Gene expression of Bacillus subtilis subtilisin E in Thermus thermophilus

The Bacillus subtilis subtilisin E gene was cloned into an expression vector of the extreme thermophile, Thermus thermophilus. Active subtilisin E was produced in E. coli, indicating that the Thermus promoter functions in E. coli. When the plasmid was further introduced into T. thermophilus, the subtilisin E gene was expressed and the gene product accumulated as an inactive pro-form, because the autoprocessing of the wild-type enzyme to the active-form did not occur at 50°C or above. Received 17 March 1999/ Accepted in revised form 28 June 1999     

The Biofilm Regulatory Network from Bacillus subtilis: A Structure-Function Analysis

Bacterial biofilms are notorious for their ability to protect bacteria from environmental challenges, most importantly the action of antibiotics. Bacillus subtilis is an extensively studied model organism used to understand the process of biofilm formation. A complex network of principal regulatory proteins including Spo0A, AbrB, AbbA, Abh, SinR, SinI, SlrR, and RemA, work in concert to transition B. subtilis from the free-swimming planktonic state to the biofilm state. In this review, we explore, connect, and summarize decades worth of structural and biochemical studies that have elucidated this protein signaling network. Since structure dictates function, unraveling aspects of protein molecular mechanisms will allow us to devise ways to exploit critical features of the biofilm regulatory pathway, such as possible therapeutic intervention. This review pools our current knowledge base of B. subtilis biofilm regulatory proteins and highlights potential therapeutic intervention points.     

Efficiency of transcriptional terminators in Bacillus subtilis

To promote more efficient synthesis of heterologous gene products in a Bacillus subtilis host, we have developed a system for rapidly testing the effect of a putative terminator on in vivo gene expression. Terminator structures from the Bacillus amyloliquefaciens amyE gene, the Bacillus licheniformis penP gene, the B. subtilis bglS gene, and the Bacillus thuringiensis cry gene were subcloned and inserted into a vector in such a way as to disrupt expression of the cat-86 gene. Comparisons are made between gene expression levels and the stabilities of the respective stem-loop structures.     

Genetic interaction observed in Bacillus subtilis

Summary Some evidence was obtained that genetic interaction occurs inBacillus subtilis K. A mixed inoculation of two doubly auxotrophic mutants onto approriate media yielded tiny colonies which seemed to be initiated by heterocaryons or heterozygotes. The tiny colonies contained not only a recombinant type which acquired two characters from one or another parent, but also some abnormal types having new characters which were not recognized in either parent. The phenomenon is similar to the genetic interaction found inStreptomyces.With 5 Figures in the Text     

Transition-state regulators: sentinels of Bacillus subtilis post-exponential gene expression

When Bacillus subtilis encounters a nutrient-depleted environment, it expresses a wide variety of genes that encode functions in alternative pathways of metabolism and energy production. Expression of these genes first occurs during the transition from active growth into stationary phase and is controlled by a class of proteins termed transition-state regulators. In several instances, a given gene is redundantly controlled by two or more of these regulators and many of these regulators control genes in numerous different pathways. The AbrB, Hpr and Sin proteins are the best-studied examples of these regulatory molecules. Their role is to prevent inappropriate and possibly detrimental functions from being expressed during exponential growth when they are not needed. They serve as elements integrating sporulation with ancillary stationary-phase phenomena and appear to participate in the timing of early sporulation events and in fine-tuning the magnitude of gene expression in response to specific environmental conditions.     

Interactions between Bacillus subtilis early spore coat morphogenetic proteins

When challenged by stresses such as starvation, the soil bacterium Bacillus subtilis produces an endospore surrounded by a proteinaceous coat composed of >70 proteins that are organized into three main layers: an amorphous undercoat, lightly staining lamellar inner coat and electron-dense outer coat. This coat protects the spore against a variety of chemicals or lysozyme. Mutual interactions of the coat's building blocks are responsible for the formation of this structurally complex and extraordinarily resistant shell. However, the assembly process of spore coat proteins is still poorly understood. In the present work, the main focus is on the three spore coat morphogenetic proteins: SpoIVA, SpoVID and SafA. Direct interaction between SpoIVA and SpoVID proteins was observed using a yeast two-hybrid assay and verified by coexpression experiment followed by Western blot analysis. Coexpression experiments also confirmed previous findings that SpoVID and SafA directly interact, and revealed a novel interaction between SpoIVA and SafA. Moreover, gel filtration analysis revealed that both SpoIVA and SpoVID proteins form large oligomers.     

The Bacillus subtilis spore coat protein interaction network

Bacterial spores are surrounded by a morphologically complex, mechanically flexible protein coat, which protects the spore from toxic molecules. The interactions among the over 50 proteins that make up the coat remain poorly understood. We have used cell biological and protein biochemical approaches to identify novel coat proteins in Bacillus subtilis and describe the network of their interactions, in order to understand coat assembly and the molecular basis of its protective functions and mechanical properties. Our analysis characterizes the interactions between 32 coat proteins. This detailed view reveals a complex interaction network. A key feature of the network is the importance of a small subset of proteins that direct the assembly of most of the coat. From an analysis of the network topology, we propose a model in which low-affinity interactions are abundant in the coat and account, to a significant degree, for the coat's mechanical properties as well as structural variation between spores.