In vivo role of FdhD and FdmE in formate metabolism in Pseudomonas putida: Redundancy and expression in the stationary phase

Abstract

In Pseudomonas putida two open reading frames (ORFs) PP0257 and PP0492 were originally annotated as accessory formate dehydrogenase proteins. The ORF PP0492 (fdmD) is at the 3' end of the ORF PP0489 through PP0491 operon that encodes one of the formate dehydrogenases of P. putida and it is transcribed with this operon preferentially at the stationary phase. The ORF PP0257 (fdhD) is unlinked to formate dehydrogenase clusters and, as to multi-component formate dehydrogenases, PP0257 is also preferentially expressed in the stationary phase, although transcription can be mediated by either RpoD or RpoS. The transcriptional level of expression of fdhD increased in response to formaldehyde/formate or chemicals that yield these cited C1 compounds through their metabolism. In spite of these correlations, inactivation of PP0257 does not produce a significant effect on in vivo formate dehydrogenase activity, while inactivation of PP0492 leads to a 60% decrease in in vivo activity. These results suggest that redundancy in formaldehyde/formate metabolism in P. putida extends to the proteins involved in maturation/location of formate dehydrogenase complexes.

Core and auxiliary functions of one-carbon metabolism in Pseudomonas putida exposed by a systems-level analysis of transcriptional and physiological responses

“…Mutants of E. coli and Salmonella enterica , lacking either fdhD or fdhE , display limited FDH activity ( 97 , 98 ). However, almost identical 14 CO 2 evolution levels have been reported for P. putida Δ fdhD compared to the parental strain when fed with 10 mM 14 C-formate or 14 C-formaldehyde—and deleting fdhE hampered 14 CO 2 release from 14 C-formate ( 33 ). These results indicate that the four FDHs of P. putida probably display different levels of substrate affinity ( K M ), responding to formate challenges in a concentration-dependent fashion.…”

“…S4 ), hinting at similar functionality of the polypeptides. The gene encoding FdhD ( PP_0257 ), a FDH accessory sulfurtransferase, was also highly overexpressed in the presence of formate ( 33 ). The sequence coverage plot of these loci at the single-nucleotide resolution suggests that PP_4596 constitutes a separate, single transcriptional unit, whereas fdhD and PP_0256 are part of the same mRNA transcript ( Fig.…”

“…As P. putida can endure relatively high formaldehyde concentrations, the direct oxidative product of methanol, aldehyde detoxification systems are expected to be active upon cultivation of the cells in the presence of the C1 alcohol. Formaldehyde detoxification involves the concerted action of formaldehyde dehydrogenases and FDHs that eventually yield CO 2 ( 33 , 79 ). P. putida KT2440 harbors two NAD + -binding, glutathione-independent formaldehyde dehydrogenases ( 32 ), encoded by fdhA ( PP_0328 ) and fdhB ( PP_3970 , also known as ybdR ).…”

“…Together with these biochemical activities, the natural resistance of P. putida toward solvents and alcohols highlights its potential to host synthetic methanol assimilation pathways ( 29 , 30 ). Additionally, the genome of P. putida encodes multiple NAD + -dependent formaldehyde (i.e., fdhA and fdhB ) and formate dehydrogenases (FDHs, e.g., fmdEFGH )—enabling the use of these C1 molecules as co-substrates to boost ATP formation ( 31 - 33 ). Again, a deep understanding of the genetic and molecular basis of the native methanol, formaldehyde, and formate oxidation capabilities of P. putida is required—especially toward informing metabolic engineering of C1 assimilation.…”

Exploiting environmental niches and the potential of environmental microbes

“…The metabolism of these compounds in P. putida KT2440 is redundant. Now these authors (Roca and Ramos, 2009) show that the proteins involved in the maturation and localization of formate dehydrogenase complexes are also redundant. Formaldehyde is produced in vivo in the metabolism of methoxylated aromatic compounds and histidine and the system formaldehyde dehydrogenase/formate dehydrogenase functions as a detoxifying system.…”