Mode of action of the Spiroplasma CpG methylase M.SssI.

The cytosine DNA methylase from the wall-less prokaryote, Spiroplasma strain MQ1 (M.SssI) methylates completely and exclusively CpG-containing sequences, thus showing sequence specificity which is similar to that of mammalian DNA methylases. M.SssI is shown here to methylate duplex DNA processively as judged by kinetic analysis of methylated intermediates. The cytosine DNA methylases, M.HpaII and M.HhaI, from other prokaryotic organisms, appear to methylate in a non-processive manner or with a very low degree of processivity. The Spiroplasma enzyme interacts with duplex DNA irrespective to the presence of CpG sequences in the substrate DNA. The enzyme proceeds along a CpG-containing DNA substrate molecule methylating one strand of DNA at a time.     

Cloning and expression of the thermostable pullulanase gene from Thermus sp. strain AMD-33 in Escherichia coli

Abstract The gene coding for a thermostable pullulanase from a thermophile, Thermus sp. strain AMD-33, was cloned in Escherichia coli using pDR540 as a vector. A restriction map was determined for the plasmid pTPS131 which contained the fragment carrying the pullulanase gene. DNA-DNA hybridisation analysis showed that the DNA fragment contained the gene from Thermus sp. strain AMD-33. The strain of E. coli harbouring the plasmid pTPS131 produced most of the pullulanase protein cellularly, whereas Thermus sp. strain AMD-33 produced pullulanase extracellularly. Comparative studies of the enzyme from the thermophile and the plasmid-encoded enzyme in E. coli demonstrated that the optimum temperature and pH of the enzymes were closely similar.     

Cloning and characterization of the Escherichia coli gene coding for alkaline phosphatase.

The Escherichia coli structural gene for alkaline phosphatase, phoA, and a promoter-like mutant of phoA, called pho-1003(Bin) phoA+, were cloned by using plasmid vectors. Initially, these genes were cloned on deoxyribonucleic acid fragments of 28.9 kilobases (kb). Subsequently, they were subcloned on fragments and 4.8 and then 2.7 kilobases. A restriction map was developed, and phoA was localized to a 1.7-kb region. The promoter end of the gene was inferred by its proximity to another gene cloned on the same deoxyribonucleic acid fragment, proC. The stability of the largest plasmid (33.3 kb) was found to be recA dependent, although the subcloned plasmids were stable in a recA+ strain. Synthesis of alkaline phosphatase directed by the phoA+ and pho-1003(Bin) phoA+ plasmids in a phoA deletion strain was assayed under repressing and derepressing levels of phosphate. These data were compared with the copy numbers of the plasmids. It was found that synthesis of alkaline phosphatase was tightly regulated, even under derepressing conditions: a copy number of 17 enabled cells to synthesize only about twofold more enzyme than did cells with 1 chromosomal copy of phoA+. Enzyme levels were also compared for cells containing pho-1003(Bin) phoA+ and phoA+.     

Cloning, sequencing, and expression of the N-acyl-D-mannosamine dehydrogenase gene from Flavobacterium sp. strain 141-8 in Escherichia coli.

The gene coding for N-acyl-D-mannosamine dehydrogenase (NAM-DH) from Flavobacterium sp. strain 141-8 was cloned and expressed under the control of a lac promoter in Escherichia coli JM109. The DNA sequence of the gene was determined, and an open reading frame encoding a polypeptide composed of 272 amino acid residues (Mr, 27,473) was identified. The E. coli transformants which showed over 200-fold higher NAM-DH activity than did the Flavobacterium strain produced the enzyme as a protein fused with beta-galactosidase. Despite being a fusion, NAM-DH produced by E. coli transformants appeared unchanged in pH optimum, Km, and substrate specificity from Flavobacterium sp. strain 141-8. This newly recombinant enzyme may be applicable to the quantitative determination of sialic acid in serum.     

Cloning, sequencing, and expression of the N-acyl-D-mannosamine dehydrogenase gene from Flavobacterium sp. strain 141-8 in Escherichia coli.

