Showing papers by "Erhard Bremer published in 2011"

Genome Sequences of the Biotechnologically Important Bacillus megaterium Strains QM B1551 and DSM319

Abstract: Bacillus megaterium is deep-rooted in the Bacillus phylogeny, making it an evolutionarily key species and of particular importance in understanding genome evolution, dynamics, and plasticity in the bacilli. B. megaterium is a commercially available, nonpathogenic host for the biotechnological production of several substances, including vitamin B(12), penicillin acylase, and amylases. Here, we report the analysis of the first complete genome sequences of two important B. megaterium strains, the plasmidless strain DSM319 and QM B1551, which harbors seven indigenous plasmids. The 5.1-Mbp chromosome carries approximately 5,300 genes, while QM B1551 plasmids represent a combined 417 kb and 523 genes, one of the largest plasmid arrays sequenced in a single bacterial strain. We have documented extensive gene transfer between the plasmids and the chromosome. Each strain carries roughly 300 strain-specific chromosomal genes that account for differences in their experimentally confirmed phenotypes. B. megaterium is able to synthesize vitamin B(12) through an oxygen-independent adenosylcobalamin pathway, which together with other key energetic and metabolic pathways has now been fully reconstructed. Other novel genes include a second ftsZ gene, which may be responsible for the large cell size of members of this species, as well as genes for gas vesicles, a second β-galactosidase gene, and most but not all of the genes needed for genetic competence. Comprehensive analyses of the global Bacillus gene pool showed that only an asymmetric region around the origin of replication was syntenic across the genus. This appears to be a characteristic feature of the Bacillus spp. genome architecture and may be key to their sporulating lifestyle.

Protection of Bacillus subtilis against Cold Stress via Compatible-Solute Acquisition

Abstract: Accumulation of compatible solutes is a strategy widely employed by bacteria to achieve cellular protection against high osmolarity. These compounds are also used in some microorganisms as thermostress protectants. We found that Bacillus subtilis uses the compatible solute glycine betaine as an effective cold stress protectant. Glycine betaine strongly stimulated growth at 15°C and permitted cell proliferation at the growth-inhibiting temperature of 13°C. Initial uptake of glycine betaine at 15°C was low but led eventually to the buildup of an intracellular pool whose size was double that found in cells grown at 35°C. Each of the three glycine betaine transporters (OpuA, OpuC, and OpuD) contributed to glycine betaine accumulation in the cold. Protection against cold stress was also accomplished when glycine betaine was synthesized from its precursor choline. Growth of a mutant defective in the osmoadaptive biosynthesis for the compatible solute proline was not impaired at low temperature (15°C). In addition to glycine betaine, the compatible solutes and osmoprotectants L-carnitine, crotonobetaine, butyrobetaine, homobetaine, dimethylsulfonioactetate, and proline betaine all served as cold stress protectants as well and were accumulated via known Opu transport systems. In contrast, the compatible solutes and osmoprotectants choline-O-sulfate, ectoine, proline, and glutamate were not cold protective. Our data highlight an underappreciated facet of the acclimatization of B. subtilis to cold environments and allow a comparison of the characteristics of compatible solutes with respect to their osmotic, heat, and cold stress-protective properties for B. subtilis cells. Bacillus subtilis can be found ubiquitously in the upper layers of the soil. Drying of the soil occurs frequently, and as a consequence, the salinity and osmolarity in this ecological niche increase. Such a rise in the external osmotic potential inevitably triggers water efflux from the B. subtilis cell (11). To prevent a reduction in water content and hence a drop of turgor, the cells initially increase the concentration of ions (primarily K) through transport processes (35, 65) and subsequently accumulate compatible solutes, such as proline and

Osmotically Controlled Synthesis of the Compatible Solute Proline Is Critical for Cellular Defense of Bacillus subtilis against High Osmolarity

Abstract: Bacillus subtilis is known to accumulate large amounts of the compatible solute proline via de novo synthesis as a stress protectant when it faces high-salinity environments. We elucidated the genetic determinants required for the osmoadaptive proline production from the precursor glutamate. This proline biosynthesis route relies on the proJ-encoded γ-glutamyl kinase, the proA-encoded γ-glutamyl phosphate reductase, and the proH-encoded Δ1-pyrroline-5-caboxylate reductase. Disruption of the proHJ operon abolished osmoadaptive proline production and strongly impaired the ability of B. subtilis to cope with high-osmolarity growth conditions. Disruption of the proA gene also abolished osmoadaptive proline biosynthesis but caused, in contrast to the disruption of proHJ, proline auxotrophy. Northern blot analysis demonstrated that the transcription of the proHJ operon is osmotically inducible, whereas that of the proBA operon is not. Reporter gene fusion studies showed that proHJ expression is rapidly induced upon an osmotic upshift. Increased expression is maintained as long as the osmotic stimulus persists and is sensitively linked to the prevalent osmolarity of the growth medium. Primer extension analysis revealed the osmotically controlled proHJ promoter, a promoter that resembles typical SigA-type promoters of B. subtilis. Deletion analysis of the proHJ promoter region identified a 126-bp DNA segment carrying all sequences required in cis for osmoregulated transcription. Our data disclose the presence of ProA-interlinked anabolic and osmoadaptive proline biosynthetic routes in B. subtilis and demonstrate that the synthesis of the compatible solute proline is a central facet of the cellular defense to high-osmolarity surroundings for this soil bacterium.

