Bacterial cancer therapy in autochthonous colorectal cancer affects tumor growth and metabolic landscape.

Bacterial cancer therapy (BCT) shows great promise for treatment of solid tumors, yet basic mechanisms of bacterial-induced tumor suppression remain undefined. Attenuated strains of Salmonella enterica serovar Typhimurium (STm) have commonly been used in mouse models of BCT in xenograft and orthotopic transplant cancer models. We aimed to better understand the tumor epithelium-targeted mechanisms of BCT by using autochthonous mouse models of intestinal cancer and tumor organoid cultures to assess the effectiveness and consequences of oral treatment with aromatase A-deficient STm (STmΔaroA). STmΔaroA delivered by oral gavage significantly reduced tumor burden and tumor load in both a colitis-associated colorectal cancer (CAC) model and in a spontaneous Apcmin/+ intestinal cancer model. STmΔaroA colonization of tumors caused alterations in transcription of mRNAs associated with tumor stemness, epithelial-mesenchymal transition, and cell cycle. Metabolomic analysis of tumors demonstrated alteration in the metabolic environment of STmΔaroA-treated tumors, suggesting that STmΔaroA imposes metabolic competition on the tumor. Use of tumor organoid cultures in vitro recapitulated effects seen on tumor stemness, mesenchymal markers, and altered metabolome. Furthermore, live STmΔaroA was required, demonstrating active mechanisms including metabolite usage. We have demonstrated that oral BCT is efficacious in autochthonous intestinal cancer models, that BCT imposes metabolic competition, and that BCT has direct effects on the tumor epithelium affecting tumor stem cells.

Syzygium samarangense Volatile Oil Inhibited Bacteria Growth and Extracellular Protease of Salmonella typhimurium.

BACKGROUND AND OBJECTIVE: Medicinal plants are fast becoming essential pharmaceuticals for disease and infection management. The vast antimicrobial properties of these plants reside in the inhibitory properties of their endogenous secondary metabolites. Therefore, this study aimed to assess if the volatile oil of Syzygium samarangense inhibits enteric bacteria growth and its effect against the caseinolytic activity of the extracellular protease of Salmonella typhimurium. MATERIALS AND METHODS: The volatile oil was extracted by hydrodistillation, while the antimicrobial assay was assessed with the microdilution method. The extracellular protease was partially purified by salting out, followed by size-exclusion chromatography. The mode of inhibition of this enzyme was deduced from the enzyme-substrate kinetics using a line-weaver burke plot. RESULTS: The antimicrobial properties of the oil were reported against ten enteric bacteria. Proteus vulgaris has the highest IC50 value of 0.75±0.004% v/v, while S. typhimurium, the most sensitive bacterium, showed the lowest IC50 value of 0.17±0.005% v/v. The extracellular protease of S. typhimurium was partially purified to achieve 3.73 purification fold and 314.2 µmol min-1 mg-1 protein. The optimal caseinolytic activity of this enzyme was found at pH 7.5 and 40 °C. The protease showed significantly higher activity in the presence of Zn2+ (9.3±0.33 U min-1) as compared to the control (7.0±0.58 U min-1) (p<0.05), however, K+, Ca2+, Co2+, Ba2+, Pb2+ and Hg2+ considerably reduced the enzyme activity. The activity of this enzyme was competitively inhibited by the volatile oil as an inhibitor. CONCLUSION: The volatile oil of S. samarangense inhibited a wide range of enteric bacteria and, therefore proposed as a potential antimicrobial agent. Inhibiting the extracellular protease of S. typhimurium may be one of its modes of action against these pathogens.

Structure-Based Virtual Screening of LsrK Kinase Inhibitors to Target Quorum Sensing.

