From Modules to Networks: a Systems-Level Analysis of the Bacitracin Stress Response in Bacillus subtilis.

Bacterial resistance against antibiotics often involves multiple mechanisms that are interconnected to ensure robust protection. So far, the knowledge about underlying regulatory features of those resistance networks is sparse, since they can hardly be determined by experimentation alone. Here, we present the first computational approach to elucidate the interplay between multiple resistance modules against a single antibiotic and how regulatory network structure allows the cell to respond to and compensate for perturbations of resistance. Based on the response of Bacillus subtilis toward the cell wall synthesis-inhibiting antibiotic bacitracin, we developed a mathematical model that comprehensively describes the protective effect of two well-studied resistance modules (BceAB and BcrC) on the progression of the lipid II cycle. By integrating experimental measurements of expression levels, the model accurately predicts the efficacy of bacitracin against the B. subtilis wild type as well as mutant strains lacking one or both of the resistance modules. Our study reveals that bacitracin-induced changes in the properties of the lipid II cycle itself control the interplay between the two resistance modules. In particular, variations in the concentrations of UPP, the lipid II cycle intermediate that is targeted by bacitracin, connect the effect of the BceAB transporter and the homeostatic response via BcrC to an overall resistance response. We propose that monitoring changes in pathway properties caused by a stressor allows the cell to fine-tune deployment of multiple resistance systems and may serve as a cost-beneficial strategy to control the overall response toward this stressor.IMPORTANCE Antibiotic resistance poses a major threat to global health, and systematic studies to understand the underlying resistance mechanisms are urgently needed. Although significant progress has been made in deciphering the mechanistic basis of individual resistance determinants, many bacterial species rely on the induction of a whole battery of resistance modules, and the complex regulatory networks controlling these modules in response to antibiotic stress are often poorly understood. In this work we combined experiments and theoretical modeling to decipher the resistance network of Bacillus subtilis against bacitracin, which inhibits cell wall biosynthesis in Gram-positive bacteria. We found a high level of cross-regulation between the two major resistance modules in response to bacitracin stress and quantified their effects on bacterial resistance. To rationalize our experimental data, we expanded a previously established computational model for the lipid II cycle through incorporating the quantitative action of the resistance modules. This led us to a systems-level description of the bacitracin stress response network that captures the complex interplay between resistance modules and the essential lipid II cycle of cell wall biosynthesis and accurately predicts the minimal inhibitory bacitracin concentration in all the studied mutants. With this, our study highlights how bacterial resistance emerges from an interlaced network of redundant homeostasis and stress response modules.

Publisher Correction: Bacillus SEVA siblings: A Golden Gate-based toolbox to create personalized integrative vectors for Bacillus subtilis.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Publisher Correction: Bacillus SEVA siblings: A Golden Gate-based toolbox to create personalized integrative vectors for Bacillus subtilis.

A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.

Sporobeads: The Utilization of the Bacillus subtilis Endospore Crust as a Protein Display Platform.

Upon starvation, the soil bacterium Bacillus subtilis forms an intracellular, metabolically inactive endospore. Its core contains the DNA, encased by three protein layers protecting it against a multitude of environmental threats. The outermost layer, the crust, harbors great potential as a protein-displaying platform: a gene of interest can be translationally fused to a crust protein gene, resulting in endospores displaying the desired protein on their surface. To unlock this potential in a standardized fashion, we designed a suite of 12 vectors (Sporovectors), based on the BioBrick cloning standard. With these vectors, proteins can easily be fused N- or C-terminally to the six crust proteins CotV, CotW, CotX, CotY, CotZ, and CgeA under the control of the strongest crust gene promoter PcotYZ. All Sporovectors were evaluated with GFP and two different laccases. On the basis of our data, CotY and CotZ represent the best anchor proteins. But there are significant differences in activity and functional stability between the two tested laccases. Our vector suite is a powerful tool to generate and evaluate a vast variety of functionalized endospores. It allows quickly identifying the best anchor and fusion site for the protein of interest. Our findings demonstrate that the crust of B. subtilis endospores is an inexpensive and easy platform for displaying different proteins of interest.

