The Cellular Response to Lanthanum Is Substrate Specific and Reveals a Novel Route for Glycerol Metabolism in Pseudomonas putida KT2440

Abstract

Ever since the discovery of the first rare earth element (REE)-dependent enzyme, the physiological role of lanthanides has become an emerging field of research due to the environmental implications and biotechnological opportunities. In Pseudomonas putida KT2440, the two pyrroloquinoline quinone-dependent alcohol dehydrogenases (PQQ-ADHs) PedE and PedH are inversely regulated in response to REE availability. This transcriptional switch is orchestrated by a complex regulatory network that includes the PedR2/PedS2 two-component system and is important for efficient growth on several alcoholic volatiles. To study whether cellular responses beyond the REE switch exist, the differential proteomic responses that occur during growth on various model carbon sources were analyzed. Apart from the Ca2+-dependent enzyme PedE, the differential abundances of most identified proteins were conditional. During growth on glycerol—and concomitant with the proteomic changes—lanthanum (La3+) availability affected different growth parameters, including the onset of logarithmic growth and final optical densities. Studies with mutant strains revealed a novel metabolic route for glycerol utilization, initiated by PedE and/or PedH activity. Upon oxidation to glycerate via glyceraldehyde, phosphorylation by the glycerate kinase GarK most likely yields glycerate-2-phosphate, which is eventually channeled into the central metabolism of the cell. This new route functions in parallel with the main degradation pathway encoded by the glpFKRD operon and provides a growth advantage to the cells by allowing an earlier onset of growth with glycerol as the sole source of carbon and energy. IMPORTANCE The biological role of REEs has long been underestimated, and research has mainly focused on methanotrophic and methylotrophic bacteria. We have recently demonstrated that P. putida, a plant growth-promoting bacterium that thrives in the rhizosphere of various food crops, possesses a REE-dependent alcohol dehydrogenase (PedH), but knowledge about REE-specific effects on physiological traits in nonmethylotrophic bacteria is still scarce. This study demonstrates that the cellular response of P. putida to lanthanum (La3+) is mostly substrate specific and that La3+ availability highly affects the growth of cells on glycerol. Further, a novel route for glycerol metabolism is identified, which is initiated by PedE and/or PedH activity and provides a growth advantage to this biotechnologically relevant organism by allowing a faster onset of growth. Overall, these findings demonstrate that lanthanides can affect physiological traits in nonmethylotrophic bacteria and might influence their competitiveness in various environmental niches.

Fatty Acid and Alcohol Metabolism in Pseudomonas putida: Functional Analysis Using Random Barcode Transposon Sequencing

“…The alcohol dehydrogenases that showed the most consistent fitness defects across multiple conditions were the two PQQ-dependent alcohol dehydrogenases PP_2674 (pedE) and PP_2679 (pedH), as well as the Fe-dependent alcohol dehydrogenase PP_2682 (yiaY) (Figure 4B). Both pedE and pedH have been extensively studied in P. putida and other related bacteria, and are known to have broad substrate specificities for alcohols and aldehydes (25,26,49). Their activity is dependent on the activity of pedF (PP_2675), a cytochrome c oxidase that regenerates the PQQ cofactor (25).…”

“…putida is also able to oxidize and catabolize a wide variety of alcohols. Much work has focused on the unique biochemistry and regulation of two pyrroloquinoline quinone (PQQ)dependent alcohol dehydrogenases (ADH), pedE and pedH, which exhibit broad substrate specificity for both alcohols and aldehydes (25,26). Specific work has also investigated the suitability of P. putida for the production of ethanol (27) and the genetic basis for its ability to catabolize butanol and 1,4-butanediol (28)(29)(30).…”

Biological Pincer Complexes

“…Lanthanide‐PQQ alcohol dehydrogenases are also found in Pseudomonas putida KT 2440 . Recent evidence suggests this microorganism possesses two dehydrogenases that convert the glycerol substrate to glyceraldehyde; PedE using a calcium‐PQQ enzyme and PedH using a lanthanide‐PQQ version . The physiological significance of lanthanide incorporation into the PQQ‐containing enzymes in various microbes remains uncertain, but probably relates to the greater Lewis acidity of this metal when compared to calcium or magnesium.…”

Biological Function of Lanthanide in Plant-Symbiotic Bacteria: Lanthanide-Dependent Methanol Oxidation System

“…2A). Kawai et al, 2007Hibi et al, 2011Nakagawa et al, 2012Fitriyanto et al, 2011aWang et al, 2019Pastawan et al, 2020Fitriyanto et al, 2011bPol et al, 2014Wehrmann et al, 2020 In the case of Mr. extorquens AM1, MxaFI is encoded on the mxa cluster ( Fig. 2B) and the gene cluster includes several genes for methanol oxidation; mxaF and mxaI, encoding subunits α and β of MDH, respectively (Nunn and Lidstrom, 1986a;1986b); mxaG, encoding cytochrome cL, which is electron acceptor for MxaFI (Amaratunga et al, 1997); mxaJ, encoding a putative chaperone for MxaFI (Featherston et al, 2019); mxaA, encoding a putative chaperone for Ca 2+ insertion to MxaF (Morris et al, 1995); and many other genes of unknown function (Anderson et al, 1990;Toyama et al, 1998).…”