Cloning of theHIS3 gene ofYarrowia lipolytica

TheHIS3 gene of the yeastYarrowia lipolytica has been cloned from a genomic library by complementation of thehis3 mutation ofSaccharomyces cerevisiae. The gene was subsequently subcloned inEscherichia coli and characterized by restriction enzyme mapping.     

Cloning and expression inEscherichia coli of entomotoxic protein gene fromBacillus thuringiensis subspecieskurstaki

A plasmid borne larvicidal crystal protein gene from B.thuringiensis subspecieskurstaki was cloned inEscherichia coli using a specific 20-mer oligonucleotide probe. The gene expressed inE. coli at a high level. TransgenicE. coli cells produced large irregular bodies which looked bright under phase contrast microscopy. The phase bright bodies released by sonic disruption of cells could be pelleted by centrifugation. Toxicity trials on the larvae ofSpodoptera litura showed that the pellet was antifeedant and toxic to the larvae. The supernatant was only mildly antifeedant. Even short term feeding of larvae on the toxin delayed the onset of pupation.     

Escherichia coli-Anacystis nidulans plasmid shuttle vecotrs containing the PL promoter from bacteriophage lambda

The gene encoding xylanase activity in the ruminal bacteriumBacteroides ruminicola D31d was cloned and expressed inEscherichia coli with the plasmid vector pUC18. The gene was isolated on a 4.7-kilobase pair partialPstI genomic DNA fragment. The xylanase activity expressed inE. coli was cell associated and could degrade both oatspelt xylan and Remazol Brilliant Blue-xylan. The xylanase did not have detectable activity against carboxymethylcellulose. Utilization of an endogenous promoter byE. coli was indicated by expression of xylanase activity after subcloning of the insert into pBR322 in opposite orientations. TheB. ruminicola D31d xylanase gene was compared by Southern hybridization analyses with xylanase genes cloned fromB. ruminocola 23 andB. ovatus V975, a human intestinal isolate. The D31d xylanase gene did not cross-hybridize with either of the other two genes. In addition, the 23 xylanase gene did not cross-hybridize with the other two genes according to the same technique. These results indicate that the three cloned genes do not share a high degree of genetic similarity, despite the similar enzymatic activities. This is the first study to compare cloned genes from ruminal and colonicBacteroides species.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

Molecular and genetic characterization of superoxide dismutase in Haemophilus influenzae type b

Oxygen free radicals present a serious potential threat to microbial survival, through their ability to inflict Indiscriminate damage on proteins and DNA. Superoxide dismutase (SOD, EC 1.15.1.1), among other oxygen-metabolizing enzymes, is essential to prevent these toxic molecules from accumulating in the bacterial cytosol during aerobic metabolism. The gene sodA, encoding manganese-containing SOD ([Mn]-SOD), has been cloned from a virulent strain of Haemophilus influenzae type b using degenerate oligonucleotides encoding regions of the gene conserved across different bacterial species. The gene product has been identified as [Mn]-SOD by its similarity at key amino acid residues to known examples of the enzyme, by expression of enzymatically active protein from cloned DNA expressed in Escherichia coli, and by demonstration that an in-frame deletion in the gene abolishes this activity. In contrast to the situation in E. coli, this [Mn]-SOD is the only active SOD detected in H. influenzae. In further contrast to E. coli, [Mn]-SOD gene expression in H. influenzae has been found to be only partially repressed under anaerobic conditions. When expressed in E. coli the gene is regulated by Fur and Fnr, and the promoter region, identified experimentally, has been found to contain nucleotide sequence motifs similar to the Fur- and Fnr-binding sequences of E. coli, suggesting the involvement of analogues of these aerobiosis- responsive activators in H. influenzae gene expression.     

