枯草芽胞杆菌基因组混组方法

基因组混组作为一种育种方法,通过循环原生质体融合等手段,使得不同菌株来源的基因组能够得到充分重组,增加将正向突变整合到同一重组子中的机会。使用4株带有4种不同标记的枯草芽胞杆菌亲本为初始菌株,通过循环转化、循环转导或循环原生质体融合的手段进行基因组混组,统计后代中非亲本类型占整个群体的比例,以衡量基因组混组的效果。分别经过5轮循环原生质体融合、循环转化或者循环转导,结果显示,重组程度较高者在后代群体中的比例较低,带有4种标记的后代未出现,带有3种标记的后代最高分别为4.53×10?4、1.64×10?4、4.47×10?3,明显低于文献报道的天蓝色链霉菌中同样实验的结果:带4种和3种标记的后代分别占2.5%、17%。对比上述实验的结果和文献报道的天蓝色链霉菌、乳杆菌基因组混组的结果,并结合计算机模拟循环融合过程,分析后认为:要达到较充分的基因组混组,需要有能够实现微生物细胞间高频重组的操作技术作为基础,重组频率应该不低于10?3～10?2数量级。     

Partial Genome Scale Analysis of Gene Expression in Human Adipose Tissue Using DNA Array

Objective: Large scale analysis of gene expression in adipose tissue provides a basis for the identification of novel candidate genes involved in the pathophysiology of obesity. Our goal was to explore gene expression in human adipose tissue at a partial genome scale using DNA array. Research Methods and Procedures: Labeled cDNA, derived from human adipose tissue poly(A⁺) RNA, was hybridized to a DNA array containing over 18,000 human expressed sequence‐tagged (EST) clones. The results were analyzed by database searches. Results: Homology searches of the 300 EST clones with highest hybridization signals revealed that 145 contained DNA sequences identical to known genes and 79 could be linked to UniGene clusters. Of the 145 identified genes, 136 were nonredundant and subsequently characterized with respect to function and chromosomal localization by searching MEDLINE, UniGene, GeneMap, OMIM, SWISS‐PROT, the Genome Database, and the Location Data Base. The identified genes were grouped according to their putative functions; cell/organism defense (9.6%), cell division (5.1%), cell signaling/communication (19.8%), cell structure/motility (12.5%), gene/protein expression (16.9%), metabolism (16.2%), and unclassified (19.8%). Less than 50% of these genes have previously been reported to be expressed in adipose tissue. The chromosomal localization of 268 genes strongly expressed in adipose tissue showed that their relative abundance was significantly increased on chromosomes 11, 19, and 22 compared to the expected distribution of the same number of random genes. Discussion: Our study resulted in the identification of numerous genes previously not reported to be expressed in adipose tissue. These results suggest that DNA array is a powerful tool in the search for novel regulatory pathways within adipose tissue on a scale that is not possible using conventional methods.     

DNA Repair and Genome Maintenance in Bacillus subtilis

Summary: From microbes to multicellular eukaryotic organisms, all cells contain pathways responsible for genome maintenance. DNA replication allows for the faithful duplication of the genome, whereas DNA repair pathways preserve DNA integrity in response to damage originating from endogenous and exogenous sources. The basic pathways important for DNA replication and repair are often conserved throughout biology. In bacteria, high-fidelity repair is balanced with low-fidelity repair and mutagenesis. Such a balance is important for maintaining viability while providing an opportunity for the advantageous selection of mutations when faced with a changing environment. Over the last decade, studies of DNA repair pathways in bacteria have demonstrated considerable differences between Gram-positive and Gram-negative organisms. Here we review and discuss the DNA repair, genome maintenance, and DNA damage checkpoint pathways of the Gram-positive bacterium Bacillus subtilis. We present their molecular mechanisms and compare the functions and regulation of several pathways with known information on other organisms. We also discuss DNA repair during different growth phases and the developmental program of sporulation. In summary, we present a review of the function, regulation, and molecular mechanisms of DNA repair and mutagenesis in Gram-positive bacteria, with a strong emphasis on B. subtilis.     