In the era of increased antibiotic resistance, targeting enzymes involved in bacterial communication (quorum sensing) represents a new strategy to fight bacterial infections. LsrK is a kinase responsible for the phosphorylation of autoinducer-2, a signaling molecule involved in quorum sensing. Inhibiting LsrK would lead to quorum sensing inactivation and interfere with the pathogenesis. In this study, we built the first LsrK 3D model and performed virtual screening of a locally available database. Selected compounds were tested against LsrK, and the analogue search conducted based on the positive hits led to the identification of low-micromolar LsrK inhibitors. These results prove the utility of the model and provide the first class of LsrK inhibitors to be further optimized as antivirulence agents.

Drug repurposing: An approach to tackle drug resistance in S. typhimurium.

Drug resistant S. typhimurium pose important public health problem. The development of effective drugs with novel mechanism(s) of action is needed to overcome issues pertaining to drug resistance. Drug repurposing based on computational analyses is considered a viable alternative strategy to circumvent this issue. In this context, 1309 FDA-approved drugs molecules from Mantra 2.0 database were analyzed for this study, against S. typhimurium. Sixteen compounds having similar profiles of gene expression as quinolones were identified from the database, Mantra 2.0. Further, the pharmacophore characteristics of each resultant molecule were identified and compared with the features of nalidixic acid, using the PharamGist program. Subsequently, the activities of these compounds against S. typhimurium DNA gyrase were identified, using molecular docking study. Side effects analysis was also performed for the identified compounds. Molecular dynamics simulation was carried out for the compound to validate its binding efficiency. Further, characterization of screened compound revealed IC50 values in micromolar concentration range, of which flufenamic acid showed comparable in vitro activity alongside ciprofloxacin and nalidixic acid. Thus represent interesting starting points for further optimization against S. typhimurium infections. It may be noted that the results we have obtained are the first experimental evidence of flufenamic acid activity against S. typhimurium.

Chemical rescue of the post-translationally carboxylated lysine mutant of allantoinase and dihydroorotase by metal ions and short-chain carboxylic acids.

Bacterial allantoinase (ALLase) and dihydroorotase (DHOase) are members of the cyclic amidohydrolase family. ALLase and DHOase possess similar binuclear metal centers in the active site in which two metals are bridged by a post-translationally carboxylated lysine. In this study, we determined the effects of carboxylated lysine and metal binding on the activities of ALLase and DHOase. Although DHOase is a metalloenzyme, purified DHOase showed high activity without additional metal supplementation in a reaction mixture or bacterial culture. However, unlike DHOase, ALLase had no activity unless some specific metal ions were added to the reaction mixture or culture. Substituting the metal binding sites H59, H61, K146, H186, H242, or D315 with alanine completely abolished the activity of ALLase. However, the K146C, K146D and K146E mutants of ALLase were still active with about 1-6% activity of the wild-type enzyme. These ALLase K146 mutants were found to have 1.4-1.7 mol metal per mole enzyme subunit, which may indicate that they still contained the binuclear metal center in the active site. The activity of the K146A mutant of the ALLase and the K103A mutant of DHOase can be chemically rescued by short-chain carboxylic acids, such as acetic, propionic, and butyric acids, but not by ethanol, propan-1-ol, and imidazole, in the presence of Co2+ or Mn2+ ions. However, the activity was still ~10-fold less than that of wild-type ALLase. Overall, these results indicated that the 20 natural basic amino acid residues were not sufficiently able to play the role of lysine. Accordingly, we proposed that during evolution, the post-translational modification of carboxylated lysine in the cyclic amidohydrolase family was selected for promoting binuclear metal center self-assembly and increasing the nucleophilicity of the hydroxide at the active site for enzyme catalysis. This kind of chemical rescue combined with site-directed mutagenesis may also be used to identify a binuclear metal center in the active site for other metalloenzymes.

The copper supply pathway to a Salmonella Cu,Zn-superoxide dismutase (SodCII) involves P(1B)-type ATPase copper efflux and periplasmic CueP.

