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Background: The phytohormone indole-3-acetic acid (IAA) is widely distributed among plant-associated bacteria. Certain strains of the Pseudomonas syringae complex can further metabolize IAA into a less biologically active amino acid conjugate, 3-indole-acetyl-ε-L-lysine, through the action of the iaaL gene. In P. syringae and Pseudomonas savastanoi strains, the iaaL gene is found in synteny with an upstream gene, here called matE, encoding a putative MATE family transporter. In P. syringae pv. tomato (Pto) DC3000, a pathogen of tomato and Arabidopsis plants, the HrpL sigma factor controls the expression of a suite of virulence-associated genes via binding to hrp box promoters, including that of the iaaL gene. However, the significance of HrpL activation of the iaaL gene in the virulence of Pto DC3000 is still unclear., Results: A conserved hrp box motif is found upstream of the iaaL gene in the genomes of P. syringae strains. However, although the promoter region of matE is only conserved in genomospecies 3 of this bacterial group, we showed that this gene also belongs to the Pto DC3000 HrpL regulon. We also demonstrated that the iaaL gene is transcribed both independently and as part of an operon with matE in this pathogen. Deletion of either the iaaL or the matE gene resulted in reduced fitness and virulence of Pto DC3000 in tomato plants. In addition, we used multicolor fluorescence imaging to visualize the responses of tomato plants to wild-type Pto DC3000 and to its ΔmatE and ΔiaaL mutants. Activation of secondary metabolism prior to the development of visual symptoms was observed in tomato leaves after bacterial challenges with all strains. However, the observed changes were strongest in plants challenged by the wild-type strain, indicating lower activation of secondary metabolism in plants infected with the ΔmatE or ΔiaaL mutants., Conclusions: Our results provide new evidence for the roles of non-type III effector genes belonging to the Pto DC3000 HrpL regulon in virulence.

Conformational changes in sigma 54 (sigma(54)) and sigma(54)-holoenzyme depend on nucleotide hydrolysis by an activator. We now show that sigma(54) and its holoenzyme bind to the central ATP-hydrolyzing domains of the transcriptional activators PspF and NifA in the presence of ADP-aluminum fluoride, an analog of ATP in the transition state for hydrolysis. Direct binding of sigma(54) Region I to activator in the presence of ADP-aluminum fluoride was shown and inferred from in vivo suppression genetics. Energy transduction appears to occur through activator contacts to sigma(54) Region I. ADP-aluminum fluoride-dependent interactions and consideration of other AAA+ proteins provide insight into activator mechanochemical action.

Transcription initiation by the sigma(54) RNA polymerase requires specialised activators and their associated nucleoside triphosphate hydrolysis. To explore the roles of sigma(54) in initiation we used random mutagenesis of rpoN and an in vivo activity screen to isolate functionally altered sigma(54) proteins. Five defective mutants, each with a different single amino acid substitution, were obtained. Three failed in transcription after forming a closed complex. One such mutant mapped to regulatory Region I of sigma(54), the other two to Region III. The Region I mutant allowed transcription independently of activator and showed reduced activator-dependent sigma(54) isomerisation. The two Region III mutants displayed altered behaviour in a sigma(54) isomerisation assay and one failed to stably bind early melted DNA as the holoenzyme; they may contribute to a communication pathway linking changes in sigma to open complex formation. Two further Region III mutants showed gross defects in overall DNA binding. For one, sufficient residual DNA binding activity remained to allow us to demonstrate that other activities were largely unaffected. Changes in DNA binding preferences and core polymerase-dependent properties were evident amongst the mutants.

Control of transcription frequently involves the direct interaction of activators with RNA polymerase. In bacteria, the formation of stable open promoter complexes by the sigma(54) RNA polymerase is critically dependent on sigma(54) amino Region I sequences. Their presence correlates with activator dependence, and removal allows the holoenzyme to engage productively with melted DNA independently of the activator. Using purified Region I sequences and holoenzymes containing full-length or Region I-deleted sigma(54), we have explored the involvement of Region I in transcription activation. Results show that Region I in trans inhibits a reversible conformational change in the holoenzyme believed to be polymerase isomerization. Evidence is presented indicating that the holoenzyme (and not the promoter DNA per se) is one interacting target used by Region I in preventing polymerase isomerization. Activator overcomes this inhibition in a reaction requiring nucleotide hydrolysis. Region I in trans is able to inhibit activated transcription by the holoenzyme containing full-length sigma(54). Inhibition appeared to be noncompetitive with respect to the activator, suggesting that a direct activator interaction occurs with parts of the holoenzyme outside Region I. Stabilization of isomerized holoenzyme bound to melted DNA by Region I in trans occurs largely independently of the initiating nucleotide, suggesting a role for Region I in maintaining the open complex.