Genome engineering reveals large dispensable regions in Bacillus subtilis

Bacterial genomes contain 250 to 500 essential genes, as suggested by single gene disruptions and theoretical considerations. If this view is correct, the remaining nonessential genes of an organism, such as Bacillus subtilis, have been acquired during evolution in its perpetually changing ecological niches. Notably, approximately 47% of the approximately 4,100 genes of B. subtilis belong to paralogous gene families in which several members have overlapping functions. Thus, essential gene functions will outnumber essential genes. To answer the question to what extent the most recently acquired DNA contributes to the life of B. subtilis under standard laboratory growth conditions, we initiated a "reconstruction" of the B. subtilis genome by removing prophages and AT-rich islands. Stepwise deletion of two prophages (SPbeta, PBSX), three prophage-like regions, and the largest operon of B. subtilis (pks) resulted in a genome reduction of 7.7% and elimination of 332 genes. The resulting strain was phenotypically characterized by metabolic flux analysis, proteomics, and specific assays for protein secretion, competence development, sporulation, and cell motility. We show that genome engineering is a feasible strategy for functional analysis of large gene clusters, and that removal of dispensable genomic regions may pave the way toward an optimized Bacillus cell factory.     

Expression of the cloned xylanases from an alkalophilic,thermophilic Bacillus in Bacillus subtilis

A recombinant plasmid construct, pLPX6.5, harbouring a 6.5 kb Hind III fragment of genomic DNA, from an alkalophilic, thermophilic Bacillus NCIM 59 and coding for xylanase activity, was electroporatically transformed into Bacillus subtilis MI 111. The expression of the recombinant xylanases was confirmed by cross-reactivity with antibodies raised against purified xylanase II (M _r 15,800) from NCIM 59. However, as there were different xylan hydrolysis products from NCIM 59 and the host B. subtilis, the two xylanases appear to have different modes of action. Xylanase expression in the transformants was 6-fold higher than in the host. There was no significant enhancement in the expression of recombinant xylanases by adding xylan to the growth medium.The authors are with the Division of Biochemical Sciences, National Chemical Laboratory, Pune-411008, India     

Genome engineering using a synthetic gene circuit in Bacillus subtilis

Genome engineering without leaving foreign DNA behind requires an efficient counter-selectable marker system. Here, we developed a genome engineering method in Bacillus subtilis using a synthetic gene circuit as a counter-selectable marker system. The system contained two repressible promoters (B. subtilis xylA (P_xyl) and spac (P_spac)) and two repressor genes (lacI and xylR). P_xyl-lacI was integrated into the B. subtilis genome with a target gene containing a desired mutation. The xylR and P_spac–chloramphenicol resistant genes (cat) were located on a helper plasmid. In the presence of xylose, repression of XylR by xylose induced LacI expression, the LacIs repressed the P_spac promoter and the cells become chloramphenicol sensitive. Thus, to survive in the presence of chloramphenicol, the cell must delete P_xyl-lacI by recombination between the wild-type and mutated target genes. The recombination leads to mutation of the target gene. The remaining helper plasmid was removed easily under the chloramphenicol absent condition. In this study, we showed base insertion, deletion and point mutation of the B. subtilis genome without leaving any foreign DNA behind. Additionally, we successfully deleted a 2-kb gene (amyE) and a 38-kb operon (ppsABCDE). This method will be useful to construct designer Bacillus strains for various industrial applications.     

富含蛋氨酸玉米醇溶蛋白在枯草芽胞杆菌中的表达

蛋氨酸是畜禽饲料中的第一限制性氨基酸,也是饲料产品进行质量控制与质量评价最为关注的指标之一。利用基因工程方法,从玉米胚乳中克隆高蛋氨酸蛋白基因（10kuδzein）,与pHT43构建重组表达质粒,将其转入枯草芽胞杆菌中,IPTG诱导其表达,发现重组菌在26ku处出现了1条明显条带。HPLC检测蛋氨酸含量,重组菌的蛋氨酸含量比野生型菌株提高了20．51％。该重组菌为以后将其做为饲料添加剂进一步应用提供了技术基础。     

Gene Expression After Transformation in Bacillus subtilis

Sensitive assays of histidase activity were used to follow the production of this enzyme as directed by a gene newly introduced into cells of Bacillus subtilis by transformation. Histidase activity can be detected in histidase-negative recipient cells within 1 hr after the addition of deoxyribonucleic acid extracted from histidase-positive donors. Enzyme production continues for one to two additional hours and then ceases. Histidase production in the transformed cells is fully sensitive to catabolite repression. Catabolite repression is rapidly established after transformation of recipient cells that are resistant to this form of regulation.