23Na NMR spectroscopy of free Na+ in the halotolerant bacterium Brevibacterium sp. and Escherichia coli

Nagata, Shinichi; Adachi, Kyoko; Shirai, Katsuhisa; Sano, Hiroshi

doi:10.1099/13500872-141-3-729

Cited by 26 publications

(22 citation statements)

References 22 publications

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“…Indeed, Na + content of 0.04-0.2 М was found in cells of Vibrio costicola и Brevibacterium sp. growing at high NaCl concentrations (Gilboa et al, 1991;Nagata et al, 1995).…”

Section: Some Properties Of the Ectoine Biosynthetic Enzymesmentioning

confidence: 99%

Genetic and Biochemical Aspects of Ectoine Biosynthesis in Moderately Halophilic and Halotolerant Methylotrophic Bacteria

Khmelenina

Mustakhimov

Reshetnikov

et al. 2010

American Journal of Agricultural and Biological Sciences

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Problem statement:The cyclic imino acid ectoine is a widely distributed compatible solute synthesizing by halophilic and halotolerant bacteria to prevent osmotic stress at high external salinity. This water-keeping compound is used in a variety of commercial cosmetics and therapeutic products. Approach: Development of integrated, predictive functional model of the metabolic and regulatory netwoks of ectoine-producing microbes is an active area of research. In this article we present a brief overview of the current knowledge on genetic and biochemical aspects of ectoine biosynthesis in aerobic halophilic and halotolerant bacteria utilizing C 1 compounds (methylotrophs). Although enzymology and genetics of the ectoine biosynthesis in methylotrophs are similar to other halophilic bacteria, the regulatory patterns are different. In all methylotrophic bacteria studied, the genes coding for specific enzymes of ectoine biosynthesis: Diaminobutyric Acid (DABA) aminotransferase (EctB), DABA acetyltransferase (EctA) and ectoine synthase (EctC) are organized into ectABC or ectABC-ask, whith is linked to gene encoding Aspartokinase isozyme (Ask). Results: Remarkably, the methylotrophic bacteria possessing a four-gene cluster showed higher halotolerance and accumulated more ectoine than bacteria with a cluster composed of three genes. The DABA acetyltransferases from three methylotrophic species have been comparatively characterized. The properties of the enzymes correlate with eco-physiological and metabolic particularities of the host. Some elements of the regulatory system governing the ectoine pathway operation have been revealed in both methane and methanol utilizing bacteria. In Methylomicrobium alcaliphilum transcription of the ectABC-ask operon is initiated from two σ 70 -like promoters and controlled by the EctR, a MarR-type negative regulator. EctR orthologs were identified in genomes of several heterotrophic halophilic bacteria. Here we present genomic data indicating that similar regulatory system may occur in diverse halophilic and halotolerant bacteria. Conclusion: Currently available data suggest that in methylotrophic bacteria the ectoine biosynthesis pathways are evolutionary well conserved, particularly with respect to the genes and enzymes involved. However, some differences in the ect-gene cluster organization and regulation could be observed.

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“…Indeed, Na + content of 0.04-0.2 М was found in cells of Vibrio costicola и Brevibacterium sp. growing at high NaCl concentrations (Gilboa et al, 1991;Nagata et al, 1995).…”

Section: Some Properties Of the Ectoine Biosynthetic Enzymesmentioning

confidence: 99%

Genetic and Biochemical Aspects of Ectoine Biosynthesis in Moderately Halophilic and Halotolerant Methylotrophic Bacteria

Khmelenina

Mustakhimov

Reshetnikov

et al. 2010

American Journal of Agricultural and Biological Sciences

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“…It has proved, however, rather difficult to quantify the NMR visibility of internal sodium, which has been variously reported to be in the range 30-60% (37,38). 23 Na is a spin 3/2 nucleus.…”

