Juhyun Kim

Metabolic and regulatory rearrangements underlying glycerol metabolism in Pseudomonas putida KT2440

While the natural niches of the soil bacterium Pseudomonas putida are unlikely to include significant amounts of free glycerol as a growth substrate, this bacterium is genetically equipped with the functions required for its metabolism. We have resorted to deep sequencing of the transcripts in glycerol-grown P. putida KT2440 cells to gain an insight into the biochemical and regulatory components involved in the shift between customary C sources (e.g. glucose or succinate) to the polyol. Transcriptomic results were contrasted with key enzymatic activities under the same culture conditions. Cognate expression profiles revealed that genes encoding enzymes of the Entner-Doudoroff route and other catabolic pathways, e.g. the gluconate and 2-ketogluconate loops, were significantly downregulated on glycerol. Yet, the compound simultaneously elicited a gluconeogenic response that indicated an efficient channelling of C skeletons back to biomass build-up through the glyoxylate shunt rather than energization of the cells through downwards pathways, i.e. tricarboxylic acid cycle and oxidative phosphorylation. The simultaneous glycolytic and gluconeogenic metabolic regimes on glycerol, paradoxical as they seem, make sense from an ecological point of view by favouring prevalence versus exploration. This metabolic situation was accompanied by a considerably low expression of stress markers as compared with other C sources.

Transcriptomic fingerprinting of Pseudomonas putida under alternative physiological regimes

Pseudomonas putida KT2440 is a metabolically versatile soil bacterium useful both as a model biodegradative organism and as a host of catalytic activities of biotechnological interest. In this report, we present the high-resolution transcriptome of P. putida cultured on different carbon sources as revealed by deep sequencing of the corresponding RNA pools. Examination of the data from growth on substrates that are processed through distinct pathways (glucose, fructose, succinate and glycerol) revealed that ≥ 20% of the P. putida genome is differentially expressed depending on the ensuing physiological regime. Changes affected not only metabolic genes but also a suite of global regulators, e.g. the rpoS sigma subunit of RNA polymerase, various cold-shock proteins and the three HU histone-like proteins. Specifically, the genes encoding HU subunit variants hupA, hupB and hupN drastically altered their expression levels (and thus their ability to form heterodimeric combinations) under the diverse growth conditions. Furthermore, we found that two small RNAs, crcZ and crcY, known to inhibit the Crc protein that mediates catabolite repression in P. putida, were both down-regulated by glucose. The raw transcriptomic data generated in this work is made available to the community through the Gene Expression Omnibus database.

The differential response of the Pben promoter of Pseudomonas putida mt‐2 to BenR and XylS prevents metabolic conflicts in m‐xylene biodegradation

Pseudomonas putida mt-2 encompasses two alternative and potentially conflicting routes for benzoate metabolism, one meta pathway encoded by xyl genes of the pWW0 plasmid and mastered by the Pm promoter and XylS, and one chromosomally encoded ortho pathway initiated by Pben and the BenR protein. Any cross-activation of Pben promoter by XylS ought to cause a metabolic conflict during the degradation of m-xylene because 3-methylbenzoate (3MBz) generated as an intermediate can be channelled through the ortho pathway and produce toxic dead-end metabolites. The activation of Pben by XylS was revisited using both reporter technology and tiling arrays targeted to the sequences of interest around messenger RNA initiation of both Pben and Pm promoters. Analysis of supersensitive luxCDABE fusions, inspection of xylX versus benA transcripts and growth tests of benR mutants indicated that the natural expression ranges of XylS under various conditions are insufficient to cause a significant cross-regulation of Pben whether cells face endogenous or exogenous 3MBz. This seems to stem from the nature of the operators for binding either transcriptional factor, which in the case of the Pben promoter of P. putida mt-2 appear to have evolved for avoiding a strong interaction with XylS.

Deconvolution of gene expression noise into spatial dynamics of transcription factor-promoter interplay

Gene expression noise is not only the mere consequence of stochasticity, but also a signal that reflects the upstream physical dynamics of the cognate molecular machinery. Soil bacteria facing recalcitrant pollutants exploit noise of catabolic promoters to deploy beneficial phenotypes such as metabolic bet-hedging and/or division of biochemical labor. Although the role of upstream promoter-regulator interplay in the origin of this noise is little understood, its specifications are probably ciphered in flow cytometry data patterns. We studied Pm promoter activity of the environmental bacterium Pseudomonas putida and its cognate regulator XylS by following expression of Pm-gfp fusions in single cells. Using mathematical modeling and computational simulations, we determined the kinetic properties of the system and used them as a baseline code to interpret promoter activity in terms of upstream regulator dynamics. Transcriptional noise was predicted to depend on the intracellular physical distance between regulator source (where XylS is produced) and the target promoter. Experiments with engineered bacteria in which this distance is minimized or enlarged confirmed the predicted effects of source/target proximity on noise patterns. This approach allowed deconvolution of cytometry data into mechanistic information on gene expression flow. It also provided a basis for selecting programmable noise levels in synthetic regulatory circuits.

