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Hopper, D. J. & Chapman, P.J. (1971) Bwchern. J. 122,19-28 Hopper, D. J. & Keat, M. J. (1975) Abstr. FEES Meet. loth. abstr. no. 1553 Hopper, D. J. &Taylor, D. G. (1975)J. Bucreriol. 122,1-6 Keat, M. J. & Hopper, D. J. (1975) Biochem. SOC. Truns.3,358-359

Ethanolamine Catabolism by Bacteria, Including Escherichiu coli CAROL M. BLACKWELL, F. ANNE SCARLETT and JOHN M. TURNER Department of Biochemistry, University of Liverpool, P.O.Box 147, Liverpool L69 3BX,

U.K. Although phosphatidylethanolamineis the major phospholipid found in most bacteria, and ethanolamine is known to be a component of lipopolysaccharides in some bacteria (Drewry etal., 1971),little is known of ethanolamine degradation by bacteria. A number of enzymes are known to act on the amino alcohol, but their occurrence and relative significanceare unknown. Ethanolamine ammonia-lyase (Bradbeer, 1965),ethanolamine oxidase (Narrod & Jakoby, 1964) and a biodegradative ethanolamine kinase (Jones & Turner, 1971;Faulkner & Turner, 1974) have been demonstrated to exist in only a few species. The ability of bacteria to deaminate ethanolamine was determined by growing them on simple synthetic media containing (per litre) 2g of glycerol, 2g of succinic acid, 7g of KzHP04,3g of KHZPO4,1g of NaZSO4,0.1 g of MgS04,7Hz0 and 1g of ethanolamine. Control media were either devoid of a fixed nitrogen source or contained l g of (NH4)zS04/litre replacing NazS04 and ethanolamine. In some cases media were supplemented with 0.01 % (w/v) yeast extract (Difco). Media were adjusted to pH7 with HCl or NaOH and sterilized by autoclaving. Bacteria capable of growth on these media are listed in Table 1. Of the bacteria capable of utilizing ethanolamine only when yeast extract was present, Escherichia coli and Klebsiella aerogenes were found to grow well in media in which yeast extract was replaced by vitamin Biz (40,ugllitre). This was not the case with Corynebacterium aquaticum, which required thiamin but not vitamin Blz for growth. In many cases a strong odour of acetaldehyde was noted during growth. In only a few cases was good growth on (NH4)zS04not paralleled by growth on ethanolamine-containing media (Table 1). Assays for enzymes of ethanolamine metabolism in cell-free extracts of bacteria revealed that C. aquaticum possessed ethanolamine ammonia-lyase activity after growth on glycerol+ethanolamine+mineral salts+yeastextract medium. The bacterium was normally harvested in the exponential phase of growth, the ammonia-lyase activity in cell-free extracts declining rapidly when the stationary phase had been reached. Enzyme activity was optimally active at about pH 8 in 0.1 M-Tris/HCl buffer, with ethanolamine at 3 m and 5'-deoxyadenosyl-cobalamin at 1 0 ~ Reactions ~ . were started by the additionofextract. Acolorimetricmethod(Pazetal., 1965)wasused tomeasurealdehyde formation. Specific activities of 1-3 nmol of acetaldehyde formed/min per mg of protein at 37°C were found. Enzyme activity was retained during storage for 24h at 4°C in the presence of Zm-dithiothreitol. Whereas substrate concentrations above about 3mM inhibited enzyme activity in crude extracts, preparations obtained by DEAE-cellulose chromatography were not inhibited by 10m-ethanolamine and the K, was found to be 1-2m~.The K , for coenzyme Blz was 0 . 2 - 0 . 5 ~ ~ . Considerably higher ethanolamine ammonia-lyase activities were found in crude extracts of E. coli and K. aerogenes grown on media containing ethanolamine and supplemented with vitamin BIZ. Specific activities of up to lOOnmol of acetaldehyde formed/min per mg of protein at 37°C were measured. The K,,, value for ethanolaminein each case was only about 0 . 2 m ~The . treatment of crude extracts with 1mg of activated charcoal/mg of protein markedly decreased activity in the absence of added coenzyme Biz. The & for the coenzymewith charcoal-treated enzyme was about 0.1 p ~Overnight . dialysis of charcoal-treated extracts against 20m~-Tris/HClbuffer, pH 8 at 4"C, led VOl. 4

