Osmotic regulation of intracellular solute pools in Lactobacillus plantarum

Glaasker, Erwin; Konings, Wil N.; Poolman, Berend

doi:10.1128/jb.178.3.575-582.1996

Cited by 102 publications

(94 citation statements)

References 35 publications

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“…subtilis accumulates significant amounts of glycine betaine in low-osmolarity media through synthesis from choline (4) or through direct uptake from the environment (21,42). The accumulation of this osmoprotectant at low osmolarity by gram-positive bacteria (4,12,16,26) appears to be of general physiological importance. It is probably connected with the high turgor maintained by gram-positive microorganisms.…”

Section: Discussionmentioning

confidence: 99%

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Three transport systems for the osmoprotectant glycine betaine operate in Bacillus subtilis: characterization of OpuD

1996

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The accumulation of the osmoprotectant glycine betaine from exogenous sources provides a high degree of osmotic tolerance to Bacillus subtilis. We have identified, through functional complementation of an Escherichia coli mutant defective in glycine betaine uptake, a new glycine betaine transport system from B. subtilis. The DNA sequence of a 2,310-bp segment of the cloned region revealed a single gene (opuD) whose product (OpuD) was essential for glycine betaine uptake and osmoprotection in E. coli. The opuD gene encodes a hydrophobic 56.13-kDa protein (512 amino acid residues). OpuD shows a significant degree of sequence identity to the choline transporter BetT and the carnitine transporter CaiT from E. coli and a BetT-like protein from Haemophilus influenzae. These membrane proteins form a family of transporters involved in the uptake of trimethylammonium compounds. The OpuD-mediated glycine betaine transport activity in B. subtilis is controlled by the environmental osmolarity. High osmolarity stimulates de novo synthesis of OpuD and activates preexisting OpuD proteins to achieve maximal glycine betaine uptake activity. An opuD mutant was constructed by marker replacement, and the OpuD-mediated glycine betaine uptake activity was compared with that of the previously identified multicomponent OpuA and OpuC (ProU) glycine betaine uptake systems. In addition, a set of mutants was constructed, each of which synthesized only one of the three glycine betaine uptake systems. These mutants were used to determine the kinetic parameters for glycine betaine transport through OpuA, OpuC, and OpuD. Each of these uptake systems shows high substrate affinity, with K m values in the low micromolar range, which should allow B. subtilis to efficiently acquire the osmoprotectant from the environment. The systems differed in their contribution to the overall glycine betaine accumulation and osmoprotection. A triple opuA, opuC, and opuD mutant strain was isolated, and it showed no glycine betaine uptake activity, demonstrating that three transport systems for this osmoprotectant operate in B. subtilis.

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Section: Discussionmentioning

confidence: 99%

“…The antibiotics ampicillin, kanamycin, tetracycline, and chloramphenicol were used with E. coli cultures at final concentrations of 100, 50, 5, and 30 g/ml, respectively. Radiolabeled [1][2][3][4][5][6][7][8][9][10][11][12][13][14] (Fig. 1) into strain JH642 and selecting for kanamycin-resistant transformants.…”

Section: Methodsmentioning

confidence: 99%

Three transport systems for the osmoprotectant glycine betaine operate in Bacillus subtilis: characterization of OpuD

1996

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“…Due to their reduced biosynthetic capabilities, LAB have to import these compounds from their (natural) environments, such as milk, meat products, or plant materials (393,394). The ability of these osmolytes to reduce the inhibitory effect of hyperosmotic situations has been established for a large number of LAB (395)(396)(397)(398)(399)(400)(401)(402)(403)(404)(405)(406).…”

