scholarly journals Enhanced Glutamate Synthesis and Export by the Thermotolerant Emerging Industrial Workhorse Bacillus methanolicus in Response to High Osmolarity

2021 ◽  
Vol 12 ◽  
Author(s):  
Christine Frank ◽  
Tamara Hoffmann ◽  
Oskar Zelder ◽  
Max F. Felle ◽  
Erhard Bremer
Keyword(s):  
High Salinity ◽  
Regulatory Gene ◽  
Compatible Solutes ◽  
Compatible Solute ◽  
Adjustment Process ◽  
Cell Factory ◽  
Biotechnological Production ◽  
High Osmolarity ◽  
Bacillus Methanolicus ◽  
Glutamate Synthesis

The thermotolerant methylotroph Bacillus methanolicus MGA3 was originally isolated from freshwater marsh soil. Due to its ability to use methanol as sole carbon and energy source, B. methanolicus is increasingly explored as a cell factory for the production of amino acids, fine chemicals, and proteins of biotechnological interest. During high cell density fermentation in industrial settings with the membrane-permeable methanol as the feed, the excretion of low molecular weight products synthesized from it will increase the osmotic pressure of the medium. This in turn will impair cell growth and productivity of the overall biotechnological production process. With this in mind, we have analyzed the core of the physiological adjustment process of B. methanolicus MGA3 to sustained high osmolarity surroundings. Through growth assays, we found that B. methanolicus MGA3 possesses only a restricted ability to cope with sustained osmotic stress. This finding is consistent with the ecophysiological conditions in the habitat from which it was originally isolated. None of the externally provided compatible solutes and proline-containing peptides affording osmostress protection for Bacillus subtilis were able to stimulate growth of B. methanolicus MGA3 at high salinity. B. methanolicus MGA3 synthesized the moderately effective compatible solute L-glutamate in a pattern such that the cellular pool increased concomitantly with increases in the external osmolarity. Counterintuitively, a large portion of the newly synthesized L-glutamate was excreted. The expression of the genes (gltAB and gltA2) for two L-glutamate synthases were upregulated in response to high salinity along with that of the gltC regulatory gene. Such a regulatory pattern of the system(s) for L-glutamate synthesis in Bacilli is new. Our findings might thus be generally relevant to understand the production of the osmostress protectant L-glutamate by those Bacilli that exclusively rely on this compatible solute for their physiological adjustment to high osmolarity surroundings.

scholarly journals OpuF, a NewBacillusCompatible Solute ABC Transporter with a Substrate-Binding Protein Fused to the Transmembrane Domain

2018 ◽  
Vol 84 (20) ◽  
Author(s):  
Laura Teichmann ◽  
Henriette Kümmel ◽  
Bianca Warmbold ◽  
Erhard Bremer
Keyword(s):  
Abc Transporters ◽  
Transmembrane Domain ◽  
Binding Protein ◽  
Substrate Binding ◽  
Compatible Solutes ◽  
Compatible Solute ◽  
Content Type ◽  
High Osmolarity ◽  
Physiological Characterization ◽  
Substrate Binding Protein

ABSTRACTThe accumulation of compatible solutes is a common defense of bacteria against the detrimental effects of high osmolarity. Uptake systems for these compounds are cornerstones in cellular osmostress responses because they allow the energy-preserving scavenging of osmostress protectants from environmental sources.Bacillus subtilisis well studied with respect to the import of compatible solutes and its five transport systems (OpuA, OpuB, OpuC, OpuD, and OpuE), for these stress protectants have previously been comprehensively studied. Building on this knowledge and taking advantage of the unabated appearance of new genome sequences of members of the genusBacillus, we report here the discovery, physiological characterization, and phylogenomics of a new member of the Opu family of transporters, OpuF (OpuFA-OpuFB). OpuF is not present inB. subtilisbut it is widely distributed in members of the large genusBacillus. OpuF is a representative of a subgroup of ATP-binding cassette (ABC) transporters in which the substrate-binding protein (SBP) is fused to the transmembrane domain (TMD). We studied the salient features of the OpuF transporters fromBacillus infantisandBacillus panaciterraeby functional reconstitution in aB. subtilischassis strain lacking known Opu transporters. A common property of the examined OpuF systems is their substrate profile; OpuF mediates the import of glycine betaine, proline betaine, homobetaine, and the marine osmolyte dimethylsulfoniopropionate (DMSP). Anin silicomodel of the SBP domain of the TMD-SBP hybrid protein OpuFB was established. It revealed the presence of an aromatic cage, a structural feature commonly present in ligand-binding sites of compatible solute importers.IMPORTANCEThe high-affinity import of compatible solutes from environmental sources is an important aspect of the cellular defense of many bacteria and archaea against the harmful effects of high external osmolarity. The accumulation of these osmostress protectants counteracts high-osmolarity-instigated water efflux, a drop in turgor to nonphysiological values, and an undue increase in molecular crowding of the cytoplasm; they thereby foster microbial growth under osmotically unfavorable conditions. Importers for compatible solutes allow the energy-preserving scavenging of osmoprotective and physiologically compliant organic solutes from environmental sources. We report here the discovery, exemplary physiological characterization, and phylogenomics of a new compatible solute importer, OpuF, widely found in members of theBacillusgenus. The OpuF system is a representative of a growing subgroup of ABC transporters in which the substrate-scavenging function of the substrate-binding protein (SBP) and the membrane-embedded substrate translocating subunit (TMD) are fused into a single polypeptide chain.

