Cloning and expression of Thiobacillus ferrooxidans mercury ion resistance genes in Escherichia coli.

A search of various domestic isolates of Thiobacillus ferrooxidans revealed that some were fairly resistant to mercury ion. A proportion of mercury-resistant clones were able to volatilize mercury, and their corresponding gene was localized not in the plasmid DNA but in chromosomal DNA. This mercury ion resistance gene was cloned in Escherichia coli. E. coli carrying the recombinant plasmid was able to grow in the presence of more than 40 micrograms of HgCl2 per ml. Deletion analysis of the recombinant plasmid showed that the entire coding sequence of the mercury ion resistance gene was located within a 2.3-kilobase fragment of the chromosomal DNA from strain E-15. At least two polypeptides (molecular mass, 56 and 16 kDa, respectively) were coded by this fragment.     

Nucleotide sequence of a chromosomal mercury resistance determinant from a Bacillus sp. with broad-spectrum mercury resistance.

A 13.5-kilobase HindIII fragment, bearing an intact mercury resistance (mer) operon, was isolated from chromosomal DNA of broad-spectrum mercury-resistant Bacillus sp. strain RC607 by using as a probe a clone containing the mercury reductase (merA) gene. The new clone, pYW33, expressed broad-spectrum mercury resistance both in Escherichia coli and in Bacillus subtilis, but only in B. subtilis was the mercuric reductase activity inducible. Sequencing of a 1.8-kilobase mercury hypersensitivity-producing fragment revealed four open reading frames (ORFs). ORF1 may code for a regulatory protein (MerR). ORF2 and ORF4 were associated with cellular transport function and the hypersensitivity phenotype. DNA fragments encompassing the merA and the merB genes were sequenced. The predicted Bacillus sp. strain RC607 MerA (mercuric reductase) and MerB (organomercurial lyase) were similar to those predicted from Staphylococcus aureus plasmid pI258 (67 and 73% amino acid identities, respectively); however, only 40% of the amino acid residues of RC607 MerA were identical to those of the mercuric reductase from gram-negative bacteria. A 69-kilodalton polypeptide was isolated and identified as the merA gene product by examination of its amino-terminal sequence.     

Functional expression of two Bacillus subtilis chromosomal genes in Escherichia coli.

EcoRI-cleaved deoxyribonucleic acid segments carrying two genes from Bacillus subtilis, pyr and leu, have been cloned in Escherichia coli by insertion into a derivative of the E. coli bacteriophage lambda. Lysogenization of pyrimidine- and leucine-requiring auxotrophs of E. coli by the hybrid phages exhibited prototrophic phenotypes, suggesting the expression of B. subtilis genes in E. coli. Upon induction, these lysogens produced lysates capable of transducing E. coli pyr and leu auxotrophs to prototrophy with high frequency. Isolated DNAs of these bacteriophages have the ability to transform B. subtilis auxotrophs to pyr and leu independence and contain EcoRI-cleaved segments which hybridize to corresponding segments of B. subtilis.     

Cloning and expression of the nitrile hydratase and amidase genes from Bacillus sp. BR449 into Escherichia coli

A moderate thermophile, Bacillus sp. BR449 was previously shown to exhibit a high level of nitrile hydratase (NHase) activity when growing on high levels of acrylonitrile at 55 degrees C. In this report, we describe the cloning of a 6.1 kb SalI DNA fragment encoding the NHase gene cluster of BR449 into Escherichia coli. Nucleotide sequencing revealed six ORFs encoding (in order), two unidentified putative proteins, amidase, NHase beta- and alpha-subunits and a small putative protein of 101 amino acids designated P12K. Spacings and orientation of the coding regions as well as their gene expression in E. coli suggest that the beta-subunit, alpha-subunit, and P12K genes are co-transcribed. Analysis of deduced amino acid sequences indicate that the amidase (348 aa, MW 38.6 kDa) belongs to the nitrilase-related aliphatic amidase family, and that the NHase beta- (229 aa, MW 26.5 kDa) and alpha- (214 aa, MW 24.5 kDa) subunits comprise a cobalt-containing member of the NHase family, which includes Rhodococcus rhodochrous J1 and Pseudomonas putida 5B NHases. The amidase/NHase gene cluster differs both in arrangement and composition from those described for other NHase-producing strains. When expressed in Escherichia coli DH5alpha, the subcloned NHase genes produced significant levels of active NHase enzyme when cobalt ion was added either to the culture medium or cell extracts. Presence of the P12K gene and addition of amide compounds as inducers were not required for this expression.     

