Extreme acidophilic bacteria like Leptospirillum sp. require an efficient enzyme system to counteract strong oxygen stress conditions in their natural habitat. The genome of Leptospirillum sp. CF-1 encodes the thioredoxin-fold protein TFP2, which exhibits a high structural similarity to the thioredoxin domain of E. coli CnoX. CnoX from Escherichia coli is a chaperedoxin that protects protein substrates from oxidative stress conditions using its holdase function and a subsequent transfer to foldase chaperones for refolding. Recombinantly produced and purified Leptospirillum sp. TFP2 possesses both thioredoxin and chaperone holdase activities in vitro. It can be reduced by thioredoxin reductase (TrxR). The tfp2 gene co-locates with genes for the chaperone foldase GroES/EL on the chromosome. The “tfp2 cluster” (ctpA-groES-groEL-hyp-tfp2-recN) was found between 1.9 and 8.8-fold transcriptionally up-regulated in response to 1 mM hydrogen peroxide (H₂O₂). Leptospirillum sp. tfp2 heterologously expressed in E. coli wild type and cnoX mutant strains lead to an increased tolerance of these E. coli strains to H₂O₂ and significantly reduced intracellular protein aggregates. Finally, a proteomic analysis of protein aggregates produced in E. coli upon exposition to oxidative stress with 4 mM H₂O₂, showed that Leptospirillum sp. tfp2 expression caused a significant decrease in the aggregation of 124 proteins belonging to fifteen different metabolic categories. These included several known substrates of DnaK and GroEL/ES. These findings demonstrate that Leptospirillum sp. TFP2 is a chaperedoxin-like protein, acting as a key player in the control of cellular proteostasis under highly oxidative conditions that prevail in extreme acidic environments.

Susanne Sievers and Daniela Zühlke were not included as authors in the original publication

Tellurite (TeO₃²⁻) is a highly soluble and toxic oxyanion that inhibits the growth of Escherichia coli at concentrations as low as ~1 µg/mL. This toxicity has been primarily attributed to the generation of reactive oxygen species (ROS) during its intracellular reduction by thiol-containing molecules and NAD(P)H-dependent enzymes. However, under anaerobic conditions, E. coli exhibits significantly increased tellurite tolerance—up to 100-fold in minimal media—suggesting the involvement of additional, ROS-independent mechanisms. In this study, we combined chemical-genomic screening, untargeted metabolomics, and targeted biochemical assays to investigate the effects of tellurite under both aerobic and anaerobic conditions. Our findings reveal that tellurite perturbs amino acid and nucleotide metabolism, leading to intracellular imbalances that impair protein synthesis. Additionally, tellurite induces notable changes in membrane lipid composition, particularly in phosphatidylethanolamine derivatives, which may influence biophysical properties of the membrane, such as fluidity or curvature. This membrane remodeling could contribute to the increased resistance observed under anaerobic conditions, although direct evidence of altered membrane fluidity remains to be established. Overall, these results demonstrate that tellurite toxicity extends beyond oxidative stress, impacting central metabolic pathways and membrane-associated functions regardless of oxygen availability.

D-tagatose is a high-value, low-calorie sweetener that can be produced from dairy lactose via a two-step enzymatic route: lactose hydrolysis to galactose followed by galactose isomerization to tagatose. Here, we combined genomics, in silico structural analysis, and biochemical assays to evaluate the lactose-to-tagatose conversion potential of an Antarctic isolate, L47, identified as Ewingella americana (NCBI accession SAMN54554459). Genome mining revealed one L-arabinose isomerase gene (araA) and three β-galactosidase genes (bgaA, bglY, lacZ), an uncommon combination in a single bacterium. Recombinant AraA was produced in Escherichia coli and biochemically characterized, showing Mn²⁺ dependence and measurable D-galactose isomerization, reaching ~18% tagatose from 100 mM galactose after 48 h under the tested conditions. In contrast, the β-galactosidases were predominantly recovered as insoluble aggregates in E. coli; therefore, β-galactosidase activity was assessed using washed inclusion-body preparations. Under these conditions, BgaA displayed the most consistent o-NPG hydrolyzing activity, whereas BglY and LacZ did not yield reproducible activity. Overall, our results identify BgaA as the most tractable lactose-hydrolyzing candidate from L47 in the current workflow and indicate that AraA performance is the principal bottleneck toward an efficient lactose-to-tagatose process, motivating future optimization at the enzyme and process levels.