Copyright 0 1996 by the Genetics Society of America Evolution of Coenzyme BI2Synthesis Among Enteric Bacteria: Evidence for Loss and Reacquisition of a Multigene Complex Jeffrey G. Lawrence and John R. Roth Department of Biology, University of Utah, Salt Lake City, Utah 84112 Manuscript received June 16, 1995 Accepted for publication October 4, 1995 ABSTRACT We have examined the distribution of cobalamin (coenzyme BI2) synthetic ability and cobalamin- dependent metabolism among entericbacteria. Most species of enteric bacteria tested synthesize cobala- min under both aerobic and anaerobic conditions and ferment glycerol in a cobalamindependent fashion. The group of species including Escha'chia coli and Salmonella typhimurium cannot ferment glyc- erol. E. coli strains cannot synthesize cobalamin de novo, and Salmonella spp. synthesize cobalamin only under anaerobic conditions. In addition, the cobalamin synthetic genes of Salmonella spp. (cob) show a regulatory pattern different from that of other enteric taxa tested. We propose that the cobalamin synthetic genes, as well asgenes providing cobalamindependent diol dehydratase, were lostby a common ancestor of E. coli and Salmonella spp. and were reintroduced as a single fragment into the Salmonella lineage from an exogenous source. Consistent with this hypothesis, the S. typhimurium cob genes do not hybridize with the genomes of other enteric species. The Salmonella cob operon may represent a class of genes characterized by periodic loss and reacquisition by host genomes. This process may be an important aspect of bacterial population genetics and evolution. OBALAMIN (coenzyme BIZ) is a large evolution- The cobalamin biosynthetic genes have been charac- C arily ancient molecule ( GEORGOPAPADAKOUand terized in S. typhimurium; most genes are located in a SCOTT 1977; ESCHENMOSER1988; SCOTT 1990, 1993) large 20-gene cluster at minute 41 on the genetic map that participates as a cofactor in both prokaryotic and ( JETER and ROTH 1987; ROTHet al. 1993). Within the eukaryotic metabolism (SCHNEIDERand STRO~NSKI 1987; cluster, genes encoding the enzymes for the threeparts STRO~NSKI 1987).Cobalamin is derived from uroporph- of the pathway are clustered (Figure 1).Mutations con- yrinogen I11 (Uro 111), a precursor in the synthesis of ferring CobI-, CobIII-, and CobII- phenotypes are lo- heme, siroheme, cobamides and chlorophylls (in pho- calized, in that order,within the cob operon ( JETER and tosynthetic organisms). As detailed in Figure 1, the con- ROTH1987; ESCALANTE-SEMERENAet al. 1992; O'TOOLE version of Uro I11 to the complex coenzyme BI2 inter- et al. 1993; ROTHet al. 1993). Additional genes outside mediate cobinamide is referred to as part I of the this cluster encode both biosynthetic enzymes for corri- biosynthetic pathway (hereafter denoted CobI) . Part noid adenosylation (ESCALANTE-SEMERENAet al. 1990) I1 of the pathway (CobII) entails the biosynthesis of and synthesis of aminopropanol, thelinker between the dimethylbenzimidazole (DMB) from probable flavin corrin ring and the lower ligand (GRABAUand ROTH precursors (HORIGand RENZ 1978;JOHNSON and ESCA- 1992); transportof cobalamin is also encoded by genes LANTE-SEMERENA1992; LINCENSet al. 1992; CHENet al. unlinked to the cob operon (RIOUX and KADNER 1989; 1995). The covalent joining of cobinamide, DMB, and RIOUX et al. 1990; BRADBEER1991). Despite this large a phosphoribosyl group donated by nicotinate mono- genetic investment in cobalamin synthesis and acquisi- nucleotide is achieved by part I11 of the pathway (Cob tion, entailing nearly 1% of the S. typhimurium chromo- 111). Among the enteric bacteria, both Salmonella typhi- some, only four known, and one likely, cobalaminde- murium ( JETER et al. 1984) and Klebsiella pneumonia pendent reactions have been characterized among the (ALBERT et al. 1980) are known to synthesize cobalamin enteric bacteria: de novo under anaerobic conditions. These organisms 1. Homocysteine methyltransferase catalyzes the final can perform all three parts of the biosynthetic pathway. step in methionine biosynthesis. The metE and metH Escherichiacoli synthesizes cobalamin only when pro- genes encode cobalamin-independent and cobalamin- vided with cobinamide (VOLCANIet al. 1961); therefore, dependent methyltransferases, respectively (SMITHand E. coli performs only parts I1 and I11 of the cobalamin CHILDS 1966;CHILDS and SMITH1969), in S. typhimu- biosynthetic pathway. rium. These genes are also found in E. coli (DAVISand MINGIOLI1950). Ethanolamine-ammonia lyase converts ethanol- Corresponding authm:John Roth, Department of Biology, University 2. of Utah, Salt Lake City, UT 84112. amineinto acetylaldehyde and ammoniaand is en- E-mail: [email protected] coded by the eutBC genes in s. typhimurium; the enzyme Genetics 142 11-24 (January, 1996) 12 J. G. Lawrence and J. R. Roth FIGURE1.