Transcription by RNA polymerase (RNAP) is often regulated by interactions with control proteins to link specific gene expression to environmental signals and temporal cues. Often activators help recruit RNAP to promoters to increase initiation rates (Busby and Ebright 1999). In contrast, activity of the bacterial 54 containing RNAP holoenzyme is regulated at the DNA melting step (for review, see Buck et al. 2000). Hydrolysis of an NTP by an activator drives a change in configuration of the 54 -holoenzyme, converting the initial closed complex to an open complex to allow interaction with the template DNA for mRNA synthesis (Wedel and Kustu 1995). Preopening of DNA templates does not overcome the requirement for NTP hydrolysis by an activator to promote engagement of the holoenzyme with the melted DNA (Wedel and Kustu 1995;Cannon et al. 1999).The activators of 54 -holoenzyme are members of the large AAA+ protein family, which use ATP binding and hydrolysis to remodel their substrates (Neuwald et al. 1999;Cannon et al. 2000Cannon et al. , 2001. The greater part of the central domain of 54 activators corresponds to the AAA core structure, and includes ATP-binding and hydrolyzing determinants. The 54 protein is known to be the primary target for the NTPase of activators, but how activators use NTP binding and hydrolysis is not well understood (Cannon et al. 2000). Similarly, the nature of the interaction between 54 and the activator is not well described, but an interaction with 54 can be detected in the case of the DctD activator by protein cross-linking (Lee and Hoover 1995). Here we show that the use of ADP-aluminum fluoride, an analog of ATP that mimics ATP in the transition state for hydrolysis, allows formation of a stable complex among the activator PspF, the PspF and NifA central activating domains, and 54 . The binding assay was used to help define determinants in 54 and the activator needed for their interaction, and to show that binding can lead to an altered 54 -DNA footprint. The need for a transition-state analog of ATP for protein-protein binding is discussed in relation to the required ATPase activity of activators of 54 -dependent transcription. In particular, it seems that altered functional states of activators exist as ATP is hydrolyzed. This suggests a parallel to some switch and motor proteins that use nucleotide binding and hydrolysis to establish alternate functional states (Hirose and Amos 1999).

In bacteria, association of the specialized N protein with the core RNA polymerase subunits forms a holoenzyme able to bind promoter DNA, but unable to melt DNA and initiate transcription unless acted on by an activator protein. The conserved amino-terminal 50 amino acids of N (Region I) are required for the response to activators. We have used pre-melted DNA templates, in which the template strand is unpaired and accessible for transcription initiation, to mimic a naturally melted promoter and explore the function of Region I. Our results indicate that one activity of Region I sequences is to inhibit productive interaction of holoenzyme with pre-melted DNA. On pre-melted DNA targets, either activation of N -holoenzyme or removal of Region I allowed efficient formation of complexes in which melted DNA was sequestered by RNA polymerase. Like natural pre-initiation complexes formed on conventional DNA templates through the action of activator, such complexes were heparin-resistant and transcriptionally active. The inhibitory N Region I domain functioned in trans to confer heparin sensitivity to complexes between Region I-deleted holoenzyme and pre-melted promoter DNA. Evidence that Region I senses the conformation of the promoter was obtained from protein footprint experiments. We suggest that one function for Region I is to mask a single-strand DNA-binding activity of the holoenzyme. On the basis of extended DNA footprints of Region I-deleted holoenzyme, we also propose that Region I prevents RNA polymerase isomerization, a conformational change necessary for access to and the subsequent stable association of holoenzyme with melted DNA.

MarA, SoxS and Rob are transcription factors belonging to the AraC family. While these proteins have been associated historically with control of multiple antibiotic resistance, and tolerance to oxidative stress agents and organic solvents, only a paucity of experimental data support a role in regulating virulence. Clinical Escherichia coli isolates, and isogenic strains lacking marA, soxS and rob, were studied in a murine model of ascending pyelonephritis, which is a clinically relevant model of urinary tract infection. Organisms lacking all three transcription factors (triple knockouts) were significantly less virulent than parental strains, and complementation studies demonstrated that the addition of marA, soxS and rob individually restored wild-type virulence in the triple-knockout strain. Deletion of soxS or rob alone was more detrimental than the removal of marA. Thus, all three proteins contribute to virulence in vivo. INTRODUCTIONThe AraC family of transcription factors is composed of more than 1000 members (Alekshun & Levy, 2004), many of which have well-known roles as virulence factors (Finlay & Falkow, 1997). ExsA from Pseudomonas aeruginosa regulates a type III secretion system (TTSS) (Hauser et al., 1998), Yersinia spp. LcrF (VirF) and YbtA control a TTSS (Flashner et al., 2004) and yersiniabactin (siderophore) (Fetherston et al., 1996) biosynthesis, respectively, and ToxT from Vibrio cholerae governs the synthesis of cholera toxin and toxin coregulated pili (Champion et al., 1997). Inactivation of genes specifying AraC family members [e.g. BfpT, ToxT, LcrF (VirF), Rv1931c, ExsA, Sp1433 and MarA] attenuates virulence in human subjects (Bieber et al., 1998) and a variety of animal infection models (Champion et al., 1997;Flashner et al., 2004; Frota et al., 2004;Hauser et al., 1998;Hava & Camilli, 2002;Randall & Woodward, 2001).Thus, in addition to a primary role in virulence, it is assumed that many members of the AraC family play larger roles in affecting the overall physiology of the bacterial cell.Notably, genomic array experiments have shown that P. aeruginosa ExsA and V. cholerae ToxT regulate the expression of a large collection of genes termed regulons (Bina et al., 2003;Wolfgang et al., 2003).Escherichia coli MarA and SoxS were originally identified based on their ability to control multiple antibiotic resistance (Mar) (George & Levy, 1983a, b), and susceptibility to superoxide and other oxidative stress agents (Wu & Weiss, 1991), respectively. Experiments with Rob, a MarA and SoxS paralogue, showed that it could function in a similar manner (Ariza et al., 1995). Subsequent data have documented multidrug-resistant clinical strains of E. coli (Linde et al., 2000; Maneewannakul & Levy, 1996) and Salmonella enterica serovar Typhimurium (S. typhimurium) (Koutsolioutsou et al., 2001) that constitutively express AraC family members. It is therefore surmised that these proteins may play a role in the infectious process.Although the soxS and mar loci are expressed by S. typhimurium within macrophag...

