Théophile Bernard

Variations in the response of salt-stressed Rhizobium strains to betaines

A total of 15 rhizobial strains representing Rhizobium meliloti, Rhizobium japonicum, Rhizobium trifolii, Rhizobium leguminosarum, Rhizobium sp. (Sesbania rostrata) and Rhizobium sp. (Hedysarum coronarium), were studied with regard to growth rate under salt stress in defined liquid media. In the presence of inhibitory concentrations of NaCl, enhancement of growth resulting from added glycine betaine was observed for R. meliloti strains and Rhizobium sp. (Hedysarum coronarium) but not for other Rhizobium species. The concentration of glycine betaine required for maximal growth stimulation was very low (1 mM) in comparison with the osmolarity of the medium. The stimulation was shown to be independent of any specific solutes. Other related compounds like proline betaine, carnitine, choline, γ-butyrobetaine and pipecolate betaine were also effective compounds in restoring the growth rate of cells grown in medium of elevated osmolarity. High rate of glycine betaine uptake was demonstrated in R. meliloti cells grown in media of increased osmotic strength. The intracellular concentration of this solute was found to be 308 mM in 0.3 M NaCl-grown cells and 17 times lower in minimal medium-grown cells. Glycine betaine was used for growth under conditions of low osmolarity but could not serve as sole carbon or nitrogen source in medium of increased osmotic strength. Experiments with [14C]glycine betaine showed that this molecule was not metabolized by cells subjected to osmotic stress, whereas it was rapidly converted to dimethylglycine, sarcosine and glycine in minimal medium-grown cells.

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

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 ofmeso-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.

Ectoine accumulation and osmotic regulation in Brevibacterium linens

Brevibacterium linens can grow in media of relatively high osmotic strength (up to 3 M-NaCl). Optimal growth in minimal medium occurred with either added NaCl (0.5-1 M) or other osmolytes developing an equivalent osmotic pressure. Above 1 M-NaCl the growth rate slowed, but was enhanced by adding the osmoprotectant glycine betaine or its precursor choline (1 mM) to the media. Neither soluble carbohydrates nor ninhydrin-reacting compounds accumulated during osmotic treatment. However, a Dragendorff-positive compound, together with K+ ions, accumulated (7-fold) as a consequence of elevating the medium osmolality. Spectral characteristics of the purified compound were similar to those of synthesized 1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine). In the absence of exogenously supplied osmoprotectants, a strict dependence between the intracellular ectoine content and the external salt concentration (up to 1 M-NaCl) was observed. From 1 to 2 M-NaCl, the ectoine content decreased, and this signalled decreased self-osmoprotection by this bacterium. Interestingly, at these salt concentrations, exogenous glycine betaine led to the greatest beneficial effect. Glycine betaine or choline added to the medium was actively taken up by the cells in an osmolality-dependent manner, and the resulting betaine accumulation caused a sharp decrease in intracellular ectoine content. Radiolabelling of ectoine occurred only when L-[U-14C]glutamate was used as a precursor; de novo synthesis was dependent on the external osmolality and was strongly inhibited by exogenously supplied glycine betaine. These results imply that ectoine may play a major role in counteracting the effects of osmotic stress in media of osmotic pressure equivalent to ⋜ 1 M-NaCl.

Glutamine, Glutamate, and α-Glucosylglycerate Are the Major Osmotic Solutes Accumulated by Erwinia chrysanthemi Strain 3937

ABSTRACT Erwinia chrysanthemi is a phytopathogenic soil enterobacterium closely related to Escherichia coli. Both species respond to hyperosmotic pressure and to external added osmoprotectants in a similar way. Unexpectedly, the pools of endogenous osmolytes show different compositions. Instead of the commonly accumulated glutamate and trehalose, E. chrysanthemi strain 3937 promotes the accumulation of glutamine and α-glucosylglycerate, which is a new osmolyte for enterobacteria, together with glutamine. The amounts of the three osmolytes increased with medium osmolarity and were reduced when betaine was provided in the growth medium. Both glutamine and glutamate showed a high rate of turnover, whereas glucosylglycerate stayed stable. In addition, the balance between the osmolytes depended on the osmolality of the medium. Glucosylglycerate and glutamate were the major intracellular compounds in low salt concentrations, whereas glutamine predominated at higher concentrations. Interestingly, the ammonium content of the medium also influenced the pool of osmolytes. During bacterial growth with 1 mM ammonium in stressing conditions, more glucosylglycerate accumulated by far than the other organic solutes. Glucosylglycerate synthesis has been described in some halophilic archaea and bacteria but not as a dominant osmolyte, and its role as an osmolyte in Erwinia chrysanthemi 3937 shows that nonhalophilic bacteria can also use ionic osmolytes.