A specialized aspartokinase enhances the biosynthesis of the osmoprotectants ectoine and hydroxyectoine in Pseudomonas stutzeri A1501.

Abstract: The compatible solutes ectoine and hydroxyectoine are widely produced by bacteria as protectants against osmotic and temperature stress. L-Aspartate-beta-semialdehyde is used as the precursor molecule for ectoine/ hydroxyectoine biosynthesis that is catalyzed by the EctABCD enzymes. L-Aspartate-beta-semialdehyde is a central intermediate in different biosynthetic pathways and is produced from L-aspartate by aspartokinase (Ask) and aspartate-semialdehyde-dehydrogenase (Asd). Ask activity is typically stringently regulated by allosteric control to avoid gratuitous synthesis of aspartylphosphate. Many organisms have evolved multiple forms of aspartokinase, and feedback regulation of these specialized Ask enzymes is often adapted to the cognate biochemical pathways. The ectoine/hydroxyectoine biosynthetic genes (ectABCD) are followed in a considerable number of microorganisms by an ask gene (ask_ect), suggesting that Ask_Ect is a specialized enzyme for this osmoadaptive biosynthetic pathway. However, none of these Ask_Ect enzymes have been functionally characterized. Pseudomonas stutzeri A1501 synthesizes both ectoine and hydroxyectoine in response to increased salinity, and it possesses two Ask enzymes: Ask_Lys and Ask_Ect. We purified both Ask enzymes and found significant differences with regard to their allosteric control: Ask_LysC was inhibited by threonine and in a concerted fashion by threonine and lysine, whereas Ask_Ect showed inhibition only by threonine. The ectABCD_ask genes from P. stutzeri A1501 were cloned and functionally expressed in Escherichia coli, and this led to osmostress protection. An E. coli strain carrying the plasmid-based ectABCD_ask gene cluster produced significantly more ectoine/hydroxyectoine than a strain expressing the ectABCD gene cluster alone. This finding suggests a specialized role for Ask_Ect in ectoine/hydroxyectoine biosynthesis.

Ectoine and Hydroxyectoine as Protectants against Osmotic and Cold Stress: Uptake through the SigB-Controlled Betaine-Choline- Carnitine Transporter-Type Carrier EctT from Virgibacillus pantothenticus

Abstract: Virgibacillus pantothenticus has been shown to synthesize the compatible solute ectoine in response to high salinity or low growth temperature. We found that exogenously provided ectoine and hydroxyectoine also serve as protectants against these challenges. Transport studies with ( 14 C)ectoine revealed that both types of stress induced a high-affinity ectoine uptake activity in V. pantothenticus. By using an Escherichia coli mutant defective in osmoprotectant uptake systems, a functional complementation approach for osmostress resistance in the presence of ectoine was employed to retrieve a gene encoding an ectoine transporter from V. pantothenticus. The cloned gene (ectT) encodes a protein (EctT) that is a member of the BCCT (betaine-choline-carnitine- transporter) family of carriers. Osmoprotection assays demonstrated that the EctT carrier mediates the preferential import of ectoine and hydroxyectoine but also possesses minor uptake activities for the compatible solutes proline and glycine betaine. Northern blot analysis with RNA isolated from V. pantothenticus revealed that a rise in the external osmolality or a reduction in growth temperature strongly increased the transcription of the ectT gene. Primer extension analysis demonstrated that ectT was transcribed under these conditions from a SigB-type promoter. SigB is the master regulator of the general stress regulon of bacilli and provides protection to cells against various challenges, including high salinity and low temperature. Both the synthesis of ectoine and the EctT-mediated uptake of ectoine and hydroxyectoine are triggered by the same environ- mental cues, high salinity and cold stress, and thereby provide, in a concerted fashion, the protection of V. pantothenticus against these challenges.

T-box-mediated control of the anabolic proline biosynthetic genes of Bacillus subtilis.

