c-di-AMP determines the hierarchical organization of bacterial RCK proteins.

In bacteria, intracellular K+ is involved in the regulation of membrane potential, cytosolic pH, and cell turgor as well as in spore germination, environmental adaptation, cell-to-cell communication in biofilms, antibiotic sensitivity, and infectivity. The second messenger cyclic-di-AMP (c-di-AMP) has a central role in modulating the intracellular K+ concentration in many bacterial species, controlling transcription and function of K+ channels and transporters. However, our understanding of how this regulatory network responds to c-di-AMP remains poor. We used the RCK (Regulator of Conductance of K+) proteins that control the activity of Ktr channels in Bacillus subtilis as a model system to analyze the regulatory function of c-di-AMP with a combination of in vivo and in vitro functional and structural characterization. We determined that the two RCK proteins (KtrA and KtrC) are neither physiologically redundant or functionally equivalent. KtrC is the physiologically dominant RCK protein in the regulation of Ktr channel activity. In explaining this hierarchical organization, we found that, unlike KtrA, KtrC is very sensitive to c-di-AMP inactivation and lack of c-di-AMP regulation results in RCK protein toxicity, most likely due to unregulated K+ flux. We also found that KtrC can assemble with KtrA, conferring c-di-AMP regulation to the functional KtrA/KtrC heteromers and potentially compensating KtrA toxicity. Altogether, we propose that the central role of c-di-AMP in the control of the K+ machinery, by modulating protein levels through gene transcription and by regulating protein activity, has determined the evolutionary selection of KtrC as the dominant RCK protein, shaping the hierarchical organization of regulatory components of the K+ machinery.

The previously uncharacterized RnpM (YlxR) protein modulates the activity of ribonuclease P in Bacillus subtilis in vitro.

Even though Bacillus subtilis is one of the most studied organisms, no function has been identified for about 20% of its proteins. Among these unknown proteins are several RNA- and ribosome-binding proteins suggesting that they exert functions in cellular information processing. In this work, we have investigated the RNA-binding protein YlxR. This protein is widely conserved in bacteria and strongly constitutively expressed in B. subtilis suggesting an important function. We have identified the RNA subunit of the essential RNase P as the binding partner of YlxR. The main activity of RNase P is the processing of 5' ends of pre-tRNAs. In vitro processing assays demonstrated that the presence of YlxR results in reduced RNase P activity. Chemical cross-linking studies followed by in silico docking analysis and experiments with site-directed mutant proteins suggest that YlxR binds to the region of the RNase P RNA that is important for binding and cleavage of the pre-tRNA substrate. We conclude that the YlxR protein is a novel interaction partner of the RNA subunit of RNase P that serves to finetune RNase P activity to ensure appropriate amounts of mature tRNAs for translation. We rename the YlxR protein RnpM for RNase P modulator.

Cyclic di-AMP Signaling in Bacteria.

The second messenger molecule cyclic di-AMP (c-di-AMP) is formed by many bacteria and archaea. In many species that produce c-di-AMP, this second messenger is essential for viability on rich medium. Recent research has demonstrated that c-di-AMP binds to a large number of proteins and riboswitches, which are often involved in potassium and osmotic homeostasis. c-di-AMP becomes dispensable if the bacteria are cultivated on minimal media with low concentrations of osmotically active compounds. Thus, the essentiality of c-di-AMP does not result from an interaction with a single essential target but rather from the multilevel control of complex homeostatic processes. This review summarizes current knowledge on the homeostasis of c-di-AMP and its function(s) in the control of cellular processes.

PAE viewer: a webserver for the interactive visualization of the predicted aligned error for multimer structure predictions and crosslinks.

The development of AlphaFold for protein structure prediction has opened a new era in structural biology. This is even more the case for AlphaFold-Multimer for the prediction of protein complexes. The interpretation of these predictions has become more important than ever, but it is difficult for the non-specialist. While an evaluation of the prediction quality is provided for monomeric protein predictions by the AlphaFold Protein Structure Database, such a tool is missing for predicted complex structures. Here, we present the PAE Viewer webserver (http://www.subtiwiki.uni-goettingen.de/v4/paeViewerDemo), an online tool for the integrated visualization of predicted protein complexes using a 3D structure display combined with an interactive representation of the Predicted Aligned Error (PAE). This metric allows an estimation of the quality of the prediction. Importantly, our webserver also allows the integration of experimental cross-linking data which helps to interpret the reliability of the structure predictions. With the PAE Viewer, the user obtains a unique online tool which for the first time allows the intuitive evaluation of the PAE for protein complex structure predictions with integrated crosslinks.

