Variations in the response of salt-stressed Rhizobium strains to betaines

A total of 15 rhizobial strains representing Rhizobium meliloti, Rhizobium japonicum, Rhizobium trifolii, Rhizobium leguminosarum, Rhizobium sp. (Sesbania rostrata) and Rhizobium sp. (Hedysarum coronarium), were studied with regard to growth rate under salt stress in defined liquid media. In the presence of inhibitory concentrations of NaCl, enhancement of growth resulting from added glycine betaine was observed for R. meliloti strains and Rhizobium sp. (Hedysarum coronarium) but not for other Rhizobium species. The concentration of glycine betaine required for maximal growth stimulation was very low (1 mM) in comparison with the osmolarity of the medium. The stimulation was shown to be independent of any specific solutes. Other related compounds like proline betaine, carnitine, choline, -butyrobetaine and pipecolate betaine were also effective compounds in restoring the growth rate of cells grown in medium of elevated osmolarity. High rate of glycine betaine uptake was demonstrated in R. meliloti cells grown in media of increased osmotic strength. The intracellular concentration of this solute was found to be 308 mM in 0.3 M NaCl-grown cells and 17 times lower in minimal medium-grown cells. Glycine betaine was used for growth under conditions of low osmolarity but could not serve as sole carbon or nitrogen source in medium of increased osmotic strength. Experiments with [¹⁴C]glycine betaine showed that this molecule was not metabolized by cells subjected to osmotic stress, whereas it was rapidly converted to dimethylglycine, sarcosine and glycine in minimal medium-grown cells.Abbreviations LAS lactate-aspartate-salts - LGS lactate-glutamate-salts - LS lactate-succinate - MSY mannitol-salts-yeast - YLS yeast-lactate-succinate     

Characteristics and osmoregulatory roles of uptake systems for proline and glycine betaine in Lactococcus lactis.

Lactococcus lactis subsp. lactis ML3 contains high pools of proline or betaine when grown under conditions of high osmotic strength. These pools are created by specific transport systems. A high-affinity uptake system for glycine betaine (betaine) with a Km of 1.5 microM is expressed constitutively. The activity of this system is not stimulated by high osmolarities of the growth or assay medium but varies strongly with the medium pH. A low-affinity proline uptake system (Km, > 5 mM) is expressed at high levels only in chemically defined medium (CDM) with high osmolarity. This transport system is also stimulated by high osmolarity. The expression of this proline uptake system is repressed in rich broth with low or high osmolarity and in CDM with low osmolarity. The accumulated proline can be exchanged for betaine. Proline uptake is also effectively inhibited by betaine (Ki of between 50 and 100 microM). The proline transport system therefore probably also transports betaine. The inhibition of proline transport by betaine results in low proline pools in cells grown in high-osmotic-strength, betaine-containing CDM. The energy and pH dependency and the influence of ionophores on the activity of both transport systems suggest that these systems are not proton motive force driven. At low osmolarities, proline uptake is low but significant. This low proline uptake is also inhibited by betaine, although to a lesser extent than in cells grown in high-osmotic-strength CDM. These data indicate that proline uptake in L. lactis is enzyme mediated and is not dependent on passive diffusion, as was previously believed.     

Functional expression of Sinorhizobium meliloti BetS, a high-affinity betaine transporter, in Bradyrhizobium japonicum USDA110

