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.

Effects of Growth at Low Water Activity on the Thermal Tolerance of Staphylococcus aureus

2000 ◽  
Vol 63 (9) ◽  
pp. 1277-1281 ◽  
Author(s):  
JOSEPH R. SHEBUSKI ◽  
ODDUR VILHELMSSON ◽  
KAREN J. MILLER
Keyword(s):  
Staphylococcus Aureus ◽  
Osmotic Stress ◽  
Water Activity ◽  
Thermal Tolerance ◽  
Stress Proteins ◽  
Foodborne Pathogen ◽  
Food Poisoning ◽  
Compatible Solute ◽  
Log Reduction ◽  
Reduced Water

Staphylococcus aureus is the most osmotolerant foodborne pathogen, and outbreaks of staphylococcal food poisoning are often linked to foods of reduced water activity (aw) values. While it is generally known that the thermal tolerance of microorganisms increases as the aw of the heating menstruum is decreased, surprisingly little research has examined the influence of growth medium aw on microbial thermal tolerance. In the present study, we show that growth of S. aureus at an aw value of 0.94 leads to the development of dramatically enhanced thermal tolerance (i.e., less than 1 log reduction after heating for 20 min at 60°C). We further show that the identity of the accumulated compatible solute within cells grown at low aw can also influence the overall level of thermal tolerance of S. aureus. Finally, we provide evidence that the synthesis of general stress and/or osmotic stress proteins is required for the development of enhanced thermal tolerance of S. aureus at low aw.

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.

scholarly journals Role of σB in Regulating the Compatible Solute Uptake Systems of Listeria monocytogenes: Osmotic Induction of opuC Is σB Dependent

2003 ◽  
Vol 69 (4) ◽  
pp. 2015-2022 ◽  
Author(s):  
Katy R. Fraser ◽  
David Sue ◽  
Martin Wiedmann ◽  
Kathryn Boor ◽  
Conor P. O'Byrne
Keyword(s):  
Listeria Monocytogenes ◽  
Solute Transport ◽  
Compatible Solutes ◽  
Strain Analysis ◽  
Compatible Solute ◽  
Transport Systems ◽  
Carnitine Transporter ◽  
Solute Uptake ◽  
Carnitine Transport

ABSTRACT The regulation of the compatible solute transport systems in Listeria monocytogenes by the stress-inducible sigma factor σB was investigated. Using wild-type strain 10403S and an otherwise isogenic strain carrying an in-frame deletion in sigB, we have examined the role of σB in regulating the ability of cells to utilize betaine and carnitine during growth under conditions of hyperosmotic stress. Cells lacking σB were defective for the utilization of carnitine but retained the ability to utilize betaine as an osmoprotectant. When compatible solute transport studies were performed, the initial rates of uptake of both betaine and carnitine were found to be reduced in the sigB mutant; carnitine transport was almost abolished, whereas betaine transport was reduced to approximately 50% of that of the parent strain. Analysis of the cytoplasmic pools of compatible solutes during balanced growth revealed that both carnitine and betaine steady-state pools were reduced in the sigB mutant. Transcriptional reporter fusions to the opuC (which encodes an ABC carnitine transporter) and betL (which encodes an a secondary betaine transporter) operons were generated by using a promoterless copy of the gus gene from Escherichia coli. Measurement of β-glucuronidase activities directed by opuC-gus and betL-gus revealed that transcription of opuC is largely σB dependent, consistent with the existence of a potential σB consensus promoter motif upstream from opuCA. The transcription of betL was found to be sigB independent. Reverse transcriptase PCR experiments confirmed these data and indicated that the transcription of all three known compatible solute uptake systems (opuC, betL, and gbu), as well as a gene that is predicted to encode a compatible solute transporter subunit (lmo1421) is induced in response to elevated osmolarity. The osmotic induction of opuCA and lmo1421 was found to be strongly σB dependent. Together these observations suggest that σB plays a major role in the regulation of carnitine utilization by L. monocytogenes but is not essential for betaine utilization by this pathogen.

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 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 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 The effect of temperature and water activity on the growth of Staphylococcus aureus

2010 ◽  
Vol 27 (Special Issue 2) ◽  
pp. 28-35 ◽  
Author(s):  
A. Medveďová ◽  
Ľ. Valík ◽  
A. Studeničová
Keyword(s):  
Staphylococcus Aureus ◽  
Growth Rate ◽  
Sodium Chloride ◽  
Water Activity ◽  
Specific Growth ◽  
Incubation Temperature ◽  
Growth Parameters ◽  
Effect Of Temperature ◽  
Extended Model ◽  
Growth Responses

The growth responses of Staphylococcus aureus 2064 as affected by water activity and incubation temperature were studied in two different laboratory media. Growth parameters at temperatures from 7 to 51&deg;C and a<sub>w</sub> in the range from 1.0 to 0.86 were fitted using Ratkowsky models. The effect of temperature within its whole range on the specific growth rate was modelled by the extended model under the following equation: &radic;&micro; = 0.0456 (T &ndash; T<sub>min</sub>) [1 &ndash; e<sup>0.447(T &ndash; T<sub>max</sub>)</sup>]. The water activity values of tested media were adjusted by sodium chloride in the range from a<sub>w</sub> = 1.0 to 0.86 and experiments were conducted at 15 and 18&deg;C. The growth responses of S. aureus on water activity at 15&deg;C and 18&deg;C in PCA broth and BHI broth was described by simplified Ratkowsky model in the form: &radic;&micro; = b &times; a<sub>w</sub>. Validation of the found relationships confirmed sound fitting of the data and thus the referred results of the isolate originated from ewes&rsquo; cheese can be used in the growth prediction of S. aureus, reliably.