Cloning and heterologous expression of ectoine biosynthesis genes from Bacillus halodurans in Escherichia coli

The genes involved in the biosynthetic pathway of ectoine (2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid) from Bacillus halodurans were cloned as an operon and expressed in E. coli. Analysis of the deduced ectoine biosynthesis cluster amino acid sequence revealed that the ectoine operon contain 2,389 bp, encoded by three genes; ectA, ectB and ectC that encode proteins of 189, 427 and 129 amino acids with deduced molecular masses of 21,048, 47,120 and 14,797 Da respectively. Extracts of induced cells showed two bands at 41 kDa and 17 kDa, possibly corresponding to the products of the later two genes. However the expression of ectA gene could not be ascertained by SDS-PAGE. The activity of the ectA protein was confirmed by an acylation assay. The transgenic E. coli accumulated upto 4.6 mg ectoine/l culture. This is the first report of an engineered E. coli strain carrying the ectoine genes of the alkaliphilic bacterium, B. halodurans.     

Cloning and expression in Escherichia coli of mercuric ion resistance coding genes from Zymomonas mobilis.

From a genomic library of Zymomonas mobilis prepared in Escherichia coli, two clones (carrying pZH4 and pZH5) resistant to the mercuric ion were isolated. On partial restriction analysis these two clones appeared to have the same 2.9 kb insert. Mercuric reductase activity was assayed from the Escherichia coli clone carrying pZH5 and it was Hg(2+)-inducible, NADH dependent and also required 2-mercaptoethanol for its activity. The plasmid pZH5 encoded three polypeptides, mercuric reductase (merA; 65 kDa), a transport protein (merT 18-17 kDa) and merC (15 kDa) as analysed by SDS-PAGE. Southern blot analysis showed the positive signal for the total DNA prepared from Hgr Z. mobilis but not with the Hgs strain which was cured for a plasmid (30 kb). These results were also confirmed by isolating this plasmid from Hgr Z. mobilis and transforming into E. coli. Moreover the plasmid pZH5 also hybridized with the mer probes derived from Tn21.     

Cloning and expression of formaldehyde resistance from Escherichia coli

Abstract Formaldehyde resistance in Escherichia coli strain VU3695 is mediated by a 94 kilobase plasmid. The genes responsible for formaldehyde resistance were identified on a 9.2 kb DNA fragment and cloned in pBR322. By minicell analysis three proteins were shown to be encoded by this fragment.     

Cloning of a tellurite resistance determinant from Bacillus stearothermophilus V in Escherichia coli

A potassium tellurite-resistance determinant was isolated from Bacillus stearothermophilus V and cloned in Escherichia coli. Transformed cells formed black colonies when grown on solid media containing permissive tellurite concentrations. The resistance determinant was contained in a B. stearothermophilus V chromosomal DNA fragment of 7 kb.     

Cloning of Clostridium acetobutylicum genes and their expression in Escherichia coli and Bacillus subtilis

Summary DNA from Clostridium acetobutylicum ABKn8 was partially digested with Sau3A and the fragments obtained were inserted into the unique BamHI site of the cloning vector pHV33. The recombinant plasmids were used to transform Escherichia coli HB101 with selection for ampicillin resistance. A collection of ampicillin-resistant, tetracycline-sensitive clones representative of the Clostridium acetobutylicum genome was made. The clones were shown to carry recombinant plasmids each containing an insert of 2 to 16 kb in size. Several of them complemented the HB101 proA2 or leuB6 auxotrophic mutations. The cloned sequences were shown by Southern blot hybridization to be homologous to the corresponding ABKn8 DNA fragments.     