DarA-the central processing unit for the integration of osmotic with potassium and amino acid homeostasis in Bacillus subtilis.

Cyclic di-adenosine monophosphate (c-di-AMP) is a second messenger involved in diverse metabolic processes including osmolyte uptake, cell wall homeostasis, as well as antibiotic and heat resistance. This study investigates the role of the c-di-AMP receptor protein DarA in the osmotic stress response in Bacillus subtilis. Through a series of experiments, we demonstrate that DarA plays a central role in the cellular response to osmotic fluctuations. Our findings show that DarA becomes essential under extreme potassium limitation as well as upon salt stress, highlighting its significance in mediating osmotic stress adaptation. Suppressor screens with darA mutants reveal compensatory mechanisms involving the accumulation of osmoprotectants, particularly potassium and citrulline. Mutations affecting various metabolic pathways, including the citric acid cycle as well as glutamate and arginine biosynthesis, indicate a complex interplay between the osmotic stress response and metabolic regulation. In addition, the growth defects of the darA mutant during potassium starvation and salt stress in a strain lacking the high-affinity potassium uptake systems KimA and KtrAB can be rescued by increased affinity of the remaining potassium channel KtrCD or by increased expression of ktrD, thus resulting in increased potassium uptake. Finally, the darA mutant can respond to salt stress by the increased expression of MleN , which can export sodium ions.IMPORTANCEEnvironmental bacteria are exposed to rapidly changing osmotic conditions making an effective adaptation to these changes crucial for the survival of the cells. In Gram-positive bacteria, the second messenger cyclic di-AMP plays a key role in this adaptation by controlling (i) the influx of physiologically compatible organic osmolytes and (ii) the biosynthesis of such osmolytes. In several bacteria, cyclic di-adenosine monophosphate (c-di-AMP) can bind to a signal transduction protein, called DarA, in Bacillus subtilis. So far, no function for DarA has been discovered in any organism. We have identified osmotically challenging conditions that make DarA essential and have identified suppressor mutations that help the bacteria to adapt to those conditions. Our results indicate that DarA is a central component in the integration of osmotic stress with the synthesis of compatible amino acid osmolytes and with the homeostasis of potassium, the first response to osmotic stress.

Control of asparagine homeostasis in Bacillus subtilis: identification of promiscuous amino acid importers and exporters.

The Gram-positive model bacterium B. subtilis is able to import all proteinogenic amino acids from the environment as well as to synthesize them. However, the players involved in the acquisition of asparagine have not yet been identified for this bacterium. In this work, we used d-asparagine as a toxic analog of l-asparagine to identify asparagine transporters. This revealed that d- but not l-asparagine is taken up by the malate/lactate antiporter MleN. Specific strains that are sensitive to the presence of l-asparagine due to the lack of the second messenger cyclic di-AMP or due to the intracellular accumulation of this amino acid were used to isolate and characterize suppressor mutants that were resistant to the presence of otherwise growth-inhibiting concentrations of l-asparagine. These screens identified the broad-spectrum amino acid importers AimA and BcaP as responsible for the acquisition of l-asparagine. The amino acid exporter AzlCD allows detoxification of l-asparagine in addition to 4-azaleucine and histidine. This work supports the idea that amino acids are often transported by promiscuous importers and exporters. However, our work also shows that even stereo-enantiomeric amino acids do not necessarily use the same transport systems.IMPORTANCETransport of amino acid is a poorly studied function in many bacteria, including the model organism Bacillus subtilis. The identification of transporters is hampered by the redundancy of transport systems for most amino acids as well as by the poor specificity of the transporters. Here, we apply several strategies to use the growth-inhibitive effect of many amino acids under defined conditions to isolate suppressor mutants that exhibit either reduced uptake or enhanced export of asparagine, resulting in the identification of uptake and export systems for l-asparagine. The approaches used here may be useful for the identification of transporters for other amino acids both in B. subtilis and in other bacteria.

Ornithine is the central intermediate in the arginine degradative pathway and its regulation in Bacillus subtilis.

