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.

Comparative genome and phenotypic analysis of three Clostridioides difficile strains isolated from a single patient provide insight into multiple infection of C. difficile.

BACKGROUND: Clostridioides difficile infections (CDI) have emerged over the past decade causing symptoms that range from mild, antibiotic-associated diarrhea (AAD) to life-threatening toxic megacolon. In this study, we describe a multiple and isochronal (mixed) CDI caused by the isolates DSM 27638, DSM 27639 and DSM 27640 that already initially showed different morphotypes on solid media. RESULTS: The three isolates belonging to the ribotypes (RT) 012 (DSM 27639) and 027 (DSM 27638 and DSM 27640) were phenotypically characterized and high quality closed genome sequences were generated. The genomes were compared with seven reference strains including three strains of the RT 027, two of the RT 017, and one of the RT 078 as well as a multi-resistant RT 012 strain. The analysis of horizontal gene transfer events revealed gene acquisition incidents that sort the strains within the time line of the spread of their RTs within Germany. We could show as well that horizontal gene transfer between the members of different RTs occurred within this multiple infection. In addition, acquisition and exchange of virulence-related features including antibiotic resistance genes were observed. Analysis of the two genomes assigned to RT 027 revealed three single nucleotide polymorphisms (SNPs) and apparently a regional genome modification within the flagellar switch that regulates the fli operon. CONCLUSION: Our findings show that (i) evolutionary events based on horizontal gene transfer occur within an ongoing CDI and contribute to the adaptation of the species by the introduction of new genes into the genomes, (ii) within a multiple infection of a single patient the exchange of genetic material was responsible for a much higher genome variation than the observed SNPs.

Structural and biochemical analysis of the essential diadenylate cyclase CdaA from Listeria monocytogenes.

The recently identified second messenger cyclic di-AMP (c-di-AMP) is involved in several important cellular processes, such as cell wall metabolism, maintenance of DNA integrity, ion transport, transcription regulation, and allosteric regulation of enzyme function. Interestingly, c-di-AMP is essential for growth of the Gram-positive model bacterium Bacillus subtilis. Although the genome of B. subtilis encodes three c-di-AMP-producing diadenlyate cyclases that can functionally replace each other, the phylogenetically related human pathogens like Listeria monocytogenes and Staphylococcus aureus possess only one enzyme, the diadenlyate cyclase CdaA. Because CdaA is also essential for growth of these bacteria, the enzyme is a promising target for the development of novel antibiotics. Here we present the first crystal structure of the L. monocytogenes CdaA diadenylate cyclase domain that is conserved in many human pathogens. Moreover, biochemical characterization of the cyclase revealed an unusual metal cofactor requirement.

High prevalence of nontoxigenic Clostridium difficile isolated from hospitalized and non-hospitalized individuals in rural Ghana.

Since data about Clostridium difficile infection in sub-Saharan Africa are scarce, we determined its epidemiology and risk factors in a cross-sectional study in Eikwe, a rural community in Ghana. We tested stool samples from 176 hospitalized patients with diarrhoea and from 131 asymptomatic non-hospitalized individuals for C. difficile and some other enteric pathogens. The overall prevalence rate of C. difficile was 4.9% with ribotype 084 being predominant. With 75% of the isolates, a high rate of nontoxigenic strains was present in symptomatic patients, most of whom had no other identified enteric pathogens. All strains were susceptible against metronidazole and vancomycin, respectively. Data on lifestyle and medical history showed that age <5years (p=0.004), and use of ceftriaxone (p=0.023) were the most important risk factors for C. difficile carriage status. Although our data suggest that C. difficile is currently not a major cause of diarrhoea in this setting, the epidemiology of C. difficile in sub-Saharan Africa awaits further investigation.

Evidence for synergistic control of glutamate biosynthesis by glutamate dehydrogenases and glutamate in Bacillus subtilis.