Cloning and expression of penicillin acylase genes from overproducing strains of Escherichia coli and Bacillus megaterium

Summary Penicillin acylase genes from Escherichia coli 194, of an overproducing mutant (194-3) of this strain, and of a similar overproducing mutant of Bacillus megaterium UN1 were cloned in E. coli DH1 on the plasmid vector pACYC184. The sizes of chromosomal DNA fragments essential for penicillin acylase production were found by Tn1000 mutagenesis and in vitro deletions to be between 2.2 and 2.5 kb in the case of both E. coli genes and between 2.3 and 2.7 kb in the case of the mutant Bacillus gene. Restriction mapping indicated substantial sequence differences between the E. coli and B. megaterium penicillin acylase genes. Enzyme production in E. coli recombinants from both overproducing mutants was found to be constitutive and higher than in the original strains. The Bacillus penicillin acylase was produced intracellularly in E. coli recombinants, which is in contrast to the normal extracellular production of this enzyme in B. megaterium. Recombinant plasmids containing penicillin acylase genes from either source were found to be unstable in the absence of selection pressure for retention of the vector.     

枯草芽孢杆菌Mn-SOD基因的克隆及原核表达

为了实现Mn-SOD基因在大肠杆菌(E.coli)中的可溶性表达,根据枯草芽孢杆菌(Bacillus subtilis)168sodA核酸序列设计引物,以枯草芽孢杆菌ATCC 9372基因组为模板,PCR扩增获得了Mn-SOD基因.将此基因重组至原核表达载体pET-28a,构建含Mn-SOD基因的重组表达质粒,并转化至大肠杆菌BL21(DE3).异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达获得Mn-SOD,蛋白分子量约为26kD,占全菌蛋白的5.6%.改良的连苯三酚自氧化法测定SOD活力,菌体可溶性总蛋白SOD比活为51.09U·mg^-1,是对照组的.8倍.枯草芽孢杆菌ATCC 9372 Mn-SOD基因在大肠杆菌BL21(DE3)中首次成功表达,产物具有较高的可溶性和活性,为大量制备Mn-SOD奠定了基础.     

Cloning and expression of Bacillus subtilis phage DNA methyltransferase genes in Escherichia coli and B. subtilis

The DNA methyltransferase (Mtase) genes of the temperate Bacillus subtilis phages SPR (wild type and various mutants), phi 3T, rho 11 and SP beta have been cloned and expressed in Escherichia coli and B. subtilis host-plasmid vector systems. Mtase activity has been quantitated in these clones by performing in vitro methylation assays of cell-free extracts. The four-phage Mtase genes differ in the amount of Mtase synthesized when transcribed from their genuine promoters. In B. subtilis as well as in E. coli the SPR Mtase is always produced in smaller amounts than the other phage Mtases. Expression levels of the SPR Mtase are dependent on the strength of the upstream vector promoter sequences. Overproduction of the SPR wild-type and mutant enzymes was achieved in E. coli (inducible expression) by fusions to the lambda pL or the tac promoter and in B. subtilis (constitutive expression) by means of the phage SP02 promoter.     

Azaserine resistance in Escherichia coli: chromosomal location of multiple genes.

Resistance to azaserine in Escherichia coli is the result of mutations in at least three different loci. All spontaneously arising azaserine-resistant mutants harbor a lesion in the aroP gene. However, a lesion in this gene is not solely responsible for resistance. All spontaneously arising intermediate-level azaserine-resistant mutants also harbor a lesion in a gene designated azaA, which lies near min 43 on the chromosome. High-level resistant mutants harbor lesions in the aroP and azaA genes and in a third gene designated azaB, which lies near min 69 on the chromosome. Transport studies demonstrate that mutants harboring lesions in the azaA gene are not defective in the transport of the aromatic amino acids, but that mutants which harbor lesions in the azaB gene are defective in phenylalanine transport but not in tyrosine or tryptophan transport.     

Cloning and expression of a Bacillus coagulans amylase gene in Escherichia coli

Summary A partial EcoRI fragment of Bacillus coagulans DNA cloned in an Escherichia coli K12 bacteriophage host-vector system was shown to direct the synthesis of a thermostable -amylase whose activity could be detected in situ on petri plates using the iodine staining method. A 3.31 kb EcoRI fragment containing the active gene with its own promoter was subcloned in pBR322; in the new clone, called pAMY2, the amylase was shown to accumulate in the periplasmic space. The molecular weight of the enzyme, confirmed by in vivo labelling of plasmid products in minicells, was estimated to be 60000.The restriction map of the plasmid was determined for five restriction enzymes and two new plasmids with smaller DNA inserts were constructed, both directing the synthesis of amylase; one of them with a 2.2 kb PstI insert was shown to be responsible for the synthesis of a fused -lactamase--amylase protein with amylase activity.