Among the Rhizobiaceae, Bradyrhizobium japonicum strain USDA110 appears to be extremely salt sensitive, and the presence of glycine betaine cannot restore its growth in medium with an increased osmolarity (E. Boncompagni, M. Osteras, M. C. Poggi, and D. Le Rudulier, Appl. Environ. Microbiol. 65:2072-2077, 1999). In order to improve the salt tolerance of B. japonicum, cells were transformed with the betS gene of Sinorhizobium meliloti. This gene encodes a major glycine betaine/proline betaine transporter from the betaine choline carnitine transporter family and is required for early osmotic adjustment. Whereas betaine transport was absent in the USDA110 strain, such transformation induced glycine betaine and proline betaine uptake in an osmotically dependent manner. Salt-treated transformed cells accumulated large amounts of glycine betaine, which was not catabolized. However, the accumulation was reversed through rapid efflux during osmotic downshock. An increased tolerance of transformant cells to a moderate NaCl concentration (80 mM) was also observed in the presence of glycine betaine or proline betaine, whereas the growth of the wild-type strain was totally abolished at 80 mM NaCl. Surprisingly, the deleterious effect due to a higher salt concentration (100 mM) could not be overcome by glycine betaine, despite a significant accumulation of this compound. Cell viability was not significantly affected in the presence of 100 mM NaCl, whereas 75% cell death occurred at 150 mM NaCl. The absence of a potential gene encoding Na(+)/H(+) antiporters in B. japonicum could explain its very high Na(+) sensitivity.     

Betaine and L-carnitine transport by Listeria monocytogenes Scott A in response to osmotic signals.

The naturally occurring compatible solutes betaine and L-carnitine allow the food-borne pathogen Listeria monocytogenes to adjust to environments of high osmotic strength. Previously, it was demonstrated that L. monocytogenes possesses an ATP-dependent L-carnitine transporter (A. Verheul, F. M. Rombouts, R. R. Beumer, and T. Abee, J. Bacteriol. 177:3205-3212, 1995). The present study reveals that betaine and L-carnitine are taken up by separate highly specific transport systems and support a secondary transport mechanism for betaine uptake in L. monocytogenes. The initial uptake rates of betaine and L-carnitine are not influenced by an osmotic upshock, but the duration of transport of both osmolytes is directly related to the osmotic strength of the medium. Regulation of uptake of both betaine and L-carnitine is subject to inhibition by preaccumulated solute. Internal betaine inhibits not only transport of external betaine but also that of L-carnitine and, similarly, internal L-carnitine inhibits transport of both betaine and L-carnitine. The inhibition is alleviated upon osmotic upshock, which suggests that alterations in membrane structure are transmitted to the allosteric binding sites for betaine and L-carnitine of both transporters at the inner surface of the membrane. Upon osmotic downshock, betaine and L-carnitine are rapidly released by L. monocytogenes as a consequence of activation of a channel-like activity. The osmolyte-sensing mechanism described is new and is consistent with various unexplained observations of osmoregulation in other bacteria.     

The BCCT family of carriers: from physiology to crystal structure

Increases in the environmental osmolarity are key determinants for the growth of microorganisms. To ensure a physiologically acceptable level of cellular hydration and turgor at high osmolarity, many bacteria accumulate compatible solutes. Osmotically controlled uptake systems allow the scavenging of these compounds from scarce environmental sources as effective osmoprotectants. A number of these systems belong to the BCCT family (betaine-choline-carnitine-transporter), sodium- or proton-coupled transporters (e.g. BetP and BetT respectively) that are ubiquitous in microorganisms. The BCCT family also contains CaiT, an L-carnitine/γ-butyrobetaine antiporter that is not involved in osmotic stress responses. The glycine betaine transporter BetP from Corynebacterium glutamicum is a representative for osmoregulated symporters of the BCCT family and functions both as an osmosensor and osmoregulator. The crystal structure of BetP in an occluded conformation in complex with its substrate glycine betaine and two crystal structures of CaiT in an inward-facing open conformation in complex with L-carnitine and γ-butyrobetaine were reported recently. These structures and the wealth of biochemical data on the activity control of BetP in response to osmotic stress enable a correlation between the sensing of osmotic stress by a transporter protein with the ensuing regulation of transport activity. Molecular determinants governing the high-affinity binding of the compatible solutes by BetP and CaiT, the coupling in symporters and antiporters, and the osmoregulatory properties are discussed in detail for BetP and various BCCT carriers.     