The Gram-positive bacterium Bacillus subtilis can utilize several proteinogenic and non-proteinogenic amino acids as sources of carbon, nitrogen, and energy. The utilization of the amino acids arginine, citrulline, and ornithine is catalyzed by enzymes encoded in the rocABC and rocDEF operons and by the rocG gene. The expression of these genes is controlled by the alternative sigma factor SigL. RNA polymerase associated with this sigma factor depends on ATP-hydrolyzing transcription activators to initiate transcription. The RocR protein acts as a transcription activator for the roc genes. However, the details of amino acid metabolism via this pathway are unknown. Here, we investigated the contributions of all enzymes of the Roc pathway to the degradation of arginine, citrulline, and ornithine. We identified the previously uncharacterized RocB protein as responsible for the conversion of citrulline to ornithine. In vitro assays with the purified enzyme suggest that RocB acts as a manganese-dependent N-carbamoyl-L-ornithine hydrolase that cleaves citrulline to form ornithine and carbamate. Moreover, the molecular effector that triggers transcription activation by RocR has not been unequivocally identified. Using a combination of transcription reporter assays and biochemical experiments, we demonstrate that ornithine is the molecular inducer of RocR activity. Taken together, our work suggests that binding of ATP to RocR triggers its hexamerization, and binding of ornithine then allows ATP hydrolysis and activation of roc gene transcription. Thus, ornithine is the central molecule of the roc degradative pathway as it is the common intermediate of arginine and citrulline degradation and the molecular effector of RocR.

Understudied proteins and understudied functions in the model bacterium Bacillus subtilis-A major challenge in current research.

Model organisms such as the Gram-positive bacterium Bacillus subtilis have been studied intensively for decades. However, even for model organisms, no function has been identified for about one fourth of all proteins. It has recently been realized that such understudied proteins as well as poorly studied functions set a limitation to our understanding of the requirements for cellular life, and the Understudied Proteins Initiative has been launched. Of poorly studied proteins, those that are strongly expressed are likely to be important to the cell and should therefore be considered high priority in further studies. Since the functional analysis of unknown proteins can be extremely laborious, a minimal knowledge is required prior to targeted functional studies. In this review, we discuss strategies to obtain such a minimal annotation, for example, from global interaction, expression, or localization studies. We present a set of 41 highly expressed and poorly studied proteins of B. subtilis. Several of these proteins are thought or known to bind RNA and/or the ribosome, some may control the metabolism of B. subtilis, and another subset of particularly small proteins may act as regulatory elements to control the expression of downstream genes. Moreover, we discuss the challenges of poorly studied functions with a focus on RNA-binding proteins, amino acid transport, and the control of metabolic homeostasis. The identification of the functions of the selected proteins not only will strongly advance our knowledge on B. subtilis, but also on other organisms since many of the proteins are conserved in many groups of bacteria.

Protein complexes in cells by AI-assisted structural proteomics.

Accurately modeling the structures of proteins and their complexes using artificial intelligence is revolutionizing molecular biology. Experimental data enable a candidate-based approach to systematically model novel protein assemblies. Here, we use a combination of in-cell crosslinking mass spectrometry and co-fractionation mass spectrometry (CoFrac-MS) to identify protein-protein interactions in the model Gram-positive bacterium Bacillus subtilis. We show that crosslinking interactions prior to cell lysis reveals protein interactions that are often lost upon cell lysis. We predict the structures of these protein interactions and others in the SubtiWiki database with AlphaFold-Multimer and, after controlling for the false-positive rate of the predictions, we propose novel structural models of 153 dimeric and 14 trimeric protein assemblies. Crosslinking MS data independently validates the AlphaFold predictions and scoring. We report and validate novel interactors of central cellular machineries that include the ribosome, RNA polymerase, and pyruvate dehydrogenase, assigning function to several uncharacterized proteins. Our approach uncovers protein-protein interactions inside intact cells, provides structural insight into their interaction interfaces, and is applicable to genetically intractable organisms, including pathogenic bacteria.

Exploring Synergistic Effects of Levan and Levan-Metabolizing Bacillaceae in Promoting Growth and Enhancing Immunity of Tomato and Wheat.