In the Gram-positive bacterium, Bacillus subtilis glutamate is synthesized by the glutamine synthetase and the glutamate synthase (GOGAT). During growth with carbon sources that exert carbon catabolite repression, the rocG glutamate dehydrogenase (GDH) gene is repressed and the transcription factor GltC activates the expression of the GOGAT encoding gltAB genes. In the presence of amino acids of the glutamate family, the GDH RocG is synthesized and the enzyme prevents GltC from binding to DNA. The dual control of glutamate biosynthesis allows the efficient utilization of the available nutrients. Here we provide genetic and biochemical evidence that, like RocG, also the paralogous GDH GudB can inhibit the transcription factor GltC, thereby controlling glutamate biosynthesis. Contradictory previous observations show that high level of GDH activity does not result in permanent inhibition of GltC. By controlling the intracellular levels of glutamate through feeding with exogenous arginine, we observed that the GDH-dependent control of GltC and thus expression of the gltAB genes inversely correlates with the glutamate pool. These results suggest that the B. subtilis GDHs RocG and GudB in fact act as glutamate sensors. In conclusion, the GDH-mediated control of glutamate biosynthesis seems to depend on the intracellular glutamate concentration.

A novel engineering tool in the Bacillus subtilis toolbox: inducer-free activation of gene expression by selection-driven promoter decryptification.

Bacillus subtilis is a Gram-positive bacterium that is easy to manipulate genetically. Several methods for genome engineering have been developed that helped to extend our understanding of how the B. subtilis cell operates. Consequently, the bacterium has become one of the best-studied organisms. B. subtilis has also been engineered for industrial applications. Moreover, great progress has been achieved in promoter engineering to improve the performance of production strains. To expand the toolbox for engineering B. subtilis, we have constructed a system for the inducer-free activation of gene expression. The system relies on spontaneous mutational activation of a cryptic promoter and selection-driven enrichment of bacteria harbouring the mutated promoter. The synthetic promoter is cryptic due to a perfect direct repeat, separating the binding motifs of the RNA polymerase housekeeping sigma factor. The promoter can be fused to genes for industrial applications and to a growth-promoting gene that, upon mutational activation of the promoter, allows enrichment of the engineered bacteria due to a selective growth advantage.

The γ-aminobutyrate permease GabP serves as the third proline transporter of Bacillus subtilis.

PutP and OpuE serve as proline transporters when this imino acid is used by Bacillus subtilis as a nutrient or as an osmostress protectant, respectively. The simultaneous inactivation of the PutP and OpuE systems still allows the utilization of proline as a nutrient. This growth phenotype pointed to the presence of a third proline transport system in B. subtilis. We took advantage of the sensitivity of a putP opuE double mutant to the toxic proline analog 3,4-dehydro-dl-proline (DHP) to identify this additional proline uptake system. DHP-resistant mutants were selected and found to be defective in the use of proline as a nutrient. Whole-genome resequencing of one of these strains provided the lead that the inactivation of the γ-aminobutyrate (GABA) transporter GabP was responsible for these phenotypes. DNA sequencing of the gabP gene in 14 additionally analyzed DHP-resistant strains confirmed this finding. Consistently, each of the DHP-resistant mutants was defective not only in the use of proline as a nutrient but also in the use of GABA as a nitrogen source. The same phenotype resulted from the targeted deletion of the gabP gene in a putP opuE mutant strain. Hence, the GabP carrier not only serves as an uptake system for GABA but also functions as the third proline transporter of B. subtilis. Uptake studies with radiolabeled GABA and proline confirmed this conclusion and provided information on the kinetic parameters of the GabP carrier for both of these substrates.

Cyclic di-AMP homeostasis in bacillus subtilis: both lack and high level accumulation of the nucleotide are detrimental for cell growth.

The genome of the Gram-positive soil bacterium Bacillus subtilis encodes three potential diadenylate cyclases that may synthesize the signaling nucleotide cyclic di-AMP (c-di-AMP). These enzymes are expressed under different conditions in different cell compartments, and they localize to distinct positions in the cell. Here we demonstrate the diadenylate cyclase activity of the so far uncharacterized enzymes CdaA (previously known as YbbP) and CdaS (YojJ). Our work confirms that c-di-AMP is essential for the growth of B. subtilis and shows that an excess of the molecule is also harmful for the bacteria. Several lines of evidence suggest that the diadenylate cyclase CdaA is part of the conserved essential cda-glm module involved in cell wall metabolism. In contrast, the CdaS enzyme seems to provide c-di-AMP for spores. Accumulation of large amounts of c-di-AMP impairs the growth of B. subtilis and results in the formation of aberrant curly cells. This phenotype can be partially suppressed by elevated concentrations of magnesium. These observations suggest that c-di-AMP interferes with the peptidoglycan synthesis machinery. The activity of the diadenylate cyclases is controlled by distinct molecular mechanisms. CdaA is stimulated by a regulatory interaction with the CdaR (YbbR) protein. In contrast, the activity of CdaS seems to be intrinsically restricted, and a single amino acid substitution is sufficient to drastically increase the activity of the enzyme. Taken together, our results support the idea of an important role for c-di-AMP in B. subtilis and suggest that the levels of the nucleotide have to be tightly controlled.