The Essential UPP Phosphatase Pair BcrC and UppP Connects Cell Wall Homeostasis during Growth and Sporulation with Cell Envelope Stress Response in Bacillus subtilis.

The bacterial cell wall separates the cell from its surrounding and protects it from environmental stressors. Its integrity is maintained by a highly regulated process of cell wall biosynthesis. The membrane-located lipid II cycle provides cell wall building blocks that are assembled inside the cytoplasm to the outside for incorporation. Its carrier molecule, undecaprenyl phosphate (UP), is then recycled by dephosphorylation from undecaprenyl pyrophosphate (UPP). In Bacillus subtilis, this indispensable reaction is catalyzed by the UPP phosphatases BcrC and UppP. Here, we study the physiological function of both phosphatases with respect to morphology, cell wall homeostasis and the resulting cell envelope stress response (CESR). We demonstrate that uppP and bcrC represent a synthetic lethal gene pair, which encodes an essential physiological function. Accordingly, cell growth and morphology were severely impaired during exponential growth if the overall UPP phosphatase level was limiting. UppP, but not BcrC, was crucial for normal sporulation. Expression of bcrC, but not uppP, was upregulated in the presence of cell envelope stress conditions caused by bacitracin if UPP phosphatase levels were limited. This homeostatic feedback renders BcrC more important during growth than UppP, particularly in defense against cell envelope stress.

The Bacillus BioBrick Box 2.0: expanding the genetic toolbox for the standardized work with Bacillus subtilis.

Standardized and well-characterized genetic building blocks allow the convenient assembly of novel genetic modules and devices, ensuring reusability of parts and reproducibility of experiments. In the first Bacillus subtilis-specific toolbox using the BioBrick standard, we presented integrative vectors, promoters, reporter genes and epitope tags for this Gram-positive model bacterium. With the Bacillus BioBrick Box 2.0, we significantly expand the range of our toolbox by providing new integrative vectors, introducing novel tools for fine-tuning protein expression, and carefully evaluating codon-adapted fluorescence proteins in B. subtilis, which cover the whole spectrum of visible light. Moreover, we developed new reporter systems to allow evaluating the strength of promoters and ribosome binding sites. This well-evaluated extension of our BioBrick-based toolbox increases the accessibility of B. subtilis and will therefore promote the use of this model bacterium and biotechnological workhorse as a host for fundamental and applied Synthetic Biology projects.

Bacillus SEVA siblings: A Golden Gate-based toolbox to create personalized integrative vectors for Bacillus subtilis.

Bacillus subtilis combines natural competence for genetic transformation with highly efficient homologous recombination. These features allow using vectors that integrate into the genome via double homologous recombination. So far, their utilization is restricted by the fixed combination of resistance markers and integration loci, as well as species- or strain-specific regions of homology. To overcome these limitations, we developed a toolbox for the creation of personalized Bacillus vectors in a standardized manner with a focus on fast and easy adaptation of the sequences specifying the integration loci. We based our vector toolkit on the Standard European Vector Architecture (SEVA) to allow the usage of their vector parts. The Bacillus SEVA siblings are assembled via efficient one-pot Golden Gate reactions from four entry parts with the choice of four different enzymes. The toolbox contains seven Bacillus resistance markers, two Escherichia coli origins of replication, and a free choice of integration loci. Vectors can be customized with a cargo, before or after vector assembly, and could be used in different B. subtilis strains and potentially beyond. Our adaptation of the SEVA-standard provides a powerful and standardized toolkit for the convenient creation of personalized Bacillus vectors.

The cell envelope stress response of Bacillus subtilis: from static signaling devices to dynamic regulatory network.