Osmoprotection of Escherichia coli by Peptone Is Mediated by the Uptake and Accumulation of Free Proline but Not of Proline-Containing Peptides

1999 ◽  
Vol 65 (12) ◽  
pp. 5272-5278 ◽  
Author(s):  
Maria-Rosario Amezaga ◽  
Ian R. Booth
Keyword(s):  
Escherichia Coli ◽  
Compatible Solutes ◽  
Complex Mixture ◽  
Compatible Solute ◽  
Logarithmic Phase ◽  
Main Role ◽  
Type I ◽  
Free Proline ◽  
High Osmolarity ◽  
E Coli

ABSTRACT The effect of meat peptone type I (Sigma) on the growth ofEscherichia coli cells under hyperosmotic stress has been investigated. Peptone is a complex mixture of peptides with a small content of free amino acids, which resembles nutrients found in natural environments. Our data showed that peptone enhances the growth ofE. coli cells in high-osmolarity medium to levels higher than those achieved with the main compatible solute in bacteria, glycine betaine. The mechanism of osmoprotection by peptone comprises the uptake and accumulation of the compatible solute, proline. The main role of the peptides contained in peptone is the provision of nutrients rather than the intracellular accumulation of osmolytes. In contrast toListeria monocytogenes (M. R. Amezaga, I. Davidson, D. McLaggan, A. Verheul, T. Abee, and I. R. Booth, Microbiology 141:41–49, 1995), E. coli does not accumulate exogenous peptides for osmoprotection and peptides containing proline do not lead to the accumulation of proline as a compatible solute. In late-logarithmic-phase cultures of E. coli growing at high osmolarity plus peptone, proline becomes the limiting factor for growth, and the intracellular pools of proline are not maintained. This is a consequence of the low concentration of free proline in peptone, the catabolism of proline by E. coli, and the inability ofE. coli to utilize proline-containing peptides as a source of compatible solutes. Our data highlight the role that natural components in food such as peptides play in undermining food preservation regimes, such as high osmolarity, and also that the specific mechanisms of osmoprotection by these compounds differ according to the organism.

scholarly journals Synthesis, Release, and Recapture of Compatible Solute Proline by Osmotically Stressed Bacillus subtilis Cells

2012 ◽  
Vol 78 (16) ◽  
pp. 5753-5762 ◽  
Author(s):  
Tamara Hoffmann ◽  
Carsten von Blohn ◽  
Agnieszka Stanek ◽  
Susanne Moses ◽  
Helena Barzantny ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
High Salinity ◽  
De Novo ◽  
Compatible Solutes ◽  
Growth Conditions ◽  
Compatible Solute ◽  
Content Type ◽  
Cellular Defense ◽  
Stressed Cells ◽  
External Salinity

ABSTRACTBacillus subtilissynthesizes large amounts of the compatible solute proline as a cellular defense against high osmolarity to ensure a physiologically appropriate level of hydration of the cytoplasm and turgor. It also imports proline for this purpose via the osmotically inducible OpuE transport system. Unexpectedly, anopuEmutant was at a strong growth disadvantage in high-salinity minimal media lacking proline. Appreciable amounts of proline were detected in the culture supernatant of theopuEmutant strain, and they rose concomitantly with increases in the external salinity. We found that the intracellular proline pool of severely salinity-stressed cells of theopuEmutant was considerably lower than that of itsopuE+parent strain. This loss of proline into the medium and the resulting decrease in the intracellular proline content provide a rational explanation for the observed salt-sensitive growth phenotype of cells lacking OpuE. None of the known MscL- and MscS-type mechanosensitive channels ofB. subtilisparticipated in the release of proline under permanently imposed high-salinity growth conditions. The data reported here show that the OpuE transporter not only possesses the previously reported role for the scavenging of exogenously provided proline as an osmoprotectant but also functions as a physiologically highly important recapturing device for proline that is synthesizedde novoand subsequently released by salt-stressedB. subtiliscells. The wider implications of our findings for the retention of compatible solutes by osmotically challenged microorganisms and the roles of uptake systems for compatible solutes are considered.

