Draft Genome Sequence of the Marine Streptomyces sp. Strain AA1529, Isolated from the Yellow Sea

Here we report the draft genome sequence of a Streptomyces strain, AA1529, isolated from marine sediment from the Yellow Sea. Its genome contains a subset of unique genes and gene clusters that encode diverse secondary metabolites, suggesting great potential as a source for the discovery of novel gene clusters and bioactive compounds.     

Identification and molecular characterization of a novel Bacillus strain capable of degrading Tween-80

A Tween-80-degrading novel marine Bacillus strain, N10, has recently been isolated in Alexandria University, Egypt. The taxonomic position of this endospore forming bacterium was investigated on the basis of fatty acid analysis and 16S rRNA gene sequencing. Comparative computer database analyses revealed that the bacterium is a Bacillus subtilis strain. The gene encoding the small acid-soluble protein gamma-type (SASP-B), sspE, was successfully utilized in this study as a tool for discrimination between the two B. subtilis subspecies W23 and 168. Based on the alignment of 16S rRNA sequences and analysis of SASP-B relatedness, it has been demonstrated that the novel marine B. subtilis strain N10 is more closely related to the B. subtilis reference strain W23 than to 168. The strain, N10, has been deposited in the Bacillus Genetic Stock Center (BGSC) and assigned the accession number 3A17.     

Draft Genome Sequence of Marine-Derived Streptomyces sp. Strain AA0539, Isolated from the Yellow Sea,China

Here, we report the draft genome sequence of Streptomyces sp. strain AA0539, isolated from marine sediment of the Yellow Sea, China. Its small genome (∼5.8 Mb) contains large, unique genes and gene clusters for diverse secondary metabolites, suggesting great potential as a source for the discovery of novel natural products.     

Draft genome sequence of the marine Streptomyces sp. strain PP-C42, isolated from the Baltic Sea

Streptomyces, a branch of aerobic Gram-positive bacteria, represents the largest genus of actinobacteria. The streptomycetes are characterized by a complex secondary metabolism and produce over two-thirds of the clinically used natural antibiotics today. Here we report the draft genome sequence of a Streptomyces strain, PP-C42, isolated from the marine environment. A subset of unique genes and gene clusters for diverse secondary metabolites as well as antimicrobial peptides could be identified from the genome, showing great promise as a source for novel bioactive compounds.     

From genome to function: systematic analysis of the soil bacterium bacillus subtilis

Bacillus subtilis is a sporulating Gram-positive bacterium that lives primarily in the soil and associated water sources. Whilst this bacterium has been studied extensively in the laboratory, relatively few studies have been undertaken to study its activity in natural environments. The publication of the B. subtilis genome sequence and subsequent systematic functional analysis programme have provided an opportunity to develop tools for analysing the role and expression of Bacillus genes in situ. In this paper we discuss analytical approaches that are being developed to relate genes to function in environments such as the rhizosphere.     

A Bacillus cereus cytolytic determinant, cereolysin AB, which comprises the phospholipase C and sphingomyelinase genes: nucleotide sequence and genetic linkage.

A cloned cytolytic determinant from the genome of Bacillus cereus GP-4 has been characterized at the molecular level. Nucleotide sequence determination revealed the presence of two open reading frames. Both open reading frames were found by deletion and complementation analysis to be necessary for expression of the hemolytic phenotype by Bacillus subtilis and Escherichia coli hosts. The 5' open reading frame was found to be nearly identical to a recently reported phospholipase C gene derived from a mutant B. cereus strain which overexpresses the respective protein, and it conferred a lecithinase-positive phenotype to the B. subtilis host. The 3' open reading frame encoded a sphingomyelinase. The two tandemly encoded activities, phospholipase C and sphingomyelinase, constitute a biologically functional cytolytic determinant of B. cereus termed cereolysin AB.     

Thermostable chitosanase from Bacillus sp. Strain CK4: cloning and expression of the gene and characterization of the enzyme

A thermostable chitosanase gene from the environmental isolate Bacillus sp. strain CK4, which was identified on the basis of phylogenetic analysis of the 16S rRNA gene sequence and phenotypic analysis, was cloned, and its complete DNA sequence was determined. The thermostable chitosanase gene was composed of an 822-bp open reading frame which encodes a protein of 242 amino acids and a signal peptide corresponding to a 30-kDa enzyme. The deduced amino acid sequence of the chitosanase from Bacillus sp. strain CK4 exhibits 76.6, 15.3, and 14.2% similarities to those from Bacillus subtilis, Bacillus ehemensis, and Bacillus circulans, respectively. C-terminal homology analysis shows that Bacillus sp. strain CK4 belongs to cluster III with B. subtilis. The gene was similar in size to that of the mesophile B. subtilis but showed a higher preference for codons ending in G or C. The enzyme contains 2 additional cysteine residues at positions 49 and 211. The recombinant chitosanase has been purified to homogeneity by using only two steps with column chromatography. The half-life of the enzyme was 90 min at 80 degrees C, which indicates its usefulness for industrial applications. The enzyme had a useful reactivity and a high specific activity for producing functional oligosaccharides as well, with trimers through hexamers as the major products.     