The protein tyrosine kinases EpsB and PtkA differentially affect biofilm formation in Bacillus subtilis.

The Gram-positive soil bacterium Bacillus subtilis is able to choose between motile and sessile lifestyles. The sessile way of life, also referred to as biofilm, depends on the formation of an extracellular polysaccharide matrix and some extracellular proteins. Moreover, a significant proportion of cells in a biofilm form spores. The first two genes of the 15-gene operon for extracellular polysaccharide synthesis, epsA and epsB, encode a putative transmembrane modulator protein and a putative protein tyrosine kinase, respectively, with similarity to the TkmA/PtkA modulator/kinase couple. Here we show that the putative kinase EpsB is required for the formation of structured biofilms. However, an epsB mutant is still able to form biofilms. As shown previously, a ptkA mutant is also partially defective in biofilm formation, but this defect is related to spore formation in the biofilm. The absence of both kinases resulted in a complete loss of biofilm formation. Thus, EpsB and PtkA fulfil complementary functions in biofilm formation. The activity of bacterial protein tyrosine kinases depends on their interaction with modulator proteins. Our results demonstrate the specific interaction between the putative kinase EpsB and its modulator protein EpsA and suggest that EpsB activity is stimulated by its modulator EpsA.

Metabolic rewiring enables ammonium assimilation via a non-canonical fumarate-based pathway.

Glutamate serves as the major cellular amino group donor. In Bacillus subtilis, glutamate is synthesized by the combined action of the glutamine synthetase and the glutamate synthase (GOGAT). The glutamate dehydrogenases are devoted to glutamate degradation in vivo. To keep the cellular glutamate concentration high, the genes and the encoded enzymes involved in glutamate biosynthesis and degradation need to be tightly regulated depending on the available carbon and nitrogen sources. Serendipitously, we found that the inactivation of the ansR and citG genes encoding the repressor of the ansAB genes and the fumarase, respectively, enables the GOGAT-deficient B. subtilis mutant to synthesize glutamate via a non-canonical fumarate-based ammonium assimilation pathway. We also show that the de-repression of the ansAB genes is sufficient to restore aspartate prototrophy of an aspB aspartate transaminase mutant. Moreover, in the presence of arginine, B. subtilis mutants lacking fumarase activity show a growth defect that can be relieved by aspB overexpression, by reducing arginine uptake and by decreasing the metabolic flux through the TCA cycle.

Control of glutamate homeostasis in Bacillus subtilis: a complex interplay between ammonium assimilation, glutamate biosynthesis and degradation.

Glutamate, the major amino group donor in anabolism, is synthesized by the combined action of the glutamine synthetase (GS) and the glutamate synthase (GOGAT) in Bacillus subtilis. The glutamate dehydrogenase (GDH) exclusively degrades glutamate. GS and GDH are both trigger enzymes, active in nitrogen metabolism and in controlling gene expression. Feedback-inhibited GS (FBI-GS) controls DNA-binding activities of two transcription factors, the repressor GlnR and TnrA, the global regulator of nitrogen metabolism. FBI-GS binds to and activates GlnR. This protein complex inhibits GS formation and thus glutamine synthesis. Moreover, FBI-GS inhibits DNA-binding activity of TnrA. Glutamate biosynthesis, the reaction linking carbon with nitrogen metabolism, is controlled by GDH. Together with glutamate GDH inhibits GltC, the transcription factor that activates expression of the GOGAT genes. Thus, GS and GDH control glutamine and glutamate synthesis, respectively, depending on the nitrogen status of the cell. B. subtilis lacking a functional GDH show a severe growth defect. Interestingly, the growth defect is suppressed by the rapid activation of an inactive GDH. Thus, maintenance of glutamate homeostasis is crucial for cellular vitality. This review covers the recent work on the complex control of glutamine and glutamate metabolism in the Gram-positive model organism B. subtilis.

A high-frequency mutation in Bacillus subtilis: requirements for the decryptification of the gudB glutamate dehydrogenase gene.