CellShape: A user-friendly image analysis tool for quantitative visualization of bacterial cell factories inside

Visualization of the intracellular constituents of individual bacteria while performing as live biocatalysts is in principle doable through more or less sophisticated fluorescence microscopy. Unfortunately, rigorous quantitation of the wealth of data embodied in the resulting images requires bioinformatic tools that are not widely extended within the community-let alone that they are often subject to licensing that impedes software reuse. In this context we have developed CellShape, a user-friendly platform for image analysis with subpixel precision and double-threshold segmentation system for quantification of fluorescent signals stemming from single-cells. CellShape is entirely coded in Python, a free, open-source programming language with widespread community support. For a developer, CellShape enhances extensibility (ease of software improvements) by acting as an interface to access and use existing Python modules; for an end-user, CellShape presents standalone executable files ready to open without installation. We have adopted this platform to analyse with an unprecedented detail the tridimensional distribution of the constituents of the gene expression flow (DNA, RNA polymerase, mRNA and ribosomal proteins) in individual cells of the industrial platform strain Pseudomonas putida KT2440. While the CellShape first release version (v0.8) is readily operational, users and/or developers are enabled to expand the platform further.

Deconvolution of gene expression noise into physical dynamics of cognate promoters

When facing recalcitrant pollutants, soil bacteria exploit noise of catabolic promoters for deploying environmentally beneficial phenotypes such as metabolic bet-hedging an/or division of biochemical labor. While the origin of such noise in terms of upstream promoter-regulator interplay is hardly understood, its dynamics has to be somehow encrypted in the patterns of flow-cytometry data delivered by transcriptional reporter fusions. On this background, we have examined the behaviour of the Pm promoter of the environmental bacterium Pseudomonas putida and its cognate 3-methylbenzoate-responsive regulator XylS under different conditions by following expression of Pm-GFP fusions in single cells. Using mathematical modeling and computational simulations we elucidated the kinetic properties of the system and use them as a baseline code to interpret the observed fluorescence output in terms of upstream regulator variability. Transcriptional noise was predicted to depend on the intracellular physical distance between the regulator source (where the e.g. XylS is being produced in the cells) and the target promoter. Experiments with engineered bacteria where this distance is either minimized or enlarged proved the effects of proximity on noise patterns as predicted by the model. This approach not only allowed deconvolution of cytometry data into mechanistic information on the gene expression flow. But it also provided a mechanistic basis for selecting a given level of noise in engineered regulatory nodes e.g. in Synthetic Biology constructs.

The interplay of EIIANtr with C-source regulation of the Pu promoter of Pseudomonas putida mt-2: Role of PtsN in Pu promoter outcome

The presence of some sugars (e.g. glucose) downregulates the activity of the Pu promoter of plasmid pWW0 of Pseudomonas putida mt-2, which drives the upper TOL operon for biodegradation of m-xylene. Genetic evidence produced 20 years ago documented an effect of the EIIANtr (PtsN) protein of the nitrogen-related phosphoenolpyruvate-dependent phosphotransferase system (PTSNtr ) in such a C-source control of Pu activity. In this study, we have exploited the wealth of recent information on the PTS of P. putida as well as transcriptomic data available in the last few years on this bacterium to revisit this question - and the role of EIIANtr as such. To this end, we examined Pu output under physiological conditions known to either phosphorylate PTS proteins to saturation or to deplete them altogether from high-energy phosphate. The results showed that Pu activity is checked by EIIANtr regardless of its phosphorylation state. However, such inhibition is intensified during growth on glucose (which correlates with more phosphate-free EIIANtr ) and partially relieved in fructose, which triggers phosphorylation of PTS proteins. These data explain former inconsistencies on the Pu-PTSNtr interplay and provides a better understanding of the metabolic and regulatory retroactivity between the TOL plasmid and its host metabolism.