Cloning and Sequencing of a 36-kb Region of the Bacillus subtilis Genome between the gnt and iol Operons

Within the framework of an international project for the sequencingof the entire Bacillus subtilis genome, a 36-kb chromosome segment,which covers the region between the gnt and iol operons, hasbeen cloned and sequenced. This region (36447 bp) contains 33complete open reading frames (ORFs; genes) including the fourgnt genes and one partial gene. A homology search for the productsof the 33 complete ORFs revealed significant homology to knownproteins in 16 of them such as tetracycline resistance protein(Clostridium perfringens), asparagine synthetase (Arabidopsisthaliana), aldehyde dehydrogenase (Pseudomonas oleovorans),2,5-dichloro-2,5-cyclohexadiene-1,4-diol dehydrogenase (P. paucimobilis),heat shock protein HtpG (Escherichia coli), galactose-protonsymporter (E. coli), auxin-induced protein (common tobacco),glucitol operon repressor (E. coli) and methylmalonate-semialdehydedehydrogenase (P. aeruginosa). Unlike the regions we sequencedso far, this region contained two short sequence multiplications:one was a tandem sequence duplication (409 and 410 bp), andthe other a triplication consisting of two highly conserved118-bp tandem sequences preceded by a less conserved similarsequence (129 bp). The reasons for the presence of these sequencemultiplications in the gnt to iol region were deduced.     

Sequence Analysis of the groESL-cotA Region of the Bacillus subtilis Genome, Containing the Restriction/Modification System Genes

We have determined a 35-kb sequence of the groESL-gutR-cotA(45°–52°) region of the Bacillus subtilis genome.In addition to the groESL, gutRB and cotA genes reported previously,we have newly identified 24 ORFs including gutA and fruC genes,encoding glucitol permease and fructokinase, respectively. Theinherent restriction/modification system genes, hsdMR and hsdMM,were mapped between groESL and gutRB, and we have identifiedtwo open reading frames (ORFs) encoding 5-methylcytosine formingDNA methyl transferase and an operon probably encoding a restrictionenzyme complex. The unusual genome structure of few ORFs andlower GC content around the restriction/modification genes stronglysuggests that the region originated from a bacteriophage integratedduring evolution.     

Cloning and Sequencing of a 23-kb Region of the Bacillus subtilis Genome between the iol and hut Operons

Within the framework of an international project for the sequencingof the entire Bacillus subtilis genome, a 23-kb chromosomalsegment, which covers the region between the iol and hut operons,has been cloned and sequenced, creating a 99-kb contig fromthe gnt operon to the wapA locus. This region (23351 bp) contains25 complete open reading frames (ORFs; genes) including deoR,dra, nupC and pdp and two partial ones. The region (5140 bp)containing these four genes, being also sequenced by H. H. Saxildet al., was sequenced by subjecting a long polymerase chainreaction product to random sequencing using phage M13mp19. However,we could detect no conflict, between two independently determinedsequences, which could be attributed to our sequencing method.A homology search for the 24 newly identified gene productsrevealed significant homology to known proteins in 14 of them.It was notable that three proteins, encoded by the successivegenes (yxeMNO), exhibited meaningful homology to the E. coliGlnHPQ products constituting a periplasmic ATP-dependent transportsystem for glutamine.     

Systematic Sequencing of the 180 Kilobase Region of the Bacillus Subtilis Chromosome Containing the Replication Origin

We have determined a 180 kb contiguous sequence in the replicationorigin region of the Bacillus subtilis chromosome. Open readingframes (ORF) in this region were unambiguously identified fromthe determined sequence, using criteria characteristic for theB. subtilis gene structure, i.e., starting with an ATG, GTGor TTG codon preceded by sequences complementary to the 3' endof the 16S rRNA. Four rRNA gene sets, 7 individual tRNA genesand 1 scRNA gene were identified, occupying 20 kb in total.In the remaining 160 kb region, 158 ORFs were identified, suggestingthat 1 ORF is coded on average by 1 kb of DNA of the B. subtilisgenome. Among the 158 ORFs, the functions of 48 ORFs were assignedand those of 11 ORFs are suggested through significant similaritiesto known proteins present in data banks. However, the functionsof more than half of the ORFs (63%) remain to be determined.     

