scholarly journals Conserved ABC Transport System Regulated by the General Stress Response Pathways of Alpha- and Gammaproteobacteria

2016 ◽  
Vol 199 (5) ◽  
Author(s):  
Julien Herrou ◽  
Jonathan W. Willett ◽  
Daniel M. Czyż ◽  
Gyorgy Babnigg ◽  
Youngchang Kim ◽  
...  
Keyword(s):  
Transport System ◽  
Brucella Abortus ◽  
Quaternary Ammonium ◽  
Sigma Factor ◽  
Glycine Betaine ◽  
Chronic Infection ◽  
Compatible Solutes ◽  
Compatible Solute ◽  
Periplasmic Binding Protein ◽  
Content Type

ABSTRACT Brucella abortus σE1 is an EcfG family sigma factor that regulates the transcription of dozens of genes in response to diverse stress conditions and is required for maintenance of chronic infection in a mouse model. A putative ATP-binding cassette transporter operon, bab1_0223-bab1_0226, is among the most highly activated gene sets in the σE1 regulon. The proteins encoded by the operon resemble quaternary ammonium-compatible solute importers but are most similar in sequence to the broadly conserved YehZYXW system, which remains largely uncharacterized. Transcription of yehZYXW is activated by the general stress sigma factor σS in Enterobacteriaceae, which suggests a functional role for this transport system in bacterial stress response across the classes Alphaproteobacteria and Gammaproteobacteria. We present evidence that B. abortus YehZYXW does not function as an importer of known compatible solutes under physiological conditions and does not contribute to the virulence defect of a σE1-null strain. The sole in vitro phenotype associated with genetic disruption of this putative transport system is reduced growth in the presence of high Li+ ion concentrations. A crystal structure of B. abortus YehZ revealed a class II periplasmic binding protein fold with significant structural homology to Archaeoglobus fulgidus ProX, which binds glycine betaine. However, the structure of the YehZ ligand-binding pocket is incompatible with high-affinity binding to glycine betaine. This is consistent with weak measured binding of YehZ to glycine betaine and related compatible solutes. We conclude that YehZYXW is a conserved, stress-regulated transport system that is phylogenetically and functionally distinct from quaternary ammonium-compatible solute importers. IMPORTANCE Brucella abortus σE1 regulates transcription in response to stressors encountered in its mammalian host and is necessary for maintenance of chronic infection in a mouse model. The functions of the majority of genes regulated by σE1 remain undefined. We present a functional/structural analysis of a conserved putative membrane transport system (YehZYXW) whose expression is strongly activated by σE1. Though annotated as a quaternary ammonium osmolyte uptake system, experimental physiological studies and measured ligand-binding properties of the periplasmic binding protein (PBP), YehZ, are inconsistent with this function. A crystal structure of B. abortus YehZ provides molecular insight into differences between bona fide quaternary ammonium osmolyte importers and YehZ-related proteins, which form a distinct phylogenetic and functional group of PBPs.

scholarly journals Investigations of Dimethylglycine, Glycine Betaine, and Ectoine Uptake by a Betaine-Carnitine-Choline Transporter Family Transporter with Diverse Substrate Specificity in Vibrio Species

2020 ◽  
Vol 202 (24) ◽  
Author(s):  
Gwendolyn J. Gregory ◽  
Anirudha Dutta ◽  
Vijay Parashar ◽  
E. Fidelma Boyd
Keyword(s):  
Amino Acid ◽  
Glycine Betaine ◽  
Vibrio Parahaemolyticus ◽  
Compatible Solutes ◽  
Binding Pocket ◽  
Compatible Solute ◽  
Amino Acid Residues ◽  
Choline Transporter ◽  
Content Type ◽  
Highly Effective

