Cloning of the Streptococcus thermophilus beta-galactosidase gene and its expression in Escherichia coli and Bacillus subtilis cells

The beta-galactosidase gene from the chromosome of Streptococcus thermophilus, strain 6 kb, has been cloned on a vector plasmid pBR322. The corresponding gene has been found to be located on the Pst1 DNA fragment. The restriction map of this 6 kb fragment has been constructed. The shortening of the DNA fragment carrying the beta-galactosidase gene has been achieved by digestion of the recombinant derivative of pBR322 by the restriction endonuclease Sau3A under the conditions of incomplete hydrolysis. The obtained fragments have been cloned into the BamHI site in the berepliconed shuttle vector pCB20 for grampositive and gramnegative bacteria. The obtained recombinant plasmids contained the beta-galactosidase gene in the inserted fragments of different length. Expression of the cloned beta-galactosidase gene in Escherichia coli and Bacillus subtilis cells has been studied.     

Cloning of the BamHI methyl transferase gene from Bacillus amyloliquefaciens

We wish to report the initial characterization of a recombinant clone containing the BamHI methylase gene. Genomic chromosomal DNA purified from Bacillus amyloliquefaciens was partially cleaved with HindIII, fractionated by size, and cloned into pSP64. Plasmid DNA from this library was challenged with BamHI endonuclease and transformed into Escherichia coli HB101. A recombinant plasmid pBamM6.5 and a subclone pBamM2.5 were shown to contain the BamHI methylase gene based on three independent observations. Both plasmids were found to be resistant to BamHI endonuclease cleavage, and chromosomal DNA isolated from E. coli HB101 cells harboring either of the plasmids pBamM6.5 or pBamM2.5 was resistant to cleavage by BamHI endonuclease. In addition, DNA isolated from lambda phage passaged through E. coli HB101 containing either plasmid was also resistant to BamHI cleavage. Expression of the BamHI methylase gene is dependent on orientation in pSP64. In these clones preliminary evidence indicates that methylase gene expression may be under the direction of the plasmid encoded LacZ promoter.     

C型产气荚膜梭菌β1毒素基因表达

利用PCR技术,从C型产气荚膜梭菌染色体DNA中扩增出β1毒素基因,然后用限制性核酸内切酶BamHI和EcoRI对其进行双酶切处理,回收0．95kb的β1毒素基因片段,最后将其定向克隆在事先经同样内切酶处理的载体pET-28c中相应位点上,转化至受体菌B121(DE3)qh。经BamHI和Eco RI双酶切鉴定和核苷酸序列测定证实,构建的重组质粒pETXBl含有B毒素基因,并且具有正确的基因序列和阅读框架。重组菌株BI21(DE3)(pETXB1)经IPIG诱导后,其表达产物经ELISA检测和SDS-PAGE分析,结果表明重组菌株可以高效表达β1毒素蛋白,该蛋白占菌体总蛋白相对含量的12．24％。     

Cloning of the modification methylase gene of Bacillus centrosporus in Escherichia coli

The gene specifying a sequence-specific modification methylase of Bacillus centrosporus has been cloned in Escherichia coli using the restriction endonuclease HindIII and the plasmid pBR322. The selection was based on detection of new methylation properties rendering recombinant plasmids carrying the methylase gene nonsusceptible to BcnI endonuclease cleavage. The presence of a 3.2-kb HindIII fragment in either orientation conferred BcnI resistance on the recombinant plasmids. These results suggest that the BcnI methylase gene is expressed in E. coli under the control of a promoter located on the cloned fragment. The relative level of BcnI methylase enzyme in E. coli was similar to that in B. centrosporus. The recombinant clones do not exhibit any BcnI restriction-endonuclease activity.     

Cloning in Streptococcus pneumoniae of the gene for DpnII DNA methylase.

The gene coding for the pneumococcal DNA adenine methylase that recognizes the sequence 5'-GATC-3' was cloned in a strain of Streptococcus pneumoniae that lacked both restriction endonucleases DpnI and DpnII. The gene was cloned as a 3.7-kilobase fragment of chromosomal DNA from a DpnII-containing strain inserted in both possible orientations in the multicopy plasmid vector pMP5 to give recombinant plasmids pMP8 and pMP10. Recombinant plasmids were selected by their resistance to DpnII cleavage. Cells carrying the recombinant plasmids modified phage in vivo so that it was restricted by DpnI- but not DpnII-containing hosts. They also showed levels of DNA methylase activity five times higher than that in cells of the original DpnII strain. No DpnII activity was observed in the clones; therefore, it was concluded that the insert did not contain an intact DpnII endonuclease gene and that methylation of host DNA did not turn on a latent form of the gene.     

Cloning the BamHI restriction modification system.

