INTRODUCTION

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Virtually all Salmonella isolates synthesize B₁₂ de novo under anaerobic conditions (27, 34, 43). The ability to synthesize and import B₁₂ requires more than 35 known genes (48)approaching 1% of the genome. However, mutations that eliminate B₁₂ synthesis from otherwise wild-type strains cause no growth defect under the standard aerobic or anaerobic lab conditions used thus far. Since evolutionary maintenance of such a large fraction of the genome requires selection, it seems inescapable that natural conditions must exist under which endogenously synthesized B₁₂ is important to growth of salmonellae. Salmonella enterica serovar Typhimurium makes B₁₂ de novo only in the absence of oxygen (27). Degradation of ethanolamine or 1,2-propanediol requires B₁₂ and provides a carbon and energy source, but growth on these compounds has been observed only under aerobic conditions requiring exogenous B₁₂ (28, 46). These paradoxical aspects of B₁₂ metabolism have been reviewed (47).

The B₁₂ paradox may be resolved by the finding, described here, that the electron acceptor tetrathionate supports anaerobic use of ethanolamine or 1,2-propanediol as the sole carbon and energy source by using endogenously synthesized B₁₂. Under anaerobic conditions, tetrathionate supports considerably better growth on these carbon sources than the other alternative electron acceptors tested. Tetrathionate plus either ethanolamine or 1,2-propanediol provides the only known conditions under which B₁₂ synthesis is essential to the growth of wild-type Salmonella.

While tetrathionate metabolism has not been studied extensively, its reduction is likely to follow the pathway diagrammed in Fig. 1 (4, 36). The enzymes encoded by the ttr operon reduce tetrathionate to thiosulfate (9, 21), which can be reduced further to sulfite plus hydrogen sulfide by enzymes encoded by the phs operon (11, 20). Sulfite can be reduced to hydrogen sulfide by the dissimilatory anaerobic sulfite reductase encoded by the asr genes (17, 25, 26). Salmonella also has an assimilatory sulfite reductase (CysJI), which acts with or without oxygen (32). It is not clear where in nature tetrathionate might be encountered, but it seems likely to occur in bacterial communities that include sulfate-reducing bacteria; it has been detected in humid soils that support growth of such bacteria (52).

View larger version (16K): [in this window] [in a new window]   FIG. 1.   Reduction of tetrathionate to sulfide. The tetrathionate reductase (Ttr) described here performs the initial reduction to thiosulfate (S2O32). This area of metabolism has been reviewed by Barrett and Clark (4).

Taken together, the three sulfur-reducing enzyme systems (encoded by ttr, phs, and asr) and the genes for 1,2-propanediol degradation (pdu) and de novo B₁₂ synthesis (cbi) comprise more than 1% of the Salmonella genome and seem to act as part of a logical pattern of metabolism. All of these genes are characteristic of S. enterica and absent from Escherichia coli. This suggests that B₁₂-dependent anaerobic catabolism of small molecules, supported by reduction of sulfur compounds, may be central to the life of salmonellae. A model is described for evolution of this system during the divergence of Salmonella and Escherichia coli. (Throughout the rest of this article, propanediol refers to 1,2-propanediol.)

We thank Eric Kofoid for insightful comments and for constructing the ttrR deletion, Edward King for taking the pictures of the bacterial cell chains and granules, David Blair for generously sharing his phase-contrast microscope, and Renee Dawson for carefully reading the manuscript.

	MATERIALS AND METHODS

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Bacterial strains and crosses. All strains are derived from S. enterica serovar Typhimurium strain LT2. Key strains and their sources are listed in Table 1. The transposable elements TPOP1 and TPOP2 were described previously (44). All transductional crosses were mediated by the high-frequency transducing mutant of phage P22 (HT105, int) (51). Growth of phage and procedures for crosses have been described previously (7). Standard methods for cell culture have been described previously (1, 6).