Cloning of genes encoding for C-P lyase fromPseudomonas isolates PG2982 and GLC11: Identification of a cryptic allele on the chromosome ofP. aeruginosa

Two isolates ofPseudomonas sp., GLC11 and PG2982, can use glyphosate as a sole source of phosphorus. This ability is indicative of enzymatic cleavage of a carbon-phosphorus bond, and the enzyme has been named C-P lyase. We have cloned, inEscherichia coli, gene/s coding for C-P lyase on a broad host range cosmid pLA2917. Restriction fragment arrangement of cloned fragments of PG2982 and GLC11 has been established. Analysis by Southern hybridization between two clones revealed a strong homology between threePstI fragments of pPG-CP-14 (derived from PG2982) and pGC-CP-4 (derived from GLC11). With the construct pGC-CP-4 as a probe, the presence of a cryptic allele for C-P lyase has been demonstrated on the chromosome of the parent isolate,pseudomonas aeruginosa PAO1. It is suggested that genetic rearrangement such as frame shift or a point mutation activated the cryptic C-P lyase gene. Metabolism of glyphosate byE. coli carrying pPG-CP-14 or pGC-CP-4 has been demonstrated by radiometric experiments.     

Construction of a sequencedClostridium perfringens-Escherichia coli shuttle plasmid

A newClostridium perfringens-Escherichia coli shuttle plasmid has been constructed and its complete DNA sequence compiled. The vector, pJIR418, contains the replication regions from theC. perfringens replicon pIP404 and theE. coli vector pUC18. The multiple cloning site and lacZ gene from pUC18 are also present, which means that X-gal screening can be used to select recombinants inE. coli. Both chloramphenicol and erythromycin resistance can be selected inC. perfringens andE. coli since pJIR418 carries theC. perfringens catP and ermBP genes. Insertional inactivation of either the catP or ermBP genes can also be used to directly screen recombinants in both organisms. The versatility of pJIR418 and its applicability for the cloning of toxin genes fromC. perfringens have been demonstrated by the manipulation of a cloned gene encoding the production of phospholipase C.     

Molecular cloning and expression ofBacillus subtilis bglS gene inSaccharomyces cerevisiae

A 2.7-kb EcoRI DNA fragment carrying aBacillus subtilis endo--1,3-1,4-glucanase gene (bglS) from theE. coli plasmid pFG1 was cloned into anEscherichia coli/yeast shuttle vector to construct a hybrid plasmid YCSH. The hybrid plasmid was used to transformSaccharomyces cerevisiae, and thebglS gene was expressed. Variation between levels ofbglS gene expression inS. cerevisiae was about 2.3-fold, depending on the orientation of the 2.7-kb DNA fragment. Assay of substrate specificity and optimal pH of the enzyme demonstrated that the enzyme encoded by YCSH (bglS) was identical with that found inB. subtilis, but the expression level ofbglS gene inS. cerevisiae (YCSH) was much lower than that inE. coli (YCSH).     

Cloning and expression of anAeromonas hydrophila chitinase gene inescherichia coli

The gene encoding an extracellular chitinase fromAeromonas hydrophila has been cloned and expressed inEscherichia coli. Plasmid pJP2512 contained the smallest DNA insert (3.9 kb) producing chitinase. The chitinase gene is transcribed from its own promoter, producing a protein of M_r 96,000. The chitinase open reading frame, an estimated 2.6 kb in length, has been subcloned to a 3.0 kb fragment; however, this fragment does not carry the functional chitinase promoter. InE. coli the chitinase enzyme is unable to transverse the outer membrane, being secreted across the cytoplasmic membrane and accumulating in the periplasmic space.     

Expression ofStreptomyces melC andchoA genes by a clonedStreptococcus thermophilus promoter STP2201

Streptococcus thermophilus (ST) chromosomal DNA (chr DNA) fragments having promoter activity were cloned and selected inEscherichia coli using a chloramphenicol acetyltransferase- (cat-) based promoter-probe vector pKK520-3. Insertion of a promoterless streptomycete melanin biosynthesis operon (melC) downstream from the promoters of the library further identified clone ST_P2201 as a strong promoter inE. coli. Subcloning of a ST_P2201-melC DNA fragment into the pMEU-seriesS. thermophilus-E. coli shuttle vectors yielded pEU5xML2201x plasmids that conferred Mel⁺ phenotype toE. coli. The pEU5aML2201a was further shown to afford a high level of tyrosinase production (2 units mg^–1 protein) inE. coli, and to produce an apparently inactivemelC gene product that reacts with anti-tyrosinase antiserum inS. thermophilus. SubstitutingmelC with a streptomycete cholesterol oxidase gene (choA) in the same orientation yielded pEU5aCH2201a that conferred ChoA activity to anE. coli transformant at a level of (1.06±0.15)×10^–7 units mg^–1 protein. Introduction of this plasmid intoS. thermophilus by electrotransformation yielded ChoA transformant that produced the enzyme at about 25% of the level found inE. coli.     

