Genetic characterization of the pdu operon: use of 1,2-propanediol in Salmonella typhimurium.

Salmonella typhimurium is able to catabolize 1,2-propanediol for use as the sole carbon and energy source; the first enzyme of this pathway requires the cofactor adenosyl cobalamin (Ado-B12). Surprisingly, Salmonella can use propanediol as the sole carbon source only in the presence of oxygen but can synthesize Ado-B12 only anaerobically. To understand this situation, we have studied the pdu operon, which encodes proteins for propanediol degradation. A set of pdu mutants defective in aerobic degradation of propanediol (with exogenous vitamin B12) defines four distinct complementation groups. Mutations in two of these groups (pduC and pduD) eliminate propanediol dehydratase activity. Based on mutant phenotypes, a third complementation group (pduG) appears to encode a cobalamin adenosyl transferase activity. No function has been assigned to the pduJ complementation group. Propionaldehyde dehydrogenase activity is eliminated by mutations in any of the four identified complementation groups, suggesting that this activity may require a complex of proteins encoded by the operon. None of the mutations analyzed affects either of the first two genes of the operon (pduA and pduB), which were identified by DNA sequence analysis. Available data suggest that the pdu operon includes enough DNA for about 15 genes and that the four genetically identified genes are the only ones required for aerobic use of propanediol.     

Purification and initial biochemical characterization of ATP:Cob(I)alamin adenosyltransferase (EutT) enzyme of Salmonella enterica

ATP:cob(I)alamin adenosyltransferase (EutT) of Salmonella enterica was overproduced and enriched to approximately 70% homogeneity, and its basic kinetic parameters were determined. Abundant amounts of EutT protein were produced, but all of it remained insoluble. Soluble active EutT protein (approximately 70% homogeneous) was obtained after treatment with detergent. Under conditions in which cobalamin (Cbl) was saturating, Km(ATP) = 10 microm, kcat = 0.03 s(-1), and Vmax = 54.5 nm min(-1). Similarly, under conditions in which MgATP was saturating, Km(Cbl) = 4.1 microm, kcat = 0.06 s(-1), and Vmax = 105 nm min(-1). Unlike other ATP:co(I)rrinoid adenosyltransferases in the cell (i.e. CobA and PduO), EutT activity was > or =50-fold higher with ATP versus GTP, and EutT retained 80% of its activity with ADP substituted for ATP and was completely inactive with AMP as substrate, indicating that the enzyme requires the beta-phosphate group of the nucleotide substrate. The data suggest that the amino group of adenine might play a role in nucleotide recognition and/or binding. Unlike the housekeeping CobA enzyme, EutT was not inhibited by inorganic tripolyphosphate (PPPi). Results from 31P NMR spectroscopy studies identified PPi and Pi as by-products of the EutT reaction. In the absence of Cbl, EutT cleaved ATP into adenosine and PPPi, suggesting that PPPi is broken down into PPi and Pi. Electron transfer protein partners for EutT were not encoded by the eut operon. EutT-dependent activity was detected in cell-free extracts of cobA strains enriched for EutT when FMN and NADH were used to reduce cob(III)alamin to cob(I)alamin.     

The 17-Gene Ethanolamine (eut) Operon of Salmonella typhimurium Encodes Five Homologues of Carboxysome Shell Proteins

The eut operon of Salmonella typhimurium encodes proteins involved in the cobalamin-dependent degradation of ethanolamine. Previous genetic analysis revealed six eut genes that are needed for aerobic use of ethanolamine; one (eutR), encodes a positive regulator which mediates induction of the operon by vitamin B12 plus ethanolamine. The DNA sequence of the eut operon included 17 genes, suggesting a more complex pathway than that revealed genetically. We have correlated an open reading frame in the sequence with each of the previously identified genes. Nonpolar insertion and deletion mutations made with the Tn10-derived transposable element T-POP showed that at least 10 of the 11 previously undetected eut genes have no Eut phenotype under the conditions tested. Of the dispensable eut genes, five encode apparent homologues of proteins that serve (in other organisms) as shell proteins of the carboxysome. This bacterial organelle, found in photosynthetic and sulfur-oxidizing bacteria, may contribute to CO2 fixation by concentrating CO2 and excluding oxygen. The presence of these homologues in the eut operon of Salmonella suggests that CO2 fixation may be a feature of ethanolamine catabolism in Salmonella.     

