Complete sequence and genetic organization of pDTG1, the 83 kilobase naphthalene degradation plasmid from Pseudomonas putida strain NCIB 9816-4

The complete 83,042 bp sequence of the circular naphthalene degradation plasmid pDTG1 from Pseudomonas putida strain NCIB 9816-4 was determined in order to examine the process by which the nah and sal operons may have been compiled and distributed in nature. Eighty-nine open reading frames were predicted using computer analyses, comprising 80.0% of the pDTG1 DNA sequence. The most distinctive feature of the plasmid is the upper and lower naphthalene degradation operons, which occupy 9.5 kb and 13.4 kb regions, respectively, bordered by numerous defective mobile genetic element fragments. Identified on this plasmid were homologues of genes required for large plasmid replication, maintenance, and conjugation, as well as transposases, resolvases, and integrases, suggesting an evolution that involved the lateral transfer of DNA between bacterial species. Also found were genes that contain a high degree of sequence similarity to other known degradation genes, as well as genes involved in chemotaxis. Although the incompatibility group designation of pDTG1 remains unresolved, striking sequence organization and homology exists between the plasmid backbones of pDTG1 and the IncP-9 toluene-degradation plasmid pWW0, which suggests a divergent evolution from a progenitor plasmid prior to degradative gene incorporation.     

Bacterial degradation of phthalate isomers and their esters

Phthalate isomers and their esters are used heavily in various industries. Excess use and leaching from the product pose them as major pollutants. These chemicals are toxic, teratogenic, mutagenic and carcinogenic in nature. Various aspects like toxicity, diversity in the aerobic bacterial degradation, enzymes and genetic organization of the metabolic pathways from various bacterial strains are reviewed here. Degradation of these esters proceeds by the action of esterases to form phthalate isomers, which are converted to dihydroxylated intermediates by specific and inducible phthalate isomer dioxygenases. Metabolic pathways of phthalate isomers converge at 3,4-dihydroxybenzoic acid, which undergoes either ortho- or meta- ring cleavage and subsequently metabolized to the central carbon pathway intermediates. The genes involved in the degradation are arranged in operons present either on plasmid or chromosome or both, and induced by specific phthalate isomer. Understanding metabolic pathways, diversity and their genetic regulation may help in constructing bacterial strains through genetic engineering approach for effective bioremediation and environmental clean up.     

Suicidal genetically engineered microorganisms for bioremediation: need and perspectives

In the past few decades, increased awareness of environmental pollution has led to the exploitation of microbial metabolic potential in the construction of several genetically engineered microorganisms (GEMs) for bioremediation purposes. At the same time, environmental concerns and regulatory constraints have limited the in situ application of GEMs, the ultimate objective behind their development. In order to address the anticipated risks due to the uncontrolled survival/dispersal of GEMs or recombinant plasmids into the environment, some attempts have been made to construct systems that would contain the released organisms. This article discusses the designing of safer genetically engineered organisms for environmental release with specific emphasis on the use of bacterial plasmid addiction systems to limit their survival thus minimizing the anticipated risk. We also conceptualize a novel strategy to construct "Suicidal Genetically Engineered Microorganisms (SGEMs)" by exploring/combining the knowledge of different plasmid addiction systems (such as antisense RNA-regulated plasmid addiction, proteic plasmid addiction etc.) and inducible degradative operons of bacteria.     

Regulation of the nah and sal operons of plasmid NAH7: evidence for a new function in nahR

The catabolism of naphthalene and salicylate is specified by two operons on an 80 Kb metabolic plasmid, NAH7. These operons, nah and sal, are carried on the contiguous 30 Kb EcoRI-A, C fragments, and are under positive control of a regulator region, nahR. Five Nah Sal Tn5 insertion mutants form two complementation groups: A = nahR203, nahR204; and B = nahR201, nahR202, nahR205. The physical and genetic maps assign the nahR location to the 15.7-17.2 Kb region of the EcoRI-A fragment, with suggestion of more than one control gene.     

