The Burkholderia cenocepacia iron starvation σ factor, OrbS, possesses an on-board iron sensor.

Burkholderia cenocepacia is an opportunistic pathogen that causes severe infections of the cystic fibrosis (CF) lung. To acquire iron, B. cenocepacia secretes the Fe(III)-binding compound, ornibactin. Genes for synthesis and utilisation of ornibactin are served by the iron starvation (IS) extracytoplasmic function (ECF) σ factor, OrbS. Transcription of orbS is regulated in response to the prevailing iron concentration by the ferric uptake regulator (Fur), such that orbS expression is repressed under iron-sufficient conditions. Here we show that, in addition to Fur-mediated regulation of orbS, the OrbS protein itself responds to intracellular iron availability. Substitution of cysteine residues in the C-terminal region of OrbS diminished the ability to respond to Fe(II) in vivo. Accordingly, whilst Fe(II) impaired transcription from and recognition of OrbS-dependent promoters in vitro by inhibiting the binding of OrbS to core RNA polymerase (RNAP), the cysteine-substituted OrbS variant was less responsive to Fe(II). Thus, the cysteine residues within the C-terminal region of OrbS contribute to an iron-sensing motif that serves as an on-board 'anti-σ factor' in the presence of Fe(II). A model to account for the presence two regulators (Fur and OrbS) that respond to the same intracellular Fe(II) signal to control ornibactin synthesis and utilisation is discussed.

Overproduction and purification of Mycobacterium tuberculosis WhiB3 in Escherichia coli is enhanced by co-expression with trigger factor chaperone.

Members of the WhiB-like (Wbl) family of proteins are found in Acintomycetes and are somewhat recalcitrant to overproduction as soluble proteins in the laboratory protein expression workhorse Esherichia coli. The aim of this study was to evaluate the effects of culture conditions and co-expression of the chaperone protein, trigger factor (TF), on the soluble production of recombinant Mycobacterium tuberculosis (Mtb) WhiB3. A pET28a derived expression plasmid coding for His6-WhiB3 was created and the effects of varying the concentration of inducer (IPTG), the timing of induction, the nature of the inducer (auto-induction medium) and the temperature of the cultivation on the production of soluble His6-WhiB3 were tested. Whilst His6-WhiB3 protein was readily detected, the overwhelming majority of the protein was present in the insoluble fraction of cell-free extracts. However, co-expression of the tig from pTf16, coding for TF, increased His6-WhiB3 solubility dramatically, facilitating its isolation by affinity chromatography. Purified His6-WhiB3 was shown to be monomeric, and UV-visible spectra suggested that ∼10% of the isolated protein possessed a [4Fe-4S] cluster. The secondary structural properties of His6-WhiB3 were altered by acquisition of an iron-sulfur cluster. By developing a protocol to readily overproduce and purify WhiB3, this study paves the way for future structure-function experiments.

Food-evoked nostalgia.

In three studies, we examined food as an elicitor of nostalgia. Study 1 participants visualised eating either a nostalgic or regularly consumed food. Study 2 participants visualised consuming 12 foods. Study 3 participants consumed 12 flavour samples. Following their food experiences, all participants responded to questions regarding the profile of food-evoked nostalgia (i.e. autobiographical relevance, arousal, familiarity, positive and negative emotions) and several psychological functions (i.e. positive affect, self-esteem, social connectedness, meaning in life). Study 2 and 3 participants also reported their state nostalgia. Results revealed that food is a powerful elicitor of nostalgia. Food-evoked nostalgia has a similar contextual profile to previously examined elicitors, but is a predominantly positive emotional experience. Food-evoked nostalgia served multiple psychological functions and predicted greater state nostalgia.

The Di-Iron Protein YtfE Is a Nitric Oxide-Generating Nitrite Reductase Involved in the Management of Nitrosative Stress.