The cell envelope stress response (CESR) encompasses all regulatory events that enable a cell to protect the integrity of its envelope, an essential structure of any bacterial cell. The underlying signaling network is particularly well understood in the Gram-positive model organism Bacillus subtilis. It consists of a number of two-component systems (2CS) and extracytoplasmic function σ factors that together regulate the production of both specific resistance determinants and general mechanisms to protect the envelope against antimicrobial peptides targeting the biogenesis of the cell wall. Here, we summarize the current picture of the B. subtilis CESR network, from the initial identification of the corresponding signaling devices to unraveling their interdependence and the underlying regulatory hierarchy within the network. In the course of detailed mechanistic studies, a number of novel signaling features could be described for the 2CSs involved in mediating CESR. This includes a novel class of so-called intramembrane-sensing histidine kinases (IM-HKs), which-instead of acting as stress sensors themselves-are activated via interprotein signal transfer. Some of these IM-HKs are involved in sensing the flux of antibiotic resistance transporters, a unique mechanism of responding to extracellular antibiotic challenge.

Anatomy of the bacitracin resistance network in Bacillus subtilis.

Protection against antimicrobial peptides (AMPs) often involves the parallel production of multiple, well-characterized resistance determinants. So far, little is known about how these resistance modules interact and how they jointly protect the cell. Here, we studied the interdependence between different layers of the envelope stress response of Bacillus subtilis when challenged with the lipid II cycle-inhibiting AMP bacitracin. The underlying regulatory network orchestrates the production of the ABC transporter BceAB, the UPP phosphatase BcrC and the phage-shock proteins LiaIH. Our systems-level analysis reveals a clear hierarchy, allowing us to discriminate between primary (BceAB) and secondary (BcrC and LiaIH) layers of bacitracin resistance. Deleting the primary layer provokes an enhanced induction of the secondary layer to partially compensate for this loss. This study reveals a direct role of LiaIH in bacitracin resistance, provides novel insights into the feedback regulation of the Lia system, and demonstrates a pivotal role of BcrC in maintaining cell wall homeostasis. The compensatory regulation within the bacitracin network can also explain how gene expression noise propagates between resistance layers. We suggest that this active redundancy in the bacitracin resistance network of B. subtilis is a general principle to be found in many bacterial antibiotic resistance networks.

A New Way of Sensing: Need-Based Activation of Antibiotic Resistance by a Flux-Sensing Mechanism.

UNLABELLED: Sensing of and responding to environmental changes are of vital importance for microbial cells. Consequently, bacteria have evolved a plethora of signaling systems that usually sense biochemical cues either via direct ligand binding, thereby acting as "concentration sensors," or by responding to downstream effects on bacterial physiology, such as structural damage to the cell. Here, we describe a novel, alternative signaling mechanism that effectively implements a "flux sensor" to regulate antibiotic resistance. It relies on a sensory complex consisting of a histidine kinase and an ABC transporter, in which the transporter fulfills the dual role of both the sensor of the antibiotic and the mediator of resistance against it. Combining systems biological modeling with in vivo experimentation, we show that these systems in fact respond to changes in activity of individual resistance transporters rather than to changes in the antibiotic concentration. Our model shows that the cell thereby adjusts the rate of de novo transporter synthesis to precisely the level needed for protection. Such a flux-sensing mechanism may serve as a cost-efficient produce-to-demand strategy, controlling a widely conserved class of antibiotic resistance systems. IMPORTANCE: Bacteria have to be able to accurately perceive their environment to allow adaptation to changing conditions. This is usually accomplished by sensing the concentrations of beneficial or harmful substances or by measuring the effect of the prevailing conditions on the cell. Here we show the existence of a new way of sensing the environment, where the bacteria monitor the activity of an antibiotic resistance transporter. Such a "flux-sensing" mechanism allows the cell to detect its current capacity to deal with the antibiotic challenge and thus precisely respond to the need for more transporters. We propose that this is a cost-efficient way of regulating antibiotic resistance on demand.

The Bacillus BioBrick Box: generation and evaluation of essential genetic building blocks for standardized work with Bacillus subtilis.