Boosting plant immunity by priming agents can lower agrochemical dependency in plant production. Levan and levan-derived oligosaccharides (LOS) act as priming agents against biotic stress in several crops. Additionally, beneficial microbes can promote plant growth and protect against fungal diseases. This study assessed possible synergistic effects caused by levan, LOS and five levan- and LOS-metabolizing Bacillaceae (Bacillus and Priestia) strains in tomato and wheat. Leaf and seed defense priming assays were conducted in non-soil (semi-sterile substrate) and soil-based systems, focusing on tomato-Botrytis cinerea and wheat-Magnaporthe oryzae Triticum (MoT) pathosystems. In the non-soil system, seed defense priming with levan, the strains (especially Bacillus velezensis GA1), or their combination significantly promoted tomato growth and protection against B. cinerea. While no growth stimulatory effects were observed for wheat, disease protective effects were also observed in the wheat-MoT pathosystem. When grown in soil and subjected to leaf defense priming, tomato plants co-applied with levan and the bacterial strains showed increased resistance to B. cinerea compared with plants treated with levan or single strains, and these effects were synergistic in some cases. For seed defense priming in soil, more synergistic effects on disease tolerance were observed in a non-fertilized soil as compared to a fertilized soil, suggesting that potential prebiotic effects of levan are more prominent in poor soils. The potential of using combinations of Bacilliaceae and levan in sustainable agriculture is discussed.

The current state of SubtiWiki, the database for the model organism Bacillus subtilis.

Bacillus subtilis is a Gram-positive model bacterium with extensive documented annotation. However, with the rise of high-throughput techniques, the amount of complex data being generated every year has been increasing at a fast pace. Thus, having platforms ready to integrate and give a representation to these data becomes a priority. To address it, SubtiWiki (http://subtiwiki.uni-goettingen.de/) was created in 2008 and has been growing in data and viewership ever since. With millions of requests every year, it is the most visited B. subtilis database, providing scientists all over the world with curated information about its genes and proteins, as well as intricate protein-protein interactions, regulatory elements, expression data and metabolic pathways. However, there is still a large portion of annotation to be unveiled for some biological elements. Thus, to facilitate the development of new hypotheses for research, we have added a Homology section covering potential protein homologs in other organisms. Here, we present the recent developments of SubtiWiki and give a guided tour of our database and the current state of the data for this organism.

Essentiality of c-di-AMP in Bacillus subtilis: Bypassing mutations converge in potassium and glutamate homeostasis.

In order to adjust to changing environmental conditions, bacteria use nucleotide second messengers to transduce external signals and translate them into a specific cellular response. Cyclic di-adenosine monophosphate (c-di-AMP) is the only known essential nucleotide second messenger. In addition to the well-established role of this second messenger in the control of potassium homeostasis, we observed that glutamate is as toxic as potassium for a c-di-AMP-free strain of the Gram-positive model bacterium Bacillus subtilis. In this work, we isolated suppressor mutants that allow growth of a c-di-AMP-free strain under these toxic conditions. Characterization of glutamate resistant suppressors revealed that they contain pairs of mutations, in most cases affecting glutamate and potassium homeostasis. Among these mutations, several independent mutations affected a novel glutamate transporter, AimA (Amino acid importer A, formerly YbeC). This protein is the major transporter for glutamate and serine in B. subtilis. Unexpectedly, some of the isolated suppressor mutants could suppress glutamate toxicity by a combination of mutations that affect phospholipid biosynthesis and a specific gain-of-function mutation of a mechanosensitive channel of small conductance (YfkC) resulting in the acquisition of a device for glutamate export. Cultivation of the c-di-AMP-free strain on complex medium was an even greater challenge because the amounts of potassium, glutamate, and other osmolytes are substantially higher than in minimal medium. Suppressor mutants viable on complex medium could only be isolated under anaerobic conditions if one of the two c-di-AMP receptor proteins, DarA or DarB, was absent. Also on complex medium, potassium and osmolyte toxicity are the major bottlenecks for the growth of B. subtilis in the absence of c-di-AMP. Our results indicate that the essentiality of c-di-AMP in B. subtilis is caused by the global impact of the second messenger nucleotide on different aspects of cellular physiology.

Bacillus subtilis, a Swiss Army Knife in Science and Biotechnology.

