Interactions of homologous and heterologous deoxyribonucleic acids and competent Bacillus subtilis cells.

Glucosylated and nonglucosylated bacteriophage T4 deoxyribonucleic acids (DNAs) are able to bind to competent cells of Bacillus subtilis, although the former does so in a rather unstable fashion, probably because of the glucosylation. Several heterologous DNAs compete with homologous DNA for the same receptors in binding and in transformation. A different pattern in competition for DNA binding was observed for homologous and T4 glucosylated DNAs in intact cells as compared with protoplasts or membrane vesicles. The results are consistent with the existence of two types of receptor sites on the membrane of competent B. subtilis cells.     

Cloning in Bacillus subtilis of temperature-dependent promoter fragments from Bacillus stearothermophilus and Bacillus subtilis

Abstract Using promoter-probe plasmids, more than 200 promoter-containing fragments from Bacillus stearothermophilus and Bacillus subtilis were cloned in B. subtilis . Among these, 15 promoter fragments were highly temperature-dependent in activity compared to the promoter sequence (TTGAAA for the −35 region, TATAAT for the −10 region) of the amylase gene, amyT , from B. stearothermophilus . Some fragments exhibited higher promoter activities at elevated temperature (48°C), others showed higher activities at lower temperature (30°C). Active promoter fragments at higher and lower temperatures were obtained mainly from the thermophile ( B. stearothermophilus ) and the mesophile ( B. subtilis ), respectively. A promoter fragment active at high temperature was sequenced, and the feature of the putative promoter region was discussed.     

SigM-responsive genes of Bacillus subtilis and their promoters

Promoters of nine Bacillus subtilis genes (bcrC, yacK, ydaH, yfnI, yjbD, ypbG, ypuA, yraA, and ysxA), all responsive to artificially induced increases in the stress-responsive extracytoplasmic function sigma factor, SigM, were mapped by rapid amplification of cDNA ends-PCR. The resulting promoter consensus suggests that overlapping control by SigX or SigW is common.     

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).     

Effect of 6-(p-hydroxyphenylazo)-uracil on the homologous and heterologous transduction processes in Bacillus subtilis.

We have studied the effect of 6-(p-hydroxyphenylazo)-uracil on the recombination processes that operate in the homologous and heterologous transduction mediated by PBS1 and SP10 phages of Bacillus subtilis. The results obtained demonstrate that the process of heterologous genetic exchange is sensitive to this compound, whereas the homologous process is not. The present data, along with those of our previous work (U. Canosi, A. G. Siccardi, A. Falaschi, and G. Mazza, J. Bacteriol. 126:108--121, 1976), suggest that the DNA polymerase III is involved in the recombination process that operates in transformation and heterologous transduction, whereas homologous transduction follows a partially independent pathway not involving this protein.     

Fate of heterologous deoxyribonucleic acid in Bacillus subtilis.

CsCl density gradient fractionation of cell lysates was employed to follow the fate of Escherichia coli, phage T6, and non-glucosylated phage T6 deoxyribonucleic acid (DNA) after uptake by competent cells of Bacillus subtilis 168 thy minus trp minus. Shortly after uptake, most of the radioactive Escherichia coli or non-glucosylated T6 DNA was found in the denatured form; the remainder of the label was associated with recipient DNA. Incubation of the cells after DNA uptake led to the disappearance of denatured donor DNA and to an increase in the amount of donor label associated with recipient DNA. These findings are analogous to those previously reported with homologous DNA. By contrast, T6 DNA, which is poorly taken up, appeared in the native form shortly after uptake and was degraded on subsequent incubation. The nature of the heterologous DNA fragments associated with recipient DNA was investigated with Escherichia coli 2-H and 3-H-labeled DNA. Association of radioactivity with recipient DNA decreased to one-fourth in the presence of excess thymidine; residual radioactivity could not be separated from recipient DNA by shearing (sonic oscillation) and/or denaturation, but was reduced by one-half in the presence of a DNA replication inhibitor. Residual radioactivity associated with donor DNA under these conditions was about 5% of that originally taken up. Excess thymidine, but not the DNA replication inhibitor, also decreased association of homologous DNA label with recipient DNA; but, even in the presence of both of these, the decrease amounted to only 60%. It is concluded that most, or all, of the Escherichia coli DNA label taken up is associated with recipient DNA in the form of mononucleotides via DNA replication.     

Functional analysis of homologous and heterologous promoters in strawberry fruits using transient expression

The isolation and characterization of fruit-specific promoters are critical for the manipulation of the nutritional value and quality of fruits by genetic engineering. The analysis of regulatory sequences of many ripening-related genes has remained elusive for many species due to their low transformation efficiency and/or lengthy regeneration of a small number of transgenic plants. Strawberry is an important crop and represents one of the most widely studied non-climacteric model systems. However, until recently, its difficult regeneration has limited the functional study of promoters by stable transformation. A protocol based on biolistic transient transformation has been developed in order to study the function of promoters in a fast and efficient manner in strawberry fruits. The protocol has been applied to the study of the GalUR promoter, a gene involved in the biosynthesis of vitamin C in this fruit. The activity of the GalUR promoter is restricted to the fruit, being strictly dependent on light. The analysis of deletion series revealed the presence of a minimum activation region 397 bp upstream of the gene with a putative G-box motif, and a negative regulatory region between -397 and -518 bp, where an I-box was identified. The transient assay has been used to study the activity of the tomato polygalacturonase and the pepper fibrillin promoters in strawberry fruits. Whereas slight activity was observed with the fibrillin promoter, no significant activity was found with the polygalacturonase promoter. The GalUR promoter in transiently transformed ripe tomato fruits showed no activity, indicating the presence of regulatory sequences specific for its function in strawberry fruit.     