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Table 1. Ability of bacteria to grow with ethanolamine as the sole or m j o r source of nitrogen in semi-synthetic media Bacterial strains are identified by their National Collection of Industrial Bacteria (N.C.I.B.) catalogue number unless otherwise indicated. N.C.P.P.B. indicates a strain from the National Collection of Plant Pathogenic Bacteria. A.T.C.C. indicates a strain from the American Type Culture Collection. N.C.T.C. indicates a strain from the National Collection of Type Cultures. Bacteria were grown on the media described in the text, in shaken liquid cultures except where noted. Growing with ethanolamine as nitrogen Growing with ethanolamine as sole source in medium supplemented with nitrogen source in simple synthetic medium yeast extract Achromobacter sp. (8250 and 9205) Arthrobacterparafineus (10699) Corynebacterium aquaticum (9460) Erwinia anam (N.C.P.P.B. 441) Escherichia coli (8114) Erwinia carotovora (N.C.P.P.B. 1280) Klebsiella aerogenes (8267) Erwinia milletiae (N.C.P.P.B. 955) Pseudomonas sp. (A.T.C.C. 13796) Flauobacterium arborescens (8185) Flavobacterium rhenanum (9157) Klebsiella aerogenes (418 ) Growing with (NH&SO4 but not Micrococcus denitrifcans (8944) Mycobacterium smegmatis* (N.C.T.C. 7017) ethanolamine as nitrogen source Alcaligenesfaecalis (8156) Pseudomonas sp. (8858) Enterobacter cloaca (8259) Pseudomonas sp. (10431) Proteus mirabilist (6389) Pseudomonas denitri3cans (8376) Proteus morganiit (232) Pseudomonas multivorans (9085) Proteus vulgarisf (365) Pseudomonas ovalis (9229) Serratia marcescens (1377) Pseudomonasputida (9304 and 10559) Xanthomonas hyacynthit (N.C.P.P.B 599) * Growth occurred only in surface culture on media solidified with agar (2%, w/v) at 37°C. t Medium supplemented with yeast extract.

to little loss of activity. Untreated extracts lost considerable activity. The poor recovery of activity obtained on ionexchange chromatography of extracts may be due to photolysis of the cofactor yielding inhibitory products, e.g. adenylcobamidehydroxide. Experiments with ['4C]ethanolamine suggest that the simultaneous presence of alternative sources of nitrogen, e.g. (N&)2so4, did not prevent the deamination of ethanolamine by bacteria shown to possess ammonia-lyaseactivity. Radioactivity was assimilated by such bacteria. Enzyme assays showed that ammonia-lyase formation was induced by the presence of ethanolamine in the growth medium even when (NH&so4 was present. An examination of Pseudomonas sp. A.T.C.C. 13796 after growth on ethanolamine (1 g/litre)+mineral salts+yeast extract failed to reveal ethanolamine ammonia-lyase activity in cell-free extracts. The presence of low ethanolamine oxidase (Narrod & Jakoby, 1964) activity was c o n b e d , however, specific activities of 0.3-l.Onm01 of glycolaldehyde formed per min/mg at 37°C being measured. Growth on glywol+ mineral salt s+yeast-extract medium containing [14C]ethanolamine resulted in the assirnilation of radioactivity by the bacteria. The uptake of radioactivity occurred after the main phase of growth unless the concentration of yeast extract was decreased, in which caseuptake occurred earlier. The addition of (NH4)2S04did not affect assimilation of radioactivity. The ability of bacteria to deaminate ethanolamineappears to be widespread. Chang & Chang (1975) have obtained nutritional evidencethat E. coliK12, Enterobacter aerogenes (A.T.C.C. 1033) and Salmonella typhimurium LT2 possess vitamin Blz-dependent ethanolamine deaminase enzymes. The fact that strains of E. coli require vitamin BIZ 1976

