48 research outputs found
A unique way of energy conservation in glutamate fermenting clostridia
Genetic analysis revealed that Rhodobacter capsulatus contains six
rnfABCDEG-genes that are responsible for the electron flow in nitrogen fixation
(rnf = Rhodobacter nitrogen fixation). Homolgous genes have been detected in
Clostridium tetani. In this work, a membrane complex has been purified from the
related Clostridium tetanomorphum that catalyses the reduction of
NAD + (E°' = −320 mV) with ferredoxin (E°' ≤ −420 mV).
The difference in the redox potential of ≥ 100 mV could be useful for
additional energy conservation in the fermentation of glutamate to ammonia,
CO 2 , acetate, butyrate, and H 2 .
The complex consists of six subunits (RnfABCDEG), of which four
N-termini (RnfCDEG) could be sequenced.
The sequences are 60-80% identical to the deduced sequences of the Rnf-subunits from C. tetani.
The rnf operon has been completely sequenced and aligned with the sequences
of C. tetani. The complex contains both non-covalently bound flavin as well
as covalently bound flavin.
The non-covalently bound flavin was identified as FMN and riboflavin in 1:1
stochiometric ratio, each 0.3 mol/mol Rnf complex (180 kDa). The subunits RnfG
and RnfD contain covalently bound flavin linked via phosphodiester bond. The
iron was determined as 25±1 mol per Rnf complex. Usually, Rnf activity was
measured with NADH and ferricyanide at 420 nm. In order to measure NAD +
reduction with reduced ferredoxin catalysed by Rnf complex, the ferredoxin was
purified from C. tetanomorphum and reduced by Ti(III)citrate at pH 7.0. High
Rnf activities were observed in the membrane preparations of Clostridium
aminobutyricum, Clostridium pascui and Clostridium propionicum. Thus,
additional energy conservation can be explained in these bacteria. However Rnf
activity was absent in Eubacterium barkeri, a nicotinate fermenting bacteria.
The soluble butyryl-CoA-dehydrogenase/electron transferring flavoprotein
(Bcd/Etf) complex was purified from C. pascui as well as from C. tetanomorphum.
The N- terminal sequences of the three subunits (αβγ) showed high identities
with the deduced sequences of C. tetani. The Bcd/Etf complex purified from C.
tetanomorphum was shown to catalyze the endergonic reduction of ferredoxin with
NADH coupled to the exergonic reduction of crotonyl-CoA to butyryl-CoA (E°' =
-10 mV) with NADH. The12 reduced ferredoxin could be used for H 2 production
catalysed by a hydrogenase or probably used for additional energy conservation
via Rnf (about 0.3 mol ATP/ mol glutamate). Experiments with [2,4,4- 2 H]
glutamate and detection of citramalate-lyase activity showed that C. pascui
and C. tetanomorphum ferment glutamate via the methylaspartate pathway
Recommended from our members
Whole Animal Automated Platform for Drug Discovery against Multi-Drug Resistant Staphylococcus aureus
Staphylococcus aureus, the leading cause of hospital-acquired infections in the United States, is also pathogenic to the model nematode Caenorhabditis elegans. The C. elegans-S. aureus infection model was previously carried out on solid agar plates where the bacteriovorous C. elegans feeds on a lawn of S. aureus. However, agar-based assays are not amenable to large scale screens for antibacterial compounds. We have developed a high throughput liquid screening assay that uses robotic instrumentation to dispense a precise amount of methicillin resistant S. aureus (MRSA) and worms in 384-well assay plates, followed by automated microscopy and image analysis. In validation of the liquid assay, an MRSA cell wall defective mutant, MW2ΔtarO, which is attenuated for killing in the agar-based assay, was found to be less virulent in the liquid assay. This robust assay with a Z’-factor consistently greater than 0.5 was utilized to screen the Biomol 4 compound library consisting of 640 small molecules with well characterized bioactivities. As proof of principle, 27 of the 30 clinically used antibiotics present in the library conferred increased C. elegans survival and were identified as hits in the screen. Surprisingly, the antihelminthic drug closantel was also identified as a hit in the screen. In further studies, we confirmed the anti-staphylococcal activity of closantel against vancomycin-resistant S. aureus isolates and other Gram-positive bacteria. The liquid C. elegans – S. aureus assay described here allows screening for anti-staphylococcal compounds that are not toxic to the host
A unique way of energy conservation in glutamate fermenting clostridia
Genetic analysis revealed that Rhodobacter capsulatus contains six
rnfABCDEG-genes that are responsible for the electron flow in nitrogen fixation
(rnf = Rhodobacter nitrogen fixation). Homolgous genes have been detected in
Clostridium tetani. In this work, a membrane complex has been purified from the
related Clostridium tetanomorphum that catalyses the reduction of
NAD + (E°' = −320 mV) with ferredoxin (E°' ≤ −420 mV).