The gene coding for N-acyl-D-mannosamine dehydrogenase (NAM-DH) from Flavobacterium sp. strain 141-8 was cloned and expressed under the control of a lac promoter in Escherichia coli JM109. The DNA sequence of the gene was determined, and an open reading frame encoding a polypeptide composed of 272 amino acid residues (Mr, 27,473) was identified. The E. coli transformants which showed over 200-fold higher NAM-DH activity than did the Flavobacterium strain produced the enzyme as a protein fused with beta-galactosidase. Despite being a fusion, NAM-DH produced by E. coli transformants appeared unchanged in pH optimum, Km, and substrate specificity from Flavobacterium sp. strain 141-8. This newly recombinant enzyme may be applicable to the quantitative determination of sialic acid in serum.     

The isolation and characterization of the Escherichia coli DNA adenine methylase (dam) gene.

The E. coli dam (DNA adenine methylase) enzyme is known to methylate the sequence GATC. A general method for cloning sequence-specific DNA methylase genes was used to isolate the dam gene on a 1.14 kb fragment, inserted in the plasmid vector pBR322. Subsequent restriction mapping and subcloning experiments established a set of approximate boundaries of the gene. The nucleotide sequence of the dam gene was determined, and analysis of that sequence revealed a unique open reading frame which corresponded in length to that necessary to code for a protein the size of dam. Amino acid composition derived from this sequence corresponds closely to the amino acid composition of the purified dam protein. Enzymatic and DNA:DNA hybridization methods were used to investigate the possible presence of dam genes in a variety of prokaryotic organisms.     

Sequence specificity of the P1 modification methylase (M.Eco P1) and the DNA methylase (M.Eco dam) controlled by the Escherichia coli dam gene.

Labeled oligonucleotides have been fractionated from pancreatic DNase digests of DNA that had been methylated in vitro with the P1 modification enzyme (M·Eco P1) or with the DNA-adenine methylase (M·Eco dam) controlled by the Escherichia coli dam gene. The sequences of methylated oligonucleotides were established for M·Eco dam modification of calf thymus DNA. The results show that M·Eco dam inethylates adenine residues contained in the twofold symmetrical sequence, 5′ … G-A-T-C … 3′. The sequence for the site methylated by M·Eco P1 has also been deduced; we propose that M·Eco P1 modification produces the following methylated pentameric sequence: 5′ … A-G-A¹-C-Py … 3′ (where $\text{A}{}^1\text{= N}{}^6$ methyladenine and Py is C or T).     

Cloning and expression of the gene encoding phospholipase A1 from Serratia sp. MK1 in Escherichia coli.

The gene encoding extracellular phospholipase A1 of Serratia sp. MK1 was cloned from a genomic DNA library. Formation of transparent halos on the PCY agar plates was used to identify E. coli carrying the phospholipase A1 gene. A 4.2 kb EcoRI fragment was isolated and sequenced. From nucleotide sequences and expression of various plasmids, two open reading frames (plaA and plaS) involved in efficient expression of phospholipase A1 in natural and recombinant host were identified. Extracellular phospholipase A1 activity was identified as the gene product of plaA encoding 321 amino acids with a predicted MW of 33,400. Analysis of the amino acid sequence revealed significant homology (around 70%) to phospholipase A1 of Serratia liquefaciens and Yersinia enterocolitica. The sequence, -Gly-X1-Ser-X2-Gly-, known as a lipase-specific consensus sequence was also found in the bacterial phospholipase A1. PlaS encoding a protein of 224 amino acids showed no enzymatic activity, but might be necessary for the efficient expression of phospholipase A1 in E. coli. To further improve the production of phospholipase A1 as a soluble and active form in E. coli, the effect of some parameters was examined. Surprisingly, a higher yield of soluble and active phospholipase A1 could be obtained under the combined conditions of a lower temperature, an enriched medium, and a lower-strength promoter.     

Cloning of a dibutyl phthalate hydrolase gene from Acinetobacter sp. strain M673 and functional analysis of its expression product in Escherichia coli

A dibutyl phthalate (DBP) transforming bacterium, strain M673, was isolated and identified as Acinetobacter sp. This strain could not grow on dialkyl phthalates, including dimethyl, diethyl, dipropyl, dibutyl, dipentyl, dihexyl, di(2-ethylhexyl), di-n-octyl, and dinonyl phthalate, but suspensions of cells could transform these compounds to phthalate via corresponding monoalkyl phthalates. During growth in Luria–Bertani medium, M673 produced the high amounts of non-DBP-induced intracellular hydrolase in the stationary phase. One DBP hydrolase gene containing an open reading frame of 1,095 bp was screened from a genomic library, and its expression product hydrolyzed various dialkyl phthalates to the corresponding monoalkyl phthalates.     

