Search

Many natural products comprise N-O containing functional groups with crucial roles for biological activity. Their enzymatic formation is predominantly achieved by oxidation of an amine to form a hydroxylamine, which enables further functionalization. N-hydroxylation by flavin-dependent enzymes has so far been attributed to a distinct group of flavoprotein monooxygenases (FPMOs) containing two dinucleotide binding domains. Here, we present three flavoprotein N-hydroxylases that exhibit a glutathione reductase 2 (GR2)-type topology with only one nucleotide binding domain, which belong to a distinct phylogenetic branch within the GR2-fold FPMOs. In addition to PqsL of Pseudomonas aeruginosa, which catalyses the N-hydroxylation of a primary aromatic amine during biosynthesis of 2-alkyl-4-hydroxyquinoline N-oxide respiratory chain inhibitors, we analysed isofunctional orthologs from Burkholderia thailandensis (HmqL) and Chryseobacterium nematophagum (PqsL

The opportunistic pathogen Pseudomonas aeruginosa can utilize unusual carbon sources, like sodium dodecyl sulfate (SDS) and alkanes. Whereas the initiating enzymatic steps of the corresponding degradation pathways have been characterized in detail, the oxidation of the emerging long-chain alcohols has received little attention. Recently, the genes for the Lao ( l ong-chain- a lcohol/ a ldehyde o xidation) system were discovered to be involved in the oxidation of long-chain alcohols derived from SDS and alkane degradation. In the Lao system, LaoA is predicted to be an alcohol dehydrogenase/oxidase; however, according to genetic studies, efficient long-chain-alcohol oxidation additionally required the Tat-dependent protein LaoB. In the present study, the Lao system was further characterized. In vivo analysis revealed that the Lao system complements the substrate spectrum of the well-described Exa system, which is required for growth with ethanol and other short-chain alcohols. Mutational analysis revealed that the Tat site of LaoB was required for long-chain-alcohol oxidation activity, strongly suggesting a periplasmic localization of the complex. Purified LaoA was fully active only when copurified with LaoB. Interestingly, in vitro activity of the purified LaoAB complex also depended on the presence of the Tat site. The copurified LaoAB complex contained a flavin cofactor and preferentially oxidized a range of saturated, unbranched primary alcohols. Furthermore, the LaoAB complex could reduce cytochrome c550-type redox carriers like ExaB, a subunit of the Exa alcohol dehydrogenase system. LaoAB complex activity was stimulated by rhamnolipids in vitro. In summary, LaoAB constitutes an unprecedented protein complex with specific properties apparently required for oxidizing long-chain alcohols. IMPORTANCE Pseudomonas aeruginosa is a major threat to public health. Its ability to thrive in clinical settings, water distribution systems, or even jet fuel tanks is linked to detoxification and degradation of diverse hydrophobic substrates that are metabolized via alcohol intermediates. Our study illustrates a novel flavoprotein long-chain-alcohol dehydrogenase consisting of a facultative two-subunit complex, which is unique among related enzymes, while the homologs of the corresponding genes are found in numerous bacterial genomes. Understanding the catalytic and compartmentalization processes involved is of great interest for biotechnological and hygiene research, as it may be a potential starting point for rationally designing novel antibacterial substances with high specificity against this opportunistic pathogen.