Next to Escherichia coli, Bacillus subtilis is the most studied and best understood organism that also serves as a model for many important pathogens. Due to its ability to form heat-resistant spores that can germinate even after very long periods of time, B. subtilis has attracted much scientific interest. Another feature of B. subtilis is its genetic competence, a developmental state in which B. subtilis actively takes up exogenous DNA. This makes B. subtilis amenable to genetic manipulation and investigation. The bacterium was one of the first with a fully sequenced genome, and it has been subject to a wide variety of genome- and proteome-wide studies that give important insights into many aspects of the biology of B. subtilis. Due to its ability to secrete large amounts of proteins and to produce a wide range of commercially interesting compounds, B. subtilis has become a major workhorse in biotechnology. Here, we review the development of important aspects of the research on B. subtilis with a specific focus on its cell biology and biotechnological and practical applications from vitamin production to concrete healing. The intriguing complexity of the developmental programs of B. subtilis, paired with the availability of sophisticated tools for genetic manipulation, positions it at the leading edge for discovering new biological concepts and deepening our understanding of the organization of bacterial cells.

Structural basis for c-di-AMP-dependent regulation of the bacterial stringent response by receptor protein DarB.

The bacterial second messenger c-di-AMP controls essential cellular processes, including potassium and osmolyte homeostasis. This makes synthesizing enzymes and components involved in c-di-AMP signal transduction intriguing as potential targets for drug development. The c-di-AMP receptor protein DarB of Bacillus subtilis binds the Rel protein and triggers the Rel-dependent stringent response to stress conditions; however, the structural basis for this trigger is unclear. Here, we report crystal structures of DarB in the ligand-free state and of DarB complexed with c-di-AMP, 3'3'-cGAMP, and AMP. We show that DarB forms a homodimer with a parallel, head-to-head assembly of the monomers. We also confirm the DarB dimer binds two cyclic dinucleotide molecules or two AMP molecules; only one adenine of bound c-di-AMP is specifically recognized by DarB, while the second protrudes out of the donut-shaped protein. This enables DarB to bind also 3'3'-cGAMP, as only the adenine fits in the active site. In absence of c-di-AMP, DarB binds to Rel and stimulates (p)ppGpp synthesis, whereas the presence of c-di-AMP abolishes this interaction. Furthermore, the DarB crystal structures reveal no conformational changes upon c-di-AMP binding, leading us to conclude the regulatory function of DarB on Rel must be controlled directly by the bound c-di-AMP. We thus derived a structural model of the DarB-Rel complex via in silico docking, which was validated with mass spectrometric analysis of the chemically crosslinked DarB-Rel complex and mutagenesis studies. We suggest, based on the predicted complex structure, a mechanism of stringent response regulation by c-di-AMP.

ListiWiki: A database for the foodborne pathogen Listeria monocytogenes.

Listeria monocytogenes is a Gram positive foodborne pathogen that regularly causes outbreaks of systemic infectious diseases. The bacterium maintains a facultative intracellular lifestyle; it thrives under a variety of environmental conditions and is able to infect human host cells. L. monocytogenes is genetically tractable and therefore has become an attractive model system to study the mechanisms employed by facultative intracellular bacteria to invade eukaryotic cells and to replicate in their cytoplasm. Besides its importance for basic research, L. monocytogenes also serves as a paradigmatic pathogen in genomic epidemiology, where the relative stability of its genome facilitates successful outbreak detection and elucidation of transmission chains in genomic pathogen surveillance systems. In both terms, it is necessary to keep the annotation of the L. monocytogenes genome up to date. Therefore, we have created the database ListiWiki (http://listiwiki.uni-goettingen.de/) which stores comprehensive information on the widely used L. monocytogenes reference strain EDG-e. ListiWiki is designed to collect information on genes, proteins and RNAs and their relevant functional characteristics, but also further information such as mutant phenotypes, available biological material, and publications. In its present form, ListiWiki combines the most recent annotation of the EDG-e genome with published data on gene essentiality, gene expression and subcellular protein localization. ListiWiki also predicts protein-protein interactions networks based on protein homology to Bacillus subtilis proteins, for which detailed interaction maps have been compiled in the sibling database SubtiWiki. Furthermore, crystallographic information of proteins is made accessible through integration of Protein Structure Database codes and AlphaFold structure predictions. ListiWiki is an easy-to-use web interface that has been developed with a focus on an intuitive access to all information. Use of ListiWiki is free of charge and its content can be edited by all members of the scientific community after registration. In our labs, ListiWiki has already become an important and easy to use tool to quickly access genome annotation details that we can keep updated with advancing knowledge. It also might be useful to promote the comprehensive understanding of the physiology and virulence of an important human pathogen.

