Riboflavin operon in Bacillus subtilis contains additional promoters

Using the methods of molecular cloning permitted to show that riboflavin operon of Bacillus subtilis contains four promoters. Three of them are functionally active in the Bacillus subtilis system. The main promoter of the operon with regulatory region was cloned in plasmid pPL603. Cells containing the constructed plasmid pGM32 are resistant to chloramphenicol. The level of resistance is regulated by concentration of riboflavin (the effector of operon). The following model of rib-operon has been proposed: (Formula: see text).     

The induction of auxotrophic mutations for riboflavin by the integration of plasmid pLRS33 into the chromosome of Bacillus subtilis

The transformation of Bacillus subtilis Lys- strains with plasmid pLRS33 containing pBR322 and the Bac. subtilis chromosomal fragment carrying the genes for lysin biosynthesis and the riboflavin operon regulatory operator region (ribO) leads to the appearance of Rib- mutants. It was shown that these mutants contained long deletions covering a great portion of the riboflavin operon.     

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.     

Cloning of the genes controlling the biosynthesis of bacitracin in Bacillus licheniformis

The DNA fragment from bacitracin-producing Bacillus licheniformis strain is cloned on pMX39 vector plasmid in Bacillus subtilis cells. Bacillus subtilis cells carrying the cloned fragment inhibit the growth of bacitracin-sensitive tester strain. The observed inhibition of growth is due to the production by Bacillus subtilis of bacteriocin substance that is identified as bacitracin by TLC-chromatography. In contrast to the data published earlier it is shown that Bacillus subtilis can in fact produce the small amounts of bacitracin. Introduction of the cloned Bacillus licheniformis DNA into Bacillus subtilis cells stimulates this bacitracin production. The restriction site map of the Bacillus licheniformis chromosomal region bearing the cloned fragment is constructed.     

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.     

Riboflavin auxotrophs of Escherichia coli]

Escherichia coli riboflavin auxotrophs having a different level of riboflavin requirement were isolated. This auxotrophic mutations are located near cysB93 and trpA62 markers. The complementary effect of Bacillus subtilis riboflavin operon linked with pPR1 hybrid plasmid with rib8-1 and rib1-1 mutations was obtained.     

Biosynthesis of riboflavin. 6,7-Dimethyl-8-ribityllumazine 5'-phosphate is not a substrate for riboflavin synthase.

Phosphotransferase from carrot is shown to catalyze the phosphorylation of 6,7-dimethyl-8-ribityllumazine specifically at position 5' of the ribityl side chain. The lumazine 5'-phosphate is neither a substrate nor an inhibitor of riboflavin synthase from Bacillus subtilis and Escherichia coli. It follows that the obligatory product of riboflavin synthase is riboflavin and not FMN.     

Uptake and binding of riboflavin by membrane vesicles of bacillus subtilis

Riboflavin uptake and membrane-associated riboflavin-binding activity have been investigated in Bacillus subtilis. The uptake and binding activity of the vitamin were found to be repressed coordinately by riboflavin present in the growth medium. The uptake of riboflavin has been shown to have properties of a carrier-mediated process, and membrane vesicles have been shown to demonstrate riboflavin counterflow and exchange. The membrane-associated binding activity for riboflavin has been solubilized with detergents, and a procedure for the partial purification of this component is described. The partially purified riboflavin-binding component has properties expected for a carrier involved in riboflavin uptake, as it shows saturation kinetics and is inhibited by riboflavin analogues. Evidence is also presented showing that reduced riboflavin binds to a greater extent than oxidized riboflavin, and the possible role of the reduced riboflavin in riboflavin uptake is discussed.     

Riboflavin biosynthesis operon of Bacillus subtilis. XIII. Genetic and biochemical study of mutants with regard to intermediate stages of biosynthesis]

New riboflavin dependent mutants of Bacillus subtilis accumulating different pteridines were studied. The data obtained show that the formation of ribityl side chain proceeds in a few steps at least on a part of riboflavin precursors. The oxidation of connected ribosyl into ribulose with subsequent restoration of it into ribityl proceeds at first. The corresponding genes are located on terminal part of riboflavin operon, as show the results of two-factor transformational crosses with different donors and recipients.     

Operon of riboflavin biosynthesis in Bacillus subtilis. XVI. Localization of the ribC-group markers on the chromosome]

Regulatory markers of ribC group were located on the chromosome of Bacillus subtilis by means of genetic transformation. Markers of this group controlling the regulation of riboflavin biosynthesis were mapped between markers of resistance to acriflavin and streptomycin (strC group). The value of cotransfer index between acriflavin-resistance markers and ribC markers was found to be 26--32%. Acriflavin inhibits the riboflavin biosynthesis. The level of inhibition depends on the genotype of riboflavin-producing strains, while the inhibition of the cell growth does not depend on it.     

