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Baby chicks administered a fecal transplant from adult chickens are resistant to Salmonella colonization by competitive exclusion. A two-pronged approach was used to investigate the mechanism of this process. First, Salmonella response to an exclusive (Salmonella competitive exclusion product, Aviguard®) or permissive microbial community (chicken cecal contents from colonized birds containing 7.85 Log10 Salmonella genomes/gram) was assessed ex vivo using a S. typhimurium reporter strain with fluorescent YFP and CFP gene fusions to rrn and hilA operon, respectively. Second, cecal transcriptome analysis was used to assess the cecal communities' response to Salmonella in chickens with low (≤5.85 Log10 genomes/g) or high (≥6.00 Log10 genomes/g) Salmonella colonization. The ex vivo experiment revealed a reduction in Salmonella growth and hilA expression following co-culture with the exclusive community. The exclusive community also repressed Salmonella's SPI-1 virulence genes and LPS modification, while the anti-virulence/inflammatory gene avrA was upregulated. Salmonella transcriptome analysis revealed significant metabolic disparities in Salmonella grown with the two different communities. Propanediol utilization and vitamin B12 synthesis were central to Salmonella metabolism co-cultured with either community, and mutations in propanediol and vitamin B12 metabolism altered Salmonella growth in the exclusive community. There were significant differences in the cecal community's stress response to Salmonella colonization. Cecal community transcripts indicated that antimicrobials were central to the type of stress response detected in the low Salmonella abundance community, suggesting antagonism involved in Salmonella exclusion. This study indicates complex community interactions that modulate Salmonella metabolism and pathogenic behavior and reduce growth through antagonism may be key to exclusion. [ABSTRACT FROM AUTHOR]

Sirtuins are [NAD.sup.+]-dependent protein deacylases that are conserved in all domains of life and are involved in diverse cellular processes, including control of gene expression and central metabolism. Eukaryotic sirtuins have N-terminal extensions that have been linked to protein multimerization and cellular localization. Here the first evidence of sirtuin isoforms in bacteria is reported. The enterobacterium Salmonella enterica synthesizes two isoforms of Cobb sirtuin, a shorter 236-amino-acid isoform (here [CobB.sub.S]) and a longer 273-amino-acid isoform (here [CobB.sub.L]). The N-terminal 37-amino-acid extension of [CobB.sub.L] is amphipathic, containing 18 basic amino acids (12 of which are Arg) and 13 hydrophobic ones; both isoforms were active in vivo and in vitro. Northern blot and transcription start site analyses revealed that cobB is primarily expressed as two monocistronic cobB mRNAs from two transcription start sites, one of which was mapped within the neighboring ycfX gene and the other of which was located within cobB. Additionally, a low-abundance ycfX-cobB bicistronic mRNA was observed which could encode up to three proteins (YcfX, [CobB.sub.L], and [CobB.sub.S]). [CobB.sub.L] isoforms are common within the family Enterobacteriaceae, but species of the genus Erwinia (including the plant pathogen Erwinia amylovora) encode only the [CobB.sub.L] isoform. The [CobB.sub.L] isoform from E. amylovora restored growth of as S. enterica cobB mutant strain on low acetate. doi: 10.1128/JB.00874-10

Strains of Salmonella enterica serovar Typhimurium LT2 lacking a functional 2-methylcitric acid cycle (2-MCC) display increased sensitivity to propionate. Previous work from our group indicated that this sensitivity to propionate is in part due to the production of 2-methylcitrate (2-MC) by the Krebs cycle enzyme citrate synthase (GltA). Here we report in vivo and in vitro data which show that a target of the 2-MC isomer produced by GltA (2-[MC.sup.ltA]) is fructose-l,6-bisphosphatase (FBPase), a key enzyme in gluconeogenesis. Lack of growth due to inhibition of FBPase by 2-[MC.sup.GltA] was overcome by increasing the level of FBPase or by micromolar amounts of glucose in the medium. We isolated an fbp allele encoding a single amino acid substitution in FBPase (S123F), which allowed a strain lacking a functional 2-MCC to grow in the presence of propionate. We show that the 2-[MC.sup.gltA] and the 2-MC isomer synthesized by the 2-MC synthase (PrpC; 2-[MC.sup.PrpC]) are not equally toxic to the cell, with 2-[MC.supGltA] being significantly more toxic than 2-[MC.sup.PrpC]. This difference in 2-MC toxicity is likely due to the fact that as a si-citrate synthase, GltA may produce multiple isomers of 2-MC, which we propose are not substrates for the 2-MC dehydratase (PrpD) enzyme, accumulate inside the cell, and have deleterious effects on FBPase activity. Our findings may help explain human inborn errors in propionate metabolism. doi: 10.1128/JB.01301-09