The multiple biological activities of 2-alkylquinolones (AQs) are crucial for virulence of Pseudomonas aeruginosa, conferring advantages during infection and in polymicrobial communities. Whereas 2-heptyl-3-hydroxyquinolin-4(1H)-one (the “Pseudomonas quinolone signal” [PQS]) is an important quorum sensing signal molecule, 2-alkyl-1-hydroxyquinolin-4(1H)-ones (also known as 2-alkyl-4-hydroxyquinoline N-oxides [AQNOs]) are antibiotics inhibiting respiration. Hydroxylation of the PQS precursor 2-heptylquinolin-4(1H)-one (HHQ) by the signal synthase PqsH boosts AQ quorum sensing. Remarkably, the same reaction, catalyzed by the ortholog AqdB, is used by Mycobacteroides abscessus to initiate degradation of AQs. The antibiotic 2-heptyl-1-hydroxyquinolin-4(1H)-one (HQNO) is hydroxylated by Staphylococcus aureus to the less toxic derivative PQS-N-oxide (PQS-NO), a reaction probably also catalyzed by a PqsH/AqdB ortholog. In this study, we provide a comparative analysis of four AQ 3-monooxygenases of different organisms. Due to the major impact of AQ/AQNO 3-hydroxylation on the biological activities of the compounds, we surmised adaptations on the enzymatic and/or physiological level to serve either the producer or target organisms. Our results indicate that all enzymes share similar features and are incapable of discriminating between AQs and AQNOs. PQS-NO, hence, occurs as a native metabolite of P. aeruginosa although the unfavorable AQNO 3-hydroxylation is minimized by export as shown for HQNO, involving at least one multidrug efflux pump. Moreover, M. abscessus is capable of degrading the AQNO heterocycle by concerted action of AqdB and dioxygenase AqdC. However, S. aureus and M. abscessus orthologs disfavor AQNOs despite their higher toxicity, suggesting that catalytic constraints restrict evolutionary adaptation and lead to the preference of non-N-oxide substrates by AQ 3-monooxygenases. IMPORTANCEPseudomonas aeruginosa, Staphylococcus aureus, and Mycobacteroides abscessus are major players in bacterial chronic infections and particularly common colonizers of cystic fibrosis (CF) lung tissue. Whereas S. aureus is an early onset pathogen in CF, P. aeruginosa establishes at later stages. M. abscessus occurs at all stages but has a lower epidemiological incidence. The dynamics of how these pathogens interact can affect survival and therapeutic success. 2-Alkylquinolone (AQ) and 2-alkylhydroxyquinoline N-oxide (AQNO) production is a major factor of P. aeruginosa virulence. The 3-position of the AQ scaffold is critical, both for attenuation of AQ toxicity or degradation by competitors, as well as for full unfolding of quorum sensing. Despite lacking signaling functionality, AQNOs have the strongest impact on suppression of Gram-positives. Because evidence for 3-hydroxylation of AQNOs has been reported, it is desirable to understand the extent by which AQ 3-monooxygenases contribute to manipulation of AQ/AQNO equilibrium, resistance, and degradation.

Light-dependent or light-stimulated catalysis provides a multitude of perspectives for implementation in technological or biomedical applications. Despite substantial progress made in the field of photobiocatalysis, the number of usable light-responsive enzymes is still very limited. Flavoproteins have exceptional potential for photocatalytic applications because the name-giving cofactor intrinsically features light-dependent reactivity, undergoing photoreduction with a variety of organic electron donors. However, in the vast majority of these enzymes, photoreactivity of the enzyme-bound flavin is limited or even suppressed. Here, we present a flavoprotein monooxygenase in which catalytic activity is controllable by blue light illumination. The reaction depends on the presence of nicotinamide nucleotide-type electron donors, which do not support the reaction in the absence of light. Employing various experimental approaches, we demonstrate that catalysis depends on a protein-mediated photoreduction of the flavin cofactor, which proceeds via a radical mechanism and a transient semiquinone intermediate., The number of usable light-responsive enzymes is limited, despite the potential biotechnological applications. Here, the authors report a flavoprotein monooxygenase which is controllable by blue light illumination, and propose a mechanism involving protein-mediated radical photoreduction of FAD via a semiquinone intermediate.