Sequence Analysis of the Bacillus subtilis 168 Chromosome Region between the sspC and odhA Loci (184{degrees}-180{degrees})

The nucleotide sequence of 45,389 bp in the 184°-;180°region of the Bacillus subtilis chromosome, containing the cgecluster, which is controlled by the sporulation regulatory proteinGerE, was determined. Fifty-four putative ORFs with putativeribosome-binding sites were recognized. Seven of them correspondto previously characterized genes: cgeB, cgeA, cgeC, cgeD, cgeE,ctpA, and odhA. The deduced products of 25 ORFs were found todisplay significant similarities to proteins in the data banks.We have identified genes involved in detoxification, cell walls,and in the metabolism of biotins, purines, fatty acids, carbohydratesand amino acids. The remaining 22 ORFs showed no similarityto known proteins. Both an attachment site of the SPßprophage and 2 new putative DNA replication terminators wereidentified in this region.     

Cloning and Sequencing of a 27.8-kb Nucleotide Sequence of the 79{degrees}-81{degrees}. Region of the Bacillus subtilis Genome Containing the sspE Locus

The nucleotide sequence of a 27830-bp DNA segment in the 79°–81°.region of the Bacillus subtilis genome has been determined.This region contains 29 complete ORFs including the sspE gene,which encodes a small acid-soluble spore protein gamma and locateson the one side terminal of our assigned region. A homologysearch for the products deduced from the 29 ORFs revealed thatnine of them exhibit significant similarity to known proteins,e.g. proteins involved in an iron uptake system, a multidrugresistance protein, a chloramphenicol resistance protein, epoxidehydrolase, adenine glycosylase, and a glucose-1-dehydrogenasehomolog.     

青霉素酶基因在枯草芽孢杆菌中的高效表达

以获得大量胞外青霉素酶为目的,将青霉素酶基因克隆至表达载体pWB980中,并转化到双蛋白酶缺陷的Bacillus subtilis DB104。重组菌在LB培养基中培养24小时后, SDS-PAGE分析发现目的蛋白分子量为28kDa,酶活力为339U/mL;通过筛选7种不同的发酵培养基发现4#培养基更利于青霉素酶的表达,最大酶活力为1580U/mL,较优化前提高了3.66倍,并对该重组菌进行了7L罐放大实验,结果显示在培养24小时产酶达到高峰,酶活力为1255.8 U/mL。     

Sequence Analysis of a 32-kb Region Including the Major Ribosomal Protein Gene Clusters from Alkaliphilic Bacillus sp. Strain C-125

Forty-one open reading frames (ORFs) were identified in a 32-kb DNA fragment of alkaliphilic Bacillus sp. C-125. A similarity search using the BSORF database found 37 ORFs with significant sequence similarity to B. subtilis RNA polymerase subunits, elongation factor G, elongation factor Tu, and ribosomal proteins. Each ORF product showed more than 70% identity to those of B. subtilis. Gene organization in the region of str, S10, spc, and the α cluster was highly conserved among three strains, C-125, B. subtilis, and B. stearothermophilus.     

Sequence Analysis of a 25-kb Segment in the 17{degrees}-19{degrees} Region of the Bacillus subtilis Chromosome Containing ada Locus

As a part of the Bacillus subtilis genome sequencing project,we have determined a 25-kb sequence covering the 17°–19°region. This region contains 26 complete open reading frames(ORFs) including the alkA and adaA/B operon, which encode genesfor adaptive response to DNA alkylation. A homology search forthe newly identified 21 ORFs revealed that 4 of them exhibita significant similarity to known proteins, e.g., methicillin-resistantStaphylococcus aureus (MRSA) protein homolog, proteins involvedin chloramphenicol resistance, glucosamine synthase and an ABCtransporter protein. The remaining 17 ORFs did not show anysignificant sequence similarities to known gene products inthe database.     