The genome of Rhodobacter sphaeroides encodes the components of two distinct pathways for salvaging cobinamide (Cbi), a precursor of adenosylcobalamin (AdoCbl, coenzyme [B.sub.12]). One pathway, conserved among bacteria, depends on a bifunctional kinase/guanylyltransferase (CobP) enzyme to convert adenosylcobinamide (AdoCbi) to AdoCbi-phosphate (AdoCbi-P), an intermediate in de novo AdoCbl biosynthesis. The other pathway, of archaeal origin, depends on an AdoCbi amidohydrolase (CbiZ) enzyme to generate adenosylcobyric acid (AdoCby), which is converted to AdoCbi-P by the AdoCbi-P synthetase (CobD) enzyme. Here we report that R. sphaeroides strain 2.4.1 synthesizes AdoCbl de novo and that it salvages Cbi using both of the predicted Cbi salvaging pathways. AdoCbl produced by R. sphaeroides was identified and quantified by high-performance liquid chromatography and bioassay. The deletion of cobB (encoding an essential enzyme of the de novo corrin ring biosynthetic pathway) resulted in a strain of R. sphaeroides that would not grow on acetate in the absence of exogenous corrinoids. The results from a nutritional analysis showed that the presence of either CbiZ or CobP was necessary and sufficient for Cbi salvaging, that CbiZ-dependent Cbi salvaging depended on the presence of CobD, and that CobP-dependent Cbi salvaging occurred in a [cbiZ.sup.+] strain. Possible reasons why R. sphaeroides maintains two distinct pathways for Cbi salvaging are discussed.

In Salmonella enterica, tricarballylate (Tcb) catabolism requires function of TcuB, a membrane-bound protein that contains [4Fe-4S] clusters and heme. TcuB transfers electrons from reduced flavin adenine dinucleotide in the Tcb dehydrogenase (TcuA) to electron acceptors in the membrane. We recently showed that functions needed to assemble [Fe-S] clusters (i.e., the iscRSUA-hscBA-fdx operon) compensate for the lack of ApbC during growth of an apbC strain on Tcb. ApbC had been linked to [Fe-S] cluster metabolism, and we showed that an apbC strain had decreased TcuB activity. Here we report findings that expand our understanding of the regulation of expression of the iscRSUA genes in Salmonella enterica. We investigated why low levels of glucose or other saccharides restored growth of an apbC strain on Tcb. Here we report the following findings. (i) A [less than or equal to] 1 mM concentration of glucose, fructose, ribose, or glycerol restores growth of an apbC strain on Tcb. (ii) The saccharide effect results in increased levels of TcuB activity. (iii) The saccharide effect depends on the global regulatory protein Cra. (iv) Putative Cra binding sites are present in the regulatory region of the iscRSUA operon. (v) Cra protein binds to all three sites in the iscRSUA promoter region in a concentrationdependent fashion. To our knowledge, this is the first report of the involvement of Cra in [Fe-S] cluster assembly.

This report provides in vivo evidence for the posttranslational control of the acetyl coenzyme A (Ac-CoA) synthetase (AcsA) enzyme of Bacillus subtilis by the acuA and acuC gene products. In addition, both in vivo and in vitro data presented support the conclusion that the yhdZ gene of B. subtilis encodes a [NAD.sup.+]-dependent protein deacetylase homologous to the yeast Sir2 protein (also known as sirtuin). On the basis of this new information, a change in gene nomenclature, from yhdZ to srtN (for sirtuin), is proposed to reflect the activity associated with the YdhZ protein. In vivo control of B. subtilis AcsA function required the combined activities of AcuC and SrtN. Inactivation of acuC or srtN resulted in slower growth and cell yield under low-acetate conditions than those of the wild-type strain, and the acuC srtN strain grew under low-acetate conditions as poorly as the acsA strain. Our interpretation of the latter result was that both deacetylases (AcuC and SrtN) are needed to maintain AcsA as active (i.e., deacetylated) so the cell can grow with low concentrations of acetate. Growth of an acuA acuC srtN strain on acetate was improved over that of the [acuA.sup.+] acuC srtN strain, indicating that the AcuA acetyltransferase enzyme modifies (i.e., inactivates) AcsA in vivo, a result consistent with previously reported in vitro evidence that AcsA is a substrate of AcuA.