ABSTRACT Fluctuations in osmolarity are one of the most prevalent stresses to which bacteria must adapt, both hypo- and hyperosmotic conditions. Most bacteria cope with high osmolarity by accumulating compatible solutes (osmolytes) in the cytoplasm to maintain the turgor pressure of the cell. Vibrio parahaemolyticus, a halophile, utilizes at least six compatible solute transporters for the uptake of osmolytes: two ABC family ProU transporters and four betaine-carnitine-choline transporter (BCCT) family transporters. The full range of compatible solutes transported by this species has yet to be determined. Using an osmolyte phenotypic microarray plate for growth analyses, we expanded the known osmolytes used by V. parahaemolyticus to include N,N-dimethylglycine (DMG), among others. Growth pattern analysis of four triple-bccT mutants, possessing only one functional BCCT, indicated that BccT1 (VP1456), BccT2 (VP1723), and BccT3 (VP1905) transported DMG. BccT1 was unusual in that it could take up both compounds with methylated head groups (glycine betaine [GB], choline, and DMG) and cyclic compounds (ectoine and proline). Bioinformatics analysis identified the four coordinating amino acid residues for GB in the BccT1 protein. In silico modeling analysis demonstrated that GB, DMG, and ectoine docked in the same binding pocket in BccT1. Using site-directed mutagenesis, we showed that a strain with all four residues mutated resulted in the loss of uptake of GB, DMG, and ectoine. We showed that three of the four residues were essential for ectoine uptake, whereas only one of the residues was important for GB uptake. Overall, we have demonstrated that DMG is a highly effective compatible solute for Vibrio species and have elucidated the amino acid residues in BccT1 that are important for the coordination of GB, DMG, and ectoine transport. IMPORTANCE Vibrio parahaemolyticus possesses at least six osmolyte transporters, which allow the bacterium to adapt to high-salinity conditions. In this study, we identified several additional osmolytes that were utilized by V. parahaemolyticus. We demonstrated that the compound DMG, which is present in the marine environment, was a highly effective osmolyte for Vibrio species. We determined that DMG is transported via BCCT family carriers, which have not been shown previously to take up this compound. BccT1 was a carrier for GB, DMG, and ectoine, and we identified the amino acid residues essential for the coordination of these compounds. The data suggest that for BccT1, GB is more easily accommodated than ectoine in the transporter binding pocket.

scholarly journals Salt Stress-Induced Changes in the Transcriptome, Compatible Solutes, and Membrane Lipids in the Facultatively Phototrophic Bacterium Rhodobacter sphaeroides

2011 ◽  
Vol 77 (21) ◽  
pp. 7551-7559 ◽  
Author(s):  
Minoru Tsuzuki ◽  
Oleg V. Moskvin ◽  
Masayuki Kuribayashi ◽  
Kiichi Sato ◽  
Susana Retamal ◽  
...  
Keyword(s):  
Salt Stress ◽  
Rhodobacter Sphaeroides ◽  
Glycine Betaine ◽  
Mutational Analysis ◽  
Compatible Solutes ◽  
Phospholipid Composition ◽  
Compatible Solute ◽  
Content Type ◽  
Genes Encoding ◽  
Induced Changes

ABSTRACTResponses to NaCl stress were investigated in phototrophically grownAlphaproteobacterium Rhodobacter sphaeroidesby transcriptome profiling, mutational analysis, and measurements of compatible solutes and membrane phospholipids. After exposure to salt stress, genes encoding two putative glycine betaine uptake systems,proVWXandbetS, were highly upregulated. Mutational analysis revealed that BetS, not ProVWX, was the primary transporter of this compatible solute. Upon the addition of salt, exogenous glycine betaine was taken up rapidly, and maximal intracellular levels were reached within minutes. In contrast, synthesis of another important compatible solute inR. sphaeroides, trehalose, increased slowly following salt stress, reaching maximal levels only after several hours. This accumulation pattern was consistent with the more gradual increase in salt-induced transcription of the trehalose biosynthesis operonotsBA. Several genes encoding putative transcription factors were highly induced by salt stress. Multiple copies of one of these factors,crpO(RSP1275), whose product is a member of the cyclic AMP receptor protein/fumarate and nitrate reduction regulator (CRP/FNR) family, improved NaCl tolerance. WhencrpOwas provided in multicopy, expression of genes for synthesis or transport of compatible solutes was unaltered, but the membrane phospholipid composition became biased toward that found in salt-stressed cells. Collectively, this study characterized transcriptional responses to salt stress, correlated changes in transcription with compatible solute accumulation rates, identified the main glycine betaine transporter and trehalose synthase, characterized salt-induced changes in phospholipid composition, and uncovered a transcription factor associated with changes in phospholipids. These findings set the stage for deciphering the salt stress-responsive regulatory network inR. sphaeroides.

