Evidence that Escherichia coli accumulates glycine betaine from marine sediments.

Escherichia coli grew faster in autoclaved marine sediment than in seawater alone. When E. coli was cultivated in sediment diluted with minimal medium M63 at 0.6 M NaCl, supplemented or not supplemented with glucose or with seawater, the osmoprotector glycine betaine was accumulated in the cells. The best growth occurred on glucose. Accumulation of glycine betaine was not observed with E. coli was grown in sterile seawater alone. The fact that E. coli grew better in the sediments than in seawater is attributed somewhat to the high content of organic matter in the sediment but mainly to the accumulation of glycine betaine. Thus, osmoprotection should be considered to be an additional factor in bacterial survival in estuarine sediments.     

Survival in seawater of Escherichia coli cells grown in marine sediments containing glycine betaine

Considering both the protective effect of glycine betaine (GB) on enteric bacteria grown at high osmolarity and the possible presence of GB in marine sediments, we have analyzed the survival, in nutrient-free seawater, of Escherichia coli cells incubated in sediments supplemented with GB or not supplemented and measured the efficiency of GB uptake systems and the expression of proP and proU genes in both seawater and sediments. We did this by using strains harboring proP-lacZ and proU-lacZ operon or gene fusions. We found that the uptake of GB and the expression of both proP and proU were very weak in seawater. The survival ability of cells in seawater supplemented with GB was a linear function of GB concentration, although the overall protection by the osmolyte was low. In sediments, proP expression was weak and GB uptake and proU expression were variable, possibly depending on the availability of organic nutrients. In a sediment with a high total organic carbon content, GB uptake was very high and proU expression was enhanced; cells previously incubated in this sediment showed a higher resistance to decay in seawater. GB might therefore play a significant role in the long-term maintenance of enteric bacterial cells in some marine sediments.     

Survival in seawater of Escherichia coli cells grown in marine sediments containing glycine betaine.

Considering both the protective effect of glycine betaine (GB) on enteric bacteria grown at high osmolarity and the possible presence of GB in marine sediments, we have analyzed the survival, in nutrient-free seawater, of Escherichia coli cells incubated in sediments supplemented with GB or not supplemented and measured the efficiency of GB uptake systems and the expression of proP and proU genes in both seawater and sediments. We did this by using strains harboring proP-lacZ and proU-lacZ operon or gene fusions. We found that the uptake of GB and the expression of both proP and proU were very weak in seawater. The survival ability of cells in seawater supplemented with GB was a linear function of GB concentration, although the overall protection by the osmolyte was low. In sediments, proP expression was weak and GB uptake and proU expression were variable, possibly depending on the availability of organic nutrients. In a sediment with a high total organic carbon content, GB uptake was very high and proU expression was enhanced; cells previously incubated in this sediment showed a higher resistance to decay in seawater. GB might therefore play a significant role in the long-term maintenance of enteric bacterial cells in some marine sediments.     

Influence of glycine betaine on the transfer of plasmid RP4 between Escherichia coli strains in marine sediments

The conjugative transfer of plasmid RP4 between two strains of Escherichia coli in a sterile marine sediment was enhanced by the presence of glycine betaine (frequency increased 20 to 40 times). The conjugation was also facilitated by the osmoprotection of donor cells. Glycine betaine is a universal osmolyte and has been found in marine sediments at high concentrations. So this phenomenon could have epidemiological and sanitary importance by increasing the possibility of dissemination of some plasmids present in enterobacteria in natural marine deposits.     

Efflux of choline and glycine betaine from osmoregulating cells of Escherichia coli

We present evidence that glycine betaine (betaine) which was synthesized from choline was excreted and reaccumulated in osmoregulating cells of Escherichia coli. Choline which was accumulated in bet mutants defective in betaine synthesis was shown to be excreted in response to betaine uptake. Our data suggest that E. coli has efflux systems for betaine and choline which are independent of the uptake systems for these metabolites. The ProU system of E. coli, but not that of Salmonella typhimurium, can mediate low-affinity choline uptake.     

