Glycine Betaine Transport System Research Articles

Genomic Analysis of Halotolerant Bacterial Strains Martelella soudanensis NC18T and NC20.

Two novel, halotolerant strains of Martelella soudanensis, NC18T and NC20, were isolated from deep subsurface sediment, deeply sequenced, and comparatively analyzed with related strains. Based on a phylogenetic analysis using 16S rRNA gene sequences, the two strains grouped with members of the genus Martelella. Here, we sequenced the complete genomes of NC18T and NC20 to understand the mechanisms of their halotolerance. The genome sizes and G+C content of the strains were 6.1 Mb and 61.8 mol%, respectively. Moreover, NC18T and NC20 were predicted to contain 5,849 and 5,830 genes, and 5,502 and 5,585 protein-coding genes, respectively. Both strains contain the identically predicted 6 rRNAs and 48 tRNAs. The harboring of halotolerant-associated genes revealed that strains NC18T and NC20 might tolerate high salinity through the accumulation of potassium ions in a "salt-in" strategy induced by K+ uptake protein (kup) and the K+ transport system (trkAH and kdpFABC). These two strains also use the ectoine transport system (dctPQM), the glycine betaine transport system (proVWX), and glycine betaine uptake protein (opu) to accumulate "compatible solutes," such as ectoine and glycine betaine, to protect cells from salt stress. This study reveals the halotolerance mechanism of strains NC18T and NC20 in high salt environments and suggests potential applications for these halotolerant and halophilic strains in environmental biotechnology.

Transcriptomic Analysis of Staphylococcus xylosus in Solid Dairy Matrix Reveals an Aerobic Lifestyle Adapted to Rind.

Staphylococcus xylosus is found in the microbiota of traditional cheeses, particularly in the rind of soft smeared cheeses. Despite its frequency, the molecular mechanisms allowing the growth and adaptation of S. xylosus in dairy products are still poorly understood. A transcriptomic approach was used to determine how the gene expression profile is modified during the fermentation step in a solid dairy matrix. S. xylosus developed an aerobic metabolism perfectly suited to the cheese rind. It overexpressed genes involved in the aerobic catabolism of two carbon sources in the dairy matrix, lactose and citrate. Interestingly, S. xylosus must cope with nutritional shortage such as amino acids, peptides, and nucleotides, consequently, an extensive up-regulation of genes involved in their biosynthesis was observed. As expected, the gene sigB was overexpressed in relation with general stress and entry into the stationary phase and several genes under its regulation, such as those involved in transport of anions, cations and in pigmentation were up-regulated. Up-regulation of genes encoding antioxidant enzymes and glycine betaine transport and synthesis systems showed that S. xylosus has to cope with oxidative and osmotic stresses. S. xylosus expressed an original system potentially involved in iron acquisition from lactoferrin.

