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Prior to the world-wide dominance of F. ×ananassa in strawberry production, native species had been cultivated in European gardens for centuries. Especially the musk strawberry (F. moschata) had been highly appreciated due to its fruit size and extraordinary aroma. Detailed studies on the diversity of the species' fruit aroma are lacking, although breeding aims to improve strawberry aroma by complex crossings during recent years. Today a few cultivars, abandoned synanthropic occurrences and native populations of this species exist in Germany. Here we characterised aroma profiles of F. moschata accessions by analysing Volatile Organic Compounds. In particular, differences between native and cultivated accessions as well as the diversity in aroma profiles of native populations were investigated. Profiles of Volatile Organic Compounds were analysed by immersion stir bar sorptive extraction-gas chromatography-quadrupol mass spectrometry (imm-SBSE-GC-qMS). These data were compared with a genetic characterisation of samples based on eight microsatellite loci using univariate and multivariate statistical analyses. High amounts of furanones and the key compound methyl anthranilate were characteristic for the aroma profile of F. moschata. We detected a considerable diversity of Volatile Organic Compounds among accessions of F. moschata, particularly among genetically distinct samples from the same population. Native accessions contained more terpenoids but less esters and were moderately differentiated from cultivated samples. The observed patterns of Volatile Organic Compounds indicate that cultivated accessions had been selected for favourable aroma profiles and thus showing traces of domestication. Moreover, native populations harbour a great diversity of Volatile Organic Compounds, which could be also considered for future breeding efforts., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Ritz et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Adenosine triphosphate (ATP)-binding cassette (ABC) transporters are multidomain transmembrane proteins, which facilitate the transport of various substances across cell membranes using energy derived from ATP hydrolysis. They are important drug targets since they mediate decreased drug susceptibility during pharmacological treatments. For the methylotrophic yeast Pichia pastoris , a model organism that is a widely used host for protein expression, the role and function of its ABC transporters is unexplored. In this work, we investigated the Pichia ABC-B transporter STE6-2p. Functional investigations revealed that STE6-2p is capable of transporting rhodamines in vivo and is active in the presence of verapamil and triazoles in vitro. A phylogenetic analysis displays homology among multidrug resistance (MDR) transporters from pathogenic fungi to human ABC-B transporters. Further, we present high-resolution single-particle electron cryomicroscopy structures of an ABC transporter from P. pastoris in the apo conformation (3.1 Å) and in complex with verapamil and adenylyl imidodiphosphate (AMP-PNP) (3.2 Å). An unknown density between transmembrane helices 4, 5, and 6 in both structures suggests the presence of a sterol-binding site of unknown function.

The treatment of infectious diseases caused by multidrug-resistant pathogens is a major clinical challenge of the 21st century. The membrane-embedded respiratory cytochrome bd -type oxygen reductase is a critical survival factor utilized by pathogenic bacteria during infection, proliferation and the transition from acute to chronic states. Escherichia coli encodes for two cytochrome bd isoforms that are both involved in respiration under oxygen limited conditions. Mechanistic and structural differences between cydABX ( Ecbd-I ) and appCBX ( Ecbd-II ) operon encoded cytochrome bd variants have remained elusive in the past. Here, we demonstrate that cytochrome bd - II catalyzes oxidation of benzoquinols while possessing additional specificity for naphthoquinones. Our data show that although menaquinol-1 (MK1) is not able to directly transfer electrons onto cytochrome bd-II from E. coli , it has a stimulatory effect on its oxygen reduction rate in the presence of ubiquinol-1. We further determined cryo-EM structures of cytochrome bd - II to high resolution of 2.1 Å. Our structural insights confirm that the general architecture and substrate accessible pathways are conserved between the two bd oxidase isoforms, but two notable differences are apparent upon inspection: (i) Ecbd-II does not contain a CydH-like subunit, thereby exposing heme b 595 to the membrane environment and (ii) the AppB subunit harbors a structural demethylmenaquinone-8 molecule instead of ubiquinone-8 as found in CydB of Ecbd-I Our work completes the structural landscape of terminal respiratory oxygen reductases of E. coli and suggests that structural and functional properties of the respective oxidases are linked to quinol-pool dependent metabolic adaptations in E. coli ., Competing Interests: The authors declare no competing interest.

