Evidence for transcription attenuation rendering cryptic a sigmaS- dependent promoter of the osmotically regulated proU operon of Salmonella typhimurium

The osmotically regulated proU locus in Escherichia coli has two promoters, P1 and P2, that are recognized, respectively, by the σ S- and σ 70-bearing RNA polymerase holoenzymes. However, the equivalent of the P1 promoter does not appear to exist in Salmonella typhimurium. We demonstrate in this study that wild-type S. typhimurium has a cryptic P1 promoter that is recognized by σ S RNA polymerase in vitro and that a 22-bp deletion from +63 to +84 (relative to the start site of transcription) confers σ S-dependent in vivo expression of a reporter gene fusion to P1. Primer extension analysis of RNA isolated from cells carrying the wild-type and mutant S. typhimurium proU constructs indicated that a primer which hybridizes proximal to +60 is able to detect P1-initiated transcripts from both constructs but a primer which hybridizes distal to +85 is able to do so only from the latter. Our results suggest that the σ S-controlled proU P1 promoter in S. typhimurium may be rendered cryptic because of factor-dependent transcription attenuation within a short distance downstream of the promoter start site

Effects of H-NS and potassium glutamate on &#963;<SUP>S</SUP>- and &#963;<SUP>70</SUP>-directed transcription in vitro from osmotically regulated P1 and P2 promoters of proU in Escherichia coli

We have used supercoiled DNA templates in this study to demonstrate that transcription in vitro from the P1 and P2 promoters of the osmoresponsive proU operon of Escherichia coli is preferentially mediated by the &#963;s and &#963;70-bearing RNA polymerase holoenzymes, respectively. Addition of potassium glutamate resulted in the activation of transcription from both P1 and P2 and also led to a pronounced enhancement of &#963;s selectivity at the P1 promoter. Transcription from P2, and to a lesser extent from P1, was inhibited by the nucleoid protein H-NS but only in the absence of potassium glutamate. This study validates the existence of dual promoters with dual specificities for proU transcription. Our results also support the proposals that potassium, which is known to accumulate in cells grown at high osmolarity, is at least partially responsible for effecting the in vivo induction of proU transcription and that it does so through two mechanisms, directly by the activation of RNA polymerase and indirectly by the relief of repression imposed by H-NS

An N-Terminally Truncated RpoS (σ(S)) Protein in Escherichia coli Is Active In Vivo and Exhibits Normal Environmental Regulation Even in the Absence of rpoS Transcriptional and Translational Control Signals

RpoS (σ(S)) in Escherichia coli is a stationary-phase-specific primary sigma factor of RNA polymerase which is 330 amino acids long and belongs to the eubacterial σ(70) family of proteins. Conserved domain 1.1 at the N-terminal end of σ(70) has been shown to be essential for RNA polymerase function, and its deletion has been shown to result in a dominant-lethal phenotype. We now report that a σ(S) variant with a deletion of its N-terminal 50 amino acids (σ(S)Δ1-50), when expressed in vivo either from a chromosomal rpoS::IS10 allele (in rho mutant strains) or from a plasmid-borne arabinose-inducible promoter, is as proficient as the wild type in directing transcription from the proU P1 promoter; at three other σ(S)-dependent promoters that were tested (osmY, katE, and csiD), the truncated protein exhibited a three- to sevenfold reduced range of activities. Catabolite repression at the csiD promoter (which requires both σ(S) and cyclic AMP [cAMP]-cAMP receptor protein for its activity) was also preserved in the strain expressing σ(S)Δ1-50. The intracellular content of σ(S)Δ1-50 was regulated by culture variables such as growth phase, osmolarity, and temperature in the same manner as that described earlier for σ(S), even when the truncated protein was expressed from a template that possessed neither the transcriptional nor the translational control elements of wild-type rpoS. Our results indicate that, unlike that in σ(70), the N-terminal domain in σ(S) may not be essential for the protein to function as a sigma factor in vivo. Furthermore, our results suggest that the induction of σ(S)-specific promoters in stationary phase and during growth under conditions of high osmolarity or low temperature is mediated primarily through the regulation of σ(S) protein degradation

Molecular cloning of an osmoregulatory locus in Escherichia coli: increased proU gene dosage results in enhanced osmotolerance.