Pseudomonas aeruginosa is an important nosocomial pathogen that is frequently recalcitrant to available antibiotics, underlining the urgent need for alternative therapeutic options against this pathogen. Targeting virulence functions is a promising alternative strategy as it is expected to generate less-selective resistance to treatment compared to antibiotics. Capitalizing on our nonligand-based benzamide-benzimidazole (BB) core structure compounds reported to efficiently block the activity of the P. aeruginosa multiple virulence factor regulator MvfR, here we report the first class of inhibitors shown to interfere with PqsBC enzyme activity, responsible for the synthesis of the MvfR activating ligands HHQ and PQS, and the first to target simultaneously MvfR and PqsBC activity. The use of these compounds reveals that inhibiting PqsBC is sufficient to block P. aeruginosa's acute virulence functions, as the synthesis of MvfR ligands is inhibited. Our results show that MvfR remains the best target of this QS pathway, as we show that antagonists of this target block both acute and persistence-related functions. The structural properties of the compounds reported in this study provide several insights that are instrumental for the design of improved MvfR regulon inhibitors against both acute and persistent P. aeruginosa infections. Moreover, the data presented offer the possibility of a polypharmacology approach of simultaneous silencing two targets in the same pathway. Such a combined antivirulence strategy holds promise in increasing therapeutic efficacy and providing alternatives in the event of a single target's resistance development.

The nosocomial pathogen Pseudomonas aeruginosa regulates its virulence via a complex quorum sensing network, which, besides N-acylhomoserine lactones, includes the alkylquinolone signal molecules 2-heptyl-3-hydroxy-4(1H)-quinolone (Pseudomonas quinolone signal [PQS]) and 2-heptyl-4(1H)-quinolone (HHQ). Mycobacteroides abscessus subsp. abscessus, an emerging pathogen, is capable of degrading the PQS and also HHQ. Here, we show that although M. abscessus subsp. abscessus reduced PQS levels in coculture with P. aeruginosa PAO1, this did not suffice for quenching the production of the virulence factors pyocyanin, pyoverdine, and rhamnolipids. However, the levels of these virulence factors were reduced in cocultures of P. aeruginosa PAO1 with recombinant M. abscessus subsp. massiliense overexpressing the PQS dioxygenase gene aqdC of M. abscessus subsp. abscessus, corroborating the potential of AqdC as a quorum quenching enzyme. When added extracellularly to P. aeruginosa cultures, AqdC quenched alkylquinolone and pyocyanin production but induced an increase in elastase levels. When supplementing P. aeruginosa cultures with QsdA, an enzyme from Rhodococcus erythropolis which inactivates N-acylhomoserine lactone signals, rhamnolipid and elastase levels were quenched, but HHQ and pyocyanin synthesis was promoted. Thus, single quorum quenching enzymes, targeting individual circuits within a complex quorum sensing network, may also elicit undesirable regulatory effects. Supernatants of P. aeruginosa cultures grown in the presence of AqdC, QsdA, or both enzymes were less cytotoxic to human epithelial lung cells than supernatants of untreated cultures. Furthermore, the combination of both aqdC and qsdA in P. aeruginosa resulted in a decline of Caenorhabditis elegans mortality under P. aeruginosa exposure.

Alkyl quinolones (AQs) are multifunctional bacterial secondary metabolites generally known for their antibacterial and algicidal properties. Certain representatives are also employed as signalling molecules of Burkholderia strains and Pseudomonas aeruginosa. The marine Gammaproteobacterium Microbulbifer sp. HZ11 harbours an AQ biosynthetic gene cluster with unusual topology but does not produce any AQ-type metabolites under laboratory conditions. In this study, we demonstrate the potential of strain HZ11 for AQ production by analysing intermediates and key enzymes of the pathway. Moreover, we demonstrate that exogenously added AQs such as 2-heptyl-1(H)-quinolin-4-one (referred to as HHQ) or 2-heptyl-1-hydroxyquinolin-4-one (referred to as HQNO) are brominated by a vanadium-dependent haloperoxidase (V-HPO HZ11), which preferably is active towards AQs with C5–C9 alkyl side chains. Bromination was specific for the third position and led to 3-bromo-2-heptyl-1(H)-quinolin-4-one (BrHHQ) and 3-bromo-2-heptyl-1-hydroxyquinolin-4-one (BrHQNO), both of which were less toxic for strain HZ11 than the respective parental compounds. In contrast, BrHQNO showed increased antibiotic activity against Staphylococcus aureus and marine isolates. Therefore, bromination of AQs by V-HPO HZ11 can have divergent consequences, eliciting a detoxifying effect for strain HZ11 while simultaneously enhancing antibiotic activity against other bacteria.

