A genetic toolbox to empower Paracoccus pantotrophus DSM 2944 as a metabolically versatile SynBio chassis.

BACKGROUND: To contribute to the discovery of new microbial strains with metabolic and physiological robustness and develop them into successful chasses, Paracoccus pantotrophus DSM 2944, a Gram-negative bacterium from the phylum Alphaproteobacteria and the family Rhodobacteraceae, was chosen. The strain possesses an innate ability to tolerate high salt concentrations. It utilizes diverse substrates, including cheap and renewable feedstocks, such as C1 and C2 compounds. Also, it can consume short-chain alkanes, predominately found in hydrocarbon-rich environments, making it a potential bioremediation agent. The demonstrated metabolic versatility, coupled with the synthesis of the biodegradable polymer polyhydroxyalkanoate, positions this microbial strain as a noteworthy candidate for advancing the principles of a circular bioeconomy. RESULTS: The study aims to follow the chassis roadmap, as depicted by Calero and Nikel, and de Lorenzo, to transform wild-type P. pantotrophus DSM 2944 into a proficient SynBio (Synthetic Biology) chassis. The initial findings highlight the antibiotic resistance profile of this prospective SynBio chassis. Subsequently, the best origin of replication (ori) was identified as RK2. In contrast, the non-replicative ori R6K was selected for the development of a suicide plasmid necessary for genome integration or gene deletion. Moreover, when assessing the most effective method for gene transfer, it was observed that conjugation had superior efficiency compared to electroporation, while transformation by heat shock was ineffective. Robust host fitness was demonstrated by stable plasmid maintenance, while standardized gene expression using an array of synthetic promoters could be shown. pEMG-based scarless gene deletion was successfully adapted, allowing gene deletion and integration. The successful integration of a gene cassette for terephthalic acid degradation is showcased. The resulting strain can grow on both monomers of polyethylene terephthalate (PET), with an increased growth rate achieved through adaptive laboratory evolution. CONCLUSION: The chassis roadmap for the development of P. pantotrophus DSM 2944 into a proficient SynBio chassis was implemented. The presented genetic toolkit allows genome editing and therewith the possibility to exploit Paracoccus for a myriad of applications.

Draft Genome Sequence and Annotation of the Halotolerant Carotenoid-Producing Strain Paracoccus bogoriensis BOG6T.

Paracoccus spp. are Gram-negative, coccoid bacteria, fascinating for their ability to grow in highly diverse environments while producing commercially relevant products. This study describes the draft genome sequence of the halotolerant, alkaliphilic, and thermotolerant carotenoid-producing type strain Paracoccus bogoriensis BOG6T.

Diversity of culturable bacteria isolated from ancestral crops of Arica and Parinacota Region, Atacama Desert.

Arica and Parinacota Region is located at the extreme north of the Atacama Desert, where the high levels of salts and boron, lack of rain, high UV radiation, among other conditions, make this zone an extreme environment. Despite these characteristics, in the transversal valleys, different types of crops are cultivated in this region, which are associated to beneficial microorganisms with specific traits that allow plants surviving and developing under extreme conditions. However, there is incomplete information related to these microorganisms. In this work, bacteria associated with ancestral crops were isolated from oregano, alfalfa, maize, potato, and grapevine samples from Belén, Codpa, Molinos, Poconchile and Socoroma localities, representing the first report of these microorganisms in those sites. Bacteria were identified, being γ-Proteobacteria, the most frequent class (~ 74.4%), with members of Pseudomonas genus the most common isolated genus. All bacteria were functionally characterized for plant growth-promoting activities, including siderophores and auxins production, phosphate solubilization, and nitrogen fixation, revealing an extraordinary potential from these microorganisms for agricultural applications under arid and semiarid conditions.

A method for filamentous fungal growth and sample preparation aimed at more consistent MALDI-TOF MS spectra despite variations in growth rates and/or incubation times.