scholarly journals Gbu Glycine Betaine Porter and Carnitine Uptake in Osmotically Stressed Listeria monocytogenes Cells

2002 ◽  
Vol 68 (11) ◽  
pp. 5647-5655 ◽  
Author(s):  
Mary Lou Mendum ◽  
Linda Tombras Smith
Keyword(s):  
Listeria Monocytogenes ◽  
Transport System ◽  
Glycine Betaine ◽  
Compatible Solutes ◽  
High Salt ◽  
Uptake System ◽  
Food Borne ◽  
Protein Mutants ◽  
Carnitine Uptake ◽  
Do So

ABSTRACT The food-borne pathogen Listeria monocytogenes grows actively under high-salt conditions by accumulating compatible solutes such as glycine betaine and carnitine from the medium. We report here that the dominant transport system for glycine betaine uptake, the Gbu porter, may act as a secondary uptake system for carnitine, with a Km of 4 mM for carnitine uptake and measurable uptake at carnitine concentrations as low as 10 μM. This porter has a Km for glycine betaine uptake of about 6 μM. The dedicated carnitine porter, OpuC, has a Km for carnitine uptake of 1 to 3 μM and a V max of approximately 15 nmol/min/mg of protein. Mutants lacking either opuC or gbu were used to study the effects of four carnitine analogs on growth and uptake of osmolytes. In strain DP-L1044, which had OpuC and the two glycine betaine porters Gbu and BetL, triethylglycine was most effective in inhibiting growth in the presence of glycine betaine, but trigonelline was best at inhibiting growth in the presence of carnitine. Carnitine uptake through OpuC was inhibited by γ-butyrobetaine. Dimethylglycine inhibited both glycine betaine and carnitine uptake through the Gbu porter. Carnitine uptake through the Gbu porter was inhibited by triethylglycine. Glycine betaine uptake through the BetL porter was strongly inhibited by trigonelline and triethylglycine. These results suggest that it is possible to reduce the growth of L. monocytogenes under osmotically stressful conditions by inhibiting glycine betaine and carnitine uptake but that to do so, multiple uptake systems must be affected.

Humectant Permeability Influences Growth and Compatible Solute Uptake by Staphylococcus aureus Subjected to Osmotic Stress

2002 ◽  
Vol 65 (6) ◽  
pp. 1008-1015 ◽  
Author(s):  
ODDUR VILHELMSSON ◽  
KAREN J. MILLER
Keyword(s):  
Staphylococcus Aureus ◽  
Sodium Chloride ◽  
Water Activity ◽  
Glycine Betaine ◽  
Compatible Solutes ◽  
Foodborne Pathogen ◽  
Compatible Solute ◽  
Solute Uptake ◽  
Stressed Cells ◽  
Carnitine Uptake

The effects of different humectants (sodium chloride, sucrose, and glycerol) on the growth of and compatible solute (glycine betaine, proline, and carnitine) uptake by the osmotolerant foodborne pathogen Staphylococcus aureus were investigated. While growth in the presence of the impermeant humectants sodium chloride and sucrose induced the accumulation of proline and glycine betaine by cells, growth in the presence of the permeant humectant glycerol did not. When compatible solutes were omitted from low-water-activity media, growth was very poor in the presence of impermeant humectants. In contrast, the addition of compatible solutes had essentially no effect on growth when cells were grown in low-water-activity media containing glycerol as the humectant. Carnitine was found to accumulate to high intracellular levels in osmotically stressed cells when proline and glycine betaine were absent, making it a potentially important compatible solute for this organism.

scholarly journals Characterization of a Sinorhizobium melilotiATP-Binding Cassette Histidine Transporter Also Involved in Betaine and Proline Uptake

2000 ◽  
Vol 182 (13) ◽  
pp. 3717-3725 ◽  
Author(s):  
Eric Boncompagni ◽  
Laurence Dupont ◽  
Tam Mignot ◽  
Magne Østeräs ◽  
Annie Lambert ◽  
...  
Keyword(s):  
Glycine Betaine ◽  
Sinorhizobium Meliloti ◽  
Binding Protein ◽  
Compatible Solutes ◽  
Site Directed Mutagenesis ◽  
Uptake System ◽  
Periplasmic Binding Protein ◽  
Gene Encoding ◽  
Inhibition Of Growth

