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Research has been conducted on the Salmonella enterica serovar Typhimurium's isc gene cluster. The study of the metabolic consequences of two insertions in this cluster is presented.

Research has been conducted on the PurF-independent thiamine synthesis in Salmonella entrica serovar Typhimurium. The results of the mutation isolation in a number of loci are dicussed.

A study concerning the structural characterization of the ApbE lipoprotein is examined. Experiments reveal that it is exposed to the periplasmic space.

Research was conducted to examine the proposed synthesis that the pyrimidine moiety of thiamine can be characterized independently of the first enzyme of the de novo purine synthesis, phosphoribosylpyrophosphate amidotransferase (PurF), in Salmonella typhimurium. Isotope labeling experiments were carried out with two compounds, (2-(super 13)C)glycine and (super 13C)formate. Results demonstrate that PurF-independent TPP synthesis did not function at the same capacity as that involving the PurF enzyme.

Research was conducted to examine the regulation of thiamine pyrophosphate synthesis in Salmonella typhimurium. Results revealed the existence of new gene products, apbE single mutants and apbE purfF double mutants, that require thiamine. The apbE locus was identified as the product used in thiamine synthesis. It has been observed to produce a 36-kDa membrane-associated lipoprotein.

A research study was conducted to determine the importance of rseC, a gene in the rpoE cluster of the Salmonella thyphimurium (S typhimurium). Some of the methods used were the insertion of rseB, genetic analysis, descriptive analysis and complementation analysis. Results revealed that rseC, the fourth gene in the rpoE cluster, plays a distinctive role in the thiamine synthesis in S thyphimurium independent from rpoE .

Thiamine pyrophosphate (TPP) in Salmonella typhimurium is a necessary cofactor for several enzymes in the cell. The identification and characterization of a 2-gene operon at 46 Cs on the s. typhimurium chromosome involved in thiamine biosynthesis is described. It shows that thiM is an operon with thiD and is transcriptionally regulated by TTA, suggesting a biosynthetic role in TPP synthesis.

The initial physical and genetic characterization of a thiI, a new locus that is necessary for 4-methyl-5-(beta-hydroxyethyl)-thiazole synthesis, is presented. This new gene is found to be a 1,449-bp open reading frame situated at minute 9.65 on the S. typhimurium chromosome. Research results also reveal that it encodes a 55-kDa 483-amino-acid protein. ThiI is not transcriptionally regulated by thiamine pyrophosphate.

Derivatives of purF mutants from Salmonella typhimurium that no longer needed panthotenate for thiamine-independent growth were isolated. Analysis of these mutants indicated that they were defective in succinate dehydrogenase (Sdh), an enzyme of the tricarboxylic acid cycle. Phenotypic studies showed that a defect in Sdh decreased the amount of thiamine needed by S. typhimurium. This reduced requirement correlated with levels of succinyl-coenzyme A.

Thiamine is thought to be synthesized by two alternative pathways, one involving the first four enzymes of the purine pathway and a second that can function independently of the purine pathway. Insertion mutations in purG and purI prevent thiamine synthesis through the alternative pyrimidine biosynthetic (APB) pathway under aerobic but not anaerobic growth conditions. In contrast, point mutations in purG and purI caused one of three distinct phenotypes: [Pur.sup.-] [Apb.sup.-], [Pur.sup.-] [Apb.sup.+], or [Pur.sup.+] [Apb.sup.-]. Analysis of these three mutant classes demonstrated two genetically separable functions for PurG and PurI in thiamine synthesis. In addition to their known enzymatic role in de novo purine synthesis, we propose that PurG and PurI play a novel, possibly nonenzymatic role in the APB pathway. Suppression analysis of Put Apb mutants identified two new genetic loci involved in the APB pathway, apbB and apbD. We show here that mutations in apbB and apbD cause distinct, allele-specific suppression of the thiamine requirement of purG and purI mutants. Our results suggest that PurG and PurI and one or more components of the APB pathway may function as a complex needed for aerobic function of the APB pathway.

The first five steps in de novo purine biosynthesis are involved in the formation of the 4-amino-5-hydroxymethyl-2-methyl pyrimidine (HMP) moiety of thiamine. We show here that the first enzyme in de novo purine biosynthesis, PurF, is required for thiamine synthesis during aerobic growth on some but not other carbon sources. We show that PurF-independent thiamine synthesis depends on the recently described alternative pyrimidine biosynthetic (APB) pathway. Null mutations in zwf (encoding glucose-6-P dehydogenase), gnd (encoding gluconate-6-P dehydrogenase), purE (encoding aminoimidazole ribo-nucleotide carboxylase), and purR (encoding a regulator of gene expression) were found to affect the function of the APB pathway. A model is presented to account for the involvement of these gene products in thiamine biosynthesis via the APB pathway. Results presented herein demonstrate that function of the APB pathway can be prevented either by blocking intermediate formation or by diverting intermediate (s) from the pathway. Strong genetic evidence supports the conclusion that aminoimidazole ribotide (AIR) is an intermediate in the APB pathway.

