Showing papers by "Erhard Bremer published in 2017"

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

Abstract: 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.

Guardians in a stressful world: the Opu family of compatible solute transporters from Bacillus subtilis.

Abstract: The development of a semi-permeable cytoplasmic membrane was a key event in the evolution of microbial proto-cells. As a result, changes in the external osmolarity will inevitably trigger water fluxes along the osmotic gradient. The ensuing osmotic stress has consequences for the magnitude of turgor and will negatively impact cell growth and integrity. No microorganism can actively pump water across the cytoplasmic membrane; hence, microorganisms have to actively adjust the osmotic potential of their cytoplasm to scale and direct water fluxes in order to prevent dehydration or rupture. They will accumulate ions and physiologically compliant organic osmolytes, the compatible solutes, when they face hyperosmotic conditions to retain cell water, and they rapidly expel these compounds through the transient opening of mechanosensitive channels to curb water efflux when exposed to hypo-osmotic circumstances. Here, we provide an overview on the salient features of the osmostress response systems of the ubiquitously distributed bacterium Bacillus subtilis with a special emphasis on the transport systems and channels mediating regulation of cellular hydration and turgor under fluctuating osmotic conditions. The uptake of osmostress protectants via the Opu family of transporters, systems of central importance for the management of osmotic stress by B. subtilis, will be particularly highlighted.

Feeding on compatible solutes: A substrate-induced pathway for uptake and catabolism of ectoines and its genetic control by EnuR.

Abstract: Ectoine and 5-hydroxyectoine are widely synthesized microbial osmostress protectants. They are also versatile nutrients but their catabolism and the genetic regulation of the corresponding genes are incompletely understood. Using the marine bacterium Ruegeria pomeroyi DSS-3, we investigated the utilization of ectoines and propose a seven steps comprising catabolic route that entails an initial conversion of 5-hydroxyectoine to ectoine, the opening of the ectoine ring, and the subsequent degradation of this intermediate to l-aspartate. The catabolic genes are co-transcribed with three genes encoding a 5-hydroxyectoine/ectoine-specific TRAP transporter. A chromosomal deletion of this entire gene cluster abolishes the utilization of ectoines as carbon and nitrogen sources. The presence of ectoines in the growth medium triggers enhanced expression of the importer and catabolic operon, a process dependent on a substrate-inducible promoter that precedes this gene cluster. EnuR, a member of the MocR/GabR-type transcriptional regulators, controls the activity of this promoter and functions as a repressor. EnuR contains a covalently bound pyridoxal-5'-phosphate, and we suggest that this co-factor is critical for the substrate-mediated induction of the 5-hydroxyectoine/ectoine import and catabolic genes. Bioinformatics showed that ectoine consumers are restricted to the Proteobacteria and that EnuR is likely a central regulator for most ectoine/5-hydroxyectoine catabolic genes.

Tinkering with Osmotically Controlled Transcription Allows Enhanced Production and Excretion of Ectoine and Hydroxyectoine from a Microbial Cell Factory.

Abstract: Ectoine and hydroxyectoine are widely synthesized by members of the Bacteria and a few members of the Archaea as potent osmostress protectants. We have studied the salient features of the osmostress-responsive promoter directing the transcription of the ectoine/hydroxyectoine biosynthetic gene cluster from the plant-root-associated bacterium Pseudomonas stutzeri by transferring it into Escherichia coli, an enterobacterium that does not produce ectoines naturally. Using ect-lacZ reporter fusions, we found that the heterologous ect promoter reacted with exquisite sensitivity in its transcriptional profile to graded increases in sustained high salinity, responded to a true osmotic signal, and required the buildup of an osmotically effective gradient across the cytoplasmic membrane for its induction. The involvement of the -10, -35, and spacer regions of the sigma-70-type ect promoter in setting promoter strength and response to osmotic stress was assessed through site-directed mutagenesis. Moderate changes in the ect promoter sequence that increase its resemblance to housekeeping sigma-70-type promoters of E. coli afforded substantially enhanced expression, both in the absence and in the presence of osmotic stress. Building on this set of ect promoter mutants, we engineered an E. coli chassis strain for the heterologous production of ectoines. This synthetic cell factory lacks the genes for the osmostress-responsive synthesis of trehalose and the compatible solute importers ProP and ProU, and it continuously excretes ectoines into the growth medium. By combining appropriate host strains and different plasmid variants, excretion of ectoine, hydroxyectoine, or a mixture of both compounds was achieved under mild osmotic stress conditions.IMPORTANCE Ectoines are compatible solutes, organic osmolytes that are used by microorganisms to fend off the negative consequences of high environmental osmolarity on cellular physiology. An understanding of the salient features of osmostress-responsive promoters directing the expression of the ectoine/hydroxyectoine biosynthetic gene clusters is lacking. We exploited the ect promoter from an ectoine/hydroxyectoine-producing soil bacterium for such a study by transferring it into a surrogate bacterial host. Despite the fact that E. coli does not synthesize ectoines naturally, the ect promoter retained its exquisitely sensitive osmotic control, indicating that osmoregulation of ect transcription is an inherent feature of the promoter and its flanking sequences. These sequences were narrowed to a 116-bp DNA fragment. Ectoines have interesting commercial applications. Building on data from a site-directed mutagenesis study of the ect promoter, we designed a synthetic cell factory that secretes ectoine, hydroxyectoine, or a mixture of both compounds into the growth medium.

