Skip to main content
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems
  • Log in
  • My alerts
  • My Cart

Main menu

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • COVID-19 Special Collection
    • Archive
    • Minireviews
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About AEM
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems

User menu

  • Log in
  • My alerts
  • My Cart

Search

  • Advanced search
Applied and Environmental Microbiology
publisher-logosite-logo

Advanced Search

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • COVID-19 Special Collection
    • Archive
    • Minireviews
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About AEM
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
Physiology

Uptake of Amino Acids and Their Metabolic Conversion into the Compatible Solute Proline Confers Osmoprotection to Bacillus subtilis

Adrienne Zaprasis, Monika Bleisteiner, Anne Kerres, Tamara Hoffmann, Erhard Bremer
A. M. Spormann, Editor
Adrienne Zaprasis
aPhilipps University Marburg, Department of Biology, Laboratory for Microbiology, Marburg, Germany
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Monika Bleisteiner
aPhilipps University Marburg, Department of Biology, Laboratory for Microbiology, Marburg, Germany
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Anne Kerres
aPhilipps University Marburg, Department of Biology, Laboratory for Microbiology, Marburg, Germany
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Tamara Hoffmann
aPhilipps University Marburg, Department of Biology, Laboratory for Microbiology, Marburg, Germany
bLOEWE–Center for Synthetic Microbiology, Philipps University Marburg, Marburg, Germany
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Erhard Bremer
aPhilipps University Marburg, Department of Biology, Laboratory for Microbiology, Marburg, Germany
bLOEWE–Center for Synthetic Microbiology, Philipps University Marburg, Marburg, Germany
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
A. M. Spormann
Roles: Editor
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
DOI: 10.1128/AEM.02797-14
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

ABSTRACT

The data presented here reveal a new facet of the physiological adjustment processes through which Bacillus subtilis can derive osmostress protection. We found that the import of proteogenic (Glu, Gln, Asp, Asn, and Arg) and of nonproteogenic (Orn and Cit) amino acids and their metabolic conversion into proline enhances growth under otherwise osmotically unfavorable conditions. Osmoprotection by amino acids depends on the functioning of the ProJ-ProA-ProH enzymes, but different entry points into this biosynthetic route are used by different amino acids to finally yield the compatible solute proline. Glu, Gln, Asp, and Asn are used to replenish the cellular pool of glutamate, the precursor for proline production, whereas Arg, Orn, and Cit are converted into γ-glutamic semialdehyde/Δ1-pyrroline-5-carboxylate, an intermediate in proline biosynthesis. The import of Glu, Gln, Asp, Asn, Arg, Orn, and Cit did not lead to a further increase in the size of the proline pool that is already present in osmotically stressed cells. Hence, our data suggest that osmoprotection of B. subtilis by this group of amino acids rests on the savings in biosynthetic building blocks and energy that would otherwise have to be devoted either to the synthesis of the proline precursor glutamate or of proline itself. Since glutamate is the direct biosynthetic precursor for proline, we studied its uptake and found that GltT, an Na+-coupled symporter, is the main uptake system for both glutamate and aspartate in B. subtilis. Collectively, our data show how effectively B. subtilis can exploit environmental resources to derive osmotic-stress protection through physiological means.

INTRODUCTION

Bacillus subtilis is a resident of the upper layers of the soil and of the rhizosphere, and it can also efficiently colonize root surfaces (1–3). The blueprint of its genome (4) bears the hallmarks of a bacterium that can exploit many plant-produced compounds for its growth. Accordingly, a considerable portion of the coding capacity of the B. subtilis chromosome (5) is devoted to high-affinity import systems (6) that allow the scavenging of a wide spectrum of nutrients. Reoccurring and persisting high osmolarity in the soil ecosystem (7) is a situation in which B. subtilis can take advantage of the import of plant-produced compounds (8–10) for its physiological adjustment to these unfavorable environmental conditions (7, 11).

As in many bacterial species (11–13), cellular adaptation of B. subtilis to both sudden and sustained increases in the external osmolarity involves a two-stage process (7, 14). It initially encompasses the uptake of large quantities of potassium as an emergency stress reaction to curb water efflux (14, 15) and, subsequently, the replacement of part of this ion by organic osmolytes, such as proline (Pro) and glycine betaine (GB), to decrease the ionic strength of the cytoplasm and to optimize its solvent properties (14, 16–19). These organic osmolytes, commonly referred to as compatible solutes, are highly compliant with cellular physiology and biochemistry (7, 12, 14). They are brought into the soil ecosystem through root exudates, decaying plant material, and osmotically downshocked or disintegrated microbial cells (8–10, 20). B. subtilis cells exposed to high salinity can capture them via osmotically inducible high-affinity transport systems to derive osmostress protection (7, 16).

Proline is the only compatible solute that B. subtilis can synthesize de novo (14), a process that is mediated through the ProJ-ProA-ProH biosynthetic route (18, 21) (Fig. 1). High-level production of proline is achieved through the osmotic induction of proHJ transcription (18) and probably through reduced feedback control (22, 23) of the activity of the ProJ enzyme by proline. Together, these two events lead to the buildup of cellular proline pools whose size is linearly related to the osmolarity prevalent in the environment at a given time (17, 18); they can reach values of about 500 mM in severely osmotically stressed cells (e.g., after growth in a minimal medium with 1.2 M NaCl) (17, 24). The genetic disruption of the osmostress-adaptive proline-biosynthetic route causes osmotic sensitivity of B. subtilis (18), highlighting the central role of compatible-solute accumulation in the cellular adjustment to high-osmolarity environments (7, 11). Relief from osmostress can also be accomplished through import of proline via the osmotically inducible OpuE transporter (25, 26), a system that is also involved in the recovery of newly synthesized proline that leaks, or is actively excreted, into the medium by B. subtilis cells continuously challenged by high osmolarity (27). Of all the compatible solutes imported by B. subtilis (7, 28, 29), proline is the only one that can also be catabolized (30) (Fig. 1), and this limits the effectiveness of an external supply of proline as an osmostress protectant in comparison to metabolically inert compatible solutes, such as glycine betaine or proline betaine (16, 24, 29). Curiously, induction of the proline-catabolic putBCP operon occurs only when the amino acid is present in the growth medium but not by the large amount of proline that is accumulated inside the cell via de novo synthesis under osmotic-stress conditions (24, 30). B. subtilis can also exploit proline-containing peptides of different lengths and compositions as osmoprotectants through uptake and their subsequent intracellular hydrolysis to yield free proline (24) (Fig. 1).

FIG 1
  • Open in new tab
  • Download powerpoint
FIG 1

Overview of the uptake, synthesis, catabolism, and generation of l-proline through the metabolic conversion of several proteogenic and nonproteogenic amino acids by B. subtilis. The dashed arrows indicate that several enzymes are involved in the indicated steps.

Here, we demonstrate that B. subtilis can achieve osmoprotection by proline in yet another way. We found that the cell imports a restricted set of proteinogenic and nonproteinogenic amino acids, all of which are present in the rhizosphere and in the soil (8–10), and subsequently converts them either into the proline-biosynthetic precursor glutamate (Glu) (31, 32) or into the intermediate in proline biosynthesis, γ-glutamic semialdehyde/Δ1-pyrroline-5-carboxylate (33, 34) (Fig. 1). Both processes involve the previously identified osmostress-adaptive ProJ-ProA-ProH proline-biosynthetic enzymes (18), but in a way that allows the preservation of precious building blocks and biosynthetic resources.

MATERIALS AND METHODS

Chemicals.Amino acids, the ninhydrin reagent for the quantification of proline by a colorimetric assay (35), and the antibiotics chloramphenicol, kanamycin, tetracycline, and spectinomycin were all purchased from Sigma-Aldrich (Steinheim, Germany) or from Carl Roth GmbH (Karlsruhe, Germany). Radiolabeled l-[U-14C]proline (269 mCi mmol−1), l-[U-14C]glutamate (253 mCi mmol−1), and l-[U-14C]aspartate (200 mCi mmol−1) were purchased from GE Healthcare Lifesciences (Munich, Germany); l-[U-14C]arginine (274.3 mCi mmol−1) was purchased from PerkinElmer (Rodgau, Germany).

Bacterial strains, media, and growth conditions.The genetic properties of the B. subtilis strains used in this study are summarized in Table 1; they are all derived from the laboratory strain JH642 (36, 37). B. subtilis strains were routinely cultivated in Spizizen′s minimal medium (SMM) (38) with 0.5% (wt/vol) glucose as the carbon source and l-tryptophan (20 mg liter−1) and l-phenylalanine (18 mg liter−1) to satisfy the auxotrophic requirements of strain JH642 (trpC2 pheA1) and its mutant derivatives (Table 1). A solution of trace elements was added to SMM to improve the growth of B. subtilis strains (38). The osmolarity of growth media was adjusted by adding NaCl from a 5 M stock solution. All B. subtilis cultures were inoculated from exponentially growing precultures in prewarmed (37°C) SMM to optical densities at 578 nm (OD578) of about 0.1, and the cultures were then propagated at 37°C in a shaking water bath set to 220 rpm; 100-ml shake flasks filled with 20-ml medium were used for these experiments. When the use of glutamate and aspartate (Asp) by B. subtilis as sole nitrogen sources was assessed, the ammonium source [(NH4)2SO4; 15 mM] was replaced by various concentrations of either l-Glu or l-Asp in preparing the SMM.