ABSTRACT The symbiotic soil bacterium Sinorhizobium melilotiuses the compatible solutes glycine betaine and proline betaine for both protection against osmotic stress and, at low osmolarities, as an energy source. A PCR strategy based on conserved domains in components of the glycine betaine uptake systems from Escherichia coli(ProU) and Bacillus subtilis (OpuA and OpuC) allowed us to identify a highly homologous ATP-binding cassette (ABC) binding protein-dependent transporter in S. meliloti. This system was encoded by three genes (hutXWV) of an operon which also contained a fourth gene (hutH2) encoding a putative histidase, which is an enzyme involved in the first step of histidine catabolism. Site-directed mutagenesis of the gene encoding the periplasmic binding protein (hutX) and of the gene encoding the cytoplasmic ATPase (hutV) was done to study the substrate specificity of this transporter and its contribution in betaine uptake. These mutants showed a 50% reduction in high-affinity uptake of histidine, proline, and proline betaine and about a 30% reduction in low-affinity glycine betaine transport. When histidine was used as a nitrogen source, a 30% inhibition of growth was observed inhut mutants (hutX and hutH2). Expression analysis of the hut operon determined using ahutX-lacZ fusion revealed induction by histidine, but not by salt stress, suggesting this uptake system has a catabolic role rather than being involved in osmoprotection. To our knowledge, Hut is the first characterized histidine ABC transporter also involved in proline and betaine uptake.

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

2000 ◽  
Vol 182 (1) ◽  
pp. 203-206 ◽  
Author(s):  
Tiemen van der Heide ◽  
Bert Poolman
Keyword(s):  
Lactococcus Lactis ◽  
Transport System ◽  
Glycine Betaine ◽  
Compatible Solutes ◽  
Hyperosmotic Stress ◽  
Transport Activity

ABSTRACT Microorganisms react upon hyperosmotic stress by accumulating compatible solutes. Here we report that Lactococcus lactisuses 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.

scholarly journals Identification of a Salt-Induced Primary Transporter for Glycine Betaine in the Methanogen Methanosarcina mazei Gö1

2002 ◽  
Vol 68 (5) ◽  
pp. 2133-2139 ◽  
Author(s):  
M. Roeßler ◽  
K. Pflüger ◽  
H. Flach ◽  
T. Lienard ◽  
G. Gottschalk ◽  
...  
Keyword(s):  
Glycine Betaine ◽  
Northern Blot Analysis ◽  
Compatible Solutes ◽  
Gene Organization ◽  
Compatible Solute ◽  
Salt Adaptation ◽  
Methanosarcina Mazei ◽  
Transport Studies ◽  
Uptake Activity ◽  
Transport Device

ABSTRACT The salt adaptation of the methanogenic archaeon Methanosarcina mazei Gö1 was studied at the physiological and molecular levels. The freshwater organism M. mazei Gö1 was able to adapt to salt concentrations up to 1 M, and the addition of the compatible solute glycine betaine to the growth medium facilitated adaptation to higher salt concentrations. Transport studies with cell suspensions revealed a salt-induced glycine betaine uptake activity in M. mazei Gö1, and inhibitor studies argue for a primary transport device. Analysis of the genome of M. mazei Gö1 identified a homolog of known primary glycine betaine transporters. This gene cluster was designated Ota (osmoprotectant transporter A). Its sequence and gene organization are very similar to those of the glycine betaine transporter OpuA of Bacillus subtilis. Northern blot analysis of otaC revealed a salt-dependent transcription of this gene. Ota is the first identified salt-induced transporter for compatible solutes in Archaea.

scholarly journals WrpA Is an Atypical Flavodoxin Family Protein under Regulatory Control of the Brucella abortus General Stress Response System

2016 ◽  
Vol 198 (8) ◽  
pp. 1281-1293 ◽  
Author(s):  
Julien Herrou ◽  
Daniel M. Czyż ◽  
Jonathan W. Willett ◽  
Hye-Sook Kim ◽  
Gekleng Chhor ◽  
...  
Keyword(s):  
Stress Response ◽  
Mouse Model ◽  
Binding Site ◽  
Brucella Abortus ◽  
Chronic Infection ◽  
Content Type ◽  
General Stress Response ◽  
General Stress

