Downloaded from www.microbiologyresearch.org by IP: 23.22.250.46 On: Sun, 31 Jan 2016 12:40:40 Acetate excretion during growth of Salmonella enterica on ethanolamine requires phosphotransacetylase (EutD) activity, and acetate recapture requires acetyl-CoA synthetase (Acs) and phosphotransacetylase (Pta) activities Vincent J. Starai,3 Jane Garrity and Jorge C. Escalante-Semerena Correspondence Jorge C. Escalante-Semerena escalante@bact.wisc.edu Department of Bacteriology, University of Wisconsin-Madison, 1710 University Ave, Madison, WI 53726-4087, USA Received 26 April 2005 Revised 12 August 2005 Accepted 1 September 2005 This report shows that Salmonella enterica catabolizes ethanolamine to acetyl-CoA (Ac-CoA), which enters the glyoxylate bypass and tricarboxylic acid cycle for the generation of energy and central metabolites. During growth on ethanolamine, S. enterica excreted acetate, whose recapture depended on Ac-CoA synthetase (Acs) and the housekeeping phosphotransacetylase (Pta) enzyme activities. The Pta enzyme did not play a role in acetate excretion during growth of S. enterica on ethanolamine. It is proposed that during growth on ethanolamine, acetate excretion is necessary to maintain a pool of free CoA. Acetate excretion requires the eut operon-encoded phosphotransacetylase (EutD) and acetate kinase (Ack) enzymes. EutD function was not required for growth on ethanolamine, and an eutD strain showed only a slight reduction in growth rate. The existence of an as-yet-unidentified system that releases acetate was revealed during growth of a strain lacking Acs, the housekeeping phosphotransacetylase (Pta), and EutD. The functions of pyruvate oxidase (PoxB), Ack and STM3118 protein [a homologue of the Saccharomyces cerevisiae Ac-CoA hydrolase (Ach1p) enzyme] were not involved in the release of acetate by the acs pta eutD strain. INTRODUCTION The enterobacterium Salmonella enterica can use ethanol- amine and 1,2-propanediol as sources of carbon, energy, and, in the case of ethanolamine, nitrogen (Blackwell & Turner, 1978; Bobik et al., 1999; Chang & Chang, 1975; Roof & Roth, 1988). Catabolism of ethanolamine and 1,2-propanediol by this bacterium is intriguing. The biochemistry for the conversion of either compound is straightforward (Fig. 1). In both cases, an alcohol function is converted to the corresponding aldehyde by either coenzyme B 12 (AdoCbl)- dependent deamination (ethanolamine to acetaldehyde) (Faust et al., 1990) or dehydration (1,2-propanediol to pro- pionaldehyde) (Bobik et al., 1997). Acetaldehyde is oxidized to acetate, and propionaldeyde is oxidized to propionate, each of which is activated to its corresponding acyl-CoA derivative (Bobik et al., 1997; Faust et al., 1990; Leal et al., 2003). In S. enterica, ethanolamine and 1,2-propanediol are likely catabolized within a carboxysome-like structure (Havemann et al., 2002; Havemann & Bobik, 2003; Kofoid et al., 1999; Stojiljkovic et al., 1995), hereafter referred to as the meta- bolosome. Ethanolamine catabolism is important to the lifestyle of S. enterica in the gut, as strains unable to grow on ethanolamine are attenuated in the mouse model of infec- tion (Stojiljkovic et al., 1995). Results from in vivo expres- sion technology (IVET) experiments have also indicated that 1,2-propanediol is relevant to the survival of S. enterica as a pathogen (Conner et al., 1998). Why these compounds are metabolized within metabolosomes remains an open question. Genetic and biochemical analyses of S. enterica strains identified the chromosomal locus required for growth of this bacterium on ethanolamine (Roof & Roth, 1988, 1989). Sequence analysis of the ethanolamine utilization (eut) operon revealed 17 open reading frames, eutSPQTDMNEJGHAB- CLKR (Kofoid et al., 1999; Stojiljkovic et al., 1995) (Fig. 2a). The eutR gene, located 39 to the operon and independently transcribed from it (Sheppard & Roth, 1994), encodes the EutR transcription activator protein, which becomes active in the presence of ethanolamine and coenzyme B 12 (Roof & Roth, 1988, 1992; Sheppard & Roth, 1994). Several of the eut genes (e.g. eutSMNLK) encode proteins with homology to cyanobacterial carboxysomal shell proteins (Kofoid et al., 3Present address: Department of Biochemistry, Dartmouth Medical School, 7200 Vail Building Hanover, NH 03755-3844, USA. Abbreviations: Ac-CoA, acetyl-CoA; Ac-P, acetyl phosphate; Ack, acetate kinase; Acs, acetyl-CoA synthetase; Pta, phosphotransacetylase. 0002-8156 G 2005 SGM Printed in Great Britain 3793 Microbiology (2005), 151, 3793–3801 DOI 10.1099/mic.0.28156-0