The putative phosphate transporter PitB (PP1373) is involved in tellurite uptake in Pseudomonas putida KT2440

Abstract

Tellurium oxyanions are chemical species of great toxicity and their presence in the environment has increased because of mining industries and photovoltaic and electronic waste. Recovery strategies for this metalloid that are based on micro-organisms are of interest, but further studies of the transport systems and enzymes responsible for implementing tellurium transformations are required because many mechanisms remain unknown. Here, we investigated the involvement in tellurite uptake of the putative phosphate transporter PitB (PP1373) in soil bacterium Pseudomonas putida KT2440. For this purpose, through a method based on the CRISPR/Cas9 system, we generated a strain deficient in the pitB gene and characterized its phenotype on exposing it to varied concentrations of tellurite. Growth curves and transmission electronic microscopy experiments for the wild-type and ΔpitB strains showed that both were able to internalize tellurite into the cytoplasm and reduce the oxyanion to black nano-sized and rod-shaped tellurium particles, although the ΔpitB strain showed an increased resistance to the tellurite toxic effects. At a concentration of 100 μM tellurite, where the biomass formation of the wild-type strain decreased by half, we observed a greater ability of ΔpitB to reduce this oxyanion with respect to the wild-type strain (~38 vs ~16 %), which is related to the greater biomass production of ΔpitB and not to a greater consumption of tellurite per cell. The phenotype of the mutant was restored on over-expressing pitB in trans. In summary, our results indicate that PitB is one of several transporters responsible for tellurite uptake in P. putida KT2440.

Phosphite synthetic auxotrophy as an effective biocontainment strategy for the industrial chassis Pseudomonas putida

“…3 , 5 ). Prior to this research, references to the native Pi transport system of P. putida KT2440 were scarce [ 45 , 46 ], and most annotations of transporters were based on their homology with their E. coli ’s counterparts. During this study, we found that deletions in what we identified by homology as PitA ( PP_4103 ) did not dramatically affect the growth of P. putida in Pi (Additional file 1 : Figure S2A).…”

“…As such, its regained ability to grow on P sources is most likely resulting from a series of acquired evolutionary mutations. Since our modifications have not affected the core Pi metabolism of the mutant but only its transport, we believe that these mutations may have altered the structural configuration of other native transporters specific to molecules such as sulphate (similar in size to phosphate), or promiscuous transporters with the ability to cotransport different molecules [ 46 ]. As a result, these mutations could either unlock new transport abilities or improve the specificity of a low affinity transporter to incorporate Pi.…”

Production of selenium nanoparticles occurs through an interconnected pathway of sulphur metabolism and oxidative stress response in Pseudomonas putida KT2440

“…For mutant complementation the protocol described by Montenegro et al ( 2021 ) was used. Briefly the DNA sequence encoding the respective gene for each mutant (see Table 1 ) of P. putida KT2440 was amplified directly from the chromosome of the bacterium with their corresponding primers (see Table S3 ).…”

“…This metabolic capacity could be used for the implementation of reduction reactions such as the transformation of oxyanions (e.g. selenite, tellurite) to their respective elemental species (Se 0 , Te 0 ) (Montenegro et al, 2021 ; Vieto et al, 2021 ).…”

Microbial Musings – February 2021

“…The soil bacterium Pseudomonas putida KT2440 is known to use the later route to detoxify tellurite, forming a black precipitate inside the cells. In this paper Rafael Montenegro ( @RafaMM19 ), Sofía Vieto ( @sofivieto ) and colleagues from the group of Max Chavarría ( @MaxChavarriaV ), at the Centro Nacional de Innovaciones Biotecnológicas (CENIBiot), Costa Rica, have investigated how the tellurite enters the cell and identified one of the transporters involved [ 6 ]. They studied the P. putida orthologue of an inorganic phosphate transporter, PitB, as the Escherichia coli PitA protein was known to be involved in tellurite uptake in E. coli [ 7 ].…”