Functional Expression of Sinorhizobium meliloti BetS, a High-Affinity Betaine Transporter, in Bradyrhizobium japonicum USDA110

Among the Rhizobiaceae, Bradyrhizobium japonicum strain USDA110 appears to be extremely salt sensitive, and the presence of glycine betaine cannot restore its growth in medium with an increased osmolarity (E. Boncompagni, M. Østerås, M. C. Poggi, and D. Le Rudulier, Appl. Environ. Microbiol. 65:2072-2077, 1999). In order to improve the salt tolerance of B. japonicum, cells were transformed with the betS gene of Sinorhizobium meliloti. This gene encodes a major glycine betaine/proline betaine transporter from the betaine choline carnitine transporter family and is required for early osmotic adjustment. Whereas betaine transport was absent in the USDA110 strain, such transformation induced glycine betaine and proline betaine uptake in an osmotically dependent manner. Salt-treated transformed cells accumulated large amounts of glycine betaine, which was not catabolized. However, the accumulation was reversed through rapid efflux during osmotic downshock. An increased tolerance of transformant cells to a moderate NaCl concentration (80 mM) was also observed in the presence of glycine betaine or proline betaine, whereas the growth of the wild-type strain was totally abolished at 80 mM NaCl. Surprisingly, the deleterious effect due to a higher salt concentration (100 mM) could not be overcome by glycine betaine, despite a significant accumulation of this compound. Cell viability was not significantly affected in the presence of 100 mM NaCl, whereas 75% cell death occurred at 150 mM NaCl. The absence of a potential gene encoding Na⁺/H⁺ antiporters in B. japonicum could explain its very high Na⁺ sensitivity.     

Differential regulation of Ota and Otb, two primary glycine betaine transporters in the methanogenic archaeon methanosarcina mazei Gö1

Methanogenic archaea accumulate glycine betaine in response to hypersalinity, but the regulation of proteins involved, their mechanism of activation and regulation of the corresponding genes are largely unknown. Methanosarcina mazei differs from most other methanoarchaea in having two gene clusters both encoding a potential glycine betaine transporter, Ota and Otb. Western blot as well as quantitative real-time PCR revealed that Otb is not regulated by osmolarity. On the other hand, cellular levels of Ota increased with increasing salt concentrations. A maximum was reached at 300-500 mM NaCl. Ota concentrations reached a maximum 4 h after an osmotic upshock. Hyperosmolarity also caused an increase in cellular Ota concentrations. In addition to osmolarity Ota expression was regulated by the growth phase. Expression of Ota as well as transport of betaine was downregulated in the presence of glycine betaine.     

Influence of Osmolarity and the Presence of an Osmoprotectant on Lactococcus lactis Growth and Bacteriocin Production

Growth inhibition of Lactococcus lactis provoked by increasing osmolarity is reversed when glycine betaine (GB) or its analogs are added to a defined medium. Lacticin 481 production increased sharply with growth medium osmolarity in the absence of osmoprotectant but remained unaffected when GB was supplied in media of increasing osmolarity.     

Transport and catabolism of proline betaine in salt-stressed Rhizobium meliloti

Exogenous proline betaine (N,N-dimethylproline or stachydrine) highly stimulated the growth rate of Rhizobium meliloti, in media of inhibitory concentration of NaCl whereas proline was ineffective. High levels of proline betaine uptake occurred in cells grown in media of elevated osmotic strength; on the contrary, only low activity was found in cells grown in minimal medium. The apparent K _m was 10 M with a maximal transport rate of 25 nmol min^-1 mg^-1 of protein in 0.3 M NaCl-grown cells. The concentrative transport was totally abolished by KCN (2 mM), 2,4-dinitrophenol (2 mM), and carbonyl cyanide-m-chlorophenyl hydrazone (CCCP 10 M) but was insensitive to arsenate (5 mM). Glycine betaine was a very potent inhibitor of proline betaine uptake while proline was not. Proline betaine transport was not reduced in osmotically shocked cells and no proline betaine binding activity was detected in the crude periplasmic shock fluid. In the absence of salt stress, Rhizobium meliloti actively catabolized proline betaine but this catabolism was blocked by increasing the osmotic strength of the medium. The osmolarity in the growth medium regulates the use of proline betaine either as a carbon and nitrogen source or as an osmoprotectant.Abbreviations LAS lactate-aspartate-salts - MSY mannitol-salts-yeast - CCCP carbonyl cyanide-m-chlorophenyl hydrazone - DCCD dicyclohexylcarbodiimide - KCN potassium cyanide - Hepes 4-(2-hydroxyethyl)-1-piperzine-ethanesulphonic acid     

