Genetic analysis of a transposon carrying toluene degrading genes on a TOL plasmid pWWO

Summary Toluene degrading (xyl) genes on a Pseudomonas TOL plasmid pWWO are located within a 39-kb DNA portion. The 56-kb region including these xyl genes and its 17-kb derivative with a deletion of the internal 39-kb portion transposed to various sites on target replicons such as pACYC184 and R388 in escherichia coli recA strains. Thus the 56- and 17-kb regions were designated Tn4651 and Tn4652, respectively. Genetic analysis of Tn4652 demonstrated that its transposition occurs by a two-step process, namely, cointegrate formation and its subsequent resolution. The presence in cis of DNA sequences of no more than 150 bp at both ends of Tn4652 was prerequisite for cointegrate formation, and this step was mediated by a trans-acting factor, transposase, which was encoded in a 3.0-kb segment at one end of the transposon. Cointegrate resolution took place site-specifically within a 200-bp fragment, which was situated 10 kb away from the transposase gene. Based on the stability of cointegrates formed by various mini-Tn4652 derivatives, it was shown that the cointergrate resolution requires two trans-acting factors encoded within 1.0- and 1.2-kb fragments that encompass the recombination site involved in the resolution.     

Segregational instability of pUB110-derived recombinant plasmids in Bacillus subtilis

To study plasmid instability in Bacillus subtilis the pUB110-derived hybrid plasmid pLB2 (3.6 kb) and the bifunctional replicon pLB5 (5.9 kb), able to replicate in B. subtilis and Escherichia coli, were constructed. In both vectors homologous B. subtilis, or heterologous E. coli DNA fragments of various lengths were inserted. Irrespective of the source of the cloned DNA, the segregational stability of the recombinant plasmids in B. subtilis was severely affected by the DNA inserts. In contrast, no instability was observed in E. coli. In B. subtilis a steep inverse relationship existed between the size of the inserts and the level of stability. Increased size of the pLB plasmids resulted in strongly reduced copy numbers. This seems to be the primary cause of the size-dependent segregational instability.     

Regulation of naphthalene catabolic genes of plasmid NAH7.

Tn5 insertion mutations defining a regulatory gene, nahR, of the naphthalene catabolic pathway encoded by the NAH7 plasmid were mapped within a small NAH7 region only a few hundred bases upstream of the nahG gene, the most promoter-proximal gene of the nahGHIJK operon. The nahR mutations blocked the induction of both the nahABCDEF and nahGHIJK operons, and the defect was completely corrected in the presence of the wild-type allele in a trans position. The pleiotropic, recessive, and negative nature of these mutations indicates that the nahR gene specifies a regulatory element which is required to activate both nah operons.     

Fate in soil of a recombinant plasmid carrying aDrosophila gene

A recombinant plasmid (C357; 3.5 Mdal) containing heterologous DNA (pBR322 [2.6 Mdal] with cDNA for an egg yolk protein fromDrosophila grimshawi) inEscherichia coli strain HB101 survived in and was recovered on selective media from sterile and nonsterile soil during 27 days at frequencies similar to those of theE. coli(pBR322) system. In sterile saline, the numbers of all cells decreased during 34 days, but the numbers of the plasmidless host declined less. There was no selective loss of the heterologous DNA in either soil or saline, as determined by colony hybridization with a³²P-labeled DNA probe for the cDNA, but the HB101(C357) appeared to be less able than HB101(pBR322) to cope with conditions of starvation. These results suggested that nonessential eucaryotic DNA inserted into plasmid DNA has little effect on the survival in soil or saline of the bacterial host and the maintenance of the vector.     

A plasmid cloning vector for the direct selection of strains carrying recombinant plasmids

A plasmid cloning vector with ampicillin-resistance and streptomycin-sensitivity markers is suitable for the direct selection of strains carrying recombinant plasmids. The selection for plasmid transformants utilizes their ampicillin resistance whereas selection for recombinant plasmids is based on the inactivation of the rpsL gene contained on the plasmid. When streptomycin-resistant Escherichia coli strains are used as recipients in transformation, transformants carrying the parental plasmid are phenotypically sensitive to streptomycin while those carrying hybrid plasmids are resistant to streptomycin.     

Lambda transducing phages carrying plasmid R100 replication genes

VAλ73, a λ bacteriophage carrying R100 replication genes, maintains itself as an independent plasmid under conditions in which the λ replication genes are inactive.     

