Salinity stress responses in the plant growth promoting rhizobacteria,Azospirillum spp

In order to adapt to the fluctuations in soil salinity/osmolarity the bacteria of the genusAzospirillum accumulate compatible solutes such as glutamate, proline, glycine betaine, trehalose, etc. Proline seems to play a major role in osmoadaptation. With increase in osmotic stress the dominant osmolyte inA. brasilense shifts from glutamate to proline. Accumulation of proline inA. brasilense occurs by both uptake and synthesis. At higher osmolarityA. brasilense Sp7 accumulates high intracellular concentration of glycine betaine which is taken up via a high affinity glycine betaine transport system. A salinity stress induced, periplasmically located, glycine betaine binding protein (GBBP) of ca. 32 kDa size is involved in glycine betaine uptake inA. brasilense Sp7. Although a similar protein is also present inA. brasilense Cd it does not help in osmoprotection. It is not known ifA. brasilense Cd can also accumulate glycine betaine under salinity stress and if the GBBP-like protein plays any role in glycine betaine uptake. This strain, under salt stress, seems to have inadequate levels of ATP to support growth and glycine betaine uptake simultaneously. ExceptA. halopraeferens, all other species ofAzospirillum lack the ability to convert choline into glycine betaine. Mobilization of thebet ABT genes ofE. coli intoA. brasilense enables it to use choline for osmoprotection. Recently, aproU-like locus fromA. lipoferum showing physical homology to theproU gene region ofE. coli has been cloned. Replacement of this locus, after inactivation by the insertion of kanamycin resistance gene cassette, inA. lipoferum genome results in the recovery of mutants which fail to use glycine betaine as osmoprotectant.     

Isolation and molecular characterization of theSinorhizobium meliloti bet locus encoding glycine betaine biosynthesis

To cope with osmotic stress,Sinorhizobium meliloti accumulates organic compatible solutes such as glutamate, trehalose, N-acetylglutaminylglutamine amide, and the most potent osmoprotectant glycine betaine. In order to study the regulation of the glycine betaine biosynthetic pathway, a genetic and molecular analysis was performed. We have selected a Tn5 mutant ofS. meliloti which was deficient in choline dehydrogenase activity. The mutation was complemented using a genomic bank ofS. meliloti. Subcloning and DNA sequencing of a 8-6 kb region from the complemented plasmid showed four open reading frames with an original structural organization of thebet locus compared to that described inE. coli. (i) ThebetB and thebetA genes which encode a glycine betaine aldehyde dehydrogenase, and a choline dehydrogenase, respectively, are separated from thebetI gene (regulatory protein) by an additional gene namedbetC. The BetC protein shares about 30% identity with various sulphatases and is involved in the conversion of choline-O-sulphate into choline. Choline-O-sulphate is used as an osmoprotectant, or as a carbon or sulphur source and this utilization is dependent on a functionalbet locus. (ii) No sequence homologous tobetT (encoding a high-affinity choline transport system inE. coli) was found in the vicinity of thebet locus. (iii) ThebetB and thebetA genes, as well as thebetI and thebetC genes are, respectively, separated by 211 and 167 bp sequences containing inverted repeats. Southern blot analysis indicated that thebet locus is located on the chromosome, and not on the megaplasmids.     

Combined effect of betaine and trehalose on osmotic tolerance of <Emphasis Type="Italic">Escherichia coli</Emphasis> in mineral salts medium

In mineral salts medium, supplementing with betaine in combination with increased production of endogenous osmoprotectant from a second copy of the trehalose biosynthetic genes (otsBA) improved growth of E. coli and increased the MIC for xylose, glucose, sodium lactate and NaCl. With these compounds, this combination was more effective than either betaine or trehalose alone. With succinate, this combination was no more effective than betaine alone. Neither approach improved tolerance to ethanol. A combination of betaine and increased trehalose may improve strain productivity for many bioproducts by promoting growth in the presence of high sugar concentrations.     