A99 IMPACT OF OSMOTIC PERTURBATIONS ON THE GUT MICROBIOTA AND HOST HEALTH

Abstract Background Osmotic diarrhea is a prevalent condition concurrent with diverse pathologies such as Crohn’s disease and is due to unabsorbed solutes in the gastrointestinal contents inhibiting normal water absorption. Osmotic laxatives such as polyethylene glycol (PEG) take advantage of this process to counteract constipation. However, short-term PEG treatment impacts the gut microbial community, and in humanized and conventional mice leads to disappearance of the abundant and prevalent bacterial family S24-7, while increasing the relative abundance of a related family, Bacteroidaceae. Excitingly, despite community re-equilibration over weeks, S24-7 is capable of superseding other bacterial members and reach its original levels when reintroduced into the environment. My central hypothesis is that the depletion of S24-7 during osmotic diarrhea is due to its deficiency of stress response pathways required to counteract increased osmolality. However, in the absence of this stress, S24-7 is capable of exploiting a unique interaction with the immune system to successfully recolonize. Aims To shed light on this, I will pursue the following two aims: Aim 1: Compare the growth, survival and expression profiles of S24-7 and B. thetaiotaomicron in a bi-colonized gnotobiotic mouse model before, during and after osmotic perturbation to identify candidate genes involved in response to osmotic perturbation. Aim 2: Identify changes in S24-7 gene expression during recolonization and evaluate the host immune response. Methods Gnotobiotic mice were first colonized with S24-7 and then B. thetaiotaomicron, treated with PEG for 6 days and monitored during recovery and recolonization for 20 days. Fecal pellets were used to quantify bacterial abundances via qPCR and bacterial gene expression analysis through metatranscriptomics. Mouse serum was used for ELISA immunoassays to compare adaptive immune responses through serum IgG as well as to quantify glycine betaine levels. Results During PEG treatment S24-7 became undetectable and recolonized to pre-treatment levels during recovery. Using genomic and metagenomic annotation we have identified that S24-7 isolates do not possess the glycine betaine transport system, which is present in B. thetaiotaomicron (Figure 1). B. thetaiotaomicron reduced host serum glycine betaine levels, unlike S24-7. In S24-7 mono-colonized mice, S24-7 specific serum IgG was not detectable. However, once mice were co-colonized with B. thetaiotaomicron, both S24-7 and B. thetaiotaomicron specific IgG is detected, identifying an interesting relationship between S24-7 and the adaptive immune system that may shed light on its unique recolonization. Conclusions By studying S24-7 sensitivity to osmotic stress and its robust colonization abilities we are shedding light on the mechanisms of microbiota response to perturbations that are commonly experienced by the human gut. Funding Agencies None

Ectoine functions as an osmoprotectant in Bacillus subtilis and is accumulated via the ABC-transport system OpuC

We report here that the cyclic amino acid ectoine functions as an osmoprotectant for the soil bacterium Bacillus subtilis. Growth experiments with a set of B. subtilis strains that carry defined mutations in the glycine betaine transport systems OpuA, OpuC and OpuD and the choline transport system OpuB revealed that ectoine was specifically accumulated via the ABC-transport system OpuC. Competition experiments employing unlabeled ectoine and radiolabeled glycine betaine showed that the OpuC transport system has a low affinity for ectoine with a Ki value of approximately 1.5 mM. Ectoine was identified by 1H NMR spectroscopy in the solute pool of cells grown in the presence of ectoine. Ectoine could not be used by B. subtilis as sole carbon or nitrogen source. Our data thus characterise ectoine as a metabolically inert stress compound for B. subtilis and establish a crucial role for the ABC-transport system OpuC for the acquisition of the osmoprotectant ectoine from the environment.

OusB, a Broad-Specificity ABC-Type Transporter fromErwinia chrysanthemi, Mediates Uptake of Glycine Betaine and Choline with a High Affinity

The ability of Erwinia chrysanthemi to cope with environments of elevated osmolality is due in part to the transport and accumulation of osmoprotectants. In this study we have identified a high-affinity glycine betaine and choline transport system in E. chrysanthemi. By using a pool of Tn5-B21 ousA mutants, we isolated a mutant that could grow in the presence of a toxic analogue of glycine betaine (benzyl-glycine betaine) at high osmolalities. This mutant was impaired in its ability to transport all effective osmoprotectants in E. chrysanthemi. The DNA sequence of the regions flanking the transposon insertion site revealed three chromosomal genes (ousVWX) that encode components of an ABC-type transporter (OusB): OusV (ATPase), OusW (permease), and OusX (periplasmic binding protein). The OusB components showed a significant degree of sequence identity to components of ProU from Salmonella enterica serovar Typhimurium and Escherichia coli. OusB was found to restore the uptake of glycine betaine and choline through functional complementation of an E. coli mutant defective in both ProU and ProP osmoprotectant uptake systems. Competition experiments demonstrated that choline, dimethylsulfoniacetate, dimethylsulfoniopropionate, and ectoine were effective competitors for OusB-mediated betaine transport but that carnitine, pipecolate, and proline were not effective. In addition, the analysis of single and double mutants showed that OusA and OusB were the only osmoprotectant transporters operating in E. chrysanthemi.

