担当区分：責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Pharmaceutical Society of Japan  

DOI： 10.1248/bpbreports.4.3_92

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Proceedings of the National Academy of Sciences  

Mechanistic studies of anaerobic gut bacteria have been hindered by the lack of a fluorescent protein system to track and visualize proteins and dynamic cellular processes in actively growing bacteria. Although underappreciated, many gut “anaerobes” are able to respire using oxygen as the terminal electron acceptor. The oxygen continually released from gut epithelial cells creates an oxygen gradient from the mucus layer to the anaerobic lumen [L. Albenberg et al., Gastroenterology 147, 1055–1063.e8 (2014)], with oxygen available to bacteria growing at the mucus layer. Here, we show that <italic>Bacteroides</italic> species are metabolically and energetically robust and do not mount stress responses in the presence of 0.10 to 0.14% oxygen, defined as nanaerobic conditions [A. D. Baughn, M. H. Malamy, Nature 427, 441–444 (2004)]. Taking advantage of this metabolic capability, we show that nanaerobic growth provides sufficient oxygen for the maturation of oxygen-requiring fluorescent proteins in <italic>Bacteroides</italic> species. Type strains of four different <italic>Bacteroides</italic> species show bright GFP fluorescence when grown nanaerobically versus anaerobically. We compared four different red fluorescent proteins and found that mKate2 yields the highest red fluorescence intensity in our assay. We show that GFP-tagged proteins can be localized in nanaerobically growing bacteria. In addition, we used time-lapse fluorescence microscopy to image dynamic type VI secretion system processes in metabolically active <italic>Bacteroides fragilis</italic>. The ability to visualize fluorescently labeled <italic>Bacteroides</italic> and fluorescently linked proteins in actively growing nanaerobic gut symbionts ushers in an age of imaging analyses not previously possible in these bacteria.

DOI： 10.1073/pnas.2009556117

PubMed

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その他リンク： https://syndication.highwire.org/content/doi/10.1073/pnas.2009556117

掲載種別：研究論文（学術雑誌）   出版者・発行元：American Society for Biochemistry & Molecular Biology (ASBMB)  

The Na⁺-pumping NADH-ubiquinone (UQ) oxidoreductase (Na⁺-NQR) is present in the respiratory chain of many pathogenic bacteria and is thought to be a promising antibiotic target. Whereas many details of Na⁺-NQR structure and function are known, the mechanisms of action of potent inhibitors is not well-understood; elucidating the mechanisms would not only advance drug design strategies but might also provide insights on a terminal electron transfer from riboflavin to UQ. To this end, we performed photoaffinity labeling experiments using photoreactive derivatives of two known inhibitors, aurachin and korormicin, on isolated <italic>Vibrio cholerae</italic> Na⁺-NQR. The inhibitors labeled the cytoplasmic surface domain of the NqrB subunit including a protruding N-terminal stretch, which may be critical to regulate the UQ reaction in the adjacent NqrA subunit. The labeling was blocked by short-chain UQs such as ubiquinone-2. The photolabile group (2-aryl-5-carboxytetrazole (ACT)) of these inhibitors reacts with nucleophilic amino acids, so we tested mutations of nucleophilic residues in the labeled region of NqrB, such as Asp⁴⁹ and Asp⁵² (to Ala), and observed moderate decreases in labeling yields, suggesting that these residues are involved in the interaction with ACT. We conclude that the inhibitors interfere with the UQ reaction in two ways: the first is blocking structural rearrangements at the cytoplasmic interface between NqrA and NqrB, and the second is the direct obstruction of UQ binding at this interfacial area. Unusual competitive behavior between the photoreactive inhibitors and various competitors corroborates our previous proposition that there may be two inhibitor binding sites in Na⁺-NQR.

DOI： 10.1074/jbc.ra120.014229

PubMed

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担当区分：筆頭著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：American Society for Microbiology  

<title>ABSTRACT</title>
In bacteria, the respiratory pathways that drive molecular transport and ATP synthesis include a variety of enzyme complexes that utilize different electron donors and acceptors. This property allows them to vary the efficiency of energy conservation and to generate different types of electrochemical gradients (H⁺ or Na⁺). We know little about the respiratory pathways in <italic>Bacteroides</italic> species, which are abundant in the human gut, and whether they have a simple or a branched pathway. Here, we combined genetics, enzyme activity measurements, and mammalian gut colonization assays to better understand the first committed step in respiration, the transfer of electrons from NADH to quinone. We found that a model gut <italic>Bacteroides</italic> species, <named-content content-type="genus-species">Bacteroides fragilis</named-content>, has all three types of putative NADH dehydrogenases that typically transfer electrons from the highly reducing molecule NADH to quinone. Analyses of NADH oxidation and quinone reduction in wild-type and deletion mutants showed that two of these enzymes, Na⁺-pumping <underline>N</underline>ADH:<underline>q</underline>uinone oxido<underline>r</underline>eductase (NQR) and <underline>N</underline>ADH <underline>d</underline>e<underline>h</underline>ydrogenase <underline>II</underline> (NDH2), have NADH dehydrogenase activity, whereas H⁺-pumping <underline>N</underline>ADH:<underline>u</underline>biquinone <underline>o</underline>xidoreductase (NUO) does not. Under anaerobic conditions, NQR contributes more than 65% of the NADH:quinone oxidoreductase activity. When grown in rich medium, none of the single deletion mutants had a significant growth defect; however, the double Δ<italic>nqr</italic> Δ<italic>ndh2</italic> mutant, which lacked almost all NADH:quinone oxidoreductase activity, had a significantly increased doubling time. Despite unaltered <italic>in vitro</italic> growth, the single <italic>nqr</italic> deletion mutant was unable to competitively colonize the gnotobiotic mouse gut, confirming the importance of NQR to respiration in <named-content content-type="genus-species">B. fragilis</named-content> and the overall importance of respiration to this abundant gut symbiont.


