Antibiotics can induce cell death via a variety of action modes, including the inhibition of transcription, ribosomal function, and cell wall biosynthesis. In this study, we demonstrated directly that iron availability is important to the action of antibiotics, and the ferric reductases of Pseudomonas putida and Pseudomonas aeruginosa could accelerate antibiotic-mediated cell death by promoting the Fenton reaction. The modulation of reduced nicotinamide-adenine dinucleotide (NADH) levels and iron chelation affected the actions of antibiotics. Interestingly, the deletion of the ferric reductase gene confers more antibiotic resistance upon cells, and its overexpression accelerates antibiotic-mediated cell death. The results of transcriptome analysis showed that both Pseudomonas species induce many oxidative stress genes under antibiotic conditions, which could not be observed in ferric reductase mutants. Our results indicate that iron homeostasis is crucial for bacterial cell survival under antibiotics and should constitute a significant target for boosting the action of antibiotics.

Acinetobacter baumannii is an aerobic and gram-negative pathogenic bacterium that is resistant to most antibiotics. Recently, A. baumannii 1656-2 exhibited the ability to form biofilms under clinical conditions. In this study, global metabolite profiling of both planktonic and biofilm forms of A. baumannii 1656-2 was performed using high-resolution nuclear magnetic resonance (NMR) spectroscopy and multivariate statistical analysis to investigate the metabolic patterns leading to biofilm formation. Principal components analysis (PCA) and orthogonal partial least-squares discriminant analysis (OPLS-DA) score plots showed a distinct separation between planktonic and biofilm cells. Metabolites including acetates, pyruvate, succinate, UDP-glucose, AMP, glutamate, and lysine were increasingly involved in the energy metabolism of biofilm formation. In particular, the ratio of N-acetyl-D-glucosamine (GlcNAc) to D-glucosamine (GlcNH₂) was significantly higher during biofilm formation than under the planktonic condition. This study demonstrates that NMR-based global metabolite profiling of bacterial cells can provide valuable insight into the metabolic changes in multidrug resistant and biofilm-forming bacteria such as A. baumannii 1656-2.

Pseudomonas putida harbors two ferredoxin-NADP ⁺ reductases (Fprs) on its chromosome, and their functions remain largely unknown. Ferric reductase is structurally contained within the Fpr superfamily. Interestingly, ferric reductase is not annotated on the chromosome of P. putida . In an effort to elucidate the function of the Fpr as a ferric reductase, we used a variety of biochemical and physiological methods using the wild-type and mutant strains. In both the ferric reductase and flavin reductase assays, FprA and FprB preferentially used NADPH and NADH as electron donors, respectively. Two Fprs prefer a native ferric chelator to a synthetic ferric chelator and utilize free flavin mononucleotide (FMN) as an electron carrier. FprB has a higher k _cat / K _m value for reducing the ferric complex with free FMN. The growth rate of the fprB mutant was reduced more profoundly than that of the fprA mutant, the growth rate of which is also lower than the wild type in ferric iron-containing minimal media. Flavin reductase activity was diminished completely when the cell extracts of the fprB mutant plus NADH were utilized, but not the fprA mutant with NADPH. This indicates that other NADPH-dependent flavin reductases may exist. Interestingly, the structure of the NAD(P) region of FprB, but not of FprA, resembled the ferric reductase (Fre) of Escherichia coli in the homology modeling. This study demonstrates, for the first time, the functions of Fprs in P. putida as flavin and ferric reductases. Furthermore, our results indicated that FprB may perform a crucial role as a NADH-dependent ferric/flavin reductase under iron stress conditions.