Cloning and characterization of theHalobacillus trueperi betHgene, encoding the transport system for the compatible solute glycine betaine

Cloning and characterization of the<i>Halobacillus trueperi betH</i>gene, encoding the transport system for the compatible solute glycine betaine

Cloning and characterization of the Halobacillus trueperi betH gene, encoding the transport system for the compatible solute glycine betaine

Halobacillus trueperi accumulates glycine betaine under condition of high osmolarity. A fragment of the glycine betaine transporter betH gene was obtained from the genome of H. trueperi with degenerate primers. Through Southern blot hybridization and inverse PCR, a 5.1 kb EcoRI fragment containing the complete betH gene was identified and subsequently sequenced. The betH gene was predicted to encode a 55.2 kDa protein (504 amino acid residues) with 12 transmembrane regions. BetH showed 56% identity to the OpuD of Bacillus subtilis which belongs to the betaine/carnitine/choline transporter (BCCT) family. Its putative promoter region was highly homologous to σ B-dependent promoter of B. subtilis. A 2.6 kb fragment containing the betH gene was cloned into pUC18 and transformed into the Escherichia coli MKH13. The accumulation of glycine betaine in transformed E. coli MKH13 bacteria was confirmed using 13C nuclear magnetic resonance spectroscopy.

Cloning and characterization of the Halobacillus trueperi betH Gene, encoding the transport system for the compatible solute glycine betaine

Cloning and characterization of the Halobacillus trueperi betH Gene, encoding the transport system for the compatible solute glycine betaine

Isolation and Characterization of ButA, a Secondary Glycine Betaine Transport System Operating in Tetragenococcus halophila

Through functional complementation of an Escherichia coli mutant defective in glycine betaine uptake, we identified a single-component glycine betaine transporter from Tetragenococcus halophila, a moderate halophilic lactic acid bacterium. DNA sequence analysis characterized the ButA protein as a member of the betaine choline carnitine transporter (BCCT) family, that includes a variety of previously characterized compatible solute transporters such as OpuD from Bacillus subtilis, EctP and BetP from Corynebacterium glutamicum, and BetL from Listeria monocytogenes. When expressed in the heterologous host E. coli, the permease is specific for glycine betaine and does not transport the other osmoprotectants previously described for T. halophila (i.e. carnitine, choline, dimethylsulfonioacetate, dimethylsulfoniopropionate, and ectoine). In E. coli, statement of ButA is mainly constitutive and maximal uptake activity may result from a weak osmotic induction. This is the first study demonstrating a role for a permease in osmoregulation, and GB uptake, of a lactic acid bacterium.

BetS is a major glycine betaine/proline betaine transporter required for early osmotic adjustment in Sinorhizobium meliloti.

Hybridization to a PCR product derived from conserved betaine choline carnitine transporter (BCCT) sequences led to the identification of a 3.4-kb Sinorhizobium meliloti DNA segment encoding a protein (BetS) that displays significant sequence identities to the choline transporter BetT of Escherichia coli (34%) and to the glycine betaine transporter OpuD of Bacillus subtilis (30%). Although the BetS protein shows a common structure with BCCT systems, it possesses an unusually long hydrophilic C-terminal extension (169 amino acids). After heterologous expression of betS in E. coli mutant strain MKH13, which lacks choline, glycine betaine, and proline transport systems, both glycine betaine and proline betaine uptake were restored, but only in cells grown at high osmolarity or subjected to a sudden osmotic upshock. Competition experiments demonstrated that choline, ectoine, carnitine, and proline were not effective competitors for BetS-mediated betaine transport. Kinetic analysis revealed that BetS has a high affinity for betaines, with K(m)s of 16 +/- 2 microM and 56 +/- 6 microM for glycine betaine and proline betaine, respectively, in cells grown in minimal medium with 0.3 M NaCl. BetS activity appears to be Na(+) driven. In an S. meliloti betS mutant, glycine betaine and proline betaine uptake was reduced by about 60%, suggesting that BetS represents a major component of the overall betaine uptake activities in response to salt stress. beta-Galactosidase activities of a betS-lacZ strain grown in various conditions showed that betS is constitutively expressed. Osmotic upshock experiments performed with wild-type and betS mutant cells, treated or not with chloramphenicol, indicated that BetS-mediated betaine uptake is the consequence of immediate activation of existing proteins by high osmolarity, most likely through posttranslational activation. Growth experiments underscored the crucial role of BetS as an emerging system involved in the rapid acquisition of betaines by S. meliloti subjected to osmotic upshock.