Metabolic engineering of carotenoid biosynthesis in Escherichia coli by ordered gene assembly in Bacillus subtilis

We attempted to optimize the production of zeaxanthin in Escherichia coli by reordering five biosynthetic genes in the natural carotenoid cluster of Pantoea ananatis. Newly designed operons for zeaxanthin production were constructed by the ordered gene assembly in Bacillus subtilis (OGAB) method, which can assemble multiple genes in one step using an intrinsic B. subtilis plasmid transformation system. The highest level of production of zeaxanthin in E. coli (820 microg/g [dry weight]) was observed in the transformant with a plasmid in which the gene order corresponds to the order of the zeaxanthin metabolic pathway (crtE-crtB-crtI-crtY-crtZ), among a series of plasmids with circularly permuted gene orders. Although two of five operons using intrinsic zeaxanthin promoters failed to assemble in B. subtilis, the full set of operons was obtained by repressing operon expression during OGAB assembly with a p(R) promoter-cI repressor system. This result suggests that repressing the expression of foreign genes in B. subtilis is important for their assembly by the OGAB method. For all tested operons, the abundance of mRNA decreased monotonically with the increasing distance of the gene from the promoter in E. coli, and this may influence the yield of zeaxanthin. Our results suggest that rearrangement of biosynthetic genes in the order of the metabolic pathway by the OGAB method could be a useful approach for metabolic engineering.     

Localization of camphor degradative plasmids on the chromosome of Pseudomonas putida strains PaW]

Camphor degradative plasmids (CAM, pRK1) are preferentially situated on chromosomes of Pseudomonas putida strains PaW. After having been transferred into Cam+ strains, the TOL plasmid pWWO dissociates into the cryptic plasmid pWWO-8 and chromosome-borne transposon Tn4651. The opposite situation, i.e. reconstruction of the TOL plasmid pWWO from the cryptic plasmid pWWO-8 and chromosome-borne catabolic operons of the pWWO plasmid has been described. Cam- derivatives of the CAM plasmid were obtained in vivo which contain the TOL plasmid transposons Tn4651 or Tn4652 as obligatory structural elements. These plasmids as well as pWWO-8 determine conjugational mobilization of chromosome-located cam operons followed by their integration into the chromosome of recipient.     

The kilE locus of promiscuous IncP alpha plasmid RK2 is required for stable maintenance in Pseudomonas aeruginosa.

Eight coordinately regulated operons constitute the kor regulon of the IncP alpha plasmid RK2. Three operons specify functions required for replication initiation, conjugative transfer, and control of gene expression. The functions of the other operons, including those of the four coregulated operons that compose the kilA, kilC, and kilE loci, have not been determined. Here, we present the first evidence that a kil determinant is involved in IncP plasmid maintenance. Elevation of KorC levels specifically to reduce the expression of the KorC-regulated kilC and kilE operons severely affected the maintenance of both the IncP alpha plasmid RK2lac and the IncP beta plasmid R751 in Pseudomonas aeruginosa but had little effect on plasmid maintenance in Escherichia coli. Precise deletion of the two kilE operons from RK2lac was achieved with the VEX mutagenesis system for large genomes. The resulting plasmid showed significant loss of stability in P. aeruginosa only. The defect could be complemented by reintroduction of kilE at a different position on the plasmid. The instability of the RK2lac delta kilE mutant did not result from a reduction in average plasmid copy number, reduced expression of kilC, decreased conjugative transfer, or loss of the korE regulator. We found that both the par and kilE loci are required for full stability of RK2lac in P. aeruginosa and that the par and kilE functions act independently. These results demonstrate a critical role for the kilE locus in the stable inheritance of RK2 in P. aeruginosa.     

Functions of the duplicated hik31 operons in central metabolism and responses to light, dark, and carbon sources in Synechocystis sp. strain PCC 6803

There are two closely related hik31 operons involved in signal transduction on the chromosome and the pSYSX plasmid in the cyanobacterium Synechocystis sp. strain PCC 6803. We studied the growth, cell morphology, and gene expression in operon and hik mutants for both copies, under different growth conditions, to examine whether the duplicated copies have the same or different functions and gene targets and whether they are similarly regulated. Phenotype analysis suggested that both operons regulated common and separate targets in the light and the dark. The chromosomal operon was involved in the negative control of autotrophic events, whereas the plasmid operon was involved in the positive control of heterotrophic events. Both the plasmid and double operon mutant cells were larger and had division defects. The growth data also showed a regulatory role for the chromosomal hik gene under high-CO(2) conditions and the plasmid operon under low-O(2) conditions. Metal stress experiments indicated a role for the chromosomal hik gene and operon in mediating Zn and Cd tolerance, the plasmid operon in Co tolerance, and the chromosomal operon and plasmid hik gene in Ni tolerance. We conclude that both operons are differentially and temporally regulated. We suggest that the chromosomal operon is the primarily expressed copy and the plasmid operon acts as a backup to maintain appropriate gene dosages. Both operons share an integrated regulatory relationship and are induced in high light, in glucose, and in active cell growth. Additionally, the plasmid operon is induced in the dark with or without glucose.     

