Adaptation of<i>Escherichia coli</i>to high osmolarity environments: Osmoregulation of the high-affinity glycine betaine transport system ProU

206

127

The aquaporin family of molecular water channels is widely expressed throughout the plant and animal kingdoms. No bacterial aquaporins are known; however, sequence-related bacterial genes have been identified that encode glycerol facilitators (glpF). By homology cloning, a novel aquaporin-related DNA (aqpZ) was identified that contained no surface N-glycosylation consensus. The aqpZ RNA was not identified in mammalian mRNA by Northern analysis and exhibited bacterial codon usage preferences. Southern analysis failed to demonstrate aqpZ in mammalian genomic DNA, whereas a strongly reactive DNA was present in chromosomal DNA from Escherichia coli and other bacterial species and did not correspond to glpF. The aqpZ DNA isolated from E. coli contained a 693-base pair open reading frame encoding a polypeptide 28 -38% identical to known aquaporins. When compared with other aquaporins, aqpZ encodes a 10-residue insert preceding exofacial loop C, truncated NH 2 and COOH termini, and no cysteines at known mercury-sensitive sites. Expression of aqpZ cRNA conferred Xenopus oocytes with a 15-fold increase in osmotic water permeability, which was maximal after 5 days of expression, was not inhibited with HgCl 2 , exhibited a low activation energy (E a ‫؍‬ 3.8 kcal/mol), and failed to transport nonionic solutes such as urea and glycerol. In contrast, oocytes expressing glpF transported glycerol but exhibited limited osmotic water permeability. Phylogenetic comparison of aquaporins and homologs revealed a large separation between aqpZ and glpF, consistent with an ancient gene divergence.

Section: And Discussionmentioning

confidence: 99%

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confidence: 99%

Molecular Cloning and Characterization of AqpZ, a Water Channel from Escherichia coli

Calamita

Bishai

Preston

et al. 1995

206

127

“…The subsequent decrease of cellular water activity together with the loss of cell turgor lead to lessen the bacterial cell expansion rate (1). Surviving such injuring conditions implies the reversion of water flux across the cell membrane; this can be achieved by amassing highly soluble compounds termed osmolytes (2,3). Thus, Escherichia coli cells rapidly take up high amounts of potassium ions (4,5) and subsequently increase their glutamate content to balance electric charges.…”

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confidence: 99%

“…To avoid the perturbing effect of elevated ionic strength, K ϩ -glutamate can be progressively replaced by organic osmolytes that behave neutral at physiological pH (6). Such compounds, termed compatible solutes (7), may be endogenously synthesized or imported from the surrounding medium (3,8). Imported compatible solutes generally confer a high degree of osmotic tolerance to injured cells.…”

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confidence: 99%

Glycine Betaine-assisted Protein Folding in a lysAMutant of Escherichia coli

Bourot¹,

Sire²,

Trautwetter³

et al. 2000

139

Osmoprotectants exogenously supplied to a hyperosmotic culture medium are efficiently imported and amassed by stressed cells of Escherichia coli. In addition to their evident role in the recovery and maintenance of osmotic balance, these solutes should play an important role on the behavior of cellular macromolecules, for example in the process of protein folding. Using a random chemical mutagenesis approach, a conditional lysine auxotrophic mutant was obtained. The growth of this mutant was restored by addition of either lysine or osmoprotectants including glycine betaine (GB) in the minimal medium. The growth rate increased proportionally with the augmentation of the intracellular GB concentration. The mutation was located in the lysA gene and resulted in the substitution of the Ser at position 384 by Phe of the diaminopimelate decarboxylase (DAPDC), which catalyzes the conversion of meso-diaminopimelate to L-lysine. We purified both the wild type DAPDC and the mutated DAPDC-sf and demonstrated that GB was capable of activating DAPDC-sf in vitro, thus confirming the in vivo results. Most importantly, we showed that the activation was correlated with a conformational change of DAPDC-sf. Taken together, these results show, for the first time, that GB may actively assist in vivo protein folding in a chaperone-like manner.Water availability is primordial for life of all organisms. Bacteria submitted to a severe hyperosmotic stress instantaneously lose a large amount of their intracellular water to balance the osmotic strength between intracellular and extracellular spaces. The subsequent decrease of cellular water activity together with the loss of cell turgor lead to lessen the bacterial cell expansion rate (1). Surviving such injuring conditions implies the reversion of water flux across the cell membrane; this can be achieved by amassing highly soluble compounds termed osmolytes (2, 3). Thus, Escherichia coli cells rapidly take up high amounts of potassium ions (4, 5) and subsequently increase their glutamate content to balance electric charges. To avoid the perturbing effect of elevated ionic strength, K ϩ -glutamate can be progressively replaced by organic osmolytes that behave neutral at physiological pH (6). Such compounds, termed compatible solutes (7), may be endogenously synthesized or imported from the surrounding medium (3,8). Imported compatible solutes generally confer a high degree of osmotic tolerance to injured cells. Among these so-called osmoprotectants, glycine betaine (GB) 1 is by far the most effective and the most commonly assayed for hyperosmotic purposes.In addition to the obvious predominant role they play in cellular osmotic adjustment, internalized and accumulated osmoprotectants should directly participate in other intracellular processes. Protective as well as stabilizing effects of betaine and other solutes on proteins denaturation because of increased salinity or temperature have been reported (9 -12). It is tempting to extrapolate these results in vivo; however, bacteria submitte...

“…GB transport in Escherichia coli is under osmotic control (27,29,30) and is mediated by two transport systems: the single component H ϩ -compatible solute cotransporter ProP (31) and the multicomponent transport system ProU (32,33). The ProU system is a member of the ATP-binding cassette superfamily (34,35) consisting of two cytoplasmic, membrane-associated ATPases, ProV; two integral membrane proteins, ProW; and a periplasmic ligand-binding protein, ProX (33).…”

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confidence: 99%

Cation-π Interactions as Determinants for Binding of the Compatible Solutes Glycine Betaine and Proline Betaine by the Periplasmic Ligand-binding Protein ProX from Escherichia coli

Schiefner

Breed

Bösser

et al. 2004

Self Cite

118

137

Compatible solutes such as glycine betaine and proline betaine are accumulated to exceedingly high intracellular levels by many organisms in response to high osmolarity to offset the loss of cell water. They are excluded from the immediate hydration shell of proteins and thereby stabilize their native structure. Despite their exclusion from protein surfaces, the periplasmic ligand-binding protein ProX from the Escherichia coli ATP-binding cassette transport system ProU binds the compatible solutes glycine betaine and proline betaine with high affinity and specificity. To understand the mechanism of compatible solute binding, we determined the high resolution structure of ProX in complex with its ligands glycine betaine and proline betaine. This crystallographic study revealed that cation-interactions between the positive charge of the quaternary amine of the ligands and three tryptophan residues forming a rectangular aromatic box are the key determinants of the high affinity binding of compatible solutes by ProX. The structural analysis was combined with site-directed mutagenesis of the ligand binding pocket to estimate the contributions of the tryptophan residues involved in binding.