Chemical reagents and growth media. Standard aerobic cell culture was conducted in Difco nutrient broth supplemented with 0.1 mM NaCl (14). Minimal medium was the No-carbon-E (NCE) medium (13), supplemented with trace metals (0.3 µM CaCl₂, 0.1 µM ZnSO₄, 0.045 µM FeSO₄, 0.2 µM Na₂Se₂O₃, 0.2 µM Na₂MoO₄, 2 µM MnSO₄, 0.1 µM CuSO₄, 3 µM CoCl₂, and 0.1 µM NiSO₄). Unless otherwise indicated, carbon sources were provided at the following concentrations: glucose, 11 mM; glycerol, 43 mM; ethanolamine (Aldrich Chemical Co.), 25 mM in solid media, 10 mM in liquid; and 1,2-propanediol and potassium acetate (Aldrich Chemical Co.), 25 mM in solid media, 50 mM in liquid. Tetrathionate (Sigma Chemical Co.) was added at the final concentrations indicated in the figure legends and tables; for growth on plates and for assay of induction of the ttrBCA genes, it was added at a final concentration of 10 mM. For growth in liquid culture, it was added at 40 mM, since under these conditions a higher growth yield was obtained by adding the fourfold excess. Trimethylamine N-oxide, potassium nitrate, fumaric acid, and dimethyl sulfoxide were added at concentrations of 5, 10, 15, and 20 mM. Cyanocobalamin (Sigma Chemical Co.) was added at a final concentration of 200 nM unless otherwise specified. AdoB₁₂ (5'-deoxyadenosylcobalamine) and Cbi (cobinamide dicyanide; Sigma Chemical Co.) were each added at a final concentration of 15 nM. Antibiotics were added to nutrient broth medium at the following concentrations: kanamycin, 50 µg/ml; tetracycline, 20 µg/ml; and chloramphenicol, 20 µg/ml. In minimal medium, tetracycline was added to a final concentration of 10 µg/ml.

Cell growth and enzyme assays. Anaerobic conditions (37°C) for petri dishes were provided by an anaerobic chamber (Forma Anaerobic System Model 1024) with a gas mixture of CO₂, H₂, and N₂ (5:6:89). For anaerobic liquid cultures, media were preincubated in the anaerobic chamber for 12 to 24 h. Cells for these cultures were pregrown aerobically to stationary phase in NCE glycerol, washed twice in NCE, and then diluted 10,000-fold into the liquid media inside the anaerobic chamber. The anaerobic culture tubes were then crimp capped, and the medium and headspace were flushed with nitrogen (7). Incubation was at 37^o with shaking. Cultures that contained AdoB₁₂ or Cbi were prepared and incubated in the dark. Turbidity was monitored in a spectrophotometer at 650 nm. Chlorate sensitivity was tested anaerobically on agar plates containing minimal E medium supplemented with 11 mM glucose with or without 0.2 mM potassium chlorate. -Galactosidase activity from liquid cell cultures was assayed as described previously (37).

Sulfur assay. Elemental sulfur was detected in cell cultures with a modified version of a method of cyanolysis (19). Soluble forms of sulfur were removed by pelleting cells and washing pellets with water. The cell pellet was dried, and elemental sulfur was dissolved in acetone by overnight incubation at 37°C with shaking. Sample dilutions were made in acetone. Elemental sulfur was detected by cyanolysis; thiocyanate derivatives were formed by adding 0.1 ml of 0.1 M KCN to a 1-ml sample at room temperature. To detect thiocyanates, 0.1 ml of an aqueous Fe(NO₃)₃ solution (Aldrich Chemical Co.) [0.25 M Fe(NO₃)₃ in 3 M HNO₃] was added and the samples were read at 460 nm in a Beckman DU 640 spectrophotometer. Final sulfur concentrations were estimated by comparing absorbance to a standard curve prepared with elemental sulfur.

Isolation of insertion mutations in and near the ttr locus. Insertion mutants unable to reduce tetrathionate were obtained by using MudJ (Kn^r-lac) or Tn10dTc; these mutants were identified by their failure to produce acid on MacConkey indicator medium (Difco) containing 10 mM tetrathionate. Mutants defective in synthesis of the molybdopterin cofactor (required for tetrathionate reductase) were identified on the basis of their resistance to chlorate and have been described previously (54). Of 18 ttr::MudJ insertions isolated (from 10,000 random insertion mutants), 4 expressed the lacZ gene from the ttr promoter. Other insertions of the MudJ (Kn^r-lac) element were obtained by screening a random insertion pool for clones whose -galactosidase level was induced by tetrathionate (white colonies on nutrient broth-X-Gal [5-bromo-4-chloro-3-indolyl--D-galactopyranoside] plates, blue colonies on nutrient broth-X-Gal tetrathionate plates).

Determination of the ttr operon sequence and insertion sites. Starting with strains carrying multiple insertions in the ttr operon, fragments of the operon were amplified by PCR using primers complementary to known sequences at the ends of the particular inserted elements. The amplified fragments were sequenced according to the method of Sanger et al. (49) at the University of Utah Health Sciences DNA Sequence Facility. This information facilitated the design of primers that were used to amplify and sequence the regions between the ttr operon and nearby genes and to determine the insertion site of transposable elements.