The nosocomial pathogen Pseudomonas aeruginosa regulates its virulence via a complex quorum sensing network, which, besides N-acylhomoserine lactones, includes the alkylquinolone signal molecules 2-heptyl-3-hydroxy-4(1H)-quinolone (Pseudomonas quinolone signal [PQS]) and 2-heptyl-4(1H)-quinolone (HHQ). Mycobacteroides abscessus subsp. abscessus, an emerging pathogen, is capable of degrading the PQS and also HHQ. Here, we show that although M. abscessus subsp. abscessus reduced PQS levels in coculture with P. aeruginosa PAO1, this did not suffice for quenching the production of the virulence factors pyocyanin, pyoverdine, and rhamnolipids. However, the levels of these virulence factors were reduced in cocultures of P. aeruginosa PAO1 with recombinant M. abscessus subsp. massiliense overexpressing the PQS dioxygenase gene aqdC of M. abscessus subsp. abscessus, corroborating the potential of AqdC as a quorum quenching enzyme. When added extracellularly to P. aeruginosa cultures, AqdC quenched alkylquinolone and pyocyanin production but induced an increase in elastase levels. When supplementing P. aeruginosa cultures with QsdA, an enzyme from Rhodococcus erythropolis which inactivates N-acylhomoserine lactone signals, rhamnolipid and elastase levels were quenched, but HHQ and pyocyanin synthesis was promoted. Thus, single quorum quenching enzymes, targeting individual circuits within a complex quorum sensing network, may also elicit undesirable regulatory effects. Supernatants of P. aeruginosa cultures grown in the presence of AqdC, QsdA, or both enzymes were less cytotoxic to human epithelial lung cells than supernatants of untreated cultures. Furthermore, the combination of both aqdC and qsdA in P. aeruginosa resulted in a decline of Caenorhabditis elegans mortality under P. aeruginosa exposure.

Bile salts such as cholate are surface-active steroid compounds with functions for digestion and signaling in vertebrates. Upon excretion into soil and water bile salts are an electron- and carbon-rich growth substrate for environmental bacteria. Degradation of bile salts proceeds via intermediates with a 3-keto-Δ1,4-diene structure of the steroid skeleton as shown for e.g. Pseudomonas spp. Recently, we isolated bacteria degrading cholate via intermediates with a 3-keto-7-deoxy-Δ4,6-structure of the steroid skeleton suggesting the existence of a second pathway for cholate degradation. This potential new pathway was investigated with Novosphingobium sp. strain Chol11. A 7α-hydroxysteroid dehydratase encoded by hsh2 was identified, which was required for the formation of 3-keto-7-deoxy-Δ4,6-metabolites. A hsh2 deletion mutant could still grow with cholate but showed impaired growth. Cholate degradation of this mutant proceeded via 3-keto-Δ1,4-diene metabolites. Heterologous expression of Hsh2 in the bile salt-degrading Pseudomonas sp. strain Chol1 led to formation of a dead-end steroid with a 3-keto-7-deoxy-Δ4,6-diene structure. Hsh2 is the first steroid dehydratase with an important function in a metabolic pathway of bacteria that use bile salts as growth substrates. This pathway contributes to a broad metabolic repertoire of Novosphingobium strain Chol11 that may be advantageous in competition with other bile salt-degrading bacteria. This article is protected by copyright. All rights reserved.