The difference in the redox potential of ≥ 100 mV could be useful for
additional energy conservation in the fermentation of glutamate to ammonia,
CO 2 , acetate, butyrate, and H 2 .
The complex consists of six subunits (RnfABCDEG), of which four
N-termini (RnfCDEG) could be sequenced.
The sequences are 60-80% identical to the deduced sequences of the Rnf-subunits from C. tetani.
The rnf operon has been completely sequenced and aligned with the sequences
of C. tetani. The complex contains both non-covalently bound flavin as well
as covalently bound flavin.
The non-covalently bound flavin was identified as FMN and riboflavin in 1:1
stochiometric ratio, each 0.3 mol/mol Rnf complex (180 kDa). The subunits RnfG
and RnfD contain covalently bound flavin linked via phosphodiester bond. The
iron was determined as 25±1 mol per Rnf complex. Usually, Rnf activity was
measured with NADH and ferricyanide at 420 nm. In order to measure NAD +
reduction with reduced ferredoxin catalysed by Rnf complex, the ferredoxin was
purified from C. tetanomorphum and reduced by Ti(III)citrate at pH 7.0. High
Rnf activities were observed in the membrane preparations of Clostridium
aminobutyricum, Clostridium pascui and Clostridium propionicum. Thus,
additional energy conservation can be explained in these bacteria. However Rnf
activity was absent in Eubacterium barkeri, a nicotinate fermenting bacteria.
The soluble butyryl-CoA-dehydrogenase/electron transferring flavoprotein
(Bcd/Etf) complex was purified from C. pascui as well as from C. tetanomorphum.
The N- terminal sequences of the three subunits (αβγ) showed high identities
with the deduced sequences of C. tetani. The Bcd/Etf complex purified from C.
tetanomorphum was shown to catalyze the endergonic reduction of ferredoxin with
NADH coupled to the exergonic reduction of crotonyl-CoA to butyryl-CoA (E°' =
-10 mV) with NADH. The12 reduced ferredoxin could be used for H 2 production
catalysed by a hydrogenase or probably used for additional energy conservation
via Rnf (about 0.3 mol ATP/ mol glutamate). Experiments with [2,4,4- 2 H]
glutamate and detection of citramalate-lyase activity showed that C. pascui
and C. tetanomorphum ferment glutamate via the methylaspartate pathway
Activity of a novel protonophore against methicillin-resistant Staphylococcus aureus
Aim: Compound 1-(4-chlorophenyl)-4,4,4-trifluoro-3-hydroxy-2-buten-1-one (compound 1) was identified as a hit against methicillin-resistant Staphylococcus aureus (MRSA) strain MW2.
Methods & results: The MIC of compound 1 against MRSA was 4 μg/ml. The compound showed enhanced activity at acidic pH by lowering bacterial intracellular pH and exhibited no lysis of human red blood cells at up to 64 μg/ml and its IC50 against HepG2 cells was 32 μg/ml. The compound reduced 1-log10 colony forming units of intracellular MRSA in macrophages and prolonged the survival of MRSA-infected Caenorhabditis elegans (p = 0.0015) and Galleria mellonella (p = 0.0002).
