apbA, a new genetic locus involved in thiamine biosynthesis in Salmonella typhimurium.

In Salmonella typhimurium, the synthesis of the pyrimidine moiety of thiamine can occur by utilization of the first five steps in de novo purine biosynthesis or independently of the pur genes through the alternative pyrimidine biosynthetic, or APB, pathway (D. M. Downs, J. Bacteriol. 174:1515-1521, 1992). We have isolated the first mutations defective in the APB pathway. These mutations define the apbA locus and map at 10.5 min on the S. typhimurium chromosome. We have cloned and sequenced the apbA gene and found it to encode a 32-kDa polypeptide whose sequence predicts an NAD/flavin adenine dinucleotide-binding pocket in the protein. The phenotypes of apbA mutants suggest that, under some conditions, the APB pathway is the sole source of the pyrimidine moiety of thiamine in wild-type S. typhimurium, and furthermore, the pur genetic background of the strain influences whether this pathway can function under aerobic and/or anaerobic growth conditions.     

The apbE Gene Encodes a Lipoprotein Involved in Thiamine Synthesis in Salmonella typhimurium

Thiamine pyrophosphate is an essential cofactor that is synthesized de novo in Salmonella typhimurium. The biochemical steps and gene products involved in the conversion of aminoimidazole ribotide (AIR), a purine intermediate, to the 4-amino-5-hydroxymethyl-2-methyl pyrimidine (HMP) moiety of thiamine have yet to be elucidated. We have isolated mutations in a new locus (Escherichia coli open reading frame designation yojK) at 49 min on the S. typhimurium chromosome. Two significant phenotypes associated with lesions in this locus (apbE) were identified. First, apbE purF double mutants require thiamine, specifically the HMP moiety. Second, in the presence of adenine, apbE single mutants require thiamine, specifically both the HMP and the thiazole moieties. Together, the phenotypes associated with apbE mutants suggest that flux through the purine pathway has a role in regulating synthesis of the thiazole moiety of thiamine and are consistent with ApbE being involved in the conversion of AIR to HMP. The product of the apbE gene was found to be a 36-kDa membrane-associated lipoprotein, making it the second membrane protein implicated in thiamine synthesis.     

Genetic Analysis of Metabolic Crosstalk and Its Impact on Thiamine Synthesis in Salmonella Typhimurium

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.     

Evidence for a new, oxygen-regulated biosynthetic pathway for the pyrimidine moiety of thiamine in Salmonella typhimurium.

The synthesis of the pyrimidine moiety of thiamine (vitamin B1) shares five reactions with the de novo purine biosynthetic pathway. Aminoimidazole ribotide (AIR) is the last common intermediate before the two pathways diverge. Evidence for the existence of a new pathway to the pyrimidine which bypasses the de novo purine biosynthetic pathway is reported here. This pathway is only expressed under anaerobic growth conditions and is denoted alternative pyrimidine biosynthesis or APB. Labeling studies are consistent with pantothenate being a precursor to the pyrimidine moiety of thiamine that is synthesized by the APB pathway. The APB pathway is independent of the alternative purF function which was proposed previously (D. M. Downs and J. R. Roth, J. Bacteriol. 173:6597-6604, 1991). The alternative purF function is shown here to be affected by temperature and exogenous pantothenate. Although the evidence suggests that the APB pathway is separate from the alternative purF function, the relationship between this function and the APB pathway is not yet clear.     

Complex Metabolic Phenotypes Caused by a Mutation in yjgF,Encoding a Member of the Highly Conserved YER057c/YjgF Family of Proteins