Section: Transmembrane Fluxesmentioning

confidence: 99%

NMR and Microorganisms

2001

Current Issues in Molecular Biology

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This article is an introduction to the use of NMR for the investigation of microbial physiology and metabolism. NMR parameters which determine the sensitivity and resolving power of the method are reviewed. A broad survey of current applications follows. Qualitative uses are described first; they include compound identification and localisation. Quantitative aspects, such as pH, concentration and flux measurements are then examined, as well as the corresponding experimental constraints. The review ends with suggestions of possible future developments in instrument capabilities aimed at improving sensitivity: higher fields, spectroscopic and imaging microprobes. IntroductionThe first report on the application of NMR to microorganisms is that of Eakin et al. on the yeast, Candida utilis (1). During the past 27 years, many developments have taken place, which make in vivo NMR a very useful tool in the study of microbial physiology and metabolism. It is the purpose of this introductory review to provide the reader with an orientation to the field. It is comprised of four parts. We first list a few characteristics of the NMR technique which are deemed important for metabolic investigation. We then describe several applications of NMR, first as a qualitative tool then as a quantitative instrument. We have taken the liberty to mention advances which have taken place in related fields, such as biofluid analysis and NMR studies of mammalian cells or whole organs, whenever they were perceived as useful and readily adaptable to the case of micro-organisms. We end by mentioning various probable or desirable future developments. Some Aspects of NMR of Importance in MicrobiologyWe assume that the reader has some knowledge of NMR. We will be mainly concerned with small molecules, observed either in homogenous solutions (supernatant or growth medium, cell extracts) or inside living cells. SensitivityThe sensitivity of the NMR experiment should probably be the foremost concern of prospective users. It depends on the following factors. 1) Nature of the nucleus: Each magnetic nucleus is characterised by a spin quantum number I > 0 and a gyromagnetic ratio γ. Several elements which play an important role in biochemistry have "low γ" nuclei (Mg, Ca) and present special problems. A further point is that nuclei with I $1 (such as 14 N) have a quadrupole moment and display very broad lines when bound in a molecule. Exceptions include alkali, alkaline earth, and chlorine ions, provided they are not bound to a carrier macromolecule. 2) Operating magnetic field: The sensitivity depends roughly on the square of the static field. As the price of a spectrometer is also a steep function of the magnetic field, there is an obvious economic limitation here. It is compounded with the requirement of a wide sample access, because magnet prices also increase with sample bore. 3) Active volume: Provided the sample has a correct shape and is relatively homogenous, sensitivity is roughly proportional to the volume inside the radio-frequency coi...

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“…In the range of 30 to 100-mM extracellular [Na ϩ ]; ⌬pNa was around Ϫ55 mV; it decreased slightly when the extracellular sodium concentration was increased. The intracellular free (visible) Na ϩ concentrations, measured by 23 Na NMR in Escherichia coli (3,12), in the halotolerant Brevibacterium sp. (12), or in the moderately halophilic Vibrio costicola (9), were similar to those measured in F. succinogenes for the same extracellular [Na ϩ ].…”

Section: Vol 67 2001 Maintenance Of a Sodium Gradient In F Succinomentioning

confidence: 99%

“…The intracellular free (visible) Na ϩ concentrations, measured by 23 Na NMR in Escherichia coli (3,12), in the halotolerant Brevibacterium sp. (12), or in the moderately halophilic Vibrio costicola (9), were similar to those measured in F. succinogenes for the same extracellular [Na ϩ ]. As a result, the calculated ⌬pNa values were comparable for these bacterial systems, although the ⌬pNa value was slightly higher in the case of E. coli (Ϫ76 mV for 75 mM extracellular [Na ϩ ]) (3).…”

Section: Vol 67 2001 Maintenance Of a Sodium Gradient In F Succinomentioning

confidence: 99%

In Vivo ²³ Na Nuclear Magnetic Resonance Study of Maintenance of a Sodium Gradient in the Ruminal Bacterium Fibrobacter succinogenes S85