sigmaN (sigma54) RNA polymerase holoenzyme closed complexes isomerize to open complexes in a reaction requiring nucleoside triphosphate hydrolysis by enhancer binding activator proteins. Here, we characterize Klebsiella pneumoniae sigmaN mutants, altered in the carboxy DNA-binding domain (F354A/F355A, F402A, F403A and F402A/F403A), that fail in activator-dependent transcription. The mutant holoenzymes have altered activator-dependent interactions with promoter sequences that normally become melted. Activator-dependent stable complexes accumulated slowly in vitro (F402A) and to a reduced final level (F403A, F402A/F403A, F354A/F355A). Similar results were obtained in an assay of activator-independent stable complex formation. Premelted templates did not rescue the mutants for stable preinitiation complex formation but did for deleted region I sigmaN, suggesting different defects. The DNA-binding domain substitutions are within sigmaN sequences previously shown to be buried upon formation of the wild-type holoenzyme or closed complex, suggesting that, in the mutants, alteration of the sigmaN-core and sigmaN-DNA interfaces has occurred to change holoenzyme activity. Core-binding assays with the mutant sigmas support this view. Interestingly, an internal deletion form of sigmaN lacking the major core binding determinant was able to assemble into holoenzyme and, although unable to support activator-dependent transcription, formed a stable activator-independent holoenzyme promoter complex on premelted DNA templates.

Sigma subunits of bacterial RNA polymerases are closely involved in many steps of promoter-specific transcription initiation. Holoenzyme formed with the specialised minor sigma-N (sigmaN) protein binds rare promoters in a transcriptionally inactive state and functions in enhancer-dependent transcription. Using competition and dissociation assays, we show that sigmaN-holoenzyme has a stability comparable to the major sigma70-holoenzyme. Purified partial sequences of sigmaN were prepared and assayed for retention of core RNA polymerase binding activity. Two discrete fragments of sigmaN which both bind the core but with significantly different affinities were identified, demonstrating that the sigmaN interface with core RNA polymerase is extensive. The low affinity segment of sigmaN included region I sequences, an amino terminal domain which mediates activator responsiveness and formation of open promoter complexes. The higher affinity site lies within a 95 residue fragment of region III. We propose that the core to region I contact mediates properties of the sigmaN-holoenzyme important for enhancer responsiveness. Heparin is shown to dissociate sigmaN and core, indicating that disruption of the holoenzyme is involved in the heparin sensitivity of the sigmaN closed complex., (Copyright 1999 Academic Press.)

In bacteria, association of the specialized sigmaN 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 sigmaN (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 sigmaN-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 sigmaN 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.

The xylR and xylS genes are divergent and control transcription of the TOL plasmid catabolic pathways for toluene metabolism. Four promoters are found in the 300-bp intergenic region: Pr1 and Pr2 are constitutive sigma70-dependent tandem promoters that drive expression of xylR, while expression of the xylS gene is driven from Ps2, a constitutive sigma70-dependent promoter, and by the regulatable sigma54 class Ps1 promoter. In Ps1 the XylR targets (upstream activator sequences [UASs]) overlap the Pr promoters, and two sites for integration host factor (IHF) binding are located at the region from positions -2 to -30 (-2/-30 region) and the -137/-156 region, the latter overlapping the Pr promoters. When the XylR protein binds to the UASs in the absence of effector, it represses expression from Pr promoters. In the XylR-plus background and in the absence of an effector, the level of expression from Ps1 is low, although detectable, whereas Ps2 is active. In this background and in the presence of an effector, XylR increases autorepression. In a sigma54-deficient Pseudomonas putida background, no expression occurred from Ps1 regardless of the presence of an effector. However, in the presence of an effector, the amount of RNA produced from Pr promoters was almost undetectable. This finding suggests that when no transcription occurred at the Ps1 promoter, clearance of XylR from the UASs was almost negligible. In this background, expression from Ps2 was very high regardless of the presence of an effector; this finding suggests that RNA polymerase containing sigma54 modulates expression from the downstream Ps2 sigma70-dependent promoter. In a P. putida IHF-minus background and in the presence of effector, Ps1 expression was the highest found; in contrast, the basal levels of this promoter were the lowest observed. This finding suggests that IHF acts in vivo as a repressor of the sigma54-dependent Ps1 promoter. In an IHF-deficient host background, expression from Ps2 in the presence of effector was negligible. Thus, binding of RNA polymerase containing sigma54 at the upstream promoter may modulate expression from the Ps2 promoter.