Stability of a recombinant plasmid carrying a-amylase gene in Bacillus subtilis

Plasmid pSB6 is a streptococcal recombinant plasmid carrying the a-amylase gene of Bacillus amyloliquefaciens and the chloramphenicol resistance gene. The segregational and structural instabilities of this plasmid were examined under non-selective conditions in Bacillus subtilis. These instabilities were modelled according to a kinetic expression derived from the difference in the growth between plasmid-bearing and plasmid-free cells. This plasmid showed slight segregational instability and much higher levels of structural instability under the conditions examined.     

Stability of a recombinant plasmid carrying a-amylase gene in Bacillus subtilis

Plasmid pSB6 is a streptococcal recombinant plasmid carrying the a-amylase gene of Bacillus amyloliquefaciens and the chloramphenicol resistance gene. The segregational and structural instabilities of this plasmid were examined under non-selective conditions in Bacillus subtilis. These instabilities were modelled according to a kinetic expression derived from the difference in the growth between plasmid-bearing and plasmid-free cells. This plasmid showed slight segregational instability and much higher levels of structural instability under the conditions examined.     

Construction of recombinant plasmid carrying the lambda DNA fragment responsible for prophage integration.

The recombinant DNA molecules were constructed from plasmid RSF2124 and the EcoRI fragment of lambda DNA containing the genes responsible for prophage integration. The presence of these genes in recombinant plasmids was detected genetically. lambda int-gene was shown to be expressed in either orientation of insertion in the plasmid. We found that recombinant plasmid was able to integrate into chromosome of lambda lysogens. The integration of plasmid into host chromosome was demonstrated by contransduction of chromosome and plasmid markers using generalized transducer P1 and by specialized transduction with lambda phages.     

Characterization of recombinant rabies virus carrying double glycoprotein genes

A recombinant rabies virus carrying double glycoprotein (G) genes, R(NPMGGL) strain, was generated by a reverse genetics system utilizing cloned cDNA of the RC-HL strain, and the biological properties of the virus were compared to those of the recombinant RC-HL (rRC-HL) strain. The extents of virus growth in cultured cells and virulence for adult mice of the R(NPMGGL) strain were almost same as those of the rRC-HL strain, while G protein content of the purified R(NPMGGL) virion and G protein expression level in R(NPMGGL)-infected cells were 1.5-fold higher than those of the rRC-HL strain. As a result of serial passages of the R(NPMGGL) strain in cultured cells, the expression level of G protein in cultured cells infected with the passaged R(NPMGGL) strain was maintained and virus titers rose with adaptation to the cultured cells. Furthermore, analysis of neutralization titers in mice immunized with UVinactivated virus suggested that the R(NPMGGL) strain had higher immunogenicity than that of the rRC-HL strain. The results suggest that the R(NPMGGL) strain carrying double G genes might be a useful candidate for development of a new inactivated rabies vaccine.     

Evidence of indigenous NAH plasmid of naphthalene degrading Pseudomonas putida PpG7 strain implicated in limonin degradation

A well characterized naphthalene-degrading strain, Pseudomonas putida PpG7 was observed to utilize limonin, a highly-oxygenated triterpenoid compound as a sole source of carbon and energy. Limonin concentrations evidenced a 64% reduction over 48 h of growth in batch cultures. Attempts were made to acquire a plasmid-less derivative via various methods (viz. Ethidium Bromide, SDS, elevated temperature & mitomycin C), among which the method involving mitomycin C (20 ug/ml) proved successful. Concomitant with the loss of plasmid in P. putida PpG7 strain, the cured derivative was identified as a lim- phenotype. The lim+ phenotype could be conjugally transferred to the cured derivative. Based on the results of curing with mitomycin C, conjugation studies and presence of ndo gene encoding naphthalene 1,2 dioxygenase, it was demonstrated that genes for the limonin utilization were encoded on an 83 kb indigenous transmissible Inc. P9 NAH plasmid in Pseudomonas putida PpG7 strain.     

Localization of structural genes of Vibrio cholerae cholerae toxin using the recombinant plasmid RP4omega elt

The recombinant plasmid RP4 omega elt carrying Escherichia coli heat-labile enterotoxin elt genes with 70-80% homology with genes vct of Vibrio cholerae has been constructed. We used this plasmid to determine localization of the cholerae toxin genes vct on the map of Vibrio cholerae cholerae. Two types of the donors were revealed in matings of 10 strains of V. cholerae cholerae 569B/RP4 omega elt with the polyauxotrophic recipients RV31 and RV175: some strains had enhanced frequency of mobilization of ilv-1 and lys-6 markers, the others--of trp-1. Our data suggest that structural vct genes are located within two regions of V. cholerae cholerae 569B chromosome: trp-1 and ilv-1--lys-6.     