Construction of ttrR and ttrS and prpRBCDE deletion-substitution (swap) mutations. The linear transformation method was that of Murphy (38) and used cells expressing recombination genes of phage lambda. The ability of these lambda enzymes to support targeted recombination with very short homologous sequences has recently been demonstrated (12, 61). Methods were optimized for Salmonella by Eric Kofoid (personal communication). The transformation recipient was a wild-type Salmonella strain carrying plasmid pPT223 (TT22236), which includes the lam, bet, and exo genes of phage lambda expressed from a lac promoter; this plasmid was constructed and supplied by Poteete and Fenton (42). Cells were pregrown in Luria broth with isopropyl--D-thiogalactopyranoside (2 mM) to induce the plasmid recombination genes and washed three times in 10% glycerol prior to electroporation.

Nucleotide sequence accession number. The accession number for the ttr operon sequence and the location of insertions in this sequence are available from GenBank (accession number AF282268).

	RESULTS

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Fermentation of 1,2-propanediol and ethanolamine. Ethanolamine is degraded by B₁₂-dependent conversion to acetyl coenzyme A (acetyl-CoA), which can enter the tricarboxylic acid (TCA) cycle and the glyoxalate shunt; this is diagrammed in Fig. 2 and has been reviewed previously (47). Propanediol is converted to propionyl-CoA, joined to oxaloacetate (the 2-methyl-citrate pathway), and converted to succinate plus pyruvate, which can be converted to acetyl-CoA and enter the standard TCA cycle (23, 24, 56, 60). In the absence of any electron acceptor, ethanolamine and propanediol might be fermented, providing an ATP source by conversion of acetyl-CoA (or propionyl-CoA) to acetyl-PO₄ (or propionyl-PO₄) and thence to acetate (or propionate) plus ATP. The latter reactions could be performed by the Ack and Pta enzymes or by similar enzymes encoded by the eut and pdu operons (5, 31). Excess reducing equivalents generated by conversion of acetaldehyde (or propionaldehyde) to acetyl-CoA (or propionyl-CoA) could, in principle, be balanced by reducing some aldehyde to ethanol (or propanol) and excreting the alcohol. This scheme might allow ethanolamine and propanediol to provide an energy source without respiration (fermentative growth). When this was tested, Salmonella seemed unable to ferment either ethanolamine or propanediol for use as both a carbon and energy source (see below).

View larger version (32K): [in this window] [in a new window]   FIG. 2.   Metabolism of ethanolamine and of propanediol. The upper part of this diagram outlines the known metabolism of ethanolamine and indicates the proposed role of various proteins encoded by the ethanolamine (eut) operon (31). The lower part of the diagram outlines the known metabolism of propanediol as described in references 5 and 24.

View larger version (16K): [in this window] [in a new window]   FIG. 3.   Stimulation of anaerobic growth by ethanolamine or propanediol. Cells of wild-type serovar Typhimurium, strain LT2, were grown anaerobically on minimal NCE medium supplemented with 0.2% yeast extract (YE) to provide a carbon source with or without 80 mM propanediol or 98 mM ethanolamine as an energy source.

Qualitative tests of respiratory electron acceptors. Salmonella is unable to use propanediol or ethanolamine as the sole carbon and energy source under anaerobic conditions even with the alternative respiratory electron acceptor fumarate, dimethyl sulfoxide, or trimethylamine N-oxide; very slight growth was seen with nitrate (data not shown). This result was seen both on solid media and when growth was scored qualitatively (as plus or minus) in anaerobic tubes. As expected, all of these electron acceptors allowed anaerobic growth on glycerol. However, only tetrathionate allowed strong anaerobic growth on all three carbon sources. No electron acceptor was required for growth on the fermentable carbon source glucose.

Growth of wild-type strains with tetrathionate. Growth was initially measured in anaerobic liquid cultures by monitoring the increase in optical density (OD) at 650 nm. Tetrathionate supported anaerobic growth on acetate, demonstrating that it is truly serving as a respiratory electron acceptor (Table 2). As can be seen in Fig. 4A and B, tetrathionate supported anaerobic growth on ethanolamine plus B₁₂ and on propanediol. (Reasons for supplying B₁₂ for ethanolamine tests are described below.) In the absence of an electron acceptor, none of these cultures reached an OD in excess of 0.01. Similarly no growth was seen on tetrathionate alone, in the absence of added carbon source. In agreement with previous qualitative results, some growth was seen with nitrate as an electron acceptor.