Cloning and characterization of a novel gene encoding L-ribose isomerase from Acinetobacter sp. strain DL-28 in Escherichia coli.

The gene encoding a novel L-ribose isomerase (L-RI) from Acinetobacter sp. was cloned into Escherichia coli and nucleotide sequence was determined. The gene corresponded to an open reading frame of 747 bp that codes for a deduced protein of 249 amino acids, which showed no amino acid sequence similarity with any other sugar isomerases. After expression of the gene in E. coli using pUC118 the recombinant L-RI was purified to homogeneity using different chromatographic methods. The overall enzymatic properties of the purified recombinant L-RI were the same as those of the authentic L-RI. To our knowledge, this is the first time report concerning the L-RI gene.     

Cloning, sequencing, and expression of thermophilic Bacillus sp. strain TB-90 urease gene complex in Escherichia coli.

The urease of thermophilic Bacillus sp. strain TB-90 is composed of three subunits with molecular masses of 61, 12, and 11 kDa. By using synthetic oligonucleotide probes based on N-terminal amino acid sequences of each subunit, we cloned a 3.2-kb EcoRI fragment of TB-90 genomic DNA. Moreover, we cloned two other DNA fragments by gene walking starting from this fragment. Finally, we reconstructed in vitro a 6.2-kb DNA fragment which expressed catalytically active urease in Escherichia coli by combining these three DNA fragments. Nucleotide sequencing analysis revealed that the urease gene complex consists of nine genes, which were designed ureA, ureB, ureC, ureE, ureF, ureG, ureD, ureH, and ureI in order of arrangement. The structural genes ureA, ureB, and ureC encode the 11-, 12-, and 61-kDa subunits, respectively. The deduced amino acid sequences of UreD, UreE, UreF, and UreG, the gene products of four accessory genes, are homologous to those of the corresponding Ure proteins of Klebsiella aerogenes. UreD, UreF, and UreG were essential for expression of urease activity in E. coli and are suggested to play important roles in the maturation step of the urease in a co- and/or posttranslational manner. On the other hand, UreH and UreI exhibited no significant similarity to the known accessory proteins of other bacteria. However, UreH showed 23% amino acid identity to the Alcaligenes eutrophus HoxN protein, a high-affinity nickel transporter.     

Cloning,DNA sequencing and heterologous expression of the gene for thermostable N-acylamino acid racemase from Amycolatopsis sp. TS-1-60 in Escherichia coli

The gene encoding the novel enzyme N-acylamino acid racemase (AAR) was cloned in recombinant phage -4 from the DNA library of Amycolatopsis sp. TS-1-60, a rare actinomycete, using antiserum against the enzyme. The cloned gene was subcloned and transformed in Escherichia coli JM105 using pUC118 as a vector. The AAR gene consists of an open-reading frame of 1104 nucleotides, which specifies a 368-amino-acid protein with a molecular mass of 39411Da. The molecular mass deduced from the AAR gene is in good agreement with the subunit molecular mass (40kDa) of AAR from Amycolatopsis sp. TS-1-60. The guanosine plus cytosine content of the AAR gene was about 70%. Although the AAR gene uses the unusual initiation codon GTG, the gene was expressed in Escherichia coli using the lac promoter of pUC118. The amount of the enzyme produced by the transformant was 16 times that produced by Amycolatopsis sp. TS-1-60. When the unusual initiation codon GTG was changed to ATG, the enzyme productivity of the transformant increased to more than 37 times that of Amycolatopsis sp. TS-1-60. In the comparison of the DNA sequence and the deduced amino acid sequence of AAR with those of known racemases and epimerases in data bases, no significant sequence homology was found. However, AAR resembles mandelate racemase in that requires metal ions for enzyme activity. Comparison of the deduced amino acid sequences of mandelate racemase and AAR revealed amino acid sequences in AAR similar to those of both the catalytic and metal-ion-binding sites of mandelate racemase.     

