New high-cloning-efficiency vectors for complementation studies and recombinant protein overproduction in Escherichia coli and Salmonella enterica.

Galloway et al. recently described a method to alter vectors to include Type IIS restriction enzymes for high efficiency cloning. Utilizing this method, the multiple cloning sites of complementation and overexpression vectors commonly used in our laboratory were altered to contain recognition sequences of the Type IIS restriction enzyme, BspQI. Use of this enzyme increased the rate of cloning success to >97% efficiency. L(+)-Arabinose-inducible complementation vectors and overexpression vectors encoding N-terminal recombinant tobacco etch virus protease (rTEV)-cleavable H6-tags were altered to contain BspQI sites that allowed for cloning into all vectors using identical primer overhangs. Additionally, a vector used for directing the synthesis of proteins with a C-terminal, rTEV-cleavable H6-tag was engineered to contain BspQI sites, albeit with different overhangs from that of the previously mentioned vectors. Here we apply a method used to engineer cloning vectors to contain BspQI sites and the use of each vector in either in vivo complementation studies or in vitro protein purifications.

Functional analysis of the nicotinate mononucleotide:5,6-dimethylbenzimidazole phosphoribosyltransferase (CobT) enzyme, involved in the late steps of coenzyme B12 biosynthesis in Salmonella enterica.

In Salmonella enterica, the CobT enzyme activates the lower ligand base during the assembly of the nucleotide loop of adenosylcobalamin (AdoCbl) and other cobamides. Previously, mutational analysis identified a class of alleles (class M) that failed to restore AdoCbl biosynthesis during intragenic complementation studies. To learn why class M cobT mutations were deleterious, we determined the nature of three class M cobT alleles and performed in vivo and in vitro functional analyses guided by available structural data on the wild-type CobT (CobT(WT)) enzyme. We analyzed the effects of the variants CobT(G257D), CobT(G171D), CobT(G320D), and CobT(C160A). The latter was not a class M variant but was of interest because of the potential role of a disulfide bond between residues C160 and C256 in CobT activity. Substitutions G171D, G257D, and G320D had profound negative effects on the catalytic efficiency of the enzyme. The C160A substitution rendered the enzyme fivefold less efficient than CobT(WT). The CobT(G320D) protein was unstable, and results of structure-guided site-directed mutagenesis suggest that either variants CobT(G257D) and CobT(G171D) have less affinity for 5,6-dimethylbenzimidazole (DMB) or access of DMB to the active site is restricted in these variant proteins. The reported lack of intragenic complementation among class M cobT alleles is caused in some cases by unstable proteins, and in others it may be caused by the formation of dimers between two mutant CobT proteins with residual activity that is so low that the resulting CobT dimer cannot synthesize sufficient product to keep up with even the lowest demand for AdoCbl.

A link between transcription and intermediary metabolism: a role for Sir2 in the control of acetyl-coenzyme A synthetase.

The silent information regulator protein (Sir2) and its homologs (collectively known as sirtuins) are NAD+-dependent deacetylase enzymes involved in chromosome stability, gene silencing and cell aging in eukaryotes and archaea. The discovery that sirtuin-dependent protein deacetylation is a NAD+-consuming reaction established a link with the energy generation systems of the cell. This link to metabolism was recently extended to the post-translational control of the activity of short-chain fatty acyl-coenzyme A (adenosine monophosphate-forming) synthetases in bacteria and yeast. The crystal structure of the Sir protein complexed with a peptide of a protein substrate provided insights into how sirtuins interact with their protein substrates.

Cloning, sequencing and overexpression of cobA which encodes ATP:corrinoid adenosyltransferase in Salmonella typhimurium.

The cobA gene of Salmonella typhimurium was cloned, sequenced and overexpressed. A 990-bp HpaI-SacI fragment was cloned into the multiple cloning site of plasmid pSU19, an intermediate-copy-number vector. DNA sequence analysis established that cobA is 588 bp in length and codes for a protein with a predicted molecular weight of 21.7 kDa. However, the CobA protein expressed from the T7 promoter migrated as a 25-kDa protein on SDS-polyacrylamide gels. A high degree of identity at the amino acid sequence level was established between the CobA, Pseudomonas denitrificans CobO and Escherichia coli BtuR proteins. P. denitrificans CobO has been shown to be a ATP:corrinoid adenosyltransferase enzyme. Based on the similarities between CobO and CobA, and the phenotypes of cobA mutants, we suggest that CobA is the ATP:corrinoid adenosyltransferase of S. typhimurium.

