Evaluation Of The Transfer Of The TOL Plasmid Pseudomonas Putida To Groundwater-Derived Biofilms In A Model Rock-Fracture Aquifer

Non-aqueous phase liquids (NAPLs) can become entrapped in subsurface rock fractures and become a long-term groundwater contaminant source. While remediation technologies exist, they can be expensive. Subsurface microorganisms can also degrade NAPLs trapped in the subsurface; however, this is a slow process. The possibility of enhancing microbial degradation of NAPLs via a plasmid transfer mechanism in a model rock fracture aquifer was explored. There was no indication that introduction of donor strain Pseudomonas putida SM1443::gfp2x-pWW0::dsRed into the model system led to transfer of the degradative TOL plasmid pWWO, or led to increased degradation of model NPL toluene. Plate matings with the donor strain and a groundwater-derived microbial consortium indicated that few potential recipients existed in the community. Nutrient concentration was ruled out as a limiting factor of plasmid transfer.

Evaluation Of The Transfer Of The TOL Plasmid Pseudomonas Putida To Groundwater-Derived Biofilms In A Model Rock-Fracture Aquifer

Non-aqueous phase liquids (NAPLs) can become entrapped in subsurface rock fractures and become a long-term groundwater contaminant source. While remediation technologies exist, they can be expensive. Subsurface microorganisms can also degrade NAPLs trapped in the subsurface; however, this is a slow process. The possibility of enhancing microbial degradation of NAPLs via a plasmid transfer mechanism in a model rock fracture aquifer was explored. There was no indication that introduction of donor strain Pseudomonas putida SM1443::gfp2x-pWW0::dsRed into the model system led to transfer of the degradative TOL plasmid pWWO, or led to increased degradation of model NPL toluene. Plate matings with the donor strain and a groundwater-derived microbial consortium indicated that few potential recipients existed in the community. Nutrient concentration was ruled out as a limiting factor of plasmid transfer.

Subcellular Architecture of the xyl Gene Expression Flow of the TOL Catabolic Plasmid of Pseudomonas putida mt-2

ABSTRACT Despite intensive research on the biochemical and regulatory features of the archetypal catabolic TOL system borne by pWW0 of Pseudomonas putida strain mt-2, the physical arrangement and tridimensional logic of the xyl gene expression flow remains unknown. In this work, the spatial distribution of specific xyl mRNAs with respect to the host nucleoid, the TOL plasmid, and the ribosomal pool has been investigated. In situ hybridization of target transcripts with fluorescent oligonucleotide probes revealed that xyl mRNAs cluster in discrete foci, adjacent but clearly separated from the TOL plasmid and the cell nucleoid. Also, they colocalize with ribosome-rich domains of the intracellular milieu. This arrangement was maintained even when the xyl genes were artificially relocated to different chromosomal locations. The same held true when genes were expressed through a heterologous T7 polymerase-based system, which likewise led to mRNA foci outside the DNA. In contrast, rifampin treatment, known to ease crowding, blurred the confinement of xyl transcripts. This suggested that xyl mRNAs exit from their initiation sites to move to ribosome-rich points for translation—rather than being translated coupled to transcription. Moreover, the results suggest the distinct subcellular motion of xyl mRNAs results from both innate properties of the sequences and the physical forces that keep the ribosomal pool away from the nucleoid in P. putida. This scenario is discussed within the background of current knowledge on the three-dimensional organization of the gene expression flow in other bacteria and the environmental lifestyle of this soil microorganism. IMPORTANCE The transfer of information between DNA, RNA, and proteins in a bacterium is often compared to the decoding of a piece of software in a computer. However, the tridimensional layout and the relational logic of the cognate biological hardware, i.e., the nucleoid, the RNA polymerase, and the ribosomes, are habitually taken for granted. In this work, we inspected the localization and fate of the transcripts that stem from the archetypal biodegradative plasmid pWW0 of soil bacterium Pseudomonas putida strain KT2440 through the nonhomogeneous milieu of the bacterial cytoplasm. The results expose that—similarly to computers—the material components that enable the expression flow are well separated physically and they decipher the sequences through a distinct tridimensional arrangement with no indication of transcription/translation coupling. We argue that the resulting subcellular architecture enters an extra regulatory layer that obeys a species-specific positional code and accompanies the environmental lifestyle of this bacterium.

The subcellular architecture of the xyl gene expression flow of the TOL catabolic plasmid of Pseudomonas putida mt-2

ABSTRACTDespite intensive research on the biochemical and regulatory features of the archetypal catabolic TOL system borne by pWW0 of Pseudomonas putida mt-2, the physical arrangement and tridimensional logic of the xyl gene expression flow remains unknown. In this work, the spatial distribution of specific xyl mRNAs with respect to the host nucleoid, the TOL plasmid and the ribosomal pool has been investigated. In situ hybridization of target transcripts with fluorescent oligonucleotide probes revealed that xyl mRNAs cluster in discrete foci, adjacent but clearly separated from the TOL plasmid and the cell nucleoid. Also, they co-localize with ribosome-rich domains of the intracellular milieu. This arrangement was kept even when the xyl genes were artificially relocated at different chromosomal locations. The same happened when genes were expressed through a heterologous T7 polymerase-based system, which originated mRNA foci outside the DNA. In contrast, rifampicin treatment, known to ease crowding, blurred the confinement of xyl transcripts. This suggested that xyl mRNAs intrinsically run away from their initiation sites to ribosome-rich points for translation—rather than being translated coupled to transcription. Moreover, the results suggest that the distinct subcellular motion of xyl mRNAs results both from innate properties of the sequence at stake and the physical forces that keep the ribosomal pool away from the nucleoid in P. putida. This scenario is discussed on the background of current knowledge on the 3D organization of the gene expression flow in other bacteria and the environmental lifestyle of this soil microorganism.IMPORTANCEThe transfer of information between DNA, RNA and proteins in a bacterium is often compared to the decoding of a piece of software in a computer. However, the tridimensional layout and the relational logic of the cognate biological hardware i.e. the nucleoid, the RNA polymerase and the ribosomes, are habitually taken for granted. In this work we inspected the localization and fate of the transcripts that stem from the archetypal biodegradative plasmid pWW0 of soil bacterium Pseudomonas putida KT2440 through the non-homogenous milieu of the bacterial cytoplasm. The results expose that— similarly to computers also—the material components that enable the expression flow are well separated physically and they decipher the sequences through a distinct tridimensional arrangement with no indication of transcription/translation coupling. We argue that the resulting subcellular architecture enters an extra regulatory layer that obeys a species-specific positional code that accompanies the environmental lifestyle of this bacterium.