View larger version (32K): [in this window] [in a new window]   FIG. 4.   Anaerobic growth on ethanolamine plus B12 or on propanediol with the electron acceptor tetrathionate (S4O6) or nitrate (NO3). Additions to NCE minimal medium were as follows: sodium tetrathionate (40 mM) or potassium nitrate (10 mM), ethanolamine (10 mM), and B12 (0.2 mM) (A and C) or propanediol (50 mM) (B and D). Growth was monitored on the basis of absorbance at 650 nm (A and B), viable cell counts (filled symbols in panels C and D), and microscopic counts (open symbols in panels C and D) using a Petroff-Hausser bacterial cell counter (C and D). The data for cells grown with S4O6 but no carbon source are replotted in graphs A and B (for turbidity) and C and D (for microscopic and viable cell counts).

View larger version (94K): [in this window] [in a new window]   FIG. 5.   Cell chain and granule formation during growth of wild-type serovar Typhimurium on ethanolamine or propanediol plus tetrathionate. Cells were viewed on a Zeiss Axioplan phase-contrast microscope. The scale bar in panel A represents 2 µm, and all photos are at the magnification indicated in panel A. Photographs are from stationary-phase cultures grown as described in the legend to Fig. 4. Panels A to C show cells (A and B) and refractile granules (B and C) observed in an ethanolamine-tetrathionate culture after 44 h of incubation at 37°C. (D and E) Cells and refractile granules observed in propanediol-tetrathionate culture after 67 h of incubation at 37°C. (F) Cells grown on propanediol plus NO3. Neither cell chains nor refractile granules were seen in either ethanolamine- or propanediol-grown cultures when nitrate was the electron acceptor.

Nature of the granules appearing in tetrathionate-grown cultures. A variety of bacteria are known to produce elemental sulfur granules during growth involving oxidation or reduction of sulfur compounds (36, 50). This suggested that Salmonella might deposit sulfur to form the granules that appear within and outside of cells during growth on tetrathionate. To test this, we assayed elemental sulfur by the method of Hazeu et al. (19) as described in Materials and Methods. The method detects sulfur that can be removed by centrifugation (inside or outside of cells); it does not detect dissolved sulfides. The assays revealed that, late during anaerobic growth on tetrathionate, elemental sulfur was accumulating in cells and as precipitable material to a concentration of about 1 mM (based on the volume of the culture).

Anaerobic respiration of ethanolamine and propanediol requires endogenous cobalamin. Tetrathionate appears to provide conditions that allow S. enterica serovar Typhimurium to use ethanolamine and propanediol under anaerobic conditions, the conditions under which cobalamin is synthesized. Mutants blocked in B₁₂ synthesis were used to show that anaerobic growth on these carbon sources did, in fact, require endogenous B₁₂. These experiments revealed some unexpected things.

View larger version (34K): [in this window] [in a new window]   FIG. 6.   Effect of cobamides on anaerobic growth on ethanolamine or propanediol. Wild-type serovar Typhimurium (A and C) and a mutant with an insertion in cbiD (Cob) (B and D) were grown anaerobically on ethanolamine plus tetrathionate (A and B) or propanediol plus tetrathionate (C and D). Additions were as follows: cyano-B12 (0.2 µM in panels A, B, and D; 15 nM in panel C), cobinamide dicyanide (15 nM), and AdoB12 (15 nM).

Defining the pathways of ethanolamine and propanediol respiration by mutant phenotypes. The present view of the aerobic degradative pathways for ethanolamine and propanediol is outlined in Fig. 2. However, the anaerobic respiration of these compounds has not been investigated. Table 2 presents effects of various mutations on anaerobic growth using tetrathionate as an electron acceptor in solid and liquid media. The results on solid media are extremely clearstrong growth on tetrathionate and no visible growth seen for conditions indicated by a minus sign. In liquid medium, some increase in turbidity and cell number was measured for conditions that produced no growth on plates. The difference may reflect the observation that cells are dividing to form smaller and smaller cells under these starvation conditions and may do so with very little increase in biomass (dividing down). This increases turbidity and cell number but is not apparent on the plates.