Previously characterized nitrite reductases fall into three classes: siroheme-containing enzymes (NirBD), cytochrome c hemoproteins (NrfA and NirS), and copper-containing enzymes (NirK). We show here that the di-iron protein YtfE represents a physiologically relevant new class of nitrite reductases. Several functions have been previously proposed for YtfE, including donating iron for the repair of iron-sulfur clusters that have been damaged by nitrosative stress, releasing nitric oxide (NO) from nitrosylated iron, and reducing NO to nitrous oxide (N2O). Here, in vivo reporter assays confirmed that Escherichia coli YtfE increased cytoplasmic NO production from nitrite. Spectroscopic and mass spectrometric investigations revealed that the di-iron site of YtfE exists in a mixture of forms, including nitrosylated and nitrite-bound, when isolated from nitrite-supplemented, but not nitrate-supplemented, cultures. Addition of nitrite to di-ferrous YtfE resulted in nitrosylated YtfE and the release of NO. Kinetics of nitrite reduction were dependent on the nature of the reductant; the lowest Km, measured for the di-ferrous form, was ∼90 µM, well within the intracellular nitrite concentration range. The vicinal di-cysteine motif, located in the N-terminal domain of YtfE, was shown to function in the delivery of electrons to the di-iron center. Notably, YtfE exhibited very low NO reductase activity and was only able to act as an iron donor for reconstitution of apo-ferredoxin under conditions that damaged its di-iron center. Thus, YtfE is a high-affinity, low-capacity nitrite reductase that we propose functions to relieve nitrosative stress by acting in combination with the co-regulated NO-consuming enzymes Hmp and Hcp.

Evolving MRSA: High-level ß-lactam resistance in Staphylococcus aureus is associated with RNA Polymerase alterations and fine tuning of gene expression.

Most clinical MRSA (methicillin-resistant S. aureus) isolates exhibit low-level ß-lactam resistance (oxacillin MIC 2-4 µg/ml) due to the acquisition of a novel penicillin binding protein (PBP2A), encoded by mecA. However, strains can evolve high-level resistance (oxacillin MIC ≥256 µg/ml) by an unknown mechanism. Here we have developed a robust system to explore the basis of the evolution of high-level resistance by inserting mecA into the chromosome of the methicillin-sensitive S. aureus SH1000. Low-level mecA-dependent oxacillin resistance was associated with increased expression of anaerobic respiratory and fermentative genes. High-level resistant derivatives had acquired mutations in either rpoB (RNA polymerase subunit ß) or rpoC (RNA polymerase subunit ß') and these mutations were shown to be responsible for the observed resistance phenotype. Analysis of rpoB and rpoC mutants revealed decreased growth rates in the absence of antibiotic, and alterations to, transcription elongation. The rpoB and rpoC mutations resulted in decreased expression to parental levels, of anaerobic respiratory and fermentative genes and specific upregulation of 11 genes including mecA. There was however no direct correlation between resistance and the amount of PBP2A. A mutational analysis of the differentially expressed genes revealed that a member of the S. aureus Type VII secretion system is required for high level resistance. Interestingly, the genomes of two of the high level resistant evolved strains also contained missense mutations in this same locus. Finally, the set of genetically matched strains revealed that high level antibiotic resistance does not incur a significant fitness cost during pathogenesis. Our analysis demonstrates the complex interplay between antibiotic resistance mechanisms and core cell physiology, providing new insight into how such important resistance properties evolve.

Multi-omic based production strain improvement (MOBpsi) for bio-manufacturing of toxic chemicals.

Robust systematic approaches for the metabolic engineering of cell factories remain elusive. The available models for predicting phenotypical responses and mechanisms are incomplete, particularly within the context of compound toxicity that can be a significant impediment to achieving high yields of a target product. This study describes a Multi-Omic Based Production Strain Improvement (MOBpsi) strategy that is distinguished by integrated time-resolved systems analyses of fed-batch fermentations. As a case study, MOBpsi was applied to improve the performance of an Escherichia coli cell factory producing the commodity chemical styrene. Styrene can be bio-manufactured from phenylalanine via an engineered pathway comprised of the enzymes phenylalanine ammonia lyase and ferulic acid decarboxylase. The toxicity, hydrophobicity, and volatility of styrene combine to make bio-production challenging. Previous attempts to create styrene tolerant E. coli strains by targeted genetic interventions have met with modest success. Application of MOBpsi identified new potential targets for improving performance, resulting in two host strains (E. coli NST74ΔaaeA and NST74ΔaaeA cpxPo) with increased styrene production. The best performing re-engineered chassis, NST74ΔaaeA cpxPo, produced ∼3 × more styrene and exhibited increased viability in fed-batch fermentations. Thus, this case study demonstrates the utility of MOBpsi as a systematic tool for improving the bio-manufacturing of toxic chemicals.

The past as a resource for the bereaved: nostalgia predicts declines in distress.