Selected amino acids protect hybridoma and CHO cells from elevated carbon dioxide and osmolality

Elevated pCO(2) inhibits cell growth. This growth inhibition is accompanied by a decrease in intracellular pH (pHi), as well as a decrease in glycolysis. Elevated concentrations (mM) of some amino acids have been shown by others to protect cells exposed to two very different environmental stresses: nutrient starvation and hyperosmolality. The fact that many of the amino acids shown to have protective effects against other stresses are transported into the cell through a pHi-sensitive transporter led us to study the possibility of using these amino acids as protective agents under elevated pCO(2). Screening experiments using 5, 15, and 25 mM of each amino acid showed that not all amino acids that protect cells from hyperosmolality protect them from elevated pCO(2). Glycine betaine and glycine were chosen for further characterization in both hybridoma and CHO cells. Asparagine and threonine were also tested in hybridoma and CHO cells, respectively. All amino acids tested under 195 mm Hg pCO(2)/435 mOsm/kg (50% growth inhibition) restored the specific growth rate (mu) in hybridoma cells to that observed under control conditions (40 mm Hg/320 mOsm/kg). Addition of each amino acid resulted in an increase in the consumption rate and intracellular accumulation of that amino acid. In CHO cells, glycine betaine also restored mu to control values, while glycine and threonine partially restored mu. In hybridoma cells, the higher specific antibody productivity obtained at elevated pCO(2) was maintained with the lowest amino acid concentration (5 mM). Productivity decreased toward control values with increasing amino acid concentrations. Elevated pCO(2) decreased the specific tPA productivity in the CHO cell line studied. Only glycine betaine resulted in a 20% increase in productivity at 195 mm Hg/435 mOsm/kg. With the exception of glycine betaine in hybridoma cells, amino acids did not mitigate the associated pHi decrease of at least 0.2 pH units at 195 mm Hg/435 mOsm/kg. pHi in hybridoma cells under elevated pCO(2) in the presence of glycine betaine was about 0.1 pH units below that of control. Amino acids had no effect on the cell size response of hybridoma cells, while they partially offset the increase in CHO cell size at elevated pCO(2). Glycine betaine, asparagine, and glycine increased the specific glucose consumption rate observed at 195 mm Hg/435 mOsm/kg (50% of control) to values greater than 70% of control in hybridoma cells. In CHO cells, only glycine betaine increased q(glc) (by 20%) under elevated pCO(2). All amino acids tested improved the cell yield from glutamine at 195 mm Hg/435 mOsm/kg in both cell lines.     

Choline-glycine betaine pathway confers a high level of osmotic tolerance in Escherichia coli.

Glycine betaine and its precursors choline and glycine betaine aldehyde have been found to confer a high level of osmotic tolerance when added exogenously to cultures of Escherichia coli at an inhibitory osmotic strength. In this paper, the following findings are described. Choline works as an osmoprotectant only under aerobic conditions, whereas glycine betaine aldehyde and glycine betaine function both aerobically and anaerobically. No endogenous glycine betaine accumulation was detectable in osmotically stressed cells grown in the absence of the osmoprotectant itself or the precursors. A membrane-bound, O2-dependent, and electron transfer-linked dehydrogenase was found which oxidized choline to glycine betaine aldehyde and aldehyde to glycine betaine at nearly the same rate. It displayed Michaelis-Menten kinetics; the apparent Km values for choline and glycine betaine aldehyde were 1.5 and 1.6 mM, respectively. Also, a soluble, NAD-dependent dehydrogenase oxidized glycine betaine aldehyde. It displayed Michaelis-Menten kinetics; the apparent Km values for the aldehyde, NAD, and NADP were 0.13, 0.06, and 0.5 mM, respectively. The choline-glycine betaine pathway was osmotically regulated, i.e., full enzymic activities were found only in cells grown aerobically in choline-containing medium at an elevated osmotic strength. Chloramphenicol inhibited the formation of the pathway in osmotically stressed cells.     