Sequence divergence of an archaebacterial gene cloned from a mesophilic and a thermophilic methanogen

Summary A 1.6-kb fragment of DNA from the thermophilic, methane-producing, anaerobic archaebacteriumMethanobacterium thermoautotrophicum H has been cloned and sequenced. This DNA complements mutations in both the purE₁ and purE₂ loci ofEscherichia coli. The sequence of theM. thermoautotrophicum DNA predicts that complementation inE. coli results from the synthesis of a polypeptide with a molecular weight of 36,249. A polypeptide apparently of this molecular weight is synthesized inE. coli minicells containing recombinant plasmids that carry the cloned fragment of methanogen DNA. We have previously cloned and sequenced a purE-complementing gene from the mesophilic methanogenMethanobrevibacter smithii. The two methanogen-derived purE-complementing genes are 53% homologous and encode polypeptides that are 45% homologous in their amino acid sequences but would be 74% homologous if conservative amino acid substitutions were considered as maintaining sequence homology. The genome ofM. thermoautotrophicum has a molar G+C content of 49.7%, whereas the genome ofM. smithii is 30.6% G+C. Conservation of encoded amino acids while accommodating the very different G+C contents is accomplished by use of different codons that encode the same amino acid. The majority of base changes occur at the third codon position. The intergenic regions of the clonedM. thermoautotrophicum DNA contain sequences previously identified as ribosome binding sites and as putative methanogen promoters. Although the two purE-complementing genes are apparently derived from a common ancestor, only the gene fromM. smithii maintains a codon usage that conforms to the RNY rule.     

Sequencing and bacterial expression of a novel murine alpha interferon gene

A murine new alpha interferon gene (mIFN-αB) was found by primer-based sequencing method in a murine genomic DNA library. The gene was cloned and its sequence was determined. It was expressed in Escherichia coli under the control of the PL promoter which resulted in antiviral activity on mouse L-cells. The sequence of mlFN-αB has been accepted by GENEBANK.     

Phospholipase D fromStreptomyces antibioticus: Cloning,sequencing, expression,and relationship to other phospholipases

The extracellular phospholipase D (PLD) gene fromStreptomyces antibioticus was cloned, sequenced, and expressed inEscherichia coli. Analysis of DNA sequence data revealed a putative ribosome-binding site and an open reading frame encoding a 556-amino-acid protein that included amino acid sequences obtained from the purified enzyme. The protein was expressed in an insoluble form inE. coli, but reacted with antibody against PLD. After solubilization of the protein with guanidine-HCI and 2-mercaptoethanol, subsequent dialysis restored the PLD activity. Comparison of the nucleotide sequence data with the N-terminal protein sequence indicates that this secreted protein is synthesized as a larger precursor with a 47-amino-acid N-terminal extension to the mature enzyme of 509 amino acids. The amino acid sequence of the S.antibioticus PLD was extensively compared with other PLDs and phospholipase C (PLC). The deduced amino acid sequence of the cloned PLD was highly homologous to PLDs from S. acidomyceticus andStreptomyces sp., and contained a conserved region with S.chromofuscus PLD. From comparisons of the structural similarity and properties of the various PLDs, a classification of PLDs into two subgroups has been proposed and the highly conserved region designated tentatively region X_PLD, which may be important in the catalytic function, has been identified. The homology comparison between our PLD and phosphatidylinositol-specific phospholipase C (PI-PLC) is also discussed.     