Pseudomonas aeruginosa uses quorum sensing (QS) as a cell-to-cell communication system to orchestrate the expression of virulence determinants. The biosynthesis of the important Pseudomonas quinolone signal (PQS) requires the pqsABCDE operon. Here, PqsE acts as a pathway-specific thioesterase, but it also contributes to the regulation of bacterial virulence via an unknown mechanism. In this manuscript, we report the discovery of PqsE inhibitors as tool compounds to gain further insights into its different functions. Differential scanning fluorimetry (DSF) was used to screen a fragment library, and isothermal titration calorimetry (ITC) was employed as a secondary filter. As proven by X-ray crystallography, hit molecules bound to the active center inhibiting PqsE's thioesterase activity in cell-based and in vitro assays. Notably, the ligands did not affect the levels of the PqsE-regulated virulence factor pyocyanin. These findings indicate that the regulatory function of PqsE is not linked to its thioesterase activity and must be encoded outside of the active center. This study highlights the potential of fragment-based screening for the discovery of tool compounds. This approach provided novel insight into complex biological systems, which could not be obtained by knockout studies.

Pseudomonas aeruginosa produces a number of alkylquinolone-type secondary metabolites best known for their antimicrobial effects and involvement in cell-cell communication. In the alkylquinolone biosynthetic pathway, the β-ketoacyl-(acyl carrier protein) synthase III (FabH)-like enzyme PqsBC catalyzes the condensation of octanoyl-coenzyme A and 2-aminobenzoylacetate (2-ABA) to form the signal molecule 2-heptyl-4(1H)-quinolone. PqsBC, a potential drug target, is unique for its heterodimeric arrangement and an active site different from that of canonical FabH-like enzymes. Considering the sequence dissimilarity between the subunits, a key question was how the two subunits are organized with respect to the active site. In this study, the PqsBC structure was determined to a 2 A resolution, revealing that PqsB and PqsC have a pseudo-2-fold symmetry that unexpectedly mimics the FabH homodimer. PqsC has an active site composed of Cys-129 and His-269, and the surrounding active site cleft is hydrophobic in character and approximately twice the volume of related FabH enzymes that may be a requirement to accommodate the aromatic substrate 2-ABA. From physiological and kinetic studies, we identified 2-aminoacetophenone as a pathway-inherent competitive inhibitor of PqsBC, whose fluorescence properties could be used for in vitro binding studies. In a time-resolved setup, we demonstrated that the catalytic histidine is not involved in acyl-enzyme formation, but contributes to an acylation-dependent increase in affinity for the second substrate 2-ABA. Introduction of Asn into the PqsC active site led to significant activity toward the desamino substrate analog benzoylacetate, suggesting that the substrate 2-ABA itself supplies the asparagine-equivalent amino function that assists in catalysis.

Alkyl hydroxyquinoline N-oxides (AQNOs) are antibiotic compounds produced by the opportunistic bacterial pathogen Pseudomonas aeruginosa. They are products of the alkyl quinolone (AQ) biosynthetic pathway, which also generates the quorum-sensing molecules 2-heptyl-4(1H)-quinolone (HHQ) and 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS). Although the enzymatic synthesis of HHQ and PQS had been elucidated, the route by which AQNOs are synthesized remained elusive. Here, we report on PqsL, the key enzyme for AQNO production, which structurally resembles class A flavoprotein monooxygenases such as p-hydroxybenzoate 3-hydroxylase (pHBH) and 3-hydroxybenzoate 6-hydroxylase. However, we found that unlike related enzymes, PqsL hydroxylates a primary aromatic amine group, and it does not use NAD(P)H as cosubstrate, but unexpectedly required reduced flavin as electron donor. We also observed that PqsL is active toward 2-aminobenzoylacetate (2-ABA), the central intermediate of the AQ pathway, and forms the unstable compound 2-hydroxylaminobenzoylacetate, which was preferred over 2-ABA as substrate of the downstream enzyme PqsBC. In vitro reconstitution of the PqsL/PqsBC reaction was feasible by using the FAD reductase HpaC, and we noted that the AQ:AQNO ratio is increased in an hpaC-deletion mutant of P. aeruginosa PAO1 compared with the ratio in the WT strain. A structural comparison with pHBH, the model enzyme of class A flavoprotein monooxygenases, revealed that structural features associated with NAD(P)H binding are missing in PqsL. Our study completes the AQNO biosynthetic pathway in P. aeruginosa, indicating that PqsL produces the unstable product 2-hydroxylaminobenzoylacetate from 2-ABA and depends on free reduced flavin as electron donor instead of NAD(P)H.