BamHI, a Type II restriction modification system from Bacillus amyloliquefaciensH recognizes the sequence GGATCC. The methylase and endonuclease genes have been cloned into E. coli in separate steps; the clone is able to restrict unmodified phage. Although within the clone the methylase and endonuclease genes are present on the same pACYC184 vector, the system can be maintained in E. coli only with an additional copy of the methylase gene present on a separate vector. The initial selection for BamHI methylase activity also yielded a second BamHI methylase gene which is not homologous in DNA sequence and hybridizes to different genomic restriction fragments than does the endonuclease-linked methylase gene. Finally, the interaction of the BamHI system with the E. coli Dam and the Mcr A and B functions, have been studied and are reported here.     

Regulation of the BamHI restriction-modification system by a small intergenic open reading frame, bamHIC, in both Escherichia coli and Bacillus subtilis.

BamHI, from Bacillus amyloliquefaciens H, is a type II restriction-modification system recognizing and cleaving the sequence G--GATCC. The BamHI restriction-modification system contains divergently transcribed endonuclease and methylase genes along with a small open reading frame oriented in the direction of the endonuclease gene. The small open reading frame has been designated bamHIC (for BamHI controlling element). It acts as both a positive activator of endonuclease expression and a negative repressor of methylase expression of BamHI clones in Escherichia coli. Methylase activity increased 15-fold and endonuclease activity decreased 100-fold when bamHIC was inactivated. The normal levels of activity for both methylase and endonuclease were restored by supplying bamHIC in trans. The BamHI restriction-modification system was transferred into Bacillus subtilis, where bamHIC also regulated endonuclease expression when present on multicopy plasmid vectors or integrated into the chromosome. In B. subtilis, disruption of bamHIC caused at least a 1,000-fold decrease in endonuclease activity; activity was partially restored by supplying bamHIC in trans.     

Cloning the genetic determinant of alpha-hemolysin and obtaining a series of insertional mutations in this determinant by the transposon Tn1000

Functionally active genetic determinant of alpha-hemolysin was cloned. Hemolytic plasmid pHly195 was used as a donor of the determinant and pBR322 plasmid served as recipient. Cloning was done with a help of HindIII restriction endonuclease. The recombinant plasmid obtained represents pBR322 plasmid with the built-in fragment of 7.4 kb containing genes of functionally active determinant of alpha-hemolysin. Restriction map was constructed using HindIII, EcoRI, BamHI and SalI restriction endonucleases. Insertional mutagenesis was carried out with the help of the Tn1000 transposon. Plasmid DNAs were isolated from insertional mutants of Hly- phenotype and treated with EcoRI, SalI and BamHI. On the basis of the sizes of restriction fragments of the mutant plasmid DNAs localization and orientation of insertions of Tn1000 into the cloned determinant of alpha-hemolysin were determined.     

苏云金芽胞杆菌大质粒pBMB165的克隆与分析

以pBeloBAC11为载体,成功构建了苏云金芽胞杆菌YBT-1765的基因组人工染色体(BAC)文库和质粒BAC文库.根据已克隆的包含复制子ori165在内的3.6kb片段中编码复制蛋白Rep165的核苷酸序列设计探针,通过染色体步移方式,对质粒文库和基因组文库进行筛选,得到13个覆盖YBT-1765菌株中质粒pBMB165不同区域的克隆子.通过Hind Ⅲ和BamH Ⅰ酶切分析,建立了质粒pBMB165的物理图谱和线状重叠连锁图,并测算出该质粒的大小为82kb.根据部分核苷酸序列初步统计了pBMB165上转座因子的存在机率.YBT-1765菌株基因组文库的构建和物理图谱的绘制为克隆苏云金芽胞杆菌大质粒提供了一套可行的方案,成功解决了大质粒难克隆的问题.     

Cloning and structure of the BepI modification methylase.

The gene coding for a CGCG specific DNA methylase has been cloned in E. coli from Brevibacterium epidermidis. The enzyme, named BepI methylase, is probably the cognate methylase of the FnuDII isoschizomer BepI endonuclease isolated from this strain. The expression of BepI methylase in E. coli is dependent on the orientation of the cloned fragment suggesting that the gene is transcribed from a promoter on the plasmid vector. No BepI endonuclease could be detected in the clones producing BepI methylase. The nucleotide sequence of the BepI methylase gene has been determined, it predicts a protein of 403 amino acids (MR: 45,447). Analysis of the amino acid sequence deduced from the nucleotide sequence revealed similarities between the BepI methylase and other cytosine methylases. M. BepI methylates the external cytosine in its recognition sequence.     

New plasmid vector and host system for genetic engineering in Bacillus sphaericus.