The oxidative pentose phosphate pathway is required for function of the alternative pyrimidine biosynthetic pathway, a pathway that allows thiamine synthesis in the absence of the PurF enzyme in Salmonella typhimurium. Mutants that no longer required function of the oxidative pentose phosphate pathway for thiamine synthesis were isolated. Further phenotypic analyses of these mutants demonstrated that they were also sensitive to the presence of serine in the medium, suggesting a partial defect in isoleucine biosynthesis. Genetic characterization showed that these pleiotropic phenotypes were caused by null mutations in yjgF, a previously uncharacterized open reading frame encoding a hypothetical 13.5-kDa protein. The YjgF protein belongs to a class of proteins of unknown function that exhibit striking conservation across a wide range of organisms, from bacteria to humans. This work represents the first detailed phenotypic characterization of yjgF mutants in any organism and provides important clues as to the function of this highly conserved class of proteins. Results also suggest a connection between function of the isoleucine biosynthetic pathway and the requirement for the pentose phosphate pathway in thiamine synthesis.The increasing number of completed genome sequences has resulted in the identification of new families of hypothetical proteins whose function has yet to be established. The lack of existing mutants defective in these conserved proteins suggests novel, complex, or subtle phenotypes. Through our work on thiamine synthesis in Salmonella typhimurium, we have isolated mutants defective in the recently identified YER057c/YjgF protein family. Our data suggest that defects in this protein result in complex phenotypes involving thiamine and isoleucine biosynthesis.Thiamine pyrophosphate (TPP) serves as an essential cofactor for a number of metabolic reactions involving the removal or transfer of C₂ units. Despite the important role of TPP in cellular metabolism, its synthesis and regulation are not well understood in any organism. TPP is formed from two precursors, 4-methyl-5-(β-hydroxyethyl)thiazole phosphate (THZ-P) and 4-amino-5-hydroxymethyl-2-methylpyrimidine pyrophosphate (HMP-PP). These compounds are joined and subsequently phosphorylated as shown in Fig. ​Fig.1A.1A. Although many of the enzymatic steps in both the THZ-P and HMP-PP pathways have not been clearly defined, the major precursor molecules for both of these compounds have been determined by labeling studies (17, 20, 28, 29). In particular, the purine pathway intermediate, aminoimidazole ribotide (AIR), has been shown to provide all of the atoms in HMP (28, 50, 51).Open in a separate windowFIG. 1Pathway schematics. (A) Biosynthetic pathway for TPP. The involvement of the purine pathway in HMP-PP synthesis is shown with structural intermediates prior to the AIR branch point. Arrows denoted with dotted lines represent proposed steps. Reactions involved in the conversion of AIR to HMP-PP and in the synthesis of THZ-P have not been clearly defined. Genes whose products are required for selected reactions are indicated next to the relevant arrows. Abbreviations: R-P, ribose-5-phosphate, PRPP, phosphoribosylpyrophosphate. (B) Biosynthetic pathways for the branched-chain amino acids isoleucine and valine. Enzymes that catalyze specific steps are as follows: 1, aspartate transaminase; 2, 3, and 4, aspartate kinases I, II, and III, respectively; 5, aspartate semialdehyde dehydrogenase; 6 and 7, homoserine dehydrogenases I and II, respectively; 8, homoserine kinase; 9, threonine synthase; 10, threonine deaminase; 11 and 12, acetohydroxy acid synthases I and II, respectively; 13, acetohydroxy acid isomeroreductase; 14, dihydroxy acid dehydratase; 15, transaminase B; 16, transaminase C. OAA, oxaloacetic acid.Although the involvement of the purine pathway in the synthesis of HMP is clear, there is substantial genetic and biochemical evidence indicating that the first enzyme of the purine pathway, phosphoribosylpyrophosphate amidotransferase (PurF) (EC 2.4.2.14), is not required for HMP synthesis in S. typhimurium under all conditions. Mutants defective in purF are able to grow in the absence of thiamine when glucose is used as a carbon source if pantothenate is also supplied in the medium (23). Similarly, purF mutants do not require thiamine when grown on a number of nonglucose carbon sources, such as gluconate or ribose (54). The pathway responsible for synthesis of HMP independent of the PurF enzyme has been defined as the alternative pyrimidine biosynthetic (APB) pathway (21, 54); recent biochemical data suggest that phosphoribosylamine (PRA), or a derivative, is an intermediate in this pathway (24).Significant progress in our understanding of the APB pathway has been made by the isolation and characterization of mutants unable to synthesize thiamine in a purF background. One class of mutants, designated apbA, was defective in a pantothenate biosynthetic enzyme (ketopantoate reductase [PanE]) (32, 33), consistent with previous results implicating a role for pantothenate in PurF-independent thiamine synthesis (23). A second class of these mutants was defective in the oxidative pentose phosphate pathway, affecting either glucose-6-phosphate dehydrogenase (Zwf) or gluconate-6-phosphate dehydrogenase (Gnd) (25, 54). Addition of ribose-5-phosphate (ribose-5-P) restored function of the APB pathway in these mutants, suggesting that the role of these enzymes in HMP synthesis was to supply ribose-5-P. These results led to the model shown in Fig. ​Fig.1A1A which implicates ribose-5-P and an amine donor as precursors to PRA. Repeated attempts have failed to identify either the predicted PRA-forming activity or mutants defective in this step (27). There are several possible explanations for this. It is possible that the correct substrates have not been identified and/or that the PRA-forming activity is required for another cellular function.In this report, we describe the isolation and characterization of mutations that allow function of the APB pathway in the absence of the pentose phosphate pathway. These mutations were found to disrupt a previously uncharacterized open reading frame (ORF) encoding a hypothetical 13.5-kDa protein. We have designated this gene yjgF based on homology to the respective ORF in Escherichia coli. The YjgF protein belongs to the YER057c/YjgF protein family, a class of proteins of unknown function that exhibit striking conservation across a wide range of organisms. Characterization of these mutants revealed that they also were sensitive to the presence of serine in the medium, exhibiting a requirement for isoleucine under this condition. The phenotypes caused by yjgF mutations suggest that the YjgF protein may be involved in regulation or function of the isoleucine biosynthetic pathway. Further, results suggest a connection between isoleucine biosynthesis and function of the APB pathway in thiamine synthesis.     

