Gram-positive Model Organism Bacillus Subtilis Research Articles

Impact of Protein Aggregates on Sporulation and Germination of Bacillus subtilis.

In order to improve our general understanding of protein aggregate (PA) management and impact in bacteria, different model systems and processes need to be investigated. As such, we developed an inducible synthetic PA model system to investigate PA dynamics in the Gram-positive model organism Bacillus subtilis. This confirmed previous observations that PA segregation in this organism seems to follow the Escherichia coli paradigm of nucleoid occlusion governing polar localization and asymmetric segregation during vegetative growth. However, our findings also revealed that PAs can readily persist throughout the entire sporulation process after encapsulation in the forespore during sporulation. Moreover, no deleterious effects of PA presence on sporulation, germination and spore survival against heat or UV stress could be observed. Our findings therefore indicate that the sporulation process is remarkably robust against perturbations by PAs and misfolded proteins.

The Novel Synthetic Antibiotic BDTL049 Based on a Dendritic System Induces Lipid Domain Formation while Escaping the Cell Envelope Stress Resistance Determinants.

The threat of antimicrobial-resistant bacteria is ever increasing and over the past-decades development of novel therapeutic counter measurements have virtually come to a halt. This circumstance calls for interdisciplinary approaches to design, evaluate and validate the mode of action of novel antibacterial compounds. Hereby, carbosilane dendritic systems that exhibit antimicrobial properties have the potential to serve as synthetic and rationally designed molecules for therapeutic use. The bow-tie type topology of BDTL049 was recently investigated against the Gram-positive model organism Bacillus subtilis, revealing strong bactericidal properties. In this study, we follow up on open questions concerning the usability of BDTL049. For this, we synthesized a fluorescent-labeled version of BDTL049 that maintained all antimicrobial features to unravel the interaction of the compound and bacterial membrane. Subsequently, we highlight the bacterial sensitivity against BDTL049 by performing a mutational study of known resistance determinants. Finally, we address the cytotoxicity of the compound in human cells, unexpectedly revealing a high sensitivity of the eukaryotic cells upon BDTL049 exposure. The insights presented here further elaborate on the unique features of BDTL049 as a promising candidate as an antimicrobial agent while not precluding that further rounds of rational designing are needed to decrease cytotoxicity to ultimately pave the way for synthetic antibiotics toward clinical applicability.

Synthesis and mechanism-of-action of a novel synthetic antibiotic based on a dendritic system with bow-tie topology.

Over the course of the last decades, the continuous exposure of bacteria to antibiotics—at least in parts due to misprescription, misuse, and misdosing—has led to the widespread development of antimicrobial resistances. This development poses a threat to the available medication in losing their effectiveness in treating bacterial infections. On the drug development side, only minor advances have been made to bring forward novel therapeutics. In addition to increasing the efforts and approaches of tapping the natural sources of new antibiotics, synthetic approaches to developing novel antimicrobials are being pursued. In this study, BDTL049 was rationally designed using knowledge based on the properties of natural antibiotics. BDTL049 is a carbosilane dendritic system with bow-tie type topology, which has antimicrobial activity at concentrations comparable to clinically established natural antibiotics. In this report, we describe its mechanism of action on the Gram-positive model organism Bacillus subtilis. Exposure to BDTL049 resulted in a complex transcriptional response, which pointed toward disturbance of the cell envelope homeostasis accompanied by disruption of other central cellular processes of bacterial metabolism as the primary targets of BDTL049 treatment. By applying a combination of whole-cell biosensors, molecular staining, and voltage sensitive dyes, we demonstrate that the mode of action of BDTL049 comprises membrane depolarization concomitant with pore formation. As a result, this new molecule kills Gram-positive bacteria within minutes. Since BDTL049 attacks bacterial cells at different targets simultaneously, this might decrease the chances for the development of bacterial resistances, thereby making it a promising candidate for a future antimicrobial agent.