Exogenous thiamine or thiamine phosphate forms thiamine pyrophosphate (TPP) that suppresses the transcription of the Salmonella typhimurium thi operon present at 90 min of the bacterial genome. The thi operon at 10 min is unaffected by TPP. Mutants unable to produce thiamine require either the thiamine thiazole moiety or thiamine for their growth. Most of the mutants have mutations in the 90 min area. These mutants require the thiazole moiety for growth and contain no thiamine phosphate kinase that is encoded by the thiL gene.

The ability of exogenous pantothenate to act as replacement for the needed thiamine by purF mutants of Salmonella typhimurium is negated by blocks in the oxidative pentose phosphate pathway, gnd which encodes gluconate 6-phosphate dehydrogenase or zwf which encodes glucose 6-phosphate dehydroenase. The defects resulting from the mutations can be remedied by adding more ribose 5-P which indicates that ribose 5-P is important in replacing thiamine by pantothenate.

Characterization of apbA, the new genetic locus identified in thiamine biosynthesis in Salmonella typhimurium, shows that a functional APB pathway is essential for thiamine synthesis. The phenotype of apbA mutants reveals that the pur genetic background controls the functioning of this pathway under aerobic or anaerobic growth conditions. Cloning and sequencing of the apbA gene shows that it contains a polypeptide, which is a predictor of the NAD/flavin adenine dinucleotide-binding pocket in the protein.

Evidence is presented for a novel synthesis pathway of the pyrimidine moiety of thiamine in Salmonella typhimurium. The pathway, designated as alternative pyrimidine biosynthesis (APB), bypasses the de novo synthesis of pyrimidine and is expressed during anoxic growth conditions. APB utilizes pantothenate as a precursor in pyrimidine biosynthesis. Another pyrimidine biosynthetic pathway, the purF function, which is responsible for growth in the presence of pantothenate, was shown to be affected by temperature and pantothenate levels.

The production of the pyrimidine moiety in thiamine synthesis, 2‐methyl‐4‐amino‐5‐hydroxymethylpyrimidine phosphate (HMP‐P), has been described to proceed through the Thi5‐dependent pathway in Saccharomyces cerevisiae and other yeast. Previous work found that ScThi5 functioned poorly in a heterologous context. Here we report a bacterial ortholog to the yeast HMP‐P synthase (Thi5) was necessary for HMP synthesis in Legionella pneumophila. Unlike ScThi5, LpThi5 functioned in vivo in Salmonella enterica under multiple growth conditions. The protein LpThi5 is a dimer that binds pyridoxal‐5′‐phosphate (PLP), apparently without a solvent‐exposed Schiff base. A small percentage of LpThi5 protein co‐purifies with a bound molecule that can be converted to HMP. Analysis of variant proteins both in vivo and in vitro confirmed that residues in sequence motifs conserved across bacterial and eukaryotic orthologs modulate the function of LpThi5. Importance: Thiamine is an essential vitamin for the vast majority of organisms. There are multiple strategies to synthesize and salvage this vitamin. The predominant pathway for synthesis of the pyrimidine moiety of thiamine involves the Fe‐S cluster protein ThiC. An alternative pathway utilizes Thi5, a novel enzyme that uses PLP as a substrate. The Thi5‐dependent pathway is poorly characterized in yeast and has not been characterized in Bacteria. Here we demonstrate that a Thi5‐dependent pathway is necessary for thiamine biosynthesis in Legionella pneumophila and provide biochemical data to extend knowledge of the Thi5 enzyme, the corresponding biosynthetic pathway, and the role of metabolic network architecture in optimizing its function. [ABSTRACT FROM AUTHOR]