The alternative electron acceptor tetrathionate supports B12-dependent anaerobic growth of Salmonella enterica serovar typhimurium on ethanolamine or 1,2-propanediol

Synthesis of cobalamin de novo by Salmonella enterica serovar Typhimurium strain LT2 and the absence of this ability in Escherichia coli present several problems. This large synthetic pathway is shared by virtually all salmonellae and must be maintained by selection, yet no conditions are known under which growth depends on endogenous B12. The cofactor is required for degradation of 1,2-propanediol and ethanolamine. However, cofactor synthesis occurs only anaerobically, and neither of these carbon sources supports anaerobic growth with any of the alternative electron acceptors tested thus far. This paradox is resolved by the electron acceptor tetrathionate, which allows Salmonella to grow anaerobically on ethanolamine or 1,2-propanediol by using endogenously synthesized B12. Tetrathionate provides the only known conditions under which simple cob mutants (unable to make B12) show a growth defect. Genes involved in this metabolism include the ttr operon, which encodes tetrathionate reductase. This operon is globally regulated by OxrA (Fnr) and induced anaerobically by a two-component system in response to tetrathionate. Salmonella reduces tetrathionate to thiosulfate, which it can further reduce to H2S, by using enzymes encoded by the genes phs and asr. The genes for 1,2-propanediol degradation (pdu) and B12 synthesis (cob), along with the genes for sulfur reduction (ttr, phs, and asr), constitute more than 1% of the Salmonella genome and are all absent from E. coli. In diverging from E. coli, Salmonella acquired some of these genes unilaterally and maintained others that are ancestral but have been lost from the E. coli lineage.     

Minimal functions and physiological conditions required for growth of salmonella enterica on ethanolamine in the absence of the metabolosome

During growth on ethanolamine, Salmonella enterica synthesizes a multimolecular structure that mimics the carboxysome used by some photosynthetic bacteria to fix CO(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(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.     

Studies of the CobA-type ATP:Co(I)rrinoid adenosyltransferase enzyme of Methanosarcina mazei strain Go1

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

Conserving a volatile metabolite: a role for carboxysome-like organelles in Salmonella enterica

Salmonellae can use ethanolamine (EA) as a sole source of carbon and nitrogen. This ability is encoded by an operon (eut) containing 17 genes, only 6 of which are required under standard conditions (37 degrees C; pH 7.0). Five of the extra genes (eutM, -N, -L, -K, and -G) become necessary under conditions that favor loss of the volatile intermediate, acetaldehyde, which escapes as a gas during growth on EA and is lost at a higher rate from these mutants. The eutM, -N, -L, and -K genes encode homologues of shell proteins of the carboxysome, an organelle shown (in other organisms) to concentrate CO(2). We propose that carboxysome-like organelles help bacteria conserve certain volatile metabolites-CO(2) or acetaldehyde-perhaps by providing a low-pH compartment. The EutG enzyme converts acetaldehyde to ethanol, which may improve carbon retention by forming acetals; alternatively, EutG may recycle NADH within the carboxysome.     