Construction,properties, and application of the pCB5 plasmid,a novel conjugative shuttle vector with a Cupriavidus basilensis origin of replication

Cupriavidus basilensis is a species with diverse metabolic capabilities, including degradation of xenobiotics and heavy metal resistance. Although the genomes of several strains of this species have been sequenced, no plasmid has yet been constructed for genetic engineering in this species. In this study, we identified a novel plasmid, designated pWS, from C. basilensis WS with a copy number of 1–3 per cell and a length of 2150 bp. pWS contained three protein-coding genes, among which only rep was required for plasmid replication. Rep showed no homology with known plasmid replication initiators. Unlike most plasmids, pWS did not have a cis-acting replication origin outside the region of rep. The minimal replicon of pWS was stable in C. basilensis WS without selection. A conjugative C. basilensis/Escherichia coli shuttle vector, pCB5, was constructed using the minimal replicon of pWS. Interestingly, the copy number of pCB5 was flexible and could be manipulated. Enhancing the expression level of Rep in pCB5 by either doubling the promoter or coding region of rep resulted in doubling of the plasmid copy number. Moreover, replacing the native promoter of rep with the lac promoter increased the copy number by over fivefold. Finally, using two different β-galactosidase reporting systems constructed with pCB5, we successfully demonstrated the different regulatory patterns of bph and dmp operons during diphenyl ether (DE) degradation in C. basilensis WS. Thus, this shuttle vector provided an efficient tool for DNA cloning and metabolic engineering in C. basilensis.

     

Metabolic Engineering of Carotenoid Biosynthesis in Escherichia coli by Ordered Gene Assembly in Bacillus subtilis

We attempted to optimize the production of zeaxanthin in Escherichia coli by reordering five biosynthetic genes in the natural carotenoid cluster of Pantoea ananatis. Newly designed operons for zeaxanthin production were constructed by the ordered gene assembly in Bacillus subtilis (OGAB) method, which can assemble multiple genes in one step using an intrinsic B. subtilis plasmid transformation system. The highest level of production of zeaxanthin in E. coli (820 μg/g [dry weight]) was observed in the transformant with a plasmid in which the gene order corresponds to the order of the zeaxanthin metabolic pathway (crtE-crtB-crtI-crtY-crtZ), among a series of plasmids with circularly permuted gene orders. Although two of five operons using intrinsic zeaxanthin promoters failed to assemble in B. subtilis, the full set of operons was obtained by repressing operon expression during OGAB assembly with a p_R promoter-cI repressor system. This result suggests that repressing the expression of foreign genes in B. subtilis is important for their assembly by the OGAB method. For all tested operons, the abundance of mRNA decreased monotonically with the increasing distance of the gene from the promoter in E. coli, and this may influence the yield of zeaxanthin. Our results suggest that rearrangement of biosynthetic genes in the order of the metabolic pathway by the OGAB method could be a useful approach for metabolic engineering.     

Design of expression systems for metabolic engineering: coordinated synthesis and degradation of glycogen

In metabolic engineering, systems which allow coordinated control of two metabolic pathways can be useful. We designed two expression systems and demonstrated their application by coordinating glycogen synthesis and degradation. The first expression vector pMSW2 expressed the glycogen synthesis genes in one operon and the glycogen degradation gene in a separate, coordinately regulated operon. The plasmid was designed to switch off expression of the first operon and activate expression of the second operon on addition of IPTG. As an alternative means to control glycogen synthesis and degradation pathways, we constructed expression vector pGTSD100, which contains the native Escherichia coli glycogen synthesis and degradation operon under control of the tac promoter. Both expression vectors work successfully to control the net synthesis and degradation of glycogen. In cultures of the E. coli strain TA3476 carrying the plasmid pMSW2, before the addition of IPTG, glycogen continued to accumulate in the culture. About three hours after IPTG was added, glycogen levels began to decrease. When no IPTG was added to cultures of TA3476:pMSW2, glycogen accumulated in the cells as before but the rate of degradation of glycogen was much lower. When IPTG was added to TA3476:pMSW2, the total cell protein at the end of batch cultivation was approximately 15% higher compared to cultures without IPTG addition. The extra biomass was formed during the glycogen degradation phase. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 419-426, 1997.