Tracking genetically engineered bacteria: monoclonal antibodies against surface determinants of the soil bacterium Pseudomonas putida 2440

Abstract

Assessment of potential risks involved in the release of genetically engineered microorganisms is facilitated by the availability of monoclonal antibodies (MAbs), a tool potentially able to monitor specific organisms. We raised a bank of MAbs against the soil bacterium Pseudomonas putida 2440, which is a host for modified TOL plasmids and other recombinant plasmids. Three MAbs, 7.3B, 7.4D, and 7.5D, were highly specific and recognized only P. putida bacteria. Furthermore, we developed a semiquantitative dot blot assay that allowed us to detect as few as 100 cells per spot. A 40-kDa cell surface protein was the target for MAbs 7.4D and 7.5D. Detection of the cell antigen depended on the bacterial growth phase and culture medium. The 0 antigen of lipopolysaccharide seems to be the target for MAb 7.3B, and its in vivo detection was independent of the bacterial growth phase and culture medium. MAb 7.3B was used successfully to track P. putida(pWWO) released in unsterile lake mesocosms.Recent achievements in molecular biology have provided efficient ways to manipulate prokaryotic and eukaryotic organisms, and a number of possible applications for these "new" organisms are envisaged in the food, health, and agricultural industries. Bioremediation is an area of increasing interest for decontamination of polluted areas; such treatments involve, among other strategies, release of large numbers of wild-type or modified microorganisms to decontaminate polluted soils, water, and sediments.Among soil bacteria, the pseudomonad group exhibits an enormous range of metabolic activities against biogenic and xenobiotic chemicals (31). Furthermore, the catabolic pathways of pseudomonads have been used as model systems to expand the range of recalcitrant chemicals that microorganisms can mineralize (34).We have centered our attention on Pseudomonas putida 2440 (12), a restriction-negative strain which was derived from P. putida mt-2, the natural host for archetypal TOL plasmid pWWO (49). P. putida 2440 has been used as the host for recombinant TOL plasmids able to eliminate aromatic hydrocarbons that cannot be degraded by non-pWWO-modified pathways (1, 35). Furthermore, P. putida 2440 has been shown to be an excellent recipient for recombinant plasmids through conjugation and transformation (4), and it has been used in several biotransformation processes, e.g., production of benzyl alcohols (9).Concerns about the potential risks associated with deliberate or accidental release of large numbers of genetically engineered microorganisms have been raised because of the unpredictability of their immediate and long-term behavior in the environment. The assessment of potential risks will be facilitated by the availability of monoclonal antibodies * Corresponding author.(MAbs), which make it possible for an organism to be specifically monitored and efficiently quantitated.We raised highly specific MAbs against surface determinants of P. putida 2440 (lipopolysaccharide [LPS] and proteins) and developed a solid semiquantitative assay that al...

Temperature and pyoverdine‐mediated iron acquisition control surface motility of Pseudomonas putida

“…In P. aeruginosa , WbpW is responsible for the synthesis of nucleotide sugar GDP‐ d ‐mannose, a precursor for the O‐antigen and the ‘common antigen’ (Lam et al ., 2004). The mechanism of O‐antigen synthesis begins through the activity of a transmembrane glycosyl transferase designated WbpL in P. putida so that a knockout in wbpL does not have O‐antigen (Ramos‐González et al ., 1992; Junker et al ., 2001). Both mutants in the synthesis of the O‐antigen were defective in swarming (not shown).…”

Fate of Pseudomonas putida after release into lake water mesocosms: Different survival mechanisms in response to environmental conditions

“…Strain-specific detection of P. putida DSM 3931 was achieved with monoclonal mouse IgM antibodies against surface lipopolysaccharides (LPS). Production and testing of these antibodies are described in detail by Ramos-Gonzalez et al [27]. Secondary fluoresceinisothiocyanate (FITC)-labeled antibodies (anti-mouse IgM, ix-chain-specific) were purchased from Sigma Chemical Co. (St. Louis, Mo., USA).…”

Cell envelope mutants of Pseudomonas putida: physiological characterization and analysis of their ability to survive in soil

“…Approximately 25% of the transposon mutants were luminescent after exposure to n-decyl aldehyde. To identify the mutants lacking the O-antigenic side-chain of the LPS, reactivity of the Lux transposon mutants against monoclonal antibody (mAb) 7.3B was tested by enzymelinked immunosorbent assay (ELISA) (Ramos-Gonza  lez et al, 1992). Only one mutant, called P. putida DOT-OX3, was isolated out of 500 isolates.…”

“…Enzyme-linked immunosorbent assays (ELISAs) using whole cells were performed as described by Ramos-Gonza  lez et al (1992). Brie¯y, luminescent derivatives of P. putida were adsorbed on microtitre plates, and clones lacking the O-speci®c polysaccharide of LPS were identi®ed as negative clones in assays with mAb 7.3B, which recognizes the O-antigenic side-chain of the LPS of P. putida (Ramos-Gonza Âlez et al, 1992).…”