Periplasmic Cu,Zn-superoxide dismutases (Cu,Zn-SODs) are implicated in bacterial virulence. It has been proposed that some bacterial P(1B)-type ATPases supply copper to periplasmic cupro-proteins and such transporters have also been implicated in virulence. Here we show that either of two P(1B)-type ATPases, CopA or GolT, is needed to activate a periplasmic Cu,Zn-SOD (SodCII) in Salmonella enterica serovar Typhimurium. A ΔcopA/ΔgolT mutant accumulates inactive Zn-SodCII which can be activated by copper-supplementation in vitro. In contrast, either single ATPase mutant accumulates fully active Cu,Zn-SodCII. A contribution of GolT to copper handling is consistent with its copper-responsive transcription mediated by DNA-binding metal-responsive activator GolS. The requirement for duplicate transcriptional activators CueR and GolS remains unclear since both have similar tight K(Cu). Mutants lacking periplasmic cupro-protein CueP also accumulate inactive Zn-SodCII and while CopA and GolT show functional redundancy, both require CueP to activate SodCII in vivo. Zn-SodCII is also activated in vitro by incubation with Cu-CueP and this coincides with copper transfer as monitored by electron paramagnetic resonance spectroscopy. These experiments establish a role for CueP within the copper supply pathway for Salmonella Cu,Zn-SodCII. Copper binding by CueP in this pathogen may confer protection of the periplasm from copper-mediated damage while sustaining vital cupro-enzyme activity.

Functional analysis of the genes encoding diaminopropionate ammonia lyase in Escherichia coli and Salmonella enterica serovar Typhimurium.

Diaminopropionate ammonia lyase (DAPAL) is a pyridoxal-5'phosphate (PLP)-dependent enzyme that catalyzes the conversion of diaminopropionate (DAP) to pyruvate and ammonia and plays an important role in cell metabolism. We have investigated the role of the ygeX gene of Escherichia coli K-12 and its ortholog, STM1002, in Salmonella enterica serovar Typhimurium LT2, presumed to encode DAPAL, in the growth kinetics of the bacteria. While Salmonella Typhimurium LT2 could grow on dl-DAP as a sole carbon source, the wild-type E. coli K-12 strain exhibited only marginal growth on dl-DAP, suggesting that DAPAL is functional in S. Typhimurium. The expression of ygeX in E. coli was low as detected by reverse transcriptase PCR (RT-PCR), consistent with the poor growth of E. coli on dl-DAP. Strains of S. Typhimurium and E. coli with STM1002 and ygeX, respectively, deleted showed loss of growth on dl-DAP, confirming that STM1002 (ygeX) is the locus encoding DAPAL. Interestingly, the presence of dl-DAP caused a growth inhibition of the wild-type E. coli strain as well as the knockout strains of S. Typhimurium and E. coli in minimal glucose/glycerol medium. Inhibition by dl-DAP was rescued by transforming the strains with plasmids containing the STM1002 (ygeX) gene encoding DAPAL or supplementing the medium with Casamino Acids. Growth restoration studies using media lacking specific amino acid supplements suggested that growth inhibition by dl-DAP in the absence of DAPAL is associated with auxotrophy related to the inhibition of the enzymes involved in the biosynthetic pathways of pyruvate and aspartate and the amino acids derived from them.

Thymus maroccanus essential oil, a membranotropic compound active on Gram-negative bacteria and resistant isolates.

AIMS: The effects of Thymus maroccanus essential oil (EO) on the integrity of the cell membranes and the permeability of the outer membrane (OM) and inner membrane (IM) of Escherichia coli, Enterobacter aerogenes and Salmonella enterica Typhimurium were investigated. METHODS AND RESULTS: The bacterial release of intracellular proteins, cytoplasmic ß-galactosidase and periplasmic ß-lactamase induced by T. maroccanus EO was compared to the membranotropic activity of polymyxin B (PB) known as an effective permeabilizer of the membrane of Gram-negative bacteria. Results showed that T. maroccanus EO increased the permeability of the OM and IM of studied bacteria and induced the release of intracellular proteins into the external medium. CONCLUSIONS: The effect of T. maroccanus EO on the outer membrane was comparable to that of PB, and both T. maroccanus EO and PB induce similar levels of ß-lactamase release. In addition, it also promoted the release of the cytoplasmic ß-galactosidase. Moreover, the lipopolysaccharide molecules and the overexpression of efflux pumps seem to play a crucial role in the level of susceptibility of studied bacteria to the permeabilizing effect of T. maroccanus EO. SIGNIFICANCE AND IMPACT OF STUDY: These results demonstrate that T. maroccanus EO can restore antibiotic activity by targeting the two bacterial membranes and would be attractive candidates for developing new adjuvants for combating resistant Gram-negative bacteria.