Phospholipid Interactions Regulate Transporter Activity

van der Heide et al. studied activation of the ABC transporter OpuA, which allows bacteria to accumulate glycine betaine in response to increased osmotic conditions, in reconstituted liposome preparations under varying osmotic conditions. Activation of the transporter by an osmotic gradient was dependent on the percentage of anionic lipids in the reconstituted liposomes, and in liposomes without any anionic lipids, the transporter failed to be activated. Lipids can form multiple structures depending on their biochemical properties, and maximal activation of OpuA depended on the presence of lipids that did not form bilayers. However, OpuA activity was relatively insensitive to changes in the length of the acyl chains, which would alter the membrane thickness of the liposomes. Inclusion of ions in the lumen of the vesicles to create a situation of high ionic strength stimulated OpuA activity even in the absence of an osmotic gradient. Thus, the authors propose that increased osmotic conditions alter the cytoplasmic ionic strength and alter the interaction between the transporter and the anionic phospholipid head groups, leading to transporter activation. T. van der Heide, M. C. A. Stuart, B. Poolman, On the osmotic signal and osmosensing mechanism of an ABC transport system for glycine betaine. EMBO J. 20 , 7022-7032 (2001). [Abstract] [Full Text]

Phospholipid Interactions Regulate Transporter Activity

van der Heide et al. studied activation of the ABC transporter OpuA, which allows bacteria to accumulate glycine betaine in response to increased osmotic conditions, in reconstituted liposome preparations under varying osmotic conditions. Activation of the transporter by an osmotic gradient was dependent on the percentage of anionic lipids in the reconstituted liposomes, and in liposomes without any anionic lipids, the transporter failed to be activated. Lipids can form multiple structures depending on their biochemical properties, and maximal activation of OpuA depended on the presence of lipids that did not form bilayers. However, OpuA activity was relatively insensitive to changes in the length of the acyl chains, which would alter the membrane thickness of the liposomes. Inclusion of ions in the lumen of the vesicles to create a situation of high ionic strength stimulated OpuA activity even in the absence of an osmotic gradient. Thus, the authors propose that increased osmotic conditions alter the cytoplasmic ionic strength and alter the interaction between the transporter and the anionic phospholipid head groups, leading to transporter activation.T. van der Heide, M. C. A. Stuart, B. Poolman, On the osmotic signal and osmosensing mechanism of an ABC transport system for glycine betaine. EMBO J. 20, 7022-7032 (2001). [Abstract] [Full Text]

On the osmotic signal and osmosensing mechanism of an ABC transport system for glycine betaine.

The osmosensing mechanism of the ATP-binding cassette (ABC) transporter OpuA of Lactococcus lactis has been elucidated for the protein reconstituted in liposomes. Activation of OpuA by osmotic upshift was instantaneous and reversible and followed changes in volume and membrane structure of the proteoliposomes. Osmotic activation of OpuA was dependent on the fraction of anionic lipids present in the lipid bilayer. Also, cationic and anionic lipophilic amphiphiles shifted the activation profile in a manner indicative of an osmosensing mechanism, in which electrostatic interactions between lipid headgroups and the OpuA protein play a major role. Further support for this notion came from experiments in which ATP-driven uptake and substrate-dependent ATP hydrolysis were measured with varying concentrations of osmolytes at the cytoplasmic face of the protein. Under iso-osmotic conditions, the transporter could be activated by high concentrations of ionic osmolytes, whereas neutral ones had no effect, demonstrating that intracellular ionic strength, rather than a specific signaling molecule or water activity, signals osmotic stress to the transporter. The data indicate that OpuA is under the control of a mechanism in which the membrane and ionic strength act in concert to signal osmotic changes.