The microbiological activity of α-keto-β,β-dimethyl-γ-butyrolactone

α-Keto-β,β-dimethyl-γ-butyrolactone is as active as pantoic acid in promoting growth of E. coli M-99-3, and is approximately three times as active as pantoic acid in promoting growth of E. coli M-99-4 and in reversing salicylate inhibition of E. coli; it is inactive in promoting growth of E. coli M-99-1 and only about 3% as effective as pantoic acid in promoting the growth of Acetobacter suboxydans.     

1-methylguanosine-deficient tRNA of Salmonella enterica serovar Typhimurium affects thiamine metabolism

In Salmonella enterica serovar Typhimurium a mutation in the purF gene encoding the first enzyme in the purine pathway blocks, besides the synthesis of purine, the synthesis of thiamine when glucose is used as the carbon source. On carbon sources other than glucose, a purF mutant does not require thiamine, since the alternative pyrimidine biosynthetic (APB) pathway is activated. This pathway feeds into the purine pathway just after the PurF biosynthetic step and upstream of the intermediate 4-aminoimidazolribotide, which is the common intermediate in purine and thiamine synthesis. The activity of this pathway is also influenced by externally added pantothenate. tRNAs from S. enterica specific for leucine, proline, and arginine contain 1-methylguanosine (m(1)G37) adjacent to and 3' of the anticodon (position 37). The formation of m(1)G37 is catalyzed by the enzyme tRNA(m(1)G37)methyltransferase, which is encoded by the trmD gene. Mutations in this gene, which result in an m(1)G37 deficiency in the tRNA, in a purF mutant mediate PurF-independent thiamine synthesis. This phenotype is specifically dependent on the m(1)G37 deficiency, since several other mutations which also affect translation fidelity and induce slow growth did not cause PurF-independent thiamine synthesis. Some antibiotics that are known to reduce the efficiency of translation also induce PurF-independent thiamine synthesis. We suggest that a slow decoding event at a codon(s) read by a tRNA(s) normally containing m(1)G37 is responsible for the PurF-independent thiamine synthesis and that this event causes a changed flux in the APB pathway.     

Different biosynthetic pathways of the pyrimidine moiety of thiamin in procaryotes and eucaryotes

[¹⁴C]Formate is incorporated into the C-2 of the pyrimidine moiety of thiamin by Escherichia coli and Salmonella typhimurium. In Saccharomyces cerevisiae, it is incorporated into C-4. Radioactive carbons of [1-¹⁴C]glycine and [2-¹⁴C]glycine are incorporated by S. typhimurium into the C-4 and C-6 of the pyrimidine, respectively, but not by S. cerevisiae. These facts suggest that procaryotes and eucaryotes have different biosynthetic pathways for pyrimidine. In this study, the procaryotes tested incorporated [¹⁴C]formate into the C-2 and the eucaryotes incorporated it into the C-4 of the pyrimidine.     