View this table:
  • View inline
  • View popup
TABLE 1

B. subtilis strains used in this study

Construction of bacterial strains.The gltP gene was amplified by PCR from chromosomal DNA of strain JH642, and the resulting 1,079-bp DNA fragment was cloned into the EcoRI-ClaI sites of plasmid pBSK(−) (Stratagene), yielding pADK1. A kanamycin resistance cassette (neo), derived from plasmid pDG783 (39), was inserted into the HindIII site present in the coding region of gltP. DNA of the resulting plasmid, pADK2, was linearized by cleaving with SacII and ClaI and used to transform strain JH642 and to subsequently select for kanamycin-resistant colonies; the resulting strain was ADB1 (Table 1). A second mutant allele of gltP was constructed by removing the neo kanamycin resistance cassette from pADK2 via HindIII digestion and replacing it with a chloramphenicol resistance cassette (cat) derived via PCR from plasmid pJMB1 (M. Jebbar and E. Bremer, unpublished data); this yielded plasmid pMD27. DNA of plasmid pMD27 was linearized by cleaving with XhoI and ScaI and transformed into strain JH642, yielding strain MDB52 (Table 1). To construct a gltT mutant, the gltT gene was amplified from chromosomal DNA of strain JH642 by PCR, and the resulting DNA fragment was digested with PstI and XmnI. The 711-bp PstI-XmnI DNA fragment internal to the gltT coding region was then cloned into the PstI and SmaI sites of plasmid pUS19 (40) carrying a spectinomycin resistance determinant; this yielded plasmid pADK5. Plasmid pUS19 cannot replicate in B. subtilis (40); hence, transformation of pADK5 into strain JH642 results in its chromosomal integration via a single-crossover event that leads to the disruption of the gltT gene (strain ADB4) (Table 1). The yveA::neo mutation (41) was moved by transformation of chromosomal DNA obtained from the B. subtilis 168 genetic background into JH642, yielding strain MDB43 (Table 1). Combinations of the gltP, gltT, and yveA mutant alleles were obtained by transformation with chromosomal DNA prepared from appropriate B. subtilis donor strains (Table 1).

Northern blot analysis.Cells of the B. subtilis wild-type strain JH642 were gown in SMM in the absence or the presence of 1.2 M NaCl until the cultures reached an OD578 of 0.8 to 1.0. After harvesting of the cells by centrifugation, total RNA was prepared using the acidic phenol method (42). Total RNA (10 μg) was separated according to size on a 1.4% agarose gel, transferred to a Schleicher & Schuell NY13N membrane, and hybridized with a digoxigenin-labeled single-stranded antisense RNA probe specific for gltT. The gltT antisense RNA probe covered 575 bp of the gltT gene and was produced by in vitro transcription of a corresponding gltT PCR fragment that carried an artificial T7 promoter sequence on one of its ends and that was introduced by the reversed DNA primer used. The PCR product was amplified using the primers gltT5-ADK1 (5′-GCCATTATTCTCGGACTAGCCC-3′) and gltT3-ADK2 (5′-TCCATGATACGCGGAAGAACCG-3′). The procedures to synthesize in vitro the gltT-specific antisense RNA probe, its labeling with digoxigenin, the detection of the gltT mRNA with a Roche digoxigenin kit (Roche Diagnostics GmbH, Mannheim, Germany), and the fluorophore substrate ECF (GE Healthcare Lifesciences, Munich, Germany) have been described previously (18).

Measurements of intracellular proline pools.The intracellular proline content of B. subtilis cells was determined by a colorimetric assay that detects l-proline as a colored proline-ninhydrin complex that can be quantified by measuring the absorption of the solution at 480 nm (35). For these assays, the B. subtilis cells were grown in SMM with 1.2 M NaCl in the absence or presence of various amino acids until they reached an OD578 of about 1.8 to 2.0; the harvesting and processing of the cells, the details of the assay conditions, and the calculation of the intracellular volume of B. subtilis cells have been described previously (17, 24).

Tracing the metabolic conversion of glutamate, aspartate, and arginine (Arg) into proline in osmotically stressed B. subtilis cells.The B. subtilis strain JH642 was grown in minimal medium in the presence of 1.2 M NaCl. When the culture had reached the early exponential growth phase (OD578, about 0.5), it was aliquoted (8-ml culture volume each) into four portions in prewarmed 100-ml Erlenmeyer flasks. The radiolabeled amino acids l-[U-14C]glutamate, l-[U-14C]aspartate, l-[U-14C]arginine, and l-[U-14C]proline were then added to the individual subcultures to a final concentration of 20 μM, corresponding to a specific radioactivity of 15 mCi per μmol. The cells were then incubated with shaking at 37°C, and 500-μl samples were withdrawn from each culture at 30-min intervals starting immediately after addition of the radiolabeled amino acid. The samples were harvested by centrifugation for 5 min at 13,000 rpm at room temperature in an Eppendorf tabletop centrifuge. The cell pellets were resuspended in 50 μl TE-lysozyme mixture (50 mM Tris-HCl, pH 8.0, 3 mg ml−1 lysozyme). After 15 min of incubation at 37°C, cell lysis was completed by the addition of 2 μl SDS (10%). Cell debris was removed by centrifugation for 10 min at 13,000 rpm and 4°C in an Eppendorf tabletop centrifuge. To separate the solutes of the cell extracts, 10-μl aliquots of the supernatants were spotted onto a 0.2-mm silica gel plate (Polygram Sil G; Macheray-Nagel, Düren, Germany) with a 20-cm path length. Separation of the compounds in the extracts was achieved overnight at room temperature with an acetone–n-butyl alcohol–glacial acetic acid–water (35:35:10:20) mixture as the mobile phase. Aliquots (1 μl) of l-[U-14C]glutamate, l-[U-14C]aspartate, l-[U-14C]arginine, and l-[U-14C]proline (50 nCi μl−1) were run as standards in parallel with the samples derived from the cell extracts. After a 3- to 5-day exposure of the dried silica gel plates to a phosphor screen, radiolabeled compounds were detected with a Storm 860 phosphorimager (Amersham Biosciences, Freiburg, Germany).

Uptake of radiolabeled glutamate and aspartate by B. subtilis.Uptake rates for radiolabeled l-[U-14C]glutamate and l-[U-14C]aspartate were compared in cells of the B. subtilis wild-type strain JH642 and mutants derived from this strain that carried gene disruptions either in gltT (strain ADB4), in gltP (strain ADB1), or in yveA (strain MDB43) (Table 1). The cultures were grown in SMM with glucose as the carbon source to early exponential growth phase (OD578, 0.5 to 0.7), and glutamate or aspartate spiked with radiolabeled l-[U-14C]glutamate or l-[U-14C]aspartate (specific activity for both solutes, 2.25 nCi nmol−1) was added to 2-ml aliquots of the cultures. Uptake of these amino acids was followed at 30-s intervals by measuring the radioactivity accumulated by the cells as described previously (43). To assess the kinetic parameters of the GltT transporter for glutamate and aspartate, the substrate concentrations were varied between 2 μM and 100 μM and the uptake rates were determined. The Michaelis-Menten kinetics was deduced by comparing the uptake velocities in relation to the substrate concentration in the uptake assays. Analysis and fitting of the transport data were performed using the GraphPad Prism 5 software (Graphpad Software, Inc., La Jolla, CA, USA). Three independent measurements were performed to determine the Km and Vmax values of the GltT carrier for its substrates, Glu and Asp.

Internet resources for B. subtilis and modeling of the GltT structure.The functional annotation of B. subtilis genes and their transcriptional profile measured across a comprehensive set of growth conditions (44) were assessed through SubtiWiki (http://subtiwiki.uni-goettingen.de/wiki/index.php/Main_Page) (45). The modeling of the B. subtilis GltT structure was carried out with resources provided by the I-TASSER protein structure prediction platform (http://zhanglab.ccmb.med.umich.edu/I-TASSER/) (46). Graphic representations of the retrieved in silico GltT model were prepared using the PyMOL software package (http://www.pymol.org).

RESULTS

Osmostress protection by amino acids.Both proteogenic and nonproteogenic amino acids can be found in the soil (8, 10), and they exhibit a high degree of turnover, a process to which microorganisms contribute substantially (47). We wondered whether the uptake of amino acids other than proline (25, 26) would aid B. subtilis in its process of adjustment to high-salinity surroundings. To test this idea, we grew the wild-type laboratory strain JH642 (Table 1) in a chemically defined minimal medium (SMM) containing 1.2 M NaCl in the absence or presence of individual proteogenic amino acids and the nonproteogenic amino acids ornithine (Orn) and citrulline (Cit). As expected, the osmostress protectants GB (16) and Pro (24, 25) enhance growth at high salinity (Fig. 2), but osmostress protection was also afforded by Glu, glutamine (Gln), Asp, asparagine (Asn), and Arg and by both Orn and Cit (Fig. 2).

