Overproduction and Characterization of the Bacillus subtilis Anti-sigma Factor FlgM

FlgM is an anti-sigma factor of the flagellar-specific sigma (sigma) subunit of RNA polymerase in Bacillus subtilis, and it is responsible of the coupling of late flagellar gene expression to the completion of the hook-basal body structure. We have overproduced the protein in soluble form and characterized it. FlgM forms dimers as shown by gel exclusion chromatography and native polyacrylamide gel electrophoresis and interacts in vitro with the cognate sigmaD factor. The FlgM.sigmaD complex is a stable heterodimer as demonstrated by gel exclusion chromatography, chemical cross-linking, native polyacrylamide gel electrophoresis, and isoelectric focusing. sigmaD belongs to the group of sigma factors able to bind to the promoter sequence even in the absence of core RNA polymerase. The FlgM.sigmaD complex gave a shift in a DNA mobility shift assay with a probe containing a sigmaD-dependent promoter sequence. Limited proteolysis studies indicate the presence of two structural motifs, corresponding to the N- and C-terminal regions, respectively

Spore-forming bacteria in soil cultivated with GM white poplars: isolation and characterization

The impact of transgenic white poplars (Populus alba L. cv. ‘Villafranca’) was assessed on the soil aerobic spore-forming bacteria (SFB). The genetically modified poplars, expressing either the StSy gene for resveratrol production or the bar gene for herbicide tolerance, were cultivated in greenhouse. The occurrence of SFB was monitored in soil samples collected at eight different timepoints over a two-year period. The total culturable bacterial population of the StSy and bar trials underwent significant seasonal fluctuations in the range of 106–2.5 × 108 CFU/g dry soil and of 104–5 × 108 CFU/g dry soil, respectively. Changes occurred also within the culturable SFB population with size varying at 103–5 × 104 CFU/g dry soil and 102–2 × 105 CFU/g dry soil in the StSy and bar trials, respectively. No significant differences in the size of the total and SFB culturable populations were observed when comparing each transgenic line with the nontransformed control line while seasonal shifts of soil bacterial populations were evident in both trials. The culturable SFB fraction included three isolates (SFB-1, SFB-2 and SFB-3) classified by 16S rDNA sequence analysis as members of the Bacillus genus. According to the reported data, cultivation of both herbicide- resistant and resveratrol-producing GM white poplars did not affect the culturable SFB population at the soil leve

Assessing the perceived effect of non-pharmaceutical interventions on SARS-Cov-2 transmission risk: an experimental study in Europe

We conduct a large (N = 6567) online experiment to measure the features of non-pharmaceutical interventions (NPIs) that citizens of six European countries perceive to lower the risk of transmission of SARS-Cov-2 the most. We collected data in Bulgaria (n = 1069), France (n = 1108), Poland (n = 1104), Italy (n = 1087), Spain (n = 1102) and Sweden (n = 1097). Based on the features of the most widely adopted public health guidelines to reduce SARS-Cov-2 transmission (mask wearing vs not, outdoor vs indoor contact, short vs 90 min meetings, few vs many people present, and physical distancing of 1 or 2 m), we conducted a discrete choice experiment (DCE) to estimate the public’s perceived risk of SARS-CoV-2 transmission in scenarios that presented mutually exclusive constellations of these features. Our findings indicate that participants’ perception of transmission risk was most influenced by the NPI attributes of mask-wearing and outdoor meetings and the least by NPI attributes that focus on physical distancing, meeting duration, and meeting size. Differentiating by country, gender, age, cognitive style (reflective or intuitive), and perceived freight of COVID-19 moreover allowed us to identify important differences between subgroups. Our findings highlight the importance of improving health policy communication and citizens’ health literacy about the design of NPIs and the transmission risk of SARS-Cov-2 and potentially future viruses

Structure and Function of Human Erythrocyte Pyruvate Kinase MOLECULAR BASIS OF NONSPHEROCYTIC HEMOLYTIC ANEMIA