The alternative bacterial N RNA polymerase holoenzyme binds promoters as a transcriptionally inactive complex that is activated by enhancer-binding proteins. Two distinct classes of prokaryotic factors are known based on sequence comparison and functional differences. One class, typified by the major vegetative sigma in Escherichia coli, 70 , binds promoter elements located at Ϫ10 and Ϫ35 with respect to the initiation site, and generally binds in a transcription competent state (1). The second class, related to the E. coli 54

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The locations of two mutations that prevent adhesion of Pseudomonas fluorescens Pf0-1 to sand columns and seeds (adn, adhesion) were identified. Both lie in a single gene showing homology to the NtrC/NifA family of transcription activators. The predicted 55 kDa protein encoded by adnA is most closely related to activators involved in expression of flagellar proteins, consistent with the lack of flagella in adnA strains. Constitutive adnA expression restored motility and adhesion to an adnA strain, demonstrating that the observed phenotypes are due to lack of AdnA and not a consequence of other mutations or polar effects of mutations in adnA on other genes.

A soil plot was inoculated with a mixture of Pseudomonas fluorescens Pf0-2, the wild type, and Pf0-5a, a Tn5 insertion mutant in adnA, at 7.84 log CFU/g of soil. Over a period of 231 days, culturable populations of both strains were measured at selected times below and away from the point of inoculation. Pf0-5a did not spread as fast and attained significantly lower populations than Pf0-2. At sample depths below the inoculation site, the adnA mutant showed a significant decrease in CFU/g of soil as compared to Pf0-2. Pf0-2 was first detected at the 1.5-cm annular site at 3 days after inoculation, whereas Pf0-5a required 7 days to travel the same distance. At this distance, the wild-type strain could be detected at a 21.5-to 25-cm depth, whereas Pf0-5a could be detected only as deep as 15.5 to 18 cm. At 4.5 cm from the site of inoculation and in soil fractions corresponding to 13 to 18 cm, Pf0-2 was the only strain detected. These results suggest that the transcription factor AdnA provides a fitness advantage in P. fluorescens, allowing it to spread and survive in soil under field conditions.The effective use of microbial agents, such as the pseudomonads, to remedy plant diseases, decontaminate soil, improve texture, and enhance plant nutrition requires that such agents be ecologically adapted to the location where they are released. However, inoculation of selected microbes into soil or on seeds often results in rapid loss of their population within a short time (2). Such phenomena and the factors influencing this decline have been reviewed (23).DeFlaun et al. (9) isolated two Tn5 insertion mutants of Pseudomonas fluorescens strain Pf0-1 that were deficient in attachment to soil particles and seeds and were nonmotile. The locus affected by these Tn5 insertions was identified as adnA. In complementation experiments, cloned wild-type adnA restored the wild-type phenotype described for the Tn5 insertion mutants (7a). Sequencing and expression experiments revealed that AdnA is a homologue of FleQ (7a), a transcriptional activator of the NtrC/NifA family in Pseudomonas aeruginosa which, in association with sigma 54, mediates transcription of structural genes for flagellar synthesis (4).Other regulation factors have been linked to fitness in natural environments, e.g., RosR in Rhizobium etli and GacS in Pseudomonas syringae (6,12,13). RosR is likely to regulate exopolysaccharide production, whereas GacS has been implicated in regulation of several activities, such as swarming, antibiotic, toxin, and siderophore production in the genus Pseudomonas (11,14,18,19,24). The rosR mutant showed a reduced ability to grow in the rhizosphere and compete for nodule occupancy under laboratory conditions (3, 6). In field experiments, the gacS mutant attained significantly lower populations than those of the parent strain in bean foliage, but there was no effect on the populations in soil surrounding germinating seeds (12). These experiments were conducted in either laboratory conditions or for the short term and studied the effects...