Escherichia coli holoenzyme RNA polymerase is composed of a core enzyme (E, 1 subunit composition ␣ 2␤␤ Ј) associated with one of seven sigma ()-factors that program the complex to engage and initiate transcription at different sets of promoters (1). Thus, the levels and binding properties of alternative -subunits together with factors that modulate their ability to associate with core RNA polymerase are critical for the relative composition of the multiple holoenzymes available for transcription of the distinct promoter classes within the prokaryotic genome. The seven different -factors of E. coli fall into two groups. The larger of these comprises six factors that share notable sequence and functional similarities to the major D

SummaryThe RNA polymerase-binding protein DksA is a cofactor required for guanosine tetraphosphate (ppGpp)-responsive control of transcription from s 70 promoters. Here we present evidence: (i) that both DksA and ppGpp are required for in vivo s 54 transcription even though they do not have any major direct effects on s 54 transcription in reconstituted in vitro transcription and s -factor competition assays, (ii) that previously defined mutations rendering the housekeeping s

Comparatively little is known about directed motility of environmental bacteria to common aromatic pollutants. Here, by expressing different parts of a (methyl)phenol-degradative pathway and the use of specific mutants, we show that taxis of Pseudomonas putida towards (methyl)phenols is dictated by its ability to catabolize the aromatic compound. Thus, in contrast to previously described chemoreceptor-mediated chemotaxis mechanisms towards benzoate, naphthalene and toluene, taxis in response to (methyl)phenols is mediated by metabolism-dependent behaviour. Here we show that P. putida differentially expresses three Aer-like receptors that are all polar-localized through interactions with CheA, and that inactivation of the most abundant Aer2 protein significantly decreases taxis towards phenolics. In addition, the participation of a sensory signal transduction protein composed of a PAS, a GGDEF and an EAL domain in motility towards these compounds is demonstrated. The results are discussed in the context of the versatility of metabolism-dependent coupling and the necessity for P. putida to integrate diverse metabolic signals from its native heterogeneous soil and water environments.

The Pseudomonas derived 54 -dependent regulators DmpR and XylR control the expression of genes involved in catabolism of aromatic compounds. Binding to distinct, nonoverlapping groups of aromatic effectors controls the activities of these transcriptional activators. Previous work has derived a common mechanistic model for these two regulators in which effector binding by the N-terminal 210 residues (the A-domain) of the protein relieves repression of an intrinsic ATPase activity essential for its transcription-promoting property and allows productive interaction with the transcriptional apparatus. Here we dissect the A-domains of DmpR and XylR by DNA shuffling to identify the region(s) that mediates the differences in the effector specificity profiles. Analysis of in vivo transcription in response to multiple aromatic effectors and the in vitro phenolbinding abilities of regulator derivatives with hybrid DmpR/XylR A-domains reveals that residues 110 to 186 are key determinants that distinguish the effector profiles of DmpR and XylR. Moreover, the properties of some mosaic DmpR/XylR derivatives reveal that high-affinity aromatic effector binding can be completely uncoupled from the ability to promote transcription. Hence, novel aromatic binding properties will only be translated into functional transcriptional activation if effector binding also triggers release of interdomain repression.

The 54 -factor controls expression of a variety of genes in response to environmental cues. Much previous work has implicated the nucleotide alarmone ppGpp and its co-factor DksA in control of 54 -dependent transcription in the gut commensal Escherichia coli, which has evolved to live under very different environmental conditions than Pseudomonas putida. Here we compared ppGpp/DksA mediated control of 54 -dependent transcription in these two organisms. Our in vivo experiments employed P. putida mutants and manipulations of factors implicated in ppGpp/DksA mediated control of 54 -dependent transcription in combination with a series of 54 -promoters with graded affinities for 54 -RNA polymerase. For in vitro analysis we used a P. putida-based reconstituted 54 -transcription assay system in conjunction with DNA-binding plasmon resonance analysis of native and heterologous 54 -RNA polymerase holoenzymes. In comparison with E. coli, ppGpp/DksA responsive 54 -transcription in the environmentally adaptable P. putida was found to be more robust under low energy conditions that occur upon nutrient depletion. The mechanism behind this difference can be traced to reduced promoter discrimination of low affinity 54 -promoters that is conferred by the strong DNA binding properties of the P. putida 54 -RNA polymerase holoenzyme.To ensure promoter specificity, the bacterial multisubunit catalytic core RNA polymerase (RNAP) 4 (␣ 2 ␤␤Ј) is guided to the distinct promoter classes within the genome by a dissociable -factor. In addition to the household 70 -factor, most bacteria encode a variable number of alternative -factors that fall into two families based on primary amino acid sequences and regulatory properties. The largest of these is the 70 -family, which encompasses most alternative -factors within four subgroups that all form RNAP holoenzymes that can spontaneously initiate transcription (1). Orthologs of the structurally distinct 54 -subunit are the only members of the second family. 54 imposes kinetic constrains that lock the 54 -RNAP holoenzyme in a non-productive closed complex. Thus, 54 -dependent transcription always requires activators, called bacterial enhancer-binding proteins or bEBPs, which use ATP catalysis to remodel 54