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Mediator is a well-known transcriptional co-regulator and serves as an adaptor between gene-specific regulatory proteins and RNA polymerase II. Studies on the chromatin-bound form of Mediator revealed interactions with additional protein complexes involved in various transcription-related processes, such as the Lsm2-8 complex that is part of the spliceosomal U6 small nuclear ribonucleoprotein complex. Here, we employ Chromatin Immunoprecipitation sequencing (ChIP-seq) of chromatin associated with the Lsm3 protein and the Med1 or Med15 Mediator subunits. We identify 86 genes co-occupied by both Lsm3 and Mediator, of which 73 were intron-containing ribosomal protein genes. In logarithmically growing cells, Mediator primarily binds to their promoter regions but also shows a second, less pronounced occupancy at their 3'-exons. During the late exponential phase, we observe a near-complete transition of Mediator from these promoters to a position in their 3'-ends, overlapping the Lsm3 binding sites ∼250 bp downstream of their last intron-exon boundaries. Using an unbiased RNA sequencing approach, we show that transition of Mediator from promoters to the last exon of these genes correlates to reduction of both their messenger RNA levels and splicing ratios, indicating that the Mediator and Lsm complexes cooperate to control growth-regulated expression of intron-containing ribosomal protein genes at the levels of transcription and splicing., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The phoB gene of Bacillus subtilis encodes an alkaline phosphatase (PhoB, formerly alkaline phosphatase III) that is expressed from separate promoters during phosphate deprivation in a PhoP-PhoR-dependent manner and at stage two of sporulation under phosphate-sufficient conditions independent of PhoP-PhoR. Isogenic strains containing either the complete phoB promoter or individual phoB promoter fusions were used to assess expression from each promoter under both induction conditions. The phoB promoter responsible for expression during sporulation, phoB-P(S), was expressed in a wild-type strain during phosphate deprivation, but induction occurred >3 h later than induction of Pho regulon genes and the levels were approximately 50-fold lower than that observed for the PhoPR-dependent promoter, phoB-P(V). E(sigma)E was necessary and sufficient for P(S) expression in vitro. P(S) expression in a phoPR mutant strain was delayed 2 to 3 h compared to the expression in a wild-type strain, suggesting that expression or activation of sigma(E) is delayed in a phoPR mutant under phosphate-deficient conditions, an observation consistent with a role for PhoPR in spore development under these conditions. Phosphorylated PhoP (PhoP approximately P) repressed P(S) in vitro via direct binding to the promoter, the first example of an E(sigma)E-responsive promoter that is repressed by PhoP approximately P. Whereas either PhoP or PhoP approximately P in the presence of E(sigma)A was sufficient to stimulate transcription from the phoB-P(V) promoter in vitro, roughly 10- and 17-fold-higher concentrations of PhoP than of PhoP approximately P were required for P(V) promoter activation and maximal promoter activity, respectively. The promoter for a second gene in the Pho regulon, ykoL, was also activated by elevated concentrations of unphosphorylated PhoP in vitro. However, because no Pho regulon gene expression was observed in vivo during P(i)-replete growth and PhoP concentrations increased only threefold in vivo during phoPR autoinduction, a role for unphosphorylated PhoP in Pho regulon activation in vivo is not likely.

Bacillus subtilis PhoP is a member of the OmpR family of response regulators that activates or represses genes of the Pho regulon upon phosphorylation by PhoR in response to phosphate deficiency. Because PhoP binds DNA and is a dimer in solution independent of its phosphorylation state, phosphorylation of PhoP may optimize DNA binding or the interaction with RNA polymerase. We describe alanine scanning mutagenesis of the PhoP alpha loop and alpha helix 3 region of PhoPC (Val190 to E214) and functional analysis of the mutated proteins. Eight residues important for DNA binding were clustered between Val202 and Arg210. Using in vivo and in vitro functional analyses, we identified three classes of mutated proteins. Class I proteins (PhoP(I206A), PhoP(R210A), PhoP(L209A), and PhoP(H208A)) were phosphorylation proficient and could dimerize but could not bind DNA or activate transcription in vivo or in vitro. Class II proteins (PhoP(H205A) and PhoP(V204A)) were phosphorylation proficient and could dimerize but could not bind DNA prior to phosphorylation. Members of this class had higher transcription activation in vitro than in vivo. The class III mutants, PhoP(V202A) and PhoP(D203A), had a reduced rate of phosphotransfer and could dimerize but could not bind DNA or activate transcription in vivo or in vitro. Seven alanine substitutions in PhoP (PhoP(V190A), PhoP(W191A), PhoP(Y193A), PhoP(F195A), PhoP(G197A,) PhoP(T199A), and PhoP(R200A)) that specifically affected transcription activation were broadly distributed throughout the transactivation loop extending from Val190 to as far toward the C terminus as Arg200. PhoP(W191A) and PhoP(R200A) could not activate transcription, while the other five mutant proteins showed decreased transcription activation in vivo or in vitro or both. The mutagenesis studies may indicate that PhoP has a long transactivation loop and a short alpha helix 3, more similar to OmpR than to PhoB of Escherichia coli.

Statistically designed experimental strategy has been performed in order to evaluate and optimize nutritional and environmental parameters that affect ferrous ion oxidation rate in Acidithiobacillus ferrooxidans ATCC 23270. Plackett-Burman design was carried out to evaluate efficiently the biological significance of 10 culture conditions influencing ferrous-ion oxidation rate of A. ferrooxidans grown for 5 days in shake-flask batch mode on the newly modified 9-K media. Among ten fermentation factors examined, the most significant variables influencing ferrous-ion oxidation rate were statistically elucidated to be pH and calcium nitrate as positive contributors, whereas trace metals solution and potassium chloride were the most significant negative contributors. The optimal levels of the most significant three nutritional factors were further predicted from a polynomial model created from the data obtained from three level factorial design, a Box-Behnken design. Predicted optimal ferrous-ion oxidation rate Q(Fe2+) was recorded to be 0.148 (g Fe2+/l/hr). On verifying the predicted value, an experiment was performed under optimal predicted conditions and showed an actual experimental Q(Fe2+) of 0.152 g/l/hr, which was 2.7% over the predicted value. Our optimized medium formula gave overall five folds increase in ferrous-ion oxidation rates over the previously published data of standard 9-K medium on batch culture of A. ferrooxidans ATCC 23270 with higher mu(max) (hr(-1)) of 0.177 which was achieved within 75 h incubation in shake-flask culture.

An enrichment and isolation program for new ethanol-producing thermotolerant yeasts as well as a screening program of some known thermotolerant strains resulted in the selection of several strains capable of growth at 40-43 degrees C. Among these strains four grew and fermented sugar cane molasses at 43 degrees C under batch conditions with sugar-conversion efficiencies >94% and ethanol concentrations 6.8-8.0% (w/v). The two best-performing strains, a Saccharomyces cerevisiae F111 and a Kluyveromyces marxianus WR12 were used in eight 87.5 m(3) fermentation runs (four using each strain) for industrial ethanol production in an Egyptian distillery using sugar cane molasses. Mean ethanol production was 7.7% and 7.4% (w/v), respectively, with an added advantage of cooling elimination during fermentation and higher ethanol yields compared to the distillery's S. cerevisiae SIIC (ATCC 24855) strain in use. The isolate S. cerevisiae F111 was subsequently adopted by the distillery for regular production with significant economical gains and water conservation., (Copyright 2000 John Wiley & Sons, Inc.)