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for growth on media containing ethanolamine as the sole nitrogen source provides a convenient microbiological assay for the vitamin. Part of this work was supported by grants from the Medical Research Council. Bradbeer, C. (1965)J. Biol. Chem. 240,46694674 Chang, G. W. & Chang, J. T. (1975)Nature (London)254,150-151 Drewry, D. T.,Gray, G. W. & Wilkinson, S. G. (1971)Eur. J. Biochem.21,400-403 Faulkner, A. &Turner, J. M. (1974)Biochem. Soc. Tram. 2,133-136 Jones, A. &, Turner, J. M. (1971)J. Gen. Microbiol. 67,379-381 Narrod, S . A. & Jakoby, W. B. (1964)J. Biol. Chem. 239,2289-2193 P a , M. A., Blumenfeld, 0. O., Rojkind, M., Henson, E., Furfine, C.& Gallop, P. M. (1965) Arch. Biochem. Biophys. 109, 548-559

GThreonine Catabolism via Aminoacetone: A Search for a Pathway in Bacteria STEPHEN C. BELL* and JOHN M. TURNER Department of Biochemistry, University of Liverpool, P.O. Box 147, Liverpool L69 3BX,

cr.K.

Aminoacetone, long known as a bacterial metabolite (Elliott, 1958), is derived from L-threonine via 2-amino-3-oxobutyrate by the action of L-threonine 3-hydrogenase (Neuberger & Tait, 1960). The amino ketone was originally believed to be metabolized by deamination to methylglyoxal (Elliott, 1959, 1960~)(Scheme 1) and although this is catalysed by monoamine oxidases of mammalian origin (Elliott, 19606; BufToni & Blaschko, 1963) only low activity was found in bacteria (Green t Elliott, 1964). Deamination by a transamination reaction has not been demonstrated conclusively. A claim that an Arthrobucter sp. growing on L-threonine degraded it via aminoacetone and methylglyoxal (Green & Lewis, 1968) was later refuted (Morris, 1969) and the significance of aminoacetone in threonine metabolism has remained uncertain. The identification of bacteria capable of metabolizing aminoacetone at a rapid rate (Higginset ul., 1968)led to the elucidation of a new catabolicpathway in a pseudomonad (Faulkner & Turner, 1974), involving L-1-aminopropan-2-01and propionate as intermediates (Scheme 1). The availability of a collection of bacteria each capable of growth on L-threonine as the major source of carbon and nitrogen, the majority of which possessed high L-threonine 3-dehydrogenase activity (Bell et ul., 1972), offered the opportunity to search for a pathway of threonine catabolism via aminoacetone, the amino alcohol and propionate. Of 83 isolates capable of growth on L-threonine, only eight grew on DL-aminopropanol and of these only five grew on propionate. The activities OfthekeyenzymesL-threonine 3-dehydrogenase, L-aminopropanol dehydrogenase (aminoacetone reductase) and amino alcohol 0-phosphate phospho-lyase were measured in extracts of the bacteria after growth on L-threonine and DL-aminopropanolmedia. Results are given in Table 1. Not only were the enzymes of aminoacetone metabolism low or absent after growth on L-threonine, but L-threonine 3-dehydrogenase activity was undetectable in all cases except one, where it was low. After growth on DL-aminopropanol the pseudomonads N3 and N6 exhibited significant phospho-lyase activity, suggesting amino alwhol metabolism via the route demonstrated in Pseuhmonus sp. N.C.I.B. 8858 (Faulkner & Turner, 1974). The presence of aminoacetone reductase (measured as L-aminopropano1 dehydrogenase) in these isolates indicated the ability to metabolize aminoacetone. Washed suspensionsof the bacteria grown on L-threonine were unable to oxidize aminoacetone, whereas the pseudomonads N3 and N6 oxidized the aminoketone after growth on DL-aminopropanol medium.

* Present ad-: Scotland, U.K. VOl. 4

Department of Biochemistry, University of Strathclyde,Glasgow G1 lXW,