Enhanced Hydrocarbons Degradation in the Rhizosphere of Mangrove Plants by a Halophilic Bacillus Subtilis Subtilis Strain

Sulaibikhat Embayment is a severely contaminated coastline in the State of Kuwait. The contaminating pollutants include hydrocarbons, heavy metals, and suspended particles. The objective of this study is to assess the ability of mangroves planted in the Sulaibikhat Embayment to enhance hydrocarbons degradation by the activities of rhizospheric hydrocarbon degrading bacteria (HDB). Accordingly, samples were collected from the rhizosphere of selected mangrove plants and from sediments in the same location but away from mangrove marshes. The samples were analyzed chemically and microbiologically before being enriched with a mixture of hydrocarbon compounds (HC) to isolate HDB.

A number of halophilic HDB were isolated from mangroves rhizosphere and the surrounding sediments such as Pseudomonas balearica, Microbacterium barkeri and Gordonia soli. On the other hand, Bacillus velezensis and Bacillus subtilis subtilis were both isolated only from mangroves rhizosphere. Among the isolated HDB, Bacillus subtilis subtilis was distinguished with its high degradation rates of the tested HC including poly aromatic hydrocarbons. According to our knowledge, this is the first Bacillus subtilis HC-degrading strain that was isolated from Kuwait Bay and from mangroves rhizosphere.     

嗜酸乳杆菌LB10.16及枯草芽孢杆菌BS7.29安全性评价

目的：评价枯草芽孢杆菌BS7.29和嗜酸乳杆菌LB10.16的安全性,为其应用研究提供技术支持。方法：本文通过对小鼠灌服、急性攻毒、腹腔注射、小鼠最大耐受剂量、急性攻毒等试验方法进行枯草芽孢杆菌BS7.29和嗜酸乳杆菌LB10.16的安全性研究。结果：1×108cfu/kg.bw的LB10.16和5×108cfu/kg.bw的BS7.29能够明显提高肠道推动力,改善小鼠肠道蠕动性。结论：两个菌种急性攻毒试验结果表明属于无毒物质,安全性比较高。     

An 8 kb Nucleotide Sequence at the 3' Flanking Region of the sspC Gene (184{degrees}) on the Bacillus subtilis 168 Chromosome Containing an Intein and an Intron

As part of the Bacillus subtilis genome sequencing project,we determined the complete nucleotide sequence of an 8000-bpfragment downstream of the sspC gene (184°) of the B. subtilis168 chromosome. The sequence analysis shows that the sspC geneis located inside of the SPß region, which differsfrom the current genetic map of B. subtilis 168. This regioncontains 12 putative ORFs (yojQ through yojZ and sspC). A homologysearch for the deduced products of the ORFs shows signi.cantsimilarities to enzymes involved in deoxyribonucleotide metabolism:ribonucleotide reductase (Nrd) E, NrdF, thioredoxinand dUTPase.Interestingly, this DNA fragment includes two split genes, yojPcontaining conserved motifs of an intein and yojQ and yojS withan 808-bp intervening sequence for a putative intron structure.In addition, the yojR gene includes a putative new DNA replicationterminator.     

环状芽孢杆菌几丁质酶基因序列分析、表达和生物活性测定

真菌病害一直是影响作物的主要病害之一 ,每年造成巨大经济损失。几丁质酶可水解许多病原真菌细胞壁所含有的主要成分—几丁质 ,是研究得最多的抗真菌蛋白质。许多几丁质酶基因已从微生物中克隆到 ,芽孢杆菌是一类重要的几丁质酶产生菌。环状芽孢杆菌可产生并分泌多种多糖降解酶类 ,包括几丁质酶、β 1 ,3 葡聚糖酶、β 1 ,6葡聚糖酶和半纤维素酶[1] 。Ｗａｔａｎａｂｅ克隆了环状芽孢杆菌ＷＬ 1 2菌株的几丁质酶基因ｃｈｉＡ和ｃｈｉＤ ,对该几丁质酶基因的结构和功能进行了深入研究[2～ 4 ] 。我国的陈三凤克隆了黄杆菌的几丁质酶基因 ,…     