scholarly journals Metabolic Engineering of Corynebacterium glutamicum for Methanol Metabolism

2015 ◽  
Vol 81 (6) ◽  
pp. 2215-2225 ◽  
Author(s):  
Sabrina Witthoff ◽  
Katja Schmitz ◽  
Sebastian Niedenführ ◽  
Katharina Nöh ◽  
Stephan Noack ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Biotechnological Production ◽  
Methanol Metabolism ◽  
Initial Oxidation ◽  
Content Type ◽  
Oxidation Of Methanol ◽  
Bacillus Methanolicus ◽  
Production Processes ◽  
Starting Point ◽  
Limited Application

ABSTRACTMethanol is already an important carbon feedstock in the chemical industry, but it has found only limited application in biotechnological production processes. This can be mostly attributed to the inability of most microbial platform organisms to utilize methanol as a carbon and energy source. With the aim to turn methanol into a suitable feedstock for microbial production processes, we engineered the industrially important but nonmethylotrophic bacteriumCorynebacterium glutamicumtoward the utilization of methanol as an auxiliary carbon source in a sugar-based medium. Initial oxidation of methanol to formaldehyde was achieved by heterologous expression of a methanol dehydrogenase fromBacillus methanolicus, whereas assimilation of formaldehyde was realized by implementing the two key enzymes of the ribulose monophosphate pathway ofBacillus subtilis: 3-hexulose-6-phosphate synthase and 6-phospho-3-hexuloisomerase. The recombinantC. glutamicumstrain showed an average methanol consumption rate of 1.7 ± 0.3 mM/h (mean ± standard deviation) in a glucose-methanol medium, and the culture grew to a higher cell density than in medium without methanol. In addition, [13C]methanol-labeling experiments revealed labeling fractions of 3 to 10% in the m + 1 mass isotopomers of various intracellular metabolites. In the background of aC. glutamicumΔaldΔadhEmutant being strongly impaired in its ability to oxidize formaldehyde to CO2, the m + 1 labeling of these intermediates was increased (8 to 25%), pointing toward higher formaldehyde assimilation capabilities of this strain. The engineeredC. glutamicumstrains represent a promising starting point for the development of sugar-based biotechnological production processes using methanol as an auxiliary substrate.

Humectant Permeability Influences Growth and Compatible Solute Uptake by Staphylococcus aureus Subjected to Osmotic Stress

2002 ◽  
Vol 65 (6) ◽  
pp. 1008-1015 ◽  
Author(s):  
ODDUR VILHELMSSON ◽  
KAREN J. MILLER
Keyword(s):  
Staphylococcus Aureus ◽  
Sodium Chloride ◽  
Water Activity ◽  
Glycine Betaine ◽  
Compatible Solutes ◽  
Foodborne Pathogen ◽  
Compatible Solute ◽  
Solute Uptake ◽  
Stressed Cells ◽  
Carnitine Uptake

The effects of different humectants (sodium chloride, sucrose, and glycerol) on the growth of and compatible solute (glycine betaine, proline, and carnitine) uptake by the osmotolerant foodborne pathogen Staphylococcus aureus were investigated. While growth in the presence of the impermeant humectants sodium chloride and sucrose induced the accumulation of proline and glycine betaine by cells, growth in the presence of the permeant humectant glycerol did not. When compatible solutes were omitted from low-water-activity media, growth was very poor in the presence of impermeant humectants. In contrast, the addition of compatible solutes had essentially no effect on growth when cells were grown in low-water-activity media containing glycerol as the humectant. Carnitine was found to accumulate to high intracellular levels in osmotically stressed cells when proline and glycine betaine were absent, making it a potentially important compatible solute for this organism.

scholarly journals Potential of temperature- and salinity-driven shifts in diatom compatible solute concentrations to impact biogeochemical cycling within sea ice

