Cloning of genes for proline biosynthesis in Escherichia coli

Restriction map of Escherichia coli chromosome fragment (7.4 MD) carrying proAB genes was constructed. Localization of proA and proB genes on the cloned chromosome fragment was determined by complementation test and the measuring of glutamylkinase activity (proB gene product). ProA and proB genes were cloned separately on multicopy plasmids of alternative orientation and their expression being, probably, under the control of their own regulatory regions, studied.     

Phasmid vectors for identification of genes by complementation of Escherichia coli mutants.

A bacteriophage lambda cloning vector was designed to facilitate the isolation of genes from procaryotic organisms by complementation of Escherichia coli mutants. This vector, lambda SE4, was constructed by attaching a very-low-copy-number replication system (from the plasmid NR1) and a spectinomycin resistance gene to the left arm of lambda 1059 (Karn et al., Proc. Natl. Acad. Sci. U.S.A. 77:5172-5176, 1980). This phasmid cloning vector is capable of growing lytically as a phage in a nonimmune host or lysogenically as a phasmid in an immune host. This phasmid utilizes the Spi- selection for insertions of DNA into the vector and has the ability to accept 2- to 19-kilobase Sau3A1, BamHI, BglII, BclI, or XhoII fragments; recombinants lysogenize immune hosts as single-copy-number selectable plasmids at 100% frequency. An E. coli library was constructed by using the initial vector lambda SE4, and clones of a number of representative genes were identified. A typical clone, lambda ant+, was shown to be readily mutagenized by a mini-Tn10 transposon. A general method for transferring cloned DNA segments onto bacteriophage lambda was developed. The method involves the use of in vivo recombination with a selection and was used to construct two derivatives of lambda SE4. Possible uses of these vectors and of the method for transferring cloned DNA onto phage lambda are discussed.     

Cloning and organization of seven arginine biosynthesis genes from Neisseria gonorrhoeae.

A genomic library for Neisseria gonorrhoeae, constructed in the lambda cloning vector EMBL4, was screened for clones carrying arginine biosynthesis genes by complementation of Escherichia coli mutants. Clones complementing defects in argA, argB, argE, argG, argIF, carA, and carB were isolated. An E. coli defective in the acetylornithine deacetylase gene (argE) was complemented by the ornithine acetyltransferase gene (argJ) from N. gonorrhoeae. This heterologous complementation is reported for the first time. The carAB operon from E. coli hybridized with the gonococcal clones that carried carA or carB genes under conditions of high stringency, detecting 80% or greater similarity and showing that the nucleotide sequence of the carbamoylphosphate synthetase genes is very similar in these two organisms. Under these conditions for hybridization, the gonococcal clones carrying argB or argF genes did not hybridize with plasmids containing the corresponding E. coli gene. Cocomplementation experiments established gene linkage between carA and carB. Clones complementing a gene defect in argE were also able to complement an argA mutation. This suggests that the enzyme ornithine acetyltransferase from N. gonorrhoeae (encoded by argJ) may be able to complement both argA and argE mutations in E. coli. The arginine biosynthesis genes in N. gonorrhoeae appear to be scattered as in members of the family Pseudomonadaceae.     

Expression of Campylobacter genes for proline biosynthesis in Escherichia coli

Cloned DNA from Campylobacter jejuni was found to complement auxotrophic defects in proline metabolism in several strains of Escherichia coli. A 4.4-kilobase fragment of Campylobacter DNA encodes the genes analogous to the proA and B genes of E. coli and Salmonella typhimurium.     

The identification of cryptic rhamnose biosynthesis genes in Neisseria gonorrhoeae and their relationship to lipopolysaccharide biosynthesis.

