The production of most factors involved in Bordetella pertussis virulence is controlled by a two-component regulatory system termed BvgA/S. In the Bvg+ phase virulence-activated genes (vags) are expressed, and virulence-repressed genes (vrgs) are down-regulated. The expression of these genes can also be modulated by MgSO(4) or nicotinic acid. In this study we used microarrays to analyse the influence of BvgA/S or modulation on the expression of nearly 200 selected genes. With the exception of one vrg, all previously known vags and vrgs were correctly assigned as such, and the microarray analyses identified several new vags and vrgs, including genes coding for putative autotransporters, two-component systems, extracellular sigma factors, the adenylate cyclase accessory genes cyaBDE, and two genes coding for components of a type III secretion system. For most of the new vrgs and vags the results of the microarray analyses were confirmed by RT-PCR analysis and/or lacZfusions. The degree of regulation and modulation varied between genes, and showed a continuum from strongly BvgA/S-activated genes to strongly BvgA/S-repressed genes. The microarray analyses also led to the identification of a subset of vags and vrgs that are differentially regulated and modulated by MgSO(4) or nicotinic acid, indicating that these genes may be targets for multiple regulatory circuits. For example, the expression of bilA, a gene predicted to encode an intimin-like protein, was found to be activated by BvgA/S and up-modulated by nicotinic acid. Furthermore, surprisingly, in the strain analysed here, which produces only type 2 fimbriae, the fim3 gene was identified as a vrg, while fim2 was confirmed to be a vag.

A43, an essential subunit of yeast RNA polymerase I (pol I), interacts with Rrn3, a class I general transcription factor required for rDNA transcription. The pol I-Rrn3 complex is the only form of enzyme competent for promoter-dependent transcription initiation. In this paper, using biochemical and genetic approaches, we demonstrate that the A43 polypeptide forms a stable heterodimer with the A14 pol I subunit and interacts with the common ABC23 subunit, the yeast counterpart of the subunit of bacterial RNA polymerase. We show by immunoelectronic microscopy that A43, ABC23, and A14 colocalize in the three-dimensional structure of the pol I, and we demonstrate that the presence of A43 is required for the stabilization of both A14 and ABC23 within the pol I. Because the N-terminal half of A43 is clearly related to the pol II Rpb7 subunit, we propose that the A43-A14 pair is likely the pol I counterpart of the Rpb7-Rpb4 heterodimer, although A14 distinguishes from Rpb4 by specific sequence and structure features. This hypothesis, combined with our structural data, suggests a new localization of Rpb7-Rpb4 subunits in the three-dimensional structure of yeast pol II.T he yeast Saccharomyces cerevisiae possesses three forms of nuclear RNA polymerase (pol I, II, and III), which are distinct by their subcellular localization, chromatographic behavior, subunit composition, sensitivity to ␣-amanitine, and promoter͞template specificity.The unique essential function of pol I is to transcribe multiple ribosomal DNA units to generate the 35S ribosomal precursor (1), which is subsequently matured into the functional 18S, 5.8S, and 25S RNA species (2). Yeast pol I contains 14 subunits that include a core of five subunits (A190, A135, AC40, AC19, and ABC23) related to the ␤Ј␤␣ 2 eubacterial core enzyme (3, 4). In addition to ABC23, four subunits (ABC27, ABC14.5, ABC10␣, and ABC10␤) are shared by the three forms of enzyme (5). Finally, five other polypeptides (A49, A43, A34.5, A14, and A12.2) are subunits of pol I (6-10).Although requirement of such a complex structure is still an open question, substantial amounts of data have highlighted functional properties of subunits. The active site is carried out by the two large subunits in bacteria, archaebacteria and eukaryotes (11)(12)(13)(14). Additional studies have provided insights into the function of smaller subunits of pol I. The nonessential A34.5 subunit may help the enzyme to overcome the topological constraints imposed on rDNA by transcription (8). The dispensable A14 subunit might cooperate with A34.5 subunit in this process (9) and is important for the stabilization of subunits A43 and ABC23 within the pol I (9). A49 subunit displays ribonuclease H activity (15), whose involvement in pol I transcription is still elusive. A12.2 subunit is the pol I counterpart of pol II and pol III subunits involved in RNA cleavage activity of both enzymes (16,17), suggesting that A12.2 may be implicated in the retraction and͞or termination of pol I. Finally, the essential A43 subunit (7), ...

BackgroundThe objective of the study was to analyse the evolution of Bordetella pertussis population and the influence of herd immunity in different areas of the world where newborns and infants are highly vaccinated.MethodologyThe analysis was performed using DNA microarray on 15 isolates, PCR on 111 isolates as well as GS-FLX sequencing technology on 3 isolates and the B. pertussis reference strain, Tohama I.Principal FindingsOur analyses demonstrate that the current circulating isolates are continuing to lose genetic material as compared to isolates circulating during the pre-vaccine era whatever the area of the world considered. The lost genetic material does not seem to be important for virulence. Our study confirms that the use of whole cell vaccines has led to the control of isolates that were similar to vaccine strains. GS-FLX sequencing technology shows that current isolates did not acquire any additional material when compared with vaccine strains or with isolates of the pre-vaccine era and that the sequenced strain Tohama I is not representative of the isolates. Furthermore, this technology allowed us to observe that the number of Insertion Sequence elements contained in the genome of the isolates is temporally increasing or varying between isolates.Conclusions B. pertussis adaptation to humans is still in progress by losing genetic material via Insertion Sequence elements. Furthermore, recent isolates did not acquire any additional material when compared with vaccine strains or with isolates of the pre-vaccine era. Herd immunity, following intensive vaccination of infants and children with whole cell vaccines, has controlled isolates similar to the vaccine strains without modifying significantly the virulence of the isolates. With the replacement of whole cell vaccines by subunit vaccines, containing only few bacterial antigens targeting the virulence of the bacterium, one could hypothesize the circulation of isolates expressing less or modified vaccine antigens.