Common laboratory strains of Bacillus subtilis encode two glutamate dehydrogenases: the enzymatically active protein RocG and the cryptic enzyme GudB that is inactive due to a duplication of three amino acids in its active center. The inactivation of the rocG gene results in poor growth of the bacteria on complex media due to the accumulation of toxic intermediates. Therefore, rocG mutants readily acquire suppressor mutations that decryptify the gudB gene. This decryptification occurs by a precise deletion of one part of the 9-bp direct repeat that causes the amino acid duplication. This mutation occurs at the extremely high frequency of 10(-4). Mutations affecting the integrity of the direct repeat result in a strong reduction of the mutation frequency; however, the actual sequence of the repeat is not essential. The mutation frequency of gudB was not affected by the position of the gene on the chromosome. When the direct repeat was placed in the completely different context of an artificial promoter, the precise deletion of one part of the repeat was also observed, but the mutation frequency was reduced by 3 orders of magnitude. Thus, transcription of the gudB gene seems to be essential for the high frequency of the appearance of the gudB1 mutation. This idea is supported by the finding that the transcription-repair coupling factor Mfd is required for the decryptification of gudB. The Mfd-mediated coupling of transcription to mutagenesis might be a built-in precaution that facilitates the accumulation of mutations preferentially in transcribed genes.

Risk Factors of Patients With Diarrhea for Having Clostridioides (Clostridium) difficile Infection.

Nosocomial infections with Clostridioides (Clostridium) difficile have become an emergent health threat. We sought to define risk factors for a C. difficile infection (CDI) beyond the widely known ones, such as antibiotic use and prior hospital stay. We therefore focused on a group of patients with diarrhea in order to identify risk factors for C. difficile infection among this symptomatic cohort. A total of 121 hospitalized patients from Seesen/Germany with diarrhea were included who submitted a stool sample and were interviewed about their socio-demographic background, lifestyle and state of health using a standardized questionnaire. Antibiotic potential of diuretics was examined by agar diffusion test. C. difficile was identified in 29 patients resulting in a prevalence of 24.0%. The infection was hospital-acquired in most cases (p < 0.001, 82.1%; n = 23/28, versus 29/91, 31.9%). The generally accepted risk factor previous antibiotic use was confirmed in this study (p = 0.002, n = 23/28 CDI patients, 82.1%, versus n = 44/91 non-CDI patients, 48.4%). The following additional risk factors were identified: regular consumption of proton pump inhibitors; PPI (p = 0.011, n = 24/29, 82.8% vs. n = 52/92, 56.5%), CDI patients ate less vegetables (p = 0.001, n = 12/29, 41.4% vs. 69/92, 75.0%). The intake of the diuretic agent torasemid in patients with CDI (p = 0.005, n = 18/29, 62.1%) was higher than in patients without (n = 30/92, 32.6%). More patients with CDI had to undergo a surgery in the previous year (p = 0.022, n = 13/29, 44.8% vs. n = 21/92, 22.8%) and held more birds (p = 0.056, n = 4/29, 13.8%) than individuals of the negative group (n = 3/92, 3.3%). In conclusion, although no antibiotic potential was detected in diuretics, especially torasemid seems to have significant influence for the occurrence of a CDI as well as a nutrition poor in vegetables. A diet rich in vegetables represented a fourfold lower risk for a CDI (OR 0.240, CI (0.0720 - 0.796]).

Allosteric regulation of glycogen breakdown by the second messenger cyclic di-GMP.

Streptomyces are our principal source of antibiotics, which they generate concomitant with a complex developmental transition from vegetative hyphae to spores. c-di-GMP acts as a linchpin in this transition by binding and regulating the key developmental regulators, BldD and WhiG. Here we show that c-di-GMP also binds the glycogen-debranching-enzyme, GlgX, uncovering a direct link between c-di-GMP and glycogen metabolism in bacteria. Further, we show c-di-GMP binding is required for GlgX activity. We describe structures of apo and c-di-GMP-bound GlgX and, strikingly, their comparison shows c-di-GMP induces long-range conformational changes, reorganizing the catalytic pocket to an active state. Glycogen is an important glucose storage compound that enables animals to cope with starvation and stress. Our in vivo studies reveal the important biological role of GlgX in Streptomyces glucose availability control. Overall, we identify a function of c-di-GMP in controlling energy storage metabolism in bacteria, which is widespread in Actinobacteria.