Recombinational transfer of 100-kilobase genomic DNA to plasmid in Bacillus subtilis 168

Transformation of Bacillus subtilis by a plasmid requires a circular multimeric form. In contrast, linearized plasmids can be circularized only when homologous sequences are present in the host genome. A recombinational transfer system was constructed with this intrinsic B. subtilis recombinational repair pathway. The vector, pGETS103, a derivative of the theta-type replicating plasmid pTB19 of thermophilic Bacillus, had the full length of Escherichia coli plasmid pBR322. A multimeric form of pGETS103 yielded tetracycline-resistant transformants of B. subtilis. In contrast, linearized pGETS103 gave tetracycline-resistant transformants only when the recipient strain had the pBR322 sequence in the genome. The efficiency and fidelity of the recombinational transfer of DNAs of up to 90 kb are demonstrated.     

抗MRSA菌株B2的鉴定和活性检测研究

目的寻找新型抗MRSA菌株并进行鉴定。方法:通过传统的形态学、生理生化鉴定,并基于16S rDNA的进一步分析,进行活性检测。结论:通过活性检测,发现本实验室分离到的菌株B2具有抑制MRSA生长活性,通过传统的形态学、生理生化鉴定,发现它与芽孢杆菌特征相符,基于16S rDNA的进一步分析表明,B2菌株与枯草芽孢杆菌有高度同源性,故鉴定为枯草芽孢杆菌B2。     

Complete genome sequence of Bacillus subtilis BSn5, an endophytic bacterium of Amorphophallus konjac with antimicrobial activity for the plant pathogen Erwinia carotovora subsp. carotovora

Here, we present the complete genome sequence of Bacillus subtilis strain BSn5, isolated from Amorphophallus konjac calli tissue and showing strong inhibitory activity to Erwinia carotovora subsp. carotovora, which causes Amorphophallus soft rot disease and affects the industry development of this organism.     

A new method for multiple gene inactivations in Bacillus subtilis 168, producing a strain free of selectable markers

This study describes a novel method for repeated gene inactivation in Bacillus subtilis 168. A B. subtilis strain (BS-PS) that is conditionally auxotrophic for lysine was obtained by replacing the PlysA promoter with the Pspac promoter. The homologous recombination integration vector PLC-T was constructed to contain lacI, which encodes a Pspac promoter repressor, and the chloromycetin resistance gene. Target genes were manipulated by generating an insertion sequence with two homologous arms and the target gene in PLC-T to create a specific integrating vector. Integration into the BS-PS chromosome occurred by a single crossover at either of the two homologous arms. The resulting transitional strain (BS-PS-PI) was chloromycetin resistant and lysine auxotrophic and had an unstable genome structure because of the duplication. Excision of lacI and chloromycetin resistance gene was achieved by a second single crossover at the duplication. Recovery of a lysine prototroph functioned as counter-selection and was identified by PCR. In this work, we inactivated nprE and aprE, two protease genes secreted by B. subtilis 168 free of selectable markers.     

Expression of a small RNA, BS203 RNA, from the yocI-yocJ intergenic region of Bacillus subtilis genome

We isolated and characterized a novel small RNA from Bacillus subtilis. We termed this molecule BS203 RNA from the length of its mature form (203 nt) and located the corresponding gene at the yocI-yocJ intergenic region on the B. subtilis genome. Northern blotting revealed that it is transcribed in vegetative growing cells and that the amount of BS203 RNA decreased in the middle of the vegetative phase. A computer-aided prediction of the BS203 RNA secondary structure revealed three characteristic stem-loop structures. Despite active expression during the vegetative phase, growth of the knockout mutant was not affected by depletion of BS203 RNA. A phylogenetic comparison of the sequence of the BS203 RNA with other Bacillus species including B. cereus and B. halodurans C-125, or Clostridium perfringens suggests that the sequence is unique to Bacillus subtilis.     

