An Agrobacterium sp. isolated from soil by selective growth on -y-butyrobetaine (y-trimethylaminobutyrate) as the sole source of both carbon and nitrogen has been shown to possess an inducible transport system for this growth substrate. This transport system has a Kt of 0.5 ,M and a maximal velocity of 3.8 nmol/min per mg (dry weight). The influx of -y-butyrobetaine is optimal at pH 8.5 and operates against a concentration gradient. The transport system shows a high specificity for trimethylamine carboxylic acid molecules of defined chain length. -y-Butyrobetaine uptake was significantly reduced in osmotically shocked cells and a y-butyrobetaine binding activity was detected in the crude shock fluid. This suggests a transport mechanism involving a periplasmic y-butyrobetaine binding protein.Since transport of a metabolite across a biological membrane is the first step of catabolism, it was of interest to investigate the permeation of -y-butyrobetaine in an Agrobacterium sp. that can use y-butyrobetaine as its sole source of carbon, nitrogen, and energy. We report here the existence of an inducible, high-affinity transport system for -y-butyrobetaine in an Agrobacterium sp., as well as some of its kinetic and specificity properties. MATERIALS AND METHODSBacterial strains and growth conditions. Agrobacterium sp. strain HK4 (DSM2938) was isolated from garden soil by selective aerobic culture on y-butyrobetaine as the sole source of carbon and nitrogen. This strain can also grow on L-carnitine, trans-crotonobetaine, or betaine under similar conditions. Strain HK47 carries a mutation induced by the acridine ICR191 mutagen followed by counterselection in a -y-butyrobetaine medium (9). This mutant has lost the ability to grow on -y-butyrobetaine but can still grow on L-carnitine, trans-crotonobetaine, or betaine. Cells were grown at 30°C with aeration in mineral medium (pH 7.0) containing 30 mM Na2HPO4, 20 mM KH2PO4, 0.4 mM MgSO4, 0.06 mM CaCl2, 0.034 mM FeSO4, 1 ,uM MnSO4, 0.8 ,uM CoCl2, 0.75 ,uM CUSO4, 0.75 ,uM ZnSO4, and 0.5 ,uM (NH4)6Mo7024. The medium was supplemented with 1 ml of a sterilized vitamin stock solution per liter containing the following (in milligrams per liter): pyridoxine hydrochloride, 10; riboflavin, 5; nicotinamide, 5; thiamine, 5; biotin, 2; pantothenic acid, 5; p-aminobenzoic acid, 5; folic acid, 2; and vitamin B12, 5. Unless otherwise stated, the carbon and nitrogen sources for the growth of HK4 and HK47 were 0.2% -y-butyrobetaine and L-carnitine, respectively. Sterilization of the medium was carried out by filtration on a Schleicher & Schuell filter holder (0.2-p.m pore size). When the cells were grown on D-glucose, 20 mM NH4Cl was added to the mineral medium.Growth was monitored by measuring the turbidity of the culture at 540 nm. Cell number was determined with a Petroff-Hausser bacteria counter.Transport assays. Transport assays were performed with cultures in mid-exponential-growth phase. Cells (optical density at 540 nm [OD540] = 0.8) were harvested by centrif-* Corresponding author...

A binding protein for y-butyrobetaine was purified from osmotic shock fluid of an Agrobacterium sp. It was a monomeric protein with an apparent molecular weight of 52,000 or 53,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, respectively. The isoelectric point was 4.3, as determined by isoelectric focusing. Amino acid analysis of the protein showed that Asx and Glx were predominant components and that the protein contained no cysteine. The dissociation constant of this protein for 'y-butyrobetaine was found to be 0.7 ,uM by equilibrium dialysis. Attempts to sequence the amino-terminal end with the Edman method failed, suggesting that this region of the protein is blocked.Agrobacterium sp. was isolated from soil by selective aerobic culture with -y-butyrobetaine (4-trimethylaminobutyrate; see inset of Fig. 5) as the sole source of carbon and nitrogen. The metabolism of -y-butyrobetaine in various bacteria has been reviewed recently (5). This quaternary ammonium base is involved in the biosynthesis of carnitine (3-hydroxy-4-trimethylaminobutyrate) or other related betaines (5). In the Agrobacterium sp., the biosynthetic pathway of -y-butyrobetaine leads to the production of the L-isomer of carnitine. This synthesis occurs in two steps:first, oxidation of y-butyrobetaine, which produces the ac4 unsaturated compound trans-crotonobetaine; and second, addition of a molecule of water to the double bond, which results in the formation of L-carnitine. L-Carnitine can be further metabolized to produce intermediates of the Krebs cycle.In mammals, L-carnitine is a normal constituent of plasma and tissues. It comes both from the diet and from biosynthesis in certain tissues, such as the liver and the kidney (12). The principal function of L-carnitine appears to be that of a carrier of long-chain fatty acids across the inner mitochondrial site of P-oxidation. This important function of Lcarnitine is underscored by skeletal muscle weakness, cardiomyopathy, and the accumulation of lipid droplets in tissues from patients diagnosed as having a carnitine deficiency. Such diseases can be treated by administration of L-carnitine.We believe that the Agrobacterium sp. could be a suitable organism for microbial biosynthesis of L-carnitine. For example, L-carnitine can be obtained from the culture medium of mutants blocked in the catabolism of this molecule. However, the first step of microbiological biosynthesis involves the transport of the substrate into the living cell. Since the lipid bilayer cytoplasmic membrane that forms the limit between the cytoplasm and the external medium is essentially impermeable to most hydrophilic substances, cells have evolved specific processes, catalyzed by proteins, that promote the transport of these substances across membranes. These transport processes, which can be the ratelimiting step of biosynthesis, are tightly coupled to the intracellular metabolism. They are subject to regulation by * Corresponding author. metabolic processes, and in turn, ...

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The photoaffinity reagent S‐(p‐azidophenacyl)thiocarnitine (PAP‐TC) has been synthesized according to Mauro et al. [(1986) Biochem. J. 237, 533–540]. This compound, originally designed for a structure‐function study of carnitine acetyl‐transferase, was used to analyze the Agrobacterium sp. γ‐butyrobetaine transport system. PAP‐TC appears to be a reagent specific to the transport system since it showed a competitive inhibition (K i = 70 μM) of γ‐butyrobetaine transport. UV irradiation of periplasmic proteins in the presence of [14C]PAP‐TC resulted in the irreversible labeling of the γ‐butyrobetaine‐binding protein. The addition of 1 mM γ‐butyrobetaine in the mixture significantly decreased the incorporation of the reagent, showing that this compound reacts specifically with the binding protein.