scholarly journals Synthesis of Pyruvate Dehydrogenase in Staphylococcus aureus Is Stimulated by Osmotic Stress

2002 ◽  
Vol 68 (5) ◽  
pp. 2353-2358 ◽  
Author(s):  
Oddur Vilhelmsson ◽  
Karen J. Miller
Keyword(s):  
Staphylococcus Aureus ◽  
Sodium Chloride ◽  
Osmotic Stress ◽  
Water Activity ◽  
Growth Medium ◽  
Pyruvate Dehydrogenase ◽  
Catabolite Repression ◽  
Bacterial Pathogen ◽  
Multienzyme Complex ◽  
High Concentrations

ABSTRACT The pyruvate dehydrogenase multienzyme complex (PDHC) was found to be upregulated by osmotic stress in the osmotolerant pathogen Staphylococcus aureus. Upregulation was detectable in the levels of both activity and protein and was judged to be about fourfold when sodium chloride was used to adjust the water activity (aw) of the growth medium to 0.94. The upregulation of the PDHC was also found to be humectant dependent and was greatest when impermeant, nonmetabolizable humectants were used to adjust aw. Further experiments provided evidence that in addition to osmotic upregulation, the PDHC complex is also subject to catabolite repression, thus providing a possible explanation for the observation that high concentrations of carbohydrates are generally more inhibitory to the growth of this bacterial pathogen than are high concentrations of salts.

scholarly journals Compatible-Solute-Supported Periplasmic Expression of Functional Recombinant Proteins under Stress Conditions

2000 ◽  
Vol 66 (4) ◽  
pp. 1572-1579 ◽  
Author(s):  
S. Barth ◽  
M. Huhn ◽  
B. Matthey ◽  
A. Klimka ◽  
E. A. Galinski ◽  
...  
Keyword(s):  
Osmotic Stress ◽  
Recombinant Proteins ◽  
Glycine Betaine ◽  
Metal Ion ◽  
Halophilic Bacteria ◽  
Compatible Solutes ◽  
Compatible Solute ◽  
Size Exclusion ◽  
High Yield ◽  
High Concentrations

ABSTRACT The standard method of producing recombinant proteins such as immunotoxins (rITs) in large quantities is to transform gram-negative bacteria and subsequently recover the desired protein from inclusion bodies by intensive de- and renaturing procedures. The major disadvantage of this technique is the low yield of active protein. Here we report the development of a novel strategy for the expression of functional rIT directed to the periplasmic space of Escherichia coli. rITs were recovered by freeze-thawing of pellets from shaking cultures of bacteria grown under osmotic stress (4% NaCl plus 0.5 M sorbitol) in the presence of compatible solutes. Compatible solutes, such as glycine betaine and hydroxyectoine, are low-molecular-weight osmolytes that occur naturally in halophilic bacteria and are known to protect proteins at high salt concentrations. Adding 10 mM glycine betaine for the cultivation of E. coliunder osmotic stress not only allowed the bacteria to grow under these otherwise inhibitory conditions but also produced a periplasmic microenvironment for the generation of high concentrations of correctly folded rITs. Protein purified by combinations of metal ion affinity and size exclusion chromatography was substantially stabilized in the presence of 1 M hydroxyecotine after several rounds of freeze-thawing, even at very low protein concentrations. The binding properties and cytotoxic potency of the rITs were confirmed by competitive experiments. This novel compatible-solute-guided expression and purification strategy might also be applicable for high-yield periplasmic production of recombinant proteins in different expression systems.

scholarly journals Osmotic Adaptation and Compatible Solute Biosynthesis of Phototrophic Bacteria as Revealed from Genome Analyses

2020 ◽  
Vol 9 (1) ◽  
pp. 46
Author(s):  
Johannes F. Imhoff ◽  
Tanja Rahn ◽  
Sven Künzel ◽  
Alexander Keller ◽  
Sven C. Neulinger
Keyword(s):  
Glycine Betaine ◽  
Compatible Solutes ◽  
Compatible Solute ◽  
Phototrophic Bacteria ◽  
Transport Systems ◽  
Phylogenetic Groups ◽  
Osmotic Adaptation ◽  
Cell Structures ◽  
Freshwater Bacteria ◽  
Genome Analyses

Osmotic adaptation and accumulation of compatible solutes is a key process for life at high osmotic pressure and elevated salt concentrations. Most important solutes that can protect cell structures and metabolic processes at high salt concentrations are glycine betaine and ectoine. The genome analysis of more than 130 phototrophic bacteria shows that biosynthesis of glycine betaine is common among marine and halophilic phototrophic Proteobacteria and their chemotrophic relatives, as well as in representatives of Pirellulaceae and Actinobacteria, but are also found in halophilic Cyanobacteria and Chloroherpeton thalassium. This ability correlates well with the successful toleration of extreme salt concentrations. Freshwater bacteria in general lack the possibilities to synthesize and often also to take up these compounds. The biosynthesis of ectoine is found in the phylogenetic lines of phototrophic Alpha- and Gammaproteobacteria, most prominent in the Halorhodospira species and a number of Rhodobacteraceae. It is also common among Streptomycetes and Bacilli. The phylogeny of glycine-sarcosine methyltransferase (GMT) and diaminobutyrate-pyruvate aminotransferase (EctB) sequences correlate well with otherwise established phylogenetic groups. Most significantly, GMT sequences of cyanobacteria form two major phylogenetic branches and the branch of Halorhodospira species is distinct from all other Ectothiorhodospiraceae. A variety of transport systems for osmolytes are present in the studied bacteria.

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