The proU locus in Escherichia coli encodes an important osmoregulatory function which mediates the growth-promoting effect of L-proline and glycine betaine in high-osmolarity media. This locus was cloned, in contiguity with a closely linked Tn10 insertion, onto a multicopy plasmid directly from the E. coli chromosome. For a given level of osmotic stress, the magnitude of osmoresponsive induction of a single-copy proU::lac fusion was reduced in strains with multiple copies of the proU+ genes; in comparison with haploid proU+ strains, strains with the multicopy proU+ plasmids also exhibited enhanced osmotolerance in media supplemented with 1 mM L-proline or glycine betaine. Experiments involving subcloning, Tn1000 mutagenesis, and interplasmid complementation in a deletion mutant provided evidence for the presence at this locus of two cistrons, both of which are necessary for the expression of ProU function. We propose the designations proU for the gene originally identified by the proU224::Mu d1(lac Ap) insertion and proV for the gene upstream (that is, counterclockwise) of proU

Multiple mechanisms contribute to osmotic inducibility of proU operon expression in Escherichia coli: demonstration of two osmoresponsive promoters and of a negative regulatory element within the first structural gene.

Transcription of the proU operon in Escherichia coli is induced several hundredfold upon growth of cells in media of elevated osmolarity. A low-copy-number promoter-cloning plasmid vector, with lacZ as the reporter gene, was used for assaying the osmoresponsive promoter activity of each of various lengths of proU DNA, generated by cloning of discrete restriction fragments and by an exonuclease III-mediated deletion approach. The results indicate that expression of proU in E. coli is directed from two promoters, one (P2) characterized earlier by other workers with the start site of transcription 60 nucleotides upstream of the initiation codon of the first structural gene (proV), and the other (P1) situated 250 nucleotides upstream of proV. Furthermore, a region of DNA within proV was shown to be involved in negative regulation of proU transcription; phage Mu dII1681-generated lac fusions in the early region of proV also exhibited partial derepression of proU regulation, in comparison with fusions further downstream in the operon. Sequences around promoter P1, sequences around P2, and the promoter-downstream negative regulatory element, respectively, conferred approximately 5-, 8-, and 25-fold osmoresponsivity on proU expression. Within the region genetically defined to encode the negative regulatory element, there is a 116-nucleotide stretch that is absolutely conserved between the proU operons of E. coli and Salmonella typhimurium and has the capability of exhibiting alternative secondary structure. Insertion of this region of DNA into each of two different plasmid vectors was associated with a marked reduction in the mean topological linking number in plasmid molecules isolated from cultures grown in high-osmolarity medium. We propose that this region of DNA undergoes reversible transition to an underwound DNA conformation under high-osmolarity growth conditions and that this transition mediates its regulatory effect on proU expression

Inoculum composition determines microbial community and function in an anaerobic sequential batch reactor

The sustainable recovery of resources from wastewater streams can provide many social and environmental benefits. A common strategy to recover valuable resources from wastewater is to harness the products of fermentation by complex microbial communities. In these fermentation bioreactors high microbial community diversity within the inoculum source is commonly assumed as sufficient for the selection of a functional microbial community. However, variability of the product profile obtained from these bioreactors is a persistent challenge in this field. In an attempt to address this variability, the impact of inoculum on the microbial community structure and function within the bioreactor was evaluated using controlled laboratory experiments. In the course of this work, sequential batch reactors were inoculated with three complex microbial inocula and the chemical and microbial compositions were monitored by HPLC and 16S rRNA amplicon analysis, respectively. Microbial community dynamics and chemical profiles were found to be distinct to initial inoculate and highly reproducible. Additionally we found that the generation of a complex volatile fatty acid profile was not specific to the diversity of the initial microbial inoculum. Our results suggest that the composition of the original inoculum predictably contributes to bioreactor community structure and function.United States. National Institutes of Health (P30-ES002109