Mutations (ttr) causing a defect in tetrathionate reduction. The results below confirm and support those of Hensel et al. (21, 22), which were reported while this work was in progress. A large set of mutants unable to reduce tetrathionate was isolated by using MudJ, Tn10dTc, Tn10dCm, and TPOP elements (see Materials and Methods). These mutations all affected a single region (ttr) whose chromosomal position was confirmed by transductional linkage to markers near the previously determined position of ttr mutants (9). The region includes an operon of three structural genes for enzymes, all of which are required for tetrathionate reduction, and two genes that are essential to expression of the three-gene operon. These regulatory proteins are homologous with proteins that are part of known two-component regulatory systems (21). Our mutant set included insertions in all genes except the small ttrR gene, for which a deletion was constructed as described below. All sequenced insertion mutations are described in GenBank under accession number AF282268. Insertions near the ttr operon were isolated to obtain mutations in immediately adjacent open reading frames; the ttr⁺ phenotypes of the rkh and nth mutants ensured that these adjacent genes are not essential to tetrathionate reduction.

View larger version (17K): [in this window] [in a new window]   FIG. 7.   Map of the Sallmonella ttr region and analogous region of the E. coli chromosome. Regions present in only one genome are represented as raised triangles. The map is not to scale; the sizes of various fragments, in kilobases, are shown in parentheses. The SPI2 region is represented by a dashed line to indicate its foreign evolutionary origin. Genes are represented by arrows that point in the direction of transcription. The hatched box represents the region of the Salmonella chromosome that was sequenced during the course of this work.

Global regulation of the ttr operon. By use of fusions to the lac operon formed by ttr::MudJ insertions, conditions for induction of the operon were tested. Results in Tables 3 and 4 show that, under aerobic conditions, tetrathionate causes a slight induction (5- to 10-fold), but its major inductive effect (100- to 900-fold) occurs anaerobically. The requirement for anaerobic conditions is mediated by the OxrA protein, homologous to Fnr of E. coli (16). An insertion in the oxrA gene eliminated anaerobic induction of a ttr::MudJ fusion by tetrathionate (Table 3) and prevented anaerobic growth on ethanolamine and propanediol (Table 2). Neither the ArcA global regulator (also responding to anaerobic conditions) nor the Crp-cyclic AMP system (responding to carbon starvation) affected ttr operon induction.

Proximal inducers of the ttr operon. Tetrathionate was the most effective inducer of the compounds tested (Table 4). The inducer seems to be tetrathionate per se, rather than its reduction products, because the strains used in these tests carry a ttr::MudJ insertion and are unable to reduce tetrathionate. Furthermore, mutations in the phs genes, which prevent further reduction of thiosulfate (Fig. 1), had little effect on induction by tetrathionate (Table 3). Sulfite (SO₃²) and thiosulfate (S₂O₃²) caused very little induction, even at high concentrations (Table 4); the small effects seen could reflect internal production of tetrathionate.

Mechanism of proximal ttr control. The two regulatory genes (ttrS and ttrR), which overlap by 26 bases (see Fig. 8), encode proteins similar to those of two-component regulatory systems (40) and are responsible for the tetrathionate-specific regulation of the ttr operon (21). The TtrS protein resembles sensor kinases, which (in other systems) act to phosphorylate another protein in response to a regulatory stimulus. The TtrR protein resembles these responsive regulatory proteins. Alignment of the TtrS sequence with that of known sensor kinases reveals two functional domains. The C-terminal sensory kinase domain (residue 325 to the end) includes all of the motifs required for autokinase activity (N box, G1 box, F box, and G2 box). The N-terminal domain of TtrS (residues 1 to 358) shows little homology to any gene in GenBank but contains the H box (autophosphorylation site) and is most likely involved in sensing tetrathionate. To examine the role of these proteins, we assayed the effects of mutations in the ttrR, ttrS, and promoter regions on transcription of the structural genes (ttrBCA). Results are shown in Table 5; normal regulation is shown in lines 1 and 10. These data support the model presented by Hensel et al. (21) and outlined in Fig. 8.

View larger version (20K): [in this window] [in a new window]   FIG. 8.   Proximal regulation of the ttrBCA operon. The key to inserted elements is shown on the lower right. In the model presented below the map, tetrathionate is sensed by the TtrS protein, which autophosphorylates and then transfers the phosphate group to activate TtrR. Activated TtrR cooperates with the global regulator OxrA (Fnr) to positively regulate expression of the ttrBCA operon.