Cloning and expression of a haloacid dehalogenase fromPseudomonas sp. strain 19 S

A dehalogenase gene specifying the utilization of a variety of haloacids byPseudomonas sp. Strain 19S has been cloned and expressed inE. coli. Our cloning strategy employed specific amplification of a fragment homologous toPseudomonas dehalogenase gene by Polymerase Chain Reaction (PCR). The PCR amplicon successfully acted as a probe to detect the dehalogenase gene in the Southern Blot of the digestedPseudomonas total DNA. Corresponding fragments were cloned into pUC 18 vector and amplified inE. coli MV 1190. One clone with a substantial dehalogenation activity carried a recombinant plasmid containing a 5.5 kb insert.Abbreviations 2-CPA 2-chloropropionate - MCA monochloro acetate - IPTG isopropyl-1-thio--D-galactoside - NBT nitroblue tetrazolium salt - PCR polymerase chain reaction - X-gal 5-bromo-4-chloro-3-indolyl--D-galactoside - X-phosphate 5-bromo-4-chloro-3-indolyl phosphate     

The metalloprotease gene of Serratia marcescens strain SM6

Summary Utilizing the DNA sequence of the metalloprotease fromSerratia strain E-15, we isolated and sequenced the homologous gene fromSerratia strain SM6. These two genes are similar at both the DNA and protein sequence level. Expression of the protease gene inEscherichia coli was achieved by use of thelac promoter. This resulted in the production and excretion of an immunologically detectable but inactive protein of slightly higher molecular weight than that fromSerratia. We introduced the cloned gene into previously described protease mutants. The observed pattern of protease expression suggested that these mutations fall into three classes.     

Functional analysis of combinations in astaxanthin biosynthesis genes from<Emphasis Type="Italic">Paracoccus haeundaensis</Emphasis>

Carotenoids are important natural pigments produced by many microorganisms and plants. We have previously reported the isolation of a new marine bacterium,Paracoccus haeundaensis, which produces carotenoids, mainly in the form of astaxanthin. The astaxanthin biosynthesis gene cluster, consisting of six carotenogenic genes, was cloned and characterized from this organism. Individual genes of the carotenoid biosynthesis gene cluster were functionally expressed inEscherichia coli and each gene product was purified to homogeneity. Their molecular characteristics, including enzymatic activities, were previously reported. Here, we report cloning the genes for crtE, crtEB, crtEBI, crtEBIY, crtEBIYZ, and crtEBI-YZW of theP. haeundaensis carotenoid biosynthesis genes inE. coli and verifying the production of the corresponding pathway intermediates. The carotenoids that accumulated in the transformed cells carrying these gene combinations were analyzed by chromatographic and spectroscopic methods.     

Molecular cloning ofTrichophyton mentagrophytes DNA sequences with promoter activity inEscherichia coli

A promoter probe library from the dermatophyte fungusTrichophyton mentagrophytes has been constructed in the pVB32 plasmid vector. Using this library, a set ofT. mentagrophytes DNA sequences with promoter activity inEscherichia coli has been cloned. The size and the resistance phenotype conferred by these DNA fragments varied. Southern blot analysis confirmed that they were derived fromT. mentagrophytes genomic DNA.     

Temperature and oxygen regulated expression of a glutamine synthetase gene fromVibrio alginolyticus cloned inEscherichia coli

Glutamine synthetase (GS) synthesis inVibrio alginolyticus was regulated by temperature, oxygen and nitrogen levels. A GS gene,glnA fromV. alginolyticus was cloned on a 5.67 kb insert in the recombinant plasmid pRM210, which enabledEscherichia coli glnA, ntrB, ntrC deletion mutants to utilize (NH₄)₂SO₄ as a sole source of nitrogen. TheV. alginolyticus glnA gene was expressed from a regulatory region contained within the cloned fragment.V. alginolyticus glnA expression from pRM210 was subject to regulation by temperature, oxygen and nitrogen levels. GS specific activity in anE. coli wild-type strain was not affected by temperature or oxygen. pRM211 was a deletion derivative of pRM210 and GS production by pRM211 was not regulated by temperature, oxygen or nitrogen levels inE. coli.Abbreviation GS glutamine synthetase     

