Plasmid Expression and Retention in Biofilm Cultures

exposure of plasmid-recombinant microorganisms to an open system environment, either inadvertently or intentionally, mandates research into those fundamental organ-ism-plasmid processes that influence plasmid retention, transfer, and expression. Use of plasmid-recombinant bacteria to impart a desired phenotypic ability to an existing ecosystem has been desired for the bioremediation of hazardous wastes in both controlled reactor systems and in waste-contaminated open environments. In open environmental systems, a majority of the microbial activity occurs associated with an interface, within biofilms. In waste-water or waste gas treatment, efficient reactor design often dictates the use of immobilized, biofilm-bound bacterial communities. Consequently, the fate and ability to manifest a plasmid-borne phenotype within a biofilm community requires basic information on the biofilm's genetic processes.

A plasmid is deemed unstable if it either undergoes molecular rearrangement (structural instability) or is not absolutely inherited by progeny (segregational instability).

Spatial differences in diffusion coefficients of dextran (MW = 10'000) in pure culture biofilm

Figure 7. Spatial differences in diffusion coefficients of dextran (MW = 10,000) in pure culture biofilms. Source. Ref. 83.

Horizontal distance (^m)

Figure 7. Spatial differences in diffusion coefficients of dextran (MW = 10,000) in pure culture biofilms. Source. Ref. 83.

Such phenomena can have significant effects on the outcome of cell cultivation processes. It has been established, both experimentally and theoretically, that plasmid maintenance and cloned gene expression can reduce the overall growth rate of the plasmid-bearing cell relative to the plasmid-free cell. Reduction of copy number and loss of plasmids from populations under continuous suspended culture have been reported in many cases, even in the presence of selective pressure.

That immobilization might stabilize a plasmid-bearing population can be shown mathematically (85,86). Plasmid persistence in suspended cultures has been observed in cases where the plasmid-bearing cell was at a growth rate disadvantage; this observation was directly attributed to biofilm formation and cell detachment from the biofilm (87). Improved plasmid stability upon artificial cell immobilization in K-carrageenan gel beads has been reported by de Taxis du Poet et al. (88), Nasri et al. (89), and Sayadi et al. (90).

Huang et al. (91,92) cultivated biofilm cultures under controlled hydrodynamic conditions in a parallel-plate flow cell reactor using E. coli DH5a containing a recombinant plasmid either with (pTKW106) or without (pMJR1750) a segregational stability factor, hok/sok. Both plasmid constructs provided for the inducible expression of j-galacto-sidase. Results in these studies suggest, for these two plas-mids, that the expression of the chromosomal gene for synthesis of the polysaccharide matrix of the biofilm commands a higher priority on cellular intermediates than the foreign genes. Consequently, a series of experiments were carried out to indirectly manipulate bacterial polysaccha-ride production in the biofilm by varying the nutrient carbon-to-nitrogen (C:N) ratio. Plasmid loss probability, total polysaccharide production, and ratios of total polysac-charide to total protein in biofilm cultures of E. coli DH5a (pMJR1750) all increased with increasing C:N ratios. Synthesis rates of total RNA, rRNA, and j-galactosidase-specific mRNA in E. coli DH5a (pTKW106) all increased after induction by IPTG, but levels of each parameter seen in biofilm cultures were 10-20 times lower than the same values observed in suspended cell cultures.

Cell-Cell Communication. Cell-cell communication involves a chemically unique molecule, produced biologically by one cell, that reacts with a set of specific receptors located at another cell's surface to subsequently alter cellular behavior. Two examples of cell-cell communication in higher life forms would be (1) the thousands of lectins that control embryonic development in plants and animals, and (2) signaling molecules, such as the pheromones, that control the behavior of adult insects.

Cell-cell communication in bacterial systems, both sessile and planktonic, has been recognized for years (93), but our understanding of the control mechanisms has entered a new era with the advent of various molecular diagnostic tools. Recent research has identified the specific chemical structures of various signaling molecules that manifest certain cellular responses. The majority of cell-cell communication studies have dealt with suspended bacteria (e.g., quorum sensing), but recent advances suggest that certain structural architecture in a biofilm arises due to molecular signaling.

Bacterial swarming, floc formation, and biofilm formation or aggregation are multicellular, cell-concentration-dependent behavior that can be induced in response to the recognition of surfaces of certain viscosity. Cells differentiate into multinucleated, elongated, and hyperflagellated form and orient themselves lengthwise across the surface. For a number of species, the ability to differentiate into swarmer cells is critical to virulence, surface adhesion, and colonization.

Acylated homoserine lactones serve as one class of signal molecule in bacterial communication. These molecules and their derivatives have been linked to control of bioluminescence, Ti plasmid transfer, production of virulence factors, antibiotic resistance, and swarming motility (94). Higher life forms have evolved mechanisms to interfere with such signaling processes. For example, certain seaweeds and sea grasses produce halogenated furanones, structurally similar to acylated homoserine lactones, that interfere with the swarming process and have been shown effective as antifoulants and antimicrobials (95). Givskov et al. (96) illustrated that two different furanones derived from the seaweed Delisea pulchra could progressively inhibit and eliminate the swarming behavior of Serratia li-quefaciens as concentrations were increased from 0 ig/mL to 100 ig/mL (concentrations far too low to affect the growth of the bacteria).

Davies et al. (unpublished results) employed homoserine lactone mutants of the classic PAO-1 strain of P. aeruginosa to determine if cell-cell communication plays any role in bacterial biofilm polysaccharide matrix formation. Wild strains and mutants were separately used to inoculate flow cells to observe cell adhesion to surfaces and subsequent formation of biofilms. Cells of the wild-type strain (PAO-1), retaining the ability to secrete homoserine lac-tone, adhered to surfaces avidly and formed complex biofilms comprising discrete microcolonies and well-developed water channels.

Cells of the double homoserine lactone mutant (JP2), which lacked the ability to synthesize either the Pseudo-momas autoinducer, PAI, or the factor U homoserine lac-tone, did adhere to surfaces avidly but failed to form the complexly structured biofilms. These mutant cells did not form detectable amounts of exopolysaccharide matrix material, did not aggregate into discrete microcolonies, and did not develop the water channels characteristic of the wild-type biofilms. Cells of the double homoserine lactone mutant simply formed unstructured masses of cells that were tightly packed together. These unstructured adherent masses of cells could be easily removed from the colonized surfaces by simple washing with surfactants. Chemical analyses proved that these biofilm contained no detectable biofilm exopolysaccharide matrix material. When a cell-free supernatant from a wild-type biofilm (PAO-1), which contained both types of homoserine lactone molecule, was added to the bulk fluid delivered to the flow cell, adherent JP2 mutant cells gradually began to produce matrix material and form complex biofilms; 12 h after the addition of the homoserine lactone molecules from the wild-type supernatant, the JP2 mutant strain biofilm was observed to be identical to the wild-type biofilm strain. Such research could potentially lead to nontoxic control strategies for disrupting detrimental biofilms, which would be economically and environmentally significant.

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