An ATP-dependent L-carnitine transporter in Listeria monocytogenes Scott A is involved in osmoprotection.

Listeria monocytogenes is a gram-positive, psychotrophic, food-borne pathogen which is able to grow in osmotically stressful environments. Carnitine (beta-hydroxy-L-tau-N-trimethyl aminobutyrate) can contribute significantly to growth of L. monocytogenes at high osmolarity (R. R. Beumer, M. C. te Giffel, L. J. Cox, F. M. Rombouts, and T. Abee, Appl. Environ. Microbiol. 60:1359-1363, 1994). Transport of L-[N-methyl-14C]carnitine in L. monocytogenes was shown to be energy dependent. Analysis of cell extracts revealed that L-carnitine was not further metabolized, which supplies evidence for its role as an osmoprotectant in L. monocytogenes. Uptake of L-carnitine proceeds in the absence of a proton motive force and is strongly inhibited in the presence of the phosphate analogs vanadate and arsenate. The L-carnitine permease is therefore most likely driven by ATP. Kinetic analysis of L-carnitine transport in glucose-energized cells revealed the presence of a high-affinity uptake system with a Km of 10 microM and a maximum rate of transport (Vmax) of 48 nmol min-1 mg of protein-1. L-[14C]carnitine transport in L. monocytogenes is significantly inhibited by a 10-fold excess of unlabelled L-carnitine, acetylcarnitine, and tau-butyrobetaine, whereas L-proline and betaine display, even at a 100-fold excess, only a weak inhibitory effect. In conclusion, an ATP-dependent L-carnitine transport system in L. monocytogenes is described, and its possible roles in cold adaptation and intracellular growth in mammalian cells are discussed.     

Utilization of osmoprotective compounds by hybridoma cells exposed to hyperosmotic stress

A search was undertaken for osmoprotective compounds for mouse hybridoma cell line 6H11 grown in culture. When the osmolality of the growth medium was increased above the normal osmolality of 330 mOsmol/kg, growth rates were decreased in a dose-dependent fashion, reaching zero when the osmolality of the medium reached approx. 435 mOsmol/kg through the addition of KCl (60 mM), or 510 mOsmol/kg through the addition of NaCl (100 mM), or sucrose (175 mM). For NaCl or sucrose-stressed cultures, the inclusion of glycine betaine, sarcosine, proline, glycine, or asparagine in the growth medium gave a moderate to strong osmoprotective effect, measured as the ability of these compounds to enhance cell growth rates under hyperosmotic conditions. Inclusion of dimethylglycine may also give a strong osmoprotective effect under these stress conditions.In KCl-stressed cell cultures, addition of glycine betaine, sarcosine, or dimethylglycine gave strong osmoprotective effects. Of 38 compounds tested during NaCl stress, 7 gave weak osmoprotective effects and 25 gave no osmoprotective effect. The osmoprotective compounds accumulated inside the stressed cells. Accumulation was completed after 4 to 8 h, reaching intracellular concentrations of approx. 0.27 pmol/cell, or 0.15 M, in NaCl stressed cells (100 mM NaCl added).Glycine betaine, dimethylglycine, and sarcosine accumulation was observed only when these protectants were included in the medium. For all osmoprotectants, a growth medium concentration between 5 and 30 mM gave the maximal protective effect, with the exception of dimethylglycine, for which the optimum concentration was approx. 65 mM. Osmoprotective effects obtained with glycine, sarcosine, dimethylglycine, and glycine betaine, indicate that the more methylated compounds are the most effective protectants.The cellular content of glycine betaine and the glycine betaine uptake rate increased with medium osmolality in a linear fashion. Glycine betaine uptake was described by a model comprising a saturable component obeying Michaelis-Menten kinetics and a nonsaturable component. K(m) and V(max) for glycine betaine uptake were determined at 420 mOsmol/kg (50 mM NaCl added) and 510 mOsmol/kg (100 mM NaCl added). A K(m) value of approx. 2.5 mM was obtained at both medium osmolalities, while V(max) increased from 0.010 pmol/cell . h to 0.018 pmol/cell . h as the osmolality of the growth medium was increased, indicating an effect of medium osmolality on the maximal rate of transport rather than on the affinity of the transporters for glycine betaine. Hybridoma cells were not able to utilize the glycine betaine precursors choline or glycine betaine aldehyde for osmoprotection, suggesting that the cells lack part, or all, of the choline-glycine betaine pathway or the appropriate uptake mechanism.The uptake rate for glycine in NaCl-stressed hybridoma cells was approx. four times higher than the uptake rate for glycine betaine. Furthermore, if equimolar amounts of glycine betaine, glycine, sarcosine, and proline were simultaneously added to NaCl-stressed cell cultures, the intracellular concentrations of glycine, proline, and sarcosine were significantly higher than the concentration of glycine betaine.A 40% increase in hybridoma cell volume was observed when the growth medium osmolality was increased from 300 to 520 mOsmol/kg. (c) 1994 John Wiley & Sons, Inc.     