Ectoine-Induced Proteins in Sinorhizobium meliloti Include an Ectoine ABC-Type Transporter Involved in Osmoprotection and Ectoine Catabolism

To understand the mechanisms of ectoine-induced osmoprotection in Sinorhizobium meliloti, a proteomic examination of S. meliloti cells grown in minimal medium supplemented with ectoine was undertaken. This revealed the induction of 10 proteins. The protein products of eight genes were identified by using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. Five of these genes, with four other genes whose products were not detected on two-dimensional gels, belong to the same gene cluster, which is localized on the pSymB megaplasmid. Four of the nine genes encode the characteristic components of an ATP-binding cassette transporter that was named ehu, for ectoine/hydroxyectoine uptake. This transporter was encoded by four genes (ehuA, ehuB, ehuC, and ehuD) that formed an operon with another gene cluster that contains five genes, named eutABCDE for ectoine utilization. On the basis of sequence homologies, eutABCDE encode enzymes with putative and hypothetical functions in ectoine catabolism. Analysis of the properties of ehuA and eutA mutants suggests that S. meliloti possesses at least one additional ectoine catabolic pathway as well as a lower-affinity transport system for ectoine and hydroxyectoine. The expression of ehuB, as determined by measurements of UidA activity, was shown to be induced by ectoine and hydroxyectoine but not by glycine betaine or by high osmolality.

Pipecolic acid is an osmoprotectant for Escherichia coli taken up by the general osmoporters ProU and Prop

Exogenously supplied L-pipecolic acid was accumulated by Escherichia coli cells and protected them while growing at inhibitory osmolarity. Using specific uptake mutants and competitive assays, we established that the imino acid enters the cells through the ProP and ProU systems with Km values of 225 and 53 microM, respectively. Surprisingly, in spite of the requirement for the wild-type proX gene for osmoprotective ability, no binding activity of labelled pipecolate with the periplasmic protein encoded by proX could be detected. In an attempt to demonstrate whether the two porters (ProP and ProU) are the only carriers involved in osmoregulation, a variety of molecules known for their intracellular osmolarity-dependent accumulation in various organisms were investigated. N-Dimethylproline (proline betaine), N-dimethylglycine, homobetaine (beta-alanine betaine), gamma-butyrobetaine and dimethylsulfoniopropionate were found to be capable of promoting the growth of osmotically stressed E. coli. All of these molecules enter bacterial cells via ProP and ProU porters. None of the osmoprotectants except N-dimethylproline was able to bind the periplasmic protein encoded by proX, while this protein was necessary for their uptake. Apparently, ProP and ProU are the sole osmoporters involved in osmolyte influx into E. coli cells.

The osmoprotectant glycine betaine inhibits salt-induced cross-tolerance towards lethal treatment in Enterococcus faecalis.