Highly efficient expression of homologous and heterologous genes in Bacillus megaterium

Summary By developing an efficient transformation system it was possible to reintroduce two different glucose dehydrogenase genes of Bacillus megaterium into this host. These genes were previously cloned, sequenced and expressed in Escherichia coli. Since the expression of one of these genes (gdhA) turned out to be extremely high in B. megaterium, an expression system for genes from closely and distantly related organisms using the controlling region of the gdhA gene was developed.Dedicated to Professor Dr. Dr. h. c. K. Esser on the occasion of his 65th birthday     

Molecular cloning of heterologous chromosomal DNA by recombination between a plasmid vector and a homologous resident plasmid in Bacillus subtilis

Summary The difficulty experienced in the shotgun cloning of chromosomal DNA on plasmid vectors in Bacillus subtilis is analyzed and an explanation for this difficulty is offered based on an inherent property of competent cells which imposes a requirement of plasmid multimers in transformation of plasmidfree recipients (Canosi et al., 1978). A stratagem which uses cloning by recombination between the vector and a resident homologous plasmid is tested and shown to be successful. Several recombinant plasmids are obtained containing Bacillus licheniformis DNA fragments which complement aromatic amino acid mutants of Bacillus subtilis. The yield of recombinant clones ranges from 6.7 to 210 per g of chromosomal DNA, depending on the selection and the restriction endonuclease. The various trp clones obtained after cutting chromosomal DNA with BglII and BclI do not complement trpE and exhibit both orientations with respect to the vector. The location of several restriction endonuclease cleavage sites in the cloned trp fragments is presented, and their relationship to the genetic map of Bacillus licheniformis is described.Abbreviations Km kanamycin - Cm chloramphenicol - Em erythromycin - CCC covalently closed circular - OC open circular - resistant - MDal megadalton In partial fulfillment of the requirements for the doctoral degree in the Department of Microbiology at the New York University School of Medicine, for S.C.     

不同启动子调控羰基还原酶基因在枯草芽胞杆菌中的表达水平

为了实现羰基还原酶基因mldh在枯草芽胞杆菌中的高效表达,以摩氏摩根菌MorganellamorganiiCMCC（B）49208染色体DNA为模板,PCR扩增得到目的基因mldh,分别与启动子PQ和启动子p43进行连接,构建不同启动子组合的表达载体PHY—p43-mldh、PHY—PQ—mldh、PHY—p43-p43-mldh和PHY—p43-PQ—mldh,化学法转化B．subtilisWb600后对重组茵细胞破碎液进行SDS-PAGE分析及全细胞生物转化反应实验发现,4种重组茵的转化能力差异显著,其中重组菌B．subtilisWb600（PHY—p43-p43-mldh）进行全细胞转化反应,转化液中d-伪麻黄碱的浓度最高,达到142．1mg／L,底物转化率为78．25％,成功实现了羰基还原酶基因mldh在枯草芽胞杆菌中的高效表达。     

Intracellular serine proteinase of Bacillus subtilis strain Marburg 168. Comparison with the homologous enzyme from Bacillus subtilis strain A-50.

Intracellular serine proteinase was isolated from sporulating cells of Bacillus subtilis Marburg 168 by gramicidin S-Sepharose 4B affinity chromatography. The enzymological characteristics, the amino acid composition and the 19 residues of the N-terminal sequence of the enzyme are reported. The isolated proteinase was closely related to, but not completely identical with, the intracellular serine proteinase of B. subtilis A-50. The divergence between these two intracellular enzymes was less than that between the corresponding extracellular serine proteinases (subtilisins) of types Carlsberg and BPN', produced by these bacterial strains. This may be connected with the more strict selection constraints imposed in intracellular enzymes during evolution.     

Function of heterologous and truncated RNase P proteins in Bacillus subtilis

Bacterial RNase P is composed of an RNA subunit and a single protein (encoded by the rnpB and rnpA genes respectively). The Bacillus subtilis rnpA knockdown strain d7 was used to screen for functional conservation among bacterial RNase P proteins from a representative spectrum of bacterial subphyla. We demonstrate conserved function of bacterial RNase P (RnpA) proteins despite low sequence conservation. Even rnpA genes from psychrophilic and thermophilic bacteria rescued growth of B. subtilis d7 bacteria; likewise, terminal extensions and insertions between beta strands 2 and 3, in the so-called metal binding loop, were compatible with RnpA function in B. subtilis. A deletion analysis of B. subtilis RnpA defined the structural elements essential for bacterial RNase P function in vivo. We further extended our complementation analysis in B. subtilis strain d7 to the four individual RNase P protein subunits from three different Archaea, as well as to human Rpp21 and Rpp29 as representatives of eukaryal RNase P. None of these non-bacterial RNase P proteins showed any evidence of being able to replace the B. subtilis RNase P protein in vivo, supporting the notion that archaeal/eukaryal RNase P proteins are evolutionary unrelated to the bacterial RnpA protein.