Arginine and lysine decarboxylases and the acid tolerance response of Salmonella Typhimurium.

Salmonella Typhimurium CECT 443 inactivation at pH 2.5 in Mineral Medium (MM) and MM supplemented with 0.01% (w/v) arginine, lysine or glutamic acid was studied using stationary-phase cells grown in buffered BHI pH 7.0 (non-acid adapted cells) and acidified BHI up to pH 4.5 with acetic, citric, lactic and hydrochloric acids (acid adapted cells). In all cases, acid adapted cells, with D-values ranging from 23.34 to 86.90 min, showed a significantly higher acid resistance than non-acid adapted cells, with D-values between 8.90 and 10.29 min. Whereas the conditions used for acid adaptation did not exert a significant effect on the acid resistance of the S. Typhimurium CECT 443 resulting cells, the inclusion of lysine and arginine in the challenge medium protected them against acid inactivation, reaching D-values of about 2 and 3 times higher, respectively, than those found in MM or MM supplemented with glutamic acid. None of these three amino acids significantly modified the acid resistance of non-acid adapted cells. The relative expression level of adiA (encoding the arginine decarboxylase), adiY (encoding the transcriptional activator of adiA), cadA (encoding the lysine decarboxylase) and cadB (encoding the lysine/cadaverine transport protein) was examined by quantitative PCR. Acid adapted cells showed higher relative expression levels for both systems, arginine decarboxylase and lysine decarboxylase, which demonstrates that the induction of specialized pH-homeostatic systems plays an important role in S. Typhimurium CECT 443 protection against acid stress. However, the increased acid resistance showed by acid adapted cells challenged in MM arginine or lysine free suggests the existence of other microbial survival strategies.

Comparative mutagenic effects of structurally similar flavonoids quercetin and taxifolin on tester strains Salmonella typhimurium TA102 and Escherichia coli WP-2 uvrA.

Quercetin (QT) and Taxifolin (TF) are structurally similar plant polyphenols. Both have been reported to have therapeutic potential as anti-cancer drugs and antioxidants. Mutagenic effects of QT and TF were evaluated using Salmonella typhimurium TA102 and Escherichia coli WP-2 uvrA tester strains. Either in the presence or absence of S9 mix, QT was mutagenic to TA102 and WP2 uvrA. However, the mutagenicity of QT was significantly enhanced in the presence of S9 mix. Likewise, in the presence of Iron (Fe2+) and NADPH generating system (NGS) and absence of S9 mix, QT induced significantly high mutations in both TA102 and WP-2 uvrA. Mutagenicity of QT decreased in both strains in the presence of Iron (Fe2+) or NGS alone. TF was not mutagenic in the presence or absence of S9 mix in both TA102 and WP-2 uvrA 2, regardless of the presence of iron or NGS. Incorporation of antioxidants (ascorbate, superoxide dismutase (SOD), catalase (CAT)) and/or iron chelators (desferroxamine (DF) and ethylenediamine-tetraacetate (EDTA)) in the test systems markedly decreased QT-induced mutations in both tester strains. These results suggest that QT but not TF, could induce mutations in the presence or absence of rat liver S9 or Iron (Fe2+) and NGS in both tester strains by redox cycling and Fenton reactions to produce oxygen free radicals. Our results indicate that a minor structural variation between the two plant polyphenols could elicit a marked difference in their genotoxicities. These results provide a basis for further study into the potential use of QT in combination with iron supplements.