Plant genomes consist, to a considerable extent, of non-coding repetitive DNA. Several studies showed that phylogenetic signals can be extracted from such repeatome data by using among-species dissimilarities from the RepeatExplorer2 pipeline as distance measures. Here, we advanced this approach by adjusting the read input for comparative clustering indirectly proportional to genome size and by summarizing all clusters into a main distance matrix subjected to Neighbor Joining algorithms and Principal Coordinate Analyses. Thus, our multivariate statistical method works as a "repeatomic fingerprint," and we proved its power and limitations by exemplarily applying it to the family Rosaceae at intrafamilial and, in the genera Fragaria and Rosa , at the intrageneric level. Since both taxa are prone to hybridization events, we wanted to show whether repeatome data are suitable to unravel the origin of natural and synthetic hybrids. In addition, we compared the results based on complete repeatomes with those from ribosomal DNA clusters only, because they represent one of the most widely used barcoding markers. Our results demonstrated that repeatome data contained a clear phylogenetic signal supporting the current subfamilial classification within Rosaceae . Accordingly, the well-accepted major evolutionary lineages within Fragaria were distinguished, and hybrids showed intermediate positions between parental species in data sets retrieved from both complete repeatomes and rDNA clusters. Within the taxonomically more complicated and particularly frequently hybridizing genus Rosa , we detected rather weak phylogenetic signals but surprisingly found a geographic pattern at a population scale. In sum, our method revealed promising results at larger taxonomic scales as well as within taxa with manageable levels of reticulation, but success remained rather taxon specific. Since repeatomes can be technically easy and comparably inexpensively retrieved even from samples of rather poor DNA quality, our phylogenomic method serves as a valuable alternative when high-quality genomes are unavailable, for example, in the case of old museum specimens., Competing Interests: KO is employed by Hansabred GmbH & Co. KG, Dresden, Germany. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Herklotz, Kovařík, Wissemann, Lunerová, Vozárová, Buschmann, Olbricht, Groth and Ritz.)

The Cbb 3 -type cytochrome c oxidases (Cbb 3 -CcOs), the second most abundant CcOs, catalyze the reduction of molecular oxygen to water, even at micromolar oxygen concentrations. In Pseudomonas stutzeri ZoBell, two tandemly organized cbb 3 -operons encode the isoforms Cbb 3 -1 and Cbb 3 -2 both possessing subunits CcoN, CcoO and CcoP. However, only the cbb 3 -2 operon contains an additional ccoQ gene. CcoQ consists of 62 amino acids and is predicted to possess one transmembrane spanning helix. The physiological role of CcoQ was investigated based on a CcoQ-deletion mutant and wild-type Cbb 3 -2 crystals not containing subunit CcoQ. Cbb 3 -2 isolated from the deletion mutant is inactive and appears as a dispersed band on blue native-PAGE gels. Surprisingly, in the absence of ccoQ, Cbb 3 -1 also shows a strongly reduced activity. Our data suggest that CcoQ primarily functions as an assembly factor for Cbb 3 -2 but is also required for correct assembly of Cbb 3 -1. In contrast, once correctly assembled, Cbb 3 -1 and Cbb 3 -2 possess a full enzymatic activity even in the absence of CcoQ., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Unlabelled: Cytochrome c oxidases (CcOs), members of the heme-copper containing oxidase (HCO) superfamily, are the terminal enzymes of aerobic respiratory chains. The cbb3-type cytochrome c oxidases (cbb3-CcO) form the C-family and have only the central catalytic subunit in common with the A- and B-family HCOs. In Pseudomonas stutzeri, two cbb3 operons are organized in a tandem repeat. The atomic structure of the first cbb3 isoform (Cbb3-1) was determined at 3.2 Å resolution in 2010 (S. Buschmann, E. Warkentin, H. Xie, J. D. Langer, U. Ermler, and H. Michel, Science 329:327-330, 2010, http://dx.doi.org/10.1126/science.1187303). Unexpectedly, the electron density map of Cbb3-1 revealed the presence of an additional transmembrane helix (TMH) which could not be assigned to any known protein. We now identified this TMH as the previously uncharacterized protein PstZoBell_05036, using a customized matrix-assisted laser desorption ionization (MALDI)-tandem mass spectrometry setup. The amino acid sequence matches the electron density of the unassigned TMH. Consequently, the protein was renamed CcoM. In order to identify the function of this new subunit in the cbb3 complex, we generated and analyzed a CcoM knockout strain. The results of the biochemical and biophysical characterization indicate that CcoM may be involved in CcO complex assembly or stabilization. In addition, we found that CcoM plays a role in anaerobic respiration, as the ΔCcoM strain displayed altered growth rates under anaerobic denitrifying conditions., Importance: The respiratory chain has recently moved into the focus for drug development against prokaryotic human pathogens, in particular, for multiresistant strains (P. Murima, J. D. McKinney, and K. Pethe, Chem Biol 21:1423-1432, 2014, http://dx.doi.org/10.1016/j.chembiol.2014.08.020). cbb3-CcO is an essential enzyme for many different pathogenic bacterial species, e.g., Helicobacter pylori, Vibrio cholerae, and Pseudomonas aeruginosa, and represents a promising drug target. In order to develop compounds targeting these proteins, a detailed understanding of the molecular architecture and function is required. Here we identified and characterized a novel subunit, CcoM, in the cbb3-CcO complex and thereby completed the crystal structure of the Cbb3 oxidase from Pseudomonas stutzeri, a bacterium closely related to the human pathogen Pseudomonas aeruginosa., (Copyright © 2016 Kohlstaedt et al.)