Purification and characterization of a glycine betaine binding protein from Escherichia coli

A major component of the Escherichia coli response to elevated medium osmolarity is the synthesis of a periplasmic protein with an Mr of 31,000. The protein was absent in mutants with lambda placMu insertions in the proU region, a locus involved in transport of the osmoprotectant glycine betaine. This periplasmic protein has now been purified to homogeneity. Antibody directed against the purified periplasmic protein crossreacts with the fusion protein produced as a result of the lambda placMu insertion, indicating that proU is the structural gene specifying the 31-kDa protein. The purified protein binds glycine betaine with high affinity but has no affinity for either proline or choline, clarifying the role of proU in osmoprotectant transport. The amino-terminal sequence of the mature glycine betaine binding protein is Ala-Asp-Leu-Pro-Gly-Lys-Gly-Ile-Thr-Val-Asn-Pro.     

Improved osmotolerance of recombinant Escherichia coli by de novo glycine betaine biosynthesis

The genes from the extreme halophile Ecto-thiorhodospira halochloris encoding the biosynthesis of glycine betaine from glycine were cloned into Escherichia coli. The accumulation of glycine betaine and its effect on osmotolerance of the cells were studied. In mineral medium with NaCl concentrations from 0.15 to 0.5 M, the accumulation of both endogenously synthesized and exogenously provided glycine betaine stimulated the growth of E. coli. The intracellular levels of glycine betaine and the cellular yields were clearly higher for cells receiving glycine betaine exogenously than for cells synthesizing it. The lower level of glycine betaine accumulation in cells synthesizing it is most likely a consequence of the limited availability of precursors (e.g. S-adenosylmethionine) rather than the result of a low expression level of the genes. Glycine betaine also stimulated the growth of E. coli and decreased acetate formation in mineral medium with high sucrose concentrations (up to 200 g.l(-1)).     

Dimethylthetin can substitute for glycine betaine as an osmoprotectant molecule for Escherichia coli.

Glycine betaine is believed to be the most active naturally occurring osmoprotectant molecule for Escherichia coli and other bacteria. It is a dipolar ion possessing a quaternary ammonimum group and a carboxylic acid group. To examine the molecular requirements for osmoprotective activity, dimethylthetin was compared with glycine betaine. Dimethylthetin is identical to glycine betaine except for substitution of dimethyl sulfonium for the quaternary nitrogen group. Dimethylthetin was found to be about equally as effective as glycine betaine in permitting E. coli to grow in hypertonic NaCl, and both compounds were recovered almost completely from bacterial cells grown in the presence of hypertonic NaCl. 3-Dimethylsulfonioproprionate, an analog of dimethylthetin observed in marine algae, and 3-Dimethylsulfonio-2-methylproprionate were found to be less active. Dimethylthetin may prove useful as a molecular probe to study betaine metabolism and as a model for the development of antibacterial agents.     

Cation-pi interactions as determinants for binding of the compatible solutes glycine betaine and proline betaine by the periplasmic ligand-binding protein ProX from Escherichia coli

Compatible solutes such as glycine betaine and proline betaine are accumulated to exceedingly high intracellular levels by many organisms in response to high osmolarity to offset the loss of cell water. They are excluded from the immediate hydration shell of proteins and thereby stabilize their native structure. Despite their exclusion from protein surfaces, the periplasmic ligand-binding protein ProX from the Escherichia coli ATP-binding cassette transport system ProU binds the compatible solutes glycine betaine and proline betaine with high affinity and specificity. To understand the mechanism of compatible solute binding, we determined the high resolution structure of ProX in complex with its ligands glycine betaine and proline betaine. This crystallographic study revealed that cation-pi interactions between the positive charge of the quaternary amine of the ligands and three tryptophan residues forming a rectangular aromatic box are the key determinants of the high affinity binding of compatible solutes by ProX. The structural analysis was combined with site-directed mutagenesis of the ligand binding pocket to estimate the contributions of the tryptophan residues involved in binding.     

Restoration, by glycine betaine, of growth and enzyme activities of Escherichia coli Lac-mutants

Lac- mutants of Escherichia coli which presented a growth triggered by adding glycine betaine to the medium were isolated and characterized. Glycine betaine restores beta-galactosidase (strain AM 12) and lactose permease (strain AT42) activities. It is suggested that the right and active conformation of these enzymes, lost during mutagenesis, is restored, in vivo, in presence of this betaine.     

