Inactivation of the conserved protease LonA increases production of xylanase and amylase in Bacillus subtilis.

Background: Bacillus subtilis is widely used for industrial enzyme production due to its capacity to efficiently secrete proteins. However, secretion efficiency of enzymes varies widely, and optimizing secretion is crucial to make production commercially viable. Previously, we have shown that overexpression of the xylanase XynA lowers expression of Clp protein chaperones, and that inactivation of CtsR, which regulates and represses clp transcription, increases the production of XynA.

Membrane depolarization kills dormant Bacillus subtilis cells by generating a lethal dose of ROS.

The bactericidal activity of several antibiotics partially relies on the production of reactive oxygen species (ROS), which is generally linked to enhanced respiration and requires the Fenton reaction. Bacterial persister cells, an important cause of recurring infections, are tolerant to these antibiotics because they are in a dormant state. Here, we use Bacillus subtilis cells in stationary phase, as a model system of dormant cells, to show that pharmacological induction of membrane depolarization enhances the antibiotics' bactericidal activity and also leads to ROS production.

Development of a split-luciferase assay to establish optimal protein secretion conditions for protein production by .

is a Gram-positive bacterium that is frequently used in the bioindustry for the production of various proteins, because of its superior protein secretion capacities. To determine optimal conditions for protein secretion by , a quick and sensitive method for measuring protein secretion is crucial. A fast and universal assay is most useful for detecting diverse proteins in a high-throughput manner.

Improving gene set enrichment analysis (GSEA) by using regulation directionality.

To infer the biological meaning from transcriptome data, it is useful to focus on genes that are regulated by the same regulator, i.e., regulons.

Induction of the CtsR regulon improves Xylanase production in Bacillus subtilis.

Background: The bacterium Bacillus subtilis is extensively used for the commercial production of enzymes due to its efficient protein secretion capacity. However, the efficiency of secretion varies greatly between enzymes, and despite many years of research, optimization of enzyme production is still largely a matter of trial-and-error. Genome-wide transcriptome analysis seems a useful tool to identify relevant secretion bottlenecks, yet to this day, only a limited number of transcriptome studies have been published that focus on enzyme secretion in B.

Metabolic and chromosomal changes in a mutant.

WhiA is a conserved DNA-binding protein that influences cell division in many Gram-positive bacteria and, in also chromosome segregation. How WhiA works in is unknown. Here, we tested three hypothetical mechanisms using metabolomics, fatty acid analysis, and chromosome confirmation capture experiments.

Classification of antimicrobial mechanism of action using dynamic bacterial morphology imaging.

Antimicrobial resistance is a major threat to human health. Basic knowledge of antimicrobial mechanism of action (MoA) is imperative for patient care and for identification of novel antimicrobials. However, the process of antimicrobial MoA identification is relatively laborious.

SepF supports the recruitment of the DNA translocase SftA to the Z-ring.

In many bacteria, cell division begins before the sister chromosomes are fully segregated. Specific DNA translocases ensure that the chromosome is removed from the closing septum, such as the transmembrane protein FtsK in Escherichia coli. Bacillus subtilis contains two FtsK homologues, SpoIIIE and SftA.

Mechanistic insights into the C-P targeting lipopeptide antibiotics revealed by structure-activity studies and high-resolution crystal structures.

The continued rise of antibiotic resistance is a global concern that threatens to undermine many aspects of modern medical practice. Key to addressing this threat is the discovery and development of new antibiotics that operate by unexploited modes of action. The so-called calcium-dependent lipopeptide antibiotics (CDAs) are an important emerging class of natural products that provides a source of new antibiotic agents rich in structural and mechanistic diversity.

A chromosomal loop anchor mediates bacterial genome organization.

Nucleoprotein complexes play an integral role in genome organization of both eukaryotes and prokaryotes. Apart from their role in locally structuring and compacting DNA, several complexes are known to influence global organization by mediating long-range anchored chromosomal loop formation leading to spatial segregation of large sections of DNA. Such megabase-range interactions are ubiquitous in eukaryotes, but have not been demonstrated in prokaryotes.

The Minimal Genome Compendium.

To better understand cellular life, it is essential to decipher the contribution of individual components and their interactions. Minimal genomes are an important tool to investigate these interactions. Here, we provide a database of 105 fully annotated genomes of a series of strains with sequential deletion steps of the industrially relevant model bacterium starting with the laboratory wild type strain 168 and ending with PG38, which lacks approximately 40% of the original genome.

Different Resource Allocation in a Bacillus subtilis Population Displaying Bimodal Motility.

To cope with sudden changes in their environment, bacteria can use a bet-hedging strategy by dividing the population into cells with different properties. This so-called bimodal or bistable cellular differentiation is generally controlled by positive feedback regulation of transcriptional activators. Due to the continuous increase in cell volume, it is difficult for these activators to reach an activation threshold concentration when cells are growing exponentially.

A flat embedding method for transmission electron microscopy reveals an unknown mechanism of tetracycline.

Transmission electron microscopy of cell sample sections is a popular technique in microbiology. Currently, ultrathin sectioning is done on resin-embedded cell pellets, which consumes milli- to deciliters of culture and results in sections of randomly orientated cells. This is problematic for rod-shaped bacteria and often precludes large-scale quantification of morphological phenotypes due to the lack of sufficient numbers of longitudinally cut cells.

Control of septum thickness by the curvature of SepF polymers.

