Showing papers by "Erhard Bremer published in 2016"

Identification of Differentially Expressed Genes during Bacillus subtilis Spore Outgrowth in High-Salinity Environments Using RNA Sequencing.

Abstract: In its natural habitat, the soil bacterium Bacillus subtilis often has to cope with fluctuating osmolality and nutrient availability. Upon nutrient depletion it can form dormant spores, which can revive to form vegetative cells when nutrients become available again. While the effects of salt stress on spore germination have been analyzed previously, detailed knowledge on the salt stress response during the subsequent outgrowth phase is lacking. In this study we investigated the changes in gene expression during B. subtilis outgrowth in the presence of 1.2 M NaCl using RNA sequencing. In total, 402 different genes were upregulated and 632 genes were downregulated during 90 min of outgrowth in the presence of salt. The salt stress response of outgrowing spores largely resembled the osmospecific response of vegetative cells exposed to sustained high salinity and included strong upregulation of genes involved in osmoprotectant uptake and compatible solute synthesis. The σB-dependent general stress response typically triggered by salt shocks was not induced, whereas the σW regulon appears to play an important role for osmoadaptation of outgrowing spores. Furthermore, high salinity induced many changes in the membrane protein and transporter transcriptome. Overall, salt stress seemed to slow down the complex molecular reorganization processes (‘ripening’) of outgrowing spores by exerting detrimental effects on vegetative functions such as amino acid metabolism.

Strangers in the archaeal world: osmostress‐responsive biosynthesis of ectoine and hydroxyectoine by the marine thaumarchaeon Nitrosopumilus maritimus

Abstract: Ectoine and hydroxyectoine are compatible solutes widely synthesized by members of the Bacteria to cope with high osmolarity surroundings. Inspection of 557 archaeal genomes revealed that only 12 strains affiliated with the Nitrosopumilus, Methanothrix or Methanobacterium genera harbour ectoine/hydroxyectoine gene clusters. Phylogenetic considerations suggest that these Archaea have acquired these genes through horizontal gene transfer events. Using the Thaumarchaeon 'Candidatus Nitrosopumilus maritimus' as an example, we demonstrate that the transcription of its ectABCD genes is osmotically induced and functional since it leads to the production of both ectoine and hydroxyectoine. The ectoine synthase and the ectoine hydroxylase were biochemically characterized, and their properties resemble those of their counterparts from Bacteria. Transcriptional analysis of osmotically stressed 'Ca. N. maritimus' cells demonstrated that they possess an ectoine/hydroxyectoine gene cluster (hyp-ectABCD-mscS) different from those recognized previously since it contains a gene for an MscS-type mechanosensitive channel. Complementation experiments with an Escherichia coli mutant lacking all known mechanosensitive channel proteins demonstrated that the (Nm)MscS protein is functional. Hence, 'Ca. N. maritimus' cells cope with high salinity not only through enhanced synthesis of osmostress-protective ectoines but they already prepare themselves simultaneously for an eventually occurring osmotic down-shock by enhancing the production of a safety-valve (NmMscS).