Neisseria gonorrhoeae synthesizes a rough lipopolysaccharide that does not contain any of the repetitive units characteristic of the smooth lipopolysaccharide of members of the family Enterobacteriaceae. Three gonococcal homologs of Salmonella serovar typhimurium genes involved in the synthesis of the rhamnose component of the repetitive subunits have been isolated. Gonococcal homologs for rfbB, rfbA, and rfbD were found downstream of the galE gene in a region of the chromosome which shows overall homology with the meningococcal capsule gene complex region D. Sequence alignment demonstrated that the gonococcal gene products have 69, 65, and 54% amino acid identity with the Salmonella proteins RfbB, RfbA, and RfbD. The gonococcal RfbB and RfbA amino acid sequences share even more identical residues (73 and 65%, respectively) with the amino acid sequences derived from Escherichia coli genes o355 and o292, respectively. These genes are clustered with the genes involved in the biosynthesis of enterobacterial common antigen, and o355 is listed in the GenBank and Swiss Protein data banks as rffE (encoding UDP-GlcNAc-2-epimerase). However, complementation studies demonstrated that o355 does not encode the enzyme UDP-GlcNAc-2-epimerase. Gonococcal strains constructed with null mutations in the rfbBAD genes were unchanged in lipopolysaccharide phenotype and in the synthesis of gonococcal carbohydrate-containing C antigen. We were unable to detect any changes in gonococcal phenotype with respect to lipopolysaccharide sialylation, monoclonal-antibody binding, serum sensitivity, or interaction with eukaryotic cells in vitro. We conclude that the absence of a homolog for rfbC precludes the existence of a functional dTDP-rhamnose biosynthesis pathway in the gonococcal strains examined and that these genes are only maintained in N. gonorrhoeae either because of the presence of the galE gene or because of another as yet unrecognized function.     

Intra-cistronic complementation among trpC mutants of Escherichia coli

Summary Some merodiploids containing two different trpC alleles grow in the absence of added tryptophan. The trpC gene product is the bifunctional enzyme indoleglycerol phosphate synthetase but the ability to grow on minimal medium does not require that the alleles be dificient in different functions. This indication of complementation is supported by the evidence that the trpC alleles, of the merodiploids, can be transferred by transduction or by the trp-colV,B episome. Genetic recombination between episome and chromosome is seen to occur, but at a rate too low to explain the observed results. The nature of the interaction between the trpC alleles is not readily explained since indoleglycerol phosphate synthetase appears to consist of a single polypeptide chain in in vitro studies.     

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 of a Bacillus subtilis restriction fragment complementing auxotrophic mutants of eight Escherichia coli genes of arginine biosynthesis

Summary Following shotgun cloning of EcoRI fragments of Bacillus subtilis 168 chromosomal DNA in pBR322 a hybrid plasmid, pUL720, was isolated which complements Escherichia coli K12 mutants defective for argA, B, C, D, E, F/I, carA and carB. Restriction analysis revealed that the insert of pUL720 comprises four EcoRI fragments, of sizes 12.0, 6.0, 5.0 and 0.8 kbp. Evidence was obtained from subcloning, Southern blot hybridisation, enzyme stability studies and transformation of B. subtilis arginine auxotrophs that the 12 kbp EcoRI fragment carries all the arg genes. It proved impossible to subclone the intact fragment in isolation in the multicopy vectors pBR322, pBR325 or pACYC184, and although it could be subcloned in the low copy vector pGV1106, propagation of the hybrid rapidly resulted in the selection of stable derivatives carrying, near one end, an insertion of 1 kbp of DNA originating from the E. coli chromosome. These and other stable derivatives resulting from subcloning the 12 kbp EcoRI fragment have lost only the ability to complement for E. coli argC, and it is suggested that sequences located close to the equivalent of argC are involved in destabilising plasmids bearing the 12 kbp fragment in E. coli in a copy number dependent manner.Abbreviations kop kilobase pairs - OcTase ornithine carbamoyl transferase - CPSase carbamoyl phosphate synthetase     

Cloning of Klebsiella pneumoniae pqq genes and PQQ biosynthesis in Escherichia coli

We have cloned genes from Klebsiella pneumoniae which are required for pyrroloquinoline quinone (PQQ) biosynthesis. The cloned 6.7 kb fragment can complement several chromosomal pqq mutants. Escherichia coli strains are unable to synthesize PQQ but E. coli strains containing the cloned 6.7 kb K. pneumoniae fragment can synthesize PQQ in large amounts and E. coli pts mutants can be complemented on minimal glucose medium by this clone.     

Cosmid cloning of five Zymomonas trp genes by complementation of Escherichia coli and Pseudomonas putida trp mutants.