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

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

Regulation of envelope protein composition during adaptation to osmotic stress in Escherichia coli.

Adaptation to osmotic stress alters the amounts of several specific proteins in the Escherichia coli K-12 envelope. The most striking feature of the response to elevated osmolarity was the strong induction of a periplasmic protein with an Mr of 31,000. This protein was absent in mutants with lambda plac Mu insertions in an osmotically inducible locus mapping near 58 min. The insertions are likely to be in proU, a locus encoding a transport activity for the osmoprotectants glycine betaine and proline. Factors affecting the extent of proU induction were identified by direct examination of periplasmic proteins on sodium dodecyl sulfate gels and by measuring beta-galactosidase activity from proU-lac fusions. Expression was stimulated by increasing additions of salt or sucrose to minimal medium, up to a maximum at 0.5 M NaCl. Exogenous glycine betaine acted as an osmoregulatory signal; its addition to the high-osmolarity medium substantially repressed the expression of the 31,000-dalton periplasmic protein and the proU-lac+ fusions. Elevated osmolarity also caused the appearance of a second periplasmic protein (Mr = 16,000), and severe reduction in the amounts of two others. In the outer membrane, the well-characterized repression of OmpF by high osmolarity was observed and was reversed by glycine betaine. Additional changes in membrane composition were also responsive to glycine betaine regulation.     

Metabolism of betaine as a carbon source by an osmotolerant bacterium isolated from the weed rhizosphere

An isolate of an osmotolerant rhizobacterium has been obtained from a weed rhizosphere which showed tolerance up to 1.0 M NaCl. The isolate has been subjected to growth analysis in a medium which contained 10 mM betaine as the sole carbon source. It was observed that betaine could be used as the sole carbon source for the growth of salt-tolerant rhizobacteria under NaCl-stress at 1.0 M concentration. Interestingly, it was found that betaine at 100 mM concentration suppressed the growth of salt-tolerant rhizobacteria. The growth of the osmotolerant rhizobacterium was stimulated when it was grown in a medium containing both glucose and betaine, demonstrating that betaine was an osmoprotectant. The presence of glucose at 10 mM concentration, however, did not alleviate the growth-suppressive effect of betaine at 100 mM concentration. The osmoprotective effect of betaine was demonstrated by the fact that the addition of betaine at different time intervals enhanced the growth accordingly. However, the growth-suppressive effect of betaine at 100 mM concentration was also noticeable when betaine was added at different time intervals.     