Evidence that rseC, a gene in the rpoE cluster, has a role in thiamine synthesis in Salmonella typhimurium.

In Salmonella typhimurium, the genetic loci and biochemical reactions necessary for the conversion of aminoimidazole ribotide (AIR) to the 4-amino-5-hydroxymethyl-2-methyl pyrimidine (HMP) moiety of thiamine remain unknown. Preliminary genetic analysis indicates that there may be more than one pathway responsible for the synthesis of HMP from AIR and that the function of these pathways depends on the availability of AIR, synthesized by the purine pathway or by the purF-independent alternative pyrimidine biosynthetic (APB) pathway (L. Petersen and D. Downs, J. Bacteriol. 178:5676-5682, 1996). An insertion in rseB, the third gene in the rpoE rseABC gene cluster at 57 min, prevented HMP synthesis in a purF mutant. Complementation analysis demonstrated that the HMP requirement of the purF rseB strain was due to polarity of the insertion in rseB on the downstream rseC gene. The role of RseC in thiamine synthesis was independent of rpoE.     

Role of Acetohydroxy Acid Isomeroreductase in Biosynthesis of Pantothenic Acid in Salmonella typhimurium

Structural genes have been identified for all of the enzymes involved in the biosynthesis of pantothenic acid in Salmonella typhimurium and Escherichia coli K-12, with the exception of ketopantoic acid reductase, which catalyzes the conversion of α-ketopantoate to pantoate. The acetohydroxy acid isomeroreductase from S. typhimurium efficiently bound α-ketopantoate (K_m = 0.25 mM) and catalyzed its reduction at 1/20 the rate at which α-acetolactate was reduced. Since two enzymes could apparently participate in the synthesis of pantoate, a S. typhimurium ilvC8 strain was mutagenized to derive strains completely blocked in the conversion of α-ketopantoate to pantoate. Several isolates were obtained that grew in isoleucine-valine medium supplemented with either pantoate or pantothenate, but not in the same medium supplemented with α-ketopantoate or β-alanine. The mutations that conferred pantoate auxotrophy (designated panE) to these isolates appeared to be clustered, but were not linked to panB or panC. All panE strains tested had greatly reduced levels of ketopantoic acid reductase (3 to 12% of the activity present in DU201). The capacity of the isomeroreductase to synthesize pantoate in vivo was assessed by determining the growth requirements of ilvC⁺ derivatives of panE ilvC8 strains. These strains required either α-ketopantoate, pantoate, or pantothenate when the isomeroreductase was present at low levels; when the synthesis of isomeroreductase was induced, panE ilvC⁺ strains grew in unsupplemented medium. These phenotypes indicate that a high level of isomeroreductase is sufficient for the synthesis of pantoate. panE ilvC⁺ strains also grew in medium supplemented with lysine and methionine. This phenotype resembles that of some S. typhimurium ilvG mutants (e.g., DU501) which are partially blocked in the biosynthesis of coenzyme A and are limited for succinyl coenzyme A. panE ilvC⁺ strains which lack the acetohydroxy acid synthases required only methionine for growth (in the presence of leucine, isoleucine, and valine). This and other evidence suggested that the synthesis of pantoic acid by isomeroreductase was blocked by the α-acetohydroxy acids and that pantoic acid synthesis was enhanced in the absence of these intermediates, even when the isomeroreductase was at low levels. panE ilvC⁺ strains reverted to pantothenate independence. Several of these revertants were shown to have elevated isomeroreductase levels under noninduced and induced conditions; the suppressing mutation in each revertant was shown to be closely linked to ilvC by P22 transduction. This procedure presents a means for obtaining mutants with altered regulation of isomeroreductase.     

A Novel Involvement of the Purg and Puri Proteins in Thiamine Synthesis via the Alternative Pyrimidine Biosynthetic (Apb) Pathway in Salmonella Typhimurium

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(-) Apb(-), Pur(-) Apb(+), or Pur(+) Apb(-). 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 Pur(-) 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.     