Transcriptional regulation of ectoine catabolism in response to multiple metabolic and environmental cues.

Abstract: Ectoine and hydroxyectoine are effective microbial osmostress protectants, but can also serve as versatile nutrients for bacteria. We have studied the genetic regulation of ectoine and hydroxyectoine import and catabolism in the marine Roseobacter species Ruegeria pomeroyi and identified three transcriptional regulators involved in these processes: the GabR/MocR-type repressor EnuR, the feast and famine-type regulator AsnC and the two-component system NtrYX. The corresponding genes are widely associated with ectoine and hydroxyectoine uptake and catabolic gene clusters (enuR, asnC), and with microorganisms predicted to consume ectoines (ntrYX). EnuR contains a covalently bound pyridoxal-5'-phosphate as a co-factor and the chemistry underlying the functioning of MocR/GabR-type regulators typically requires a system-specific low molecular mass effector molecule. Through ligand binding studies with purified EnuR, we identified N-(alpha)-L-acetyl-2,4-diaminobutyric acid and L-2,4-diaminobutyric acid as inducers for EnuR that are generated through ectoine catabolism. AsnC/Lrp-type proteins can wrap DNA into nucleosome-like structures, and we found that the asnC gene was essential for use of ectoines as nutrients. Furthermore, we discovered through transposon mutagenesis that the NtrYX two-component system is required for their catabolism. Database searches suggest that our findings have important ramifications for an understanding of the molecular biology of most microbial consumers of ectoines.

From substrate specificity to promiscuity: hybrid ABC transporters for osmoprotectants

Abstract: Summary The ABC-transporters OpuB and OpuC from Bacillus subtilis function as osmoprotectant import systems. Their structural genes have most likely evolved through a duplication event but the two transporters are remarkably different in their substrate profile. OpuB possesses narrow substrate specificity, while OpuC is promiscuous. We assessed the functionality of hybrids between these two ABC-transporters by reciprocally exchanging the coding regions for the OpuBC and OpuCC substrate-binding proteins between the corresponding opuB and opuC operons. Substantiating the critical role of the binding protein in setting the substrate specificity of ABC transporters, OpuB::OpuCC turned into a promiscuous system, while OpuC::OpuBC now exhibited narrow substrate specificity. Both hybrid transporters possessed a high affinity for their substrates but the transport capacity of the OpuB::OpuCC system was moderate due to the synthesis of only low amounts of the xenogenetic OpuCC protein. Suppressor mutations causing single amino acid substitutions in the GbsR repressor controlling the choline to glycine betaine biosynthesis pathway greatly improved OpuB::OpuCC-mediated compatible solute import through transcriptional up-regulation of the hybrid opuB::opuCC operon. Collectively, we demonstrate for the first time that one can synthetically switch the substrate specificity of a given ABC transporter by combining its core components with a xenogenetic ligand-binding protein. This article is protected by copyright. All rights reserved.

Synthesis of the compatible solute proline by Bacillus subtilis: point mutations rendering the osmotically controlled proHJ promoter hyperactive.