Biochemical and structural characterization of Salmonella typhimurium glyoxalase II: new insights into metal ion selectivity.

Glyoxalase II is a hydrolytic enzyme part of the glyoxalase system, responsible for detoxifying several cytotoxic compounds employing glutathione. Glyoxalase II belongs to the superfamily of metallo-beta-lactamases, with a conserved motif able to bind up to two metal ions in their active sites, generally zinc. Instead, several eukaryotic glyoxalases II have been characterized with different ratios of iron, zinc, and manganese ions. We have expressed a gene coding for a putative member of this enzyme superfamily from Salmonella typhimurium that we demonstrate, on the basis of its activity, to be a glyoxalase II, named GloB. Recombinant GloB expressed in Escherichia coli was purified with variable amounts of iron, zinc, and manganese. All forms display similar activities, as can be shown from protein expression in minimal medium supplemented with specific metal ions. The crystal structure of GloB solved at 1.4 A shows a protein fold and active site similar to those of its eukaryotic homologues. NMR and EPR experiments also reveal a conserved electronic structure at the metal site. GloB is therefore able to accommodate these different metal ions and to carry out the hydrolytic reaction with similar efficiencies in all cases. The metal promiscuity of this enzyme (in contrast to other members of the same superfamily) can be accounted for by the presence of a conserved Asp residue acting as a second-shell ligand that is expected to increase the hardness of the metal binding site, therefore favoring iron uptake in glyoxalases II.

Alteration of epithelial structure and function associated with PtdIns(4,5)P2 degradation by a bacterial phosphatase.

Elucidation of the role of PtdIns(4,5)P(2) in epithelial function has been hampered by the inability to selectively manipulate the cellular content of this phosphoinositide. Here we report that SigD, a phosphatase derived from Salmonella, can effectively hydrolyze PtdIns(4,5)P(2), generating PtdIns(5)P. When expressed by microinjecting cDNA into epithelial cells forming confluent monolayers, wild-type SigD induced striking morphological and functional changes that were not mimicked by a phosphatase-deficient SigD mutant (C462S). Depletion of PtdIns(4,5)P(2) in intact SigD-injected cells was verified by detachment from the membrane of the pleckstrin homology domain of phospholipase Cdelta, used as a probe for the phosphoinositide by conjugation to green fluorescent protein. Single-cell measurements of cytosolic pH indicated that the Na(+)/H(+) exchange activity of epithelia was markedly inhibited by depletion of PtdIns(4,5)P(2). Similarly, anion permeability, measured using two different halide-sensitive probes, was depressed in cells expressing SigD. Depletion of PtdIns(4,5)P(2) was associated with marked alterations in the actin cytoskeleton and its association with the plasma membrane. The junctional complexes surrounding the injected cells gradually opened and the PtdIns(4,5)P(2)-depleted cells eventually detached from the monolayer, which underwent rapid restitution. Similar observations were made in intestinal and renal epithelial cultures. In addition to its effects on phosphoinositides, SigD has been shown to convert inositol 1,3,4,5,6-pentakisphosphate (IP(5)) into inositol 1,4,5,6-tetrakisphosphate (IP(4)), and the latter has been postulated to mediate the diarrhea caused by Salmonella. However, the effects of SigD on epithelial cells were not mimicked by microinjection of IP(4). In contrast, the cytoskeletal and ion transport effects were replicated by hydrolyzing PtdIns(4,5)P(2) with a membrane-targeted 5-phosphatase or by occluding the inositide using high-avidity tandem PH domain constructs. We therefore suggest that opening of the tight junctions and inhibition of Na(+)/H(+) exchange caused by PtdIns(4,5)P(2) hydrolysis combine to account, at least in part, for the fluid loss observed during Salmonella-induced diarrhea.

One-bead-one-inhibitor-one-substrate screening of neuraminidase activity.