Biosynthesis of the Pyrimidine Moiety of Thiamine Independent of the PurF Enzyme (Phosphoribosylpyrophosphate Amidotransferase) in Salmonella typhimurium: Incorporation of Stable Isotope-Labeled Glycine and Formate

Genetic analyses have suggested that the pyrimidine moiety of thiamine can be synthesized independently of the first enzyme of de novo purine synthesis, phosphoribosylpyrophosphate amidotransferase (PurF), in Salmonella typhimurium. To obtain biochemical evidence for and to further define this proposed synthesis, stable isotope labeling experiments were performed with two compounds, [2-¹³C]glycine and [¹³C]formate. These compounds are normally incorporated into thiamine pyrophosphate (TPP) via steps in the purine pathway subsequent to PurF. Gas chromatography-mass spectrometry analyses indicated that both of these compounds were incorporated into the pyrimidine moiety of TPP in a purF mutant. This result clearly demonstrated that the pyrimidine moiety of thiamine was being synthesized in the absence of the PurF enzyme and strongly suggested that this synthesis utilized subsequent enzymes of the purine pathway. These results were consistent with an alternative route to TPP that bypassed only the first enzyme in the purine pathway. Experiments quantitating cellular thiamine monophosphate (TMP) and TPP levels suggested that the alternative route to TPP did not function at the same capacity as the characterized pathway and determined that levels of TMP and TPP in the wild-type strain were significantly altered by the presence of purines in the medium.     

Involvement of the L-cysteine biosynthetic pathway in azide-induced mutagenesis in Salmonella typhimurium

L-Cystine and L-cysteine specifically reverse the mutagenic action of azide in Salmonella typhimurium and Escherichia coli. To establish whether the L-cysteine biosynthetic pathway is involved in azide-induced mutagenesis, several derivatives of a mutagen tester-strain of S. typhimurium bearing mutations in different cys genes were isolated. No mutagenic effect of azide was observed in a strain carrying mutation in the cysE gene, unless the incubation medium was supplemented with exogenous O-acetylserine. Out of 16 cysK mutants 14 were mutagenized by azide very poorly or not at all. These results indicate that the activity of O-acetylserine sulfhydrylase A, and the availability of O-acetylserine, one of the two co-substrates of the enzyme, are essential for the mutagenic action of azide in S. typhimurium     

Interallelic complementation at the pyrF locus and the homodimeric nature of orotate phosphoribosyltransferase (OPRTase) in Mucor circinelloides

Using 5-fluoroorotic acid (5-FOA) as a positive selection system we isolated mutants of Mucor circinelloides altered in the pyrimidine biosynthetic pathway. These mutants were found to be deficient either in orotidine-5′-monophosphate decarboxylase (OMPdecase), or in orotate phosphoribosyltransferase (OPRTase) activity. Complementation tests among mutants lacking OPRTase activity classified them into three groups, thus suggesting the possibility of interallelic complementation. To investigate this hypothesis a cDNA clone corresponding to the OPRTase-encoding gene of M. circinelloides was isolated by direct complementation of E. coli. The genomic copy transformed to prototrophy one member of each of the three classes of OPRTase-deficient mutants. We therefore concluded that they were all altered at the same locus, the pyrF locus. The corresponding alleles were cloned and sequenced. Comparisons of the amino acid sequence of M.?circinelloides OPRTase with those of E. coli and S.?typhimurium revealed a high degree of similarity in secondary and tertiary structure. As the two bacterial enzymes exist as dimers, a homodimeric quaternary structure of the M. circinelloides mature protein can be assumed. This would also explain the interallelic complementation between some pyrF mutants. The mutations found could affect either the active site or the structure of the dimer interface of the OPRTase.     

Transport of thiamine and 4-methyl-5-hydroxyethylthiazole by Salmonella typhimurium

The transport of thiamine and 4-methyl-5-hydroxyethylthiazole (MHET), its thiazole moiety, was studied using whole cells of Salmonella typhimurium. It was found that the bacteria possessed an active transport system for thiamine that had K_m 0.21 μM and V_max 33 nmol·min^?1·(mg dry wt. cells)^?1. Transport of thiamine was glucose dependent, whereas MHET uptake was dependent on both glucose and 2-methyl-4-amino-5-hydroxymethylpyrimidine (MAHMP), the pyrimidine moiety of thiamine. Uptake of both thiamine and MHET was severely curtailed by cyanide, azide, N-ethylmaleimide and carbonyl cyanide m-chlorophenylhydrazone. Oxythiamine inhibited thiamine, but not MHET, uptake and thiamine slightly inhibited MHET uptake. 2-Methyl-4-amino-5-methoxymethylpyrimidine and 4-amino-5-hydroxymethylpyrimidine were unable to replace MAHMP as stimulators of MHET uptake, but 2-methyl-4-amino-5-aminomethylpyrimidine was marginally effective in this regard. Similar results were obtained with attempts to replace MAHMP as a growth requirement for a purD mutant of Salmonella typhimurium. MHET uptake showed saturation kinetics only in the presence of MAHMP, and is not otherwise actively transported.     

