Selection, mapping, and characterization of osmoregulatory mutants of Escherichia coli blocked in the choline-glycine betaine pathway.

ABSTRACT Glycine betaine is accumulated in cells living in high salt concentrations to balance the osmotic pressure. Glycine sarcosineN-methyltransferase (GSMT) and sarcosine dimethylglycineN-methyltransferase (SDMT) of Ectothiorhodospira halochloris catalyze the threefold methylation of glycine to betaine, with S-adenosylmethionine acting as the methyl group donor. These methyltransferases were expressed inEscherichia coli and purified, and some of their enzymatic properties were characterized. Both enzymes had high substrate specificities and pH optima near the physiological pH. No evidence of cofactors was found. The enzymes showed Michaelis-Menten kinetics for their substrates. The apparent Km andV max values were determined for all substrates when the other substrate was present in saturating concentrations. Both enzymes were strongly inhibited by the reaction productS-adenosylhomocysteine. Betaine inhibited the methylation reactions only at high concentrations.

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Corynebacterium glutamicum Is Equipped with Four Secondary Carriers for Compatible Solutes: Identification, Sequencing, and Characterization of the Proline/Ectoine Uptake System, ProP, and the Ectoine/Proline/Glycine Betaine Carrier, EctP

Journal of Bacteriology ◽

10.1128/jb.180.22.6005-6012.1998 ◽

1998 ◽

Vol 180 (22) ◽

pp. 6005-6012 ◽

Cited By ~ 76

Author(s):

Heidi Peter ◽

Brita Weil ◽

Andreas Burkovski ◽

Reinhard Krämer ◽

Susanne Morbach

Keyword(s):

Escherichia Coli ◽

Corynebacterium Glutamicum ◽

Glycine Betaine ◽

Compatible Solutes ◽

Major Facilitator Superfamily ◽

Uptake System ◽

Hyperosmotic Shock ◽

Secondary Carriers ◽

Major Facilitator

ABSTRACT Gram-positive soil bacterium Corynebacterium glutamicumuses the compatible solutes glycine betaine, proline, and ectoine for protection against hyperosmotic shock. Osmoregulated glycine betaine carrier BetP and proline permease PutP have been previously characterized; we have identified and characterized two additional osmoregulated secondary transporters for compatible solutes in C. glutamicum, namely, the proline/ectoine carrier, ProP, and the ectoine/glycine betaine/proline carrier, EctP. A ΔbetP ΔputP ΔproP ΔectP mutant was unable to respond to hyperosmotic stress, indicating that no additional uptake system for these compatible solutes is present. Osmoregulated ProP consists of 504 residues and preferred proline (Km , 48 μM) to ectoine (Km , 132 μM). The proP gene could not be expressed from its own promoter in C. glutamicum; however, expression was observed in Escherichia coli. ProP belongs to the major facilitator superfamily, whereas EctP, together with the betaine carrier, BetP, is a member of a newly established subfamily of the sodium/solute symporter superfamily. The constitutively expressed ectP codes for a 615-residue transporter. EctP preferred ectoine (Km , 63 μM) to betaine (Km , 333 μM) and proline (Km , 1,200 μM). Its activity was regulated by the external osmolality. The related betaine transporter, BetP, could be activated directly by altering the membrane state with local anesthetics, but this was not the case for EctP. Furthermore, the onset of osmotic activation was virtually instantaneous for BetP, whereas it took about 10 s for EctP.

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