Osmoregulated ABC-transport system of Lactococcus lactis senses water stress via changes in the physical state of the membrane.

An osmoregulated ABC transporter (OpuA) with novel structural features has been identified that responds to water stress. This glycine betaine transport system consists of an ATP-binding/hydrolyzing subunit (OpuAA) and a protein (OpuABC) that contains both the translocator and the substrate-binding domain. The components of OpuA have been overexpressed, purified, and functionally incorporated into liposomes with an ATP-regenerating system in the vesicle lumen. A transmembrane osmotic gradient (outside hyperosmotic relative to the inside) of both ionic and nonionic compounds was able to osmotically activate OpuA in the proteoliposomal system. Hypoosmotic medium conditions inhibited the basal activity of the system. The data show that OpuAA and OpuABC are sufficient for osmoregulated transport, indicating that OpuA can act both as osmosensor and osmoregulator. Strikingly, OpuA could also be activated by low concentrations of cationic and anionic amphipaths, which interact with the membrane. This result indicates that activation by a transmembrane osmotic gradient is mediated by changes in membrane properties/protein-lipid interactions.

Glycine Betaine Transport in Lactococcus lactis Is Osmotically Regulated at the Level of Expression and Translocation Activity

Microorganisms react upon hyperosmotic stress by accumulating compatible solutes. Here we report that Lactococcus lactis uses a transport system for glycine betaine that, contrary to earlier observations (D. Molenaar et al., J. Bacteriol. 175:5438-5444, 1993), is osmotically regulated at the levels of both expression and transport activity.

Identification of an ATP-Driven, Osmoregulated Glycine Betaine Transport System in Listeria monocytogenes

The ability of the gram-positive, food-borne pathogen Listeria monocytogenes to tolerate environments of elevated osmolarity and reduced temperature is due in part to the transport and accumulation of the osmolyte glycine betaine. Previously we showed that glycine betaine transport was the result of Na(+)-glycine betaine symport. In this report, we identify a second glycine betaine transporter from L. monocytogenes which is osmotically activated but does not require a high concentration of Na(+) for activity. By using a pool of Tn917-LTV3 mutants, a salt- and chill-sensitive mutant which was also found to be impaired in its ability to transport glycine betaine was isolated. DNA sequence analysis of the region flanking the site of transposon insertion revealed three open reading frames homologous to opuA from Bacillus subtilis and proU from Escherichia coli, both of which encode glycine betaine transport systems that belong to the superfamily of ATP-dependent transporters. The three open reading frames are closely spaced, suggesting that they are arranged in an operon. Moreover, a region upstream from the first reading frame was found to be homologous to the promoter regions of both opuA and proU. One unusual feature not shared with these other two systems is that the start codons for two of the open reading frames in L. monocytogenes appear to be TTG. That glycine betaine uptake is nearly eliminated in the mutant strain when it is assayed in the absence of Na(+) is an indication that only the ATP-dependent transporter and the Na(+)-glycine betaine symporter occur in L. monocytogenes.

Identification and disruption of BetL, a secondary glycine betaine transport system linked to the salt tolerance of Listeria monocytogenes LO28.