A new plasmid, pNQ116, was constructed in Bacillus sphaericus by cloning a promoter fragment from B. sphaericus Ts-1 into pNQ112. The plasmid (CmrKmr, 5.23 kb) contains a restriction endonuclease polylinker used for cloning foreign genes, and its cat-86 gene is expressed at high levels from the Ts-1 promoter. This plasmid vector has been transformed into B. sphaericus AS 1.270, AS 1.465, AS 1.469, and 2362, at frequencies of 10(2)-10(3) transformants per microgram of DNA, and is maintained stably under nonselective conditions in these host strains. The presence of pNQ116 in B. sphaericus 2362 does ot interfere with the mosquito larvicidal activity of the organism.     

Cloning and expression of 7.55 kb KPNI fragment of Burkholderia pseudomallei PPM1 plasmid in Escherichia coli

BamHI, SalI, PstI, and KpnI fragments of pPM1 (B. pseudomallei 12.95 kb plasmid) were cloned in E. coli. The recombinant clones carrying a 7.55 kb KpnI fragment of pPM1 were highly resistant to several aminoglycosides (streptomycin, kanamycin, and gentamycin) and fluoroguinolones (perfloxacin, ofloxacin). Two outer membrane proteins (23 and 27 kDa) absent in E. coli and capable to form 120 kDa oligomer complex were detected by the Western blot method in the strain carrying recombinant pS19 plasmid. The integration of a cloned 7.55 kb sequence in the chromosome was observed by the dot and Southern hybridization analysis in the clones carrying recombinant plasmids pS12 and pS14.     

Cloning and expression of the MspI restriction and modification genes

The genes for the MspI restriction (R) and modification enzymes (recognition sequence CCGG) have been cloned into Escherichia coli using the vector pBR322. Clones carrying both genes have been isolated from libraries prepared with EcoRI, HindIII and BamHI. The smallest fragment that encodes both activities is a 3.6-kb HindIII fragment. Plasmids purified from the clones are fully resistant to digestion by MspI, indicating that the modification gene is functional in E. coli. The clones remain sensitive to phage infection, however, indicating that the endonuclease is dysfunctional. When the R gene is brought under the control of the inducible leftward promoter from phage lambda, the level of endonuclease increases and the level of methylase decreases, suggesting that the genes are transcribed in opposite directions.     

生米卡链霉菌丙酰化酶基因定位及其核苷酸序列测定

我们已往的工作已经确定在重组质粒ｐＩＪＭ９中,生米卡链霉菌来源的４．１６ｋｂ插入ＤＮＡ片段带有丙酰化酶基因,为了进一步确定该基因在４．１６ｋｂ插入片段中的位置,我们以ＢａｍＨＩ对ｐＩＪＭ９进行酶切,在此基础上进行缺失重组亚克隆,在所获得的转化子中,Ｎｏ．５转化子含重组质粒ｐＩＪＭ９５,分子量为５．０ｋｂ,插入ＤＮＡ片段为０．５３ｋｂ,以Ｎｏ．５转化子对螺旋霉素进行生物转化实验,其转化产物经薄层层析     

Cloning the DdeI restriction-modification system using a two-step method.

DdeI, a Type II restriction-modification system from the gram-negative anaerobic bacterium Desulfovibrio desulfuricans, recognizes the sequence CTNAG. The system has been cloned into E. coli in two steps. First the methylase gene was cloned into pBR322 and a derivative expressing higher levels was constructed. Then the endonuclease gene was located by Southern blot analyses; BamHI fragments large enough to contain the gene were cloned into pACYC184, introduced into a host containing the methylase gene, and screened for endonuclease activity. Both genes are stably maintained in E. coli on separate but compatible plasmids. The DdeI methylase is shown to be a cytosine methylase. DdeI methylase clones decrease in viability as methylation activity increases in E. coli RR1 (our original cloning strain). Therefore the DdeI system has been cloned and maintained in ER1467, a new E. coli cloning strain engineered to accept cytosine methylases. Finally, it has been demonstrated that a very high level of methylation was necessary in the DdeI system for successful introduction of the active endonuclease gene into E. coli.     

Control of photosynthetic membrane assembly in Rhodobacter sphaeroides mediated by puhA and flanking sequences.