FIG 2
  • Open in new tab
  • Download powerpoint
FIG 2

Effects of amino acids on the growth of B. subtilis at high osmolarity. Cultures of the B. subtilis wild-type strain JH642 were grown in SMM with 1.2 M NaCl in the absence or presence of individual proteogenic and nonproteogenic amino acids (final concentration, 1 mM). The growth yields of the cultures were determined after 16 h of incubation at 37°C by measuring the OD578. The values shown represent the means of two independently grown cultures, and the error bars indicate standard deviations. The gray bars represent the amino acids that promote growth of B. subtilis at high salinity. The osmoprotectant GB (hatched bar) was used as a control for these experiments.

Osmostress protection by amino acids is dependent on the ProJ-ProA-ProH proline-biosynthetic route.The proline and arginine synthesis and degradation routes of B. subtilis are highly interconnected (32, 34, 48, 49). By consulting descriptions of the appropriate synthesis and metabolic pathways and uptake systems in the literature (6, 34, 48, 49) (Fig. 1 shows a summary), it became apparent that each of the osmostress-relieving amino acids identified above could be converted into proline, whereas this is not the case for each of the amino acids that did not confer osmostress protection. Hence, it seemed possible that Glu, Gln, Asp, Asn, Arg, Orn, and Cit were not osmostress protectants per se but instead served as resources for proline synthesis (Fig. 1).

The key players in the osmostress-adaptive synthesis of proline from the precursor glutamate by B. subtilis are the ProJ-ProA-ProH enzymes (18, 21, 31) (Fig. 1). We therefore used a ΔproHJ mutant that is unable to synthesize osmostress-protective levels of proline to experimentally verify the anticipated roles of Glu, Gln, Asp, Asn, Arg, Orn, and Cit as precursors for osmoprotective proline biosynthesis. Except for Pro itself (25, 26), osmostress protection by each of these amino acids was lost when the ProJ-ProH enzymes were nonoperational (Fig. 3A). This was caused by the failure of the cells to build up the large proline pool required for adjustment to high external salinity (33) (Fig. 3C). Hence, the osmostress potentials of the seven tested amino acids were indeed dependent on their metabolic conversion into proline (Fig. 1).

FIG 3
  • Open in new tab
  • Download powerpoint
FIG 3

Osmoprotection by amino acids depends on the osmostress-adaptive proline-biosynthetic route. (A) Cultures of the B. subtilis wild-type strain JH642 (black bars) and of strain JSB8 [Δ(proHJ::tet)1] (gray bars) were grown in SMM with 1.2 M NaCl in the absence or presence of individual proteogenic and nonproteogenic amino acids (final concentration, 1 mM). The growth yields of the cultures were determined after 16 h of incubation at 37°C by measuring the OD578. (B) Influence of the PutBCP catabolic system on the levels of osmostress protection by proline, glutamate, and arginine. The following strains were used: JH642 (black bars), JSB8 [Δ(proHJ::tet)1] (gray bars), and ABB1 [Δ(proHJ::tet)1 Δ(putBCP::tet)2] (hatched bars). (C) Proline content of osmotically stressed cells. Cells grown in SMM containing 1.2 M NaCl were harvested once they reached an OD578 of about 1.8 and were then assayed for their intracellular l-proline pools. The following strains were used: JH642 (black bars) and JSB8 [Δ(proHJ::tet)1] (gray bars). (D) Influence of the PutBCP catabolic system on the buildup of intracellular proline pools. The following strains (grown as described above) were used: JH642 (black bars), JSB8 [Δ(proHJ::tet)1] (gray bars), and ABB1 [Δ(proHJ::tet)1 Δ(putBCP::tet)2] (hatched bars). (C and D) The values shown represent the means of two independently grown B. subtilis cultures, and in each of these samples, the l-proline content was determined twice; the error bars indicate standard deviations.

It is apparent from the interconnected proline and arginine synthesis and degradation routes in B. subtilis (34, 48) that stress relief by Glu, Gln, Asn, and Asp should be dependent on a functional γ-glutamyl-phosphate reductase (ProA) (Fig. 1). In contrast, osmostress protection afforded by Arg, Orn, and Cit should be independent of the functioning of the ProA enzyme, since these amino acids can be metabolized to γ-glutamic semialdehyde/Δ1-pyrroline-5-carboxylate, a process that requires the activity of the ornithine aminotransferase (RocD) (33, 34). This intermediate can be fed into the proline synthesis pathway at an intersection subsequent to the ProA-catalyzed step (Fig. 1).

To substantiate this hypothesis, we used growth experiments with the B. subtilis strain GWB120, a mutant that is defective in the proBA genes and simultaneously carries a promoter mutation that allows enhanced rocDEF transcription in the absence of its natural RocR-dependent inducers (33). We note in this context that in this strain the function of ProB can be substituted for by its paralogue ProJ (Fig. 1); however, only a single ProA-type enzyme is present in B. subtilis (18, 21, 31). Fully consistent with the working hypothesis outlined above, osmostress protection by Glu, Gln, Asn, and Asp was dependent on a functional ProA protein, whereas that afforded by Arg, Orn, and Cit was not (see Fig. S1 in the supplemental material). Consequently, osmostress protection by Glu, Gln, Asp, and Asn on one hand and Arg, Orn, and Cit on the other hand is dependent on different entry points into the osmostress-adaptive ProJ-ProA-ProH proline biosynthesis route (Fig. 1).

As noted previously (24), externally provided proline confers osmostress protection (Fig. 3A), but surprisingly, the cells do not build up a sizable proline pool in a ΔproHJ mutant (18) (Fig. 3C). This phenomenon is not fully understood but is related to proline catabolism via the PutBCP system, a proline import (PutP) and degradation (PutBC) route that can be transcriptionally induced by externally provided proline but not by proline synthesized by the cell (24, 30, 50). Accordingly, the disruption of the PutBCP system in a ΔproHJ genetic mutant background led to the buildup of a very large proline pool in osmotically stressed cells that were grown in the presence of 1 mM proline in the growth medium. The size of this proline pool matched that of the pool formed through de novo synthesis in the wild-type strain in the absence of an external proline supply (Fig. 3D).

Influence of the uptake of osmostress-protective amino acids on the size of the intracellular proline pool.We wondered whether growth of the B. subtilis cells under high-salinity conditions in the presence of the osmostress protectants Glu, Gln, Asp, Asn, Arg, Orn, and Cit (Fig. 3A) would increase the cellular proline pool size and would thereby enhance growth under osmotically unfavorable conditions. However, this was not the case. When presented with an osmostress-protective amino acid, the cells accumulated the same amount of proline as cells provided with no amino acid (Fig. 3C). Consequently, the question arose as to how osmostress protection by Glu, Gln, Asp, Asn, Arg, Orn, and Cit is achieved by B. subtilis.

Tracing the metabolic conversion of osmostress-protective amino acids into proline.Synthesis of the direct proline precursor glutamate (Fig. 1) is energetically demanding (32, 34), and osmotically stressed B. subtilis cells successively drain the substantial glutamate pool (14), concomitant with the onset of enhanced proline production (18). It thus seems possible that the cells would save energy and biosynthetic building blocks for the de novo production of glutamate (32, 51) either by importing it directly or by metabolically converting exogenously provided Gln, Asp, and Asn into glutamate (Fig. 1). Conversely, Arg, Orn, and Cit could exert their osmostress-protective effects through their conversion into γ-glutamic semialdehyde/Δ1-pyrroline-5-carboxylate through the metabolic activities of the RocDEF enzymes (Fig. 1) (34, 48, 52).

To follow the anticipated metabolic conversion of these amino acids into proline, we separately fed radiolabeled Pro, Glu, Asp, and Arg (final concentration of each amino acid, 20 μM) to cells that were grown in SMM with 1.2 M NaCl. We harvested the cells at 30-min intervals over a time span of 5 h and then separated the soluble fraction of cell extracts by thin-layer chromatography, along with appropriate radiolabeled reference standards. l-[U-14C]Pro was accumulated in unmodified form (Fig. 4A), whereas l-[U-14C]Glu, l-[U-14C]Asp, and l-[U-14C]Arg were taken up by the cells and were then metabolically converted into proline (Fig. 4B, C, and D). These metabolic-labeling experiments are thus consistent with the working hypothesis outlined above, which invokes the metabolic conversion of osmostress-protective amino acids into the compatible solute proline. We note that in each of the experiments, part of the imported radiolabeld amino acid was incorporated into cellular material that did not migrate in the thin-layer chromatography, and the amount of this material increased over time (Fig. 5). At least part of the material might be newly synthesized proteins.

FIG 4
  • Open in new tab
  • Download powerpoint
FIG 4

Conversion of Glu, Asp, and Arg into Pro in B. subtilis wild-type cells growing at high salinity. Cultures of the B. subtilis strain JH642 were grown in SMM with 1.2 M NaCl. When the cultures reached an OD578 of 0.5, l-[U-14C]proline (A), l-[U-14C]glutamate (B), l-[U-14C]aspartate (C), and l-[U-14C]arginine (D) were separately added to the cells (final concentration, 20 μM). Cell samples were withdrawn at the indicated time points, and the cells were harvested by centrifugation. Soluble extracts of the cell pellets were prepared and separated by thin-layer chromatography; spots corresponding to Pro, Glu, Asn, and Arg were identified through comigrating radiolabeled reference standards. Unidentified radiolabeled compounds, in all likelihood metabolic intermediates of the imported amino acids, are indicated by asterisks.