Quasi-essentiality of RNase Y in Bacillus subtilis is caused by its critical role in the control of mRNA homeostasis.

RNA turnover is essential in all domains of life. The endonuclease RNase Y (rny) is one of the key components involved in RNA metabolism of the model organism Bacillus subtilis. Essentiality of RNase Y has been a matter of discussion, since deletion of the rny gene is possible, but leads to severe phenotypic effects. In this work, we demonstrate that the rny mutant strain rapidly evolves suppressor mutations to at least partially alleviate these defects. All suppressor mutants had acquired a duplication of an about 60 kb long genomic region encompassing genes for all three core subunits of the RNA polymerase-α, ß, ß'. When the duplication of the RNA polymerase genes was prevented by relocation of the rpoA gene in the B. subtilis genome, all suppressor mutants carried distinct single point mutations in evolutionary conserved regions of genes coding either for the ß or ß' subunits of the RNA polymerase that were not tolerated by wild type bacteria. In vitro transcription assays with the mutated polymerase variants showed a severe decrease in transcription efficiency. Altogether, our results suggest a tight cooperation between RNase Y and the RNA polymerase to establish an optimal RNA homeostasis in B. subtilis cells.

How To Deal with Toxic Amino Acids: the Bipartite AzlCD Complex Exports Histidine in Bacillus subtilis.

The Gram-positive model bacterium Bacillus subtilis can use several amino acids as sources of carbon and nitrogen. However, some amino acids inhibit the growth of this bacterium. This amino acid toxicity is often enhanced in strains lacking the second messenger cyclic dimeric adenosine 3',5'-monophosphate (c-di-AMP). We observed that the presence of histidine is also toxic for a B. subtilis strain that lacks all three c-di-AMP synthesizing enzymes. However, suppressor mutants emerged, and whole-genome sequencing revealed mutations in the azlB gene that encode the repressor of the azl operon. This operon encodes an exporter and an importer for branched-chain amino acids. The suppressor mutations result in an overexpression of the azl operon. Deletion of the azlCD genes encoding the branched-chain amino acid exporter restored the toxicity of histidine, indicating that this exporter is required for histidine export and for resistance to otherwise toxic levels of the amino acid. The higher abundance of the amino acid exporter AzlCD increased the extracellular concentration of histidine, thus confirming the new function of AzlCD as a histidine exporter. Unexpectedly, the AzlB-mediated repression of the operon remains active even in the presence of amino acids, suggesting that the expression of the azl operon requires the mutational inactivation of AzlB. IMPORTANCE Amino acids are building blocks for protein biosynthesis in each living cell. However, due to their reactivity and the similarity between several amino acids, they may also be involved in harmful reactions or in noncognate interactions and thus may be toxic. Bacillus subtilis can deal with otherwise toxic histidine by overexpressing the bipartite amino acid exporter AzlCD. Although encoded in an operon that also contains a gene for an amino acid importer, the corresponding genes are not expressed, irrespective of the availability of amino acids in the medium. This suggests that the azl operon is a last resort by which to deal with histidine stress that can be expressed due to the mutational inactivation of the cognate repressor AzlB.

Unchaining miniBacillus Strain PG10: Relief of FlgM-Mediated Repression of Autolysin Genes.

