Grantee Research Project Results
Final Report: Use of Pseudomonas Starvation Promoters in In-Situ Bioremediation
EPA Grant Number: R823390Title: Use of Pseudomonas Starvation Promoters in In-Situ Bioremediation
Investigators: Matin, Abdul , Park, C. H. , Hahm, D. H. , Rivkina, Marian , Keyhan, Mimi , Pandza, S.
Institution: Stanford University
EPA Project Officer: Hahn, Intaek
Project Period: August 15, 1995 through August 14, 1998
Project Amount: $401,960
RFA: Exploratory Research - Environmental Biology (1995) RFA Text | Recipients Lists
Research Category: Biology/Life Sciences , Human Health , Aquatic Ecosystems
Objective:
To construct Pseudomonas putida strains capable of expressing toluene monooxygenase at high levels during slow growth.Summary/Accomplishments (Outputs/Outcomes):
Toluene monooxygenase (TMO) can degrade trichloroethylene (TCE) and phenol. The former compound, especially, is a wide spread pollutant and poses serious health hazards. TMO catalyzes the critical reaction that in a mixed microbial population leads to complete mineralization of TCE.Scientists have exploited this property of TMO and related enzymes to bring about in-situ bioremediation of TCE at the contaminated sites, relying on the indigenous bacteria to produce these enzymes. However, in natural bacteria, the expression of these enzymes is linked to rapid growth. But nutrients are generally scarce in nature, precluding rapid growth and thus significant expression of TMO. To overcome this hurdle, the approach of "biostimulation" is employed, wherein nutrients to support the growth of the relevant bacteria are fed to the contaminated site. For instance, to stimulate TMO producing bacterial growth, addition of phenol has been practiced (1).
Biostimulation has proved successful in affecting significant remediation of TCE. There are problems, however, with this approach resulting from the growth of the indigenous population. A biofilm tends to form around the port used for injecting the nutrients, confining remediation to a narrow zone. Furthermore, addition of large amounts of nutrients is logistically difficult.
In our previous work, we constructed strains of Escherichia coli that expressed TMO without the need for rapid growth. These strains were able to remediate more than 100-fold more TCE per unit biomass formed than the natural bacteria (2). The recombinant E. coli strains were constructed by making use of special regulatory elements, termed the "starvation promoters." These elements are unique in cell physiology in that while most other regulatory elements determining gene expression in bacteria tend to shut down at the end of rapid growth, the starvation promoters are switched on maximally in non- (or very slowly-)growing bacteria. These promoters were discovered in the P.I. laboratory (3) during studies on the starvation response of bacteria.
In the current project, we constructed strains of an environmental bacterium, P. putida, that have also acquired this capacity. Transposon mutagenesis was used to identify starvation genes in P. putida. The gene-encoding and the promoter regions were cloned (4). Surprisingly, it was found that the cloned carbon starvation promoter was transcribed by Es54,although this holoenzyme has never before been implicated in carbon starvation response. This starvation promoter controls at least eight open reading frames, and work in progress has provided strong suggestive evidence that one of these codes for a GTP-binding protein. Evidence is accumulating in my lab. that this protein has a role in sensing starvation and perhaps other stresses, and works in concert with the other genes of this operon. These findings are highly relevant to optimizing the expression of starvation promoter-controlled genes and are being pursued in my laboratory by support from other sources.
A major accomplishment in the grant period was to construct a plasmid (pAM103) in which the tmo gene is regulated by the P. putida starvation promoter (termed Pstarv1) that we cloned. This was accomplished as follows.
Pseudomonas molecular manipulation is more complex than that of E. coli. One needs broad host range plasmids, and to employ both E. coli and the Pseudomonas sp. for the needed steps. The making of pAM103 necessitated the use of several different plasmids, assay systems, and generation of unique restrictions sites.
The following steps were employed to make a broad host range plasmid that would express the Pstarv1-tmo construct in P. putida . The initial steps of this cloning relied on tmo expression in E. coli. Whether TMO was made or not was detected using the following rationale. The tryptophanase of E. coli converts tryptophan present in the Luria Bertani medium into indole, which in the presence of TMO, is converted into indigo that is easily detectable by its deep blue color. For directional cloning, the tmo operon was flanked by the ApaI EcoRI restriction sites, using the PCR technique; the tmo gene cluster in plasmid pMKY341 served as template. The resulting tmo fragment was cloned downstream of the T7 promoter of pGEM7Zf(+) digested with ApaI EcoRI restriction enzymes, generating the plasmid, pAM101. Upon the addition of IPTG, the E. coli strains bearing this plasmid produced deep blue-colored colonies on LB agar plates, indicating the successful cloning of the tmo operon.
The tmo operon was excised from pAM101 by ApaI and EcoRI digestion and cloned into the corresponding sites of pMKU101, previously generated in this laboratory, which contains the starv1 promoter (9). The resulting plasmid, designated pAM102, has the tmo operon immediately downstream of the starv1 promoter; this was confirmed by restriction analysis. E. coli, bearing this plasmid did not produce indigo in LB medium during starvation, confirming the previous results that Pstarv1 is not expressed in E. coli (4).
To transfer the starv1-tmo construct to a broad host range plasmid for expression in P. putida, we used the plasmid pMMB67HE. Deletion of ca. 1.5 kb PvuII-EcoRI fragment generated the plasmid pMMB67HES, lacking the tac promoter and the lacIq gene. The starv1-tmo construct was excised from pAM102 by SphI and EcoRI digestion and transferred to pMMB67HES digested with the same enzymes, generating the plasmid, pAM103. The ligation efficiency was low, and it took the screening of a thousand E. coli colonies to yield three colonies containing the recombinant plasmid (identified by carbenicillin resistance, which is encoded by the bla gene in pMMB67HES).
The plasmid pAM103 was transformed into several Pseudomonas strains by triparental mating. The recA mutants of P. putida (JS388) and P. fluorescence (FAJ 2025) were among the recipients. Since the isogenic recA and recA+ strains gave similar results, we transformed pAM103 in P. putida MK1 (rec+), generating strain AMS1001. This was done because Pstarv1 was cloned from this strain, and previous results showed that plasmid integration is not a problem in this strain. AMS1001 produced a deep blue color upon starvation in glucose-M9 solid or liquid media. Since Pseudomonas species lack tryptophanase, 1 mM indole was added to these media. All the recipient strains generated indole in the decelerating phase of growth. Their characterization with respect to trichloroethylene degradation is in progress, as are studies to improve the promoter expression, and the ability of the recombinant strains to compete with indigenous flora.
Supplemental Keywords:
G-protein; flagellar synthesis; flagellar placement; chemotaxis; motility., Scientific Discipline, Toxics, Waste, Environmental Chemistry, Chemistry, Bioremediation, Biology, 33/50, Toluene, nutrients, dehalogenate, Trichloroethylene, contaminants in soil, in-situ bioremediation, recombinant P. putida strains, contaminant release, 1, 1, 1-Trichloroethane, degrade trichloroethylene, pseudomonas starvation promotersRelevant Websites:
Department of Microbiology & Immunology, Stanford University; Professor A.C. MatinProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.