嗜酸乳杆菌LB10.16及枯草芽孢杆菌BS7.29安全性评价

目的:评价枯草芽孢杆菌BS7.29和嗜酸乳杆菌LB10.16的安全性,为其应用研究提供技术支持。方法:本文通过对小鼠灌服、急性攻毒、腹腔注射、小鼠最大耐受剂量、急性攻毒等试验方法进行枯草芽孢杆菌BS7.29和嗜酸乳杆菌LB10.16的安全性研究。结果:1×108cfu/kg.bw的LB10.16和5×108cfu/kg.bw的BS7.29能够明显提高肠道推动力,改善小鼠肠道蠕动性。结论:两个菌种急性攻毒试验结果表明属于无毒物质,安全性比较高。     

枯草芽孢杆菌二联活菌的生物学特性及其基因组分析

摘要 目的：枯草芽孢杆菌二联活菌是我国临床上应用比较广泛的微生态制剂。为了评估该种微生态制剂对肠道健康的影响,对其含有的两种细菌枯草芽孢杆菌R-179和屎肠球菌R-026的生物学特性和耐药性展开研究。方法：通过细菌生长曲线的测定,分析了两种细菌在好氧和厌氧条件下生长状况和氧化还原电位,并对两种菌的抗生素耐药特性进行了检测;同时采用三代测序技术对两株细菌进行了基因组测序和生物信息学分析。结果：枯草芽孢杆菌R-179在好氧条件下生长,厌氧情况下并不生长,其在肠道环境中可以有效的消耗氧气;两种菌混合培养可以有效降低氧化还原电位,耐药性分析表明该两种菌对青霉素、万古霉素敏感,基因组分析表明该两种菌并不含有耐药转移元件,而且枯草芽孢杆菌还分泌多种抗菌活性物质。结论：利用现代研究手段,结合最新测序技术,挖掘和探讨了枯草芽孢杆菌二联活菌在肠道中的作用机制,为枯草杆菌二联活菌用于临床消化道疾病治疗的机理研究和安全应用评价提供了重要的临床参考。     

Nucleotide Sequence and Features of the Bacillus licheniformis gnt Operon

Bacillus licheniformis was able to utilize gluconate as thesole carbon source as efficiently as Bacillus subtilis did.Southern analysis indicated that B. licheniformis likely possessesonly one gnt determinant. The nucleotide sequence (6278 bp)of the B. licheniformis DNA containing the gnt operon was determined,revealing the five complete open reading frames (ORF; genes).The putative product of the first gene, oug, did not show anysignificant homology to known proteins, but those of the secondto fifth genes exhibited striking homology to the gntRKPZ genesof B. subtilis, respectively, indicating that they are the correspondinggnt genes of B. licheniformis. Not only is the organizationof the gnt genes of these two Bacilli highly conserved, butso are the cis regulatory elements of their gnt operon. Sequenceanalysis of the upstream regions of these two gnt operons impliedthat a chromosome rearrangement in B. subtilis might have occurredimmediately upstream of the gnt operon during evolution, causingit to diverge from a common ancestor into B. licheniformis andB. subtilis.     

Bacillus subtilis WHNB02植酸酶phyC基因的克隆及序列分析

采用PCR法获得产植酸酶芽孢杆菌(Bacillus subtilis)WHNB02株植酸酶的全长phyc基因,并将其克隆到pUC18-T载体。序列分析表明该基因全长1152bp,编码一个383个氨基酸的多肽,信号肽切割位点位于第26个氨基酸残基之后。系统进化树表明,来源于7株芽孢杆菌的植酸酶在遗传上分为两大类。将Bacillus subtilis WHNB02植酸酶phyC基因序列及其氨基酸序列在GenBank中登录,登录号分别为AF220075和AA043434．1。