The cbb3 cytochrome c oxidases are distant members of the superfamily of heme copper oxidases. These terminal oxidases couple O2 reduction with proton transport across the plasma membrane and, as a part of the respiratory chain, contribute to the generation of an electrochemical proton gradient. Compared with other structurally characterized members of the heme copper oxidases, the recently determined cbb3 oxidase structure at 3.2 Å resolution revealed significant differences in the electron supply system, the proton conducting pathways and the coupling of O2 reduction to proton translocation. In this paper, we present a detailed report on the key steps for structure determination. Improvement of the protein quality was achieved by optimization of the number of lipids attached to the protein as well as the separation of two cbb3 oxidase isoenzymes. The exchange of n-dodecyl-β-D-maltoside for a precisely defined mixture of two α-maltosides and decanoylsucrose as well as the choice of the crystallization method had a most profound impact on crystal quality. This report highlights problems frequently encountered in membrane protein crystallization and offers meaningful approaches to improve crystal quality., (© 2014 The Protein Society.)

The cbb3-type cytochrome c oxidases (cbb3-CcOs) are members of the heme-copper oxidase superfamily that couple the reduction of oxygen to translocation of protons across the membrane. The cbb3-CcOs are present only in bacteria and play a primary role in microaerobic respiration, being essential for nitrogen-fixing endosymbionts and for some human pathogens. As frequently observed in Pseudomonads, Pseudomonas stutzeri contains two independent ccoNO(Q)P operons encoding the two cbb3 isoforms, Cbb3-1 and Cbb3-2. While the crystal structure of Cbb3-1 from P. stutzeri was determined recently and cbb3-CcOs from other organisms were characterized functionally, less emphasis has been placed on the isoform-specific differences between the cbb3-CcOs. In this work, both isoforms were homologously expressed in P. stutzeri strains from which the genomic version of the respective operon was deleted. We purified both cbb3 isoforms separately by affinity chromatography and increased the yield of Cbb3-2 to a similar level as Cbb3-1 by replacing its native promoter. Mass spectrometry, UV-visible (UV-Vis) spectroscopy, differential scanning calorimetry, as well as oxygen reductase and catalase activity measurements were employed to characterize both cbb3 isoforms. Differences were found concerning the thermal stability and the presence of subunit CcoQ. However, no significant differences between the two isoforms were observed otherwise. Interestingly, a surprisingly high turnover of at least 2,000 electrons s(-1) and a high Michaelis-Menten constant (Km ~ 3.6 mM) using ascorbate-N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride (TMPD) as the electron donor were characteristic for both P. stutzeri cbb3-CcOs. Our work provides the basis for further mutagenesis studies of each of the two cbb3 isoforms specifically.

The heme-copper oxidases (HCOs) accomplish the key event of aerobic respiration; they couple O2 reduction and transmembrane proton pumping. To gain new insights into the still enigmatic process, we structurally characterized a C-family HCO--essential for the pathogenicity of many bacteria--that differs from the two other HCO families, A and B, that have been structurally analyzed. The x-ray structure of the C-family cbb3 oxidase from Pseudomonas stutzeri at 3.2 angstrom resolution shows an electron supply system different from families A and B. Like family-B HCOs, C HCOs have only one pathway, which conducts protons via an alternative tyrosine-histidine cross-link. Structural differences around hemes b and b3 suggest a different redox-driven proton-pumping mechanism and provide clues to explain the higher activity of family-C HCOs at low oxygen concentrations.