Mutants of Salmonella enterica lacking apbC have nutritional and biochemical properties indicative of defects in [Fe-S] cluster metabolism. Here we show that apbC is required for S. enterica to use tricarballylate as a carbon and energy source. Tricarballylate catabolism requires three gene products, TcuA, TcuB, and TcuC. Of relevance to this work is the TcuB protein, which has two [4Fe-4S] clusters required for function, making it a logical target for the apbC effect. TcuB activity was 100-fold lower in an apbC mutant than in the isogenic [apbC.sup.+] strain. Genetic data show that derepression of the iscRSUA-hscAB-fdx-orf3 operon or overexpression of iscU from a plasmid compensates for the lack of ApbC during growth on tricarballylate. The studies described herein provide evidence that the scaffold protein IscU has a functional overlap with ApbC and that ApbC function is involved in the synthesis of active TcuB.

We report results of studies of the conversion of adenosylcobyric acid (AdoCby) to adenosylcobinamidephosphate, the last step of the de novo corrin ring biosynthetic branch of the adenosylcobalamin (coenzyme [B.sub.12]) pathway of Salmonella enterica serovar Typhimurium LT2. Previous reports have implicated the CbiB protein in this step of the pathway. Hydropathy analysis predicted that CbiB would be an integral membrane protein. We used a computer-generated topology model of the primary sequence of CbiB to guide the construction of CbiB-LacZ and CbiB-PhoA protein fusions, which were used to explore the general topology of CbiB in the cell membrane. A refined model of CbiB as an integral membrane protein is presented. In vivo analyses of the effect of single-amino-acid changes showed that periplasm- and cytosol-exposed residues are critical for CbiB function. Results of in vivo studies also show that ethanolamine-phosphate (EA-P) is a substrate of CbiB, but e-Thr-P is not, and that CbiB likely activates AdoCby by phosphorylation. The latter observation leads us to suggest that CbiB is a synthetase not a synthase enzyme. Results from mass spectrometry and bioassay experiments indicate that serovar Typhimurium synthesizes norcobalamin (cobalamin lacking the methyl group at C176) when EA-P is the substrate of CbiB.

Cobinamide (Cbi) salvaging is impaired, hut not abolished, in a Salmonella enterica strain lacking a functional cobU gene. CobU is a bifunctional enzyme (NTP:adenosylcobinamide [NTP:AdoCbi] kinase, GTP: adenosyleobinamide-phosphate [GTP:AdoCbi-P] guanylyltransferase) whose AdoCbi kinase activity is necessary for Cbi salvaging in this bacterium. Inactivation of the ycfN gene in a [DELTA]cobU strain abrogated Cbi salvaging. Introduction of a plasmid carrying the ycf[N.sup.+] allele into a [DELTA]cobU [DELTA]ycfN strain substantially restored Cbi salvaging. Mass spectrometry data indicate that when YcfN-enriched cell extracts were incubated with AdoCbi and ATP, the product of the reaction was AdoCbi-P. Results from bioassays confirmed that YcfN converted AdoCbi to AdoCbi-P in an ATP-dependent manner. YcfN is a good example of enzymes that are used by the cell in multiple pathways to ensure the salvaging of valuable precursors.