A novel factor controlling bistability in Bacillus subtilis: the YmdB protein affects flagellin expression and biofilm formation.

Cells of Bacillus subtilis can either be motile or sessile, depending on the expression of mutually exclusive sets of genes that are required for flagellum or biofilm formation, respectively. Both activities are coordinated by the master regulator SinR. We have analyzed the role of the previously uncharacterized ymdB gene for bistable gene expression in B. subtilis. We observed a strong overexpression of the hag gene encoding flagellin and of other genes of the σ(D)-dependent motility regulon in the ymdB mutant, whereas the two major operons for biofilm formation, tapA-sipW-tasA and epsA-O, were not expressed. As a result, the ymdB mutant is unable to form biofilms. An analysis of the individual cells of a population revealed that the ymdB mutant no longer exhibited bistable behavior; instead, all cells are short and motile. The inability of the ymdB mutant to form biofilms is suppressed by the deletion of the sinR gene encoding the master regulator of biofilm formation, indicating that SinR-dependent repression of biofilm genes cannot be relieved in a ymdB mutant. Our studies demonstrate that lack of expression of SlrR, an antagonist of SinR, is responsible for the observed phenotypes. Overexpression of SlrR suppresses the effects of a ymdB mutation.

Glutamate metabolism in Bacillus subtilis: gene expression and enzyme activities evolved to avoid futile cycles and to allow rapid responses to perturbations of the system.

Glutamate is a central metabolite in all organisms since it provides the link between carbon and nitrogen metabolism. In Bacillus subtilis, glutamate is synthesized exclusively by the glutamate synthase, and it can be degraded by the glutamate dehydrogenase. In B. subtilis, the major glutamate dehydrogenase RocG is expressed only in the presence of arginine, and the bacteria are unable to utilize glutamate as the only carbon source. In addition to rocG, a second cryptic gene (gudB) encodes an inactive glutamate dehydrogenase. Mutations in rocG result in the rapid accumulation of gudB1 suppressor mutations that code for an active enzyme. In this work, we analyzed the physiological significance of this constellation of genes and enzymes involved in glutamate metabolism. We found that the weak expression of rocG in the absence of the inducer arginine is limiting for glutamate utilization. Moreover, we addressed the potential ability of the active glutamate dehydrogenases of B. subtilis to synthesize glutamate. Both RocG and GudB1 were unable to catalyze the anabolic reaction, most probably because of their very high K(m) values for ammonium. In contrast, the Escherichia coli glutamate dehydrogenase is able to produce glutamate even in the background of a B. subtilis cell. B. subtilis responds to any mutation that interferes with glutamate metabolism with the rapid accumulation of extragenic or intragenic suppressor mutations, bringing the glutamate supply into balance. Similarly, with the presence of a cryptic gene, the system can flexibly respond to changes in the external glutamate supply by the selection of mutations.

Prevalence and Strain Characterization of Clostridioides (Clostridium) difficile in Representative Regions of Germany, Ghana, Tanzania and Indonesia - A Comparative Multi-Center Cross-Sectional Study.

Clostridioides (Clostridium) difficile infections (CDI) are considered worldwide as emerging health threat. Uptake of C. difficile spores may result in asymptomatic carrier status or lead to CDI that could range from mild diarrhea, eventually developing into pseudomembranous colitis up to a toxic megacolon that often results in high mortality. Most epidemiological studies to date have been performed in middle- and high income countries. Beside others, the use of antibiotics and the composition of the microbiome have been identified as major risk factors for the development of CDI. We therefore postulate that prevalence rates of CDI and the distribution of C. difficile strains differ between geographical regions depending on the regional use of antibiotics and food habits. A total of 593 healthy control individuals and 608 patients suffering from diarrhea in communities in Germany, Ghana, Tanzania and Indonesia were selected for a comparative multi-center cross-sectional study. The study populations were screened for the presence of C. difficile in stool samples. Cultured C. difficile strains (n = 84) were further subtyped and characterized using PCR-ribotyping, determination of toxin production, and antibiotic susceptibility testing. Prevalence rates of C. difficile varied widely between the countries. Whereas high prevalence rates were observed in symptomatic patients living in Germany and Indonesia (24.0 and 14.7%), patients from Ghana and Tanzania showed low detection rates (4.5 and 6.4%). Differences were also obvious for ribotype distribution and toxin repertoires. Toxin A+/B+ ribotypes 001/072 and 078 predominated in Germany, whereas most strains isolated from Indonesian patients belonged to toxin A+/B+ ribotype SLO160 and toxin A-/B+ ribotype 017. With 42.9-73.3%, non-toxigenic strains were most abundant in Africa, but were also found in Indonesia at a rate of 18.2%. All isolates were susceptible to vancomycin and metronidazole. Mirroring the antibiotic use, however, moxifloxacin resistance was absent in African C. difficile isolates but present in Indonesian (24.2%) and German ones (65.5%). This study showed that CDI is a global health threat with geographically different prevalence rates which might reflect distinct use of antibiotics. Significant differences for distributions of ribotypes, toxin production, and antibiotic susceptibilities were observed.