Improved tolerance to salinity and low temperature in transgenic tobacco producing glycine betaine

Glycine betaine is an osmoprotectant found in many organisms, including bacteria and higher plants. The bacterium Escherichia coli produces glycine betaine by a two-step pathway where choline dehydrogenase (CDH), encoded by betA, oxidizes choline to betaine aldehyde which is further oxidized to glycine betaine by the same enzyme. The second step, conversion of betaine aldehyde into glycine betaine, can also be performed by the second enzyme in the pathway, betaine aldehyde dehydrogenase (BADH), encoded by betB. Transformation of tobacco (Nicotiana tabacum), a species not accumulating glycine betaine, with the E. coli genes for glycine betaine biosynthesis, resulted in transgenic plants accumulating glycine betaine. Plants producing CDH were found to accumulate glycine betaine as did F1 progeny from crosses between CDH- and BADH-producing lines. Plants producing both CDH and BADH generally accumulated higher amounts of glycine betaine than plants producing CDH alone, as determined by 1H NMR analysis. Transgenic tobacco lines accumulating glycine betaine exhibited increased tolerance to salt stress as measured by biomass production of greenhouse-grown intact plants. Furthermore, experiments conducted with leaf discs from glycine betaine-accumulating plants indicated enhanced recovery from photoinhibition caused by high light and salt stress as well as improved tolerance to photoinhibition under low temperature conditions. In conclusion, introduction of glycine betaine production into tobacco is associated with increased stress tolerance probably partly due to improved protection of the photosynthetic apparatus.     

Effect of novel compound, 1-methyl-1-piperidino methane sulfonate (MPMS), on the osmoprotectant activity of glycine betaine,cholien and l-proline in Escherichia coli

A novel compound, 1-methyl-1-piperidino methane sulfonate (MPMS), was found to block the osmoprotectant activity of choline and L-proline, but not glycine betaine in Escherichia coli. MPMS was more active against salt-sensitive than salt-resistant strains, but had no effect on the salt tolerance of a mutant which was unable to transport choline, glycine betaine and proline. Growth of E. coli in NaCl was inhibited by MPMS and restored by glycine betaine, but not by choline or L-proline. Uptake of radiolabeled glycine betaine, choline or L-proline by cells grown at high osmolarity was not inhibited when MPMS and the radioactive substrates were added simultaneously. Preincubation for 5 min with MPMS reduced the uptake of choline and L-proline, but not glycine betaine. Similar incubation with MPMS had no effect on the uptake of radiolabeled glucose or succinate. The toxicity of MPMS was much lower than that of the L-proline analogues L-azetidine-2-carboxylic acid and 3,4-dehydro-DL-proline. The exact mechanism by which MPMS exerts its effect is not entirely clear. MPMS or a metabolite may interfere with the activity of several independent permeases involved in the uptake of osmoprotective compounds, or the conversion of choline to glycine betaine, or effect the expression of some of the osmoregulatory genes.Abbreviations MPMS 1-methyl-1-piperidino-methane sulfonate     

Increased glycine betaine synthesis and salinity tolerance in AhCMO transgenic cotton lines

Glycine betaine is an osmoprotectant that plays an important role and accumulates rapidly in many plants during salinity or drought stress. Choline monooxygenase (CMO) is a major catalyst in the synthesis of glycine betaine. In our previous study, a CMO gene (AhCMO) cloned from Atriplex hortensis was introduced into cotton (Gossypium hirsutum L.) via Agrobacterium mediation to enhance resistance to salinity stress. However, there is little or no knowledge of the salinity tolerance of the transgenic plants, particularly under saline-field conditions. In the present study, two transgenic AhCMO cotton lines of the T₃ generation were used to study the AhCMO gene expression, and to determine their salinity tolerance in both greenhouse and field under salinity stress. Molecular analysis confirmed that the transgenic plants expressed the AhCMO gene. Greenhouse study showed that on average, seedlings of the transgenic lines accumulated 26 and 131% more glycine betaine than those of non-transgenic plants (SM3) under normal and salt-stress (150 mmol l⁻¹ NaCl) conditions, respectively. The osmotic potential, electrolyte leakage and malondialdehyde (MDA) accumulation were significantly lower in leaves of the transgenic lines than in those of SM3 after salt stress. The net photosynthesis rate and Fv/Fm in transgenic cotton leaves were less affected by salinity than in non-transgenic cotton leaves. Therefore, transgenic cotton over-expressing AhCMO was more tolerant to salt stress due to elevated accumulation of glycine betaine, which provided greater protection of the cell membrane and photosynthetic capacity than in non-transgenic cotton. The seed cotton yield of the transgenic plants was lower under normal conditions, but was significantly higher than that of non-transgenic plants under salt-stressed field conditions. The results indicate that over-expression of AhCMO in cotton enhanced salt stress tolerance, which is of great value in cotton production in the saline fields.     