P1 B -ATPases are among the most common resistance factors to metal-induced stress. Belonging to the superfamily of P-type ATPases, they are capable of exporting transition metal ions at the expense of adenosine triphosphate (ATP) hydrolysis. P1 B -ATPases share a conserved structure of three cytoplasmic domains linked by a transmembrane domain. In addition, they possess a unique class of domains located at the N-terminus. In bacteria, these domains are primarily associated with metal binding and either occur individually or as serial copies of each other. Within this study, the roles of the two adjacent metal-binding domains (MBDs) of CopA, the copper export ATPase of Escherichia coli were investigated. From biochemical and physiological data, we deciphered the protein-internal pathway of copper and demonstrate the distal N-terminal MBD to possess a function analogous to the metallochaperones of related prokaryotic copper resistance systems, that is its involvement in the copper transfer to the membrane-integral ion-binding sites of CopA. In contrast, the proximal domain MBD2 has a regulatory role by suppressing the catalytic activity of CopA in absence of copper. Furthermore, we propose a general functional divergence of tandem MBDs in P1 B -ATPases, which is governed by the length of the inter-domain linker.

Summary Pseudomonas aeruginosa uses the alkylquinolones PQS (2-heptyl-3-hydroxy-4(1 H )-quinolone) and HHQ (2-heptyl-4(1 H )-quinolone) as quorum-sensing signal molecules, controlling the expression of many virulence genes as a function of cell population density. The biosynthesis of HHQ is generally accepted to require the pqsABCD gene products. We now reconstitute the biosynthetic pathway in vitro, and demonstrate that in addition to PqsABCD, PqsE has a role in HHQ synthesis. PqsE acts as thioesterase, hydrolyzing the biosynthetic intermediate 2-aminobenzoylacetyl-coenzyme A to form 2-aminobenzoylacetate, the precursor of HHQ and 2-aminoacetophenone. The role of PqsE can be taken over to some extent by the broad-specificity thioesterase TesB, explaining why the pqsE deletion mutant of P. aeruginosa still synthesizes HHQ. Interestingly, the pqsE mutant produces increased levels of 2,4-dihydroxyquinoline, resulting from intramolecular cyclization of 2-aminobenzoylacetyl-coenzyme A. Overall, our data suggest that PqsE promotes the efficiency of alkylquinolone signal molecule biosynthesis in P. aeruginosa and balances the levels of secondary metabolites deriving from the alkylquinolone biosynthetic pathway.

Programmed ribosomal frameshifting (PRF) is a translational anomaly causing the ribosome to shift into an alternative reading frame. PRFs are common in viral genomes, using a single nucleotide sequence to code for two proteins in overlapping frames. In bacteria and eukaryota, PRFs are less frequent. We report on a PRF in the copper detoxification system of Escherichia coli where a metallochaperone is generated out of the first 69 amino acids and a C-terminal out-of-frame glycine of the gene copA. copA besides codes for the P1B -ATPase CopA, a membrane-integral protein and principal interaction target of the chaperone. To enhance the production of the frameshift-generated cytosolic copper binding protein a truncated transcript is produced from the monocistronic copA gene. This shorter transcript is essential for producing sufficient amounts of the chaperone to support the membrane pump. The findings close the gap in our understanding of the molecular physiology of cytoplasmic copper transport in E. coli, revealing that a chaperone-like entity is required for full functionality of the P1B -ATPase copper pump. We, moreover, demonstrate that the primary transcriptional response to copper results in formation of the small transcript and concurrently, the metallochaperone plays a key role in resistance against copper shock.