Elem Sci Anth ◽  
2020 ◽  
Vol 8 ◽  
Author(s):  
Hannah M. Dawson ◽  
Katherine R. Heal ◽  
Angela K. Boysen ◽  
Laura T. Carlson ◽  
Anitra E. Ingalls ◽  
...  
Keyword(s):  
Sea Ice ◽  
Compatible Solutes ◽  
Biogeochemical Cycling ◽  
Compatible Solute ◽  
Arctic Sea Ice ◽  
Polar Regions ◽  
Antarctic Sea Ice ◽  
Metabolite Profiles ◽  
Metabolite Pool ◽  
Cellular Metabolite

Sea-ice algae are an important source of primary production in polar regions, yet we have limited understanding of their responses to the seasonal cycling of temperature and salinity. Using a targeted liquid chromatography-mass spectrometry-based metabolomics approach, we found that axenic cultures of the Antarctic sea-ice diatom, Nitzschia lecointei, displayed large differences in their metabolomes when grown in a matrix of conditions that included temperatures of –1 and 4°C, and salinities of 32 and 41, despite relatively small changes in growth rate. Temperature exerted a greater effect than salinity on cellular metabolite pool sizes, though the N- or S-containing compatible solutes, 2, 3-dihydroxypropane-1-sulfonate (DHPS), glycine betaine (GBT), dimethylsulfoniopropionate (DMSP), and proline responded strongly to both temperature and salinity, suggesting complexity in their control. We saw the largest (> 4-fold) response to salinity for proline. DHPS, a rarely studied but potential compatible solute, had the highest intracellular concentrations among all compatible solutes of ~85 mM. When comparing the culture findings to natural Arctic sea-ice diatom communities, we found extensive overlap in metabolite profiles, highlighting the relevance of culture-based studies to probe environmental questions. Large changes in sea-ice diatom metabolomes and compatible solutes over a seasonal cycle could be significant components of biogeochemical cycling within sea ice.

scholarly journals Growth and nitrogen removal characteristics of Halomonas sp. B01 under high salinity

2019 ◽  
Vol 69 (13) ◽  
pp. 1425-1433 ◽  
Author(s):  
Te Wang ◽  
Zhengzhong Jiang ◽  
Wenbo Dong ◽  
Xiaoya Liang ◽  
Linghua Zhang ◽  
...  
Keyword(s):  
Mrna Expression ◽  
High Salinity ◽  
Halophilic Bacteria ◽  
Compatible Solutes ◽  
Aerobic Denitrification ◽  
Protective Mechanism ◽  
Sod Activity ◽  
N Removal ◽  
Nacl Concentration ◽  
Expression Abundance

Abstract Purpose At present, the nitrogen (N) removal efficiency of the microbial treatment in the high-salinity nitrogenous wastewaters is relatively low. Study on the N removal behavior and properties of moderately halophilic bacteria Halomonas under high salinity is of great significance for the microbial treatment of high-salinity nitrogenous wastewater. Methods The response mechanism of Halomonas sp. B01 to high osmotic pressure stress was investigated by measuring the compatible solute ectoine concentration and superoxide dismutase (SOD) activity. The salt tolerance during growth and N removal of the strain was evaluated by measuring the activities of growth-related and N removal–related enzymes and the mRNA expression abundance of ammonia monooxygenase-encoding gene (amoA). The process of simultaneous heterotrophic nitrification and aerobic denitrification (SND) under high salinity was described by measuring the concentration of inorganic N. Result Halomonas sp. B01 synthesized ectoine under NaCl stress, and the intracellular ectoine concentration increased with increased NaCl concentration in the growth medium. When the NaCl concentration of the medium reached 120 g L−1, the malondialdehyde concentration and SOD activity were significantly increased to 576.1 μg mg−1 and 1.7 U mg−1, respectively. The growth-related and N removal–related enzymes of the strain were active or most active in medium with 30–60 g L−1 NaCl. The amoA of the strain cultured in medium with 60 g L−1 NaCl had the highest mRNA expression abundance. In the N removal medium containing 60 g L−1 NaCl and 2121 mg L−1 NH4+-N, SND by Halomonas sp. B01 was performed over 96 h and the N removal rate reached 98.8%. Conclusion In addition to the protective mechanism of synthetic compatible solutes, Halomonas sp. B01 had the repair mechanism of SOD for lipid peroxidation. The growth-related and N removal–related enzymes of the strain were most active at a certain salt concentration; amoA also had the highest mRNA expression abundance under high salinity. Halomonas sp. B01 could efficiently perform N removal by SND under high salinity.

scholarly journals Identification and Characterization of an ATP Binding Cassette l-Carnitine Transporter inListeria monocytogenes