-The metabolism of tetrapyr- roles in Salmonella typhimunum. Heavy arrows Mmute 41 Miule 34 denote reactions specific for cobalamin bio- synthesis. Gray arrows denote regulatory in- pdu operon 11 IlpocR I I cbiARCDEFFGHJKLM~QP cobUS cob7 teractions. Abbreviations are as follows: Ado, (cobalamin-dependent) adenosyl; Cbi, cobinamide; CN, cyano; DMB, dimethylbenzimidazole; FMN, flavin mono- L nucleotide; Uro 111, uroporphyrinogen 111. CobI CobIII CobII The CobI, CobII, and CobIII pathways are ~RNAC‘” ,- UroIII -b t2: 7DMB +FMN discussed in the text. The hagenes are de- I scribed by Xu et al. (1992); the cbi and cob hemEFGIjN 4ACNCobalamin genes are described by ROTH et al. (1993) and CHENet al. (1995). is also present in strains of E. coli and K. aerogenes minsynthesis andcobalamindependent metabolism (CHANCand CHANC1975; SCARLETT and TURNER1976; among enteric bacteria. In this manner, the selective ROOF and ROTH 1988, 1989). influences operating on cobalamin synthetic genes may 3. Propanediol dehydratase catalyzes the conversion be elucidated. We suggest that the Salmonella cob and of 1,2-propanediol to propionaldehyde and is encoded pdu operons were acquired by horizontal transfer after in the pdu operon in S. typhimurium ( JETER 1990; BOBIK the loss ofthese genes by a common ancestor of both E. et al. 1992); the pdu operon maps adjacent to the cob coli and Salmonella spp. The evolution of the cobalamin operon described here. In this paper, the generalterm synthesis and utilization may provide a general model propanediol will always refer to the 1,2 isomer. Cobala- for understanding the evolution of functions and phe- rnindependent diol dehydratases, activewith 1,2 notypes under selection that is temporally or spatially ethanediol and propanediol, have also been described heterogeneous. in Citrobacter and Klebsiella species (ABELES and LEE 1961; FORAGEand FOSTER1979; TORAYAet al. 1979, MATERIALSAND METHODS 1980; OBRADORSet al. 1988). 4. Glycerol dehydratase is an enzyme distinct from Bacterial strains and culture conditions: Strains of enteric diol dehydratase (TORAYAand FUKUI 1977),allowing bacteria employed in this study are listed in Table 1. Strains of the SARA collection (BELTRANet al. 1991) of natural iso- anaerobic fermentation of glycerol in species of Kleb lates of Salmonella spp. were kindly provided by K. SANDERSON siella and Citrobacter (SCHNEIDERet al. 1970; FORAGE and H. OCHMAN.Strains of the ECOR (OCHMANand SEL and FOSTER 1979,1982); this enzyme has not been de- ANDER 1984) and SARB (BOYD et al. 1993) collections of natu- scribed among strains of Salmonella or E. coli. ral isolates of E. coli and Salmonella spp, were kindly provided 5. Queuosine synthetase catalyzes the final step in the by H. OCHMAN.An E. coli strain harboring a mtE:: Tn IOinser- tion was provided by G. STAUFFER;the wild-type K. aerogenes synthesis of queuosine, a hypermodified base present strain M5al was provided by G. ROBERTS.Additional species in four tRNA andhas been reportedto be acobalamin- of Salmonella and other species of enteric bacteria were ob- dependent enzyme in E. coli (NOGUCHIet al. 1982; FREY tained from laboratory collections. The rich medium used was et al. 1988). LB defined media included E medium (VOGELand BONNER The known cobalamindependent reactions in S. typh- 1956) as well as its derivative NCE, which lacks carbon com- pounds. MacConkey indicator agarwas used with the addition imunum do not obviously justify this organism’s large of specific carbon sources to 1% concentrations. For anaero- genetic investment in cobalamin biosynthesis and trans- bic growth on solid medium, cultures were grown under an port. The cobalamindependentmethionine synthetase atmosphere of 89% NP, 6% COP,and 5% HP. Liquid culture is redundant, and the queuosine synthetase is appar- media were prepared, degassed for 24 hr, inoculated, and ently nonessential under laboratory conditions. Pro- sealed in tubes with aluminum caps under the anoxic atmo- sphere described. Headspace gas was exchanged three times panediol utilization appears to be the primary use for with 100% N2 under a pressure of 2 atmospheres. Loss of cobalamin in S. typhimun’um since the adjacent cobala- pressure in a sealed tube during incubation indicated a possi- min biosynthetic genes (cob) and the propanediol deg- ble decay of anoxic conditions and the experiment was re- radative operon (pdu) are coregulated by the same pro- peated. tein (PocR) and are both induced in the presence of Cobalamin synthesis bioassay: To assay ability to synthesize cobalamin, strains were grown to stationary phase on a de- propanediol (BOBIK et al. 1992; CHENet al. 1994). A fined medium