The de-repression of thiamine biosynthesis by adenosine. A tool for investigating this biosynthetic pathway

1. Growth of Salmonella typhimurium LT2 in the presence of adenosine was shown to cause enormous synthesis of thiamine in washed-cell suspensions. 2. Evidence that this was due to de-repression and not an accumulation of precursors was obtained by using a mutant blocked in the biosynthesis of the thiazole moiety, which showed a similarly large synthesis of the pyrimidine of thiamine. 3. The specific requirements for a source of energy, nitrogen and sulphur were investigated, and indicated new synthesis in this system.     

Improved biotin,thiamine, and lipoic acid biosynthesis by engineering the global regulator IscR

Biotin, thiamine, and lipoic acid are industrially important molecules naturally synthesized by microorganisms via biosynthetic pathways requiring iron-sulfur (FeS) clusters. Current production is exclusively by chemistry because pathway complexity hinders development of fermentation processes. For biotin, the main bottleneck is biotin synthase, BioB, a S-adenosyl methionine-dependent radical enzyme that converts dethiobiotin (DTB) to biotin. BioB overexpression is toxic, though the mechanism remains unclear. We identified single mutations in the global regulator IscR that substantially improve cellular tolerance to BioB overexpression, increasing Escherichia coli DTB-to-biotin biocatalysis by more than 2.2-fold. Based on proteomics and targeted overexpression of FeS-cluster biosynthesis genes, FeS-cluster depletion is the main reason for toxicity. We demonstrate that IscR mutations significantly affect cell viability and improve cell factories for de novo biosynthesis of thiamine by 1.3-fold and lipoic acid by 1.8-fold. We illuminate a novel engineering target for enhancing biosynthesis of complex FeS-cluster-dependent molecules, paving the way for industrial fermentation processes.     

Evolutionary implications of potential frameshift hot-spots, methylation sites, and codon usage differences between genes and between organisms in Escherichia coli and Salmonella typhimurium

The published nucleotide sequences of the E. coli and S. typhimurium trp A and trp B genes show a high degree of similarity between homologous genes of the two organisms, and an even greater degree of similarity between the amino acid sequences of the gene products. In spite of this, analysis of the nucleotide sequences reveals that there are marked differences between E. coli and S. typhimurium genes with respect to potential frameshift mutation hot-spots and dam and mec, mutationally important, methylation sites. Such existing differences may well lead to divergent evolution of these two, presently closely related, bacteria. Codon usage patterns in the trp A and trp B genes of E. coli and S. typhimurium, and the lac I gene of E. coli, have been re-analysed in terms of AT-rich, GC-rich, neutral, or unique codons and marked preferences found. In some cases particular amino acids are most often specified by AT-rich, in others by the GC-rich, alternative codons. In still other cases the codon preference depends on the gene studied. These patterns can be interpreted in terms of enteric bacterial evolution, via hybridizations, from ancestral bacteria with AT- or GC-rich DNA.     

Elucidation of a carboxylate O-methyltransferase NcmP in nocamycin biosynthetic pathway

Nocamycins belong to the tetramic acid family natural products and show potent antimicrobial activity. Recently, the biosynthetic gene cluster of nocamycin was identified from the rare actinomycete Saccharothrix syringae and an S-adenosylmethionine (SAM) dependent methyltransferase gene NcmP was found to be located within the gene cluster. In this report, the methyltransferase gene NcmP was disrupted and a new nocamycin intermediate nocamycin E was isolated from the mutant strain. Meanwhile, NcmP was heterologously expressed in Escherichia coli BL21 (DE3) and biochemically characterized as a carboxylate O-methyltransferase in nocamycin biosynthetic pathway. Compared to nocamycin I, nocamycin E showed inferior antibacterial activity, indicating the methyl group is essential to antibacterial activity.