A bioluminescent reporter plasmid for naphthalene catabolism (pUTK21) was developed by transposon (Tn4431) insertion of the lux gene cassette from Vibrio fischeri into a naphthalene catabolic plasmid in Pseudomonas fluorescens. The insertion site of the lux transposon was the nahG gene encoding for salicylate hydroxylase. Luciferasemediated light production from P. fluorescens strains harboring this plasmid was induced on exposure to naphthalene or the regulatory inducer metabolite, salicylate. In continuous culture, light induction was rapid (15 minutes) and was highly responsive to dynamic changes in naphthalene exposure. Strains harboring pUTK21 were responsive to aromatic hydrocarbon contamination in Manufactured Gas Plant soils and produced sufficient light to serve as biosensors of naphthalene exposure and reporters of naphthalene biodegradative activity. The robust and sensitive nature of the bioluminescent reporter technology suggests that new sensing methods can be developed for on-line process monitoring and control in complex environmental matrices.
Biosensors for the detection of pollutants in the environment can complement analytical methods by distinguishing bioavailable from inert, unavailable forms of contaminants. By using fusions of the wellunderstood Tn2l mercury resistance operon (mer) with promoterless luxCDABE from Vibriofischeri, we have constructed and tested three biosensors for Hg(II). Bioluminescence specified by pRB28, carrying merRoipT', by pOS14, mediating active transport of Hg(II), and by pOS15, containing an intact mer operon, was measured in rich and minimal media. The highest sensitivities were achieved in minimal medium and were 1, 0.5, and 25 nM Hg(II) for pRB28, pOS14, and pOS15, respectively. The utility of the biosensors in natural waters was demonstrated with freshwater, rain, and estuarine samples supplemented with Hg(II). mer-lux carried by pRB28 and pOS14 responded to Hg(II) in mercury-contaminated water samples collected from a freshwater * Corresponding author.
We have demonstrated the efficacy of a light-generating genetic construction in describing the induction of a nah operon for the catabolism of naphthalene. A fragment from plasmid NAH7, which contains the promoter for the upper pathway of degradation, was transcriptionally fused to the lux genes of Vibrio fischeri. A Pseudomonas strain containing this construction is inducible to high levels of light production in the presence of a suitable substrate and the nahR regulatory gene product. This system was used to examine catabolic activity in a unique manner under a variety of growth conditions. Induction of bioluminescence was demonstrated to coincide with naphthalene degradation in all cases through the use of mineralization assays. A significant delay in bioluminescence and biodegradation was observed when naphthalene was added to batch cultures that were growing exponentially. These results suggest that the metabolism of naphthalene by this Pseudomonas strain is optimal when the growth rate of the culture is slow and is greatly reduced during exponential growth.
Pseudomonas fluorescens HK44 represents the first genetically engineered microorganism approved for field testing in the United States for bioremediation purposes. Strain HK44 harbors an introduced lux gene fused within a naphthalene degradative pathway, thereby allowing this recombinant microbe to bioluminesce as it degrades specific polyaromatic hydrocarbons such as naphthalene. The bioremediation process can therefore be monitored by the detection of light. P. fluorescens HK44 was inoculated into the vadose zone of intermediate-scale, semicontained soil lysimeters contaminated with naphthalene, anthracene, and phenanthrene, and the population dynamics were followed over an approximate 2-year period in order to assess the long-term efficacy of using strain HK44 for monitoring and controlling bioremediation processes. Results showed that P. fluorescens HK44 was capable of surviving initial inoculation into both hydrocarbon contaminated and uncontaminated soils and was recoverable from these soils 660 days post inoculation. It was also demonstrated that strain HK44 was capable of generating bioluminescence in response to soil hydrocarbon bioavailability. Bioluminescence approaching 166 000 counts/s was detected in fiber optic-based biosensor devices responding to volatile polyaromatic hydrocarbons, while a portable photomultiplier module detected bioluminescence at an average of 4300 counts/s directly from soil-borne HK44 cells within localized treatment areas. The utilization of lux-based bioreporter microorganisms therefore promises to be a viable option for in situ determination of environmental contaminant bioavailability and biodegradation process monitoring and control.