Effects of 4-Br-A23187 on Bacillus subtilis cells and unilamellar vesicles reveal it to be a potent copper ionophore.

Ionophores are small molecules or peptides that transport metal ions across biological membranes. Their transport capabilities are typically characterized in vitro using vesicles and single ion species. It is difficult to infer from these data which effects ionophores have on living cells in a complex environment (e.g., culture medium), since net ion movement is influenced by many factors including ion composition of the medium, concentration gradients, pH gradient, and protein-mediated transport processes across the membrane. To gain insights into the antibacterial mechanism of action of the semisynthetic polyether ionophore 4-Br-A23187, known to efficiently transport zinc and manganese in vitro, we investigated its effects on the gram-positive model organism Bacillus subtilis. In addition to monitoring cellular ion concentrations, the physiological impact of treatment was assessed on the proteome level. 4-Br-A23187 treatment resulted in an increase in intracellular copper levels, the extent of which depended on the copper concentration of the medium. Effects of copper accumulation mirrored by the proteomic response included oxidative stress, disturbance of proteostasis, metal and sulfur homeostasis. The antibiotic effect of 4-Br-A23187 is further aggravated by a decrease in intracellular manganese and magnesium. A liposome model confirmed that 4-Br-A23187 acts as copper ionophore in vitro.

Update on the Protein Homeostasis Network in Bacillus subtilis.

Protein homeostasis is fundamental to cell function and survival. It relies on an interconnected network of processes involving protein synthesis, folding, post-translational modification and degradation as well as regulators of these processes. Here we provide an update on the roles, regulation and subcellular localization of the protein homeostasis machinery in the Gram-positive model organism Bacillus subtilis. We discuss emerging ideas and current research gaps in the field that, if tackled, increase our understanding of how Gram-positive bacteria, including several human pathogens, maintain protein homeostasis and cope with stressful conditions that challenge their survival.

Isolation of Persister Cells of Bacillus subtilis and Determination of Their Susceptibility to Antimicrobial Peptides.

Persister cells are growth-arrested subpopulations that can survive possible fatal environments and revert to wild types after stress removal. Clinically, persistent pathogens play a key role in antibiotic therapy failure, as well as chronic, recurrent, and antibiotic-resilient infections. In general, molecular and physiological research on persister cells formation and compounds against persister cells are much desired. In this study, we firstly demonstrated that the spore forming Gram-positive model organism Bacillus subtilis can be used to generate persister cells during exposure to antimicrobial compounds. Interestingly, instead of exhibiting a unified antibiotic tolerance profile, different number of persister cells and spores were quantified in various stress conditions. qPCR results also indicated that differential stress responses are related to persister formation in various environmental conditions. We propose, for the first time to the best of our knowledge, an effective method to isolate B. subtilis persister cells from a population using fluorescence-activated cell sorting (FACS), which makes analyzing persister populations feasible. Finally, we show that alpha-helical cationic antimicrobial peptides SAAP-148 and TC-19, derived from human cathelicidin LL-37 and human thrombocidin-1, respectively, have high efficiency against both B. subtilis vegetative cells and persisters, causing membrane permeability and fluidity alteration. In addition, we confirm that in contrast to persister cells, dormant B. subtilis spores are not susceptible to the antimicrobial peptides.

FtsZ treadmilling is essential for Z-ring condensation and septal constriction initiation in Bacillus subtilis cell division

Despite the central role of division in bacterial physiology, how division proteins work together as a nanoscale machine to divide the cell remains poorly understood. Cell division by cell wall synthesis proteins is guided by the cytoskeleton protein FtsZ, which assembles at mid-cell as a dense Z-ring formed of treadmilling filaments. However, although FtsZ treadmilling is essential for cell division, the function of FtsZ treadmilling remains unclear. Here, we systematically resolve the function of FtsZ treadmilling across each stage of division in the Gram-positive model organism Bacillus subtilis using a combination of nanofabrication, advanced microscopy, and microfluidics to measure the division-protein dynamics in live cells with ultrahigh sensitivity. We find that FtsZ treadmilling has two essential functions: mediating condensation of diffuse FtsZ filaments into a dense Z-ring, and initiating constriction by guiding septal cell wall synthesis. After constriction initiation, FtsZ treadmilling has a dispensable function in accelerating septal constriction rate. Our results show that FtsZ treadmilling is critical for assembling and initiating the bacterial cell division machine.