Optimal fed-batch operation of recombinant cells subject to plasmid instability and death

Optimal feed rate strategy is studied for fed-batch culture of recombinant cells with plasmid instability and with different death rates for the plasmid-free cells (PFC) and plasmid-bearing cells (PBC). Most of the fed-batch fermentation is known to have first-order singularity and therefore a single singular arc. However, this study shows that a singular arc with second-order singularity and therefore two distinct singular arcs are possible for a recombinant cell process if PFC and PBC are subjected to death, and their specific growth rates are proportional to each other. Two types of singular arcs are elucidated and analyzed. The optimal policies over the singular arcs are theoretically explored as these findings reveal qualitative information on the singular arc, which is critically important in providing the optimal initial conditions in numerical computation of optimal feed rate profile.     

Fermentation of lactose by Zymomonas mobilis carrying a Lac+ recombinant plasmid

Lac⁺ recombinant plasmids encoding a β-galactosidase fused protein and lactose permease of Escherichia coli were introduced Zymomonas mobilis. The fused protein was expressed with 450 to 5,860 Miller units of β-galactosidase activity, and functioned as lactase. Raffinose uptake by Z. mobilis CP4 was enhanced in the plasmid-carrying strain over the plasmid-free strain, suggesting that the lactose permease was functioning in the organism. Z. mobilis carrying the plasmid could produce ethanol from lactose and whey, but could not grow on lactose as the sole carbon source. It was found that the growth of the organism was inhibited by either galactose of the galactose liberated from lactose.     

Development of a molecular detection method for naphthalene degrading pseudomonads

Abstract: A combined polymerase chain reaction amplification and reverse dot blot assay was designed for the detection of bacterial genes from soil and sediments. Total nucleic acids were directly extracted from soil using a lysozyme/sodium dodecyl sulfate/freeze-thaw method followed by rapid purification through gel electrophoresis. DNA was amplified using a highly stringent polymerase chain reaction with primers directed against the nahR regulatory gene present in plasmid NAH7 of Pseudomonas putida G7. The resulting amplification product was detected colorimetrically by reverse dot blot with an improved sensitivity ten-fold greater than traditional ethidium bromide staining after gel electrophersis. A lower limit of 10³, P. putida G7 cfu (g soil)⁻¹ was detected. This method was successfully employed to detect indigenous naphthalene-degrading bacteria from subsurface sediment collected from different locations of a naphthalene-contaminated site. Similar approaches could be developed for other soil-borne genetic markers.     

Cloning and expression in Escherichia coli of the naphthalene degradation genes from plasmid NAH7

The genes encoding the enzymes responsible for conversion of naphthalene to 2-hydroxymuconic acid (nahA through nahI) are contained on a 25-kilobase EcoRI fragment of an 85-kilobase NAH plasmid of Pseudomonas putida. These genes were cloned into the plasmid vectors pBR322 and RSF1010 to obtain the recombinant plasmids pKGX505 and pKGX511, respectively. To facilitate cloning and analysis, an NAH7 plasmid containing a Tn5 transposon in the salicylate hydroxylase gene (nahG) was used to derive the EcoRI fragment. The genes for naphthalene degradation were expressed at a low level in Escherichia coli strains containing the fragment on the recombinant plasmids pKGX505 or pKGX511. This was shown by the ability of whole cells to convert naphthalene to salicylic acid and by in vitro enzyme assays. The expression of at least two of these genes in E. coli appeared to be regulated by the presence of the inducer salicylic acid. In addition, high-level expression and induction appear to be mediated by an NAH plasmid promoter and a regulatory gene located on the fragment. A restriction endonuclease cleavage map of the cloned fragment was generated, and the map positions of several nah genes were determined by analysis of various subcloned DNA fragments.     

Isolation and characterization of recombinant DNA plasmids carrying Drosophila tRNA genes.

Recombinant plasmids carrying Drosophila melanogaster tRNA genes were constructed by ligation of HindIII-cleaved Drosophila DNA to HindIII cut pBR322 DNA. 90 clones were isolated that contained genes for one or more of eleven tRNAs. 43 of the plasmids were characterized by a number of methods: restriction nuclease digestion; agarose gel electrophoresis; hybridization with individual, purified, 125I-labelled Drosophila tRNA molecules and in situ hybridization to Drosophila chromosomes. The results show that several different tRNA genes have been isolated which code for single, specific isoacceptors. The DNAs from 8 plasmids each hybridize to single sites on Drosophila polytene chromosomes. In addition, the data show examples of two different plasmids hybridizing to different loci coding for the same tRNA; this means that we have isolated representatives of tRNA genes which map at widely separated points on the Drosophila genome.