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, 02-dependent, and electron transfer-linked dehydrogenase *as 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 ch4iline-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. Chloramnphenicol inhibited the formation of the pathway in osmotically stressed cells.

It has been shown previously that externally added glycine betaine is accumulated in Escherichia coli in response to the external osmotic strength. Here we have shown, by using nuclear magnetic resonance spectroscopy and radiochemical methods, that E. coli growing in a glucose-mineral medium of elevated osmotic strength generated with NaCl, had the same capacity to accumulate proline betaine and glycine betaine. Its capacity to accumulate gamma-butyrobetaine was, however, 40 to 50% lower. Accordingly, externally added proline betaine and glycine betaine stimulated aerobic growth of osmotically stressed cells equally well, and they were more osmoprotective than gamma-butyrobetaine. In cells grown at an osmotic strength of 0.64, 1.01, or 1.47 osmolal, respectively, the molal cytoplasmic concentration of the two former betaines corresponded to 29, 38, or 58% of the external osmotic strength. Nuclear magnetic resonance spectroscopy revealed that trehalose and glutamic acid were the only species of organic osmolytes accumulated in significant amounts in cells grown under osmotic stress in glucose-mineral medium without betaines. Their combined molal concentration in the cytoplasm of cells grown at 1.01 osmolal corresponded to 27% of the external osmotic strength.

Osmotically stressed Escherichia coli cells synthesize the osmoprotectant glycine betaline by oxidation of cholibe through glycine betaine aldehyde (choline -* glycine betalne aldehyde glycine betaine;Landfald and A. R. Str0m, J. Ilacteriol. 165:849-855, 1986. Mutants blocked at the level of choline dehydrogenase were isolated by selection of strains which did not grow at elevated osmotic strength in the presence of choline but grew when supplemented with glycine betaine. A gene governing the choline dehydrogenase activity was named betA. Mapping by P1 transduction, F' complementation, and deletion mutagenesis showed the betA gene to be located at 7.5 min in the argF-codB region of the chromosome. Mutants carrying deletions of this region also lacked glycine betaine aidehyde dehydrogeniase activity and high-affinity uptake activity for choline; these deletions did not influence the activities of glycine betalne uptake or low-affinity choline uptake, both of which were osmotically regulated.Glycine betaine is accumulated under osmotic stress in widely different-organisms such as chemoheterotrophic bacteria (16, 22, 26-28, 33. 35), halotolerant photosynthetic bacteria (12, 29, 34), marine animals, and halophytic plants (36,37). Whereas the biological activity of glycine betaine in osmoregulation cannot -be easily demonstrated for higher organisms, this is possible for Esckerichia coli and other enteric bacteria. Addition of relatively low concentrations (e.g., 1 mM) of glycine betaine or its precursors choline or glycine betaine aldehyde to medium of inhibitory osmotic strength stimulates the growth of these bacteria (11,22,(26)(27)(28)35). In the companion paper (22) we show that the choline-glycine betaine pathway of E. coli contains two enzymes: a membrane-bound, 02-dependent choline dehydrogenase and a soluble, NAD-dependent glycine betaine aldehyde dehydrogenase. The pathway is osmotically regulated, i.e., appreciable dehydrogenase activities are synthesized only in cells grown -under osmotic stress. Thus, glycine betaine synthesis ard accumulation in E. coli represent an intriguing system for studies of the molecular mechanism of osmotic tolerance (27). In this communication we describe the selection and characterization of mutants blocked in the choline-glycine betaine pathway. Genes governing several critical activities including choline and glycine betaine aldehyde oxidation and choline uptake were mapped -on -the chromosome of E. coli. MATERIALS AND METHODSBacterial and bacteriophage strains. The strains used are listed in Table 1. The betA mutations-generated in K10 were transferred to CSH7 by P1 transduction and conjugation.

Escherichia coli cells growing under osmotic stress activate systems for the transport or synthesis of several organic osmolytes. Glutamate and trehalose syntheses seem to represent mechanisms for achieving a low level of osmotic tolerance. The uptake and synthesis of betaines represent mechanisms of achieving a high level of osmotic tolerance. The osmotically‐controlled ProP and ProU systems are involved in the uptake of glycine betaine and proline. However, glycine betaine synthesis occurs only in the presence of the precursor molecule choline. The osmotically controlled genes governing the high‐affinity uptake of choline are located in close proximity to those encoding the dehydrogenases involved in the oxidation of choline to glycine betaine, but represent a different transcriptional unit. It is not known if each of these systems has its own osmotic sensor or whether a common osmotic sensor regulates all cell osmolytes.

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