FIG 5
  • Open in new tab
  • Download powerpoint
FIG 5

Uptake of radiolabeled glutamate and aspartate by B. subtilis and its gltP, gltT, and yveA mutant derivatives. (A and C) Strains JH642 (wild type) (●), ADB1 (gltP) (■), ADB4 (gltT) (▲), and MDB43 (yveA) (◆) were grown in SMM, and the initial uptake of l-[U-14C]glutamate (A) and l-[U-14C]aspartate (C) was measured at a final substrate concentration of 20 μM. (B and D) Michaelis-Menten kinetics were deduced from uptake rates for the GltT substrates l-[U-14C]glutamate (B) and l-[U-14C]aspartate (D) in strain MDB53, which possesses an intact GltT system but is defective in the GltP and YveA transporters.

GltT-mediated import of glutamate and aspartate.Since glutamate is the direct precursor for the synthesis of proline (32, 34, 48), we wondered how the amino acid is imported by B. subtilis. The GltP and YveA transporters have been implicated in glutamate uptake by B. subtilis, but the process is not satisfactorily understood. GltP, a glutamate/aspartate H+ symporter, has been cloned by functional complementation in Escherichia coli, and the purified protein has also been reconstituted in membrane vesicles (53); however, its physiological and transport properties have not been directly assessed in B. subtilis through mutant analysis. YveA, a member of the amino acid/polyamine/organocation (APC) superfamily, has been implicated in glutamate uptake, but the YveA transporter is not substrate specific, since it mediates the import of a broad range of amino acids, including that of aspartate (41). A third glutamate/aspartate transporter, GltT, is predicted from the B. subtilis genome sequence (5, 6), but its physiological contribution to glutamate import has not been studied. The amino acid sequence of this putative glutamate/aspartate Na+ symporter (6) is related to biochemically studied GltT homologs from Bacillus caldotenax and Bacillus stearothermophilus (54, 55), and it possesses overall degrees of amino acid sequence identity of 65% and 66%, respectively, to these proteins.

To characterize the contribution of the GltP, GltT, and YveA transporters to glutamate uptake, we constructed an isogenic set of B. subtilis mutant strains in which the corresponding genes were separately disrupted. We then measured the initial uptake of radiolabeled glutamate at a low substrate concentration (20 μM) in cells that were grown at 37°C in SMM. Loss of the GltP and YveA systems did not affect the uptake of glutamate, whereas the inactivation of GltT practically abrogated its import (Fig. 5A). Since the GltP, YveA, and GltT transporters are also potential uptake systems for aspartate (41, 53), we tested the uptake of radiolabeled aspartate in the same set of strains and found that only the disruption of gltT affected its uptake (Fig. 5C). Hence, GltT is an important uptake system for Glu and Asp when they are present at low (μM) external substrate concentrations and presented to B. subtilis cells cultured to early exponential phase in a minimal medium with glucose as the carbon and (NH4)2SO4 as the nitrogen source.

We determined the kinetic parameters of GltT for glutamate and aspartate in a strain (MDB53) that was simultaneously defective in the GltP and YveA transporters by varying the substrate concentration between 2 μM and 100 μM. GltT-mediated import of glutamate and aspartate exhibited Michaelis-Menten kinetics, and the affinities of the transporter for the two substrates were very similar, with Km values of 37 ± 5 μM for glutamate and 41 ± 9 μM for aspartate (Fig. 5B and D). GltT is not only a high-affinity transporter, it also possesses a substantial transport capacity, with Vmax values of 107 ± 6 nmol (min mg protein)−1 for glutamate and 60 ± 6 nmol (min mg protein)−1 for aspartate (Fig. 5B and D).

The B. subtilis GltT protein is related to the structurally characterized GltPh and GltTk proteins from the archaea Pyrococcus horikoshii (56) and Thermococcus kodakarensis (57), with an overall degree of amino acid sequence identity of approximately 32% (see Fig. S2 in the supplemental material). The last two proteins are aspartate transporters, and the residues that have been implicated in substrate binding are for the most part functionally conserved in the B. subtilis GltT protein (see Fig. S2 in the supplemental material). The GltPh and GltTk proteins are trimers, with each monomer possessing structurally separable transport and trimerization domains (56–59). A modeling study of the B. subtilis GltT protein using the I-TASSER Web server resources (46) automatically chose the P. horikoshii GltPh crystal structure containing the aspartate ligand (Protein Data Bank [PDB; http://www.rcsb.org/pdb/home/home.do] accession code 2NWX) (56) as an appropriate template. In Fig. S3 in the supplemental material, we show an overlay of a GltT monomer with one protomer of the GltPh trimeric crystal structure. This in silico model suggests that the overall fold of the B. subtilis GltT protein resembles that of GltPh and GltTk (56–59), with structurally distinct transport and trimerization domains.

GltT is involved in osmostress protection afforded by glutamate and aspartate.Having identified GltT as a system for glutamate and aspartate in exponentially growing B. subtilis cells in a minimal medium with glucose as the carbon source, we studied the role of GltT in glutamate- and aspartate-mediated osmostress protection. For these experiments, we conducted growth studies in SMM containing 1.2 M NaCl and compared the performance of these osmoprotectants with that of the compatible solutes glycine betaine (16) and proline (26, 33). Glutamate and aspartate exhibited an osmostress-protective potential similar to that afforded by proline in the wild-type strain JH642, but glycine betaine was a considerably better osmoprotectant than these compounds (Fig. 6A). In a gltT mutant strain (ADB4), osmoprotection by glutamate and aspartate was significantly reduced, and the growth of these cultures resembled that of a culture that had not received any osmostress protectant. In contrast, the osmoprotective effects of proline and glycine betaine were not affected by a gltT mutation (Fig. 6B). As expected from the data presented above, osmoprotection by glutamate and aspartate was abolished in the proHJ mutant strain JSB8 (Fig. 6C), whereas that afforded by proline and glycine betaine was unaffected (Fig. 6B and C). Taken together, our data show that the GltT transporter is an important contributor for the use of glutamate and aspartate as osmostress protectants by B. subtilis.

FIG 6
  • Open in new tab
  • Download powerpoint
FIG 6

Osmostress protection of B. subtilis by glutamate and aspartate is dependent on the GltT transporter and the functioning of the ProHJ proline-biosynthetic system. Cells of the wild-type strain JH642 (A), strain ADB4 (gltT × pUS19) (B), and strain JSB8 [Δ(proHJ::tet)1] (C) were grown at high salinity (SMM with 1.2 M NaCl) in the absence (○) or the presence of glutamate (●), aspartate (■), proline (▲), and glycine betaine (◆); the final concentration of these compounds in the growth medium was 1 mM.

Transcription of gltT is not induced in response to osmotic stress.The expression of opuE, the dominant importer for the use of exogenously provided proline as an osmostress protectant (25, 26), is upregulated in response to increases in the environmental osmolarity (25, 60). Given the involvement of GltT in the use of glutamate and aspartate as osmostress protectants (Fig. 6), we wondered whether gltT transcription would be induced in response to sustained high salinity, as well. However, Northern blot analysis with a gltT-specific antisense mRNA probe showed that this is not the case (see Fig. S4 in the supplemental material).

GltT contributes to glutamate or aspartate import when they are used as the sole nitrogen source.Amino acids can be used by B. subtilis as sole nitrogen sources (49), and we verified that this was also the case for glutamate and aspartate (Fig. 7). SMM contains 15 mM (NH4)2SO4 as the ammonium source. When the ammonium source was reduced to 2.5 mM, growth of the cells was limited (Fig. 7); the same growth yield was afforded through the supply of either glutamate or aspartate when an equivalent nitrogen concentration of these amino acids (5 mM was added to the medium) was used. Under these nitrogen-limiting conditions, loss of the GltT system had a strong effect on the growth yields of the cultures, whereas the simultaneous disruption of the glutamate/aspartate transporters GltP and YveA (41, 53) in a strain with an intact GltT transporter had no influence on glutamate and aspartate utilization as a nitrogen source (Fig. 7). Of note is our observation that glutamate and aspartate could both still be used as sole nitrogen sources in a triple-mutant strain (MDB54) simultaneously lacking the GltT, GltP, and YveA systems (Fig. 7), and this effect became more pronounced when the concentrations of these amino acids were raised to either 15 or 30 mM (Fig. 7). Hence, our growth assays not only revealed an important role of GltT in the use of glutamate and aspartate as nitrogen sources, but also uncovered the existence of a yet undisclosed glutamate and aspartate transport system in B. subtilis.

FIG 7
  • Open in new tab
  • Download powerpoint
FIG 7

The B. subtilis wild-type strain JH642 (gray bars) and its mutant derivatives that were defective in the indicated glutamate/aspartate transporter GltT, GltP, or YveA (black bars) were tested for the ability to use glutamate or aspartate as the sole nitrogen source. We grew these strains in minimal medium without a nitrogen source in the presence of either 2.5 mM, 7.5 mM, or 15 mM (NH4)2SO4 or with 5 mM, 15 mM, or 30 mM glutamate or aspartate, respectively, and then measured the growth yields of the cultures after 15 h of incubation at 37°C. For each growth condition tested, four independent cultures were used, and the means and standard deviations are shown.