Nostalgia, a sentimental longing for one's past, can serve as a resource for individuals coping with discomforting experiences. The experience of bereavement poses psychological and physical risks. In a longitudinal study, we examined whether dispositional nostalgia predicted reductions in distress associated with the death of a loved one. Undergraduate students (N = 133) provided information regarding their loss (time elapsed since loss, expectedness) and levels of initial grief, nostalgia, and distress (hyperarousal, intrusion, avoidance) at three time points over a one-month period (Times 2 and 3 occurred one week and one month after the initial session, respectively). Individuals experiencing higher nostalgia reported a decrease in intrusive thoughts across time, whereas those experiencing lower nostalgia reported no change in intrusive thoughts across time. Hyperarousal (physical symptoms, negative feelings) decreased across time among individuals with higher initial grief who experienced greater nostalgia, but increased across time among those with higher initial grief who experienced lesser nostalgia. No changes occurred in avoidance. Nostalgia can palliate bereavement.

A Phase II Single Arm Pilot Study of the CHK1 Inhibitor Prexasertib (LY2606368) in BRCA Wild-Type, Advanced Triple-Negative Breast Cancer.

LESSONS LEARNED: Monotherapy with prexasertib demonstrated modest activity in BRCA wild-type, recurrent triple-negative breast cancer, highlighting the unmet need for combination treatment strategies. Neutropenia, anemia, and thrombocytopenia are common with the use of prexasertib but are manageable with supportive care measures. Prophylactic use of granulocyte colony stimulating factor should be considered to avoid dose reductions or treatment delays. Pharmacodynamic studies showed prexasertib treatment induced DNA damage in peripheral immune cells. BACKGROUND: Cell cycle checkpoint kinase 1 (CHK1) is a major G2/M cell cycle regulator in tumors with p53 dysfunction, such as triple-negative breast cancer (TNBC). We hypothesized the second-generation CHK1 inhibitor, prexasertib, would yield clinical activity in sporadic TNBC. METHODS: This single arm, phase II trial evaluated prexasertib at 105 mg/m2 IV every 2 weeks in patients with metastatic/recurrent TNBC. The primary endpoint was overall response rate (ORR). RESULTS: All nine patients enrolled were germline BRCA wild-type (BRCAwt) and had at least one prior treatment. One partial response (PR) was observed (ORR of 11.1%). Four patients experienced stable disease. The median progression-free survival (PFS) was 86 days (range 17 to 159 days). Grade 3/4 treatment-related adverse events included afebrile neutropenia (n = 8; 88.9%), anemia (n = 3; 33.3%), and thrombocytopenia (n = 1; 11.1%). Pharmacodynamic studies showed prexasertib treatment induced DNA damage in peripheral immune cells and demonstrated a decrease in activated/reinvigorated CD8 T cells; however, the one patient with a PR showed evidence of T-cell recovery. CONCLUSION: Prexasertib monotherapy had modest clinical efficacy in BRCAwt TNBC. Further studies of prexasertib in combination with other agents are needed.

From formulation to in vivo model: A comprehensive study of a synergistic relationship between vancomycin, carvacrol, and cuminaldehyde against Enterococcus faecium.

Vancomycin-resistant Enterococcus faecium (VRE) has become endemic in healthcare settings, reducing treatment options for enterococcal infections. New antimicrobials for VRE infections are a high priority, but the development of novel antibiotics is time-consuming and expensive. Essential oils (EOs) synergistically enhance the activity of some existing antibiotics, suggesting that EO-antibiotic combinations could resensitise resistant bacteria and maintain the antibiotic repertoire. The mechanism of resensitisation of bacteria to antibiotics by EOs is relatively understudied. Here, the synergistic interactions between carvacrol (1.98 mM) and cuminaldehyde (4.20 mM) were shown to reestablish susceptibility to vancomycin (0.031 mg/L) in VRE, resulting in bactericidal activity (4.73 log10 CFU/ml reduction). Gene expression profiling, coupled with ß-galactosidase leakage and salt tolerance assays, suggested that cell envelope damage contributes to the synergistic bactericidal effect against VRE. The EO-vancomycin combination was also shown to kill clinical isolates of VRE (2.33-5.25 log10 CFU/ml reduction), and stable resistance did not appear to develop even after multiple passages. The in vivo efficacy of the EO-vancomycin combination was tested in a Galleria mellonella larvae assay; however, no antimicrobial action was observed, indicating that further drug development is required for the EO-vancomycin combination to be clinically useful for treatment of VRE infections.

Anticipated nostalgia: Looking forward to looking back.

