PilC1, a pilus‐associated protein in Neisseria meningitidis, is a key element in initial meningococcal adhesion to target cells. A promoter element (CREN, contact regulatory element of Neisseria) is responsible for the transient induction of this gene upon cell contact. crgA (contact‐regulated gene A) encodes a transcriptional regulator whose expression is also induced upon cell contact from a promoter region similar to the CREN of pilC1. CrgA shows significant sequence homologies to LysR‐type transcriptional regulators. Its inactivation in meningococci provokes a dramatic reduction in bacterial adhesion to epithelial cells. Moreover, this mutant is unable to undergo intimate adhesion to epithelial cells or to provoke effacing of microvilli on infected cells. Purified CrgA is able to bind to pilC1 and crgA promoters, and CrgA seems to repress the expression of pilC1 and crgA. Our results support a dynamic model of bacteria–cell interaction involving a network of regulators acting in cascade. CrgA could be an intermediate regulator in such a network.

SummaryPilus-mediated adherence makes an essential contribution to the pathogenesis of Neisseria meningitidis by allowing the initial localized adherence. Pili are assembled from a protein subunit called pilin. Two proteins, PilC1 and PilC2, are also key elements in the formation of pili as the production of at least one PilC protein is required for pilus assembly. In addition, PilC1 but not PilC2 modulates adhesiveness, most probably by being the adhesin. Recently, both genes have been demonstrated to be controlled by different promoters, pilC2 is expressed from a single transcription starting point (TSP), whereas pilC1 has three TSPs. One of these, PC1.1, corresponds to the unique TSP of pilC2, and two others, PC1.2 and PC1.3, are located in a region upstream of pilC1 but not pilC2. This suggests that both genes may be under the control of separate regulatory pathways. In this work, by engineering pilC1-lacZ and pilC2-lacZ transcriptional fusions, we provide evidence that expression of pilC1, but not that of pilC2, is transiently induced by bacterial cell contact. This induction required viable cells, did not need the presence of pili and relied on the expression of pilC1 from PC1.3. Destruction of this TSP by site-directed mutagenesis did not significantly diminish the piliation level or the basal expression of PilC1, but led to the loss of cell contact-dependent upregulation of pilC1 and to a dramatic decrease in bacterial adhesiveness. Taken together, these data demonstrate that cell contact-dependent upregulation of the transcription of pilC1 at PC1.3 is essential for meningococcal pilusmediated adhesion.

Metabolomics is considered as an emerging new tool for functional proteomics in the identification of new protein function or in projects aiming at modeling whole cell metabolism. When combined with proteome studies, metabolite-profiling analyses revealed unanticipated insights into the yeast sulfur pathway. In response to cadmium, the observed overproduction of glutathione, essential for the detoxification of the metal, can be entirely accounted for by a marked drop in sulfur-containing protein synthesis and a redirection of sulfur metabolite fluxes to the glutathione pathway. A kinetic analysis showed sequential and dramatic changes in intermediate sulfur metabolite pools within the first hours of the treatment. Strikingly, whereas proteome and metabolic data were positively correlated under cadmium conditions, proteome and metabolic data were negatively correlated during other growth conditions, i.e. methionine supplementation or sulfate starvation. These differences can be explained by alternative mechanisms in the regulation of Met4, the activator of the sulfur pathway. Whereas Met4 activity is controlled by the cellular cysteine content in response to sulfur source and availability, the present study suggests that Met4 activation under cadmium conditions is cysteine-independent. The results clearly indicate that the metabolic state of a cell cannot be safely predicted based solely on proteomic and/or gene expression data. Combined metabolome and proteome studies are necessary to draw a comprehensive and integrated view of cell metabolism.Assimilable sulfur is essential for all living organisms. The cell requirement for sulfur can be fulfilled by the uptake of sulfur-containing amino acids or by assimilation of inorganic sulfur into organic compounds such as cysteine or homocysteine (1, 2). In yeast, homocysteine is the precursor of methionine through the methyl cycle and of cysteine through the transsulfuration pathway ( Fig. 1) (3). Cysteine is the sensor of the metabolic state in the sulfur amino acid pathway (4) and is required for the synthesis of GSH, an essential antioxidant molecule also important for detoxification.The yeast sulfur pathway has been extensively investigated at the genetic, enzymatic, and regulatory levels (3). The pools of most metabolites of the pathway have been analyzed (5, 6), and the K m values of many enzymes have been measured (3). However, some metabolic data such as the metabolite fluxes in the pathway and the concentration of the metabolites of the transsulfuration pathway (homocysteine and cystathionine) are lacking. Moreover, the levels of some sulfur metabolites are presumed to be modified in different mutants and under different physiological conditions (i.e. sulfur starvation, the presence of a sulfur metabolite, or a toxic metal in the medium), but the few quantitative data that are available are restricted to a small part of the pathway (5). Thus, it has been shown that cadmium (Cd 2ϩ ) strongly increases GSH synthesis (7), which is consistent with the primary impor...

SummaryThe spores of Bacillus anthracis, the agent of anthrax disease, germinate within professional phagocytes, such as murine macrophage-like RAW264.7 cells and alveolar macrophages. We identi®ed a cluster of germination genes extending for 3608 nucleotides between the pag and atxA genes on the B. anthracis virulence plasmid pXOl. The three predicted proteins (40, 55 and 37 kDa in size) have signi®cant sequence similarities to B. subtilis, B. cereus and B. megaterium germination proteins. Northern blot analysis of total RNA from sporulating cells indicated that the gerX locus was organized as a tricistronic operon (gerXB, gerXA and gerXC ). Primer extension analysis identi®ed a major potential transcriptional start site 31 bp upstream from the translation initiation codon of gerXB. Expression of the gerX operon was studied using a gerXB±lacZ transcriptional fusion. Expression began 2.5±3 h after the initiation of sporulation and was detected exclusively in the forespore compartment. A gerX null mutant was constructed. It was less virulent than the parental strain and did not germinate ef®ciently in vivo or in vitro within phagocytic cells. These data strongly suggest that gerXencoded proteins are involved in the virulence of B. anthracis.