Molecular cloning of thehom—thrC—thrB cluster fromBacillus sp. ULM1: Expression of thethrC gene inEscherichia coli and corynebacteria,and evolutionary relationships of the threonine genes

A 6.5 kb DNA fragment containing the gene (thrC) encoding threonine synthase, the last enzyme of the threonine biosynthetic pathway, has been cloned from the DNA ofBacillus sp. ULM1 by complementation ofEscherichia coli andBrevibacterium lactofermentum thrC auxotrophs. Complementation studies showed that thethrB gene (encoding homoserine kinase) is found downstream from thethrC gene, and analysis of nucleotide sequences indicated that thehom gene (encoding homoserine dehydrogenase) is located upstream of thethrC gene. The organization of this cluster of genes is similar to theBacillus subtilis threonine operon (hom—thrC—thrB). An 1.9 kbBclI, fragment from theBacillus sp. ULM1 DNA insert that complementedthrC mutations both inE. coli and in corynebacteria was sequenced, and an ORF encoding a protein of 351 amino acids was found corresponding to a protein of 37462 Da. ThethrC gene showed a low G+C content (39.4%) and the encoded threonine synthase is very similar to theB. subtilis enzyme. Expression of the 1.9 kbBclI DNA fragment inE. coli minicells resulted in the formation of a 37 kDa protein. The upstream region of this gene shows promoter activity inE. coli but not in corynebacteria. A peptide sequence, including a lysine that is known to bind the pyridoxal phosphate cofactor, is conserved in all threonine synthase sequences and also in the threonine and serine dehydratase genes. Amino acid comparison of nine threonine synthases revealed evolutionary relationships between different groups of bacteria. Dedicated to Dr. J. Spížek on the occasion of his 60th birthday     

Characterization of DNA binding activities of over-expressedKpnI restriction endonuclease and modification methylase

The genes encoding theKpnI restriction endonuclease and methyltransferase fromKlebsiella pneumoniae have been cloned and expressed inEscherchia coli using a two plasmid strategy. The gene forKpnI methylase with its promoter was cloned and expressed in pACYC184. Even though the methylase clone is in a low copy number plasmid pACMK, high level expression of methylase is achieved. A hyper-expressing clone ofKpnI endonuclease, pETRK was engineered by cloning the R gene into the T7 expression system. This strategy resulted in over-expression ofKpnI endonuclease to about 15–30% of cellular protein. Both the enzymes were purified using a single Chromatographic step to apparent homogeneity. The yield of purified endonuclease and methylase from one liter of culture was approximately 30 and 6 mg respectively. Electrophoretic mobility shift assays show that both the enzymes are capable of binding to specific recognition sequence in the absence of any cofactors. The complexes ofKpnI methyl transferase and endonuclease with their cognate site exhibit distinctive behaviour with respect to ionic requirement.     

Conjugal Transfer of Plasmid DNA fromEscherichia colito Enterococci: A Method to Make Insertion Mutations

Shuttle vector pAT18 was transferred by conjugation fromEscherichia coliS17-1 toEnterococcus faecalisOG1RF andEnterococcus faeciumSE34. Transfer was mediated by the transfer functions of plasmid RK2 inE. coliS17-1 and the origin of conjugal transfer (oriT) located on pAT18. TheoriTsequence was then inserted into two plasmids to generate vectors pTEX5235 and pTEX5236. These two vectors cannot replicate in gram-positive bacteria and can be used to make insertion mutants in gram-positive bacteria. An internal sequence from an autolysin gene ofE. faecalisOG1RF was cloned into pTEX5235 and transferred by conjugation fromE. coliS17-1 toE. faecalisOG1RF. The plasmid was found to integrate into the chromosome of OG1RF by a single crossover event, resulting in a disrupted autolysin gene. A cosmid carrying the pyrimidine gene cluster fromE. faecalis,with a transposon insertion inpyrC,was also transferred fromE. coliS17-1 toE. faecalisOG1RF. After selection for the transposon, it was found to have recombined into the recipient chromosome by a double crossover between the cosmid and the chromosome of OG1RF. This resulted in apyrCknockout mutant showing an auxotrophic phenotype.