Anticipated nostalgia is a new construct that has received limited empirical attention. It concerns the anticipation of having nostalgic feelings for one's present and future experiences. In three studies, we assessed its prevalence, content, emotional profile, and implications for self-regulation and psychological functioning. Study 1 revealed that anticipated nostalgia most typically concerns interpersonal relationships, and also concerns goals, plans, current life, and culture. Further, it is affectively laden with happiness, sadness, bittersweetness, and sociality. Studies 2 and 3 assessed the implications of anticipated nostalgia for self-regulation and psychological functioning. In both studies, positive evaluation of a personal experience was linked to stronger anticipated nostalgia, and anticipated nostalgia was linked to savouring of the experience. In Study 3, anticipated nostalgia measured prior to an important life transition predicted nostalgia a few months after the transition, and post-transition nostalgia predicted heightened self-esteem, social connectedness, and meaning in life.

BPG: Seamless, automated and interactive visualization of scientific data.

BACKGROUND: We introduce BPG, a framework for generating publication-quality, highly-customizable plots in the R statistical environment. RESULTS: This open-source package includes multiple methods of displaying high-dimensional datasets and facilitates generation of complex multi-panel figures, making it suitable for complex datasets. A web-based interactive tool allows online figure customization, from which R code can be downloaded for integration with computational pipelines. CONCLUSION: BPG provides a new approach for linking interactive and scripted data visualization and is available at http://labs.oicr.on.ca/boutros-lab/software/bpg or via CRAN at https://cran.r-project.org/web/packages/BoutrosLab.plotting.general.

Staphylococcus aureus adaptation to aerobic low-redox-potential environments: implications for an intracellular lifestyle.

Methicillin-resistant Staphylococcus aureus is a 'superbug' that is responsible for extensive death and morbidity. Chronic S. aureus infections are associated with the presence of intracellular bacteria and the host cytosol is an aerobic low-redox-potential (Eh) environment. How S. aureus adapts to aerobic low-Eh environments is understudied. A low external Eh, imposed by the non-metabolizable reductant dithiothreitol, resulted in transcriptional reprogramming mediated by the redox-responsive transcription factors AgrA, Rex and SrrBA, resulting in a shift towards fermentative metabolism. Accordingly, in the presence of the host cytoplasmic reductant glutathione, the aerobic respiration of S. aureus was impaired, the intracellular NADH:NAD+ ratio increased, lactate dehydrogenase was induced, resistance to the aminoglycoside antibiotic gentamicin was enhanced and greater numbers of small-colony variants (SCVs) were detected. These observations suggest that entry of S. aureus into the aerobic low-Eh environment of the host cytosol could result in adaptive responses that promote the formation of SCVs.

Cmr is a redox-responsive regulator of DosR that contributes to M. tuberculosis virulence.

Mycobacterium tuberculosis (MTb) is the causative agent of pulmonary tuberculosis (TB). MTb colonizes the human lung, often entering a non-replicating state before progressing to life-threatening active infections. Transcriptional reprogramming is essential for TB pathogenesis. In vitro, Cmr (a member of the CRP/FNR super-family of transcription regulators) bound at a single DNA site to act as a dual regulator of cmr transcription and an activator of the divergent rv1676 gene. Transcriptional profiling and DNA-binding assays suggested that Cmr directly represses dosR expression. The DosR regulon is thought to be involved in establishing latent tuberculosis infections in response to hypoxia and nitric oxide. Accordingly, DNA-binding by Cmr was severely impaired by nitrosation. A cmr mutant was better able to survive a nitrosative stress challenge but was attenuated in a mouse aerosol infection model. The complemented mutant exhibited a ∼2-fold increase in cmr expression, which led to increased sensitivity to nitrosative stress. This, and the inability to restore wild-type behaviour in the infection model, suggests that precise regulation of the cmr locus, which is associated with Region of Difference 150 in hypervirulent Beijing strains of Mtb, is important for TB pathogenesis.

Release of nitric oxide by the Escherichia coli YtfE (RIC) protein and its reduction by the hybrid cluster protein in an integrated pathway to minimize cytoplasmic nitrosative stress.

