Preclinical Safety Assessment of Bacillus subtilis BS50 for Probiotic and Food Applications.

Despite the commercial rise of probiotics containing Bacillaceae spp., it remains important to assess the safety of each strain before clinical testing. Herein, we performed preclinical analyses to address the safety of Bacillus subtilis BS50. Using in silico analyses, we screened the 4.15 Mbp BS50 genome for genes encoding known Bacillus toxins, secondary metabolites, virulence factors, and antibiotic resistance. We also assessed the effects of BS50 lysates on the viability and permeability of cultured human intestinal epithelial cells (Caco-2). We found that the BS50 genome does not encode any known Bacillus toxins. The BS50 genome contains several gene clusters involved in the biosynthesis of secondary metabolites, but many of these antimicrobial metabolites (e.g., fengycin) are common to Bacillus spp. and may even confer health benefits related to gut microbiota health. BS50 was susceptible to seven of eight commonly prescribed antibiotics, and no antibiotic resistance genes were flanked by the complete mobile genetic elements that could enable a horizontal transfer. In cell culture, BS50 cell lysates did not diminish either Caco-2 viability or monolayer permeability. Altogether, BS50 exhibits a robust preclinical safety profile commensurate with commercial probiotic strains and likely poses no significant health risk to humans.

First complete genome sequence of Bacillus glycinifermentans B-27.

The first complete genome sequence of Bacillus glycinifermentans B-27 was determined by SMRT sequencing generating a genome sequence with a total length of 4,607,442 bases. Based on this sequence 4738 protein-coding sequences were predicted and used to identify gene clusters that are related to the production of secondary metabolites such as Lichenysin, Bacillibactin and Bacitracin. This genomic potential combined with the ability of B. glycinifermentans B-27 to grown in bile containing media might contribute to a future application of this strain as probiotic in productive livestock potentially inhibiting competing and pathogenic organisms.

Genetic analysis of the Bacillus licheniformis degSU operon and the impact of regulatory mutations on protease production.

Disruption experiments targeted at the Bacillus licheniformis degSU operon and GFP-reporter analysis provided evidence for promoter activity immediately upstream of degU. pMutin mediated concomitant introduction of the degU32 allele--known to cause hypersecretion in Bacillus subtilis-- resulted in a marked increase in protease activity. Application of 5-fluorouracil based counterselection through establishment of a phosphoribosyltransferase deficient Δupp strain eventually facilitated the marker-free introduction of degU32 leading to further protease enhancement achieving levels as for hypersecreting wild strains in which degU was overexpressed. Surprisingly, deletion of rapG--known to interfere with DegU DNA-binding in B. subtilis--did not enhance protease production neither in the wild type nor in the degU32 strain. The combination of degU32 and Δupp counterselection in the type strain is not only equally effective as in hypersecreting wild strains with respect to protease production but furthermore facilitates genetic strain improvement aiming at biological containment and effectiveness of biotechnological processes.

Transcriptome profiling of degU expression reveals unexpected regulatory patterns in Bacillus megaterium and discloses new targets for optimizing expression.

The first whole transcriptome assessment of a Bacillus megaterium strain provides unanticipated insights into the degSU regulon considered to be of central importance for exo-enzyme production. Regulatory patterns as well as the transcription of degSU itself deviate from the model organism Bacillus subtilis; the number of DegU-regulated secretory enzymes is rather small. Targets for productivity optimization, besides degSU itself, arise from the unexpected DegU-dependent induction of the transition-state regulator AbrB during exponential growth. Induction of secretion-assisting factors, such as the translocase subunit SecY or the signal peptidase SipM, promote hypersecretion. B. megaterium DegSU transcriptional control is advantageous for production purposes, since the degU32 constitutively active mutant conferred hypersecretion of a heterologous Bacillus amyloliquefaciens amylase without the detrimental rise, as for B. subtilis and Bacillus licheniformis, in extracellular proteolytic activities.

Systems biology of recombinant protein production using Bacillus megaterium.

The Gram-negative bacterium Escherichia coli is the most widely used production host for recombinant proteins in both academia and industry. The Gram-positive bacterium Bacillus megaterium represents an increasingly used alternative for high yield intra- and extracellular protein synthesis. During the past two decades, multiple tools including gene expression plasmids and production strains have been developed. Introduction of free replicating and integrative plasmids into B. megaterium is possible via protoplasts transformation or transconjugation. Using His(6)- and StrepII affinity tags, the intra- or extracellular produced proteins can easily be purified in one-step procedures. Different gene expression systems based on the xylose controlled promoter P(xylA) and various phage RNA polymerase (T7, SP6, K1E) driven systems enable B. megaterium to produce up to 1.25g of recombinant protein per liter. Biomass concentrations of up to 80g/l can be achieved by high cell density cultivations in bioreactors. Gene knockouts and gene replacements in B. megaterium are possible via an optimized gene disruption system. For a safe application in industry, sporulation and protease-deficient as well as UV-sensitive mutants are available. With the help of the recently published B. megaterium genome sequence, it is possible to characterize bottle necks in the protein production process via systems biology approaches based on transcriptome, proteome, metabolome, and fluxome data. The bioinformatical platform (Megabac, http://www.megabac.tu-bs.de) integrates obtained theoretical and experimental data.

Functional analysis of the response regulator DegU in Bacillus megaterium DSM319 and comparative secretome analysis of degSU mutants.

We functionally analysed the two-component regulatory system DegSU (historically SacU) in Bacillus megaterium DSM319 by generating a genetic knock out as well as a sacU32 mutation. The latter--known to cause a hypersecretion phenotype in Bacillus subtilis--had no influence on extracellular protease and amylase activity in B. megaterium. Since the B. megaterium DegU complemented a Bacillus licheniformis ∆degSU mutant, functionality of the protein was proven. Expression of the sacB encoded levansucrase was found to be dependent on DegSU in B. megaterium. Consistently, the fusion of the sacB promoter to gfp revealed a strong increase in GFP-expression in the sacU32 strain. On 2 D-gels of the secretome, a large number of intracellular proteins was seen. The culture medium contained only 42 secreted proteins which can be assigned to polypeptides involved in the metabolism of the cell wall, polypeptides with proteolytic activities and those with unknown functions. Though overall protease activity matches with the wild type, two proteolytic enzymes (Vpr and YwaD) are missing in the secretome of the ∆degSU strain, while other degradative enzymes are not affected. In line with such findings, no increase of proteolytic or other degradative enzymes was seen in the sacU32 mutant. Thus, compared to B. subtilis and B. licheniformis, the number of extracellular proteins influenced by DegSU is surprisingly low in B. megaterium, a feature, probably advantageous as to the use of the sacU32 mutant for production of secreted proteins.