A previously established method, based on a two-plasmid system, was used to identify promoters recognized by RNA polymerase containing the extracytoplasmic stress response sigma factor sigmaE in Escherichia coli. In addition to previously identified rpoE-dependent promoters, 11 new promoters potentially directing the expression of 15 genes were identified that were active only after over-expression of rpoE. The promoters were confirmed and transcriptional start points of the promoters were determined by primer extension analysis and S1-nuclease mapping. All the promoters contained sequences similar to the consensus sequence of rpoE-dependent promoters. The new rpoE-dependent promoters governed expression of genes encoding proteins involved in primary metabolism (fusA, tufA, recR), phospholipid and lipopolysaccharide biosynthesis (psd, lpxP), signal transduction (sixA), proposed inner or outer membrane proteins (bacA, sbmA, smpA, yeaY), and proteins with unknown function (ybaB, yaiW, yiiS, yiiT, yfeY).

The extracytoplasmic function sigma factor, s E , has been shown to play a critical role in virulence of Salmonella enterica serovar Typhimurium (S. Typhimurium). The previously optimized two-plasmid system has been used to identify S. Typhimurium promoters recognized by RNA polymerase containing s E . This method allowed identification of 34 s E -dependent promoters that direct expression of 62 genes in S. Typhimurium, 23 of which (including several specific for S. Typhimurium) have not been identified previously to be dependent upon s E in Escherichia coli. The promoters were confirmed in S. Typhimurium and transcriptional start points of the promoters were determined by S1-nuclease mapping. All the promoters contained sequences highly similar to the consensus sequence of s E -dependent promoters. The identified genes belonging to the S.Typhimurium s E -regulon encode proteins involved in primary metabolism, DNA repair systems and outer-membrane biogenesis, and regulatory proteins, periplasmic proteases and folding factors, proposed lipoproteins, and inner-and outer-membrane proteins with unknown functions. Several of these s E -dependent genes have been shown to play a role in virulence of S. Typhimurium.

The alternative sigma factor B of Staphylococcus aureus controls the expression of a variety of genes, including virulence determinants and global regulators. Genetic manipulations and transcriptional start point (TSP) analyses showed that the sigB operon is transcribed from at least two differentially controlled promoters: a putative A -dependent promoter, termed sigB p1 , giving rise to a 3.6-kb transcript covering sa2059-sa2058-rsbU-rsbV-rsbW-sigB, and a B -dependent promoter, sigB p3 , initiating a 1.6-kb transcript covering rsbV-rsbWsigB. TSP and promoter-reporter gene fusion experiments indicated that a third promoter, tentatively termed sigB p2 and proposed to lead to a 2.5-kb transcript, including rsbU-rsbV-rsbW-sigB, might govern the expression of the sigB operon. Environmental stresses, such as heat shock and salt stress, induced a rapid response within minutes from promoters sigB p1 and sigB p3 . In vitro, the sigB p1 promoter was active in the early growth stages, while the sigB p2 and sigB p3 promoters produced transcripts throughout the growth cycle, with sigB p3 peaking around the transition state between exponential growth and stationary phase. The amount of sigB transcripts, however, did not reflect the concentration of B measured in cell extracts, which remained constant over the entire growth cycle. In a guinea pig cage model of infection, sigB transcripts were as abundant 2 and 8 days postinoculation as values found in vitro, demonstrating that sigB is indeed transcribed during the course of infection. Physical interactions between staphylococcal RsbU-RsbV, RsbV-RsbW, and RsbW-B were inferred from a yeast (Saccharomyces cerevisiae) two-hybrid approach, indicating the presence of a partner-switching mechanism in the B activation cascade similar to that of Bacillus subtilis. The finding that overexpression of RsbU was sufficient to trigger an immediate and strong activation of B , however, signals a relevant difference in the regulation of B activation between B. subtilis and S. aureus in the cascade upstream of RsbU.Staphylococcus aureus is one of the leading causes for nosocomial-and community-acquired infections (11,46). Its capacity to cause a wide spectrum of diseases and to survive in unfavorable conditions is due to a network of global regulatory elements enabling it to rapidly sense changes and to respond appropriately. These elements comprise two-component regulatory systems, including the agr locus, the SarA protein family, and alternative factors (reviewed in reference 16 and references within).Computational analysis of the published staphylococcal genomes suggests that S. aureus harbors only two alternative sigma factors, B and H (45). B of S. aureus was demonstrated to influence the expression of a variety of genes (6,26,33,43,78,79), including virulence factors (23,27,34,38,43,50,51,52,62,78,79) and regulatory elements (5,6,21,26,34,47,60,66). Moreover, it affects methicillin and glycopeptide resistance (4,56,65,74), biofilm production (58), and internalization into endothelial ce...

The alternative transcription factor B of Staphylococcus aureus affects the transcription of the cap gene cluster, required for the synthesis of capsular polysaccharide (CP), although this operon is lacking an apparent B -dependent promoter. Regulation of cap expression and CP production in S. aureus strain Newman was shown here to be influenced by B , the two-component signal transduction regulatory system ArlRS, and the yabJ-spoVG locus to different extents. Inactivation of arlR or deletion of the sigB operon strongly suppressed capA (CP synthesis enzyme A) transcription. Deletion of spoVG had a polar effect on yabJ-spoVG transcription and resulted in a two-to threefold decrease in capA transcription. Interestingly, immunofluorescence showed that CP production was strongly impaired in all three mutants, signaling that the yabJ-spoVG inactivation, despite its only partial effect on capA transcription, abolished capsule formation. trans-Complementation of the ⌬spoVG mutant with yabJ-spoVG under the control of its native promoter restored CP-5 production and capA expression to levels seen in the wild type. Northern analyses revealed a strong impact of B on arlRS and yabJ-spoVG transcription. We hypothesize that ArlR and products of the yabJ-spoVG locus may serve as effectors that modulate B control over B -dependent genes lacking an apparent B promoter.Staphylococcus aureus is a major nosocomial pathogen with the ability to cause a variety of diseases, including life-threatening infections. Like most microorganisms that are able to cause invasive diseases, S. aureus produces extracellular capsular polysaccharides (CPs), which are thought to be of importance in pathogenesis (reviewed in reference 35). Although 11 serologically distinct CPs were identified in S. aureus, the majority of clinical isolates produce CPs of serotype 5 (CP-5) or serotype 8 (CP-8). CPs protect S. aureus against opsonophagocytic killing by polymorphonuclear leukocytes (16,17,25,53,56) and enhance virulence in a number of animal models of staphylococcal infection (34,40,53,54,57). Expression of CPs is known to be influenced by various environmental signals in vitro and in vivo (reviewed in references 35 and 56), and transcription of the cap operon was shown to be modulated by regulatory elements, such as arlRS, agr, ccpA, mgr, sae, and sarA (7,8,23,24,26,27,39,48,52,55). Recent microarray analyses added the alternative factor B to the regulatory network controlling cap operon expression (3, 38) and indicated B to control capA transcription in a growth phasedependent manner (3). However, the lack of an apparent B consensus sequence in the promoter of capA suggested that B regulates cap transcription indirectly. Candidates for such downstream-acting regulators might be ArlRS and SarA, which are positively controlled by B in S. aureus (2, 3), although SarA was previously shown to have only a minor effect on cap expression and CP production in S. aureus (24). RNAIII of the agr locus, known to positively affect capA expression (7, 24, 55), could ...