Section: Protecting the Cell Envelopementioning

confidence: 99%

Stress Physiology of Lactic Acid Bacteria

Papadimitriou

Alegría

Bron

et al. 2016

Microbiol Mol Biol Rev

511

404

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SUMMARYLactic acid bacteria (LAB) are important starter, commensal, or pathogenic microorganisms. The stress physiology of LAB has been studied in depth for over 2 decades, fueled mostly by the technological implications of LAB robustness in the food industry. Survival of probiotic LAB in the host and the potential relatedness of LAB virulence to their stress resilience have intensified interest in the field. Thus, a wealth of information concerning stress responses exists today for strains as diverse as starter (e.g.,Lactococcus lactis), probiotic (e.g., severalLactobacillusspp.), and pathogenic (e.g.,EnterococcusandStreptococcusspp.) LAB. Here we present the state of the art for LAB stress behavior. We describe the multitude of stresses that LAB are confronted with, and we present the experimental context used to study the stress responses of LAB, focusing on adaptation, habituation, and cross-protection as well as on self-induced multistress resistance in stationary phase, biofilms, and dormancy. We also consider stress responses at the population and single-cell levels. Subsequently, we concentrate on the stress defense mechanisms that have been reported to date, grouping them according to their direct participation in preserving cell energy, defending macromolecules, and protecting the cell envelope. Stress-induced responses of probiotic LAB and commensal/pathogenic LAB are highlighted separately due to the complexity of the peculiar multistress conditions to which these bacteria are subjected in their hosts. Induction of prophages under environmental stresses is then discussed. Finally, we present systems-based strategies to characterize the “stressome” of LAB and to engineer new food-related and probiotic LAB with improved stress tolerance.

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“…Because Hyp is activated poorly in vitro by ProRS compared with proline, we anticipated that high in vivo concentrations of hydroxyproline would be necessary to result in enough Hyp-charged tRNA Pro to support protein synthesis. To achieve this goal, we took advantage of the phenomenon that proline and other "compatible" solutes are actively accumulated intracellularly in response to hyperosmotic shock in E. coli and other prokaryotes (24,25). Proline porters encoded by the putP, proP, and proU genes mediate accumulation.…”

Section: Trans-4-hydroxyproline Incorporation In Bacteriamentioning

confidence: 99%

Co-translational Incorporation ofTrans-4-Hydroxyproline into Recombinant Proteins in Bacteria

Buechter¹,

Paolella²,

Leslie³

et al. 2003

Journal of Biological Chemistry

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Trans-4-hydroxyproline (Hyp) in eukaryotic proteins arises from post-translational modification of proline residues. Because the modification enzyme is not present in prokaryotes, no natural means exists to incorporate Hyp into proteins synthesized in Escherichia coli. We show here that under appropriate culture conditions Hyp is incorporated co-translationally directly at proline codons in genes expressed in E. coli. Amino acids not specified by the genetic code are common in native proteins and can be essential to both structure and function. Proteins that contain novel non-natural amino acid analogues, furthermore, are of value in structure and function studies and may possess beneficial therapeutic properties. Noncoded amino acids in proteins arise either by enzymatic modification of a transfer RNA (tRNA) aminoacylated with one of the 20 coded amino acids (e.g. selenocysteine from serine) or by post-translational modifications. Reproduction of these reactions when recombinant proteins are being expressed in heterologous hosts is often either inefficient or not possible. Because of these difficulties, many proteins that contain noncoded amino acids are not available in quantities sufficient for detailed biophysical and biochemical studies.Current methodology to make proteins that contain noncoded amino acids in in vitro systems is limited to the production of relatively small amounts of protein. Use of in vitro acylated suppressor tRNAs to insert amino acid analogues at termination codons in genes translated in vitro results in sitespecific incorporation (1) but suffers from inefficiency and low yield. These problems occur in part because of limitations in producing acylated tRNA, constraints in achieving appropriate concentrations of other translational components, and variability in suppression efficiency. In vivo approaches that use an aminoacyl-tRNA synthetase with both altered amino acid and tRNA recognition and suppressor tRNAs are promising (2-4), but experimental hurdles inherent to this approach have not yet been fully overcome.In a general strategy to produce proteins containing novel amino acids, the use of DNA coding triplets to insert the amino acid analogue should be more efficient than use of termination codons. If an aminoacyl-tRNA synthetase is sufficiently promiscuous, it will aminoacylate cognate wild-type tRNA with an amino acid analogue, and the misacylated-tRNA can be used as usual by the translational machinery. This phenomenon has been exploited to produce proteins in prokaryotic systems that contain, for example, analogues of phenylalanine (5) and tryptophan (6). Two requirements must be met for success in these experiments: (1) the analogue must be acylated onto a tRNA at a demonstrable rate, and (2) the analogue must accumulate in the cell at concentrations high enough to give adequate acylation. The first requirement can, in theory, be met either by exploiting the promiscuity of a wild-type synthetase or through mutagenesis to alter the substrate specificity of a wild-type syntheta...

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Osmotic regulation of intracellular solute pools in Lactobacillus plantarum

Cited by 102 publications

References 35 publications

Three transport systems for the osmoprotectant glycine betaine operate in Bacillus subtilis: characterization of OpuD

Three transport systems for the osmoprotectant glycine betaine operate in Bacillus subtilis: characterization of OpuD

Stress Physiology of Lactic Acid Bacteria

Co-translational Incorporation ofTrans-4-Hydroxyproline into Recombinant Proteins in Bacteria

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