Salt-sensitive in vitro protein synthesis by a moderately halophilic bacterium

Wydro, Robert; Madira, W.; Hiramatsu, Takashi; Kogut, Margot; Kushner, D. J.

doi:10.1038/269824a0

Cited by 24 publications

(22 citation statements)

References 9 publications

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“…1 centrations reported in earlier studies of this and similar organisms (5,20,23,28,33). Our results here also confirm the hypothesis (15,18,28,35) that these organisms are able to keep their free intracellular concentrations of Na+ at levels (i.e., <0.15 M) which are tolerated by their intracellular enzymes (12), including those of protein biosynthesis (35). Such enzymes, therefore, may be largely similar to those of the nonhalophilic relatives of these organisms (12,17).…”

supporting

confidence: 79%

“…One of the most important questions still unresolved involves the true intracellular ion concentrations relative to those in the medium and how the organisms deal with the osmotic and ionic gradients across their cell membranes. This problem has been highlighted by studies which showed that in vitro protein-synthesizing systems from such organisms (35) and most of their intracellular enzymes were inhibited by NaCl concentrations at which the cells had been grown (12,17,35). On the other hand, earlier studies of intracellular salt concentrations in such organisms suggested that the total concentration of intracellular cations was more or less equal to the cation concentration in the extracellular milieu (3,5,15,20,23,27,28,33).…”

mentioning

confidence: 97%

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Use of 23Na nuclear magnetic resonance spectroscopy to determine the true intracellular concentration of free sodium in a halophilic eubacterium

et al. 1991

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We present new data obtained by 23Na nuclear magnetic resonance spectroscopy, which can distinguish free intracellular sodium from cell-bound sodium, showing that the intracellular concentration of Na+ the halophilic eubacterium Vibrio costicola is only 5 to 20% of that in the extracellular medium. Previous methods could not distinguish free intracellular Nn.' from that bound to cell structures, and it was believed that in halophilic eubacteria the total monovalent cation concentration inside matched that of the NaCl outside. Information obtained by the newer technology raises fundamental questions about the ways in which these organisms and others which live in hypersaline environments function and cope with osmotic stress.Halophilic bacteria belong to two major kingdoms. The archaebacterial extreme halophiles generally require 2 to 3 M NaCl and can grow in saturated brines (16,25). The composition of their cell envelopes differs from that of eubacterial cells, and they have high proportions of negatively charged proteins in their membranes, ribosomes, and other cell components. They are known to accumulate intracellular potassium ions to balance the extracellular sodium concentrations (16,25).The second group, the eubacterial halophiles, are far more widely distributed, both environmentally and among families and genera (19,22,32), and they are of great ecological and economic importance in agriculture, food industry, and other more specialized processes. The eubacterial halophiles also show more wide-ranging salt requirements and tolerances, i.e., they are more adaptable. The mechanisms and components which enable them to grow over such a wide range (from 3-to 10-fold, according to species) of salt concentrations are still largely unknown. One of the most important questions still unresolved involves the true intracellular ion concentrations relative to those in the medium and how the organisms deal with the osmotic and ionic gradients across their cell membranes. This problem has been highlighted by studies which showed that in vitro protein-synthesizing systems from such organisms (35) and most of their intracellular enzymes were inhibited by NaCl concentrations at which the cells had been grown (12,17,35). On the other hand, earlier studies of intracellular salt concentrations in such organisms suggested that the total concentration of intracellular cations was more or less equal to the cation concentration in the extracellular milieu (3,5,15,20,23,27,28,33

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confidence: 79%

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confidence: 97%

Use of 23Na nuclear magnetic resonance spectroscopy to determine the true intracellular concentration of free sodium in a halophilic eubacterium

et al. 1991

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“…These results confirm the toxic nature of the Cl-ions on in vitro protein synthesis by V. costicola, effects previously observed with poly(U) as artificial mRNA (3,9,38). The greater sensitivity of the R17-directed system than the endogenous system to added Cl-ions suggests that these ions are more toxic to initiation than to elongation.…”

supporting

confidence: 78%

“…Unfortunately, none have been isolated from this or any other moderate halophile, although some studies have been done with a mixture of unidentified endogenous mRNAs (38). We have now found that a natural mRNA which functions well in the cell-free translation system of E. coli, that of bacteriophage R17 (2), can function in vitro with V. costicola.…”

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confidence: 82%

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Use of natural mRNAs in the cell-free protein-synthesizing systems of the moderate halophile Vibrio costicola