Deficiency of human erythrocyte isozyme (RPK) is, together with glucose-6-phosphate dehydrogenase deficiency, the most common cause of the nonspherocytic hemolytic anemia. To provide a molecular framework to the disease, we have solved the 2.7 A resolution crystal structure of human RPK in complex with fructose 1,6-bisphosphate, the allosteric activator, and phosphoglycolate, a substrate analogue, and we have functionally and structurally characterized eight mutants (G332S, G364D, T384M, D390N, R479H, R486W, R504L, and R532W) found in RPK-deficient patients. The mutations target distinct regions of RPK structure, including domain interfaces and catalytic and allosteric sites. The mutations affect to a different extent thermostability, catalytic efficiency, and regulatory properties. These studies are the first to correlate the clinical symptoms with the molecular properties of the mutant enzymes. Mutations greatly impairing thermostability and/or activity are associated with severe anemia. Some mutant proteins exhibit moderate changes in the kinetic parameters, which are sufficient to cause mild to severe anemia, underlining the crucial role of RPK for erythrocyte metabolism. Prediction of the effects of mutations is difficult because there is no relation between the nature and location of the replaced amino acid and the type of molecular perturbation. Characterization of mutant proteins may serve as a valuable tool to assist with diagnosis and genetic counseling

The molecular function of SwrA: an auxiliary factor modulating DegU transcriptional activity

We recently demonstrated that the main fla/che promoter PA(fla/che) can be bound by the phosphorylated form of DegU (DegU~P) with two opposite outcomes: complete repression if DegU~P alone is bound to PA(fla/che) DNA; transcriptional stimulation if DNA is bound by DegU~P complexed with SwrA. Thus SwrA, which is necessary for swarming motility, constitutes an auxiliary factor that modulates the transcriptional activity of the response regulator DegU turning it from a repressor into an activator of PA(fla/che). Evidences indicate that SwrA might modulate other DegU~P-regulated promoters. Also, we demonstrated that DegU32(Hy) is a mutant protein unable to functionally interact with SwrA at the fla/che promoter; the phenotype of degU32(Hy) strains differs from that of the wild type DegU~P and we suggest the use of degS200(Hy) mutant strains for studies aimed at analyzing the effect of the level of DegU phosphorylation. SwrA is coded by a gene containing a slippery poly-adenine tract that allows phase variations between a functional and a non-functional allelic state. In swrA+ cells (typically in undomesticated strains) fla/che transcription oscillates from the basal/medium level to the activated state that is required for swarming. When swrA is in the non-functional form (e.g. in the 168 laboratory strain) fla/che transcription can oscillate between a repressed state in which no flagella are made and a basal/medium level of transcription sufficient for a limited swimming motility. While in both swrA- and swrA+ strains oscillations depend on phosphorylation of DegU mediated by environmental stimuli, in swrA- cells the secondary fla/che promoter PD3(fla/che) plays an important role that might constitute the bistable switch acting on motility

DegU-P Represses Expression of the Motility fla-che Operon in Bacillus subtilis

Bacillus subtilis implements several adaptive strategies to cope with nutrient limitation experienced at the end of exponential growth. The DegS-DegU two-component system is part of the network involved in the regulation of postexponential responses, such as competence development, the production of exoenzymes, and motility. The degU32(Hy) mutation extends the half-life of the phosphorylated form of DegU (DegU-P); this in turn increases the production of alkaline protease, levan-sucrase, and other exoenzymes and inhibits motility and the production of flagella. The expression of the flagellum-specific sigma factor SigD, of the flagellin gene hag, and of the fla-che operon is strongly reduced in a degU32(Hy) genetic background. To investigate the mechanism of action of DegU-P on motility, we isolated mutants of degU32(Hy) that completely suppressed the motility deficiency. The mutations were genetically mapped and characterized by PCR and sequencing. Most of the mutations were found to delete a transcriptional termination signal upstream of the main flagellar operon, fla-che, thus allowing transcriptional readthrough from the cod operon. Two additional mutations improved the σ(A)-dependent promoter sequence of the fla-che operon. Using an electrophoretic mobility shift assay, we have demonstrated that purified DegU binds specifically to the P(A) promoter region of the fla-che operon. The data suggest that DegU represses transcription of the fla-che operon, and they indicate a central role of the operon in regulating the synthesis and assembly of flagella

Poly-gamma-glutamate production in Bacillus subtilis

Poly-gamma-glutamic acid (gamma-PGA) is an anionic polymer of increasing industrial interest, composed of thousands of glutamic acid residues linked by gamma-glutamyl bonds. Secretion of the polymer into the medium confers a mucoid colony morphology to the Bacillus producer strains grown on LB agar plates. Although Bacillus subtilis possesses the functional biosynthetic pgs operon, containing four genes, laboratory strains do not have the ability to produce the polymer because pgs transcription is not active. We dissected the genetic elements involved in the conversion of laboratory non-producer strains into gamma-PGA producers and established that the synergic action of two gene products is required. The co-presence of the wild-type swrAA allele, a gene involved in swarming motility, and the hyperphosphorylated form of the transcriptional factor degU, belonging to the two-component system degS/degU, is sufficient to drive pgs transcription and gamma-PGA production