Resonance assignments of the cytoplasmic domain of ECF sigma factor W pathway protein YsdB from Bacillus subtilis.

Bacterial sigma (σ) factor, along with RNA polymerase core enzyme, initiates gene transcription from specific promoter regions and therefore regulates clusters of genes in response to a particular situation. The extracytoplasmic function (ECF) σ factors are a class of alternative σ factors that are often associated with environmental signal transduction across the bacterial membrane, in which external signal triggers the release of active σ from the membrane-anchored anti-σ factor. Gram-positive model organism Bacillus subtilis (B. subtilis) has seven ECF σ factors: σM, σV, σX, σW, σY, σZ and σYlaC. Although all these ECF σ factors were found to be involved in B. subtilis antibiotic resistance, σW is among the most studied and considered to play a pivotal role in responding to antimicrobial stresses. σW is under tight control and remains deactivated until exposure to external stimuli, after which proteases PrsW and RasP cleave the specific anti-sigma factor-RsiW to release and activate σW. Membrane anchored protein YsdB is a negative regulator of this activation, possibly via its direct interaction with PrsW and/or RsiW. Importantly, YsdB is well conserved among Bacilli, including pathogenic bacteria like Bacillus cereus. In this study, we describe the chemical shift assignments of the cytoplasmic domain of YsdB (29-130) of B. subtilis in solution as a basis for further interaction studies and structure determination. The near-complete assignment and the solution structure that will follow could provide a further understanding in σW regulation.

Physiology of guanosine-based second messenger signaling in Bacillus subtilis.

The guanosine-based second messengers (p)ppGpp and c-di-GMP are key players of the physiological regulation of the Gram-positive model organism Bacillus subtilis. Their regulatory spectrum ranges from key metabolic processes over motility to biofilm formation. Here we review our mechanistic knowledge on their synthesis and degradation in response to environmental and stress signals as well as what is known on their cellular effectors and targets. Moreover, we discuss open questions and our gaps in knowledge on these two important second messengers.

Optimized Proteomics Workflow for the Detection of Small Proteins.

Small open reading frame encoded proteins (SEPs) gained increasing interest during the last few years because of their broad range of important functions in both prokaryotes and eukaryotes. In bacteria, signaling, virulence, and regulation of enzyme activities have been associated with SEPs. Nonetheless, the number of SEPs detected in large-scale proteome studies is often low as classical methods are biased toward the identification of larger proteins. Here, we present a workflow that allows enhanced identification of small proteins compared to traditional protocols. For this aim, the steps of small protein enrichment, proteolytic digest, and database search were reviewed and adjusted to the special requirement of SEPs. Enrichment by the use of small-pore-sized solid-phase material increased the number of identified SEPs by a factor of 2, and utilization of alternative proteases to trypsin reduced the spectral counts for larger proteins. The application of the optimized protocol allowed the detection of 210 already annotated proteins up to 100 amino acids (aa) length, including 16 proteins below 51 aa in the Gram-positive model organism Bacillus subtilis. Moreover, 12% of all identified proteins were up to 100 aa, which is a significantly larger fraction than that reported in studies involving traditional proteomics workflows. Finally, the application of an integrated proteogenomics search database and extensive subsequent validation resulted in the confident identification of three novel, not yet annotated, SEPs, which are 21, 26, and 42 aa long.

Intermolecular Communication in Bacillus subtilis: RNA-RNA, RNA-Protein and Small Protein-Protein Interactions.