<bold>IMPORTANCE</bold> <italic>Bacteroides</italic> species are abundant in the human intestine and provide numerous beneficial properties to their hosts. The ability of <italic>Bacteroides</italic> species to convert host and dietary glycans and polysaccharides to energy is paramount to their success in the human gut. We know a great deal about the molecules that these bacteria extract from the human gut but much less about how they convert those molecules into energy. Here, we show that <named-content content-type="genus-species">B. fragilis</named-content> has a complex respiratory pathway with two different enzymes that transfer electrons from NADH to quinone and a third enzyme complex that may use an electron donor other than NADH. Although fermentation has generally been believed to be the main mechanism of energy generation in <italic>Bacteroides</italic>, we found that a mutant lacking one of the NADH:quinone oxidoreductases was unable to compete with the wild type in the mammalian gut, revealing the importance of respiration to these abundant gut symbionts.

DOI： 10.1128/mbio.03238-19

PubMed

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掲載種別：研究論文（学術雑誌）   出版者・発行元：American Society for Microbiology  

<title>ABSTRACT</title>
Korormicin is an antibiotic produced by some pseudoalteromonads which selectively kills Gram-negative bacteria that express the Na⁺-pumping NADH:quinone oxidoreductase (Na⁺-NQR.) We show that although korormicin is an inhibitor of Na⁺-NQR, the antibiotic action is not a direct result of inhibiting enzyme activity. Instead, perturbation of electron transfer inside the enzyme promotes a reaction between O₂ and one or more redox cofactors in the enzyme (likely the flavin adenine dinucleotide [FAD] and 2Fe-2S center), leading to the production of reactive oxygen species (ROS). All <italic>Pseudoalteromonas</italic> contain the <italic>nqr</italic> operon in their genomes, including <italic>Pseudoalteromonas</italic> strain J010, which produces korormicin. We present activity data indicating that this strain expresses an active Na⁺-NQR and that this enzyme is not susceptible to korormicin inhibition. On the basis of our DNA sequence data, we show that the Na⁺-NQR of <italic>Pseudoalteromonas</italic> J010 carries an amino acid substitution (NqrB-G141A; <named-content content-type="genus-species">Vibrio cholerae</named-content> numbering) that in other Na⁺-NQRs confers resistance against korormicin. This is likely the reason that a functional Na⁺-NQR is able to exist in a bacterium that produces a compound that typically inhibits this enzyme and causes cell death. Korormicin is an effective antibiotic against such pathogens as <named-content content-type="genus-species">Vibrio cholerae</named-content>, <named-content content-type="genus-species">Aliivibrio fischeri</named-content>, and <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content> but has no effect on <named-content content-type="genus-species">Bacteroides fragilis</named-content> and <named-content content-type="genus-species">Bacteroides thetaiotaomicron</named-content>, microorganisms that are important members of the human intestinal microflora.


<bold>IMPORTANCE</bold> As multidrug antibiotic resistance in pathogenic bacteria continues to rise, there is a critical need for novel antimicrobial agents. An essential requirement for a useful antibiotic is that it selectively targets bacteria without significant effects on the eukaryotic hosts. Korormicin is an excellent candidate in this respect because it targets a unique respiratory enzyme found only in prokaryotes, the Na⁺-pumping NADH:quinone oxidoreductase (Na⁺-NQR). Korormicin is synthesized by some species of the marine bacterium <italic>Pseudoalteromonas</italic> and is a potent and specific inhibitor of Na⁺-NQR, an enzyme that is essential for the survival and proliferation of many Gram-negative human pathogens, including <named-content content-type="genus-species">Vibrio cholerae</named-content> and <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content>, among others. Here, we identified how korormicin selectively kills these bacteria. The binding of korormicin to Na⁺-NQR promotes the formation of reactive oxygen species generated by the reaction of the FAD and the 2Fe-2S center cofactors with O₂.

DOI： 10.1128/jb.00718-18

PubMed

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掲載種別：研究論文（学術雑誌）   出版者・発行元：American Chemical Society (ACS)  

DOI： 10.1021/acs.biochem.7b00612

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1074/jbc.m117.781393

Web of Science

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担当区分：筆頭著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：American Chemical Society (ACS)  

DOI： 10.1021/acs.biochem.5b00385

PubMed

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掲載種別：研究論文（学術雑誌）   出版者・発行元：American Chemical Society (ACS)  

DOI： 10.1021/acs.biochem.5b00187

PubMed

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Informa UK Limited  

DOI： 10.1080/09168451.2015.1010479

PubMed

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