We report that cobC strains of Salmonella enterica serovar Typhimurium are impaired in the ability to salvage cobyric acid (Cby), a de novo coffin ring biosynthetic intermediate, under aerobic growth conditions. In vivo and in vitro evidence support the conclusion that this new phenotype of cobC strains is due to the inability of serovar Typhimurium to dephosphorylate adenosyleobalamin-5'-phosphate (AdoCbl-5'-P), the product of the condensation of [alpha]-ribazole-5'-phosphate [alpha]-RP) and adenosylcobinamide-GDP by the AdoCbl-5'-P synthase (CobS, EC 2.7.8.26) enzyme. Increased flux through the 5,6-dimethyibenzimidazole and cobinamide (Cbi) activation branches of the nucleotide loop assembly pathway in cobC strains restored AdoCbl-5'-P synthesis from Cby in a cobC strain. The rate of the CobS-catalyzed reaction was at least 2 orders of magnitude higher with [alpha]-RP than with [alpha]-ribazole as substrate. On the basis of the data reported herein, we conclude that removal of the phosphoryl group from AdoCbl-5'-P is the last step in AdoCbl biosynthesis in serovar Typhimurium and that the reaction is catalyzed by the AdoCbl-5'-P phosphatase (CobC) enzyme. Explanations for the correction of the Cby salvaging phenotype are discussed.

Posttranslational modification is an efficient mechanism for controlling the activity of structural proteins, gene expression regulators, and enzymes in response to rapidly changing physiological conditions. Here we report in vitro and in vivo evidence that the acuABC operon of the gram-positive soil bacterium Bacillus subtilis encodes a protein acetyltransferase (AcuA) and a protein deacetylase (AcuC), which may control the activity of acetyl-coenzyme A (CoA) synthetase (AMP-forming, AcsA) in this bacterium. Results from in vitro experiments using purified proteins show that AcsA is a substrate for the acetyl-CoA-dependent AcuA acetyltransferase. Mass spectrometry analysis of a tryptic digest of acetylated AcsA (Acs[A.sup.Ac]) identified residue Lys549 as the sole modification site in the protein. Unlike sirtuins, the AcuC protein did not require NA[D.sup.+] as cosubstrate to deacetylate Acs[A.sup.Ac]. The function of the putative AcuB protein remains unknown.

Tricarballylate is the causative agent of grass tetany, a ruminant disease characterized by acute magnesium deficiency. Tricarballylate toxicity has been attributed to its ability to chelate magnesium and to inhibit aconitase, a Krebs cycle enzyme. Neither the ruminant nor the normal rumen flora can catabolize tricarballylate to ameliorate its toxic effects. However, the gram-negative enterobacterium Salmonella enterica can use tricarballylate as a carbon and energy source, providing an opportunity to study the genes and enzymes required for tricarballylate catabolism. The tricarballylate utilization (tcu) genes are organized into two transcriptional units, i.e., tcuR and tcuABC. Here, we report the initial biochemical analysis of TcuA. TcuA catalyzed the oxidation of tricarballylate to cis-aconitate. The apparent [K.sub.m] of TcuA for tricarballylate was 3.8 [+ or -] 0.4 mM, with a [V.sub.max] of 7.9 [+ or -] 0.3 mM [min.sup.-1], turnover number ([k.sub.cat]) of 6.7 x [10.sup.-2] [s.sup.-1], and a catalytic efficiency ([k.sub.cat]/[K.sub.m]) of 17.8 [M.sup.-1] [s.sup.-1]. Optimal activity was measured at pH 7.5 and 30[degrees]C. The enzyme was inactivated at 45[degrees]C. One mole of FAD was present per mole of TcuA. We propose a role for TcuB as an electron shuttle protein responsible for oxidizing FAD[H.sub.2] back to FAD in TcuA.

Here we report the initial biochemical characterization of the bifunctional [alpha]-ribazole-P ([alpha]-RP) phosphatase, adenosylcobinamide (AdoCbi) amidohydrolase CbiS enzyme from the hyperthermophilic methanogenic archaeon Methanopyrus kandleri AVI9. The cbiS gene encodes a 39-kDa protein with two distinct segments, one of which is homologous to the AdoCbi amidohydrolase (CbiZ, EC 3.5.1.90) enzyme and the other of which is homologous to the recently discovered archaeal [alpha]-RP phosphatase (CobZ, EC 3.1.3.73) enzyme. CbiS function restored AdoCbi salvaging and [alpha]-RP phosphatase activity in strains of the bacterium Salmonella enterica where either step was blocked. The two halves of the cbiS genes retained their function in vivo when they were cloned separately. The CbiS enzyme was overproduced in Escherichia coli and was isolated to >95% homogeneity. High-performance liquid chromatography, UV-visible spectroscopy, and mass spectroscopy established [alpha]-ribazole and cobyric acid as the products of the phosphatase and amidohydrolase reactions, respectively. Reasons why the CbiZ and CobZ enzymes are fused in some archaea are discussed.