In bacterial cells we find a variety of interacting macromolecules, among them RNAs and proteins. Not only small regulatory RNAs (sRNAs), but also small proteins have been increasingly recognized as regulators of bacterial gene expression. An average bacterial genome encodes between 200 and 300 sRNAs, but an unknown number of small proteins. sRNAs can be cis- or trans-encoded. Whereas cis-encoded sRNAs interact only with their single completely complementary mRNA target transcribed from the opposite DNA strand, trans-encoded sRNAs are only partially complementary to their numerous mRNA targets, resulting in huge regulatory networks. In addition to sRNAs, uncharged tRNAs can interact with mRNAs in T-box attenuation mechanisms. For a number of sRNA-mRNA interactions, the stability of sRNAs or translatability of mRNAs, RNA chaperones are required. In Gram-negative bacteria, the well-studied abundant RNA-chaperone Hfq fulfils this role, and recently another chaperone, ProQ, has been discovered and analyzed in this respect. By contrast, evidence for RNA chaperones or their role in Gram-positive bacteria is still scarce, but CsrA might be such a candidate. Other RNA-protein interactions involve tmRNA/SmpB, 6S RNA/RNA polymerase, the dual-function aconitase and protein-bound transcriptional terminators and antiterminators. Furthermore, small proteins, often missed in genome annotations and long ignored as potential regulators, can interact with individual regulatory proteins, large protein complexes, RNA or the membrane. Here, we review recent advances on biological role and regulatory principles of the currently known sRNA-mRNA interactions, sRNA-protein interactions and small protein-protein interactions in the Gram-positive model organism Bacillus subtilis. We do not discuss RNases, ribosomal proteins, RNA helicases or riboswitches.

Expanding and understanding the CRISPR toolbox for Bacillus subtilis with MAD7 and dMAD7.

The CRISPR-Cas9 system has become increasingly popular for genome engineering across all fields of biological research, including in the Gram-positive model organism Bacillus subtilis. A major drawback for the commercial use of Cas9 is the IP landscape requiring a license for its use, as well as reach-through royalties on the final product. Recently an alternative CRISPR nuclease, free to use for industrial R&D, MAD7 was released by Inscripta (CO). Here we report the first use of MAD7 for gene editing in B. subtilis, in which editing rates of 93% and 100% were established. Additionally, we engineer the first reported catalytically inactive MAD7 (dMAD7) variant (D877A, E962A, and D1213A) and demonstrate its utility for CRISPR interference (CRISPRi) at up to 71.3% reduction of expression at single and multiplexed target sites within B. subtilis. We also confirm the CRISPR-based editing mode of action in B. subtilis providing evidence that the nuclease-mediated DNA double-strand break acts as a counterselection mechanism after homologous recombination of the donor DNA.

TRNA Maturation Defects Lead to Inhibition of rRNA Processing via Synthesis of pppGpp.

rRNAs and tRNAs universally require processing from longer primary transcripts to become functional for translation. Here, we describe an unsuspected link between tRNA maturation and the 3' processing of 16S rRNA, a key step in preparing the small ribosomal subunit for interaction with the Shine-Dalgarno sequence in prokaryotic translation initiation. We show that an accumulation of either 5' or 3' immature tRNAs triggers RelA-dependent production of the stringent response alarmone (p)ppGpp in the Gram-positive model organism Bacillus subtilis. The accumulation of (p)ppGpp and accompanying decrease in GTP levels specifically inhibit 16S rRNA 3' maturation. We suggest that cells can exploit this mechanism to sense potential slowdowns in tRNA maturation and adjust rRNA processing accordingly to maintain the appropriate functional balance between these two major components of the translation apparatus.

Hydrogen Peroxide Production of Group A Streptococci (GAS) is emm-Type Dependent and Increased at Low Temperatures.