A reaction center H- strain (RCH-) of Rhodobacter sphaeroides, PUHA1, was made by in vitro deletion of an XhoI restriction endonuclease fragment from the puhA gene coupled with insertion of a kanamycin resistance gene cartridge. The resulting construct was delivered to R. sphaeroides wild-type 2.4.1, with the defective puhA gene replacing the wild-type copy by recombination, followed by selection for kanamycin resistance. When grown under conditions known to induce intracytoplasmic membrane development, PUHA1 synthesized a pigmented intracytoplasmic membrane. Spectral analysis of this membrane showed that it was deficient in B875 spectral complexes as well as functional reaction centers and that the level of B800-850 spectral complexes was greater than in the wild type. The RCH- strain was photosythetically incompetent, but photosynthetic growth was restored by complementation with a 1.45-kilobase (kb) BamHI restriction endonuclease fragment containing the puhA gene carried in trans on plasmid pRK404. B875 spectral complexes were not restored by complementation with the 1.45-kb BamHI restriction endonuclease fragment containing the puhA gene but were restored along with photosynthetic competence by complementation with DNA from a cosmid carrying the puhA gene, as well as a flanking DNA sequence. Interestingly, B875 spectral complexes, but not photosynthetic competence, were restored to PUHA1 by introduction in trans of a 13-kb BamHI restriction endonuclease fragment carrying genes encoding the puf operon region of the DNA. The effect of the puhA deletion was further investigated by an examination of the levels of specific mRNA species derived from the puf and puc operons, as well as by determinations of the relative abundances of polypeptides associated with various spectral complexes by immunological methods. The roles of puhA and other genetic components in photosynthetic gene expression and membrane assembly are discussed.     

Thermo-Inducible Expression of δ Endotoxin Gene of Bacillus thuringiensis HD1 Derived under Lambda PL Promoter in Escherichia coli

The insecticidal crystal protein gene cryIA(a) from Bacillus thuringiensis HD1 has been cloned as a single 3.765 kb Ndel fragment on the expression vector pRE1. The pBR322 based clone pES1 was digested with restriction endonuclease Ndel and the 3.765 kb fragment carrying the intact gene was eluted and cloned on pUC18 to confirm its functional integrity. This Ndel fragment was then cloned on the vector pRE1 carrying strong promoter PL of lambda upstream to the cloning site. The recombinant construct pUSR14.1 carried crystal protein (CP) gene under PL and was temperature inducible at 42°C in MZ1 host strain of Escherichia coli because of temperature sensitive CI857 gene carried by it as lysogen. Dilution based insect bioassays showed hyper-expression of toxin in these constructs. SDS-PAGE analysis indicated that polypeptides corresponding to 132 kD and 66 kD bands of HD1 endotoxin constituted 20.1% of the total soluble protein in this recombinant strain to be delta-endotoxin.     

Cloning of a restriction-modification system from Proteus vulgaris and its use in analyzing a methylase-sensitive phenotype in Escherichia coli.

A 4.84-kilobase-pair plasmid was isolated from Proteus vulgaris (ATCC 13315) and cloned into the plasmid vector pBR322. Plasmid pBR322 contains substrate sites for the restriction endonucleases PvuI and PvuII. The recombinant plasmids were resistant to in vitro cleavage by PvuII but not PvuI endonuclease and were found to cause production of PvuII endonuclease or methylase activity or both in Escherichia coli HB101. The approximate endonuclease and methylase gene boundaries were determined through subcloning, Bal 31 resection, insertional inactivation, DNA-dependent translation, and partial DNA sequencing. The two genes are adjacent and appear to be divergently transcribed. Most E. coli strains tested were poorly transformed by the recombinant plasmids, and this was shown by subcloning and insertional inactivation to be due to the PvuII methylase gene. At a low frequency, stable methylase-producing transformants of a methylase-sensitive strain were obtained, and efficiently transformed cell mutants were isolated from them.     

Cloning and sequence analysis of the genes coding for Eco57I type IV restriction-modification enzymes.

A 6.3 kb fragment of E.coli RFL57 DNA coding for the type IV restriction-modification system Eco57I was cloned and expressed in E.coli RR1. A 5775 bp region of the cloned fragment was sequenced which contains three open reading frames (ORF). The methylase gene is 1623 bp long, corresponding to a protein of 543 amino acids (62 kDa); the endonuclease gene is 2991 bp in length (997 amino acids, 117 kDa). The two genes are transcribed convergently from different strands with their 3'-ends separated by 69 bp. The third short open reading frame (186 bp, 62 amino acids) has been identified, that precedes and overlaps by 7 nucleotides the ORF encoding the methylase. Comparison of the deduced Eco57I endonuclease and methylase amino acid sequences revealed three regions of significant similarity. Two of them resemble the conserved sequence motifs characteristic of the DNA[adenine-N6] methylases. The third one shares similarity with corresponding regions of the PaeR7I, TaqI, CviBIII, PstI, BamHI and HincII methylases. Homologs of this sequence are also found within the sequences of the PaeR7I, PstI and BamHI restriction endonucleases. This is the first example of a family of cognate restriction endonucleases and methylases sharing homologous regions. Analysis of the structural relationship suggests that the type IV enzymes represent an intermediate in the evolutionary pathway between the type III and type II enzymes.