Synthesis of the Escherichia coli YtfE protein, also known as RIC, for the repair of damaged iron centres, is highly induced during anaerobic growth under conditions of nitrosative stress. How YtfE repairs nitrosative damage remains unclear. Contrary to previous reports, we show that strains defective in YtfE that lack the high-affinity NO reductase activity of the hybrid cluster protein (Hcp) are less sensitive to nitrosative stress than isogenic ytfE+ strains, which are extremely sensitive. Evidence that this sensitivity is due to YtfE-dependent release of NO into the cytoplasm includes: relief of growth inhibition by PTIO (2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide), which degrades NO; relief of nitrosative stress by deletion of narG encoding the nitrate reductase that is the major source of NO from nitrite; partial suppression of nitrosative stress due to loss of Hcp function by a further mutation in ytfE; YtfE-dependent loss of aconitase and fumarase activities in the absence of Hcp; and YtfE-dependent relief of NsrR repression of the hcp promoter in response to cytoplasmic NO. We suggest that a major role for YtfE is to reverse nitrosative damage by releasing, directly or indirectly, NO from nitrosylated proteins into the cytoplasm where the high-affinity NO reductase activity of Hcp ensures its reduction to N2O. If so, the concerted action of YtfE and Hcp would not only maintain the cytoplasmic concentration of NO in the low nM range, but also provide a rationalization for the coordinate regulation of Hcp and YtfE synthesis by NsrR.

CBMNet: the 'Crossing Biological Membranes' network in industrial biotechnology and bioenergy.

The ∼1300 academic and industry members of the BBSRC (Biotechnology and Biological Sciences Research Council) Network in Industrial Biotechnology and Bioenergy (NIBB) Crossing Biological Membranes Network (CBMNet) are motivated to explore how knowledge of the roles of biological membranes can be exploited to enhance the productivity of cell factories. Improving existing, and developing new, cell factories requires a deep understanding of the mechanisms by which substances are transported into, within, and out of the cells. Embedding consideration of membrane function into the design of cell factories is crucial for the future of almost all cell-based Industrial Biotechnology and Bioenergy (IBBE) applications. CBMNet provides a forum for: knowledge exchange between academics and companies; developing new interactions in the context of responsible innovation; forming, and then supporting, new multi-disciplinary teams to develop innovative membrane-based solutions to overcome IBBE bottlenecks; and funding consortia to carry out feasibility studies with the target of generating competitive bids for further research funding. More broadly, CBMNet is working with other NIBB to raise the profile of IBBE among policymakers and develop a national strategy for IBBE in the U.K.

Regulation, sensory domains and roles of two Desulfovibrio desulfuricans ATCC27774 Crp family transcription factors, HcpR1 and HcpR2, in response to nitrosative stress.

In silico analyses identified a Crp/Fnr family transcription factor (HcpR) in sulfate-reducing bacteria that controls expression of the hcp gene, which encodes the hybrid cluster protein and contributes to nitrosative stress responses. There is only one hcpR gene in the model sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough, but two copies in Desulfovibrio desulfuricans 27774, which can use nitrate as an alternative electron acceptor to sulfate. Structures of the D. desulfuricans hcpR1, hcpR2 and hcp operons are reported. We present evidence that hcp expression is regulated by HcpR2, not by HcpR1, and that these two regulators differ in both their DNA-binding site specificity and their sensory domains. HcpR1 is predicted to be a b-type cytochrome. HcpR1 binds upstream of the hcpR1 operon and its synthesis is regulated coordinately with hcp in response to NO. In contrast, hcpR2 expression was not induced by nitrate, nitrite or NO. HcpR2 is an iron-sulfur protein that reacts with NO and O2 . We propose that HcpR1 and HcpR2 use different sensory mechanisms to regulate subsets of genes required for defense against NO-induced nitrosative stress, and that diversification of signal perception and DNA recognition by these two proteins is a product of D. desulfuricans adaptation to its particular environmental niche.

Regulation of the Escherichia coli ydhY-T operon in the presence of alternative electron acceptors.

The Escherichia coli K-12 ydhY-T operon, coding for a predicted oxidoreductase complex, is activated under anaerobic conditions and repressed in the presence of nitrate or nitrite. Anaerobic activation is mediated by the transcription factor FNR, and nitrate/nitrite repression is mediated by NarXL and NarQP. In vitro transcription reactions revealed that the DNA upstream of ydhY-T contains sufficient information for RNA polymerase alone to initiate transcription from five locations. FNR severely inhibited synthesis of two of these transcripts (located upstream of, and within, the FNR binding site) and activated the FNR-dependent promoter previously identified in vivo. Enhanced expression of ydhY-T in an hns mutant was consistent with the location of ydhY-T within a promoter island and the FNR-independent transcription observed in vitro. FNR-dependent transcription in vitro was decreased in the presence of NarL~P. DNaseI footprinting indicated that FNR and NarL~P simultaneously bound at the ydhY-T promoter region and that NarL~P-mediated repression was due to occupation of the 7-2-7 site located downstream of the FNR-dependent promoter. Expression of ydhY-T during the anaerobic growth cycle was repressed when nitrate was present but less so in the presence of nitrite. In vivo transcription measurements indicated that the alternative electron acceptors, DMSO and fumarate, could also lower ydhY-T expression, whereas trimethylamine-N-oxide (TMAO) permitted high expression. Therefore, expression of ydhY-T is subject to complex regulation in response to electron acceptor availability that involves at least three transcription factors, FNR (anaerobic activation), NarL~P (nitrate repression) and H-NS (repression in the absence of an antagonist; e.g. FNR).