Choquet

Kushner

1990

J Bacteriol

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In vitro protein synthesis was studied in extracts of the moderate halophile Vibrio costicola by using as mRNAs the endogenous mRNA of V. costicola and the RNA of the R17 bacteriophage of Escherichwa coli. Protein synthesis (amino acid incorporation) was dependent on the messenger, ribosomes, soluble cytoplasmic factors, energy source, and tRNAmet (in the R17 RNA system) and was inhibited by certain antibiotics. These properties indicated de novo protein synthesis. In the V. costicola system directed by R17 RNA, a protein of the same electrophoretic mobility as the major coat protein of the R17 phage was synthesized. Antibiotic action and the response to added tRNA4et showed that protein synthesis in the R17 RNA system, but not in the endogenous messenger system, absolutely depended on initiation. Optimal activity of both systems was observed in 250 to 300 mM NH4' (as glutamate). Higher salt concentrations, especially those with Cl-as anion, were generally inhibitory. The R17 RNA-directed system was more sensitive to Cl-ions than the endogenous system was. Glycine betaine stimulated both systems and partly overcame the toxic effects of Cl-ions. Both systems required Mg2+, but in lower concentrations than the polyuridylic acid-directed system previously studied. Initiation factors were removed from ribosomes by washing with 3.0 to 3.5 M NH4C1, concentrations about three times as high as that needed to remove initiation factors from E. coli ribosomes. Washing with 4.0 M NH4Cl damaged V. costicola ribosomes, although the initiation factors still functioned. Cl-ions inhibited the attachment of initiation factors to tRNAmet but had little effect on binding of initiation factors to R17 RNA.Polyuridylic acid [poly(U)]-directed incorporation of phenylalanine by cell extracts of the moderate halophile Vibrio costicola is inhibited by Cl-ions (9), which prevent ribosome binding to the artificial mRNA (3).There are a number of differences between a translation system directed by poly(U) and that directed by a natural mRNA. Poly(U) has no Shine-Dalgarno sequence, which normally plays an important role in the initial attachment of natural mRNAs to ribosomes. At MATERIALS AND METHODS Bacterial culture. V. costicola NRC 37001 was grown and harvested as described previously (9).Preparation of cellular extracts, ribosomes, and initiation factors. S-30 extract, that is, the cellular material remaining in the supernatant after centrifuging for 30 min at 30,000 x gmax (Beckman type 42.1 rotor), was prepared as described previously (9).The S-150 extract and the low-salt-washed ribosomes were obtained by centrifuging the S-30 extract at 150,000 x gmax for 3 h at 4°C. The soluble S-150 fraction and the ribosomal pellet were then dialyzed or washed and stored according to the procedure of Kamekura and Kushner (9).A different low-salt extraction buffer than the one described by Kamekura and Kushner (9) was used in the preparation of the cellular extracts and of the ribosomes. It contained 124 mM ammonium glutamate, 8 mM magnesium ace...

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A crypticBacillusisolate exhibited narrow 16S rRNA gene sequence divergence withBacillus thuringiensisand showed low maintenance requirements in hyper‐osmotic complex substrate cultivations

Sachidanandham

Al-Shayji

Al-Awadhi

et al. 2005

Biotech & Bioengineering

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A cryptic Bacillus (K90) isolate obtained from soil samples from the Kuwait desert exhibited lower maintenance requirements in complex substrate cultivations than Bacillus thuringiensis. A mathematical model was used to estimate apparent maintenance coefficients (m(c)) and these were found to be 0.336 and 0.041/h for B. thuringiensis and K90, respectively. The results also showed that the values of apparent maintenance coefficients were inversely related to the specific growth rates. Furthermore, 16S rRNA gene sequencing showed that K90 exhibited 99.81% sequence similarity to that of B. mojavensis and 92.9% with B. thuringiensis. It is evident from the dendrogram that the evolution of B. mojavensis (K90) (B. subtilis group), which may have originated after B. licheniformis could have been influenced by prolonged hyper-osmotic conditions, while B. thuringiensis that evolved before B. oleronius exhibited greater sensitivity as implied by the higher maintenance coefficient obtained for the hyper-osmotic cultures. As K90 exhibited low maintenance requirements in hyperosmotic cultures, close phylogenetic relationship with B. thuringiensis, along with the reported property of encapsulation of insecticidal crystal proteins (Cry) in Bacillus strains and endophytic nature of B. mojavensis, strongly suggest that K90 could be a promising surrogate host for the transgenic delivery of "Cry" proteins.

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Salt-sensitive in vitro protein synthesis by a moderately halophilic bacterium

Cited by 24 publications

References 9 publications

Use of 23Na nuclear magnetic resonance spectroscopy to determine the true intracellular concentration of free sodium in a halophilic eubacterium

Use of 23Na nuclear magnetic resonance spectroscopy to determine the true intracellular concentration of free sodium in a halophilic eubacterium

Use of natural mRNAs in the cell-free protein-synthesizing systems of the moderate halophile Vibrio costicola

A crypticBacillusisolate exhibited narrow 16S rRNA gene sequence divergence withBacillus thuringiensisand showed low maintenance requirements in hyper‐osmotic complex substrate cultivations

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