Schwaab

Matheron

Delort

et al. 2001

Appl Environ Microbiol

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Sodium gradients (⌬pNa) were measured in resting cells of Fibrobacter succinogenes by in vivo23 Na nuclear magnetic resonance using Tm(DOTP) 5؊ [thulium(III) 1,4,7,10-tetraazacyclododecane-N,N,N-tetramethylenephosphonate] as the shift reagent. This bacterium was able to maintain a ⌬pNa of ؊55 to ؊40 mV for extracellular sodium concentrations ranging from 30 to 200 mM. Depletion of Na ؉ ions during the washing steps led to irreversible damage (modification of glucose metabolism and inability to maintain a sodium gradient).In the ruminal bacterium Fibrobacter succinogenes S85, glucose and cellobiose uptake was shown to be driven by an artificial electrical gradient (⌬⌿) or sodium gradient (⌬pNa) in de-energized cells (5), suggesting a Na ϩ cotransport of these sugars that could explain the requirement of sodium for growth of the bacterium (2, 7, 10). However, the presence of a sodium transmembrane gradient has never been demonstrated in F. succinogenes as well as it has been demonstrated in other rumen bacteria. The objective of this work was to demonstrate and measure such a sodium gradient in F. succinogenes and to monitor its variation according to that of the extracellular sodium concentration.For that purpose, an in vivo 23 Na nuclear magnetic resonance (NMR) methodology that distinguishes the resonances of intracellular and extracellular sodium has been used to measure sodium gradients directly on living cells. The technique consists of using shift reagents. The reagents are anionic complexes of lanthanides (Dy 3ϩ , Tm 3ϩ , and Tb 3ϩ ) whose paramagnetic properties, when the reagents are exchanged with external sodium, induce a chemical shift of external Na ϩ resonance. As these reagents cannot cross the cytoplasmic membrane, the intracellular sodium is not shifted. These reagents have been widely used on various biological systems, but few studies have been performed using them with bacteria. In this work we present the application of the Tm(DOTP) 5Ϫ complex thulium(III) 1,4,7,10-tetraazacyclododecane-NЈ,NЈЈ, NЈЈЈ-tetramethylenephosphonate (1) to the study of sodium gradients in a bacterium.F. succinogenes S85 (ATCC 19169) was grown for 15 h on a chemically defined medium (8) with 3 g of cellobiose ⅐ liter Ϫ1. The cells were harvested and suspended under anaerobic conditions (8, 11) in variable sodium buffers at a concentration of 10 mg of protein ⅐ ml Ϫ1 . Tm(DOTP) 5Ϫ (Macrocyclics, Richardson, Tex.) was freshly added at a final concentration of 1, 5, or 6 mM just before the experiments were performed. The bacterial suspension was transferred to 10-mm-diameter tubes in each of which was centered a capillary containing [Na 10 ϩ Dy 3ϩ (PPPi) 2 7Ϫ ], used for intensity calibration. In vivo 23 Na NMR experiments were carried out anaerobically at 39°C with a Bruker MSL 300 spectrometer operating at 79.39 MHz. 23 Na NMR spectra (60°pulse: 16.5 s; repetition time, 350 ms; 900 scans, 1K) were collected every 5 min for 15 min, and then ionophores (monensin at 90 M and valinomycin and [carbonyl cyanide m-chlo...

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23Na NMR spectroscopy of free Na+ in the halotolerant bacterium Brevibacterium sp. and Escherichia coli

Cited by 26 publications

References 22 publications

Genetic and Biochemical Aspects of Ectoine Biosynthesis in Moderately Halophilic and Halotolerant Methylotrophic Bacteria

Genetic and Biochemical Aspects of Ectoine Biosynthesis in Moderately Halophilic and Halotolerant Methylotrophic Bacteria

NMR and Microorganisms

In Vivo ²³ Na Nuclear Magnetic Resonance Study of Maintenance of a Sodium Gradient in the Ruminal Bacterium Fibrobacter succinogenes S85

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23Na NMR spectroscopy of free Na+ in the halotolerant bacterium Brevibacterium sp. and Escherichia coli

Cited by 26 publications

References 22 publications

Genetic and Biochemical Aspects of Ectoine Biosynthesis in Moderately Halophilic and Halotolerant Methylotrophic Bacteria

Genetic and Biochemical Aspects of Ectoine Biosynthesis in Moderately Halophilic and Halotolerant Methylotrophic Bacteria

NMR and Microorganisms

In Vivo 23 Na Nuclear Magnetic Resonance Study of Maintenance of a Sodium Gradient in the Ruminal Bacterium Fibrobacter succinogenes S85

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In Vivo ²³ Na Nuclear Magnetic Resonance Study of Maintenance of a Sodium Gradient in the Ruminal Bacterium Fibrobacter succinogenes S85