High levels of transcription factor RpoS (sigma S) in mviA mutants negatively affect 1,2-propanediol-dependent transcription of the cob/pdu regulon of Salmonella typhimurium LT2.

Expression of the cobalamin biosynthetic (cob) and 1,2-propanediol utilization (cob/pdu) regulon of Salmonella typhimurium LT2 is controlled at the transcriptional level by global and specific regulatory proteins. In this paper we show that mutations in the mviA gene negatively affect cob/pdu transcription in response to 1,2-propanediol in the environment. The effects of mviA mutations were consistent with its role in the regulation of RpoS levels in the cell. Null mutations in rpoS eliminated the negative effect of mviA mutations on cob/pdu transcription, and restored growth on succinate, propionate and 1,2-propanediol. In addition, mviA mutants were deficient in the utilization of succinate, propionate and 1,2-propanediol as carbon and energy sources.

Identification of a new prp locus required for propionate catabolism in Salmonella typhimurium LT2.

A new propionate (prp) locus of S. typhimurium was defined by mutation, was located to minute 8 of the chromosome, and was shown to be transcribed in the clockwise direction. A plasmid carrying the wild-type prp+ locus was isolated by complementation and its initial physical characterization is presented. Transcriptional regulation of prp was studied using MudI1734(lacZ+) operon fusions. Propionate stimulated prp transcription in a merodiploid strain containing prp+ and a prp::MudI1734 fusion, but failed to stimulate transcription of the same fusion in a haploid genetic background. prp transcription was reduced by a factor of 2 in strains deficient in the synthesis of the global regulatory protein FruR; fruR mutants failed to grow on propionate. Propionate blocked growth of prp mutants on medium containing succinate as carbon/energy source.

Construction and use of new cloning vectors for the rapid isolation of recombinant proteins from Escherichia coli.

We describe the construction and use of two sets of vectors for the over-expression and purification of protein from Escherichia coli. The set of pTEV plasmids (pTEV3, 4, 5) directs the synthesis of a recombinant protein with a N-terminal hexahistidine (His(6)) tag that is removable by the tobacco etch virus (TEV) protease. The set of pKLD plasmids (pKLD66, 116) directs the synthesis of a recombinant protein that contains a N-terminal His(6) and maltose-binding protein tag in tandem, which can also be removed with TEV protease. The usefulness of these plasmids is illustrated by the rapid, high-yield purification of the 2-methylcitrate dehydratase (PrpD) protein of Salmonella enterica, and the 2-methylaconitate isomerase (PrpF) protein of Shewanella oneidensis, two enzymes involved in the catabolism of propionate to pyruvate via the 2-methylcitric acid cycle.

Purification and initial characterization of the ATP:corrinoid adenosyltransferase encoded by the cobA gene of Salmonella typhimurium.

The cobA gene of Salmonella typhimurium and its product were overexpressed to approximately 20% of the total cell protein. CobA was purified to 98% homogeneity; N-terminal sequence analysis (21 residues) of homogeneous protein confirmed the predicted amino acid sequence. ATP:corrinoid adenosyltransferase activity was demonstrated in vitro to be associated with CobA. This activity was optimal at pH 8 and 37 degrees C. A quantitative preference was determined for Mn(II) cations and ATP. The apparent Km of CobA for ATP was 2.8 microM, and that for cob(I)alamin was 5.2 microM. Vmax was measured at 0.43 nmol/min. Cobinamide served as the substrate for CobA to yield adenosylcobinamide. Activity was stable at 4 degrees C for several weeks but was lost rapidly at room temperature (50% overnight). Dithiothreitol was required to maintain the enzymatic activity of CobA.

prpR, ntrA, and ihf functions are required for expression of the prpBCDE operon, encoding enzymes that catabolize propionate in Salmonella enterica serovar typhimurium LT2.