Molecular cloning of a major cell wall protein gene from protein-producing Bacillus brevis 47 and its expression in Escherichia coli and Bacillus subtilis

Bacillus brevis 47 contains two major cell wall proteins. Each protein forms a hexagonal array in the cell wall. A 4.8-kilobase HindIII fragment of B. brevis 47 DNA cloned into Escherichia coli with pBR322 as a vector directed the synthesis of polypeptides cross-reactive with antibody to the middle wall protein. A 700-base-pair BamHI-HpaI fragment was shown to be the essential region for the synthesis of immunoreactive polypeptides. Furthermore, this fragment appeared to contain the promoter activity. The 3.5-kilobase BamHI fragment covering the essential region as well as its downstream sequence was subcloned into the corresponding restriction site of pUB110 by using Bacillus subtilis as the cloning host. Both E. coli and B. subtilis carrying the cloned DNA synthesized several immunoreactive polypeptides which were mainly found in the cytoplasm. B. subtilis secreted polypeptides cross-reactive with antibody to the middle wall protein. These extracellular polypeptides were degraded upon prolonged culture.     

Phosphoenolpyruvate:sugar phosphotransferase system of Bacillus subtilis: cloning of the region containing the ptsH and ptsI genes and evidence for a crr-like gene.

The genes ptsI and ptsH, which encode, respectively, enzyme I and Hpr, cytoplasmic proteins involved in the phosphoenolpyruvate:sugar phosphotransferase system, were cloned from Bacillus subtilis. A plasmid containing a 4.1-kilobase DNA fragment was shown to complement Escherichia coli mutations affecting the ptsH and ptsI genes. In minicells this plasmid expressed two proteins with the molecular weights expected for Hpr and enzyme I. Therefore, ptsH and ptsI are adjacent in B. subtilis, as in E. coli. In E. coli a third gene (crr), involved in glucose translocation and also in catabolite repression, is located downstream from the ptsHI operon. The 4.1-kilobase fragment from B. subtilis was shown to contain a gene that enables an E. coli crr mutant to use glucose. This gene, unlike the E. coli crr gene, was located to the left of ptsH.     

核黄素发酵菌种改造研究进展

核黄素是一种水溶性维生素,与动植物的生长密切相关,人体不能合成核黄素,需从外部摄取,因此核黄素的生产具有重要意义。介绍了核黄素的生产发展历程和产核黄素的微生物种类。对枯草芽孢杆菌的核黄素代谢途径及其诱变育种进行了总结,重点介绍了菌种的基因重组改造方法,主要是提高核黄素操纵子表达以及提高Ru5P和GTP两种前体供应量合成途径通量,介绍了近些年新的改造方法,并对未来的发展方向进行了展望。     

核黄素基因工程研究进展

核黄素 (维生素B2 )为天然水溶性的B族维生素 ,是维持机体正常代谢所必须的物质 ,具有重要的生理功能。目前核黄素的生产方法主要有化学合成法和微生物发酵法。其中微生物发酵法是后来发展起来的一种十分经济有效的方法 ,并在核黄素主产中开始占据主导地位。为进一步获得核黄素高产菌株 ,人们对核黄素合成基因及其表达调控的机制做了深入细致的研究 ,并以此为依据 ,通过基因工程手段构建出了核黄素高产菌株 ,大大提高了核黄素的产量 ,其中尤以枯草芽孢杆菌最为成功。综述发酵法生产核黄素的现状、核黄素生物合成的分子生物学以及基因工程研究进展 ,讨论了其进一步的发展方向。     

蜡样芽孢杆菌ATCC14579核黄素操纵子的克隆及在枯草芽孢杆菌中的表达

利用BLAST从B．cereus ATCC14579的基因组中找到一段与枯草芽孢杆茵核黄素操纵子具有较高相似性的4．6kb大小的基因组DNA片段,该片段中含有完整的核黄素操纵子。该操纵子结构基因的编码产物的氨基酸序列与枯草芽孢杆菌核黄素操纵子相应结构基因的编码产物的氨基酸序列具有99％的同源性。该片段被克隆到大肠杆茵一枯草芽孢杆茵穿梭载体pHP13M中。表达分析的结果表明B．cereus ATCC14579核黄素操纵子可在大肠杆茵和枯草芽孢杆菌中表达。利用PCR方法用来自枯草杆菌的sac B基因的启动子替换B．cereus ATCC14579核黄素操纵子原有的启动子使其更好表达。替换启动子后的核黄素操纵子在本文使用的发酵条件下有较好的表达,核黄素产量从39．5mg／L增加到61．7mg/L.