Gram-positive bacteria divide by forming a thick cross wall. How the thickness of this septal wall is controlled is unknown. In this type of bacteria, the key cell division protein FtsZ is anchored to the cell membrane by two proteins, FtsA and/or SepF.

Extreme slow growth as alternative strategy to survive deep starvation in bacteria.

Bacteria can become dormant or form spores when they are starved for nutrients. Here, we find that non-sporulating Bacillus subtilis cells can survive deep starvation conditions for many months. During this period, cells adopt an almost coccoid shape and become tolerant to antibiotics.

Assessing Membrane Fluidity and Visualizing Fluid Membrane Domains in Bacteria Using Fluorescent Membrane Dyes.

Membrane fluidity is a key parameter of bacterial membranes that undergoes quick adaptation in response to environmental challenges and has recently emerged as an important factor in the antibacterial mechanism of membrane-targeting antibiotics. The specific level of membrane fluidity is not uniform across the bacterial cell membrane. Rather, specialized microdomains associated with different cellular functions can exhibit fluidity values that significantly deviate from the average.

The Multifaceted Antibacterial Mechanisms of the Pioneering Peptide Antibiotics Tyrocidine and Gramicidin S.

Cyclic β-sheet decapeptides from the tyrocidine group and the homologous gramicidin S were the first commercially used antibiotics, yet it remains unclear exactly how they kill bacteria. We investigated their mode of action using a bacterial cytological profiling approach. Tyrocidines form defined ion-conducting pores, induce lipid phase separation, and strongly reduce membrane fluidity, resulting in delocalization of a broad range of peripheral and integral membrane proteins.

Exploring the Nonconserved Sequence Space of Synthetic Expression Modules in Bacillus subtilis.

Increasing protein expression levels is a key step in the commercial production of enzymes. Predicting promoter activity and translation initiation efficiency based solely on consensus sequences have so far met with mixed results. Here, we addressed this challenge using a "brute-force" approach by designing and synthesizing a large combinatorial library comprising ∼12 000 unique synthetic expression modules (SEMs) for Bacillus subtilis.

Bactericidal activity of amphipathic cationic antimicrobial peptides involves altering the membrane fluidity when interacting with the phospholipid bilayer.

Background: Amphipathic cationic antimicrobial peptides (AMPs) TC19 and TC84, derived from the major AMPs of human blood platelets, thrombocidins, and Bactericidal Peptide 2 (BP2), a synthetic designer peptide showed to perturb the membrane of Bacillus subtilis. We aimed to determine the means by which the three AMPs cause membrane perturbation in vivo using B. subtilis and to evaluate whether the membrane alterations are dependent on the phospholipid composition of the membrane.

Synthetic antimicrobial peptides delocalize membrane bound proteins thereby inducing a cell envelope stress response.

Background: Three amphipathic cationic antimicrobial peptides (AMPs) were characterized by determining their effect on Gram-positive bacteria using Bacillus subtilis strain 168 as a model organism. These peptides were TC19 and TC84, derivatives of thrombocidin-1 (TC-1), the major AMPs of human blood platelets, and Bactericidal Peptide 2 (BP2), a synthetic designer peptide based on human bactericidal permeability increasing protein (BPI).

Methods: To elucidate the possible mode of action of the AMPs we performed a transcriptomic analysis using microarrays.

Membrane Curvature and the Tol-Pal Complex Determine Polar Localization of the Chemoreceptor Tar in Escherichia coli.

Chemoreceptors are localized at the cell poles of and other rod-shaped bacteria. Over the years, different mechanisms have been put forward to explain this polar localization, including stochastic clustering, membrane curvature-driven localization, interactions with the Tol-Pal complex, and nucleoid exclusion. To evaluate these mechanisms, we monitored the cellular localization of the aspartate chemoreceptor Tar in different deletion mutants.

The novel antibiotic rhodomyrtone traps membrane proteins in vesicles with increased fluidity.

The acylphloroglucinol rhodomyrtone is a promising new antibiotic isolated from the rose myrtle Rhodomyrtus tomentosa, a plant used in Asian traditional medicine. While many studies have demonstrated its antibacterial potential in a variety of clinical applications, very little is known about the mechanism of action of rhodomyrtone. Preceding studies have been focused on intracellular targets, but no specific intracellular protein could be confirmed as main target.

The Conserved DNA Binding Protein WhiA Influences Chromosome Segregation in Bacillus subtilis.

The DNA binding protein WhiA is conserved in Gram-positive bacteria and is present in the genetically simple cell wall-lacking mycoplasmas. The protein shows homology to eukaryotic homing endonucleases but lacks nuclease activity. WhiA was first characterized in streptomycetes, where it regulates the expression of key differentiation genes, including the cell division gene , which is essential for sporulation.

Free SepF interferes with recruitment of late cell division proteins.

The conserved cell division protein SepF aligns polymers of FtsZ, the key cell division protein in bacteria, during synthesis of the (Fts)Z-ring at midcell, the first stage in cytokinesis. In addition, SepF acts as a membrane anchor for the Z-ring. Recently, it was shown that SepF overexpression in Mycobacterium smegmatis blocks cell division.

In-Culture Cross-Linking of Bacterial Cells Reveals Large-Scale Dynamic Protein-Protein Interactions at the Peptide Level.

Identification of dynamic protein-protein interactions at the peptide level on a proteomic scale is a challenging approach that is still in its infancy. We have developed a system to cross-link cells directly in culture with the special lysine cross-linker bis(succinimidyl)-3-azidomethyl-glutarate (BAMG). We used the Gram-positive model bacterium Bacillus subtilis as an exemplar system.