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

Juhyun Kim,Angel Goñi-Moreno,Víctor De Lorenzo,Caroline S Harwood

doi:10.1128/mbio.03685-20

Abstract

ABSTRACTDespite 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.

Highlights

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
The starting point for tackling the issue is the earlier observation that RNA polymerase (RNAP) of P. putida fully colocalizes with the chromosomal DNA of the nucleoid while being entirely apart from the bulk of the ribosomal pool [20]
AND DISCUSSION mRNAs of catabolic genes expressed from the TOL plasmid are spatially organized

Summary

Introduction

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. By merging genetic analyses with in situ RNA-FISH (fluorescence in situ hybridization) and DNAFISH technology, we could faithfully locate the relative positions of the nucleoid DNA, the pWW0 plasmid, the xyl transcripts, and the ribosomes to predict the motion of cognate mRNAs through the cell interior.

Results

Conclusion