Unusual structure of the regulatory region of the riboflavin biosynthesis operon in Bacillus subtilis

In vitro mutagenesis with methylhydroxylamine and nitrosomethylurea was used to obtain a number of Bacillus subtilis mutants impaired in flavin-dependent response. Mutants displayed varying degree of flavin-dependent repression of riboflavin synthase and of 6,7-dimethyl-8-ribityl-lumasine accumulation. Single nucleotide substitutions were detected by DNA sequencing in all of the mutants, affecting the 48 b.p. target area between the mRNA start and the AUG of the first gene.     

枯草芽孢杆菌核黄素操纵子rib operon的克隆与表达

目的:构建产核黄素的枯草芽孢杆菌基因工程菌.方法:以穿梭载体pEB03构建核黄素操纵子的表达质粒载体pGJB13和pGJB14,与质粒pMX45分别转化产核黄素的枯草芽孢杆菌GJ07,并通过发酵摇瓶实验检测核黄素的产量.结果:得到产核黄素的工程菌GJ13 、GJ14和GJ08,在以蔗糖为碳源的发酵条件下,GJ08可产核黄素820mg/L,提高了约55%.结论:得到了产核黄素的高产菌种G J08.     

The bounds of the riboflavin operon in Bacillus subtilis

All the structural genes of riboflavin biosynthesis are shown to be located on the 2.8 MD DNA fragment, using the collection of plasmids, carrying the Bacillus subtilis riboflavin operon fragments and Bacillus subtilis strains, containing various deletions of rib-operon for analysis. The proximal Bgl II site is shown to be located between promoter P1 and the first structural gene ribG. The distal Hind III site of fragment C is the left bound of the rib-operon.     

Genetic mapping of regulatory mutations of Bacillus subtilis riboflavin operon

Summary Seven mutations leading to riboflavin overproduction inBacillus subtilis were found to be linked to the markerdnaF133 (145° on theB. subtilis genetic map) by transformation. Cotransfer indexes (42.5%–61.7%) suggest that theribC mutations are alleles of the same locus. Results of transduction and transformation crosses suggest the following order of markers:pyrD26 –ts-6 –dnaF133 –ribC –recA1.     

Amplification of the riboflavin operon genes of Bacillus subtilis in Escherichia coli cells]

Amplification of Bacillus subtilis DNA fragments was performed in Escherichia coli using plasmid RSF2124. The main principle of isolation and cloning hybrid plasmids was described using genes of riboflavin operon as a model. Bac. subtilis DNA was treated with restriction endonuclease EcoR; followed by the agarose gel electrophoretic separation of the resulting fragments. Gels were sliced, DNA was eluted from the corresponding slices and used to transform Bac. subtilis auxotrophs rib A72, rib S110 and rib D107. DNA fraction with the molecular weight 7 . 10(6) daltons restored prototrophy of these mutants. DNA of this fraction was ligated with EcoRI treated plasmid RSF2124 DNA and used for transformation of E. coli rk-mk+. Ampicillin resistant transformants which had lost the colicin production ability, were selected. The presence of riboflavin genes within the hybrid plasmids was detected by transformation of B. subtilis auxotrophs. Three hybrid plasmids (pPR1, pPR2 and pPR3), containing a fragment of Bac. subtilis DNA with the molecular weight 6.8 . 10(-6) daltons including riboflavin operon, were selected. The analysis of the transformation activity of Bac. subtilis DNA and plasmid pPR1 DNA revealed, that there was no restriction activity of Bac. subtilis cells against plasmid DNA amplified in E. coli. Heteroduplex analysis has shown that plasmids pPR1 and pPR2 differ in the orientation of Bac. subtilis DNA fragment. DNA of these plasmids restored prototrophy of the several studied E. coli riboflavin auxotrophs.     

Kinetic modeling of riboflavin biosynthesis in Bacillus subtilis under production conditions

To study the network dynamics of the riboflavin biosynthesis pathway and to identify potential bottlenecks in the system, an ordinary differential equation-based model was constructed using available literature data for production strains. The results confirmed that the RibA protein is rate limiting in the pathway. Under the conditions investigated, we determined a potential limiting order of the remaining enzymes under increased RibA concentration (>0.102 mM) and therefore higher riboflavin production (>0.045 mmol g _CDW ^?1 h^?1 and 0.0035 mM s^?1, respectively). The reductase activity of RibG and lumazine synthase (RibH) might be the next most limiting steps. The computational minimization of the enzyme concentrations of the pathway suggested the need for a greater RibH concentration (0.251 mM) compared with the other enzymes (RibG: 0.188 mM, RibB: 0.023 mM).     