BACKGROUND: Standardized and well-characterized genetic building blocks are a prerequisite for the convenient and reproducible assembly of novel genetic modules and devices. While numerous standardized parts exist for Escherichia coli, such tools are still missing for the Gram-positive model organism Bacillus subtilis. The goal of this study was to develop and thoroughly evaluate such a genetic toolbox. RESULTS: We developed five BioBrick-compatible integrative B. subtilis vectors by deleting unnecessary parts and removing forbidden restriction sites to allow cloning in BioBrick (RFC10) standard. Three empty backbone vectors with compatible resistance markers and integration sites were generated, allowing the stable chromosomal integration and combination of up to three different devices in one strain. In addition, two integrative reporter vectors, based on the lacZ and luxABCDE cassettes, were BioBrick-adjusted, to enable ß-galactosidase and luciferase reporter assays, respectively. Four constitutive and two inducible promoters were thoroughly characterized by quantitative, time-resolved measurements. Together, these promoters cover a range of more than three orders of magnitude in promoter strength, thereby allowing a fine-tuned adjustment of cellular protein amounts. Finally, the Bacillus BioBrick Box also provides five widely used epitope tags (FLAG, His10, cMyc, HA, StrepII), which can be translationally fused N- or C-terminally to any protein of choice. CONCLUSION: Our genetic toolbox contains three compatible empty integration vectors, two reporter vectors and a set of six promoters, two of them inducible. Furthermore, five different epitope tags offer convenient protein handling and detection. All parts adhere to the BioBrick standard and hence enable standardized work with B. subtilis. We believe that our well-documented and carefully evaluated Bacillus BioBrick Box represents a very useful genetic tool kit, not only for the iGEM competition but any other BioBrick-based project in B. subtilis.

Breaking the DNA-binding code of Ralstonia solanacearum TAL effectors provides new possibilities to generate plant resistance genes against bacterial wilt disease.

Ralstonia solanacearum is a devastating bacterial phytopathogen with a broad host range. Ralstonia solanacearum injected effector proteins (Rips) are key to the successful invasion of host plants. We have characterized Brg11(hrpB-regulated 11), the first identified member of a class of Rips with high sequence similarity to the transcription activator-like (TAL) effectors of Xanthomonas spp., collectively termed RipTALs. Fluorescence microscopy of in planta expressed RipTALs showed nuclear localization. Domain swaps between Brg11 and Xanthomonas TAL effector (TALE) AvrBs3 (avirulence protein triggering Bs3 resistance) showed the functional interchangeability of DNA-binding and transcriptional activation domains. PCR was used to determine the sequence of brg11 homologs from strains infecting phylogenetically diverse host plants. Brg11 localizes to the nucleus and activates promoters containing a matching effector-binding element (EBE). Brg11 and homologs preferentially activate promoters containing EBEs with a 5' terminal guanine, contrasting with the TALE preference for a 5' thymine. Brg11 and other RipTALs probably promote disease through the transcriptional activation of host genes. Brg11 and the majority of homologs identified in this study were shown to activate similar or identical target sequences, in contrast to TALEs, which generally show highly diverse target preferences. This information provides new options for the engineering of plants resistant to R. solanacearum.

Ca(2+)-binding reduces conformational flexibility of RC-LH1 core complex from thermophile Thermochromatium tepidum.

The light-harvesting complex, LH1, of thermophile purple bacteria Thermochromatium tepidum consists of an array of α- and ß-polypeptides which assemble the photoactive bacteriochlorophyll and closely interact with the membrane-lipids. In this study, we investigated the effect of calcium and manganese ions on the protein structure and thermostability of the reaction centre (RC)-LH1/lipid complex. The binding of Ca(2+), but not Mn(2+) is shown to shift the LH1 Q ( y ) absorption maximum from ~889 to 915 nm and to significantly raise the thermostability of the RC-LH1 complex. The ATR-FTIR spectra indicate that interaction of Ca(2+) as monitored by the carboxylates' vibration of aspartate residues, but not Mn(2+) induces changes in the α-helix packing arrangement. The reduced rate of (1)H/(2)H exchange of proteins' amide protons shows that the accessibility to (2)H(2)O is significantly lowered in Ca(2+)-substituted RC-LH1/lipid complexes. In particular, exchange with the associated lipid molecules, is significantly retarded. These results suggest that the thermostability of the RC-LH1 complex is raised by the distinct interaction with calcium cations which reduces the RC-LH1/lipid dynamics, particularly, at the membrane-water interface.