Mechanisms of Very Long Abortive Transcript Release during Promoter Escape.

A phage T5 N25 promoter variant, DG203, undergoes the escape transition at the +16 to +19 positions after transcription initiation. By specifically examining the abortive activity of the initial transcribing complex at position +19 (ITC19), we observe the production of both GreB-sensitive and GreB-resistant VLAT19. This suggests that ITC19, which is perched on the brink of escape, is highly unstable and can achieve stabilization through either backtracking or forward translocation. Of the forward-tracked fraction, only a small percentage escapes normally (followed by stepwise elongation) to produce full-length RNA; the rest presumably hypertranslocates to release GreB-resistant VLATs. VLAT formation is dependent not only on consensus -35/-10 promoters with 17 bp spacing but also on sequence characteristics of the spacer DNA. Analysis of DG203 promoter variants containing different spacer sequences reveals that AT-rich spacers intrinsically elevate the level of VLAT formation. The AT-rich spacer of DG203 joined to the -10 box presents an UP element sequence capable of interacting with the polymerase α subunit C-terminal domain (αCTD) during the escape transition, which in turn enhances VLAT release. Utilization of the spacer/-10 region UP element by αCTD subunits requires a 10-15 bp hypertranslocation. We document the physical occurrence of hyper forward translocation using ExoIII footprinting analysis.

Species-specific residues calibrate SoxR sensitivity to redox-active molecules.

In enterics, the transcription factor SoxR triggers a global stress response by sensing a broad spectrum of redox-cycling compounds. In the non-enteric bacteria Pseudomonas aeruginosa and Streptomyces coelicolor, SoxR is activated by endogenous redox-active small molecules and only regulates a small set of genes. We investigated if the more general response in enterics is reflected in the ability of SoxR to sense a wider range of redox-cycling compounds. Indeed, while Escherichia coli SoxR is tuned to structurally diverse compounds that span a redox range of -450 to +80 mV, P. aeruginosa and S. coelicolor SoxR are less sensitive to viologens, which have redox potentials below -350 mV. Using a mutagenic approach, we pinpointed three amino acids that contribute to the reduced sensitivity of P. aeruginosa and S. coelicolor SoxR. Notably these residues are not conserved in homologues of the Enterobacteriaceae. We further identified a motif within the sensor domain that tunes the activity of SoxR from enterics - inhibiting constitutive activity while allowing sensitivity to drugs with low redox potentials. Our findings highlight how small alterations in structure can lead to the evolution of proteins with distinct specificities for redox-active small molecules.

Expression of the Streptomyces coelicolor SoxR regulon is intimately linked with actinorhodin production.

The [2Fe-2S]-containing transcription factor SoxR is conserved in diverse bacteria. SoxR is traditionally known as the regulator of a global oxidative stress response in Escherichia coli, but recent studies suggest that this function may be restricted to enteric bacteria. In the vast majority of nonenterics, SoxR is predicted to mediate a response to endogenously produced redox-active metabolites. We have examined the regulation and function of the SoxR regulon in the model antibiotic-producing filamentous bacterium Streptomyces coelicolor. Unlike the E. coli soxR deletion mutant, the S. coelicolor equivalent is not hypersensitive to oxidants, indicating that SoxR does not potentiate antioxidant defense in the latter. SoxR regulates five genes in S. coelicolor, including those encoding a putative ABC transporter, two oxidoreductases, a monooxygenase, and a possible NAD-dependent epimerase/dehydratase. Expression of these genes depends on the production of the benzochromanequinone antibiotic actinorhodin and requires intact [2Fe-2S] clusters in SoxR. These data indicate that actinorhodin, or a redox-active precursor, modulates SoxR activity in S. coelicolor to stimulate the production of a membrane transporter and proteins with homology to actinorhodin-tailoring enzymes. While the role of SoxR in S. coelicolor remains under investigation, these studies support the notion that SoxR has been adapted to perform distinct physiological functions to serve the needs of organisms that occupy different ecological niches and face different environmental challenges.

RNA-Seq analysis reveals a six-gene SoxR regulon in Streptomyces coelicolor.