Certain heavy metal ions such as copper and zinc serve as essential cofactors of many enzymes, but are toxic at high concentrations. Thus, intracellular levels have to be subtly balanced. P-type ATPases of the P(IB)-subclass play a major role in metal homeostasis. The thermoacidophile Sulfolobus solfataricus possesses two P(IB)-ATPases named CopA and CopB. Both enzymes are present in cells grown in copper-depleted medium and are accumulated upon an increase in the external copper concentration. We studied the physiological roles of both ATPases by disrupting genes copA and copB. Neither of them affected the sensitivity of S. solfataricus to reactive oxygen species, nor were they a strict prerequisite to the biosynthesis of the copper protein cytochrome oxidase. Deletion mutant analysis demonstrated that CopA is an effective copper pump at low and high copper concentrations. CopB appeared to be a low-affinity copper export ATPase, which was only relevant if the media copper concentration was exceedingly high. CopA and CopB thus act as resistance factors to copper ions at overlapping concentrations. Moreover, growth tests on solid media indicated that both ATPases are involved in resistance to silver.

P1 B -ATPases are among the most common resistance factors to metal-induced stress. Belonging to the superfamily of P-type ATPases, they are capable of exporting transition metal ions at the expense of adenosine triphosphate (ATP) hydrolysis. P1 B -ATPases share a conserved structure of three cytoplasmic domains linked by a transmembrane domain. In addition, they possess a unique class of domains located at the N-terminus. In bacteria, these domains are primarily associated with metal binding and either occur individually or as serial copies of each other. Within this study, the roles of the two adjacent metal-binding domains (MBDs) of CopA, the copper export ATPase of Escherichia coli were investigated. From biochemical and physiological data, we deciphered the protein-internal pathway of copper and demonstrate the distal N-terminal MBD to possess a function analogous to the metallochaperones of related prokaryotic copper resistance systems, that is its involvement in the copper transfer to the membrane-integral ion-binding sites of CopA. In contrast, the proximal domain MBD2 has a regulatory role by suppressing the catalytic activity of CopA in absence of copper. Furthermore, we propose a general functional divergence of tandem MBDs in P1 B -ATPases, which is governed by the length of the inter-domain linker.

The study by Dulcey and colleagues in this issue of Chemistry & Biology changes our perception of the pathway of 2-alkyl-4-hydroxyquinoline biosynthesis by the opportunistic pathogen Pseudomonas aeruginosa and suggests that the biosynthetic protein complex PqsBC is a potential antibacterial target.

The extraction of disease specific information from Fourier transform infrared (FTIR) spectra of human body fluids demands the highest standards of accuracy and reproducibility of measurements because the expected spectral differences between healthy and diseased subjects are very small in relation to a large background absorbance of the whole sample. Here, we demonstrate that with the increased sensitivity of modern FTIR spectrometers, automatisation of sample preparation and modern bioinformatics, it is possible to identify and validate spectral biomarker candidates for distinguishing between urinary bladder cancer (UBC) and inflammation in suspected bladder cancer patients. The current dataset contains spectra of blood serum and plasma samples of 135 patients. All patients underwent cytology and pathological biopsy characterization to distinguish between patients without UBC (46) and confirmed UBC cases (89). A minimally invasive blood test could spare control patients a repeated cystoscopy including a transurethral biopsy, and three-day stationary hospitalisation. Blood serum, EDTA and citrate plasma were collected from each patient and processed following predefined strict standard operating procedures. Highly reproducible dry films were obtained by spotting sub-nanoliter biofluid droplets in defined patterns, which were compared and optimized. Particular attention was paid to the automatisation of sample preparation and spectral preprocessing to exclude errors by manual handling. Spectral biomarker candidates were identified from absorbance spectra and their 1(st) and 2(nd) derivative spectra using an advanced Random Forest (RF) approach. It turned out that the 2(nd) derivative spectra were most useful for classification. Repeat validation on 21% of the dataset not included in predictor training with Linear Discriminant Analysis (LDA) classifiers and Random Forests (RFs) yielded a sensitivity of 93 ± 10% and a specificity of 46 ± 18% for bladder cancer. The low specificity can be most likely attributed to the unbalanced and small number of control samples. Using this approach, spectral biomarker candidates in blood-derived biofluids were identified, which allow us to distinguish between cancer and inflammation, but the observed differences were tiny. Obviously, a much larger sample number has to be investigated to reliably validate such candidates.