Complex structure of Bacillus subtilis RibG: the reduction mechanism during riboflavin biosynthesis

Bacterial RibG is a potent target for antimicrobial agents, because it catalyzes consecutive deamination and reduction steps in the riboflavin biosynthesis. In the N-terminal deaminase domain of Bacillus subtilis RibG, structure-based mutational analyses demonstrated that Glu51 and Lys79 are essential for the deaminase activity. In the C-terminal reductase domain, the complex structure with the substrate at 2.56-A resolution unexpectedly showed a ribitylimino intermediate bound at the active site, and hence suggested that the ribosyl reduction occurs through a Schiff base pathway. Lys151 seems to have evolved to ensure specific recognition of the deaminase product rather than the substrate. Glu290, instead of the previously proposed Asp199, would seem to assist in the proton transfer in the reduction reaction. A detailed comparison reveals that the reductase and the pharmaceutically important enzyme, dihydrofolate reductase involved in the riboflavin and folate biosyntheses, share strong conservation of the core structure, cofactor binding, catalytic mechanism, even the substrate binding architecture.     

Crystal structure of a bifunctional deaminase and reductase from Bacillus subtilis involved in riboflavin biosynthesis

Bacterial RibG is an attractive candidate for development of antimicrobial drugs because of its involvement in the riboflavin biosynthesis. The crystal structure of Bacillus subtilis RibG at 2.41-A resolution displayed a tetrameric ring-like structure with an extensive interface of approximately 2400 A(2)/monomer. The N-terminal deaminase domain belongs to the cytidine deaminase superfamily. A structure-based sequence alignment of a variety of nucleotide deaminases reveals not only the unique signatures in each family member for gene annotation but also putative substrate-interacting residues for RNA-editing deaminases. The strong structural conservation between the C-terminal reductase domain and the pharmaceutically important dihydrofolate reductase suggests that the two reductases involved in the riboflavin and folate biosyntheses evolved from a single ancestral gene. Together with the binding of the essential cofactors, zinc ion and NADPH, the structural comparison assists substrate modeling into the active-site cavities allowing identification of specific substrate recognition. Finally, the present structure reveals that the deaminase and the reductase are separate functional domains and that domain fusion is crucial for the enzyme activities through formation of a stable tetrameric structure.     

Riboflavin biosynthesis operon of Bacillus subtilis. XIV. Operator-constitutive mutants]

Numerous operator-constitutive mutants of riboflavin biosynthesis were selected. All of them map in a short region of the Bacillus subtilis chromosome. The yield of riboflavin synthetase from this mutant is different, but in most cases much lower than the maximal yield from a repressor minus strain. Our tentative explanation is a partial overlap of the sites for the adsorption of repressor and RNA-polymerase. Therefore the affinity to the transcribing enzyme is diminished in the operator constitutive strains. The affinity of the repressor-effector complex to the operator depends on the effector structure.     

Cloning of the Bacillus subtilis DNA fragment containing the genes for lysine and riboflavin biosynthesis

The SalI fragment of chromosomal DNA of Bacillus subtilis carrying the gene for lysine biosynthesis and the regulatory operator region (ribO) from the riboflavin gene was cloned into Escherichia coli cells. This fragment was shown to contain the gene coding for lysine synthesizing enzyme. Localization of this gene in Bac. subtili was determined. New plasmids pLRS33 and pLRB4 were constructed using pBR322; they carry a fragment homologous to pLP102 plasmid containing the operon for riboflavin biosynthesis.     

Functional expression of enterobacterial O-polysaccharide biosynthesis enzymes in Bacillus subtilis

The expression of heterologous bacterial glycosyltransferases is of interest for potential application in the emerging field of carbohydrate engineering in gram-positive organisms. To assess the feasibility of using enzymes from gram-negative bacteria, the functional expression of the genes wbaP (formerly rfbP), wecA (formerly rfe), and wbbO (formerly rfbF) from enterobacterial lipopolysaccharide O-polysaccharide biosynthesis pathways was examined in Bacillus subtilis. WbaP and WecA are initiation enzymes for O-polysaccharide formation, catalyzing the transfer of galactosyl 1-phosphate from UDP-galactose and N-acetylglucosaminyl 1-phosphate from UDP-N-acetylglucosamine, respectively, to undecaprenylphosphate. The WecA product (undecaprenylpyrophosphoryl GlcNAc) is used as an acceptor to which the bifunctional wbbO gene product sequentially adds a galactopyranose and a galactofuranose residue from the corresponding UDP sugars to form a lipid-linked trisaccharide. Genes were cloned into the shuttle vectors pRB374 and pAW10. In B. subtilis hosts, the genes were effectively transcribed under the vegII promoter control of pRB374, but the plasmids were susceptible to rearrangements and deletion. In contrast, pAW10-based constructs, in which genes were cloned downstream of the tet resistance cassette, were stable but yielded lower levels of enzyme activity. In vitro glycosyltransferase assays were performed in Escherichia coli and B. subtilis, using membrane preparations as sources of enzymes and endogenous undecaprenylphosphate as an acceptor. Incorporation of radioactivity from UDP-alpha-D-(14)C-sugar into reaction products verified the functionality of WbaP, WecA, and WbbO in either host. Enzyme activities in B. subtilis varied between 20 and 75% of those measured in E. coli.