Abstract: Summary The ProJ and ProH enzymes of Bacillus subtilis catalyze together with ProA (ProJ-ProA-ProH), osmostress-adaptive synthesis of the compatible solute proline. The proA-encoded gamma-glutamyl phosphate reductase is also used for anabolic proline synthesis (ProB-ProA-ProI). Transcription of the proHJ operon is osmotically inducible whereas that of the proBA operon is not. Targeted and quantitative proteome analysis revealed that the amount of ProA is not limiting for the interconnected anabolic and osmostress-responsive proline production routes. A key player for enhanced osmostress-adaptive proline production is the osmotically regulated proHJ promoter. We used site-directed mutagenesis to study the salient features of this stress-responsive promoter. Two important features were identified: (i) deviations of the proHJ promoter from the consensus sequence of SigA-type promoters serve to keep transcription low under non-inducing growth conditions, while still allowing a finely-tuned induction of transcriptional activity when the external osmolarity is increased, and (ii) a suboptimal spacer length for SigA-type promoters of either 16-bp (the natural proHJ promoter), or 18-bp (a synthetic promoter variant) is strictly required to allow regulation of promoter activity in proportion to the external salinity. Collectively, our data suggest that changes in the local DNA structure at the proHJ promoter are important determinants for osmostress-inducibility of transcription. This article is protected by copyright. All rights reserved.

Clostridium difficile: A bad bug goes into defensive mode.

Abstract: Clostridium difficile was first isolated in 1935 from the stool sample of a healthy infant and was originally described as Bacillus difficilus (Hall and O’Toole, 1935). The difficulties experienced during the isolation and maintenance of this microorganism in the laboratory stuck with its name when it was re-classified as a member of the genus Clostridium. Recent taxonomic considerations that are based upon an expanded 16S gene sequence hierarchical framework (Collins et al., 1994) lead to its further re-classification as Clostridioides difficile (Lawson et al., 2016). This name was chosen to reflect (i) its similarity to Clostridium (Clostridioides 5 organisms similar to Clostridium) but (ii) without causing wide-ranging ramifications that would ensue when the use of the well-established abbreviations (C. difficile, or C. diff) for its name would no longer be possible in commercial and clinical setting (Lawson et al., 2016). This will happen if the proposal to taxonomically affiliate C. difficile with the genus Peptoclostridium is followed (Yutin and Galperin, 2013). Leaving taxonomic considerations and controversies aside, C. difficile is a rising star among unsavory microorganisms, causing hundreds of thousands of infections and thousands of deaths and burdening European and North American health care systems with billions of dollars for its treatment. About half a million cases of infections with C. difficile are estimated for the United States alone for the year 2011, leading to about 29 000 deaths and costs of 4.8 billion dollars for acute care facilities (Lessa et al., 2015). This dire situation is acerbated by the appearance of hyper-virulent variants of C. difficile that spread into human populations and the increase in the number of strains resistant to commonly used antibiotics (Abt et al., 2016; Dingle et al., 2017). The determination of a very large number of C. difficile genome sequences paints a picture of a rather diverse gene content of this species, with an estimated pan-genome of about 9600 genes but only a restricted (15–20%) core genome (Knight et al., 2015). The genus-level core genome includes about 550 protein families. Based on these data, a metabolic network comprising proteins, RNAs and metabolites has recently been constructed that is crucial for a deeper understanding of the varied biology and pathogenic potential of members of the genus Clostridium (Udaondo et al., 2017). C. difficile is a Gram-positive anaerobic spore-forming rod-shaped bacterium (Fig. 1) that can be found both in terrestrial and marine ecosystems and in the mammalian intestinal tract. Most human infants are colonized with it without exhibiting any negative symptoms, and the number of C. difficile carriers subsequently drops to about 3% in healthy adults (Bartlett and Perl, 2005). However, in hospital settings, a very large percentage of patients (20–40%) are carriers of C. difficile, and the ability of C. difficile to form highly stressand desiccation-resistant endospores (Fimlaid and Shen, 2015; Shen, 2015; Bhattacharjee et al., 2016) certainly contributes greatly to its dissemination in this environment and to the ensuing infection cycle (Abt et al., 2016). As a enteropathogen, C. difficile is a major cause of antibiotic-treatment-associated diarrhoea and the potentially deadly disease pseudomembranous colitis (Abt et al., 2016). Although great attention is focused on the considerable number of hospital acquired infections, the majority of reported cases of C. difficile infections actually occur outside clinical settings and in the absence of antibiotic use (Warriner et al., 2017), a treatment that fosters the colonization of the intestine by C. difficile (Shen, 2015; Abt et al., 2016). The sources of community-acquired C. difficile infections are open for debate but food-based reservoirs seem likely (Warriner et al., 2017). Since C. difficile is a strict anaerobe that used Stickland reactions for the generation of its energy, its metabolism needs to be carefully taken into considerations when issues related to its persistence in the environment, infection, and virulence are discussed (Bouillaut et al., 2015). Received 17 April, 2017; accepted 19 April, 2017. *For correspondence. E-mail bremer@staff.uni-marburg.de; Tel. (149)-64212821529; Fax (149)-6421-2828979.