Cloning and expression of the putative gene coding for GTP cyclohydrolase I from Escherichia coli

The putative gene coding for GTP cyclohydrolase I of Escherichia coli was isolated from a lambda gt11 expression vector library by using antibodies as a probe and has been subcloned on a 3.8 kb Bam HI fragment in the plasmid vector pUC13. E. coli cells carrying the recombinant plasmid designated pCYH express 100-fold increased levels of the enzyme. The protein formed under the control of the plasmid appears electrophoretically and immunochemically identical with the wild type enzyme.     

Cloning, expression, and characterization of the Anabaena thioredoxin gene in Escherichia coli.

The gene encoding thioredoxin in Anabaena sp. strain PCC 7119 was cloned in Escherichia coli based on the strategy that similarity between the two thioredoxins would be reflected both in the gene sequence and in functional cross-reactivity. DNA restriction fragments containing the Anabaena thioredoxin gene were identified by heterologous hybridization to the E. coli thioredoxin gene following Southern transfer, ligated with pUC13, and used to transform an E. coli strain lacking functional thioredoxin. Transformants that complemented the trxA mutation in E. coli were identified by increased colony size and confirmed by enzyme assay. Expression of the cloned Anabaena thioredoxin gene in E. coli was substantiated by subsequent purification and characterization of the algal protein from E. coli. The amino acid sequence derived from the DNA sequence of the Anabaena gene was identical to the known amino acid sequence of Anabaena thioredoxin. The E. coli strains which expressed Anabaena thioredoxin complemented the TrxA- phenotype in every respect except that they did not support bacteriophage T7 growth and had somewhat decreased ability to support bacteriophages M13 and f1.     

Cloning, sequence analysis, and expression in Escherichia coli of the gene encoding phenylacetaldehyde reductase from styrene-assimilating Corynebacterium sp. strain ST-10

The gene encoding phenylacetaldehyde reductase (PAR), a useful biocatalyst for producing chiral alcohols, was cloned from the genomic DNA of the styrene-assimilating Corynebacterium sp. strain ST-10. The gene contained an opening reading frame consisting of 1,158 nucleotides corresponding to 385 amino acid residues. The subunit molecular weight was calculated to be 40,299, which was in agreement with that determined by polyacrylamide gel electrophoresis. The enzyme was sufficiently expressed in recombinant Escherichia coli cells for practical use and purified to homogeneity by three-column chromatography steps. The predicted amino acid sequence displayed only 20–29% identity with zinc-containing, NAD⁺-dependent, long-chain alcohol dehydrogenases. Nevertheless, the probable NAD⁺- and zinc-binding sites are conserved although one of the three catalytic zinc-binding residues of the zinc-containing, long-chain alcohol dehydrogenases was substituted by Asp in PAR. The protein contains 7.6 mol zinc/mol tetramer. Therefore, the enzyme was considered as a new member of zinc-containing, long-chain alcohol dehydrogenases with a particular and broad substrate specificity. Received: 5 March 1999 / Received last revision: 10 May 1999 / Accepted: 16 May 1999     

A cellulase gene from a new alkalophilic Bacillus sp. (strain N186-1). Its cloning,nucleotide sequence and expression in Escherichia coli

 Several alkalophilic Bacillus spp. strains were selected for their capacity to produce alkaline cellulases. Culture supernatants of these strains showed optimal cellulase activities between pH 8 and 9 and they were stable from pH 6 to pH 12. A cellulase gene (celB1) from the alkalophilic Bacillus sp. strain N186-1 was cloned in Escherichia coli using polymerase chain reaction techniques. The cloned gene was present in a 2.539-bp HindIII fragment and its nucleotide sequence was determined. The coding sequence showed an open-reading frame encoding 389 amino acids. The amino acid sequence, deduced from the nucleotide sequence, permitted us to include it in family 5 (or A) of the glycosyl hydrolases. The complete open-reading frame of celB1 was cloned in the plasmid pET-11d and expressed in E. coli BL21 (DE3), in which a protein of 39 kDa was obtained in the cytoplasm; however, no endoglucanase activity was detected. A second construction in pET-12a allowed the production of a 39-kDa protein located in the periplasmic space of E. coli that had endoglucanase activity. The protein produced has optimal activity at pH 7 and 50°C and it retains more than 70% of its activity after incubation for 1 h at pH 12. Received: 27 December 1995/Received revision: 14 March 1996/Accepted: 25 March 1996