Cell chaining in Bacillus subtilis is naturally observed in a subset of cells during exponential growth and during biofilm formation. However, the recently constructed large-scale genome-minimized B. subtilis strain PG10 displays a severe and permanent defect in cell separation, as it exclusively grows in the form of long filaments of nonseparated cells. In this study, we investigated the underlying mechanisms responsible for the incomplete cell division of PG10 by genomic and transcriptomic analyses. Repression of the SigD regulon, including the major autolysin gene lytF, was identified as the cause for the cell separation problem of PG10. It appeared that SigD-regulated genes are downregulated in PG10 due to the absence of the flagellar export apparatus, which normally is responsible for secretion of FlgM, the anti-sigma factor of SigD. Although mild negative effects on growth and cell morphology were observed, deletion of flgM could revert the aberrant cell-chaining phenotype and increased transformation efficiency. Interestingly, our work also demonstrates the occurrence of increased antisense transcription of slrR, a transcriptional repressor of autolysin genes, in PG10 and provides further understanding for this observation. In addition to revealing the molecular basis of the cell separation defect in PG10, our work provides novel targets for subsequent genome reduction efforts and future directions for further optimization of miniBacillus PG10. IMPORTANCE Reduction of the size of bacterial genomes is relevant for understanding the minimal requirements for cellular life as well as from a biotechnological point of view. Although the genome-minimized Bacillus subtilis strain PG10 displays several beneficial traits as a microbial cell factory compared to its parental strain, a defect at the final stage of cell division was introduced during the genome reduction process. By genetic and transcriptomic analyses, we identified the underlying reasons for the cell separation problem of PG10. In addition to enabling PG10 to grow in a way similar to that of B. subtilis wild-type strains, our work points toward subsequent targets for fine-tuning and further reduction of the genome of PG10. Moreover, solving the cell separation defect facilitates laboratory handling of PG10 by increasing the transformation efficiency, among other means. Overall, our work contributes to understanding and improving biotechnologically attractive minimal bacterial cell factories.

Topoisomerase IV can functionally replace all type 1A topoisomerases in Bacillus subtilis.

DNA topoisomerases play essential roles in chromosome organization and replication. Most bacteria possess multiple topoisomerases which have specialized functions in the control of DNA supercoiling or in DNA catenation/decatenation during recombination and chromosome segregation. DNA topoisomerase I is required for the relaxation of negatively supercoiled DNA behind the transcribing RNA polymerase. Conflicting results have been reported on the essentiality of the topA gene encoding topoisomerase I in the model bacterium Bacillus subtilis. In this work, we have studied the requirement for topoisomerase I in B. subtilis. All stable topA mutants carried different chromosomal amplifications of the genomic region encompassing the parEC operon encoding topoisomerase IV. Using a fluorescent amplification reporter system we observed that each individual topA mutant had acquired such an amplification. Eventually, the amplifications were replaced by a point mutation in the parEC promoter region which resulted in a fivefold increase of parEC expression. In this strain both type I topoisomerases, encoded by topA and topB, were dispensable. Our results demonstrate that topoisomerase IV at increased expression is necessary and sufficient to take over the function of type 1A topoisomerases.

Two Ways To Convert a Low-Affinity Potassium Channel to High Affinity: Control of Bacillus subtilis KtrCD by Glutamate.

Potassium and glutamate are the major cation and anion, respectively, in every living cell. Due to the high concentrations of both ions, the cytoplasm of all cells can be regarded as a potassium glutamate solution. This implies that the concentrations of both ions need to be balanced. While the control of potassium uptake by glutamate is well established for eukaryotic cells, much less is known about the mechanisms that link potassium homeostasis to glutamate availability in bacteria. Here, we have discovered that the availability of glutamate strongly decreases the minimal external potassium concentration required for the highly abundant Bacillus subtilis potassium channel KtrCD to accumulate potassium. In contrast, the inducible KtrAB and KimA potassium uptake systems have high apparent affinities for potassium even in the absence of glutamate. Experiments with mutant strains revealed that the KtrD subunit responds to the presence of glutamate. For full activity, KtrD synergistically requires the presence of the regulatory subunit KtrC and of glutamate. The analysis of suppressor mutants of a strain that has KtrCD as the only potassium uptake system and that experiences severe potassium starvation identified a mutation in the ion selectivity filter of KtrD (Gly282 to Val) that similarly results in a strongly glutamate-independent increase of the apparent affinity for potassium. Thus, this work has identified two conditions that increase the apparent affinity of KtrCD for potassium, i.e., external glutamate and the acquisition of a single point mutation in KtrD.IMPORTANCE In each living cell, potassium is required for maintaining the intracellular pH and for the activity of essential enzymes. Like most other bacteria, Bacillus subtilis possesses multiple low- and high-affinity potassium uptake systems. Their activity is regulated by the second messenger cyclic di-AMP. Moreover, the pools of the most abundant ions potassium and glutamate must be balanced. We report two conditions under which the low-affinity potassium channel KtrCD is able to mediate potassium uptake at low external potassium concentrations: physiologically, the presence of glutamate results in a severely increased potassium uptake. Moreover, this is achieved by a mutation affecting the selectivity filter of the KtrD channel. These results highlight the integration between potassium and glutamate homeostasis in bacteria.