A fragment of the Salmonella typhimurium ethanolamine utilization operon was cloned and characterized. The 6.3-kb nucleotide sequence encoded six complete open reading frames, termed cchA, cchB, eutE, eutJ, eutG, and eutH. In addition, the nucleotide sequences of two incomplete open reading frames, termed eutX and eutI, were also determined. Comparison of the deduced amino acid sequences and entries in the GenBank database indicated that eutI encodes a phosphate acetyltransferase-like enzyme. The deduced amino acid sequences of the EutE and EutG proteins revealed a significant degree of homology with the Escherichia coli alcohol dehydrogenase AdhE sequence. Mutations in eutE or eutG completely abolished the ability of mutants to utilize ethanolamine as a carbon source and reduced the ability to utilize ethanolamine as a nitrogen source. The product of eutE is most probably an acetaldehyde dehydrogenase catalyzing the conversion of acetaldehyde into acetyl coenzyme A. The product of the eutG gene, an uncommon iron-containing alcohol dehydrogenase, may protect the cell from unconverted acetaldehyde by converting it into an alcohol. The deduced amino acid sequence of cchA resembles that of carboxysome shell proteins from Thiobacillus neapolitanus and Synechococcus sp. as well as that of the PduA product from S. typhimurium. CchA and CchB proteins may be involved in the formation of an intracellular microcompartment responsible for the metabolism of ethanolamine. The hydrophobic protein encoded by the eutH gene possesses some characteristics of bacterial permeases and might therefore be involved in the transport of ethanolamine. Ethanolamine-utilization mutants were slightly attenuated in a mouse model of S. typhimurium infection, indicating that ethanolamine may be an important source of nitrogen and carbon for S. typhimurium in vivo.Ethanolamine is, in the form of phosphatidylethanolamine, an essential component of both prokaryotic and eukaryotic membranes (32). The efficient recycling of components such as membrane lipids and proteins is of great importance for survival of microorganisms living in natural habitats (3,23,25,39). A metabolic pathway for ethanolamine utilization might therefore be an important survival strategy against the constant famine that microorganisms face in nature, although so far there is no experimental evidence to support this hypothesis (13,24). In addition, ethanolamine found in the mammalian gastrointestinal tract may present an important alternative source of nitrogen and carbon for bacteria living in the gut. Therefore, it should not be surprising that many enterobacteria, including Escherichia coli and Salmonella typhimurium, can use ethanolamine as a source of both carbon and nitrogen (5, 43). Roof and Roth isolated a large number of S. typhimurium mutants which were deficient in different stages of ethanolamine utilization (36). An elegant genetic analysis of these mutants revealed six complementation groups in the ethanolamine utilization operon located at 50 min on the S. typhi...