Identification of new members of the Escherichia coli K-12 MG1655 SlyA regulon.

SlyA is a member of the MarR family of bacterial transcriptional regulators. Previously, SlyA has been shown to directly regulate only two operons in Escherichia coli K-12 MG1655, fimB and hlyE (clyA). In both cases, SlyA activates gene expression by antagonizing repression by the nucleoid-associated protein H-NS. Here, the transcript profiles of aerobic glucose-limited steady-state chemostat cultures of E. coli K-12 MG1655, slyA mutant and slyA over-expression strains are reported. The transcript profile of the slyA mutant was not significantly different from that of the parent; however, that of the slyA expression strain was significantly different from that of the vector control. Transcripts representing 27 operons were increased in abundance, whereas 3 were decreased. Of the 30 differentially regulated operons, 24 have previously been associated with sites of H-NS binding, suggesting that antagonism of H-NS repression is a common feature of SlyA-mediated transcription regulation. Direct binding of SlyA to DNA located upstream of a selection of these targets permitted the identification of new operons likely to be directly regulated by SlyA. Transcripts of four operons coding for cryptic adhesins exhibited enhanced expression, and this was consistent with enhanced biofilm formation associated with the SlyA over-producing strain.

Systematic Nomenclature for GGDEF and EAL Domain-Containing Cyclic Di-GMP Turnover Proteins of Escherichia coli.

In recent years, Escherichia coli has served as one of a few model bacterial species for studying cyclic di-GMP (c-di-GMP) signaling. The widely used E. coli K-12 laboratory strains possess 29 genes encoding proteins with GGDEF and/or EAL domains, which include 12 diguanylate cyclases (DGC), 13 c-di-GMP-specific phosphodiesterases (PDE), and 4 "degenerate" enzymatically inactive proteins. In addition, six new GGDEF and EAL (GGDEF/EAL) domain-encoding genes, which encode two DGCs and four PDEs, have recently been found in genomic analyses of commensal and pathogenic E. coli strains. As a group of researchers who have been studying the molecular mechanisms and the genomic basis of c-di-GMP signaling in E. coli, we now propose a general and systematic dgc and pde nomenclature for the enzymatically active GGDEF/EAL domain-encoding genes of this model species. This nomenclature is intuitive and easy to memorize, and it can also be applied to additional genes and proteins that might be discovered in various strains of E. coli in future studies.

Three Pseudomonas putida FNR Family Proteins with Different Sensitivities to O2.

The Escherichia coli fumarate-nitrate reduction regulator (FNR) protein is the paradigm for bacterial O2-sensing transcription factors. However, unlike E. coli, some bacterial species possess multiple FNR proteins that presumably have evolved to fulfill distinct roles. Here, three FNR proteins (ANR, PP_3233, and PP_3287) from a single bacterial species, Pseudomonas putida KT2440, have been analyzed. Under anaerobic conditions, all three proteins had spectral properties resembling those of [4Fe-4S] proteins. The reactivity of the ANR [4Fe-4S] cluster with O2 was similar to that of E. coli FNR, and during conversion to the apo-protein, via a [2Fe-2S] intermediate, cluster sulfur was retained. Like ANR, reconstituted PP_3233 and PP_3287 were converted to [2Fe-2S] forms when exposed to O2, but their [4Fe-4S] clusters reacted more slowly. Transcription from an FNR-dependent promoter with a consensus FNR-binding site in P. putida and E. coli strains expressing only one FNR protein was consistent with the in vitro responses to O2. Taken together, the experimental results suggest that the local environments of the iron-sulfur clusters in the different P. putida FNR proteins influence their reactivity with O2, such that ANR resembles E. coli FNR and is highly responsive to low concentrations of O2, whereas PP_3233 and PP_3287 have evolved to be less sensitive to O2.