The genes required for the catabolism of propionate in Salmonella enterica serovar Typhimurium are organized as two transcriptional units (prpR and prpBCDE) that are divergently transcribed from one another. Sequence homology to genes encoding members of the sigma-54 family of transcriptional activators and the identification of a consensus sigma-54 promoter 5' to the prpBCDE operon suggested that PrpR was required to activate expression of this operon. We isolated insertions in prpR and showed that prpR function was needed for growth on propionate as a carbon and energy source. A medium-copy-number plasmid carrying the lacZ gene under the control of the native sigma-54 promoter of prpBCDE was used to study prpBCDE operon expression. Transcription of the lacZ reporter in prpR, ntrA, and ihfB mutants was 85-, 83-, and 15-fold lower than the level of transcription measured in strains carrying the wild-type allele of the gene tested. These data indicated that PrpR, IHF, and transcription sigma factor RpoN were required for the expression of the prpBCDE operon. Further analysis of the involvement of the integration host factor (IHF) protein in the expression of this operon is required due to the well-documented pleiotropic effect the lack of this global regulator has on gene expression. Deletion of the 5' 615-bp portion of the prpR gene resulted in a PrpR(c) mutant protein that activated prpBCDE transcription regardless of the ability of the strain to synthesize 2-methylcitrate, the putative coactivator of PrpR. These results indicate that the N terminus of PrpR is the coactivator-sensing domain of the protein. When placed under the control of the arabinose-inducible promoter P(araBAD), expression of prpR(c) allele by arabinose had a strong negative effect on growth of the cell. It is proposed that this deleterious effect of PrpR(c) may be due to an uncontrolled ATPase activity of PrpR or to cross-activation of genes whose functions negatively affect cell growth under the conditions tested.

cobU-dependent assimilation of nonadenosylated cobinamide in cobA mutants of Salmonella typhimurium.

The cobA locus of Salmonella typhimurium is involved in the assimilation of nonadenosylated cobinamide, (CN)2CBI, into cobalamin (CBL) under aerobic and anaerobic growth conditions. Aerobically, cobA mutants are unable to assimilate (CN)2CBI into CBL. However, under anaerobic conditions, cobA mutants assimilate (CN)2CBI into CBL as efficiently as cobA+ strains. On the basis of this observation, we postulated the existence of a cobA-independent pathway for the assimilation of (CN)2CBI into CBL that is functional under anaerobic growth conditions (J. C. Escalante-Semerena, S.-J. Suh, and J. R. Roth, J. Bacteriol. 172:273-280, 1990). In this paper, we report the isolation and initial genetic characterization of derivatives of cobA mutants that are unable to assimilate (CN)2CBI into CBL during anaerobic growth. As demonstrated by complementation analysis, marker rescue, and DNA sequencing data, these mutations are alleles of cobU, a gene involved in the assembly of the nucleotide loop of CBL. We have shown that the block in CBL synthesis in these cobU cobA double mutant strains can be corrected by exogenous adenosyl-CBI. Our data indicate that this new class of cobU mutations blocks CBL biosynthesis but does not destroy the putative kinase-guanylyltransferase activities of the CobU protein. We propose that this new class of cobU mutations may affect an as yet unidentified ATP:corrinoid adenosyltransferase activity of the CobU protein. Alternatively, such mutations may alter the ability of CobU to use nonadenosylated CBI as a substrate.

Identification and initial characterization of the eutF locus of Salmonella typhimurium.

We report the isolation and initial characterization of mutations in the newly described eutF locus of Salmonella typhimurium LT2. Mutations in eutF render a strain unable to utilize ethanolamine as a source of carbon and/or energy and impair growth on ethanolamine as a sole nitrogen source. Strains carrying eutF mutations exhibit a 2-order-of-magnitude decrease in transcription of the unlinked eutDEABCR operon (50 min), which codes for the enzymes needed to catabolize ethanolamine; have only 10% of the ethanolamine ammonia-lyase activity found in the wild type; and show a marked reduction in the rate of ethanolamine uptake. Deletion mapping and three-factor cross analysis results are consistent with the gene order cobA trp eutF tonB at 34 min on the linkage map. We discuss two possible roles for the EutF protein: (i) as an ethanolamine permease or (ii) as a transcription factor required for the expression of the eutDEABCR operon.