Trehalose metabolism in Escherichia coli: stress protection and stress regulation of gene expression

Endogenously synthesized trehalose is a stress protectant in Escherichia coli. Externally supplied trehalose does not serve as a stress protectant, but it can be utilized as the sole source of carbon and energy. Mutants defective in trehalose synthesis display an impaired osmotic tolerance in minimal growth media without glycine betaine, and an impaired stationary-phaseinduced heat tolerance. Mechanisms for stress protection by trehalose are discussed. The genes for trehalose-6-phosphate synthase (otsA) and anabolic trehalose-6-phosphate phosphatase (otsB) constitute an operon. Their expression is induced both by osmotic stress and by growth into the stationary phase and depend on the sigma factor encoded by rpoS (katF). rpoS is amber-mutated in E. coli K-12 and its DNA sequence varies among K-12 strains. For trehalose catabolism under osmotic stress E. coli depends on the osmoticcally inducible periplasmic trehalase (TreA). In the absence of osmotic stress, trehalose induces the formation of an enzyme II^Tre (TreB) of the group translocation system, a catabolic trehalose-6-phosphate phosphatase (TreE), and an amylotrehalase (TreC) which converts trehalose to free glucose and a glucose polymer.     

<Emphasis Type="Italic">Marinococcus halophilus</Emphasis> DSM 20408<Superscript>T </Superscript>encodes two transporters for compatible solutes belonging to the betaine-carnitine-choline transporter family: identification and characterization of ectoine transporter EctM and glycine betaine transporter BetM

In response to osmotic stress, the halophilic, Gram-positive bacterium Marinococcus halophilus accumulates compatible solutes either by de novo synthesis or by uptake from the medium. To characterize transport systems responsible for the uptake of compatible solutes, a plasmid-encoded gene bank of M. halophilus was transferred into the transport-deficient strain Escherichia coli MKH13, and two genes were cloned by functional complementation required for ectoine and glycine betaine transport. The ectoine transporter is encoded by an open reading frame of 1,578 bp named ectM. The gene ectM encodes a putative hydrophobic, 525-residue protein, which shares significant identity to betaine-carnetine-choline transporters (BCCTs). The transporter responsible for the uptake of glycine betaine in M. halophilus is encoded by an open reading frame of 1,482 bp called betM. The potential, hydrophobic BetM protein consists of 493 amino acid residues and belongs, like EctM, to the BCCT family. The affinity of whole cells of E. coli MKH13 for ectoine (K_s=1.6 M) and betaine (K_s=21.8 M) was determined, suggesting that EctM and BetM exhibit a high affinity for their substrates. An elevation of the salinity in the medium resulted in an increased uptake of ectoine via EctM and glycine betaine via BetM in E. coli MKH13 cells, demonstrating that both systems are osmoregulated.Communicated by W.D. Grant     

Cloning of theHIS3 gene ofYarrowia lipolytica

TheHIS3 gene of the yeastYarrowia lipolytica has been cloned from a genomic library by complementation of thehis3 mutation ofSaccharomyces cerevisiae. The gene was subsequently subcloned inEscherichia coli and characterized by restriction enzyme mapping.     