Group A streptococcus (GAS) is an important human pathogen whose clinical isolates differ in their ability to produce hydrogen peroxide (H2O2). H2O2 is primarily produced by the enzyme lactate oxidase (LctO), an in depth in silico research revealed that all genome-sequenced GAS possess the required gene lctO. The importance of lctO for GAS is underlined by its highly conserved catabolite control element (cre box) as well as its perfect promotor sequence in comparison to the known consensus sequences of the Gram-positive model organism Bacillus subtilis. In this study, we provide further insight in the function and regulation of lactate oxidase by analyzing a large group of clinical GAS isolates. We found that H2O2 production increased over time in the late stationary phase; after 4days of incubation, 5.4% of the isolates showed a positive result at 37°C, while the rate increased to 16.4% at 20°C. This correlation between H2O2 production and low temperatures suggests additional regulatory mechanisms for lctO besides catabolite control protein A (CcpA) and indicates that lctO might play a role for GAS energy metabolism at sub-body temperatures. Furthermore, we could identify that H2O2 production was different among clinical isolates; we could correlate H2O2 production to emm-types, indicating that emm-types 6 and 75 had the highest rate of H2O2 production. The emm-type- and temperature-dependent H2O2 production of clinical GAS isolates might contribute to their different survival strategies.

YocM a small heat shock protein can protect Bacillus subtilis cells during salt stress.

Small heat shock proteins (sHsp) occur in all domains of life. By interacting with misfolded or aggregated proteins these chaperones fulfill a protective role in cellular protein homeostasis. Here, we demonstrate that the sHsp YocM of the Gram-positive model organism Bacillussubtilis is part of the cellular protein quality control system with a specific role in salt stress response. In the absence of YocM the survival of salt shocked cells is impaired, and increased levels of YocM protect B. subtilis exposed to heat or salt. We observed a salt and heat stress-induced localization of YocM to intracellular protein aggregates. Interestingly, purified YocM appears to accelerate protein aggregation of different model substrates in vitro. In addition, the combined presence of YocM and chemical chaperones, which accumulate in salt stressed cells, can facilitate in vitro a synergistic protective effect on protein misfolding. Therefore, the beneficial role of YocM during salt stress could be related to a mutual functional relationship with chemical chaperones and adds a new possible functional aspect to sHsp chaperone activities.

The Gram-positive model organism Bacillus subtilis does not form microscopically detectable cardiolipin-specific lipid domains.

Rather than being homogenous diffusion-dominated structures, biological membranes can exhibit areas with distinct composition and characteristics, commonly termed as lipid domains. Arguably the most comprehensively studied examples in bacteria are domains formed by cardiolipin, which have been functionally linked to protein targeting, the cell division process and the mode of action of membrane-targeting antimicrobials. Cardiolipin domains were originally identified in the Gram-negative model organism Escherichia coli based on preferential staining by the fluorescent membrane dye nonylacridine orange (NAO), and later reported to also exist in other Gram-negative and -positive bacteria. Recently, the lipid-specificity of NAO has been questioned based on studies conducted in E. coli. This prompted us to reanalyse cardiolipin domains in the Gram-positive model organism Bacillus subtilis. Here we show that logarithmically growing B. subtilis does not form microscopically detectable cardiolipin-specific lipid domains, and that NAO is not a specific stain for cardiolipin in this organism.

Inactivation of the dnaK gene in Clostridium difficile 630 \u0394erm yields a temperature-sensitive phenotype and increases biofilm-forming ability

Clostridium difficile infection is a growing problem in healthcare settings worldwide and results in a considerable socioeconomic impact. New hypervirulent strains and acquisition of antibiotic resistance exacerbates pathogenesis; however, the survival strategy of C. difficile in the challenging gut environment still remains incompletely understood. We previously reported that clinically relevant heat-stress (37–41 °C) resulted in a classical heat-stress response with up-regulation of cellular chaperones. We used ClosTron to construct an insertional mutation in the dnaK gene of C. difficile 630 Δerm. The dnaK mutant exhibited temperature sensitivity, grew more slowly than C. difficile 630 Δerm and was less thermotolerant. Furthermore, the mutant was non-motile, had 4-fold lower expression of the fliC gene and lacked flagella on the cell surface. Mutant cells were some 50% longer than parental strain cells, and at optimal growth temperatures, they exhibited a 4-fold increase in the expression of class I chaperone genes including GroEL and GroES. Increased chaperone expression, in addition to the non-flagellated phenotype of the mutant, may account for the increased biofilm formation observed. Overall, the phenotype resulting from dnaK disruption is more akin to that observed in Escherichia coli dnaK mutants, rather than those in the Gram-positive model organism Bacillus subtilis.