Effects of betaine on enumeration of airborne bacteria

The osmoprotectant betaine was incorporated into collection fluid and enumeration medium to determine its effects on the colony-forming abilities of airborne bacteria, which were collected from three separate locations: a wastewater treatment plant, the roof of a laboratory building, and an unobstructed farmland. At all locations, addition of 2 to 5 mM betaine caused a significant increase (from 21.6 to 61.3%) in colonial outgrowth, compared with the growth rate of controls without betaine. The presence of betaine in both the collection fluid and the enumeration medium had an additive effect on the colony-forming ability of airborne bacteria compared with the presence of betaine in either one alone. The effect of various betaine concentrations on the enumeration of aerosolized Pseudomonas syringae was determined. Betaine showed a threshold for maximum effect at a concentration of 2 to 5 mM. At higher concentrations (10 to 20 mM), the effects of betaine were negligible or possibly inhibitory. The significance of these results with respect to the development of protocols for monitoring airborne microorganisms, including genetically engineered microorganisms, is discussed.     

Characterization of an osmoregulated periplasmic glycine betaine-binding protein in Azospirillum brasilense sp7.

Azospirillum brasilense is able to use glycine betaine as a powerful osmoprotectant; the uptake of this compound is strongly stimulated by salt stress, but significantly reduced by cold osmotic shock. Non-denaturing PAGE in the presence of [methyl-14C] glycine betaine and autoradiography demonstrated the presence of one glycine betaine-binding protein (GBBP) in periplasmic shock fluid obtained from high-osmolarity-grown cells. The binding activity was absent in periplasmic fractions from cells grown at low osmolarity. SDS-PAGE analysis showed that the osmotically inducible GBBP has an apparent molecular weight of 32,000. The isoelectric point was between 5.9 and 6.6, as determined by isoelectric focusing. This protein bound glycine betaine with high affinity (KD of 3 microM), but had no affinity for either other betaines (proline betaine, gamma-butyrobetaine, pipecolate betaine, trigonelline, homarine) or related compounds (choline, glycine betaine aldehyde, glycine and proline). Optimum binding activity occurred at pH 7.0 to 7.5, and was not altered whether or not the binding assays were done at low or high osmolarity. Immunoprecipitation and Western blotting showed that immunoadsorbed anti-GBBP antibody from E coli cross-reacted with the GBBP produced by A brasilense cells grown at high osmolarity.     

Effects of betaine on enumeration of airborne bacteria.

The osmoprotectant betaine was incorporated into collection fluid and enumeration medium to determine its effects on the colony-forming abilities of airborne bacteria, which were collected from three separate locations: a wastewater treatment plant, the roof of a laboratory building, and an unobstructed farmland. At all locations, addition of 2 to 5 mM betaine caused a significant increase (from 21.6 to 61.3%) in colonial outgrowth, compared with the growth rate of controls without betaine. The presence of betaine in both the collection fluid and the enumeration medium had an additive effect on the colony-forming ability of airborne bacteria compared with the presence of betaine in either one alone. The effect of various betaine concentrations on the enumeration of aerosolized Pseudomonas syringae was determined. Betaine showed a threshold for maximum effect at a concentration of 2 to 5 mM. At higher concentrations (10 to 20 mM), the effects of betaine were negligible or possibly inhibitory. The significance of these results with respect to the development of protocols for monitoring airborne microorganisms, including genetically engineered microorganisms, is discussed.     

Conversion of D-carnitine into L-carnitine with stereospecific carnitine dehydrogenases

D-Carnitine was converted to L-carnitine by resting and permeabilized cells as well as with purified stereospecific carnitine dehydrogenases from Agrobacterium sp. With permeabilized cells only 11% of D-carnitine was converted into L-carnitine. Using highly stereospecific D- and L-carnitine dehydrogenases from Agrobacterium sp. (pH 8.5, 50 mM D-carnitine, 1 mM NAD + , 0.1 mM NADH, 25-fold excess of L-carnitine dehydrogenase) almost 50% of the D-carnitine could be converted into L-carnitine.