Stringent promoter recognition and autoregulation by the group 3 σ-factor SigF in the cyanobacterium Synechocystis sp. strain PCC 6803

The cyanobacteirum Synechocystis sp. strain PCC 6803 possesses nine species of the sigma (σ)-factor gene for RNA polymerase (RNAP). Here, we identify and characterize the novel-type promoter recognized by a group 3 σ-factor, SigF. SigF autoregulates its own transcription and recognizes the promoter of pilA1 that acts in pilus formation and motility in PCC 6803. The pilA1 promoter (PpilA1-54) was recognized only by SigF and not by other σ-factors in PCC 6803. No PpilA1-54 activity was observed in Escherichia coli cells that possess RpoF (σ28) for fragellin and motility. Studies of in vitro transcription for PpilA1-54 identified the region from −39 to −7 including an AG-rich stretch and a core promoter with TAGGC (−32 region) and GGTAA (−12 region) as important for transcription. We also confirmed the unique PpilA1-54 architecture and further identified two novel promoters, recognized by SigF, for genes encoding periplasmic and phytochrome-like phototaxis proteins. These results and a phylogenetic analysis suggest that the PCC 6803 SigF is distinct from the E. coli RpoF or RpoD (σ70) type and constitutes a novel eubacterial group 3 σ-factor. We discuss a model case of stringent promoter recognition by SigF. Promoter types of PCC 6803 genes are also summarized

Rapid acquisition and model-based analysis of cell-free transcription–translation reactions from nonmodel bacteria

Native cell-free transcription–translation systems offer a rapid route to characterize the regulatory elements (promoters, transcription factors) for gene expression from nonmodel microbial hosts, which can be difficult to assess through traditional in vivo approaches. One such host, Bacillus megaterium, is a giant Gram-positive bacterium with potential biotechnology applications, although many of its regulatory elements remain uncharacterized. Here, we have developed a rapid automated platform for measuring and modeling in vitro cell-free reactions and have applied this to B. megaterium to quantify a range of ribosome binding site variants and previously uncharacterized endogenous constitutive and inducible promoters. To provide quantitative models for cell-free systems, we have also applied a Bayesian approach to infer ordinary differential equation model parameters by simultaneously using time-course data from multiple experimental conditions. Using this modeling framework, we were able to infer previously unknown transcription factor binding affinities and quantify the sharing of cell-free transcription–translation resources (energy, ribosomes, RNA polymerases, nucleotides, and amino acids) using a promoter competition experiment. This allows insights into resource limiting-factors in batch cell-free synthesis mode. Our combined automated and modeling platform allows for the rapid acquisition and model-based analysis of cell-free transcription–translation data from uncharacterized microbial cell hosts, as well as resource competition within cell-free systems, which potentially can be applied to a range of cell-free synthetic biology and biotechnology applications

Semi-Autonomous Revenue Authorities in Sub-Saharan Africa: Silver Bullet or White Elephant

A major component of tax administration reform in sub-Saharan Africa for the last 30 years has been the creation of semi-autonomous revenue authorities (SARAs). The effects of their creation on revenue performance have been much debated, although there are only a few quantitative studies. The core argument of this paper is that existing research suggesting diverse and contradictory outcomes has not taken account of trends in revenue performance in the years before the establishment of SARAs. Allowing for this revenue history our analysis based on 46 countries over the period 1980-2015 provides no robust evidence that SARAs induce an increase in revenue performance. This does not imply that SARAs may not provide benefits for tax collection, but they do not demonstrably increase (or decrease) revenue collected

The σ\u3csup\u3eB\u3c/sup\u3e-Dependent Promoter of the \u3ci\u3eBacillus subtilis sigB\u3c/i\u3e Operon Is Induced by Heat Shock

σB, a secondary sigma factor of Bacillus subtilis, was found to increase 5- to 10-fold when cultures were shifted from 37 to 48°C. Western blot (immunoblot) analyses, in which monoclonal antibodies specific for the sigB operon products RsbV, RsbW, and σB were used to probe extracts from wild-type and mutant B. subtilis strains, revealed that all three proteins increased coordinately after heat shock and that this increase was dependent on σB but not RsbV, a positive regulator normally essential for σB-dependent sigB expression. Nuclease protection experiments of RNA synthesized after heat shock supported the notion that the shift to 48°C enhanced transcription from the sigB operon\u27s σB-dependent promoter. The level of mRNA initiating at the σB-dependent ctc promoter was also seen to increase approximately 5- to 10-fold after heat shock. Pulse-labeling of the proteins synthesized after a shift to 48°C demonstrated that sigB wild-type and mutant strains produced the maijor heat-inducible proteins in similar amounts; however, at least seven additional proteins were present after the temperature shift in the wild-type strain but absent in the sigB null mutant. Thus, although σB is not required for the expression of essential heat shock genes, it is activated by heat shock to elevate its own synthesis and possibly the synthesis of several other heat-inducible proteins

Sporulation Phenotype of a Bacillus subtilis Mutant Expressing an Unprocessable but Active σ(E) Transcription Factor

σ(E), a sporulation-specific sigma factor of Bacillus subtilis, is formed from an inactive precursor (pro-σ(E)) by a developmentally regulated processing reaction that removes 27 amino acids from the proprotein's amino terminus. A sigE variant (sigE335) lacking 15 amino acids of the prosequence is not processed into mature σ(E) but is active without processing. In the present work, we investigated the sporulation defect in sigE335-expressing B. subtilis, asking whether it is the bypass of proprotein processing or a residual inhibition of σ(E) activity that is responsible. Fluorescence microscopy demonstrated that sigE335-expressing B. subtilis progresses further into sporulation (stage III) than do strains lacking σ(E) activity (stage II). Consistent with its stage III phenotype, and a defect in σ(E) activity rather than its timing, the sigE335 allele did not disturb early sporulation gene expression but did inhibit the expression of late sporulation genes (gerE and sspE). The Spo(−) phenotype of sigE335 was found to be recessive to wild-type sigE. In vivo assays of σ(E) activity in sigE, sigE335, and merodiploid strains indicate that the residual prosequence on σ(E335), still impairs its activity to function as a transcription factor. The data suggest that the 11-amino-acid extension on σ(E335) allows it to bind RNA polymerase and direct the resulting holoenzyme to σ(E)-dependent promoters but reduces the enzyme's ability to initiate transcription initiation and/or exit from the promoter