Management of Osmotic Stress by Bacillus Subtilis: Genetics and Physiology

Abstract: Water is the foundation of life (Stevenson et al., 2015). The development of a semipermeable membrane through which water can pass freely, but ions, nutrients, and metabolites cannot, was a key event in the evolution of microbial proto-cells (Booth et al., 2015). Due to the considerable osmotic potential of the cytoplasm caused by nucleic acids, proteins, and metabolites, water enters the cell and creates an outward-directed hydrostatic pressure, the turgor (Bremer and Krämer, 2000; Wood, 2011). Turgor is rather difficult to measure experimentally, and values between 3 and 5 atm have been reported for Gram-negative bacteria such as Escherichia coli and between 20 and 30 atm for Gram-positive bacteria such as Bacillus subtilis and Staphylococcus aureus. However, recently published data suggest that the magnitude of turgor, at least for E. coli, might have been substantially overestimated (Deng et al., 2011). Essentially, all free-living bacteria have to cope in their natural habitats with fluctuations in the osmolarity of their surroundings. Caused by the biophysical properties of the cytoplasmic membrane, fluctuations in the environmental osmolarity will inevitably trigger water fluxes along the osmotic gradient into or out of the cell; hence, the magnitude of turgor will be affected (Booth, 2014; Booth et al., 2015). Turgor is considered to be critical for the expansion of microbial cells during growth and for their viability. Consequently, cellular adjustment processes that aim to maintain turgor and the hydration of the cytoplasm within physiological acceptable boundaries are cornerstones of the stress response to osmotic changes (Bremer and Krämer, 2000; Wood, 2011). This is true for members of the Bacteria and Archaea alike (Csonka, 1989; Kempf and Bremer, 1998; Roesser and Müller, 2001; Wood et al., 2001). Water influx at low external osmolarity can potentially drive up turgor in milliseconds to such an extent that the stability of the stress-bearing peptidoglycan sacculus is no longer sufficient to resist the internal hydrostatic pressure; hence, the integrity of the cell is threatened (Booth, 2014). Conversely, water efflux in high-osmolarity habitats causes dehydration of the cytoplasm, and an ensuing drop in turgor, and the cell will experience plasmolysis; hence, growth is restricted or even prevented (Bremer and Krämer, 2000; Wood, 2011). Although a considerable number of microorganisms possess AqpZ-type aquaporins that mediate accelerated water fluxes across the cytoplasmic membrane (Calamita, 2000; Delamarche et al., 1999), it is important to recall that no bacterium can actively and vectorially transport water. Accordingly, microorganisms can only achieve control of water fluxes across their cytoplasmic membrane indirectly. They do so by actively modulating the intracellular concentration of osmotically active solutes (Bremer and Krämer, 2000; Csonka, 1989; Wood, 2011), and corresponding osmotically driven water fluxes will then ensue (Booth, 2014). Under hypotonic conditions, the cell rapidly jettisons ions and organic compounds through the transient opening of mechanosensitive channels (Naismith and Booth, 2012) and thereby reduces the osmotic potential of the cytoplasm; as a consequence, water influx and the concomitant raise in turgor is curbed. Conversely, it actively accumulates ions or organic solutes to increase the osmotic potential of the cytoplasm and thereby creates a driving force for water influx to stabilize turgor (Csonka, 1989; Galinski and Trüper, 1994; Kempf and Bremer, 1998). Here, we present an overview on the genetic and cellular adaptation mechanisms of B. subtilis, the model organism for Grampositive bacteria, to fluctuating osmolarities (Figure 11.2.1). B. subtilis can be found widely in nature and in many different habitats (Logan and De Vos, 2009), but the upper layers of the soil comprise one of the prime ecological niches in which

Biochemistry and Crystal Structure of Ectoine Synthase: A Metal-Containing Member of the Cupin Superfamily.

Abstract: Ectoine is a compatible solute and chemical chaperone widely used by members of the Bacteria and a few Archaea to fend-off the detrimental effects of high external osmolarity on cellular physiology and growth. Ectoine synthase (EctC) catalyzes the last step in ectoine production and mediates the ring closure of the substrate N-gamma-acetyl-L-2,4-diaminobutyric acid through a water elimination reaction. However, the crystal structure of ectoine synthase is not known and a clear understanding of how its fold contributes to enzyme activity is thus lacking. Using the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis (Sa), we report here both a detailed biochemical characterization of the EctC enzyme and the high-resolution crystal structure of its apo-form. Structural analysis classified the (Sa)EctC protein as a member of the cupin superfamily. EctC forms a dimer with a head-to-tail arrangement, both in solution and in the crystal structure. The interface of the dimer assembly is shaped through backbone-contacts and weak hydrophobic interactions mediated by two beta-sheets within each monomer. We show for the first time that ectoine synthase harbors a catalytically important metal co-factor; metal depletion and reconstitution experiments suggest that EctC is probably an iron-dependent enzyme. We found that EctC not only effectively converts its natural substrate N-gamma-acetyl-L-2,4-diaminobutyric acid into ectoine through a cyclocondensation reaction, but that it can also use the isomer N-alpha-acetyl-L-2,4-diaminobutyric acid as its substrate, albeit with substantially reduced catalytic efficiency. Structure-guided site-directed mutagenesis experiments targeting amino acid residues that are evolutionarily highly conserved among the extended EctC protein family, including those forming the presumptive iron-binding site, were conducted to functionally analyze the properties of the resulting EctC variants. An assessment of enzyme activity and iron content of these mutants give important clues for understanding the architecture of the active site positioned within the core of the EctC cupin barrel.