A library of Zymomonas mobilis genomic DNA was constructed in the broad-host-range cosmid pLAFR1. The library was mobilized into a variety of Escherichia coli and Pseudomonas putida trp mutants by using the helper plasmid pRK2013. Five Z. mobilis trp genes were identified by the ability to complement the trp mutants. The trpF, trpB, and trpA genes were on one cosmid, while the trpD and trpC genes were on two separate cosmids. The organization of the Z. mobilis trp genes seems to be similar to the organization found in Rhizobium spp., Acinetobacter calcoaceticus, and Pseudomonas acidovorans. The trpF, trpB, and trpA genes appeared to be linked, but they were not closely associated with trpD or trpC genes.     

Confirmatory identification of Neisseria gonorrhoeae by slide coagglutination

Neisseria gonorrhoeae was identified by the Phadebact gonococcus test, a rapid slide coagglutination technique, and the results obtained were compared with those obtained by conventional methods (Gram stain morphology, oxidase reaction, and carbohydrate utilization tests) for the confirmatory identification of gonococci. Of 308 clinical isolates examined, the coagglutination procedure correctly identified 97.8% of the isolates tested as N. gonorrhoeae and 93.9% of other bacteria as not N. gonorrhoeae. The coagglutination procedure also identified 29 laboratory strains correctly as not N. gonorrhoeae. The slide coagglutination test is easy to perform and offers a valuable alternative to other techniques for the confirmatory identification of N. gonorrhoeae.     

Cloning and expression in Escherichia coli of Serratia marcescens genes encoding prodigiosin biosynthesis

Prodigiosin, the bright red pigment produced by many strains of Serratia marcescens, is synthesized by a bifurcated pathway that terminates in the enzymatic condensation of the two final products, a monopyrrole and a bipyrrole . Sau3A fragments of S. marcescens ( Nima ) DNA were introduced into a strain of Escherichia coli K-12 by use of the cosmid vector pHC79 , and transformed clones were selected based on resistance to ampicillin. Among 879 transformants screened, 2 could be induced to synthesize prodigiosin when supplied with either one or both terminal products of the bifurcated pathway. Data are presented to support the idea that production of prodigiosin is not usually mediated by a plasmid.     

Cloning of yeast glycolysis genes by complementation

In hepatocytes isolated from fed rats, low concentrations of oxalate (50 to 100 μM) greatly enhance ketogenesis and decrease fatty acid synthesis. These metabolic changes are due to pyruvate carboxylase inhibition. Dichloroacetate, which can be catabolized into oxalate enhances ketogenesis only when cells are enriched with lactate and pyruvate and has no obvious effect on lipogenesis. The enhancement of ketogenesis, in both cases, is due to an imbalance between pyruvate dehydrogenase and pyruvate carboxylase but with oxalate, the primary event is oxaloacetate shortage and with dichloroacetate, mitochondrial acetyl CoA excess. This work demonstrates that the studied effects of dichloroacetate are not mediated by oxalate and that low concentrations of oxalate alter the lipid metabolism of hepatocytes.     

Conjugational complementation analysis of transfer-deficient mutants of Flac in Escherichia coli

A series of 102 transfer-deficient (tra(-)) mutants of Flac (84 of which have been previously described) were classified as carrying frameshift, amber, ochre, UGA, and nonsuppressible mutations. Techniques were evolved for using cultures of F prime strains as efficient recipients in matings, for measuring the number of (F'/F') heterozygote cells in transient populations, and for measuring complementation between different Flac tra(-) mutants. These techniques were used to define nine tra cistrons and to assign most of the tra(-) mutations to one or other of these.     

Cloning of a cDNA encoding adenylosuccinate lyase by functional complementation in Escherichia coli

Adenylosuccinate lyase was cloned by functional complementation of an Escherichia coli purB mutant using an avian liver cDNA expression library. The derived amino acid sequence is homologous to the bacterial purB-encoded adenylosuccinate lyase which catalyzes the same two steps in purine biosynthesis as the enzyme from animals. Avian adenylosuccinate lyase also shows regions of extensive sequence similarity to the urea cycle enzyme, argininosuccinate lyase. This homology suggests a similar mechanism for catalysis. Homology of adenylosuccinate and argininosuccinate lyases is intriguing because chickens do not utilize the urea cycle in nitrogen excretion. This is the first report of the cloning of a eukaryotic cDNA encoding adenylosuccinate lyase, and it affords a route to isolate the corresponding human gene which has been suggested to be defective in autistic children.