Biodesulfurization gene expression by promoter replacement in Rhodococcus

NRC publication: Ye

Characterization of the Basic Replicon of Rhodococcus Plasmid pSOX and Development of a Rhodococcus-Escherichia coli Shuttle Vector

The replication region of a 100-kb desulfurization plasmid (pSOX) from Rhodococcus sp. strain X309 was localized to a 4-kb KpnI fragment, and its sequence was determined. The amino acid sequence of one of the predicted open reading frames (ORFs) was related to the putative replication (Rep) protein sequences of the mycobacterial pLR7 family of plasmids. Three of the five predicted ORF products were identified by radiolabelling with the Escherichia coli T7 polymerase/promoter system. In E. coli, the Rep protein of pSOX was apparently synthesized in a shortened form, 21.3 kDa instead of the predicted 41.3 kDa, as a result of an internal initiation. This situation is reminescent of that for some bacterial Rep proteins. A shuttle plasmid was constructed with the pSOX origin, pBluescript II KS−, and the chloramphenicol resistance (Cm(r)) gene from pRF29. This new shuttle plasmid was used to demonstrate expression of the Bacillus subtilis sacB gene in a strain of Rhodococcus, rendering it sensitive to the presence of sucrose

Characterization of the basic replicon of Rhodococcus plasmid pSOX and development of a Rhodococcus-Escherichia coli shuttle vector

The replication region of a 100-kb desulfurization plasmid (pSOX) from Rhodococcus sp. strain X309 was localized to a 4-kbKpnI fragment, and its sequence was determined. The amino acid sequence of one of the predicted open reading frames (ORFs) was related to the putative replication (Rep) protein sequences of the mycobacterial pLR7 family of plasmids. Three of the five predicted ORF products were identified by radiolabelling with the Escherichia coli T7 polymerase/promoter system. In E. coli, the Rep protein of pSOX was apparently synthesized in a shortened form, 21.3 kDa instead of the predicted 41.3 kDa, as a result of an internal initiation. This situation is reminescent of that for some bacterial Rep proteins. A shuttle plasmid was constructed with the pSOX origin, pBluescript II KS 12, and the chloramphenicol resistance (Cmr) gene from pRF29. This new shuttle plasmid was used to demonstrate expression of the Bacillus subtilis sacB gene in a strain of Rhodococcus, rendering it sensitive to the presence of sucrose.NRC publication: Ye

Identification of a complete dibenzothiophene biodesulfurization operon and its regulator by functional metagenomics

Functional screening for aromatic ring oxygenases of an oil contaminated soil metagenome identified 25 different clones bearing monooxygenases coding genes. One fosmid bore an operon containing four tightly linked genes coding for a complete dibenzothiophene biodesulfurization pathway, which included the predicted monooxygenases DszC and DszA, the desulfinase DszB, and an FMN‐oxidoreductase designated DszE. The dszEABC operon provided Escherichia coli with the ability to use dibenzothiophene as the only sulfur source. Transcription of the operon is driven from a σN‐dependent promoter and regulated by an activator that was designated dszR. DszR has been purified and characterized in vitro and shown to be a constitutively active σN‐dependent activator of the group IV, which binds to two contiguous sequences located upstream of the promoter. The dsz promoter and dszE and dszR genes have apparently been recruited from an aliphatic sulfonate biodegradation pathway. If transcribed from a heterologous upstream promoter, the σN‐dependent promoter region functions as an ‘insulator’ that prevents translation of dszE, by binding with its ribosome binding site. Translational coupling, in turn, prevents translation of the downstream dszABC genes. The silencer combined with translational coupling thus represents an effective way of preventing expression of operons when spuriously transcribed from upstream promoters.This project has received funding from the European Union's Horizon 2020 research and innovation program (Blue Growth: Unlocking the potential of Seas and Oceans) through the Project ‘INMARE’ under grant agreement No. 634486 and ERA‐IB 5 ‘METACAT’. This work was further funded by grants PCIN‐2014‐107 (within ERA NET IB2 grant nr. ERA‐IB‐14‐030—MetaCat), PCIN‐2017‐078 (within the Marine Biotechnology ERA‐NET (ERA‐MBT) funded under the European Commission's Seventh Framework Programme, 2013–2017, Grant agreement 604814), BIO2014‐54494‐R and BIO2017‐85522‐R from the Ministerio de Ciencia, Innovación y Universidades, formerly Ministerio de Economía, Industria y Competitividad

Structure, fluctuation and magnitude of a natural grassland soil metagenome.

International audienceThe soil ecosystem is critical for human health, affecting aspects of the environment from key agricultural and edaphic parameters to critical influence on climate change. Soil has more unknown biodiversity than any other ecosystem. We have applied diverse DNA extraction methods coupled with high throughput pyrosequencing to explore 4.88 × 10(9) bp of metagenomic sequence data from the longest continually studied soil environment (Park Grass experiment at Rothamsted Research in the UK). Results emphasize important DNA extraction biases and unexpectedly low seasonal and vertical soil metagenomic functional class variations. Clustering-based subsystems and carbohydrate metabolism had the largest quantity of annotated reads assigned although <50% of reads were assigned at an E value cutoff of 10(-5). In addition, with the more detailed subsystems, cAMP signaling in bacteria (3.24±0.27% of the annotated reads) and the Ton and Tol transport systems (1.69±0.11%) were relatively highly represented. The most highly represented genome from the database was that for a Bradyrhizobium species. The metagenomic variance created by integrating natural and methodological fluctuations represents a global picture of the Rothamsted soil metagenome that can be used for specific questions and future inter-environmental metagenomic comparisons. However, only 1% of annotated sequences correspond to already sequenced genomes at 96% similarity and E values of <10(-5), thus, considerable genomic reconstructions efforts still have to be performed