Given the eminent threat of a 21st century flu pandemic, the search for novel antiviral compounds is an increasingly important area of research. Recent developments in antiviral research have established the viability of targeting viral neuraminidase (NA), an enzyme that cleaves sialic acid from the cell-surface-mediating passage of the virus in the respiratory tract. N-acetyl neuraminic acid (NeuAc) is the substrate for NA, and analogues of this core structure have been commercialized as antiviral therapeutics. Recent studies have established that this system is well suited for combinatorial approaches to drug discovery. An important step in the process is to develop solid-phase screening technologies. The feasibility of performing competitive solid-phase NA assays is reported herein. Initially, a fluorogenic NeuAc substrate was immobilized on solid support, and the ability of three NAs (Clostridium perfringens, Salmonella typhimurium, and Vibrio cholerae) to cleave the substrate was shown to be analogous to solution-phase assays. The solid support was then bifunctionalized with the fluorogenic NeuAc substrate and one of two known inhibitors (DANA and Zanamivir). The ability of NA to cleave NeuAc from the solid support when simultaneously presented with an inhibitor was shown to be enzyme dependent. As expected, simultaneous presentation of NeuAc and DANA, a nonspecific inhibitor, led to diminished activity for all three enzymes tested. In contrast, dual presentation of NeuAc and the selective inhibitor Zanamivir only showed significant activity against Vibrio cholerae.

Discrimination between different methylation states of chemotaxis receptor Tar by receptor methyltransferase CheR.

Bacterial chemotaxis receptors are posttranslationally modified by carboxyl methylation of specific glutamate residues within their cytoplasmic domains. This highly regulated, reversible modification counterbalances the signaling effects of ligand binding and contributes to adaptation. On the basis of the crystal structure of the gamma-glutamyl methyltransferase CheR, we have postulated that positively charged residues in helix alpha2 in the N-terminal domain of the enzyme may be complementary to the negatively charged methylation region of the methyltransferase substrates, the bacterial chemotaxis receptors. Several altered CheR proteins, in which positively charged arginine or lysine residues were substituted with alanines, were constructed and assayed for their methylation activities toward wild-type receptor and a series of receptor variants containing different glutamates available for methylation. One of the CheR mutant proteins (Arg53Ala) showed significantly lower activity toward all receptor constructs, suggesting that Arg53 may play a general role in catalysis of methyl transfer. The rest of the mutant proteins exhibited different patterns of relative methylation rates toward different receptor substrates, indicating specificity, probably through interaction of CheR with the receptor at sites distal to the specific site of methylation. The findings imply complementarity between positively charged residues of the alpha2 helix of CheR and the negatively charged glutamates of the receptor. It is likely that this complementarity is involved in discriminating different methylation states of the receptors.

Synthesis and evaluation as sialidase inhibitors of xylo-configured cyclohexenephosphonates carrying glycerol side-chain mimics.

Based on a strategy previously reported by us, we have synthesized D-xylo configured cyclohexenephosphonates designed to mimic the transition state of the sialidase reaction. The double bond orientation corresponds to the benchmark inhibitor Neu5Ac2en and we could selectively introduce hydroxyalkyl substituents in order to simulate the glycerol side-chain of neuraminic acid. The inhibitory activity of a set of compounds towards bacterial sialidases was tested and interesting differences in activity were found.

The crystal structure of phosphinothricin in the active site of glutamine synthetase illuminates the mechanism of enzymatic inhibition.