Isolation of vitamin B 12 transport mutants of Escherichia coli

Escherichia coli KBT001, a methionine-vitamin B(12) auxotroph, was found to require a minimum of 20 molecules of vitamin B(12) (CN-B(12)) per cell for aerobic growth in the absence of methionine. After mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine and penicillin selection, two kinds of B(12) transport mutant were isolated from this strain. Mutants of class I, such as KBT069, were defective in the initial rapid binding of CN-B(12) to the cell and were unable to grow in the absence of methionine even with CN-B(12) concentrations as high as 100 ng/ml. The class II mutants possessed intact initial phases of CN-B(12) uptake but were defective in the secondary energy-dependent phase. These strains were also unable to convert the CN-B(12) taken up into other cobalamins. In the absence of methionine, some of these strains (e.g., KBT103) were able to grow on media containing 1 ng of CN-B(12)/ml, whereas others (e.g., KBT041) were unable to grow with any of the CN-B(12) concentrations used. Osmotic shock treatment did not affect the initial rate of uptake of CN-B(12) but gave a substantial decrease in the secondary rate. Trace amounts of B(12)-binding macromolecules were released from the cells by the osmotic shock, but only from strains such as KBT001 and KBT041 which possessed an active initial phase of CN-B(12) uptake. These results are interpreted as being consistent with the view that the initial CN-B(12) binding site which functions in this transport system is probably bound to the cell membrane.     

Propanediol utilization genes (pdu) of Salmonella typhimurium: three genes for the propanediol dehydratase.

The propanediol utilization (pdu) operon of Salmonella typhimurium encodes proteins required for the catabolism of propanediol, including a coenzyme B12-dependent propanediol dehydratase. A clone that expresses propanediol dehydratase activity was isolated from a Salmonella genomic library. DNA sequence analysis showed that the clone included part of the pduF gene, the pduABCDE genes, and a long partial open reading frame (ORF1). The clone included 3.9 kbp of pdu DNA which had not been previously sequenced. Complementation and expression studies with subclones constructed via PCR showed that three genes (pduCDE) are necessary and sufficient for propanediol dehydratase activity. The function of ORF1 was not determined. Analyses showed that the S. typhimurium propanediol dehydratase was related to coenzyme B12-dependent glycerol dehydratases from Citrobacter freundii and Klebsiella pneumoniae. Unexpectedly, the S. typhimurium propanediol dehydratase was found to be 98% identical in amino acid sequence to the Klebsiella oxytoca propanediol dehydratase; this is a much higher identity than expected, given the relationship between these organisms. DNA sequence analyses also supported previous studies indicating that the pdu operon was inherited along with the adjacent cobalamin biosynthesis operon by a single horizontal gene transfer.     

Identification of the human and bovine ATP:Cob(I)alamin adenosyltransferase cDNAs based on complementation of a bacterial mutant

In humans, deficiencies in coenzyme B12-dependent methylmalonyl-CoA mutase (MCM) lead to methylmalonyl aciduria, a rare disease that is often fatal in newborns. Such deficiencies can result from inborn errors in the MCM structural gene or from mutations that impair the assimilation of dietary cobalamins into coenzyme B12 (Ado-B12), the required cofactor for MCM. ATP:cob(I)alamin adenosyltransferase (ATR) catalyzes the terminal step in the conversion of cobalamins into Ado-B12. Substantial evidence indicates that inherited defects in this enzyme lead to methylmalonyl aciduria, but the corresponding ATR gene has not been identified. Here we report the identification of the bovine and human ATR cDNAs as well as the corresponding human gene. A bovine liver cDNA expression library was screened for clones that complemented an ATR-deficient bacterial strain for color formation on aldehyde indicator medium, and four positive clones were isolated. The DNA sequences of two clones were determined and found to be identical. Sequence similarity searching was then used to identify a homologous human cDNA (89% identity) and its corresponding gene that is located on chromosome XII. The bovine and human cDNAs were independently cloned and expressed in Escherichia coli. Enzyme assays showed that expression strains produced 87 and 98 nmol/min/mg ATR activity, respectively. These specific activities are in line with values reported previously for bacterial ATR enzymes. Subsequent studies showed that the human cDNA clone complemented an ATR-deficient bacterial mutant for Ado-B12-dependent growth on 1,2-propanediol. This demonstrated that the human ATR is active under physiological conditions albeit in a heterologous host. In addition, Western blots were used to show that ATR expression is altered in cell lines derived from cblB methylmalonyl aciduria patients compared with cell lines from normal individuals. We propose that inborn errors in the human ATR gene identified here result in methylmalonyl aciduria. The identification of genes involved in this disorder will allow improvements in the diagnosis and treatment of this serious disease.     