The redox-regulated transcription factor SoxR is conserved in diverse bacteria, but emerging studies suggest that this protein plays distinct physiological roles in different bacteria. SoxR regulates a global oxidative stress response (involving > 100 genes) against exogenous redox-cycling drugs in Escherichia coli and related enterics. In the antibiotic producers Streptomyces coelicolor and Pseudomonas aeruginosa, however, SoxR regulates a smaller number of genes that encode membrane transporters and proteins with homology to antibiotic-tailoring enzymes. In both S. coelicolor and P. aeruginosa, SoxR-regulated genes are expressed in stationary phase during the production of endogenously-produced redox-active antibiotics. These observations suggest that SoxR evolved to sense endogenous secondary metabolites and activate machinery to process and transport them in antibiotic-producing bacteria. Previous bioinformatics analysis that searched the genome for SoxR-binding sites in putative promoters defined a five-gene SoxR regulon in S. coelicolor including an ABC transporter, two oxidoreductases, a monooxygenase and an epimerase/dehydratase. Since this in silico screen may have missed potential SoxR-targets, we conducted a whole genome transcriptome comparison of wild type S. coelicolor and a soxR-deficient mutant in stationary phase using RNA-Seq. Our analysis revealed a sixth SoxR-regulated gene in S. coelicolor that encodes a putative quinone oxidoreductase. Knowledge of the full complement of genes regulated by SoxR will facilitate studies to elucidate the function of this regulatory molecule in antibiotic producers.

An alternate mechanism of abortive release marked by the formation of very long abortive transcripts.

The Esigma70-dependent N25 promoter is rate-limited at promoter escape. Here, RNA polymerase repeatedly initiates and aborts transcription, giving rise to a ladder of short RNAs 2-11 nucleotides long. Certain mutations in the initial transcribed sequence (ITS) of N25 lengthen the abortive initiation program, resulting in the release of very long abortive transcripts (VLATs) 16-19 nucleotides long. This phenomenon is completely dependent on sequences within the first 20 bases of the ITS since altering sequences downstream of +20 has no effect on their formation. VLAT formation also requires strong interactions between RNA polymerase and the promoter. Mutations that change the -35 and -10 hexamers and the intervening 17 base pair spacer away from consensus decrease the probability of aborting at positions +16 to +19. An unusual characteristic of the VLATs is their undiminished levels in the presence of GreB, which rescues abortive RNAs (

Functional analysis of SoxR residues affecting transduction of oxidative stress signals into gene expression.

SoxR protein, a member of the MerR family of transcriptional activators, mediates a global oxidative stress response in Escherichia coli. Upon oxidation or nitrosylation of its [2Fe-2S] centers SoxR activates its target gene, soxS, by mediating a structural transition in the promoter DNA that stimulates initiation by RNA polymerase. We explored the molecular basis of this signal transduction by analyzing mutant SoxR proteins defective in responding to oxidative stress signals in vivo.We have confirmed that the DNA binding domain of SoxR is highly conserved compared with other MerR family proteins and functions in a similar manner to activate transcription. Several mutations in the dimerization domain of SoxR disrupted intersubunit communication, and the resulting proteins were unable to propagate redox signals to the soxS promoter. Mutations scattered throughout the polypeptide yielded proteins that were under-responsive to in vivo redox signals, which indicates that the redox properties of the [2Fe-2S] centers are influenced by global protein structure. These findings indicate that SoxR functions as a redox-responsive molecular switch in which subunit interactions transduce a subtle alteration in oxidation state into a profound change in DNA structure.

The products of the spoVA operon are involved in dipicolinic acid uptake into developing spores of Bacillus subtilis.

Bacillus subtilis cells with mutations in the spoVA operon do not complete sporulation. However, a spoVA strain with mutations that remove all three of the spore's functional nutrient germinant receptors (termed the ger3 mutations) or the cortex lytic enzyme SleB (but not CwlJ) did complete sporulation. ger3 spoVA and sleB spoVA spores lack dipicolinic acid (DPA) and have lower core wet densities and levels of wet heat resistance than wild-type or ger3 spores. These properties of ger3 spoVA and sleB spoVA spores are identical to those of ger3 spoVF and sleB spoVF spores that lack DPA due to deletion of the spoVF operon coding for DPA synthetase. Sporulation in the presence of exogenous DPA restored DPA levels in ger3 spoVF spores to 53% of the wild-type spore levels, but there was no incorporation of exogenous DPA into ger3 spoVA spores. These data indicate that one or more products of the spoVA operon are involved in DPA transport into the developing forespore during sporulation.

Transcription-defective soxR mutants of Escherichia coli: isolation and in vivo characterization.

The soxRS regulon protects Escherichia coli from superoxide and nitric oxide stress. SoxR protein, a transcription factor that senses oxidative stress via its [2Fe-2S] centers, transduces the signal to the soxS promoter to stimulate RNA polymerase. Here we describe 29 mutant alleles of soxR that cause defects in the activation of soxS transcription in response to paraquat, a superoxide stress agent. Owing to the selection and screen used in their isolation, most of these mutant alleles encode proteins that retained specific binding activity for the soxS promoter in vivo. The mutations were found throughout the SoxR polypeptide, although those closer to the N terminus typically exhibited greater defects in DNA binding. The degree of the defect in the transcriptional response to superoxide caused by each mutation was closely paralleled by its impaired response to nitric oxide. This work begins the general identification of the residues in the SoxR polypeptide that are critical for transducing oxidative stress signals into gene activation.