Pyridoxal 59-phosphate (PLP) is the biologically active form of vitamin B6, essential for cellular function in all domains of life. In many organisms, such as Salmonella enterica serovar Typhimurium and Escherichia coli, this cofactor can be synthesized de novo or salvaged from B6 vitamers in the environment. Unexpectedly, S. enterica strains blocked in PLP biosynthesis were able to use exogenous PLP and pyridoxine 59-phosphate (PNP) as the source of this required cofactor, while E. coli strains of the same genotype could not. Transposon mutagenesis found that phoN was essential for the salvage of PLP and PNP under the conditions tested. phoN encodes a class A nonspecific acid phosphatase (EC 3.1.3.2) that is transcriptionally regulated by the PhoPQ two-component system. The periplasmic location of PhoN was essential for PLP and PNP salvage, and in vitro assays confirmed PhoN has phosphatase activity with PLP and PNP as substrates. The data suggest that PhoN dephosphorylates B6 vitamers, after which they enter the cytoplasm and are phosphorylated by kinases of the canonical PLP salvage pathway. The connection of phoN with PhoPQ and the broad specificity of the gene product suggest S. enterica is exploiting a moonlighting activity of PhoN for PLP salvage. [ABSTRACT FROM AUTHOR]

Summary: The YggS/Ybl036c/PLPBP family includes conserved pyridoxal 5′‐phosphate (PLP)‐binding proteins that play a critical role in the homeostasis of vitamin B6 and amino acids. Disruption of members of this family causes pleiotropic effects in many organisms by unknown mechanisms. In Escherichia coli, conditional lethality of the yggS and glyA (encoding serine hydroxymethyltransferase) has been described, but the mechanism of lethality was not determined. Strains lacking yggS and serA (3‐phosphoglycerate dehydrogenase) were conditionally lethality in the M9‐glucose medium supplemented with Gly. Analyses of vitamin B6 pools found the high‐levels of pyridoxine 5′‐phosphate (PNP) in the two yggS mutants. Growth defects of the double mutants could be eliminated by overexpressing PNP/PMP oxidase (PdxH) to decrease the PNP levels. Further, a serA pdxH strain, which accumulates PNP in the presence of yggS, exhibited similar phenotype to serA yggS mutant. Together these data suggested the inhibition of the glycine cleavage (GCV) system caused the synthetic lethality. Biochemical assays confirmed that PNP disrupts the GCV system by competing with PLP in GcvP protein. Our data are consistent with a model in which PNP‐dependent inhibition of the GCV system causes the conditional lethality observed in the glyA yggS or serA yggS mutants. [ABSTRACT FROM AUTHOR]

EPR spectroscopic studies are conducted to understand the interaction of AdoMet with active ThiC from Salmonella enterica, which generates a free radical on the [alpha]-carbon of an amino acid residue. The AdoMet-dependent generation of free radical provides evidence for the complicated rearrangement catalyzed by ThiC, which employ free radical chemistry for radical SAM enzymes.

The purification and characterization of the ThiC protein from Salmonella enterica is reported. The results obtained provide insight into the activity of ThiC, which carries an oxygen labile [Fe-S] cluster critical for generation of 4-amino-5-hydroxymethyl-2-methylpyrimidine pyrophosphate (HMP-PP).

The first nutritional and biochemical activity of mutants of Salmonella enterica ApbC in the context of iron-sulfur ([Fe-S]) cluster metabolism is described. The result data shed light on the ability of ApbC to coordinate an [4Fe-4S] cluster across its dimer interface and transfer the cluster to an apoprotein acting as an [Fe-S] cluster scaffold protein, a function discovered for its eukaryotic homologues.

Research has been conducted on Salmonella enterica mutant strain which uses exogenous aminoimidazole riboside as a thiamine source. The characterization of this strain has been carried out, and the details are reported.

Research has been conducted on Fe-S cluster metabolism defect in Salmonella enterica serovar Typhimurium. Results demonstrate that this defect is affected by biochemical and nutritional properties associated with apbC or apbE mutant strains.

Building a robust, stable network must include strategies to minimize perturbations caused by environmental stress, while optimizing cellular fitness. The introduction of oxygen into the Earth's atmosphere brought challenges for the microbes that had evolved enzyme machinery and metabolic network stability in the anoxic world. Unable to generate new enzyme paradigms and metabolic networks de novo, organisms have evolved strategies to neutralize the impact of oxygen that can be added to and integrated into the existing metabolic framework. This issue of Molecular Microbiology includes a paper by Korshunov et al. in which the authors describe an elegant strategy that Escherichia coli has evolved to minimize metabolic stress that results from the acquisition and use of cystine, the oxidized form of cysteine, as a source of cellular sulfur. This study highlights how a strategy involving both cost and benefit can result in a functional, but energy intensive mechanism for this bacterium to thrive in an oxic world. [ABSTRACT FROM AUTHOR]