DISCUSSION

B. subtilis is resourceful in the ways in which it can derive osmostress protection by the compatible solute proline. Four routes are now known through which this can occur: (i) through osmotically stimulated de novo synthesis (14, 18), (ii) through osmostress-induced import of free proline via the OpuE transporter (25, 27), (iii) through the import of proline-containing peptides and their subsequent intracellular hydrolysis (24), and, as shown here for the first time, (iv) through the uptake of proteogenic and nonproteogenic amino acids that can be metabolically converted into proline (Fig. 2 and 3). For the last process, the osmotically controlled ProJ-ProA-ProH biosynthetic route (18) (Fig. 3) is required, but different entry points into this pathway are used by different osmostress-protective amino acids to finally yield the compatible solute proline (Fig. 1).

The feeding of osmoprotective amino acids does not enhance the proline pool beyond the size that is already found in osmotically stressed cells (Fig. 3C). This finding rules out a model that would rely on a larger than normal cytoplasmic pool of proline to explain the osmostress-protective effects of an external supply of certain amino acids (Fig. 2 and 3). We are thus led to the conclusion that the higher growth yield of osmotically stressed cells cultured in the presence of Glu, Gln, Asp, Asn, Arg, Orn, and Cit than of those that did not receive an osmoprotective amino acid (Fig. 2) rests on the saving of precious biosynthetic building blocks and energy sources that would otherwise have to be devoted under high-osmolarity growth conditions (61) to the synthesis of the proline precursor glutamate (31, 32) or of proline itself (31, 51). The highly interconnected proline and arginine synthesis and degradation pathways of B. subtilis (34, 48, 49) (Fig. 1) thus provide the physiological foundation for the osmoprotective effects of a selected set of proteogenic and nonproteogenic amino acids.

We note that each of the amino acids that we have identified as osmoprotectants has been detected in the soil (8–10, 47), one of the prime habitats of B. subtilis (1). High-affinity transport systems should allow their scavenging from scarce environmental resources. Transporters for Pro, Gln, and Arg have been identified in B. subtilis (25, 26, 30, 52, 62–65), whereas the identities of the import systems for Asn, Orn, and Cit are either less certain or unknown (52, 62) (Fig. 1 shows an overview). A full understanding of the import routes for Glu and Asp in B. subtilis is also lacking. We found that the activity of the Na+-coupled symporter GltT, a transporter that has not been previously functionally studied in B. subtilis, is an important contributor to the uptake of Glu and Asp by exponentially growing cells when their external concentrations are either low (20 μM) (Fig. 4), moderate (1 mM) (Fig. 6), or high (5 mM to 15 mM) (Fig. 6 and 7).

Transcription of the gene (opuE) for the main uptake system (OpuE) of the osmoprotectant proline in B. subtilis is induced by high salinity (25, 60), but this was not the case for the gltT gene (see Fig. S3 in the supplemental material). Inspection of the transcriptional profile of gltT obtained through a genome-wide tiling array study (44) revealed that it is expressed under a large set of growth conditions at similar levels, except in stationary or sporulating cells, where gltT expression is downregulated (44). Notably, expression of gltT is under the control of CodY, a globally acting transcription factor regulating the expression of a large set of metabolic genes in B. subtilis, in particular, those involved in amino acid metabolism (66). This fits nicely with our finding that the GltT transporter plays an important role in the import of Glu and Asp when these amino acids are used as sole nitrogen sources under conditions where they are supplied in growth-limiting amounts (Fig. 7).

The GltP and YveA transporters have also been implicated in the uptake of these amino acids by B. subtilis (41, 53). However, transport assays conducted by us in exponentially growing cells (Fig. 5) and osmostress protection assays (Fig. 6) did not uncover a substantial contribution of these carriers to the import of Glu and Asp. Furthermore, when these amino acids were used as sole nitrogen sources, effects of the GltP and YveA transporters were also not noticeable (Fig. 7). The transport activities of GltP and YveA might thus be physiologically relevant under growth conditions different from those tested by us. Indeed, the level of the gltP transcript varies in response to the type of carbon source available (67). Furthermore, transcriptional data obtained in a tiling array study examining more than 100 growth conditions (44) demonstrate that gltP expression is upregulated in cells that swarm, grow on solid media, enter stationary phase, or proceed to form spores. Under these conditions, very strong upregulation in the transcription of yveA is also observed, and the sporulation-specific transcription factor SigG has been implicated in its genetic control (68). Moreover, upregulation of yveA is observed under anaerobic growth conditions when the B. subtilis cells respire nitrate or undergo fermentation (44).

The cellular adjustment of B. subtilis to high-osmolarity environments is a well-staged and complex process (61, 69–71). However, the effective management of water fluxes in or out of the cell (12, 72) and the fine-tuning of the solvent properties of the cytoplasm (19, 73) are key events that allow cell proliferation under otherwise growth-inhibiting conditions (7, 11, 13). The amassing of the compatible solute proline plays an important part in the acclimatization process of the B. subtilis cell to unfavorably osmotic conditions. We surmise that the import of proteogenic and nonproteogenic amino acids that can be metabolically converted into the osmoprotectant proline should enhance the competitiveness of the bacterium in its varied natural habitats (1–3).

ACKNOWLEDGMENTS

We greatly value the expert technical assistance of Jutta Gade and thank Milton Saier (UC San Diego, La Jolla, CA, USA) for providing us with the B. subtilis yveA mutant strain. We are indebted to Vickie Koogle for her kind help in the language editing of our manuscript. E.B. greatly appreciated the hospitality of Tom Silhavy during a sabbatical at the Department of Molecular Biology of Princeton University (Princeton, NJ, USA).

Funding for this study was provided by a grant from the BMBF via the Bacell-SysMo2 consortium, by contributions from the LOEWE program of the state of Hessen (via the Center for Synthetic Microbiology, Marburg, Germany), and by the Fonds der Chemischen Industrie. A.Z. was an associate member of the International Max Planck Graduate School for Environmental, Cellular and Molecular Microbiology (IMPRS-Mic; Marburg, Germany) and gratefully acknowledges its support.

FOOTNOTES

    • Received 26 August 2014.
    • Accepted 15 October 2014.
    • Accepted manuscript posted online 24 October 2014.
  • Supplemental material for this article may be found at http://dx.doi.org/10.1128/AEM.02797-14.

  • Copyright © 2015, American Society for Microbiology. All Rights Reserved.