Although methanogenic archaea use [B.sub.12] extensively as a methyl carrier for methanogenesis, little is known about [B.sub.12] metabolism in these prokaryotes or any other archaea. To improve our understanding of how [B.sub.12] metabolism differs between bacteria and archaea, the gene encoding the ATP:co(I)rrinoid adenosyltransferase in Methanosarcina mazei strain Go1 (open reading frame MM3138, referred to as cob[A.sub.Mm] here) was cloned and used to restore coenzyme [B.sub.12] synthesis in a Salmonella enterica strain lacking the housekeeping CobA enzyme, cob[A.sub.Mm] protein was purified and its initial biochemical analysis performed. In vitro, the activity is enhanced 2.5-fold by the addition of [Ca.sup.2+] ions, but the activity was not enhanced by [Mg.sup.2+] and, unlike the S. enterica CobA enzyme, it was >50% inhibited by [Mn.sup.2+]. The Cob[A.sub.Mm] enzyme had a [K.sub.m.sup.ATP] of 3 [mu]M and a [K.sub.m.sup.HOCbl] of 1 [micro]M. Unlike the S. enterica enzyme, Cob[A.sub.Mm] used cobalamin (Cbl) as a substrate better than cobinamide (Cbi; a Cbl precursor); the [beta] phosphate of ATP was required for binding to the enzyme. A striking difference between Cob[A.sub.se] and Cob[A.sub.Mm] was the use of ADP as a substrate by Cob[A.sub.Mm], suggesting an important role for the [gamma] phosphate of ATP in binding. The results from [sup.31]P-nuclear magnetic resonance spectroscopy experiments showed that triphosphate (PP[P.sub.i]) is the reaction by-product; no cleavage of PP[P.sub.i] was observed, and the enzyme was only slightly inhibited by pyrophosphate (P[P.sub.i]). The data suggested substantial variations in ATP binding and probably corrinoid binding between Cob[A.sub.se]. and Cob[A.sub.Mm] enzymes.

Open reading frame (ORF) Mm2058 of the methanogenic archaeon Methanosarcina mazei strain Go1 was shown in vivo and in vitro to encode the nonorthologous replacement of the [alpha]-ribazole-phosphate phosphatase (CobC; EC 3.1.3.73) enzyme of Salmonella enterica serovar Typhimurium LT2. Bioinformatics analysis of sequences available in databases tentatively identified ORF Mm2058, which was cloned under the control of an inducible promoter and was used to support growth of an S. enterica strain under conditions that demanded CobC-like activity. The Mm2058 protein was expressed with a decahistidine tag at its N terminus and was purified to homogeneity using nickel affinity chromatography. High-performance liquid chromatography followed by electrospray ionization mass spectrometry showed that the Mm2058 protein had phosphatase activity that converted [alpha]-ribazole-5'-phosphate to oL-ribazole, as reported for the bacterial CobC enzyme. On the basis of the data reported here, we refer to ORF Mm2058 as cobZ. We tested the prediction by Rodionov et al. (D. A. Rodionov, A. G. Vitreschak, A. A. Mironov, and M. S. Gelfand, J. Biol. Chem. 278:41148-41159, 2003) that ORF HSL01294 (also called Vng1577) encoded the nonorthologous replacement of the bacterial CobC enzyme in the extremely halophilic archaeon Halobacterium sp. strain NRC-1. A strain of the latter carrying an in-frame deletion of ORF Vng1577 was not a cobalamin auxotroph, suggesting that either there is redundancy of this function in Halobacterium or the gene was misannotated.