Salt-sensitivity of σ(H) and Spo0A prevents sporulation of Bacillus subtilis at high osmolarity avoiding death during cellular differentiation.

Abstract: The spore-forming bacterium Bacillus subtilis frequently experiences high osmolarity as a result of desiccation in the soil. The formation of a highly desiccation-resistant endospore might serve as a logical osmostress escape route when vegetative growth is no longer possible. However, sporulation efficiency drastically decreases concomitant with an increase in the external salinity. Fluorescence microscopy of sporulation-specific promoter fusions to gfp revealed that high salinity blocks entry into the sporulation pathway at a very early stage. Specifically, we show that both Spo0A- and SigH-dependent transcription are impaired. Furthermore, we demonstrate that the association of SigH with core RNA polymerase is reduced under these conditions. Suppressors that modestly increase sporulation efficiency at high salinity map to the coding region of sigH and in the regulatory region of kinA, encoding one the sensor kinases that activates Spo0A. These findings led us to discover that B. subtilis cells that overproduce KinA can bypass the salt-imposed block in sporulation. Importantly, these cells are impaired in the morphological process of engulfment and late forespore gene expression and frequently undergo lysis. Altogether our data indicate that B. subtilis blocks entry into sporulation in high-salinity environments preventing commitment to a developmental program that it cannot complete.

EctD-mediated biotransformation of the chemical chaperone ectoine into hydroxyectoine and its mechanosensitive channel-independent excretion

Abstract: Ectoine and its derivative 5-hydroxyectoine are cytoprotectants widely synthesized by microorganisms as a defense against the detrimental effects of high osmolarity on cellular physiology and growth. Both ectoines possess the ability to preserve the functionality of proteins, macromolecular complexes, and even entire cells, attributes that led to their description as chemical chaperones. As a consequence, there is growing interest in using ectoines for biotechnological purposes, in skin care, and in medical applications. 5-Hydroxyectoine is synthesized from ectoine through a region- and stereo-specific hydroxylation reaction mediated by the EctD enzyme, a member of the non-heme-containing iron(II) and 2-oxoglutarate-dependent dioxygenases. This chemical modification endows the newly formed 5-hydroxyectoine with either superior or different stress- protecting and stabilizing properties. Microorganisms producing 5-hydroxyectoine typically contain a mixture of both ectoines. We aimed to establish a recombinant microbial cell factory where 5-hydroxyectoine is (i) produced in highly purified form, and (ii) secreted into the growth medium. We used an Escherichia coli strain (FF4169) defective in the synthesis of the osmostress protectant trehalose as the chassis for our recombinant cell factory. We expressed in this strain a plasmid-encoded ectD gene from Pseudomonas stutzeri A1501 under the control of the anhydrotetracycline-inducible tet promoter. We chose the ectoine hydroxylase from P. stutzeri A1501 for our cell factory after a careful comparison of the in vivo performance of seven different EctD proteins. In the final set-up of the cell factory, ectoine was provided to salt-stressed cultures of strain FF4169 (pMP41; ectD +). Ectoine was imported into the cells via the osmotically inducible ProP and ProU transport systems, intracellularly converted to 5-hydroxyectoine, which was then almost quantitatively secreted into the growth medium. Experiments with an E. coli mutant lacking all currently known mechanosensitive channels (MscL, MscS, MscK, MscM) revealed that the release of 5-hydroxyectoine under osmotic steady-state conditions occurred independently of these microbial safety valves. In shake-flask experiments, 2.13 g l−1 ectoine (15 mM) was completely converted into 5-hydroxyectoine within 24 h. We describe here a recombinant E. coli cell factory for the production and secretion of the chemical chaperone 5-hydroxyectoine free from contaminating ectoine.