Computer-assisted docking of flavodoxin with the ATP:Co(I)rrinoid adenosyltransferase (CobA) enzyme reveals residues critical for protein-protein interactions but not for catalysis

The activity of the housekeeping ATP:co(I)rrinoid adenosyltransferase (CobA) enzyme of Salmonella enterica sv. Typhimurium is required to adenosylate de novo biosynthetic intermediates of adenosylcobalamin and to salvage incomplete and complete corrinoids from the environment of this bacterium. In vitro, reduced flavodoxin (FldA) provides an electron to generate the co(I)rrinoid substrate in the CobA active site. To understand how CobA and FldA interact, a computer model of a CobA.FldA complex was generated. This model was used to guide the introduction of mutations into CobA using site-directed mutagenesis and the synthesis of a peptide mimic of FldA. Residues Arg-9 and Arg-165 of CobA were critical for FldA-dependent adenosylation but were catalytically as competent as the wild-type protein when cob(I)alamin was provided as substrate. These results indicate that Arg-9 and Arg-165 are important for CobA.FldA docking but not to catalysis. A truncation of the 9-amino acid N-terminal helix of CobA reduced its FldA-dependent cobalamin adenosyltransferase activity by 97.4%. The same protein, however, had a 4-fold higher specific activity than the native enzyme when cob(I)alamin was generated chemically in situ.     

A B12-responsive internal ribosome entry site (IRES) element in human methionine synthase

Regulation of homocysteine, a sulfur-containing amino acid that is a risk factor for cardiovascular diseases, is poorly understood. Methionine synthase (MS) is a key enzyme that clears intracellular homocysteine, and its activity is induced by its cofactor, vitamin B12, at a translational level. In this study, we demonstrate that translation of MS, which has a long and highly structured 5'-untranslated region, is initiated from an internal ribosome entry site (IRES), which is modulated by B12. The minimal IRES element spans 71 bases immediately upstream of the initiation codon. Electrophoretic mobility shift analysis reveals the presence of a B12 -dependent protein-RNA complex and suggests the possibility that B12-dependent increase of IRES efficiency is mediated via a protein. Modulation of the IRES-dependent translation of an essential gene by the cofactor of the encoded enzyme represents a novel example of a gene-nutrient interaction.     

Global regulation by CsrA in Salmonella typhimurium

CsrA is a regulator of invasion genes in Salmonella enterica serovar Typhimurium. To investigate the wider role of CsrA in gene regulation, we compared the expression of Salmonella genes in a csrA mutant with those in the wild type using a DNA microarray. As expected, we found that expression of Salmonella pathogenicity island 1 (SPI-1) invasion genes was greatly reduced in the csrA mutant, as were genes outside the island that encode proteins translocated into eukaryotic cells by the SPI-1 type III secretion apparatus. The flagellar synthesis operons, flg and fli, were also poorly expressed, and the csrA mutant was aflagellate and non-motile. The genes of two metabolic pathways likely to be used by Salmonella in the intestinal milieu also showed reduced expression: the pdu operon for utilization of 1,2-propanediol and the eut operon for ethanolamine catabolism. Reduced expression of reporter fusions in these two operons confirmed the microarray data. Moreover, csrA was found to regulate co-ordinately the cob operon for synthesis of vitamin B12, required for the metabolism of either 1,2-propanediol or ethanolamine. Additionally, the csrA mutant poorly expressed the genes of the mal operon, required for transport and use of maltose and maltodextrins, and had reduced amounts of maltoporin, normally a dominant protein of the outer membrane. These results show that csrA controls a number of gene classes in addition to those required for invasion, some of them unique to Salmonella, and suggests a co-ordinated bacterial response to conditions that exist at the site of bacterial invasion, the intestinal tract of a host animal.