REFERENCES

  1. 1.↵
    1. Earl AM,
    2. Losick R,
    3. Kolter R
    . 2008. Ecology and genomics of Bacillus subtilis. Trends Microbiol 16:269–275. doi:10.1016/j.tim.2008.03.004.
    OpenUrlCrossRefPubMedWeb of Science
  2. 2.↵
    1. Logan N,
    2. De Vos P
    . 2009. Bacillus, p 21–128. In De Vos P, Garrity GM, Jones D, Krieg NR, Ludwig W, Rainey FR, Schleifer K-H, Whitman WB (ed), Bergey's manual of systematic bacteriology, vol 3. Springer, Heidelberg, Germany.
    OpenUrl
  3. 3.↵
    1. Beauregard PB,
    2. Chai Y,
    3. Vlamakis H,
    4. Losick R,
    5. Kolter R
    . 2013. Bacillus subtilis biofilm induction by plant polysaccharides. Proc Natl Acad Sci U S A 110:E1621–E1630. doi:10.1073/pnas.1218984110.
    OpenUrlAbstract/FREE Full Text
  4. 4.↵
    1. Belda E,
    2. Sekowska A,
    3. Le Fevre F,
    4. Morgat A,
    5. Mornico D,
    6. Ouzounis C,
    7. Vallenet D,
    8. Medigue C,
    9. Danchin A
    . 2013. An updated metabolic view of the Bacillus subtilis 168 genome. Microbiology 159:757–770. doi:10.1099/mic.0.064691-0.
    OpenUrlCrossRefPubMedWeb of Science
  5. 5.↵
    1. Barbe V,
    2. Cruveiller S,
    3. Kunst F,
    4. Lenoble P,
    5. Meurice G,
    6. Sekowska A,
    7. Vallenet D,
    8. Wang T,
    9. Moszer I,
    10. Medigue C,
    11. Danchin A
    . 2009. From a consortium sequence to a unified sequence: the Bacillus subtilis 168 reference genome a decade later. Microbiology 155:1758–1775. doi:10.1099/mic.0.027839-0.
    OpenUrlCrossRefPubMedWeb of Science
  6. 6.↵
    1. Saier MH, Jr,
    2. Goldman SR,
    3. Maile RR,
    4. Moreno MS,
    5. Weyler W,
    6. Yang N,
    7. Paulsen IT
    . 2002. Overall transport capabilities of Bacillus subtilis, p 113–128. In Sonenshein AL, Hoch JA, Losick R (ed), Bacillus subtilis and its closest relatives: from genes to cells. ASM Press, Washington, DC.
  7. 7.↵
    1. Bremer E
    . 2002. Adaptation to changing osmolarity, p 385–391. In Sonenshein AL, Hoch JA, Losick R (ed), Bacillus subtilis and its closest relatives: from genes to cells. ASM Press, Washington, DC.
  8. 8.↵
    1. Moe LA
    . 2013. Amino acids in the rhizosphere: from plants to microbes. Am J Bot 100:1692–1705. doi:10.3732/ajb.1300033.
    OpenUrlAbstract/FREE Full Text
  9. 9.↵
    1. Warren CR
    . 2013. Quaternary ammonium compounds can be abundant in some soils and are taken up as intact molecules by plants. New Phytol 198:476–485. doi:10.1111/nph.12171.
    OpenUrlCrossRefPubMed
  10. 10.↵
    1. Warren CR
    . 2014. Response of osmolytes in soil to drying and rewetting. Soil Biol Biochem 70:22–32. doi:10.1016/j.soilbio.2013.12.008.
    OpenUrlCrossRef
  11. 11.↵
    1. Bremer E,
    2. Krämer R
    . 2000. Coping with osmotic challenges: osmoregulation through accumulation and release of compatible solutes, p 79–97. In Storz G, Hengge-Aronis R (ed), Bacterial stress responses. ASM Press, Washington, DC.
  12. 12.↵
    1. Kempf B,
    2. Bremer E
    . 1998. Uptake and synthesis of compatible solutes as microbial stress responses to high osmolality environments. Arch Microbiol 170:319–330. doi:10.1007/s002030050649.
    OpenUrlCrossRefPubMedWeb of Science
  13. 13.↵
    1. Csonka LN,
    2. Hanson AD
    . 1991. Prokaryotic osmoregulation: genetics and physiology. Annu Rev Microbiol 45:569–606. doi:10.1146/annurev.mi.45.100191.003033.
    OpenUrlCrossRefPubMedWeb of Science
  14. 14.↵
    1. Whatmore AM,
    2. Chudek JA,
    3. Reed RH
    . 1990. The effects of osmotic upshock on the intracellular solute pools of Bacillus subtilis. J Gen Microbiol 136:2527–2535. doi:10.1099/00221287-136-12-2527.
    OpenUrlCrossRefPubMedWeb of Science
  15. 15.↵
    1. Holtmann G,
    2. Bakker EP,
    3. Uozumi N,
    4. Bremer E
    . 2003. KtrAB and KtrCD: two K+ uptake systems in Bacillus subtilis and their role in adaptation to hypertonicity. J Bacteriol 185:1289–1298. doi:10.1128/JB.185.4.1289-1298.2003.
    OpenUrlAbstract/FREE Full Text
  16. 16.↵
    1. Boch J,
    2. Kempf B,
    3. Bremer E
    . 1994. Osmoregulation in Bacillus subtilis: synthesis of the osmoprotectant glycine betaine from exogenously provided choline. J Bacteriol 176:5364–5371.
    OpenUrlAbstract/FREE Full Text
  17. 17.↵
    1. Hoffmann T,
    2. Wensing A,
    3. Brosius M,
    4. Steil L,
    5. Völker U,
    6. Bremer E
    . 2013. Osmotic control of opuA expression in Bacillus subtilis and its modulation in response to intracellular glycine betaine and proline pools. J Bacteriol 195:510–522. doi:10.1128/JB.01505-12.
    OpenUrlAbstract/FREE Full Text
  18. 18.↵
    1. Brill J,
    2. Hoffmann T,
    3. Bleisteiner M,
    4. Bremer E
    . 2011. Osmotically controlled synthesis of the compatible solute proline is critical for cellular defense of Bacillus subtilis against high osmolarity. J Bacteriol 193:5335–5346. doi:10.1128/JB.05490-11.
    OpenUrlAbstract/FREE Full Text
  19. 19.↵
    1. Wood JM
    . 2011. Bacterial osmoregulation: a paradigm for the study of cellular homeostasis. Annu Rev Microbiol 65:215–238. doi:10.1146/annurev-micro-090110-102815.
    OpenUrlCrossRefPubMedWeb of Science
  20. 20.↵
    1. Welsh DT
    . 2000. Ecological significance of compatible solute accumulation by micro-organisms: from single cells to global climate. FEMS Microbiol Rev 24:263–290. doi:10.1111/j.1574-6976.2000.tb00542.x.
    OpenUrlCrossRefPubMedWeb of Science
  21. 21.↵
    1. Brill J,
    2. Hoffmann T,
    3. Putzer H,
    4. Bremer E
    . 2011. T-box-mediated control of the anabolic proline biosynthetic genes of Bacillus subtilis. Microbiology 157:977–987. doi:10.1099/mic.0.047357-0.
    OpenUrlCrossRefPubMedWeb of Science
  22. 22.↵
    1. Fujita T,
    2. Maggio A,
    3. Garcia-Rios M,
    4. Stauffacher C,
    5. Bressan RA,
    6. Csonka LN
    . 2003. Identification of regions of the tomato gamma-glutamyl kinase that are involved in allosteric regulation by proline. J Biol Chem 278:14203–14210. doi:10.1074/jbc.M212177200.
    OpenUrlAbstract/FREE Full Text
  23. 23.↵
    1. Chen M,
    2. Wei H,
    3. Cao J,
    4. Liu R,
    5. Wang Y,
    6. Zheng C
    . 2007. Expression of Bacillus subtilis proBA genes and reduction of feedback inhibition of proline synthesis increases proline production and confers osmotolerance in transgenic Arabidopsis. J Biochem Mol Biol 40:396–403. doi:10.5483/BMBRep.2007.40.3.396.
    OpenUrlCrossRefPubMed
  24. 24.↵
    1. Zaprasis A,
    2. Brill J,
    3. Thüring M,
    4. Wünsche G,
    5. Heun M,
    6. Barzantny H,
    7. Hoffmann T,
    8. Bremer E
    . 2013. Osmoprotection of Bacillus subtilis through import and proteolysis of proline-containing peptides. Appl Environ Microbiol 79:576–587. doi:10.1128/AEM.01934-12.
    OpenUrlAbstract/FREE Full Text
  25. 25.↵
    1. von Blohn C,
    2. Kempf B,
    3. Kappes RM,
    4. Bremer E
    . 1997. Osmostress response in Bacillus subtilis: characterization of a proline uptake system (OpuE) regulated by high osmolarity and the alternative transcription factor sigma B. Mol Microbiol 25:175–187. doi:10.1046/j.1365-2958.1997.4441809.x.
    OpenUrlCrossRefPubMed
  26. 26.↵
    1. Zaprasis A,
    2. Hoffmann T,
    3. Stannek L,
    4. Gunka K,
    5. Commichau FM,
    6. Bremer E
    . 2014. The gamma-aminobutyrate permease GabP serves as the third proline transporter of Bacillus subtilis. J Bacteriol 196:515–526. doi:10.1128/JB.01128-13.
    OpenUrlAbstract/FREE Full Text
  27. 27.↵
    1. Hoffmann T,
    2. von Blohn C,
    3. Stanek A,
    4. Moses S,
    5. Barzantny S,
    6. Bremer E
    . 2012. Synthesis, release, and recapture of the compatible solute proline by osmotically stressed Bacillus subtilis cells. Appl Environ Microbiol 78:5753–5762. doi:10.1128/AEM.01040-12.
    OpenUrlAbstract/FREE Full Text
  28. 28.↵
    1. Hoffmann T,
    2. Bremer E
    . 2011. Protection of Bacillus subtilis against cold stress via compatible-solute acquisition. J Bacteriol 193:1552–1562. doi:10.1128/JB.01319-10.
    OpenUrlAbstract/FREE Full Text
  29. 29.↵
    1. Bashir A,
    2. Hoffmann T,
    3. Kempf B,
    4. Xie X,
    5. Smits SH,
    6. Bremer E
    . 2014. The plant-derived compatible solutes proline betaine and betonicine confer enhanced osmotic and temperature stress tolerance to Bacillus subtilis. Microbiology 160:2283–2294. doi:10.1099/mic.0.079665-0.
    OpenUrlCrossRefPubMed
  30. 30.↵
    1. Moses S,
    2. Sinner T,
    3. Zaprasis A,
    4. Stöveken N,
    5. Hoffmann T,
    6. Belitsky BR,
    7. Sonenshein AL,
    8. Bremer E
    . 2012. Proline utilization by Bacillus subtilis: uptake and catabolism. J Bacteriol 194:745–758. doi:10.1128/JB.06380-11.
    OpenUrlAbstract/FREE Full Text
  31. 31.↵
    1. Belitsky BR,
    2. Brill J,
    3. Bremer E,
    4. Sonenshein AL
    . 2001. Multiple genes for the last step of proline biosynthesis in Bacillus subtilis. J Bacteriol 183:4389–4392. doi:10.1128/JB.183.14.4389-4392.2001.
    OpenUrlAbstract/FREE Full Text
  32. 32.↵
    1. Gunka K,
    2. Commichau FM
    . 2012. Control of glutamate homeostasis in Bacillus subtilis: a complex interplay between ammonium assimilation, glutamate biosynthesis and degradation. Mol Microbiol 85:213–224. doi:10.1111/j.1365-2958.2012.08105.x.
    OpenUrlCrossRefPubMed
  33. 33.↵
    1. Zaprasis A,
    2. Hoffmann T,
    3. Wünsche G,
    4. Florez LA,
    5. Stülke J,
    6. Bremer E
    . 2014. Mutational activation of the RocR activator and of a cryptic rocDEF promoter bypass loss of the initial steps of proline biosynthesis in Bacillus subtilis. Environ Microbiol 16:701–717. doi:10.1111/1462-2920.12193.
    OpenUrlCrossRef
  34. 34.↵
    1. Belitsky BR
    . 2002. Biosynthesis of amino acids of the glutamate and aspartate families, alanine, and polyamines, p 203–231. In Sonenshein AL, Hoch JA, Losick R (ed), Bacillus subtilis and its closest relatives. ASM Press, Washington, DC.
  35. 35.↵
    1. Bates SL,
    2. Waldren RP,
    3. Teare ID
    . 1973. Rapid determination of free proline for water-stress studies. Plant Soil 39:205–207. doi:10.1007/BF00018060.
    OpenUrlCrossRefWeb of Science
  36. 36.↵
    1. Brehm SP,
    2. Staal SP,
    3. Hoch JA
    . 1973. Phenotypes of pleiotropic-negative sporulation mutants of Bacillus subtilis. J Bacteriol 115:1063–1070.
    OpenUrlAbstract/FREE Full Text
  37. 37.↵
    1. Smith JL,
    2. Goldberg JM,
    3. Grossman AD
    . 2014. Complete genome sequences of Bacillus subtilis subsp. subtilis laboratory strains JH642 (AG174) and AG1839. Genome Announc 2:e00663-00614. doi:10.1128/genomeA.00663-14.
    OpenUrlCrossRefPubMed
  38. 38.↵
    1. Harwood CR,
    2. Archibald AR
    . 1990. Growth, maintenance and general techniques, p 1–26. In Harwood CR, Cutting SM (ed), Molecular biological methods for Bacillus. John Wiley & Sons, Chichester, United Kingdom.
  39. 39.↵
    1. Guerout-Fleury AM,
    2. Shazand K,
    3. Frandsen N,
    4. Stragier P
    . 1995. Antibiotic-resistance cassettes for Bacillus subtilis. Gene 167:335–336. doi:10.1016/0378-1119(95)00652-4.
    OpenUrlCrossRefPubMedWeb of Science
  40. 40.↵
