependent nucleosome remodeling in vitro. The purification of the Ino80-associated complex identified a highly conserved complex and the presence of a novel zinc finger protein with similarities to the mammalian transcriptional regulator Yin Yang 1 (YY1) and other members of the GLI-Krüppel family of proteins. Deletion of this Iec1 protein or the Ino80 complex subunit arp8, ies6, or ies2 causes defects in DNA damage repair, the response to replication stress, and nucleotide metabolism. We show that Iec1 is important for the correct expression of genes involved in nucleotide metabolism, including the ribonucleotide reductase subunit cdc22 and phosphate-and adenineresponsive genes. We find that Ino80 is recruited to a large number of promoter regions on phosphate starvation, including those of phosphate-and adenine-responsive genes that depend on Iec1 for correct expression. Iec1 is required for the binding of Ino80 to target genes and subsequent histone loss at the promoter and throughout the body of these genes on phosphate starvation. This suggests that the Iec1-Ino80 complex promotes transcription through nucleosome eviction.The structure of chromatin is modulated by chromatin-remodeling factors, such as histone-modifying enzymes and ATP-dependent chromatin-remodeling complexes (7,35,45,54). The latter are usually multisubunit protein complexes containing an ATPase from the SWI2/SNF2 superfamily, which uses the energy derived from ATP hydrolysis to disrupt histone-DNA interactions (6,13,26).Ino80 is a member of this SWI2/SNF superfamily and is the catalytic subunit of the Ino80 chromatin-remodeling complex (65). Initially characterized in budding yeast (Saccharomyces cerevisiae), the Ino80 complex plays a central role in DNA-mediated processes, such as DNA doublestrand break repair, homologous recombination, and the regulation of the DNA damage cell cycle checkpoint response, chromosomal-DNA replication, and transcription (5,10,18,23,24,38,39,52,56,57,65,69,74,76,80,82; reviewed in reference 4). Ino80 complexes purified from budding yeast and mammalian cells contain core subunits, which are conserved across species, as well as species-specific proteins (36,65). In budding yeast, the core Ino80 complex is composed of INO80, ARP5, ARP8, ARP4, RVB1, RVB2, IES2, and IES6 (63,65). The actin-like proteins ARP5 and ARP8 are unique to the Ino80 complex and are required for an active complex in budding yeast (38,63). ARP4 plays a role in the response to DNA damage in budding yeast and is also a member of the chromatin-remodeling complex SWR1 and the histone acetyltransferase complex NuA4 (16,42,52,76). The Rvb1 and Rvb2 proteins (also called Pontin/Tip49 and Reptin/Tip48, respectively, in mammalian cells) are AAA ϩ ATPases related to the Hol...

The transfer (tra) operon of the conjugative F plasmid of Escherichia coli is a polycistronic 33-kb operon which encodes most of the proteins necessary for F-plasmid transfer. Here, we report that transcription from P Y , the tra operon promoter, is repressed by the host nucleoid-associated protein, H-NS. Electrophoretic mobility shift assays indicate that H-NS binds preferentially to the tra promoter region, while Northern blot and transcriptional fusion analyses indicate that transcription of traY, the first gene in the tra operon, is derepressed in an hns mutant throughout growth. The plasmid-encoded regulatory protein TraJ is essential for transcription of the tra operon in wild-type Escherichia coli; however, TraJ is not necessary for plasmid transfer or traY operon transcription in an hns mutant. This indicates that H-NS represses transcription from P Y directly and not indirectly via its effects on TraJ levels. These results suggest that TraJ functions to disrupt H-NS silencing at P Y , allowing transcription of the tra operon.

The Cpx (conjugative plasmid expression) stress response of Escherichia coli is induced in response to extracytoplasmic signals generated in the cell envelope, such as misfolded proteins in the periplasm. Detection of stress is mediated by the membrane-bound histidine kinase, CpxA. Signaling of the response regulator CpxR by activated CpxA results in the expression of several factors required for responding to cell envelope stress. CpxA was originally thought to be required for the expression of the positive regulator of the F plasmid transfer (tra) operon, TraJ. It was later determined that constitutive gain-of-function mutations in cpxA led to activation of the Cpx envelope stress response and decreased TraJ expression. In order to determine the nature of the downregulation of TraJ, the level of expression of TraJ, TraM, and TraY, the F-encoded regulatory proteins of the F tra region, was determined both in a cpxA* background and in a wild-type background in which the Cpx stress response was induced by overexpression of the outer membrane lipoprotein, NlpE. Our results suggest that TraJ downregulation is controlled by a posttranscriptional mechanism that operates in the cytoplasm in response to upregulation of the Cpx stress response by both the cpxA* gain-of-function mutation and the overexpression of NlpE.

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