Phosphinothricin is a potent inhibitor of the enzyme glutamine synthetase (GS). The resolution of the native structure of GS from Salmonella typhimurium has been extended to 2.5 A resolution, and the improved model is used to determine the structure of phosphinothricin complexed to GS by difference Fourier methods. The structure suggests a noncovalent, dead-end mechanism of inhibition. Phosphinothricin occupies the glutamate substrate pocket and stabilizes the Glu327 flap in a position which blocks the glutamate entrance to the active site, trapping the inhibitor on the enzyme. One oxygen of the phosphinyl group of phosphinothricin appears to be protonated, because of its proximity to the carboxylate group of Glu327. The other phosphinyl oxygen protrudes into the negatively charged binding pocket for the substrate ammonium, disrupting that pocket. The distribution of charges in the glutamate binding pocket is complementary to those of phosphinothricin. The presence of a second ammonium binding site within the active site is confirmed by its analogue thallous ion, marking the ammonium site and its protein ligands. The inhibition of GS by methionine sulfoximine can be explained by the same mechanism. These models of inhibited GS further illuminate its catalytic mechanism.

In vitro conversion of propionate to pyruvate by Salmonella enterica enzymes: 2-methylcitrate dehydratase (PrpD) and aconitase Enzymes catalyze the conversion of 2-methylcitrate to 2-methylisocitrate.

Salmonella enterica serovar Typhimurium LT2 catabolizes propionate through the 2-methylcitric acid cycle, but the identity of the enzymes catalyzing the conversion of 2-methylcitrate into 2-methylisocitrate is unclear. This work shows that the prpD gene of the prpBCDE operon of this bacterium encodes a protein with 2-methylcitrate dehydratase enzyme activity. Homogeneous PrpD enzyme did not contain an iron-sulfur center, displayed no requirements for metal cations or reducing agents for activity, and did not catalyze the hydration of 2-methyl-cis-aconitate to 2-methylisocitrate. It was concluded that the gene encoding the 2-methyl-cis-aconitate hydratase enzyme is encoded outside the prpBCDE operon. Computer analysis of bacterial genome databases identified the presence of orthologues of the acnA gene (encodes aconitase A) in a number of putative prp operons. Homogeneous AcnA protein of S. enterica had strong aconitase activity and catalyzed the hydration of the 2-methyl-cis-aconitate to yield 2-methylisocitrate. The purification of this enzyme allows the complete reconstitution of the 2-methylcitric acid cycle in vitro using homogeneous preparations of the PrpE, PrpC, PrpD, AcnA, and PrpB enzymes. However, inactivation of the acnA gene did not block growth of S. enterica on propionate as carbon and energy source. The existence of a redundant aconitase activity (encoded by acnB) was postulated to be responsible for the lack of a phenotype in acnA mutant strains. Consistent with this hypothesis, homogeneous AcnB protein of S. enterica also had strong aconitase activity and catalyzed the conversion of 2-methyl-cis-aconitate into 2-methylisocitrate. To address the involvement of AcnB in propionate catabolism, an acnA and acnB double mutant was constructed, and this mutant strain cannot grow on propionate even when supplemented with glutamate. The phenotype of this double mutant indicates that the aconitase enzymes are required for the 2-methylcitric acid cycle during propionate catabolism.

Functional genomic, biochemical, and genetic characterization of the Salmonella pduO gene, an ATP:cob(I)alamin adenosyltransferase gene.

Salmonella enterica degrades 1,2-propanediol by a pathway dependent on coenzyme B12 (adenosylcobalamin [AdoCb1]). Previous studies showed that 1,2-propanediol utilization (pdu) genes include those for the conversion of inactive cobalamins, such as vitamin B12, to AdoCbl. However, the specific genes involved were not identified. Here we show that the pduO gene encodes a protein with ATP:cob(I)alamin adenosyltransferase activity. The main role of this protein is apparently the conversion of inactive cobalamins to AdoCbl for 1,2-propanediol degradation. Genetic tests showed that the function of the pduO gene was partially replaced by the cobA gene (a known ATP:corrinoid adenosyltransferase) but that optimal growth of S. enterica on 1,2-propanediol required a functional pduO gene. Growth studies showed that cobA pduO double mutants were unable to grow on 1,2-propanediol minimal medium supplemented with vitamin B(12) but were capable of growth on similar medium supplemented with AdoCbl. The pduO gene was cloned into a T7 expression vector. The PduO protein was overexpressed, partially purified, and, using an improved assay procedure, shown to have cob(I)alamin adenosyltransferase activity. Analysis of the genomic context of genes encoding PduO and related proteins indicated that particular adenosyltransferases tend to be specialized for particular AdoCbl-dependent enzymes or for the de novo synthesis of AdoCbl. Such analyses also indicated that PduO is a bifunctional enzyme. The possibility that genes of unknown function proximal to adenosyltransferase homologues represent previously unidentified AdoCbl-dependent enzymes is discussed.