Functional Diversity of AAA+ Protease Complexes in Bacillus subtilis.

Here, we review the diverse roles and functions of AAA+ protease complexes in protein homeostasis, control of stress response and cellular development pathways by regulatory and general proteolysis in the Gram-positive model organism Bacillus subtilis. We discuss in detail the intricate involvement of AAA+ protein complexes in controlling sporulation, the heat shock response and the role of adaptor proteins in these processes. The investigation of these protein complexes and their adaptor proteins has revealed their relevance for Gram-positive pathogens and their potential as targets for new antibiotics.

New Functions and Subcellular Localization Patterns of c-di-GMP Components (GGDEF Domain Proteins) in B. subtilis.

The universal and pleiotropic cyclic dinucleotide second messenger c-di-GMP is most prominently known to inversely regulate planktonic and sessile lifestyles of Gram-negative species. In the Gram-positive model organism Bacillus subtilis, intracellular c-di-GMP levels are modulated by a concise set of three diguanylate cylases (DgcK, DgcP, DgcW) and one phosphodiesterase (PdeH). Two recent studies have reported the negative influence of the c-di-GMP receptor DgrA (PilZ domain protein) on swarming motility indicating a conserved role of this second messenger across the bacterial domain. However, it has been suggested that the degenerated GGDEF protein YdaK and the inactive EAL domain protein YkuI may also function as c-di-GMP receptors regulating potentially other processes than motility. Here we describe a novel c-di-GMP dependent signaling network in B. subtilis regulating the production of an unknown exopolysaccharide (EPS) that leads to strongly altered colony morphologies upon overproduction. The network consists of the c-di-GMP receptor YdaK and the c-di-GMP synthetase DgcK. Both proteins establish a spatially close signal-effector cluster at the membrane. The cytoplasmic DgcP synthetase can complement for DgcK only upon overproduction, while the third c-di-GMP synthetase, DgcW, of B. subtilis is not part of the signaling pathway. Removal of the regulatory EAL domain from DgcW reveals a distinct function in biofilm formation. Therefore, our study is compatible with the “local pool signaling” hypothesis, but shows that in case of the yda operon, this can easily be overcome by overproduction of non-cognate DGCs, indicating that global pools can also confer signals to regulatory circuits in a Gram-positive bacterium.

Pentapeptide-rich peptidoglycan at the Bacillus subtilis cell-division site.

Peptidoglycan (PG), the major component of the bacterial cell wall, is one large macromolecule. To allow for the different curvatures of PG at cell poles and division sites, there must be local differences in PG architecture and eventually also chemistry. Here we report such local differences in the Gram-positive rod-shaped model organism Bacillus subtilis. Single-cell analysis after antibiotic treatment and labeling of the cell wall with a fluorescent analogue of vancomycin or the fluorescent D-amino acid analogue (FDAA) HCC-amino-D-alanine revealed that PG at the septum contains muropeptides with unprocessed stem peptides (pentapeptides). Whereas these pentapeptides are normally shortened after incorporation into PG, this activity is reduced at division sites indicating either a lower local degree of PG crosslinking or a difference in PG composition, which could be a topological marker for other proteins. The pentapeptides remain partially unprocessed after division when they form the new pole of a cell. The accumulation of unprocessed PG at the division site is not caused by the activity of the cell division specific penicillin-binding protein 2B. To our knowledge, this is the first indication of local differences in the chemical composition of PG in Gram-positive bacteria.