The TOL plasmid pWW53 encodes a catabolic pathway for the metabolism of toluene. It bears an upper-pathway operon for the oxidation of toluene to benzoate and a copy of the gene that encodes regulatory protein XylR. For metabolism of the aromatic carboxylic acid, it bears two functional homologous meta-pathway operons, together with two functional copies of the xylS regulatory gene (xylS1 and xylS3). In cells growing in the absence of pathway substrates, no mRNA from upper- and meta-pathway operons were found; however, the xylR gene was expressed from two sigma70-dependent tandem promoters, and the xylS1 and the xylS3 genes were also expressed from their sigma70-dependent promoters, called Ps2 and Ps3, respectively. In cells grown in the presence of o-xylene, the XylR protein became active and stimulated transcription from the Pu promoter for the upper pathway. Expression from xylS1 but not from xylS3 was also stimulated by XylR; this was due to activation of transcription from the xylS1 Ps1 promoter, which is sigma54 dependent, and the lack of effect on expression from the Ps2 sigma70-dependent promoter. As a result of overexpression of the xylS1 gene, the XylS1 protein was overproduced and activated transcription from Pm1 and Pm2. In cells growing on benzoate, the upper-pathway operon was not expressed, but both meta operons were expressed. Given that XylS1 but not XylS3 recognized benzoate as an effector, stimulation of transcription was found to be mediated by XylS1. This was confirmed with cloned meta-pathway promoters and regulators. When 3-methylbenzoate was present in the medium, both meta operons were also expressed and stimulation of transcription was mediated by both XylS1 and XylS3, which both recognized 3-methylbenzoate as an effector.

Growth of Pseudomonas putida (pWWO) on alkylbenzoates requires the expression of the meta pathway operon, which is mediated by the XylS protein after binding of a benzoate effector. Alternatively, in cells growing on toluene or its aromatic alcohols, overexpression of xylS mediated by XylR activated by these compounds leads to overproduction of the XylS regulator, which even in the absence of benzoate effectors stimulates transcription from the meta cleavage pathway operon promoter. We show here that in bacteria growing on glycerol or alkylbenzoates, the xylS gene is expressed at a low but constitutive level from a newly found sigma 70-dependent promoter called Ps2. The amount of XylS protein made from the transcript originated from Ps2 was sufficient to allow high levels of expression from the meta cleavage pathway operon promoter when the cells were grown in the presence of 3-methylbenzoate. The transcription initiation point of the transcript generated from Ps2 mapped 9 bp upstream from the proposed ATG of the xylS gene; this transcript contains the ribosome-binding site. The Ps2 promoter was located 110 bp downstream from a previously described sigma54-dependent promoter located upstream from the xylS open reading frame, now called Ps1. In cells growing on toluene or benzyl alcohols, the XylS regulator is overproduced as a consequence of increased expression of the gene through the effect of the two promoters working in tandem: the newly found sigma 70-dependent promoter, whose expression is XylR and toluene independent, and the sigma 54-dependent promoter, whose expression is dependent on XylR activated by its effectors. This expression pathway of the xylS gene explains why sigma 54-deficient P. putida bearing the wild-type TOL plasmid, or the wild-type P. putida strain bearing a TOL plasmid with a knocked-out xylR gene, can grow on alkylbenzoates. Until now this has been one of the unresolved paradoxes in the transcriptional control of the TOL meta cleavage pathway.