2000 ◽  
Vol 66 (11) ◽  
pp. 4696-4704 ◽  
Author(s):  
Katy R. Fraser ◽  
Duncan Harvie ◽  
Peter J. Coote ◽  
Conor P. O'Byrne
Keyword(s):  
Binding Protein ◽  
Substrate Binding ◽  
Compatible Solutes ◽  
Brain Heart Infusion ◽  
Defined Medium ◽  
Compatible Solute ◽  
Atp Binding ◽  
Insertional Inactivation ◽  
Substrate Binding Protein ◽  
Atp Binding Cassette

ABSTRACT We identified an operon in Listeria monocytogenes EGD with high levels of sequence similarity to the operons encoding the OpuC and OpuB compatible solute transporters from Bacillus subtilis, which are members of the ATP binding cassette (ABC) substrate binding protein-dependent transporter superfamily. The operon, designated opuC, consists of four genes which are predicted to encode an ATP binding protein (OpuCA), an extracellular substrate binding protein (OpuCC), and two membrane-associated proteins presumed to form the permease (OpuCB and OpuCD). The operon is preceded by a potential SigB-dependent promoter. An opuC-defective mutant was generated by the insertional inactivation of theopuCA gene. The mutant was impaired for growth at high osmolarity in brain heart infusion broth and failed to grow in a defined medium. Supplementation of the defined medium with peptone restored the growth of the mutant in this medium. The mutant was found to accumulate the compatible solutes glycine betaine and choline to same extent as the parent strain but was defective in the uptake ofl-carnitine. We conclude that the opuC operon in L. monocytogenes encodes an ABC compatible solute transporter which is capable of transporting l-carnitine and which plays an important role in osmoregulation in this pathogen.

scholarly journals Impact of high salinity and the compatible solute glycine betaine on gene expression of Bacillus subtilis

2020 ◽  
Vol 22 (8) ◽  
pp. 3266-3286
Author(s):  
Hermann Rath ◽  
Praveen K. Sappa ◽  
Tamara Hoffmann ◽  
Manuela Gesell Salazar ◽  
Alexander Reder ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bacillus Subtilis ◽  
Glycine Betaine ◽  
High Salinity ◽  
Compatible Solute

scholarly journals Role of σB in Regulating the Compatible Solute Uptake Systems of Listeria monocytogenes: Osmotic Induction of opuC Is σB Dependent

2003 ◽  
Vol 69 (4) ◽  
pp. 2015-2022 ◽  
Author(s):  
Katy R. Fraser ◽  
David Sue ◽  
Martin Wiedmann ◽  
Kathryn Boor ◽  
Conor P. O'Byrne
Keyword(s):  
Listeria Monocytogenes ◽  
Solute Transport ◽  
Compatible Solutes ◽  
Strain Analysis ◽  
Compatible Solute ◽  
Transport Systems ◽  
Carnitine Transporter ◽  
Solute Uptake ◽  
Carnitine Transport

ABSTRACT The regulation of the compatible solute transport systems in Listeria monocytogenes by the stress-inducible sigma factor σB was investigated. Using wild-type strain 10403S and an otherwise isogenic strain carrying an in-frame deletion in sigB, we have examined the role of σB in regulating the ability of cells to utilize betaine and carnitine during growth under conditions of hyperosmotic stress. Cells lacking σB were defective for the utilization of carnitine but retained the ability to utilize betaine as an osmoprotectant. When compatible solute transport studies were performed, the initial rates of uptake of both betaine and carnitine were found to be reduced in the sigB mutant; carnitine transport was almost abolished, whereas betaine transport was reduced to approximately 50% of that of the parent strain. Analysis of the cytoplasmic pools of compatible solutes during balanced growth revealed that both carnitine and betaine steady-state pools were reduced in the sigB mutant. Transcriptional reporter fusions to the opuC (which encodes an ABC carnitine transporter) and betL (which encodes an a secondary betaine transporter) operons were generated by using a promoterless copy of the gus gene from Escherichia coli. Measurement of β-glucuronidase activities directed by opuC-gus and betL-gus revealed that transcription of opuC is largely σB dependent, consistent with the existence of a potential σB consensus promoter motif upstream from opuCA. The transcription of betL was found to be sigB independent. Reverse transcriptase PCR experiments confirmed these data and indicated that the transcription of all three known compatible solute uptake systems (opuC, betL, and gbu), as well as a gene that is predicted to encode a compatible solute transporter subunit (lmo1421) is induced in response to elevated osmolarity. The osmotic induction of opuCA and lmo1421 was found to be strongly σB dependent. Together these observations suggest that σB plays a major role in the regulation of carnitine utilization by L. monocytogenes but is not essential for betaine utilization by this pathogen.

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