ABSTRACTThe general stress response (GSR) system of the intracellular pathogenBrucella abortuscontrols the transcription of approximately 100 genes in response to a range of stress cues. The core genetic regulatory components of the GSR are required forB. abortussurvival under nonoptimal growth conditionsin vitroand for maintenance of chronic infection in anin vivomouse model. The functions of the majority of the genes in the GSR transcriptional regulon remain undefined.bab1_1070is among the most highly regulated genes in this regulon: its transcription is activated 20- to 30-fold by the GSR system under oxidative conditionsin vitro. We have solved crystal structures of Bab1_1070 and demonstrate that it forms a homotetrameric complex that resembles those of WrbA-type NADH:quinone oxidoreductases, which are members of the flavodoxin protein family. However,B. abortusWrbA-relatedprotein (WrpA) does not bind flavin cofactors with a high affinity and does not function as an NADH:quinone oxidoreductasein vitro. Soaking crystals with flavin mononucleotide (FMN) revealed a likely low-affinity binding site adjacent to the canonical WrbA flavin binding site. Deletion ofwrpA(ΔwrpA) does not compromise cell survival under acute oxidative stressin vitroor attenuate infection in cell-based or mouse models. However, a ΔwrpAstrain does elicit increased splenomegaly in a mouse model, suggesting that WrpA modulatesB. abortusinteraction with its mammalian host. Despite high structural homology with canonical WrbA proteins, we propose thatB. abortusWrpA represents a functionally distinct member of the diverse flavodoxin family.IMPORTANCEBrucella abortusis an etiological agent of brucellosis, which is among the most common zoonotic diseases worldwide. The general stress response (GSR) regulatory system ofB. abortuscontrols the transcription of approximately 100 genes and is required for maintenance of chronic infection in a murine model; the majority of GSR-regulated genes remain uncharacterized. We presentin vitroandin vivofunctional and structural analyses of WrpA, whose expression is strongly induced by GSR under oxidative conditions. Though WrpA is structurally related to NADH:quinone oxidoreductases, it does not bind redox cofactors in solution, nor does it exhibit oxidoreductase activityin vitro. However, WrpA does affect spleen inflammation in a murine infection model. Our data provide evidence that WrpA forms a new functional class of WrbA/flavodoxin family proteins.

scholarly journals CosR is a repressor of compatible solute biosynthesis and transporter systems

2019 ◽  
Author(s):  
Gwendolyn J. Gregory ◽  
Daniel P. Morreale ◽  
E. Fidelma Boyd
Keyword(s):  
Glycine Betaine ◽  
Vibrio Parahaemolyticus ◽  
Turgor Pressure ◽  
Regulatory Region ◽  
Compatible Solutes ◽  
Compatible Solute ◽  
Negative Regulator ◽  
Low Salinity ◽  
Transporter Systems ◽  
Ectoine Biosynthesis