The response of Enterococcus faecalis ATCC 19433 to salt stress has been characterized previously in complex media. In this report, it has been demonstrated that this bacterium actively accumulates the osmoprotectant glycine betaine (GB) from salt-enriched complex medium BHI. To further understand the specific effects of GB and other osmoprotective compounds in salt adaptation and salt-induced cross-tolerance to lethal challenges, a chemically defined medium lacking putative osmoprotectants was used. In this medium, bacterial growth was significantly reduced by increasing concentrations of NaCl. At 0.75 M NaCl, 90% inhibition of the growth rate was observed; GB and its structural analogues restored growth to the non-salt-stressed level. In contrast, proline, pipecolate and ectoine did not allow growth recovery of stressed cells. Kinetic studies showed that the uptake of betaines shows strong structural specificity and occurs through a salt-stress-inducible high-affinity porter [Km = 3.3 microM; Vmax = 130 nmol min(-1) (mg protein)(-1); the uptake activity increased 400-fold in the presence of 0.5 M NaCl]. Moreover, GB and its analogues were accumulated as non-metabolizable cytosolic osmolytes and reached intracellular levels ranging from 1-3 to 1.5 micromol (mg protein)(-1). In contrast to the beneficial effect of GB on the growth of salt-stressed cultures of E. faecalis, its accumulation inhibits the salt-induced cross-tolerance to a heterologous lethal challenge. Indeed, pretreatment of bacterial cells with 0.5 M NaCl induced resistance to 0.3% bile salts (survival of adapted cells increased by a factor of 6800). The presence of GB in the adaptation medium reduced the acquisition of bile salts resistance 680-fold. The synthesis of 11 of the 13 proteins induced during salt adaptation was significantly reduced in the presence of GB. These results raise questions about the actual beneficial effect of GB in natural environments where bacteria are often subjected to various stresses.

Osmotic adjustment in Brevibacterium ammoniagenes: pipecolic acid accumulation at elevated osmolalities

SUMMARY: Brevibacterium ammoniagenes ATCC 6872 was grown aerobically in minimal defined glucose media of different osmolalities induced either by NaCl, other eletrolytes or non-electrolytes. Growth rate was slightly affected by elevation of medium osmolality up to 1 M-NaCl, but severely decreased at 1.5 M; however even at 2 M-NaCl, slow growth still occurred. Glycine betaine (or its precursor choline) did not stimulate growth in high osmotic media, although it accumulated intracellularly and was not metabolized. An organic solute which increased substantially in concentration during osmotic treatment with various osmolytes, was isolated and identified as pipecolic acid. No accumulation of this imino acid was observed when the medium concentration was raised by adding the permeant glycerol or when the culture medium was supplemented with glycine betaine (1 mM). While this non-proteic cyclic amino acid may play an important role in bacterial adaptation to environmental stress, it did not accumulate to a high level. Preliminary data suggest that the biosynthesis of pipecolic acid from lysine is strongly regulated by external osmolality.

Osmoprotection by pipecolic acid in Sinorhizobium meliloti: specific effects of D and L isomers.

ABSTRACT dl-Pipecolic acid (dl-PIP) promotes growth restoration of Sinorhizobium meliloti cells facing inhibitory hyperosmolarity. Surprisingly, d andl isomers of this imino acid supplied separately were not effective. The uptake of l-PIP was significantly favored in the presence of the d isomer and by a hyperosmotic stress. Chromatographic analysis of the intracellular solutes showed that stressed cells did not accumulate radiolabeled l-PIP. Rather, it participates in the synthesis of the main endogenous osmolytes (glutamate and the dipeptideN-acetylglutaminylglutamine amide) during the lag phase, thus providing a means for the stressed cells to recover the osmotic balance. 13C nuclear magnetic resonance analysis was used to determine the fate of d-PIP taken into the cells. In the absence of l-PIP, the imported d isomer was readily degraded. Supplied together with its l isomer,d-PIP was accumulated temporarily and thus might contribute together with the endogenous osmolytes to enhance the internal osmotic strength. Furthermore, it started to disappear from the cytosol when the l isomer was no longer available in the culture medium (during the late exponential phase of growth). Together, these results show an uncommon mechanism of protection of osmotically stressed cells of S. meliloti. It was proved, for the first time, that the presence of the two isomers of the same molecule is necessary for it to manifest an osmoprotective activity. Indeed, d-PIP seems to play a major role in cellular osmoadaptation through both its own accumulation and improvement of the utilization of the lisomer as an immediate precursor of endogenous osmolytes.

Synthesis of trimethylated phosphonium and arsonium analogues of the osmoprotectant glycine betaine; contrasted biological activities in two bacterial species

Abstract Phosphoniobetaine and arsenobetaine, the P and As analogues of glycine betaine (trimethylammonioacetate) were synthesized and assayed for activity in bacterial osmoprotection biotests, using Escherichia coli and Rhizobium meliloti as model organisms. The P- and As-betaines displayed similar osmoprotective activities in E. coli , but were highly toxic in the betaine-demethylating bacterium R. meliloti .