    1. Benson AK,
    2. Haldenwang WG
    . 1993. Regulation of sigma B levels and activity in Bacillus subtilis. J Bacteriol 175:2347–2356.
    OpenUrlAbstract/FREE Full Text
  41. 41.↵
    1. Lorca G,
    2. Winnen B,
    3. Saier MH, Jr
    . 2003. Identification of the l-aspartate transporter in Bacillus subtilis. J Bacteriol 185:3218–3222. doi:10.1128/JB.185.10.3218-3222.2003.
    OpenUrlAbstract/FREE Full Text
  42. 42.↵
    1. Holtmann G,
    2. Bremer E
    . 2004. Thermoprotection of Bacillus subtilis by exogenously provided glycine betaine and structurally related compatible solutes: involvement of Opu transporters. J Bacteriol 186:1683–1693. doi:10.1128/JB.186.6.1683-1693.2004.
    OpenUrlAbstract/FREE Full Text
  43. 43.↵
    1. Kappes RM,
    2. Kempf B,
    3. Bremer E
    . 1996. Three transport systems for the osmoprotectant glycine betaine operate in Bacillus subtilis: characterization of OpuD. J Bacteriol 178:5071–5079.
    OpenUrlAbstract/FREE Full Text
  44. 44.↵
    1. Nicolas P,
    2. Mäder U,
    3. Dervyn E,
    4. Rochat T,
    5. Leduc A,
    6. Pigeonneau N,
    7. Bidnenko E,
    8. Marchadier E,
    9. Hoebeke M,
    10. Aymerich S,
    11. Becher D,
    12. Bisicchia P,
    13. Botella E,
    14. Delumeau O,
    15. Doherty G,
    16. Denham EL,
    17. Fogg MJ,
    18. Fromion V,
    19. Goelzer A,
    20. Hansen A,
    21. Hartig E,
    22. Harwood CR,
    23. Homuth G,
    24. Jarmer H,
    25. Jules M,
    26. Klipp E,
    27. Le Chat L,
    28. Lecointe F,
    29. Lewis P,
    30. Liebermeister W,
    31. March A,
    32. Mars RA,
    33. Nannapaneni P,
    34. Noone D,
    35. Pohl S,
    36. Rinn B,
    37. Rugheimer F,
    38. Sappa PK,
    39. Samson F,
    40. Schaffer M,
    41. Schwikowski B,
    42. Steil L,
    43. Stülke J,
    44. Wiegert T,
    45. Devine KM,
    46. Wilkinson AJ,
    47. van Dijl JM,
    48. Hecker M,
    49. Völker U,
    50. Bessieres P,
    51. Noirot P
    . 2012. Condition-dependent transcriptome reveals high-level regulatory architecture in Bacillus subtilis. Science 335:1103–1106. doi:10.1126/science.1206848.
    OpenUrlAbstract/FREE Full Text
  45. 45.↵
    1. Michna RH,
    2. Commichau FM,
    3. Todter D,
    4. Zschiedrich CP,
    5. Stülke J
    . 2014. SubtiWiki—a database for the model organism Bacillus subtilis that links pathway, interaction and expression information. Nucleic Acids Res 42:D692–D698. doi:10.1093/nar/gkt1002.
    OpenUrlCrossRefPubMedWeb of Science
  46. 46.↵
    1. Roy A,
    2. Kucukural A,
    3. Zhang Y
    . 2010. I-TASSER: a unified platform for automated protein structure and function prediction. Nat Protoc 5:725–738. doi:10.1038/nprot.2010.5.
    OpenUrlCrossRefPubMedWeb of Science
  47. 47.↵
    1. Farrell R,
    2. Macdonald LM,
    3. Hill PW,
    4. Wanniarachchi SD,
    5. Farrar J,
    6. Bardgett RD,
    7. Jones DL
    . 2014. Amino acid dynamics across a grassland altitudinal gradients. Soil Biol Biochem 76:179–182. doi:10.1016/j.soilbio.2014.05.015.
    OpenUrlCrossRef
  48. 48.↵
    1. Baumberg S,
    2. Klingel U
    . 1993. Biosynthesis of arginine, proline and related compounds, p 299–306. In Sonenshein AL, Hoch JA, Losick R (ed), Bacillus subtilis and other Gram-positive bacteria. ASM Press, Washington, DC.
  49. 49.↵
    1. Fisher SH,
    2. Débarbouillé M
    . 2002. Nitrogen source utilization and its regulation, p 181–191. In Sonenshein AL, Hoch JA, Losick R (ed), Bacillus subtilis and its closest relatives. ASM Press, Washington, DC.
  50. 50.↵
    1. Belitsky BR
    . 2011. Indirect repression by Bacillus subtilis CodY via displacement of the activator of the proline utilization operon. J Mol Biol 413:321–336. doi:10.1016/j.jmb.2011.08.003.
    OpenUrlCrossRefPubMed
  51. 51.↵
    1. Akashi H,
    2. Gojobori T
    . 2002. Metabolic efficiency and amino acid composition in the proteomes of Escherichia coli and Bacillus subtilis. Proc Natl Acad Sci U S A 99:3695–3700. doi:10.1073/pnas.062526999.
    OpenUrlAbstract/FREE Full Text
  52. 52.↵
    1. Gardan R,
    2. Rapoport G,
    3. Debarbouille M
    . 1995. Expression of the rocDEF operon involved in arginine catabolism in Bacillus subtilis. J Mol Biol 249:843–856. doi:10.1006/jmbi.1995.0342.
    OpenUrlCrossRefPubMedWeb of Science
  53. 53.↵
    1. Tolner B,
    2. Ubbink-Kok T,
    3. Poolman B,
    4. Konings WN
    . 1995. Characterization of the proton/glutamate symport protein of Bacillus subtilis and its functional expression in Escherichia coli. J Bacteriol 177:2863–2869.
    OpenUrlAbstract/FREE Full Text
  54. 54.↵
    1. Yernool D,
    2. Boudker O,
    3. Folta-Stogniew E,
    4. Gouaux E
    . 2003. Trimeric subunit stoichiometry of the glutamate transporters from Bacillus caldotenax and Bacillus stearothermophilus. Biochemistry 42:12981–12988. doi:10.1021/bi030161q.
    OpenUrlCrossRefPubMed
  55. 55.↵
    1. Slotboom DJ,
    2. Sobczak I,
    3. Konings WN,
    4. Lolkema JS
    . 1999. A conserved serine-rich stretch in the glutamate transporter family forms a substrate-sensitive reentrant loop. Proc Natl Acad Sci U S A 96:14282–14287. doi:10.1073/pnas.96.25.14282.
    OpenUrlAbstract/FREE Full Text
  56. 56.↵
    1. Boudker O,
    2. Ryan RM,
    3. Yernool D,
    4. Shimamoto K,
    5. Gouaux E
    . 2007. Coupling substrate and ion binding to extracellular gate of a sodium-dependent aspartate transporter. Nature 445:387–393. doi:10.1038/nature05455.
    OpenUrlCrossRefPubMed
  57. 57.↵
    1. Jensen S,
    2. Guskov A,
    3. Rempel S,
    4. Hänelt I,
    5. Slotboom DJ
    . 2013. Crystal structure of a substrate-free aspartate transporter. Nat Struct Mol Biol 20:1224–1226. doi:10.1038/nsmb.2663.
    OpenUrlCrossRefPubMed
  58. 58.↵
    1. Reyes N,
    2. Ginter C,
    3. Boudker O
    . 2009. Transport mechanism of a bacterial homologue of glutamate transporters. Nature 462:880–885. doi:10.1038/nature08616.
    OpenUrlCrossRefPubMedWeb of Science
  59. 59.↵
    1. Yernool D,
    2. Boudker O,
    3. Jin Y,
    4. Gouaux E
    . 2004. Structure of a glutamate transporter homologue from Pyrococcus horikoshii. Nature 431:811–818. doi:10.1038/nature03018.
    OpenUrlCrossRefPubMedWeb of Science
  60. 60.↵
    1. Spiegelhalter F,
    2. Bremer E
    . 1998. Osmoregulation of the opuE proline transport gene from Bacillus subtilis: contributions of the sigma A- and sigma B-dependent stress-responsive promoters. Mol Microbiol 29:285–296. doi:10.1046/j.1365-2958.1998.00929.x.
    OpenUrlCrossRefPubMed
  61. 61.↵
    1. Kohlstedt M,
    2. Sappa PK,
    3. Meyer H,
    4. Maass S,
    5. Zaprasis A,
    6. Hoffmann T,
    7. Becker J,
    8. Steil L,
    9. Hecker M,
    10. van Dijl JM,
    11. Lalk M,
    12. Mäder U,
    13. Stülke J,
    14. Bremer E,
    15. Völker U,
    16. Wittmann C
    . 2014. Adaptation of Bacillus subtilis carbon core metabolism to simultaneous nutrient limitation and osmotic challenge: a multi-omics perspective. Environ Microbiol 16:1898–1917. doi:10.1111/1462-2920.12438.
    OpenUrlCrossRefPubMed
  62. 62.↵
    1. Klingel U,
    2. Miller CM,
    3. North AK,
    4. Stockley PG,
    5. Baumberg S
    . 1995. A binding site for activation by the Bacillus subtilis AhrC protein, a repressor/activator of arginine metabolism. Mol Gen Genet 248:329–340. doi:10.1007/BF02191600.
    OpenUrlCrossRefPubMedWeb of Science
  63. 63.↵
    1. Sekowska A,
    2. Robin S,
    3. Daudin JJ,
    4. Henaut A,
    5. Danchin A
    . 2001. Extracting biological information from DNA arrays: an unexpected link between arginine and methionine metabolism in Bacillus subtilis. Genome Biol. 2:research0019.0011-0019.0012. doi:10.1186/gb-2001-2-6-research0019.
    OpenUrlCrossRef
  64. 64.↵
    1. Satomura T,
    2. Shimura D,
    3. Asai K,
    4. Sadaie Y,
    5. Hirooka K,
    6. Fujita Y
    . 2005. Enhancement of glutamine utilization in Bacillus subtilis through the GlnK-GlnL two-component regulatory system. J Bacteriol 187:4813–4821. doi:10.1128/JB.187.14.4813-4821.2005.
    OpenUrlAbstract/FREE Full Text
  65. 65.↵
    1. Quentin Y,
    2. Fichant G,
    3. Denizot F
    . 1999. Inventory, assembly and analysis of Bacillus subtilis ABC transport systems. J Mol Biol 287:467–484. doi:10.1006/jmbi.1999.2624.
    OpenUrlCrossRefPubMedWeb of Science
  66. 66.↵
    1. Belitsky BR,
    2. Sonenshein AL
    . 2013. Genome-wide identification of Bacillus subtilis CodY-binding sites at single-nucleotide resolution. Proc Natl Acad Sci U S A 110:7026–7031. doi:10.1073/pnas.1300428110.
    OpenUrlAbstract/FREE Full Text
  67. 67.↵
    1. Chubukov V,
    2. Uhr M,
    3. Le Chat L,
    4. Kleijn RJ,
    5. Jules M,
    6. Link H,
    7. Aymerich S,
    8. Stelling J,
    9. Sauer U
    . 2013. Transcriptional regulation is insufficient to explain substrate-induced flux changes in Bacillus subtilis. Mol Syst Biol 9:709. doi:10.1038/msb.2013.66.
    OpenUrlCrossRefPubMed
  68. 68.↵
    1. Wang ST,
    2. Setlow B,
    3. Conlon EM,
    4. Lyon JL,
    5. Imamura D,
    6. Sato T,
    7. Setlow P,
    8. Losick R,
    9. Eichenberger P
    . 2006. The forespore line of gene expression in Bacillus subtilis. J Mol Biol 358:16–37. doi:10.1016/j.jmb.2006.01.059.
    OpenUrlCrossRefPubMedWeb of Science
  69. 69.↵
    1. Steil L,
    2. Hoffmann T,
    3. Budde I,
    4. Völker U,
    5. Bremer E
    . 2003. Genome-wide transcriptional profiling analysis of adaptation of Bacillus subtilis to high salinity. J Bacteriol 185:6358–6370. doi:10.1128/JB.185.21.6358-6370.2003.
    OpenUrlAbstract/FREE Full Text
  70. 70.↵
    1. Hahne H,
    2. Mäder U,
    3. Otto A,
    4. Bonn F,
    5. Steil L,
    6. Bremer E,
    7. Hecker M,
    8. Becher D
    . 2010. A comprehensive proteomics and transcriptomics analysis of Bacillus subtilis salt stress adaptation. J Bacteriol 192:870–882. doi:10.1128/JB.01106-09.
    OpenUrlAbstract/FREE Full Text
  71. 71.↵
    1. Höper D,
    2. Bernhardt J,
    3. Hecker M
    . 2006. Salt stress adaptation of Bacillus subtilis: a physiological proteomics approach. Proteomics 6:1550–1562. doi:10.1002/pmic.200500197.
    OpenUrlCrossRefPubMedWeb of Science
  72. 72.↵
    1. Hoffmann T,
    2. Boiangiu C,
    3. Moses S,
    4. Bremer E
    . 2008. Responses of Bacillus subtilis to hypotonic challenges: physiological contributions of mechanosensitive channels to cellular survival. Appl Environ Microbiol 74:2454–2460. doi:10.1128/AEM.01573-07.
    OpenUrlAbstract/FREE Full Text
  73. 73.↵
    1. Cayley S,
    2. Lewis BA,
    3. Record MT, Jr
    . 1992. Origins of the osmoprotective properties of betaine and proline in Escherichia coli K-12. J Bacteriol 174:1586–1595.
    OpenUrlAbstract/FREE Full Text
PreviousNext
Back to top
Download PDF
Citation Tools
Uptake of Amino Acids and Their Metabolic Conversion into the Compatible Solute Proline Confers Osmoprotection to Bacillus subtilis
Adrienne Zaprasis, Monika Bleisteiner, Anne Kerres, Tamara Hoffmann, Erhard Bremer
Applied and Environmental Microbiology Dec 2014, 81 (1) 250-259; DOI: 10.1128/AEM.02797-14

