Biology subjects

Martinez-Garcia, E.

Publications and source records attributed to Martinez-Garcia, E..

3 recordsLinked to original sources

bioRxiv2020

The naked cell: emerging properties of a surfome-streamlined Pseudomonas putida strain

ABSTRACTEnvironmental bacteria are most often endowed with native surface-attachment programs that frequently conflict with efforts to engineer biofilms and synthetic communities with given tridimensional architectures. In this work we report the editing of the genome of Pseudomonas putida KT2440 for stripping the cells of most outer-facing structures of the bacterial envelope that mediate motion, binding to surfaces and biofilm formation. To this end, 23 segments of the P. putida chromosome encoding a suite of such functions were deleted, resulting in the surface-naked strain EM371, the physicochemical properties of which changed dramatically in respect to the wild type counterpart. As a consequence, surface-edited P. putida cells were unable to form biofilms on solid supports and—because of the swimming deficiency and other physicochemical alterations—showed a much faster sedimentation in liquid media. Surface-naked bacteria were then used as carriers of interacting partners (e.g. Jun-Fos domains) ectopically expressed by means of an autotransporter display system on the now easily accessible cell envelope. Abstraction of individual bacteria as adhesin-coated spherocylinders enabled rigorous quantitative description of the multi-cell interplay brought about by thereby engineered physical interactions. The model was then applied to parameterize the data extracted from automated analysis of confocal microscopy images of the experimentally assembled bacterial flocks for analyzing their structure and distribution. The resulting data not only corroborated the value of P. putida EM371 over the parental strain as a platform for display artificial adhesins but also provided a strategy for rational engineering of distributed biocatalysis.Competing Interest StatementThe authors have declared no competing interest.View Full Text

synthetic biology↗

bioRxiv2019

High-efficiency multi-site genomic editing (HEMSE) of Pseudomonas putida through thermoinducible ssDNA recombineering

While single-stranded DNA recombineering is a powerful strategy for higher-scale genome editing, its application to species other than enterobacteria is typically limited by the efficiency of the recombinase and the action of native mismatch repair (MMR) systems. By building on [i] availability of the Erf-like single-stranded DNA-annealing protein Rec2, [ii] adoption of tightly-regulated thermoinducible device and [iii] transient expression of a MMR-supressing mutL allele, we have set up a coherent genetic platform for entering multiple changes in the chromosome of Pseudomononas putida with an unprecedented efficacy and reliability. The key genetic construct to this end is a broad host range plasmid encoding co-transcription of rec2 and P. putidas mutLE36KPP at high levels under the control of the PL/cI857 system. Cycles of short thermal shifts of P. putida cells followed by transformation with a suite of mutagenic oligos delivered different types of high-fidelity genomic changes at frequencies up to 10% per single change--that can be handled without selection. The same approach was instrumental to super-diversify short chromosomal portions for creating libraries of functional genomic segments--as shown in this case with ribosomal binding sites. These results enable the multiplexing of genome engineering of P. putida, as required for metabolic engineering of this important biotechnological chassis.

microbiology↗

bioRxiv2019

Mismatch repair hierarchy of Pseudomonas putida revealed by mutagenic ssDNA recombineering of the pyrF gene

The mismatch repair (MMR) system is one of the key molecular devices that prokaryotic cells have for ensuring fidelity of DNA replication. While the canonical MMR of E. coli involves 3 proteins (encoded by mutS, mutL and mutH), the soil bacterium Pseudomonads putida has only 2 bona fide homologues (mutS and mutL) and the sensitivity of this abridged system to different types of mismatches is unknown. On this background, sensitivity to MMR of this bacterium was inspected through single stranded (ss) DNA recombineering of the pyrF gene (the prokaryotic equivalent to yeasts URA3) with mutagenic oligos representative of every possible mispairing under either wild-type conditions, permanent deletion of mutS or transient loss of mutL activity (brought about by the thermoinducible dominant negative allele mutLE36K). Analysis of single nucleotide mutations borne by clones resistant to fluoroorotic acid (5FOA, the target of wild type PyrF) pinpointed prohibited and tolerated single-nucleotide replacements and exposed a clear grading of mismatch recognition. The resulting data unequivocally established the hierarchy A:G< C:C< G:A< C:A, A:A, G:G, T:T, T:G, A:C, C:T< G:T, T:C as the one prevalent in Pseudomonas putida. This information was vital for enabling recombineering strategies aimed at single-nucleotide changes in this biotechnologically important species.\n\nOriginality-Significance StatementSingle-stranded DNA (ssDNA) recombineering has emerged in recent years as one of the most powerful technologies of genome editing in E. coli and other Enterobacteria. However, the efforts to expand the concept and the methods towards environmental microorganisms such as Pseudomonas putida have been limited thus far by several gaps in our fundamental knowledge of how nucleotide mismatch repair (MMR) operates in such non-model species. One critical bottleneck is the hierarchy of recognition of different types of base mispairings as well as the need of setting up strategies for counteracting MMR and thus enabling tolerance to all types of changes. The work presented here tackles both issues and makes P. putida amenable to sophisticated genetic manipulations that were impossible before.

microbiology↗