AbstractBacteria accumulate small, organic compounds, called compatible solutes, via uptake from the environment or biosynthesis from available precursors to maintain the turgor pressure of the cell in response to osmotic stress. Vibrio parahaemolyticus has biosynthesis pathways for the compatible solutes ectoine (ectABCasp_ect) and glycine betaine (betIBAproXWV), four betaine-carnitine-choline transporters (bcct1-bcct4) and a second ProU transporter (proVWX). Most of these systems are induced in high salt. CosR, a MarR-type regulator, which is divergently transcribed from bcct3, was previously shown to be a direct repressor of ectABCasp_ect in Vibrio species. In this study, we investigated the role of CosR in glycine betaine biosynthesis and compatible solute transporter gene regulation. Expression analyses demonstrated that betIBAproXWV, bcct1, bcct3, and proVWX are repressed in low salinity. Examination of an in-frame cosR deletion mutant shows induced expression of these systems in the mutant at low salinity compared to wild-type. DNA binding assays demonstrate that purified CosR binds directly to the regulatory region of each system. In Escherichia coli GFP reporter assays, we demonstrate that CosR directly represses transcription of betIBAproXWV, bcct3, and proVWX. Similar to V. harveyi, we show betIBAproXWV is positively regulated by the LuxR homolog OpaR. Bioinformatics analysis demonstrates that CosR is widespread within the genus, present in over 50 species. In several species, the cosR homolog was clustered with the betIBAproXWV operon, which again suggests the importance of this regulator in glycine betaine biosynthesis. Incidentally, in four Aliivibrio species that contain ectoine biosynthesis genes, we identified another MarR-type regulator, ectR, clustered with these genes, which suggests the presence of a novel ectoine regulator. Homologs of EctR in this genomic context were present in A. fischeri, A. finisterrensis, A. sifiae and A. wodanis.ImportanceVibrio parahaemolyticus can accumulate compatible solutes via biosynthesis and transport, which allow the cell to survive in high salinity conditions. There is little need for compatible solutes under low salinity conditions, and biosynthesis and transporter systems are repressed. However, the mechanism of this repression is not fully elucidated. CosR plays a major role in the repression of multiple compatible solute systems in V. parahaemolyticus as a direct negative regulator of ectoine and glycine betaine biosynthesis systems and four transporters. Homology analysis suggests that CosR functions in this manner in many other Vibrio species. In Aliivibrio species, we identified a new MarR family regulator EctR that clusters with the ectoine biosynthesis genes.

scholarly journals Quorum Sensing Regulators AphA and OpaR Control Expression of the Operon Responsible for Biosynthesis of the Compatible Solute Ectoine

2019 ◽  
Vol 85 (22) ◽  
Author(s):  
Gwendolyn J. Gregory ◽  
Daniel P. Morreale ◽  
Megan R. Carpenter ◽  
Sai S. Kalburge ◽  
E. Fidelma Boyd
Keyword(s):  
Quorum Sensing ◽  
Vibrio Parahaemolyticus ◽  
De Novo ◽  
Turgor Pressure ◽  
Compatible Solutes ◽  
Compatible Solute ◽  
Feed Forward ◽  
Content Type ◽  
Feed Forward Loop ◽  
Ectoine Biosynthesis

ABSTRACT To maintain the turgor pressure of the cell under high osmolarity, bacteria accumulate small organic compounds called compatible solutes, either through uptake or biosynthesis. Vibrio parahaemolyticus, a marine halophile and an important human and shellfish pathogen, has to adapt to abiotic stresses such as changing salinity. Vibrio parahaemolyticus contains multiple compatible solute biosynthesis and transporter systems, including the ectABC-asp_ect operon required for de novo ectoine biosynthesis. Ectoine biosynthesis genes are present in many halotolerant bacteria; however, little is known about the mechanism of regulation. We investigated the role of the quorum sensing master regulators OpaR and AphA in ect gene regulation. In an opaR deletion mutant, transcriptional reporter assays demonstrated that ect expression was induced. In an electrophoretic mobility shift assay, we showed that purified OpaR bound to the ect regulatory region indicating direct regulation by OpaR. In an aphA deletion mutant, expression of the ect genes was repressed, and purified AphA bound upstream of the ect genes. These data indicate that AphA is a direct positive regulator. CosR, a Mar-type regulator known to repress ect expression in V. cholerae, was found to repress ect expression in V. parahaemolyticus. In addition, we identified a feed-forward loop in which OpaR is a direct activator of cosR, while AphA is an indirect activator of cosR. Regulation of the ectoine biosynthesis pathway via this feed-forward loop allows for precise control of ectoine biosynthesis genes throughout the growth cycle to maximize fitness. IMPORTANCE Accumulation of compatible solutes within the cell allows bacteria to maintain intracellular turgor pressure and prevent water efflux. De novo ectoine production is widespread among bacteria, and the ect operon encoding the biosynthetic enzymes is induced by increased salinity. Here, we demonstrate that the quorum sensing regulators AphA and OpaR integrate with the osmotic stress response pathway to control transcription of ectoine biosynthesis genes in V. parahaemolyticus. We uncovered a feed-forward loop wherein quorum sensing regulators also control transcription of cosR, which encodes a negative regulator of the ect operon. Moreover, our data suggest that this mechanism may be widespread in Vibrio species.

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