The trimethylammonium compound glycine betaine (N,N, N-trimethylglycine) can be accumulated to high intracellular concentrations, conferring enhanced osmo- and cryotolerance upon Listeria monocytogenes. We report the identification of betL, a gene encoding a glycine betaine uptake system in L. monocytogenes, isolated by functional complementation of the betaine uptake mutant Escherichia coli MKH13. The betL gene is preceded by a consensus sigmaB-dependent promoter and is predicted to encode a 55-kDa protein (507 amino acid residues) with 12 transmembrane regions. BetL exhibits significant sequence homologies to other glycine betaine transporters, including OpuD from Bacillus subtilis (57% identity) and BetP from Corynebacterium glutamicum (41% identity). These high-affinity secondary transporters form a subset of the trimethylammonium transporter family specific for glycine betaine, whose substrates possess a fully methylated quaternary ammonium group. The observed Km value of 7.9 microM for glycine betaine uptake after heterologous expression of betL in E. coli MKH13 is consistent with values obtained for L. monocytogenes in other studies. In addition, a betL knockout mutant which is significantly affected in its ability to accumulate glycine betaine in the presence or absence of NaCl has been constructed in L. monocytogenes. This mutant is also unable to withstand concentrations of salt as high as can the BetL+ parent, signifying the role of the transporter in Listeria osmotolerance.

Streptococcus mutans ffh, a gene encoding a homologue of the 54 kDa subunit of the signal recognition particle, is involved in resistance to acid stress.

The ability of Streptococcus mutans, a bacterial pathogen associated with dental caries, to tolerate rapid drops in plaque pH (acidurance), is considered an important virulence factor. To study this trait, Tn917 mutants of S. mutans strain JH1005 which display acid sensitivity have been isolated and partially characterized. In this paper, the characterization of one of these mutants, AS17, is reported. Preliminary sequence analysis revealed that the transposon insertion in AS17 occurred in the intergenic region of a two-gene locus which has been named sat for secretion and acid tolerance. This locus displays a high degree of homology to the ylxM-ffh operon of Bacillus subtilis. The sat+ locus was cloned by complementation of a conditional Escherichia coli ffh mutant with an S. mutans genomic library. Sequencing of the complementing clone identified the intact ylxM and ffh genes as well as a partial ORF with homology to the proUlopuAC gene of B. subtilis which encodes the binding protein of the ProU/OpuA osmoregulated glycine betaine transport system. RNA dot blot experiments indicated steady-state levels of ffh mRNA in the mutant that were approximately eightfold lower compared to parental levels. This suggests a partial polar effect of the sat-1::Tn917 mutation on ffh expression. Upon acid shock (pH 5), wild-type ffh mRNA levels were found to increase approximately four- to eightfold compared to unstressed (pH 7.5) levels. Mutant levels remained unaltered under the same conditions. Experiments designed to investigate the origins of the acid-sensitivity of the mutant revealed a lack of an acid-adaptive/tolerance response. Assays of proton-extruding ATPase (H+/ATPase) specific activity measured with purified membranes derived from acid-shocked AS17 showed twofold lower levels compared to the parent strain. Also, AS17 was found to be unable to ferment sorbitol although it was able to grow in glucose and a variety of other sugar substrates. These findings suggest that Ffh may be involved in the maintenance of a functional membrane protein composition during adaptation of S. mutans to changing environmental conditions.

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 cloning of a Azospirillum lipoferum locus that complements Escherichia coli proU mutant

Glycine betaine relieved sodium chloride-mediated inhibition of growth in Azospirillum lipoferum ATCC 29708. 35S-methionine labelling of proteins after salinity up-shock revealed strong induction of a 30 kDa protein which cross-reacted with the anti-glycine betaine binding protein antibody from Escherichia coli. This suggested that A. lipoferum had a salinity-induced ProU-like high-affinity glycine betaine transport system. A genomic library of A. lipoferum ATCC 29708 was screened for the proU-like gene by complementation of a proU mutant of E. coli. Four recombinant cosmids, capable of restoring growth of the proU mutant on plates containing 600 mM NaCl and 1 mM glycine betaine were selected. Selected recombinant cosmids hybridized with a proU gene probe from E. coli. Complementation of E. coli proU mutant with the A. lipoferum genomic DNA was evident by the ability of proU mutant (containing selected recombinant cosmids) to grow on minimal medium supplemented with 600 mM NaCl and 1 mM glycine betaine.