Abstract: Bacillus subtilis possesses interlinked routes for the synthesis of proline. The ProJ–ProA–ProH route is responsible for the production of proline as an osmoprotectant, and the ProB–ProA–ProI route provides proline for protein synthesis. We show here that the transcription of the anabolic proBA and proI genes is controlled in response to proline limitation via a T-box-mediated termination/antitermination regulatory mechanism, a tRNA-responsive riboswitch. Primer extension analysis revealed mRNA leader transcripts of 270 and 269 nt for the proBA and proI genes, respectively, both of which are synthesized from SigA-type promoters. These leader transcripts are predicted to fold into two mutually exclusive secondary mRNA structures, forming either a terminator or an antiterminator configuration. Northern blot analysis allowed the detection of both the leader and the full-length proBA and proI transcripts. Assessment of the level of the proBA transcripts revealed that the amount of the full-length mRNA species strongly increased in proline-starved cultures. Genetic studies with a proB–treA operon fusion reporter strain demonstrated that proBA transcription is sensitively tied to proline availability and is derepressed as soon as cellular starvation for proline sets in. Both the proBA and the proI leader sequences contain a CCU proline-specific specifier codon prone to interact with the corresponding uncharged proline-specific tRNA. By replacing the CCU proline specifier codon in the proBA T-box leader with UUC, a codon recognized by a Phe-specific tRNA, we were able to synthetically re-engineer the proline-specific control of proBA transcription to a control that was responsive to starvation for phenylalanine.

Cellular Adjustments of Bacillus subtilis and Other Bacilli to Fluctuating Salinities

Abstract: The soil-dwelling bacterium Bacillus subtilis inhabits an ecological niche subjected to frequent changes in osmotic and saline conditions that are caused by rainfall and desiccation. Such changes elicit water fluxes across the cytoplasmic membrane and can drive up turgor under hypo-osmotic conditions to such an extent that the cell will rupture, or under hyper-osmotic conditions, cause the dehydration of the cytoplasm, a reduction in turgor and eventually growth arrest and cell death. Proteome and genome-wide transcriptional profiling studies have highlighted the complexity and multifaceted nature of the osmotic stress response systems of B. subtilis. However, it is beyond doubt that effective water management by the cell is the cornerstone of its acclimatization to either sudden or sustained rises in the environmental osmolarity and the osmotic downshift that inevitably will follow hyperosmotic growth conditions. The accumulation and expulsion of ions and compatible solutes play key roles in these cellular osmotic adjustment processes.

Bacterial abl-like genes: production of the archaeal osmolyte N(ε)-acetyl-β-lysine by homologous overexpression of the yodP-kamA genes in Bacillus subtilis.

Abstract: \( {N^{\varepsilon }}{\text{ - acetyl - }}\beta {\text{ - lysine}} \) is an archaeal compatible solute whose synthesis is mediated by the sequential reactions of the lysine-2,3-aminomutase AblA and the acetyltransferase AblB. α-Lysine serves as the precursor and is converted by AblA to β-lysine, and AblB then acetylates this intermediate to \( {N^{\varepsilon }}{\text{ - acetyl - }}\beta {\text{ - lysine}} \). The biochemical and biophysical properties of \( {N^{\varepsilon }}{\text{ - acetyl - }}\beta {\text{ - lysine}} \) have so far not been studied intensively due to restrictions in the supply of this compound. A search for ablAB-like genes in the genomes of members of the family Bacillaceae revealed the yodP–kamA genes that encode a AblA-related lysine-2,3-aminomutase and AblB-related putative acetyltransferase. In Bacillus subtilis, the yodP–kamA genes are part of a transcriptional unit (yodT–yodS–yodR–yodQ–yodP–kamA) whose expression is upregulated during sporulation and controlled by the mother-cell-specific transcription factor SigE. \( {N^{\varepsilon }}{\text{ - acetyl - }}\beta {\text{ - lysine}} \) was not detectable in vegetatively growing or osmotically stressed B. subtilis cells, and the deletion of the yodT–yodS–yodR–yodQ–yodP–kamA region had no noticeable effects on growth in rich or minimal media or osmotic stress resistance. However, when we expressed the yodP–kamA genes outside their natural genetic context from an isopropyl β-d-1-thiogalactopyranoside-inducible promoter on a plasmid in B. subtilis, the recombinant strain synthesized considerable amounts (0.28 μmol/mg protein) of \( {N^{\varepsilon }}{\text{ - acetyl - }}\beta {\text{ - lysine}} \). The data reported here thus open the bottleneck for the large-scale production of \( {N^{\varepsilon }}{\text{ - acetyl - }}\beta {\text{ - lysine}} \) to investigate its properties as a compatible solute.