Spatial organization of the gene expression hardware in <i>Pseudomonas putida</i>

Kim, Ju-Hyun; Goñi-Moreno, Ángel; Calles, Belén

doi:10.1111/1462-2920.14544

Cited by 15 publications

(28 citation statements)

References 69 publications

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“…In an early stage, mRNA is necessarily tethered to its DNA template in the plasmid and therefore the gene and its transcript occupy the same spot. But at a later stage the xyl mRNA could move away from the plasmid towards nucleoid-free ribosome-rich domains of the cell inside (20). Note that the sizes of the upper-and lower TOL operons are ~ 8 Kb and 11 Kb respectively and, if linearly stretched, the 5' ends of their fulllength mRNAs could be found well away from the plasmid while still tethered to the template.…”

Section: Resultsmentioning

confidence: 99%

“…6C). Given that such transcripts originate in single promoters per bacterium and that nucleoid-free domains of P. putida cells are filled with ribosomes (20) it is possible that the images of…”

Section: The Drive Of Xyl Mrnas To Escape the Nucleoid Is Detached Ofmentioning

confidence: 99%

“…Pseudomonas sp. has also a low NC ratio (65) and the ribosomes are spatially segregated from the nucleoid (20). It is thus conceivable that TOL transcripts are not coupled to translation as they are produced but they instead move after complete transcription towards ribosome-rich cytoplasmic spots.…”

Section: Disruption Of Intracellular Crowding Enables Xyl Transcriptsmentioning

confidence: 99%

“…In this work we have inspected the localization of mRNAs initiated in the catabolic promoters of the TOL plasmid pWW0 of P. putida mt-2. The starting point for tackling the issue is the earlier observation that RNA polymerase (RNAP) of P. putida fully co-localizes with the chromosomal DNA of the nucleoid while being entirely apart from the bulk of the ribosomal pool (20). This observation suggested that rather than being coupled to translation ab initio, many (if not most) of the mRNAs initiated at chromosomal promoters need to move to ribosome-rich, nucleoid-free domains of the intracellular space for translation.…”

Section: Introductionmentioning

confidence: 99%

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The subcellular architecture of thexylgene expression flow of the TOL catabolic plasmid ofPseudomonas putidamt-2

Kim

Goñi-Moreno

2020

Preprint

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Despite 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.

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Section: Resultsmentioning

confidence: 99%

Section: The Drive Of Xyl Mrnas To Escape the Nucleoid Is Detached Ofmentioning

confidence: 99%

Section: Disruption Of Intracellular Crowding Enables Xyl Transcriptsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

The subcellular architecture of thexylgene expression flow of the TOL catabolic plasmid ofPseudomonas putidamt-2

Kim

Goñi-Moreno

2020

Preprint

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“…Finally, a co-localization effect of the target biosynthetic genes with the transcription machinery is thinkable: it is known that the transcription and translation machinery represent limited resources in bacterial cells which are subject to competitive interaction by different modules of the cellular circuitry 46 . Moreover, in P. putida , transcriptional and translational machinery are located in spatially distinct positions in the cell 47 and it has been discussed, at least for E. coli , that rrn operons substantially recruit the transcription machinery thus forming transcription foci with high RNA polymerase availability 48 . Therefore, the transcription level of genes integrated in the rrn operons A–D, which occur in relative proximity to one another, may be especially positively influenced by a local focus of the transcription machinery that is generated here.…”

Section: Discussionmentioning

confidence: 99%

Pseudomonas putida rDNA is a favored site for the expression of biosynthetic genes

Domröse

Hage-Hülsmann

Thies

et al. 2019

Sci Rep

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Since high-value bacterial secondary metabolites, including antibiotics, are often naturally produced in only low amounts, their efficient biosynthesis typically requires the transfer of entire metabolic pathways into suitable bacterial hosts like Pseudomonas putida . Stable maintenance and sufficient expression of heterologous pathway-encoding genes in host microbes, however, still remain key challenges. In this study, the 21 kb prodigiosin gene cluster from Serratia marcescens was used as a reporter to identify genomic sites in P. putida KT2440 especially suitable for maintenance and expression of pathway genes. After generation of a strain library by random Tn5 transposon-based chromosomal integration of the cluster, 50 strains exhibited strong prodigiosin production. Remarkably, chromosomal integration sites were exclusively identified in the seven rRNA-encoding rrn operons of P. putida . We could further demonstrate that prodigiosin production was mainly dependent on (i) the individual rrn operon where the gene cluster was inserted as well as (ii) the distance between the rrn promoter and the inserted prodigiosin biosynthetic genes. In addition, the recombinant strains showed high stability upon subculturing for many generations. Consequently, our findings demonstrate the general applicability of rDNA loci as chromosomal integration sites for gene cluster expression and recombinant pathway implementation in P. putida KT2440.

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RNA localization in prokaryotes: Where, when, how, and why

Irastortza-Olaziregi

Amster‐Choder

2020

WIREs RNA

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Only recently has it been recognized that the transcriptome of bacteria and archaea can be spatiotemporally regulated. All types of prokaryotic transcripts—rRNAs, tRNAs, mRNAs, and regulatory RNAs—may acquire specific localization and these patterns can be temporally regulated. In some cases bacterial RNAs reside in the vicinity of the transcription site, but in many others, transcripts show distinct localizations to the cytoplasm, the inner membrane, or the pole of rod‐shaped species. This localization, which often overlaps with that of the encoded proteins, can be achieved either in a translation‐dependent or translation‐independent fashion. The latter implies that RNAs carry sequence‐level features that determine their final localization with the aid of RNA‐targeting factors. Localization of transcripts regulates their posttranscriptional fate by affecting their degradation and processing, translation efficiency, sRNA‐mediated regulation, and/or propensity to undergo RNA modifications. By facilitating complex assembly and liquid–liquid phase separation, RNA localization is not only a consequence but also a driver of subcellular spatiotemporal complexity. We foresee that in the coming years the study of RNA localization in prokaryotes will produce important novel insights regarding the fundamental understanding of membrane‐less subcellular organization and lead to practical outputs with biotechnological and therapeutic implications. This article is categorized under: RNA Export and Localization > RNA Localization Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs RNA Interactions with Proteins and Other Molecules > Protein‐RNA Interactions: Functional Implications

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Spatial organization of the gene expression hardware in Pseudomonas putida

Cited by 15 publications

References 69 publications

The subcellular architecture of thexylgene expression flow of the TOL catabolic plasmid ofPseudomonas putidamt-2

The subcellular architecture of thexylgene expression flow of the TOL catabolic plasmid ofPseudomonas putidamt-2

Pseudomonas putida rDNA is a favored site for the expression of biosynthetic genes

RNA localization in prokaryotes: Where, when, how, and why

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