During growth on ethanolamine, Salmonella enterica synthesizes a multimolecular structure that mimics the carboxysome used by some photosynthetic bacteria to fix C[O.sub.2]. In S. enterica, this carboxysome-like structure (hereafter referred to as the ethanolamine metabolosome) is thought to contain the enzymatic machinery needed to metabolize ethanolamine into acetyl coenzyme A (acetyl-CoA). Analysis of the growth behavior of mutant strains of S. enterica lacking specific functions encoded by the 17-gene ethanolamine utilization (eut) operon established the minimal biochemical functions needed by this bacterium to use ethanolamine as a source of carbon and energy. The data obtained support the conclusion that the ethanolamine ammnonia-lyase (EAL) enzyme (encoded by the eutBC genes) and coenzyme [B.sub.12] are necessary and sufficient to grow on ethanolamine. We propose that the EutD phosphotransacetylase and EutG alcohol dehydrogenase are important to maintain metabolic balance. Glutathione (GSH) had a strong positive effect that compensated for the lack of the EAL reactivase EutA protein under aerobic growth on ethanolamine. Neither GSH nor EutA was needed during growth on ethanolamine under reduced-oxygen conditions. GSH also stimulated growth of a strain lacking the acetaldehyde dehydrogenase (EutE) enzyme. The role of GSH in ethanolamine catabolism is complex and requires further investigation. Our data show that the ethanolamine metabolosome is not involved in the biochemistry of ethanolamine catabolism. We propose the metabolosome is needed to concentrate low levels of ethanolamine catabolic enzymes, to keep the level of toxic acetaldehyde low, to generate enough acetyl-CoA to support cell growth, and to maintain a pool of free CoA.

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 (Ach1 p) enzyme] were not involved in the release of acetate by the acs pta eutD strain.

We report evidence for the existence of a putative ABC transporter for corrinoid utilization in the extremely halophilic archaeon Halobacterium sp. strain NRC-1. Results from genetic and nutritional analyses of Halobacterium showed that mutants with lesions in open reading frames (ORFs) Vng1370G, Vng1371Gm, and Vng1369G required a [10.sup.5]-fold higher concentration of cobalamin for growth than the wild-type or parent strain. The data support the conclusion that these ORFs encode orthologs of the bacterial cobalamin ABC transporter permease (btuC; Vng1370G), ATPase (btuD; Vng1371Gm), and substrate-binding protein (btuF; Vng1369G) components. Mutations in the Vng1370G, Vng1371Gm, and Vng1369G genes were epistatic, consistent with the hypothesis that their products work together to accomplish the same function. Extracts of btuF mutant strains grown in the presence of cobalamin did not contain any cobalamin molecules detectable by a sensitive bioassay, whereas btuCD mutant strain extracts did. The data are consistent with the hypothesis that the BtuF protein is exported to the extracellular side of the cell membrane, where it can bind cobalamin in the absence of BtuC and BtuD. Our data also provide evidence for the regulation of corrinoid transport and biosynthesis. Halobacterium synthesized cobalamin in a chemically defined medium lacking corrinoid precursors. To the best of our knowledge, this is the first genetic analysis of an archaeal corrinoid transport system.

The function of the PrpR protein of Salmonella enterica serovar Typhimurium LT2 was studied in vitro and in vivo. The PrpR protein is a sensor of 2-methylcitrate (2-MC), an intermediate of the 2-methylcitric acid cycle used by this bacterium to convert propionate to pyruvate. PrpR was unresponsive to citrate (a close structural analogue of 2-MC) and to propionate, suggesting that 2-MC, not propionate, is the metabolite that signals the presence of propionate in the environment to S. enterica, prpR alleles encoding mutant proteins with various levels of 2-MC-independent activity were isolated. All lesions causing constitutive PrpR activity were mapped to the N-terminal domain of the protein. Removal of the entire sensing domain resulted in a protein (Prp[R.sup.c]) with the highest 2-MC-independent activity. Residue A162 is critical to 2-MC sensing, since the mutant PrpR protein Prp[R.sup.A162T] was as active as the Prp[R.sup.c] protein in the absence of 2-MC. DNA footprinting studies identified the site in the region between prpR and the prpBCDE operon to which the PrpR protein binds. Analysis of the binding-site sequence revealed two sites with dyed symmetry. Results from DNase I footprinting assays suggested that the PrpR protein may have higher affinity for the site proximal to the [P.sub.prpBCDE] promoter.