The poc locus is required for 1,2-propanediol-dependent transcription of the cobalamin biosynthetic (cob) and propanediol utilization (pdu) genes of Salmonella typhimurium.

In this communication we present evidence that indicates that 1,2-propanediol (1,2-PDL) is a positive effector of the transcription of the cobalamin (vitamin B12) biosynthetic (cob) operon and of the 1,2-PDL utilization (pdu) genes. The stimulatory effects of 1,2-PDL were demonstrated using Mu d-lac transcriptional fusions to cob and pdu. Significantly increased levels of transcription of the cob and pdu operon fusions were measured in cultures grown under both anoxic and highly aerated conditions when 1,2-PDL was present in the culture medium. We have isolated mutants that carry lesions that render both pdu and cob transcription unresponsive to 1,2-PDL. These mutations were mapped to the region between cob and pdu (41 min), and they define the poc locus PDL and cobalamin). The poc locus is required for the positive regulatory effects of 1,2-PDL to be exerted. Complementation of Poc function was achieved in trans by an F' plasmid carrying the poc+ locus, suggesting that at least one diffusible product regulates the expression of cob and pdu in S. typhimurium.

In vitro synthesis of the nucleotide loop of cobalamin by Salmonella typhimurium enzymes.

In Salmonella typhimurium, the CobU, CobS, CobT, and CobC proteins have been proposed to catalyze the late steps in adenosylcobalamin biosynthesis, which define the nucleotide loop assembly pathway. This paper reports the in vitro assembly of the nucleotide loop of adenosylcobalamin from its precursors adenosylcobinamide, 5, 6-dimethylbenzimidazole, nicotinate mononucleotide, and GTP. Incubation of these precursors with the CobU, CobS, and CobT proteins resulted in the synthesis of adenosylcobalamin-5'-phosphate. This cobamide was isolated by HPLC, identified by UV-visible spectroscopy and mass spectrometry, and shown to support growth of a cobalamin auxotroph. Adenosylcobalamin-5'-phosphate was also isolated from reaction mixtures containing adenosylcobinamide-GDP (the product of the CobU reaction) and alpha-ribazole-5'-phosphate (the product of the CobT reaction) as substrates and CobS. These results allowed us to conclude that CobS is the cobalamin(-5'-phosphate) synthase enzyme in S. typhimurium. The CobC enzyme, previously shown to dephosphorylate alpha-ribazole-5'-phosphate to alpha-ribazole, was shown to dephosphorylate adenosylcobalamin-5'-phosphate to adenosylcobalamin. Adenosylcobinamide was converted to adenosylcobalamin in reactions where all four enzymes were present in the reaction mixture. This in vitro system offers a unique opportunity for the rapid synthesis and isolation of cobamides with structurally different lower-ligand bases that can be used to investigate the contributions of the lower-ligand base to cobalamin-dependent reactions.

Salmonella typhimurium LT2 catabolizes propionate via the 2-methylcitric acid cycle.

We previously identified the prpBCDE operon, which encodes catabolic functions required for propionate catabolism in Salmonella typhimurium. Results from (13)C-labeling experiments have identified the route of propionate breakdown and determined the biochemical role of each Prp enzyme in this pathway. The identification of catabolites accumulating in wild-type and mutant strains was consistent with propionate breakdown through the 2-methylcitric acid cycle. Our experiments demonstrate that the alpha-carbon of propionate is oxidized to yield pyruvate. The reactions are catalyzed by propionyl coenzyme A (propionyl-CoA) synthetase (PrpE), 2-methylcitrate synthase (PrpC), 2-methylcitrate dehydratase (probably PrpD), 2-methylisocitrate hydratase (probably PrpD), and 2-methylisocitrate lyase (PrpB). In support of this conclusion, the PrpC enzyme was purified to homogeneity and shown to have 2-methylcitrate synthase activity in vitro. (1)H nuclear magnetic resonance spectroscopy and negative-ion electrospray ionization mass spectrometry identified 2-methylcitrate as the product of the PrpC reaction. Although PrpC could use acetyl-CoA as a substrate to synthesize citrate, kinetic analysis demonstrated that propionyl-CoA is the preferred substrate.