The Ectoine Hydroxylase: A Nonheme-Containing Iron(II) and 2-Oxoglutarate-Dependent Dioxygenase

Abstract: Ectoine and 5-hydroxyectoine are well-recognized members of the compatible solutes and are widely employed by microorganisms as osmostress protectants. The EctABC enzymes catalyze the synthesis of ectoine. A subgroup of the ectoine producers can convert ectoine into 5-hydroxyectoine through a region-selective and stereospecific hydroxylation reaction catalyzed by the EctD protein, a member of the nonheme-containing iron(II) and 2-oxoglutarate-dependent dioxygenase superfamily. Structures of EctD are known from the Virgibacillus salexigens EctD protein in its apo- and iron-bound forms. Furthermore, a set of crystal structures is known from the EctD protein from Sphingopyxis alaskensis in its apo- and Fe(II)-bound forms and also from a dead-end complex of EctD together with the substrate 2-oxogluterate and the product 5-hydroxyectoine. In this article, we describe the purification and biochemically characterization of nine different EctD proteins from different species. Furthermore, the importance of the Fe(II) ligand and its binding site is described. The octahedral geometry of Fe(II) binding is mediated via two histidine and one aspartate side chain together with water molecules. The latter are replaced by the binding of the 2-oxoglutarate substrate to the EctD enzyme. The binding mechanism of Fe(II) is strictly conserved within the EctD protein superfamily. 3D Structure Structure of the dimeric ectoine hydroxylase from S. alaskensis (SaEctD). One monomer is represented as cartoon representation. The other monomer is highlighted as a surface with the bound 2-oxoglutarate substrate (shown as blue sticks) and the 5-hydroxyectoine product (shown in orange). The bound Fe(II) ligand is shown as spheres colored in magenta (PDB code: 4Q5O). The signature sequence motif of SaEctD is colored in green. These figures were prepared using PyMOL (www.pymol.org). Keywords: compatible solutes; osmostress protectants; cupin; mononuclear iron center

Mechanistic and transcriptomic salt stress effects on Bacillus subtilis spore germination and outgrowth

Abstract: The spore-forming soil bacterium Bacillus subtilis frequently encounters changes in environmental conditions within its natural habitat. Flooding, desiccation, and agricultural activities can, for instance, cause strong fluctuations in osmolality. While the complex osmotic stress response of vegetative cells is increasingly understood, less is known about salt stress effects on germinating and outgrowing spores, which, however, is of applied interested for soil ecology, food microbiology, and astrobiology. Although negative effects of high salinity have been reported, they have been poorly investigated on a mechanistic level in the past. Thus, we have investigated the effects of NaCl and other salts on spore germination and outgrowth of B. subtilis and could gain interesting new mechanistic, structural, and transcriptomic insights. While the initiation of germination is still possible despite high salinity, increasing NaCl concentration cause an increasing delay of germination commitment, increasing heterogeneity of germination onset, slower germination on single spore and population levels, and decreasing germination efficiency. Data from broad analyses using various techniques suggest NaCl-inhibition of at least one early process in germination (e.g. commitment or germination initiation), and of at least one subsequent event. The most likely processes to be inhibited are ion, Ca2+-DPA and water fluxes, which might be negatively affected by direct interactions of Na+ and Cl- with transporters or channels, annihilation of a chemical gradient, and/or low extracellular water activity. We have strong evidence that NaCl-inhibition is due to a combination of ionic and osmotic effects. Moreover, the spore coat plays an important role for successful germination at high salinity, likely being involved in protection from ionic stress. Outgrowth, defined by the onset of metabolism after germination, can also be detected at high salt concentrations (≤ 4.8 M NaCl), although the ability to proliferate at high salinity strongly depends on the composition of the germination medium. To determine underlying physiological and genetic mechanisms, the salt stress response of outgrowing B. subtilis spores was investigated using RNA-seq. While the transcriptomic salt stress response during outgrowth revealed similarities to the response of vegetative cells, we could also identify several interesting differences.