The eutT gene of Salmonella enterica was cloned and overexpressed, and the function of its product was established in vivo and in vitro. The EutT protein has an oxygen-labile, metal-containing ATP:co(I)rrinoid adenosyltransferase activity associated with it. Functional redundancy between EutT and the housekeeping ATP: co(I)rrinoid adenosyltransferase CobA enzyme was demonstrated through phenotypic analyses of mutant strains. Lack of CobA and EutT blocked ethanolamine utilization. EutT was necessary and sufficient for growth of an S. enterica cobA eutT strain on ethanolamine as a carbon and energy or nitrogen source. A [eutT.sup.+] gene provided in trans corrected the adenosylcobalamin-dependent transcription of a eut-lacZ operon fusion in a cobA strain. Cell extracts enriched for EutT protein contained strong, readily detectable ATP:co(I)rrinoid adenosyltransferase activity. The activity was only detected in extracts maintained under anoxic conditions, with complete loss of activity upon exposure to air or treatment with the [Fe.sup.2+] ion chelator bathophenanthroline. While the involvement of another metal ion cannot be ruled out, the observed sensitivity to air and bathophenanthroline suggests involvement of [Fe.sup.2+]. We propose that the EutT protein is a unique metal-containing ATP:co(I)rrinoid adenosyltransferase. It is unclear whether the metal ion plays a structural or catalytic role.

In Salmonella enterica, the last step of the synthesis of adenosylcobamide is catalysed by the cobalamin synthase enzyme encoded by the cobs gene of this bacterium. Overexpression of the S. enterica cobS gene in Escherichia coil elicited the accumulation of the phage shock protein PspA, a protein whose expression has been linked to membrane stress. Resolution of inner and outer membranes of S. enterica by isopycnic density ultracentrifugation showed Cobs activity associated with the inner membrane, a result that was confirmed using antibodies against CobS. Computer analysis of the predicted amino acid sequence of CobS suggested it was an integral membrane protein. Results of experiments performed with strains carrying plasmids encoding CobS-alkaline phosphatase or CobS-[beta]-galactosidase protein fusions were consistent with the membrane localization of the CobS protein. Modifications to the predicted model were made based on data obtained from experiments using protein fusions. The function encoded by the cobS orthologue in the methanogenic archaeon Methanobacterium thermoautotrophicum strain [DELTA]H compensated for the lack of CobS during cobalamin synthesis in cobS strains of S. enterica. Cobalamin synthase activity was also detected in a membrane preparation of M. thermoautotrophicum. It was concluded that the assembly of the nucleotide loop of adenosylcobamides in archaea and bacteria is a membrane-associated process. Possible reasons for the association of adenosylcobamide biosynthetic enzymes with the cell membrane are discussed.

The propionate utilization operons of several bacteria differ from each other in the occurrence of two genes, acnD and prpF, in place of or in addition to the prpD gene encoding an Fe/S-independent 2-methylcitrate dehydratase enzyme. We cloned the acnD and prpF genes from two organisms, Shewanella oneidensis and Vibrio cholerae, and found that, together, the AcnD and PrpF proteins restored the ability of a prpD mutant strain of Salmonella enterica to grow on propionate as a source of carbon and energy. However, neither acnD nor prpF alone was able to substitute for prpD. The AcnD and PrpF proteins were isolated and biochemically analyzed. The AcnD protein required reconstitution of an Fe/S cluster for activity. All detectable AcnD activity was lost after incubation with iron-chelating agents, and no AcnD activity was observed after attempted reconstitution without iron. Nuclear magnetic resonance spectroscopy and in vitro activity assay data showed that AcnD dehydrated 2-methylcitrate and citrate to 2-methyl-cis-aconitate and cis-aconitate, respectively; AcnD also hydrated cis-aconitate. However, 2-methylisocitrate and isocitrate were not substrates for AcnD, indicating that AcnD only catalyzes the first half of the aconitase-like dehydration reactions. No aconitase-like activity was found for PrpF. It is hypothesized that, in vivo, PrpF is an accessory protein required to prevent oxidative damage of the Fe/S center of active AcnD enzyme or that it may be involved in synthesis or repair of the Fe/S cluster present in AcnD.