Integration host factor is required for 1,2-propanediol-dependent transcription of the cob/pdu regulon in Salmonella typhimurium LT2.

We show that integration host factor (IHF) is required for the activation of transcription of the cobalamin biosynthetic (cob) and 1,2-propanediol (1,2-PDL) utilization (pdu) operons in Salmonella typhimurium LT2. A lack of IHF affected transcription of the cob/pdu regulon in at least two ways. First, the level of the regulatory protein PocR was decreased in ihfB (formerly himD) mutants, as judged by Western blot analysis with polyclonal antiserum raised against PocR. Second, even when PocR was available, in the absence of IHF, PocR was unable to activate transcription of cob/pdu in response to 1,2-PDL. This result suggested an additional role for IHF in PocR-dependent transcription activation. Consistent with these findings, ihfB mutants of this bacterium were unable to use 1,2-PDL as a carbon or energy source.

Purification and characterization of CobT, the nicotinate-mononucleotide:5,6-dimethylbenzimidazole phosphoribosyltransferase enzyme from Salmonella typhimurium LT2.

We report the purification and biochemical characterization of the cobalamin biosynthetic enzyme nicotinate-mononucleotide:5, 6-dimethylbenzimidazole phosphoribosyltransferase (CobT) from Salmonella typhimurium. cobT was overexpressed and the protein purified to approximately 97% homogeneity. NH2-terminal sequence analysis confirmed that the protein encoded by cobT was purified. Homogeneous CobT catalyzed the synthesis of N1-(5-phospho-alpha-D-ribosyl)-5,6-dimethylbenzimidazole. The identity of high performance liquid chromatography-purified product was confirmed by fast atom bombardment mass spectrometry. CobT activity was optimal at 45 degrees C and pH 10.0. The apparent Km for nicotinate mononucleotide was 680 microM; the apparent Km for 5, 6-dimethylbenzimidazole was less than 10 microM. CobT used nicotinamide mononucleotide as a ribose phosphate donor. CobT phosphoribosylated alternative base substrates including benzimidazole, 4,5-dimethyl-1,2-phenylenediamine, imidazole, histidine, adenine, and guanine in vitro. The resulting ribotides were incorporated into cobamides that were differentially utilized by methionine synthase (EC 2.1.1.13), ethanolamine ammonia-lyase (EC 4.3.1.7), and 1,2-propanediol dehydratase (EC 4.2.1.28) in vivo. The lack of base substrate specificity by CobT may explain the inability to isolate mutants blocked in the synthesis of 5, 6-dimethylbenzimidazole in this bacterium.

Tetrahydromethanopterin-dependent methanogenesis from non-physiological C1 donors in Methanobacterium thermoautotrophicum.

Methanogenesis from the non-physiological C1 donors thioproline, thiazolidine, hexamethylenetetramine, formaldehyde (HCHO), and HOCH2-S-coenzyme M (CoM) was catalyzed by cell extracts of Methanobacterium thermoautotrophicum under a hydrogen atmosphere. Tetrahydromethanopterin (H4MPT) and HS-CoM were required in the reaction mixture. The non-physiological compounds were found to be in chemical equilibrium with HCHO, which has been shown to react spontaneously with H4MPT to form methylene-H4MPT, an intermediate of the methanogenic pathway at the formaldehyde level of oxidation. Highfield (360 MHZ) 1H and 13C nuclear magnetic resonance studies performed on the interaction between HCHO and HS-CoM showed that these compounds are in equilibrium with HOCH2-S-CoM and that the equilibrium is pH dependent. When methanogenesis from the non-physiological donors was followed under a nitrogen atmosphere, the C1 moiety from each compound underwent a disproportionation, forming methenyl-H4MPT+ and methane. The compounds tested served as substrates for the enzymatic synthesis of methenyl-H4MPT+.

CobB, a new member of the SIR2 family of eucaryotic regulatory proteins, is required to compensate for the lack of nicotinate mononucleotide:5,6-dimethylbenzimidazole phosphoribosyltransferase activity in cobT mutants during cobalamin biosynthesis in Salmonella typhimurium LT2.