Matrix-assisted laser-desorption and ionization time-of-flight mass spectrometry can be used for the characterization and identification of filamentous fungi, for which it is desirable to have a means of growth in which the resulting spectra remain as consistent as possible over time. To this end, we initially opted for growth in oil-overlaid small-volume liquid culture, using a medium (Czapek Dox) not containing significant amount of proteins or peptides, and with protein extraction from the entire culture volume. For both 3-week and 10-day time courses, however, we observed marked spectral changes over growth time, along with lower peak richness compared to agar-plate controls. Guided by the above, we next employed a more nutrient-rich MALDI-TOF MS-compatible liquid-culture medium, now used without an oil overlay. For a 10-day time course, we again observed marked spectral changes over growth time, along with lower peak richness compared to agar-plate controls. Finally, we opted for a method employing filter-paper-supported growth in the same MALDI-TOF MS-compatible rich medium within sealed 1.5 ml Eppendorf tubes, again with protein extraction from the entire culture volume. Using this final method, while we observed significant spectral changes between 2 days and 3 days, from 3 days to 10 days the spectra remained very consistent, with comparable peak richness to agar-plate controls. This method gave slightly better identifications and lower spectral variance compared to agar-plate controls, and the use of this method for the construction of growth-time-point-specific databases for fungal identification is discussed.

MALDI-TOF MS protein fingerprinting of mixed samples.

Analytical techniques currently available for the characterization of mixtures of microorganisms are generally based on next-generation sequencing. Motivated to develop practical and less-expensive methods for characterizing such mixtures, we propose, as an alternative or complement, the use of matrix-assisted laser-desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS), which is capable of high-resolution discrimination between species and even between biotypes within species. Potential approaches employing this technique for such characterization are discussed along with impediments to their successful employment. As a consequence, our rationale has been to capitalize on the powerful algorithms currently available for spectral comparison. Following this rationale, the first priority is to ensure the generation of MALDI-TOF MS spectra from mixtures of microorganisms that contain manageable peak complexities and that can be handled by the existing spectral comparison algorithms, preferably with the option to archive and re-run sample preparations and to pipette replicates of these onto MALDI-TOF MS sample plates. The second priority is to ensure that database entry is comparably facile to sample preparation so that large databases of known microorganism mixture MALDI-TOF MS spectra could be readily prepared for comparison with the spectra of unknown mixtures. In this article, we address the above priorities and generate illustrative MALDI-TOF MS spectra to demonstrate the utility of this approach. In addition, we investigate methods aimed at chemically modulating the peak complexity of the obtained MALDI-TOF MS spectra.

Identification of Penicillium species by MALDI-TOF MS analysis of spores collected by dielectrophoresis.

In matrix-assisted laser-desorption and ionization mass spectrometry, spectral differences are frequently observed using different growth media on agar plates and/or different growth times in culture, which add undesirable analytical variance. In this article, we explore an approach to the above problem based upon the rationale that, while protein expression in fungal mycelium may well vary under different growth conditions, this might not apply to the same extent in fungal spores. To this end, we have exploited the fact that while mycelium is generally anchored to the fungal-growth substrate, some fungi produce physically-isolated spores which, as such, are amenable to manipulation using dielectrophoresis (the translational motion of charged or uncharged matter caused by polarization effects in a non-uniform electrical field). Such fields can be conveniently generated through the charging of an insulator using the triboelectric effect (the transfer of charge between two objects through friction when they are rubbed together). In this study, polystyrene microbiological inoculating loops were used in combination with nylon-fabric rubbing to harvest fungal spores from five species from within the genus Penicillium, which were grown on agar plates containing two different media over an extended time course. In terms of average Bruker spectral-comparison scores, our method generated higher scores in 80% of cases tested and, in terms of average coefficients of variation, our method generated lower spectral variability in 93% of cases tested. Harvesting of spores using a rapid, inexpensive and simple dielectrophoretic method, therefore, facilitates improved fungal identification for the Penicillium species tested.