	DISCUSSION

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The alternative electron acceptor tetrathionate allows Salmonella to grow anaerobically on ethanolamine or propanediol by using endogenously synthesized B₁₂. These are the only conditions we know under which wild-type Salmonella requires B₁₂ for growth. Almost 2% of the Salmonella genome (88 genes) is dedicated to the metabolism discussed here. Synthesis and import of B₁₂ requires at least 30 genes; the eut, the propanediol (pdu), and the proprionate (prp) operons contain 17, 23, and 5 genes, respectively. Operons for sulfur reduction are ttr (five genes), phs (five genes), and asr (three genes). We presume that the natural environment of Salmonella must frequently include anaerobic conditions with tetrathionate, ethanolamine, and/or propanediol; these conditions select for maintenance of the B₁₂ synthesis (cob) genes. The importance of B₁₂ to propanediol use is supported by the fact that propanediol induces the cob operon (7, 45). This complex of abilities is found in virtually every Salmonella isolate; most are absent from E. coli. Aspects of this complex are used to enrich for and identify salmonellae in natural isolates and distinguish them from E. coli (8, 21, 39, 41, 43). The metabolism described here appears to be an important aspect of a Salmonella-specific lifestyle.

The mechanism of tetrathionate reduction has not been studied extensively in Salmonella (4, 15). By analogy with similar systems in other bacteria, we presume that reduction of tetrathionate can support electron transport and generate a proton gradient. Salmonella can grow anaerobically on tetrathionate plus glycerol, and use of glycerol as a carbon source is known to require electron transport (15). Furthermore, tetrathionate allows anaerobic growth on acetate, the catabolism of which provides no means of substrate-linked phosphorylation. Surprisingly, tetrathionate can serve as an electron acceptor even in strains with mutations in synthesis of both ubiquinone and menaquinone (Tom Fazzio, personal communication), suggesting that tetrathionate reductase may accept electrons directly from NADH or FADH₂. It is not clear why tetrathionate should be superior to nitrate in supporting anaerobic growth on ethanolamine or propanediol or acetate.

Cell chains and lethality were noted at late stages of growth on tetrathionate. We suspect that these phenomena reflect toxic effects of thiosulfate, sulfite, or sulfide in the medium or sulfur accumulation within cells. Salmonella possesses the phs and asr systems, which can reduce thiosulfate completely to sulfide. A mutational block in the first step (phs) neither relieves nor exacerbates the toxicity. However, the effects of such mutants may be masked by the assimilatory thiosulfate reductase (CysM), which converts thiosulfate to sulfite, or by rhodanese, which can, in principle, convert thiosulfate to sulfite plus sulfide. While little work has been done on rhodanese in Salmonella, the activity has been reported in E. coli (18), and genomes of both E. coli and serovar Typhimurium include rhodanese homologues. Under natural conditions, toxic accumulations might be diluted more than was possible in the growth tubes used for these experiments.

A surprising aspect of anaerobic growth on tetrathionate was the appearance of sulfur granules. We presume that the sulfur granules are generated by the chemical reaction of sulfide ions with tetrathionate, which has been described previously (30). It is not known whether Salmonella enzymes contribute to this process. However, serovar Typhimurium (but not E. coli) can reduce mineral sulfur, and Salmonella mutants are known which fail in this (K. Nealson and D. Lies, personal communication; M. Price-Carter, unpublished results).

Most of the activities mentioned here are found in Salmonella but not in E. coli. (The ethanolamine operon is shared by both species.) We suggest that the Salmonella pattern evolved by acquisition of foreign genes and mutational loss of ancestral genes. The ability to synthesize B₁₂ and catabolize propanediol was acquired by Salmonella (but not E. coli) as a single DNA fragment from an organism having a guanosine-plus-cytosine content and codon usage atypical for Salmonella (47). This occurred about 70 million years ago, perhaps 30 million years after the divergence of salmonellae and E. coli (33-35). In contrast, the ttr operon was probably carried by the common ancestor of E. coli and Salmonella and unilaterally lost from the E. coli lineage in the course of their divergence. The guanosine-plus-cytosine content and codon usage of ttr are typical of ancestral genes shared by Salmonella and E. coli (21). Tetrathionate reduction is found in many other enteric bacteria, suggesting that it was present in the common ancestor of enteric lineages (11). The phs and asr genes also seem likely to be ancestral genes lost from the E. coli lineage. The pathogenicity island SPI2, close to the ttr operon on the Salmonella chromosome (Fig. 7), is clearly of foreign origin and appears to have been added to the genome of Salmonella but not to that of E. coli (22). The evolution of the genes described here exemplifies the divergence of Salmonella and E. coli by genomic fluxlineage-specific events of gene loss and acquisition (35).