Metabolism of dimethylsulfoniopropionate and glycine betaine by a marine bacterium

Abstract The metabolism of the methylated osmolytes glycine betaine (GB) and dimethylsulfoniopropionate (DMSP) was studied in a bacterium (strain MD 14–50) isolated from a colony of the cyanobacterium Trichodesmium . MD 14–50 when grown on DMSP cleaved dimethylsulfide (DMS) from DMSP and oxidized acrylate. In contrast to DMSP, GB was metabolized by sequential N-demethylations. Low concentrations (100 μM) of DMSP or GB allowed the growth of MD 14–50 on glucose at higher salinities than in their absence. At elevated salinities, DMSP was accumulated intracellularly with less catabolism and DMS production. Thus, DMSP and GB were catabolized by different mechanisms but functioned interchangeably as osmolytes.     

Osmotically induced intracellular trehalose, but not glycine betaine accumulation promotes desiccation tolerance in Escherichia coli

Trehalose considerably increased the tolerance of Escherichia coli to air drying, whether added as an excipient prior to drying or accumulated as a compatible solute in response to osmotic stress. The protective effect of exogenously added trehalose was concentration dependent, up to a threshold value of 350 mM. However, trehalose alone cannot explain the intrinsically greater desiccation tolerance of stationary compared to exponential phase E. coli cells, although their tolerance was also enhanced by exogenous or endogenously accumulated trehalose. In contrast, glycine betaine whether added as an excipient or accumulated intracellularly had no influence on desiccation tolerance. These data demonstrate that the protection provided by compatible solutes to cells subjected to desiccation differs from that during osmotic stress, due to the much greater reduction in available cell water. The protective effects of trehalose during desiccation appear to be due to its stabilising influence on membrane structure, its chemically inert nature and the propensity of trehalose solutions to form glasses upon drying, properties which are not shared by glycine betaine.     

New sequence types and multidrug resistance among pathogenic Escherichia coli isolates from coastal marine sediments

The spread of antibiotic-resistant microorganisms is widely recognized, but data about their sources, presence, and significance in marine environments are still limited. We examined 109 Escherichia coli strains from coastal marine sediments carrying virulence genes for antibiotic susceptibility, specific resistance genes, prevalence of class 1 and 2 integrons, and sequence type. Antibiotic resistance was found in 35% of strains, and multiple resistances were found in 14%; the resistances detected most frequently were against tetracycline (28%), ampicillin (16.5%), trimethoprim-sulfamethoxazole (13%), and streptomycin (7%). The highest prevalence of resistant strains was in phylogenetic group A, whereas phylogroup B2 exhibited a significantly lower frequency than all the other groups. Sixty percent of multiresistant strains harbored class 1 or 2 integrase genes, and about 50% carried resistance genes (particularly dfrA and aadA) linked to a class 1 integron. Multilocus sequence typing of 14 selected strains identified eight different types characteristic of extraintestinal pathogens and three new allelic combinations. Our data suggest that coastal marine sediment may be a suitable environment for the survival of pathogenic and antimicrobial-resistant E. coli strains capable of contributing to resistance spread via integrons among benthic bacteria, and they highlight a role for these strains in the emergence of new virulent genotypes.     

Evidence that TraT interacts with OmpA of Escherichia coli

The OmpA protein is one of the major outer membrane proteins of Escherichia coli. Among other functions the protein serves as a receptor for several phages and increases the efficiency of F-mediated conjugation when present in recipient cells. TraT is an F-factor-coded outer membrane lipoprotein involved in surface exclusion, the mechanism by which E. coli strains carrying F-factors become poor recipients in conjugation. To determine a possible interaction of TraT with OmpA, the influence of TraT on phage binding to cells was measured. Because TraT inhibits inactivation of OmpA-specific phages it is suggested that TraT interacts directly with OmpA. Sequence homology of TraT with proteins 38, the phage proteins recognizing outer membrane proteins, supports this finding. A model of protein interactions is discussed.     

Adaptation of Escherichia coli to high osmolarity environments: Osmoregulation of the high-affinity glycine betaine transport system ProU