The cobB gene of Salmonella typhimurium LT2 has been isolated and genetically and biochemically characterized. cobB was located by genetic means to the 27-centisome region of the chromosome. Genetic crosses established the gene order to be cobB pepT phoQ, and the direction of cobB transcription was shown to be clockwise. The nucleotide sequence of cobB (711 base pairs) predicted a protein of 237 amino acids length with a molecular mass of 26.3 kDa, a mass consistent with the experimentally determined one of approximately 28 kDa. The cobB gene was defined genetically by deletions (10), insertions (5), and point mutations (15). The precise location of a Tn10d(Tc) element within cobB was established by sequencing. DNA sequence analysis of the region flanking cobB located it 81 base pairs 3' of the potABCD operon, with the potABCD operon and cobB being divergently transcribed. cobB was overexpressed to approximately 30% of the total soluble protein using a T7 overexpression system. In vitro activity assays showed that cell-free extracts enriched for CobB catalyzed the synthesis of the cobalamin biosynthetic intermediate N1-(5-phospho-alpha-D-ribosyl)-5, 6-dimethylbenzimidazole (also known as alpha-ribazole-5'-phosphate) from nicotinate mononucleotide and 5,6-dimethylbenzimidazole, the reaction known to be catalyzed by the CobT phosphoribosyltransferase enzyme (EC 2.4.2.21) (Trzebiatowski, J. R. and Escalante-Semerena, J. C. (1997) J. Biol. Chem. 272, 17662-17667). Computer analysis of the primary amino acid sequence of the CobB protein identified the sequences GAGISAESGIRTFR and YTQNID which are diagnostic of members of the SIR2 family of eucaryotic transcriptional regulators. Possible roles of CobB as a regulator are discussed within the context of the catabolism of propionate, a pathway known to require cobB function (Tsang, A. W. and Escalante-Semerena, J. C. (1996) J. Bacteriol. 178, 7016-7019).

Glutathione is required for maximal transcription of the cobalamin biosynthetic and 1,2-propanediol utilization (cob/pdu) regulon and for the catabolism of ethanolamine, 1,2-propanediol, and propionate in Salmonella typhimurium LT2.

Transcription of the cob/pdu regulon of Salmonella typhimurium is activated by the PocR regulatory protein in response to 1,2-propanediol (1,2-PDL) in the environment. Nutritional analysis and DNA sequencing confirmed that a strain defective in expression of the cob/pdu regulon in response to 1,2-PDL lacked a functional gshA gene. gshA encodes gamma-glutamylcysteine synthetase (L-glutamate:L-cysteine gamma-ligase [ADP forming]; EC 6.3.2.2), the enzyme that catalyzes the first step in the synthesis of glutathione (GSH). The DNA sequence of gshA was partially determined, and the location of gshA in the chromosome was established by two-factor crosses. P22 cotransduction of gshA with nearby markers showed 21% linkage to srl and 1% linkage to hyd; srl was 9% cotransducible with hyd. In light of these data, the gene order gshA srl hyd is suggested. The level of reduced thiols in the gshA strain was 87% lower than the levels measured in the wild-type strain in both aerobically and anaerobically grown cells. 1,2-PDL-dependent transcription of cob/pdu was studied by using M. Casadaban's Mu-lacZ fusions. In aerobically grown cells, transcription of a cbi-lacZ fusion (the cbi genes are the subset of cob genes that encode functions needed for the synthesis of the corrin ring) was 4-fold lower and transcription of a pdu-lacZ fusion was 10-fold lower in a gshA mutant than in the wild-type strain. Expression of the cob/pdu regulon in response to 1,2-PDL was restored when GSH was included in the medium. In anaerobically grown cells, cbi-lacZ transcription was only 0.4-fold lower than in the gshA+ strain; pdu-lacZ transcription was reduced only by 0.34-fold, despite the lower thiol levels in the mutant. cobA-lacZ transcription was used as negative control of gene whose transcription is not controlled by the PocR/1,2-PDL system; under both conditions, cobA transcription remained unaffected. The gshA mutant strain was unable to utilize 1,2-PDL, ethanolamine, or propionate as a carbon and energy source. The defect in ethanolamine utilization appears to be at the level of ethanolamine ammonia-lyase activity, not at the transcriptional level. Possible roles for GSH in ethanolamine, 1,2-PDL, and propionate catabolism are proposed and discussed.