一株海洋链霉菌MMHS020的抗菌活性代谢产物

【背景】海洋微生物因其生存环境的多样性与独特性,已成为天然产物研究的重要来源。【目的】以一株太平洋海泥来源链霉菌MMHS020为出发菌株,筛选可促进其产生丰富代谢产物的发酵条件,挖掘菌株在抗菌抗肿瘤方面的潜力。【方法】采用单菌株多次级代谢产物策略对MMHS020菌株进行培养诱导,使其产生更丰富的活性代谢产物。双层平板法测定发酵产物对6种指示菌的抑菌活性。以硅胶柱层析、葡聚糖凝胶层析和制备层析等方法对代谢产物进行分离纯化,再通过质谱技术和~1H-NMR和~(13)C-NMR对化合物进行结构解析。【结果】链霉菌属MMHS020菌株可在较高浓度盐离子环境中产生丰富的抑菌活性代谢产物,显示出对枯草芽孢杆菌、结核分枝杆菌和藤黄微球菌等多种指示菌的抑制活性。从发酵产物中分离鉴定了3个化合物,分别是诺卡胺素(1)、麦角甾醇(2)和星形孢菌素(3)。其中星形孢菌素表现出白色念珠菌的抑制活性,而诺卡胺素则对其他几个指示菌表现出较强的抑制活性。【结论】海洋链霉菌MMHS020菌株可代谢产生丰富多样的生物活性物质,具有开发成为新型抑菌生物制剂的潜力。     

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.     

一种快速分离检测产脂肽类生物表面活性剂枯草芽孢杆菌的方法

脂肽(Lipopeptide)是由枯草芽孢杆菌(Bacillus subtilis)等微生物产生的一类具有较强表面活性的生物表面活性剂.枯革杆菌磷酸泛酰巯基转移酶基因(afp)是枯草芽孢杆菌中参与脂肽代谢的功能性基因.采用sfp基因PCR对从环境中得到的一组产生表面活性剂的微生物进行筛选,结合Tricine-SDS-PAGE电泳对PCR结果呈阳性的菌蛛的代谢粗初提物进行检测,初步鉴定得到两株枯草芽孢杆菌.进一步利用16S rDNA序列的系统发育学分析确定这两种菌株为枯草芽孢杆菌,并利用TLC、HPLC鉴定其产物为脂肽类表面活性剂,从而建立了一套快速分离检测产生脂肽类生物表面活性剂的枯草芽孢杆菌方法.     

From gene regulation to gene function: regulatory networks in bacillus subtilis

Bacillus subtilis is a sporulating Gram-positive bacterium that lives primarily in the soil and associated water sources. The publication of the B. subtilis genome sequence and subsequent systematic functional analysis and gene regulation programmes, together with an extensive understanding of its biochemistry and physiology, makes this micro-organism a prime candidate in which to model regulatory networks in silico. In this paper we discuss combined molecular biological and bioinformatical approaches that are being developed to model this organism's responses to changes in its environment.     

A gene encoding a tyrosine tRNA synthetase is located near sacS in Bacillus subtilis.

Within the frame of an attempt to sequence the whole Bacillus subtilis genome, a region of 5.5 kbp of the B. subtilis chromosome near the sacS locus has been sequenced. It contains five complete coding sequences, including the sequence of sacY, three unknown CDS and a sequence coding for a tyrosine tRNA synthetase. That the corresponding CDS encodes a functional synthetase has been demonstrated by complementation of an Escherichia coli mutant possessing a thermosensitive tRNA synthetase. Insertion of a kanamycin resistance cassette in the B. subtilis chromosome at the corresponding locus resulted, however, in no apparent phenotype, demonstrating that this synthetase is dispensable. Finally phylogenetic relationships between known tyrosine and tryptophan tRNA synthetases are discussed.     

一株以葡萄糖为底物产2,3-丁二醇微生物菌株的筛选及鉴定

从自然界中筛选出一批以葡萄糖为底物发酵产2,3-丁二醇的菌株,经初步发酵测定发酵液中2,3-丁二醇含量,其中菌株6-7的2,3-丁二醇产量最高达49.6g/L。对其进行常规生理生化鉴定实验,并结合16SrDNA序列分析,比对结果表明,菌株6-7与Bacillus subtilis strain BIHB332相似性达99%。在细菌分类学上属于枯草芽孢杆菌属,将其命名为Bacillussubtilis6-7。其特点是属于环境友好和食品安全型菌株,因此,利用Bacillus subtilis6-7生产2,3-丁二醇具有良好的工业应用价值。