Abstract: A sudden increase in the osmolarity of the environment is highly detrimental to the growth and survival of Fscherichia coli and Salmonella typhimurium since it triggers a rapid efflux of water from the cell, resulting in a decreased turgor. Changes in the external osmolarity must therefore be sensed by the microorganisms and this information must be converted into an adaptation process that aims at the restoration of turgor. The physiological reaction of the cell to the changing environmental condition is a highly coordinated process. Loss of turgor triggers a rapid influx of K⁺ ions into the cell via specific transporters and the concomitant synthesis of counterions, such as glutamate. The increased intracellular concentration of K⁺-glutamate allows the adaptation of the cell to environments of moderately high osmolarities. At high osmolarity, K⁺-glutamate is insufficient to ensure cell growth, and the bacteria therefore replace the accumulated K⁺ ions with compounds that are less d eleterious for the cell's physiology. These compatible solutes include polyoles such as trehalose, amino acids such as proline, and methyl-amines such as glycine betaine. One of the most important compatible solutes for bacteria is glycine betaine. This potent osmoprotectant is widespread in nature, and its intracellular accumulation is achieved through uptake from the environment or synthesis from its precursor choline. In this overview, we discuss the properties of the high-affinity glycine betaine transport system ProU and the osmotic regulation of its structural genes.     

Effect of sediments on the survival of Escherichia coli in marine waters.

Escherichia coli, a fecal coliform, was found to survive for longer periods of time in unsterile natural seawater when sediment material was present than in seawater alone, and at least on one occasion growth was observed to occur. This enteric bacterium was found to increase rapidly in number in autoclaved natural seawater and autoclaved sediment taken from areas receiving domestic wastes, even when the seawater had salinities as high as 34 g/kg. However, in autoclaved seawater, growth was always more gradual and never reached numbers as high as those observed when sediment was present. It was found that nutrients were easily eluted from the sediment after autoclaving or upon addition to artificial seawater, but little elution occured during mixing of the sediments with unsterile natural seawater. The longer survival of E. coli in the sediment is attributed to the greater content of organic matter present in the sediment than the sweater. These laboratory results, in part, could explain why on a volume basis larger numbers of coliforms and fecal coliforms and fecal coliforms were found in estuarine sediments than the overlaying water at field sites.     

Extreme halophiles synthesize betaine from glycine by methylation

Glycine betaine is a compatible solute, which is able to restore and maintain osmotic balance of living cells. It is synthesized and accumulated in response to abiotic stress. Betaine acts also as a methyl group donor and has a number of important applications including its use as a feed additive. The known biosynthetic pathways of betaine are universal and very well characterized. A number of enzymes catalyzing the two-step oxidation of choline to betaine have been isolated. In this work we have studied a novel betaine biosynthetic pathway in two phylogenically distant extreme halophiles, Actinopolyspora halophila and Ectothiorhodospira halochloris. We have identified a three-step series of methylation reactions from glycine to betaine, which is catalyzed by two methyltransferases, glycine sarcosine methyltransferase and sarcosine dimethylglycine methyltransferase, with partially overlapping substrate specificity. The methyltransferases from the two organisms show high sequence homology. E. halochloris methyltransferase genes were successfully expressed in Escherichia coli, and betaine accumulation and improved salt tolerance were demonstrated.     

Nanomolar levels of dimethylsulfoniopropionate, dimethylsulfonioacetate, and glycine betaine are sufficient to confer osmoprotection to Escherichia coli.

We combined the use of low inoculation titers (300 +/- 100 CFU/ml) and enumeration of culturable cells to measure the osmoprotective potentialities of dimethylsulfoniopropionate (DMSP), dimethylsulfonioacetate (DMSA), and glycine betaine (GB) for salt-stressed cultures of Escherichia coli. Dilute bacterial cultures were grown with osmoprotectant concentrations that encompassed the nanomolar levels of GB and DMSP found in nature and the millimolar levels of osmoprotectants used in standard laboratory osmoprotection bioassays. Nanomolar concentrations of DMSA, DMSP, and GB were sufficient to enhance the salinity tolerance of E. coli cells expressing only the ProU high-affinity general osmoporter. In contrast, nanomolar levels of osmoprotectants were ineffective with a mutant strain (GM50) that expressed only the low-affinity ProP osmoporter. Transport studies showed that DMSA and DMSP, like GB, were taken up via both ProU and ProP. Moreover, ProU displayed higher affinities for the three osmoprotectants than ProP displayed, and ProP, like ProU, displayed much higher affinities for GB and DMSA than for DMSP. Interestingly, ProP did not operate at substrate concentrations of 200 nM or less, whereas ProU operated at concentrations ranging from 1 nM to millimolar levels. Consequently, proU(+) strains of E. coli, but not the proP(+) strain GM50, could also scavenge nanomolar levels of GB, DMSA, and DMSP from oligotrophic seawater. The physiological and ecological implications of these observations are discussed.