Pseudomonas putida is a metabolically versatile saprophytic soil bacterium that has been certified as a biosafety host for the cloning of foreign genes. The bacterium also has considerable potential for biotechnological applications. Sequence analysis of the 6.18 Mb genome of strain KT2440 reveals diverse transport and metabolic systems. Although there is a high level of genome conservation with the pathogenic Pseudomonad Pseudomonas aeruginosa (85% of the predicted coding regions are shared), key virulence factors including exotoxin A and type III secretion systems are absent. Analysis of the genome gives insight into the non-pathogenic nature of P. putida and points to potential new applications in agriculture, biocatalysis, bioremediation and bioplastic production.

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Complete genome sequence and comparative analysis of the metabolically versatile Pseudomonas putida KT2440

The genus Pseudomonas currently contains 144 species, making it the genus of Gram-negative bacteria that contains the largest number of species. Currently, multilocus sequence analysis (MLSA) is the preferred method for establishing the phylogeny between species and genera. Four partial gene sequences of housekeeping genes (16S rRNA, gyrB, rpoB, and rpoD) were obtained from 112 complete or draft genomes of strains related to the genus Pseudomonas that were available in databases. These genes were analyzed together with the corresponding sequences of 133 Pseudomonas type strains of validly published species to assess their correct phylogenetic assignations. We confirmed that 30% of the sequenced genomes of non-type strains were not correctly assigned at the species level in the accepted taxonomy of the genus and that 20% of the strains were not identified at the species level. Most of these strains had been isolated and classified several years ago, and their taxonomic status has not been updated by modern techniques. MLSA was also compared with indices based on the analysis of whole-genome sequences that have been proposed for species delineation, such as tetranucleotide usage patterns (TETRA), average nucleotide identity (ANIm, based on MUMmer and ANIb, based on BLAST) and genome-to-genome distance (GGDC). TETRA was useful for discriminating Pseudomonas from other genera, whereas ANIb and GGDC clearly separated strains of different species. ANIb showed the strongest correlation with MLSA. The correct species classification is a prerequisite for most diversity and evolutionary studies. This work highlights the necessity for complete genomic sequences of type strains to build a phylogenomic taxonomy and that all new genome sequences submitted to databases should be correctly assigned to species to avoid taxonomic inconsistencies.

/pdf/phylogenomics-and-systematics-in-pseudomonas-4z06titkab.pdf

Phylogenomics and systematics in Pseudomonas

Much of synthetic biology research makes use of model organisms, such as Escherichia coli. Here, Victor de Lorenzo and colleagues emphasize the need for a wider choice of model organisms and advocate the use of environmental Pseudomonas strains as model organisms that possess the necessary metabolic traits required to meet current and future synthetic biology and biotechnological needs.

/pdf/biotechnological-domestication-of-pseudomonads-using-dqs5uz1vq3.pdf

Biotechnological domestication of pseudomonads using synthetic biology

Summary
The genome of the soil bacterium Pseudomonas putida strain KT2440 has been erased of various determinants of resistance to antibiotics encoded in its extant chromosome To this end, we employed a coherent genetic platform that allowed the precise deletion of multiple genomic segments in a large variety of Gram-negative bacteria including (but not limited to) P putida The method is based on the obligatory recombination between free-ended homologous DNA sequences that are released as linear fragments generated upon the cleavage of the chromosome with unique I-SceI sites, added to the segment of interest by the vector system Despite the potential for a SOS response brought about by the appearance of double stranded DNA breaks during the process, fluctuation experiments revealed that the procedure did not increase mutation rates – perhaps due to the protection exerted by I-SceI bound to the otherwise naked DNA termini With this tool in hand we made sequential deletions of genes mexC, mexE, ttgA and ampC in the genome of the target bacterium, orthologues of which are known to determine various degrees of antibiotic resistance in diverse microorganisms Inspection of the corresponding phenotypes demonstrated that the efflux pump encoded by ttgA sufficed to endow P putida with a high-level of tolerance to β-lactams, chloramphenicol and quinolones, but had little effect on, eg aminoglycosides Analysis of the mutants revealed also a considerable diversity in the manifestation of the resistance phenotype within the population and suggested a degree of synergism between different pumps The directed edition of the P putida chromosome shown here not only enhances the amenability of this bacterium to deep genomic engineering, but also validates the corresponding approach for similar handlings of a large variety of Gram-negative microorganisms

Engineering multiple genomic deletions in Gram-negative bacteria: analysis of the multi-resistant antibiotic profile of Pseudomonas putida KT2440.

https://backend.orbit.dtu.dk/ws/files/160753786/1_s2.0_S1096717618301502_main.pdf

D. Regenhardt

Papers

Pedigree and taxonomic credentials of Pseudomonas putida strain KT2440.