Novel subtype of type IIs restriction enzymes. BfiI endonuclease exhibits similarities to the EDTA-resistant nuclease Nuc of Salmonella typhimurium.

The type IIs restriction enzyme BfiI recognizes the non-palindromic nucleotide sequence 5'-ACTGGG-3' and cleaves complementary DNA strands 5/4 nucleotides downstream of the recognition sequence. The genes coding for the BfiI restriction-modification (R-M) system were cloned/sequenced and biochemical characterization of BfiI restriction enzyme was performed. The BfiI R-M system contained three proteins: two N4-methylcytosine methyltransferases and a restriction enzyme. Sequencing of bisulfite-treated methylated DNA indicated that each methyltransferase modifies cytosines on opposite strands of the recognition sequence. The N-terminal part of the BfiI restriction enzyme amino acid sequence revealed intriguing similarities to an EDTA-resistant nuclease of Salmonella typhimurium. Biochemical analyses demonstrated that BfiI, like the nuclease of S. typhimurium, cleaves DNA in the absence of Mg(2+) ions and hydrolyzes an artificial substrate bis(p-nitrophenyl) phosphate. However, unlike the nonspecific S. typhimurium nuclease, BfiI restriction enzyme cleaves DNA specifically. We propose that the DNA-binding specificity of BfiI stems from the C-terminal part of the protein. The catalytic N-terminal subdomain of BfiI radically differs from that of type II restriction enzymes and is presumably similar to the EDTA-resistant nonspecific nuclease of S. typhimurium; therefore, BfiI did not require metal ions for catalysis. We suggest that BfiI represents a novel subclass of type IIs restriction enzymes that differs from the archetypal FokI endonuclease by the fold of its cleavage domain, the domain location, and reaction mechanism.

The active form of the R2F protein of class Ib ribonucleotide reductase from Corynebacterium ammoniagenes is a diferric protein.

Corynebacterium ammoniagenes contains a ribonucleotide reductase (RNR) of the class Ib type. The small subunit (R2F) of the enzyme has been proposed to contain a manganese center instead of the dinuclear iron center, which in other class I RNRs is adjacent to the essential tyrosyl radical. The nrdF gene of C. ammoniagenes, coding for the R2F component, was cloned in an inducible Escherichia coli expression vector and overproduced under three different conditions: in manganese-supplemented medium, in iron-supplemented medium, and in medium without addition of metal ions. A prominent typical tyrosyl radical EPR signal was observed in cells grown in rich medium. Iron-supplemented medium enhanced the amount of tyrosyl radical, whereas cells grown in manganese-supplemented medium had no such radical. In highly purified R2F protein, enzyme activity was found to correlate with tyrosyl radical content, which in turn correlated with iron content. Similar results were obtained for the R2F protein of Salmonella typhimurium class Ib RNR. The UV-visible spectrum of the C. ammoniagenes R2F radical has a sharp 408-nm band. Its EPR signal at g = 2.005 is identical to the signal of S. typhimurium R2F and has a doublet with a splitting of 0.9 millitesla (mT), with additional hyperfine splittings of 0.7 mT. According to X-band EPR at 77-95 K, the inactive manganese form of the C. ammoniagenes R2F has a coupled dinuclear Mn(II) center. Different attempts to chemically oxidize Mn-R2F showed no relation between oxidized manganese and tyrosyl radical formation. Collectively, these results demonstrate that enzymatically active C. ammoniagenes RNR is a generic class Ib enzyme, with a tyrosyl radical and a diferric metal cofactor.