Transfer of E. coli gutD gene into maize and regeneration of salt-tolerant transgenic plants

GutD gene, encoding a key enzyme (glucitol-6-phosphate dehydrogenase) of sugar alcohol metabolic pathway inE. coli, was transferred into maize. Results of Southern and Western blotting analysis certified that this gene had integrated and been expressed in transgenic maize plants and their progeny. The synthesis and accumulation of sorbitol were detected in transgenic maize plants and a preliminary nutrient solution culture experiment showed thatgutD transgenic maize plants had an increased tolerance to salt stress compared with nontransgenic ones. Project supported by the National Natural Science Foundation of China (Grant No. 39670413) and “863” State High Technology Development Program.     

Quorum Sensing Regulates the Osmotic Stress Response in Vibrio harveyi

Bacteria use a chemical communication process called quorum sensing to monitor cell density and to alter behavior in response to fluctuations in population numbers. Previous studies with Vibrio harveyi have shown that LuxR, the master quorum-sensing regulator, activates and represses >600 genes. These include six genes that encode homologs of the Escherichia coli Bet and ProU systems for synthesis and transport, respectively, of glycine betaine, an osmoprotectant used during osmotic stress. Here we show that LuxR activates expression of the glycine betaine operon betIBA-proXWV, which enhances growth recovery under osmotic stress conditions. BetI, an autorepressor of the V. harveyi betIBA-proXWV operon, activates the expression of genes encoding regulatory small RNAs that control quorum-sensing transitions. Connecting quorum-sensing and glycine betaine pathways presumably enables V. harveyi to tune its execution of collective behaviors to its tolerance to stress.     

Trehalose/2-sulfotrehalose biosynthesis and glycine-betaine uptake are widely spread mechanisms for osmoadaptation in the Halobacteriales

We investigated the mechanisms of osmoadaptation in the order Halobacteriales, with special emphasis on Haladaptatus paucihalophilus, known for its ability to survive in low salinities. H. paucihalophilus genome contained genes for trehalose synthesis (trehalose-6-phosphate synthase/trehalose-6-phosphatase (OtsAB pathway) and trehalose glycosyl-transferring synthase pathway), as well as for glycine betaine uptake (BCCT family of secondary transporters and QAT family of ABC transporters). H. paucihalophilus cells synthesized and accumulated ∼1.97–3.72 μmol per mg protein of trehalose in a defined medium, with its levels decreasing with increasing salinities. When exogenously supplied, glycine betaine accumulated intracellularly with its levels increasing at higher salinities. RT-PCR analysis strongly suggested that H. paucihalophilus utilizes the OtsAB pathway for trehalose synthesis. Out of 83 Halobacteriales genomes publicly available, genes encoding the OtsAB pathway and glycine betaine BCCT family transporters were identified in 38 and 60 genomes, respectively. Trehalose (or its sulfonated derivative) production and glycine betaine uptake, or lack thereof, were experimentally verified in 17 different Halobacteriales species. Phylogenetic analysis suggested that trehalose synthesis is an ancestral trait within the Halobacteriales, with its absence in specific lineages reflecting the occurrence of gene loss events during Halobacteriales evolution. Analysis of multiple culture-independent survey data sets demonstrated the preference of trehalose-producing genera to saline and low salinity habitats, and the dominance of genera lacking trehalose production capabilities in permanently hypersaline habitats. This study demonstrates that, contrary to current assumptions, compatible solutes production and uptake represent a common mechanism of osmoadaptation within the Halobacteriales.     

Trehalose Biosynthesis in Response to Abiotic Stresses

Trehalose is a non‐reducing disaccharide that is present in diverse organisms ranging from bacteria and fungi to invertebrates, in which it serves as an energy source, osmolyte or protein/membrane protectant. The occurrence of trehalose and trehalose biosynthesis pathway in plants has been discovered recently. Multiple studies have revealed regulatory roles of trehalose‐6‐phosphate, a precursor of trehalose, in sugar metabolism, growth and development in plants. Trehalose levels are generally quite low in plants but may alter in response to environmental stresses. Transgenic plants overexpressing microbial trehalose biosynthesis genes have been shown to contain increased levels of trehalose and display drought, salt and cold tolerance. In‐silico expression profiling of all Arabidopsis trehalose‐6‐phosphate synthases (TPSs) and trehalose‐6‐phosphate phosphatases (TPPs) revealed that certain classes of TPS and TPP genes are differentially regulated in response to a variety of abiotic stresses. These studies point to the importance of trehalose biosynthesis in stress responses.     