Plasmids that encode genes for the degradation of recalcitrant compounds are often examined only for characteristics of the degradative pathways and ignore regions that are necessary for plasmid replication, incompatibility, and conjugation. If these characteristics were known, then the mobility of the catabolic genes between species could be predicted and different catabolic pathways might be combined to alter substrate range. Two catabolic plasmids, pSS50 and pSS60, isolated from chlorobiphenyl-degrading strains and a 3-chlorobenzoate-degrading plasmid, pBR60, were compared with the previously described IncP group (Pseudomonas group P-i) plasmids pJP4 and R751. All three of the former plasmids were also members of the IncP group, although pBR60 is apparently more distantly related. DNA probes specific for known genetic loci were used to determine the order of homologous loci on the plasmids. In all of these plasmids the order is invariant, demonstrating the conservation of this "backbone" region. In addition, all five plasmids display at least some homology with the mercury resistance transposon, Tn501, which has been suggested to be characteristic of the 13 subgroup of the IncP plasmids. Plasmids pSS50 and pSS60 have been mapped in detail, and repeat sequences that surround the suspected degradation genes are described.
Broad-host-range plasmid RK2 encodes several different kil genes which are potentially lethal to an Escherichia coli host. The kil genes and the essential RK2 replication gene trfA are regulated by the products of kor genes. We have shown previously that kilA can be controlled by a constitutively expressed korA gene. In this study, we have found that the wild-type, autoregulated korA gene is insufficient for control of kilA cloned on high-copy-number plasmids. One of two other genes must also be present with korA. One gene is korB, originally discovered by its ability to control the determinants in the kilB region and later found to affect expression of both trfA and korA. The other is a new gene, korE, which has been cloned from the 2.2' to 4.1' region located between korC and kilA. Studies with a kilA-cat fusion suggest that korA, korB, and korE all participate in the control of kilA gene expression.
Broad-host-range plasmid RK2 encodes several kil operons (kiLA, kiLB, ki4C, kilE) whose expression is potentially lethal to Escherichia coli host cells. The kil operons and the RK2 replication initiator gene (trfA) are coregulated by various combinations of kor genes (korA, korB, korC, korE). This regulatory network is called the kU-kor regulon. Presented here are studies on the structure, product, and expression of korC. Genetic mnpping revealed the precise location of korC in a region near transposon Tnl. We determined the nucleotide sequence of'this region and identified the korC structural gene by analysis of korC mutants. Sequence analysis predicts the korC product to be a polypeptide of 85 amino acids with a molecular mass of 9,150 daltons. The KorC polypeptide was identified in vivo by expressing wild-type and mutant korC allel,s from a bacteriophage T7 RNA' polymerase-dependent promoter. The predicted structure of KorC polypeptide has a net positive charge and a helix-turn-helix region similar to those of known DNA-binding proteins. These properties 'are consistent with the repressorlike function of KorC protein, and we discuss the evidence that KorA'and KorC proteins act as corepressors in-the control of the kilC and kilE operons. Finally, we show that koiC is expressed from the bla promoters within the upstream transposon Tnl, suggesting that insertion of Tnl interrupted a plasmid operon that may have originally included korC and kilC.Plasmids of incompatibility group P (IncP) can replicate in many different species of gram-negative bacteria (17,44,80). The genetic and molecular basis for this extensive host range is not yet understood. However, studies on the IncP plasmid RK2 (30) have revealed an unusual system of genetic interactions involved in the control of plasmid replication and maintenance (22, 80).Two genetic determinants are required for RK2 replication: oriV, the origin of unidirectional replication, and trfA, a gene that encodes a polypeptide needed for initiation of replication at oriV (20,33,41,46,47,53,54,59,64,66,74,78,81). The trfA operon is controlled as part of a complex regulatory network. This network, designated the kil-kor regulon, also includes several potentially host-lethal kil operons (kilA, kilB, kilC, kilE) whose functions are unknown (21,48,62; J. Kornacki, C. Chang, and D. Figurski, unpublished data). The kil operons and the trfA operon are negatively regulated by various kor genes (korA, korB, korC, korE) (4,5,21,55,60,72,76,77,(86)(87)(88) Kornacki et al., unpublished data). The regulation of trfA by kor genes directly links the kil-kor regulon to control of plasmid replication. Furthermore, coregulation of the trfA and kil operons hints that the kil determinants may be involved in plasmid maintenance or host range.A distinctive feature of the kil-kor regulon is that the operons are regulated by combinations of kor genes (Fig. 1). korA and korB functions inhibit expression of the trfA operon (55, 60), the kilA operon (4, 21, 87, 88), and the korA-korB operon (5, 72, ...
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