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Genetic differentiation by natural selection is readily observed among microbial populations, but a more comprehensive understanding of evolutionary forces, genetic causes, and resulting phenotypic advantages is not often sought. Recently, a surface population of Pseudomonas putida bacteria was shown to evolve rapidly by natural selection of better-adapted variants in a mixed-species biofilm consortium (S. K. Hansen, P. B. Rainey, J. A. Haagensen, and S. Molin, Nature 445:533-536, 2007). Adaptation was caused by mutations in a wapH homolog (PP4943) involved in core lipopolysaccharide biosynthesis. Here we investigate further the biofilm physiology and the phenotypic characteristics of the selected P. putida rough colony variants. The coexistence of the P. putida population in a mixed-species biofilm with Acinetobacter sp. strain C6 is dependent on the benzoate excreted from Acinetobacter during the catabolism of benzyl alcohol, the sole carbon source. Examination of biofilm development and the dynamics of the wild-type consortium revealed that the biofilm environment became oxygen limited, possibly with low oxygen concentrations around Acinetobacter microcolonies. In contrast to P. putida wild-type cells, which readily dispersed from the mixed-species biofilm in response to oxygen starvation, the rough variant cells displayed a nondispersal phenotype. However, in monospecies biofilms proliferating on benzoate, the rough variant (like the wild-type population) dispersed in response to oxygen starvation. A key factor explaining this conditional, nondispersal phenotype is likely to be the acquired ability of the rough variant to coaggregate specifically with Acinetobacter cells. We further show that the P. putida rough variant displayed enhanced production of a cellulose-like polymer as a consequence of the mutation in wapH. The resulting phenotypic characteristics of the P. putida rough variant explain its enhanced fitness and ability to form tight structural associations with Acinetobacter microcolonies.

Research has been conducted on the Pseudomonas putada KT2442 genes. The generation of the random transcriptional fusions of the promoterless bacterial luciferase to P. putida genes has been examined.

A study was conducted to show that the expression of the Gef protein or the X174 E protein in Pseudomonas putida brings about cell death resulting from the formation of membrane holes. Killing efficiency was evaluated with a liquid medium while P. putida cells were determined by centrifugation. Results indicated that the expression of the proteins in P. putida cells made the cell envelope of the heterologous host lose its integrity. It also facilitated the release of cytoplasmic content to the extracellular medium.

A study was conducted to characterize an efficient biologically contained Pseudomonas putida strain. Bacterial strains were prepared with shaking at 30 degrees C while Escherichia coli strains were determined in a modified M9 minimal medium supplemented with glucose, benzoate and p-hydroxyphenylacetic acid. Results showed that a laboratory-based active biological containment system based on killing genes can perform under outdoor conditions.

Toluene-degrading Pseudomonas putida shows reduced activity in the biofilm environment, but is responsible for 65% of the toluene degradation by the entire toluene-degrading community in a biofilter for waste gas treatment. The fully hydrated biofilm has a highly heterogeneous structure, and the cell-free channels extend from the biofilm surface into the matrix. This indicates that toluene reaches deep into the biofilm and gets degraded by P. putida which is present throughout the biofilm matrix.

A recombinant biological containment system may be used to control the growth of Pseudomonas putida in response to the presence or absence of the alkylbenzoates that it degrades. In the absence of alkylbenzoates, a regulatory cascade drives expression from the Plac promoter fused to the gef gene, whose product is a killing protein. Contained strains were genetically stable and survived well as long as alkylbenzoates were present.

Two-dimensional polyacrylamide electrophoretic analysis of the responses of Pseudomonas putida KT2442 to nutrient starvation shows that carbon deprivation causes the production of starvation- and stress-induced proteins. The recovery phase begins when glucose is added to this carbon-starved culture and is characterized by the introduction of a new type of transiently expressed proteins and the suppression of starvation-induced proteins.

Change in cell shapes, reduction of cell size and reduced total protein synthesis are the typical Pseudomonas putida KT2442 response to nutrient starvation. The cells in culture survive temperature peaks and troughs, increased osmolarity, presence of solvents and oxidative chemicals by a cross-protection mechanism. The culture survives for at least a full month under conditions of carbon, nitrogen and multiple-nutrient starvation by maintaining a nondormant state. This survival response is not seen when sulfates and phosphates are absent.

A model substrate dependent suicide system was developed to biologically contain Pseudomonas putida KT2440. The system comprises two elements, one of which is a fusion between a synthetic lac promoter and the gef gene which has a killing mechanism. The other element regulates the first element and is a fusion between the promoter of the P. putida TOL plasmid encoded meta-cleavage pathway operon and the lac I gene.

Results show that in Pseudomonas putida strain IsoF a bioluminescent N-acylhomoserine lactone (AHL) reporter plasmid can be induced, which contain a unique gene ppuA in a cluster of four genes whose expression is AHL dependent and associated with quorum-sensing system. Data indicate that the ppu gene is unique for the P. putida strain IsoF and the strain influences biofilm formation through its quorum-sensing system.

A novel reporter system was constructed for the monitoring of cellular growth activity. By constructing an expression cassette consisting of the novel green fluorescent protein gene expressed from an Escherichia coli rRNA promoter, the reporter system was derived, generating green fluorescence in fast-growing cells of both E coli and Pseudomonas putida. Results indicate that the dynamics of biofilm growth activity can be traced to the level of individual cells, cell clusters and microcolonies.