A novel potassium deficiency-induced stimulon in<Emphasis Type="Italic">Anabaena torulosa</Emphasis>

Potassium deficiency enhanced the synthesis of fifteen proteins in the nitrogen-fixing cyanobacteriumAnabaena torulosa and of nine proteins inEscherichia coli. These were termed potassium deficiency-induced proteins or PDPs and constitute hitherto unknown potassium deficiency-induced stimulons. Potassium deficiency also enhanced the synthesis of certain osmotic stress-induced proteins. Addition of K⁺ repressed the synthesis of a majority of the osmotic stress-induced proteins and of PDPs in these bacteria. These proteins contrast with the dinitrogenase reductase ofA. torulosa and the glycine betaine-binding protein ofE. coli, both of which were osmo-induced to a higher level in potassium-supplemented conditions. The data demonstrate the occurrence of novel potassium deficiency-induced stimulons and a wider role of K+ in regulation of gene expression and stress responses in bacteria.     

Osmotic adaptation in Brevibacterium linens: differential effect of proline and glycine betaine on cytoplasmic osmolyte pool

In the coryneform Brevibacterium linens, ectoine constitutes the major intracellular solute accumulated under elevated medium osmolarity. Here we report that exogenously supplied proline, choline, glycine betaine, and even ectoine, protected bacterial cells against deleterious effects of a hyperosmotic constraint (i.e. 1.5 M NaCl). In all cases, a significant improvement of growth was observed; in parallel, intracellular osmolyte pools composed mainly of glutamate and ectoine substantially increased, either with added glycine betaine (under limiting supply) or with proline. However, these two osmoprotectants behaved differently: glycine betaine acted as a genuine osmoprotectant, whereas proline was accumulated only transiently and participated actively in the biosynthesis of glutamate, ectoine, and trehalose. The strategy developed by B. linens cells allows the proposal of a novel role for proline in the osmoprotection process through its conversion to the apparently preferred endogenous osmolyte ectoine.     

Metabolic engineering of glycine betaine synthesis: plant betaine aldehyde dehydrogenases lacking typical transit peptides are targeted to tobacco chloroplasts where they confer betaine aldehyde resistance

Certain higher plants synthesize and accumulate glycine betaine, a compound with osmoprotectant properties. Biosynthesis of glycine betaine proceeds via the pathway choline betaine aldehyde glycine betaine. Plants such as tobacco (Nicotiana tabacum L.) which do not accumulate glycine betaine lack the enzymes catalyzing both reactions. As a step towards engineering glycine betaine accumulation into a non-accumulator, spinach and sugar beet complementary-DNA sequences encoding the second enzyme of glycine-betaine synthesis (betaine aldehyde dehydrogenase, BADH, EC 1.2.1.8) were expressed in tobacco. Despite the absence of a typical transit peptide, BADH was targeted to the chloroplast in leaves of transgenic plants. Levels of extractable BADH were comparable to those in spinach and sugar beet, and the molecular weight, isoenzyme profile and K _m for betaine aldehyde of the BADH enzymes from transgenic plants were the same as for native spinach or sugar beet BADH. Transgenic plants converted supplied betaine aldehyde to glycine betaine at high rates, demonstrating that they were able to transport betaine aldehyde across both the plasma membrane and the chloroplast envelope. The glycine betaine produced in this way was not further metabolized and reached concentrations similar to those in plants which accumulate glycine betaine naturally. Betaine aldehyde was toxic to non-transformed tobacco tissues whereas transgenic tissues were resistant due to detoxification of betaine aldehyde to glycine betaine. Betaine aldehyded ehydrogenase is therefore of interest as a potential selectable marker, as well as in the metabolic engineering of osmoprotectant biosynthesis.Abbreviations BADH betaine aldehyde dehydrogenase - bp base pairs - FAB-MS fast atom bombardment-mass spectrometry - GAPDH NADP-linked glyceraldehyde-3-phosphate dehydrogenase We thank Dr. G. An for the gift of the vector pGA643 and Mr. Sylvain Lebeurier for help in maintaining plants. This work was supported, in part, by grants from the Natural Sciences and Engineering Research Council of Canada, the Rockefeller Foundation, and the U.S. Department of Agriculture, and by gifts from CIBAGEIGY Biotechnology.     