Conclusion: Compound 1 is a protonophore with potent in vitro and in vivo activity against MRSA and no toxicity in mammalian cells up to 8 μg/ml that warrants further investigation as a novel antibacterial
Dissection of the Caffeate Respiratory Chain in the Acetogen Acetobacterium woodii: Identification of an Rnf-Type NADH Dehydrogenase as a Potential Coupling Siteâ–¿
The anaerobic acetogenic bacterium Acetobacterium woodii couples caffeate reduction with electrons derived from hydrogen to the synthesis of ATP by a chemiosmotic mechanism with sodium ions as coupling ions, a process referred to as caffeate respiration. We addressed the nature of the hitherto unknown enzymatic activities involved in this process and their cellular localization. Cell extract of A. woodii catalyzes H2-dependent caffeate reduction. This reaction is strictly ATP dependent but can be activated also by acetyl coenzyme A (CoA), indicating that there is formation of caffeyl-CoA prior to reduction. Two-dimensional gel electrophoresis revealed proteins present only in caffeate-grown cells. Two proteins were identified by electrospray ionization-mass spectrometry/mass spectrometry, and the encoding genes were cloned. These proteins are very similar to subunits α (EtfA) and β (EtfB) of electron transfer flavoproteins present in various anaerobic bacteria. Western blot analysis demonstrated that they are induced by caffeate and localized in the cytoplasm. Etf proteins are known electron carriers that shuttle electrons from NADH to different acceptors. Indeed, NADH was used as an electron donor for cytosolic caffeate reduction. Since the hydrogenase was soluble and used ferredoxin as an electron acceptor, the missing link was a ferredoxin:NAD+ oxidoreductase. This activity could be determined and, interestingly, was membrane bound. A search for genes that could encode this activity revealed DNA fragments encoding subunits C and D of a membrane-bound Rnf-type NADH dehydrogenase that is a potential Na+ pump. These data suggest the following electron transport chain: H2 → ferredoxin → NAD+ → Etf → caffeyl-CoA reductase. They also imply that the sodium motive step in the chain is the ferredoxin-dependent NAD+ reduction catalyzed by Rnf
Antibacterial properties of 3-(phenylsulfonyl)-2-pyrazinecarbonitrile
The emergence of multidrug-resistant bacterial strains has heightened the need for new antimicrobial agents based on novel chemical scaffolds that are able to circumvent current modes of resistance. We recently developed a whole-animal drug-screening methodology in pursuit of this goal and now report the discovery of 3-(phenylsulfonyl)-2-pyrazinecarbonitrile (PSPC) as a novel antibacterial effective against resistant nosocomial pathogens. The minimum inhibitory concentrations (MIC) of PSPC against Staphylococcus aureus and Enterococcus faecium were 4 μg/mL and 8 μg/mL, respectively, whereas the MICs were higher against the Gram-negative bacteria Klebsiella pneumoniae (64 μg/mL), Acinetobacter baumannii (32 μg/mL), Pseudomonas aeruginosa (\u3e64 μg/mL), and Enterobacter spp. (\u3e64 μg/mL). However, co-treatment of PSPC with the efflux pump inhibitor phenylalanine arginyl β-naphthylamide (PAβN) or with sub-inhibitory concentrations of the lipopeptide antibiotic polymyxin B reduced the MICs of PSPC against the Gram-negative strains by \u3e4-fold. A sulfide analog of PSPC (PSPC-1S) showed no antibacterial activity, whereas the sulfoxide analog (PSPC-6S) showed identical activity as PSPC across all strains, confirming structure-dependent activity for PSPC and suggesting a target-based mechanism of action. PSPC displayed dose dependent toxicity to both Caenorhabditis elegans and HEK-293 mammalian cells, culminating with a survival rate of 16% (100 μg/mL) and 8.5% (64 μg/mL), respectively, at the maximum tested concentration. However, PSPC did not result in hemolysis of erythrocytes, even at a concentration of 64 μg/mL. Together these results support PSPC as a new chemotype suitable for further development of new antibiotics against Gram-positive and Gram-negative bacteria