The Highly Conserved Asp23 Family Protein YqhY Plays a Role in Lipid Biosynthesis in Bacillus subtilis.

In most bacteria, fatty acid biosynthesis is an essential process that must be controlled by the availability of precursors and by the needs of cell division. So far, no mechanisms controlling synthesis of malonyl-coenzyme A (CoA), the committed step in fatty acid synthesis, have been identified in the Gram-positive model bacterium Bacillus subtilis. We have studied the localization and function of two highly expressed proteins of unknown function, YqhY and YloU. Both proteins are members of the conserved and widespread Asp23 family. While the deletion of yloU had no effect, loss of the yqhY gene induced the rapid acquisition of suppressor mutations. The vast majority of these mutations affect subunits of the acetyl-CoA carboxylase (ACCase) complex, the enzyme that catalyzes the formation of malonyl-CoA. Moreover, lack of yqhY is accompanied by the formation of lipophilic clusters in the polar regions of the cells indicating an increased activity of ACCase. Our results suggest that YqhY controls the activity of ACCase and that this control results in inhibition of ACCase activity. Hyperactivity of the enzyme complex in the absence of YqhY does then provoke mutations that cause reduced ACCase activity.

Gut bacterial communities of diarrheic patients with indications of Clostridioides difficile infection.

We present bacterial 16S rRNA gene datasets derived from stool samples of 44 patients with diarrhea indicative of a Clostridioides difficile infection. For 20 of these patients, C. difficile infection was confirmed by clinical evidence. Stool samples from patients originating from Germany, Ghana, and Indonesia were taken and subjected to DNA isolation. DNA isolations of stool samples from 35 asymptomatic control individuals were performed. The bacterial community structure was assessed by 16S rRNA gene analysis (V3-V4 region). Metadata from patients and control individuals include gender, age, country, presence of diarrhea, concomitant diseases, and results of microbiological tests to diagnose C. difficile presence. We provide initial data analysis and a dataset overview. After processing of paired-end sequencing data, reads were merged, quality-filtered, primer sequences removed, reads truncated to 400 bp and dereplicated. Singletons were removed and sequences were sorted by cluster size, clustered at 97% sequence similarity and chimeric sequences were discarded. Taxonomy to each operational taxonomic unit was assigned by BLASTn searches against Silva database 123.1 and a table was constructed.

Control of potassium homeostasis is an essential function of the second messenger cyclic di-AMP in Bacillus subtilis.

The second messenger cyclic di-adenosine monophosphate (c-di-AMP) is essential in the Gram-positive model organism Bacillus subtilis and in related pathogenic bacteria. It controls the activity of the conserved ydaO riboswitch and of several proteins involved in potassium (K+) uptake. We found that the YdaO protein was conserved among several different bacteria and provide evidence that YdaO functions as a K+ transporter. Thus, we renamed the gene and protein KimA (K+ importer A). Reporter activity assays indicated that expression beyond the c-di-AMP-responsive riboswitch of the kimA upstream regulatory region occurred only in bacteria grown in medium containing low K+ concentrations. Furthermore, mass spectrometry analysis indicated that c-di-AMP accumulated in bacteria grown in the presence of high K+ concentrations but not in low concentrations. A bacterial strain lacking all genes encoding c-di-AMP-synthesizing enzymes was viable when grown in medium containing low K+ concentrations, but not at higher K+ concentrations unless it acquired suppressor mutations in the gene encoding the cation exporter NhaK. Thus, our results indicated that the control of potassium homeostasis is an essential function of c-di-AMP.