Improved salt tolerance in tobacco plants by co-transformation of a betaine synthesis gene <Emphasis Type="Italic">BADH</Emphasis> and a vacuolar Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter gene <Emphasis Type="Italic">SeNHX1</Emphasis>

Three types of transgenic tobacco plants were acquired by separate transformation or co-transformation of a vacuolar Na⁺/H⁺ antiporter gene, SeNHX1, and a betaine synthesis gene, BADH. When exposed to 200 mM NaCl, the dual gene-transformed plants displayed greater accumulation of betaine and Na⁺ than their wild-type counterparts. Photosynthetic rate and photosystem II activity in the transgenic plants were less affected by salt stress than wild-type plants. Transgenic plants exhibited a greater increase in osmotic pressure than wild-type plants when exposed to NaCl. More importantly, the dual gene transformed plants accumulated higher biomass than either of the single transgenic plants under salt stress. Taken together, these findings indicate that simultaneous transformation of BADH and SeNHX1 genes into tobacco plants can enable plants to accumulate betaine and Na⁺, thus conferring them more tolerance to salinity than either of the single gene transformed plants or wild-type tobacco plants. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Transport and metabolism of trehalose in Escherichia coli and Salmonella typhimurium

The metabolism of trehalose in wild type cells of Escherichia coli and Salmonella typhimurium has been investigated. Intact cells of Escherichia coli (grown on trehalose) accumulated [¹⁴C]-trehalose as [¹⁴C]-trehalose 6-phosphate. Toluene-treated cells catalyzed the synthesis of the [¹⁴C]-sugar phosphate from [¹⁴C]-trehalose and phosphoenolpyruvate; ATP did not serve as phosphoryl donor. Trehalose 6-phosphate could subsequently be hydrolyzed by trehalose 6-phosphate hydrolase, an enzyme which catalyzes the hydrolysis of the disaccharide phosphate into glucose and glucose 6-phosphate. Both Escherichia coli and Salmonella typhimurium induced this enzyme when they grew on trehalose.These findings suggest that trehalose is transported in these bacteria by an inducible phosphoenolpyruvate:trehalose phosphotransferase system.The presence of a constitutive trehalase was also detected.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethanosulfonic acid - PEP phosphoenolpyruvate - PTS phosphoenolpyruvate: glycose phosphotransferase system - O.D. optical density     

betaine modulates intracellular thiol and potassium levels in Escherichia coli in medium with high osmolarity and alkaline pH

Glycine betaine stimulates the growth rate of various bacteria in high osmolarity medium. In our studies, glycine betaine stimulated the growth rate of Escherichia coli K 12 in minimal medium with normal osmolarity at alkaline pH (pH 8.2). Betaine also caused a reduction in the intracellular pools of K⁺ and low molecular weight thiols in E. coli growing both in medium with high osmolarity and at alkaline pH. These effects of betaine were absent at pH 7.0. In cells growing in high osmolarity medium, 10 mM sodium acetate or 10 M N-ethylmaleimide reduced expression of the osmosensitive gene proU to the same extent as treatment with betaine; however, under these conditions, sodium acetate and N-ethylmaleimide did not stimulate the growth of E. coli. It is proposed that low molecular weight thiols and intracellular pH may participate in the response of E. coli to betaine.