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Print

Alerts
Sign In to Email Alerts with your Email Address
Email

Thank you for sharing this Applied and Environmental Microbiology article.

NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.

Enter multiple addresses on separate lines or separate them with commas.
Uptake of Amino Acids and Their Metabolic Conversion into the Compatible Solute Proline Confers Osmoprotection to Bacillus subtilis
(Your Name) has forwarded a page to you from Applied and Environmental Microbiology
(Your Name) thought you would be interested in this article in Applied and Environmental Microbiology.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
Uptake of Amino Acids and Their Metabolic Conversion into the Compatible Solute Proline Confers Osmoprotection to Bacillus subtilis
Adrienne Zaprasis, Monika Bleisteiner, Anne Kerres, Tamara Hoffmann, Erhard Bremer
Applied and Environmental Microbiology Dec 2014, 81 (1) 250-259; DOI: 10.1128/AEM.02797-14
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Top
  • Article
    • ABSTRACT
    • INTRODUCTION
    • MATERIALS AND METHODS
    • RESULTS
    • DISCUSSION
    • ACKNOWLEDGMENTS
    • FOOTNOTES
    • REFERENCES
  • Figures & Data
  • Info & Metrics
  • PDF

Related Articles

Cited By...

About

  • About AEM
  • Editor in Chief
  • Editorial Board
  • Policies
  • For Reviewers
  • For the Media
  • For Librarians
  • For Advertisers
  • Alerts
  • RSS
  • FAQ
  • Permissions
  • Journal Announcements

Authors

  • ASM Author Center
  • Submit a Manuscript
  • Article Types
  • Ethics
  • Contact Us

Follow #AppEnvMicro

@ASMicrobiology

       

ASM Journals

ASM journals are the most prominent publications in the field, delivering up-to-date and authoritative coverage of both basic and clinical microbiology.

About ASM | Contact Us | Press Room

 

ASM is a member of

Scientific Society Publisher Alliance

 

American Society for Microbiology
1752 N St. NW
Washington, DC 20036
Phone: (202) 737-3600

Copyright © 2021 American Society for Microbiology | Privacy Policy | Website feedback

 

Print ISSN: 0099-2240; Online ISSN: 1098-5336