Complex biosynthetic pathways are required for the assembly and insertion of iron-sulfur (Fe-S) cluster cofactors. Each of the four cluster biogenesis systems that have been discovered requires at ...least one ATPase. Generally, the function of nucleotide hydrolysis in Fe-S cluster biogenesis is understudied. For example, the cytosolic Fe-S cluster assembly (CIA) pathway is proposed to begin with a scaffold, which assembles nascent Fe-S clusters destined for cytosolic and nuclear enzymes. This scaffold, comprised of Nbp35 and Cfd1 in yeast, possesses an ATPase site that is necessary for CIA function, but the role of nucleotide hydrolysis is poorly understood. Herein, we describe the in vitro methods that have been developed to uncover how the ATPase site of the scaffold regulates interaction with one of its partner proteins, Dre2. We describe a qualitative affinity copurification assay and a quantitative assay for evaluating the dissociation constant for the scaffold-partner protein complex. Finally, we describe kinetic methods to measure the k
and K
values for ATP hydrolysis by the scaffold-partner protein complex and the execution of the ATPase assays in an anaerobic environment. These methods could be applied to study other ATPases to advance our mechanistic understanding of nucleotide hydrolases involved in metallocluster biogenesis.
Robert Hungate, considered the father of rumen microbiology, was the fi rst to initiate a systematic exploration of the microbial ecosystem of the rumen, but he was not alone. The techniques he ...developed to isolate and identify cellulose-digesting bacteria from the rumen have had a major impact not only in delineating the complex ecosystem of the rumen but also in clinical microbiology and in the exploration of a number of other anaerobic ecosystems, including the human hindgut. Rumen microbiology has pioneered our understanding of much of microbial ecology and has broadened our knowledge of ecology in general, as well as improved the ability to feed ruminants more efficiently. The discovery of anaerobic fungi as a component of the ruminal fl ora disproved the central dogma in microbiology that all fungi are aerobic organisms. Further novel interactions between bacterial species such as nutrient cross feeding and interspecies H2 transfer were fi rst described in ruminal microorganisms. The complexity and diversity present in the rumen make it an ideal testing ground for microbial theories (e.g., the effects of nutrient limitation and excess) and techniques(such as 16S rRNA), which have rewarded the investigators that have used this easily accessed ecosystem to understand larger truths. Our understanding of characteristics of the ruminal microbial population has opened new avenues of microbial ecology, such as the existence of hyperammonia-producing bacteria and how they can be used to improve N efficiency in ruminants. In this review, we examine some of the contributions to science that were fi rst made in the rumen, which have not been recognized in a broader sense.
Background: NiFeSe hydrogenases are metalloenzymes that catalyze the reaction H
2↔2H
++2e
-.They are generally heterodimeric, contain three iron–sulfur clusters in their small subunit and a ...nickel–iron-containing active site in their large subunit that includes a selenocysteine (SeCys) ligand.
Results: We report here the X-ray structure at 2.15 Å resolution of the periplasmic NiFeSe hydrogenase from
Desulfomicrobium baculatum in its reduced, active form. A comparison of active sites of the oxidized, as-prepared,
Desulfovibrio gigas and the reduced
D. baculatum hydrogenases shows that in the reduced enzyme the nickel-iron distance is 0.4 Å shorter than in the oxidized enzyme. In addition, the putative oxo ligand, detected in the as-prepared
D.gigas enzyme, is absent from the
D.baculatum hydrogenase. We also observe higher-than-average temperature factors for both the active site nickel–selenocysteine ligand and the neighboring Glu18 residue, suggesting that both these moieties are involved in proton transfer between the active site and the molecular surface. Other differences between NiFeSe and NiFe hydrogenases are the presence of a third 4Fe4S cluster replacing the 3Fe4S cluster found in the
D.gigas enzyme, and a putative iron center that substitutes the magnesium ion that has already been described at the C terminus of the large subunit of two NiFe hydrogenases.
Conclusions: The heterolytic cleavage of molecular hydrogen seems to be mediated by the nickel center and the selenocysteine residue. Beside modifying the catalytic properties of the enzyme, the selenium ligand might protect the nickel atom from oxidation. We conclude that the putative oxo ligand is a signature of inactive ‘unready’ NiFe hydrogenases.
Our current understanding of the evolution of mutualisms is limited partly because there have been relatively few model systems for studying it in real time. A model mutualistic interaction between ...the bacterium D. vulgaris and the archaeaon M. maripaludis was developed to allow for rigorous tests of general hypotheses about the evolution and ecology of mutualisms. This model system also allows us to develop an evolutionary genetics perspective on an interaction that plays a key ecological role in many oxygen-free microbial communities. Here, we describe the techniques used to make anoxic media for propagating these species alone or in conditions that require their cooperation.
Anaerobic cell culture Speers, Allison M; Cologgi, Dena L; Reguera, Gemma
Current protocols in microbiology (Online),
February 2009, Volume:
Appendix 4
Journal Article
Although the ability of some microorganisms to grow without O(2) has long been recognized, the application of new methodologies has greatly expanded the known diversity and potential of anaerobic ...microorganisms and processes. In particular, anaerobic techniques that permit the successful cultivation of microorganisms on solid media have opened new avenues for the study of the physiology and metabolic potential of many new microorganisms using molecular, genomic, and proteomic tools. One technique above all has proven instrumental for anaerobic studies over the years: the use of the anaerobic chamber. This unit gives a brief description of the methods used for the cultivation of anaerobic microorganisms, and describes in detail the principles and applications of anaerobic chambers, with special emphasis on vinyl glove boxes. The methodologies described in this unit should provide the interested but inexperienced investigator with the basic tools to successfully cultivate anaerobic microorganisms and study anaerobic processes.
The Anoxomat®system provides an automated evacuation–replacement technique to create an anaerobic or microaerophilic environment in a jar. We evaluated the Anoxomat®system for the growth of obligate ...anaerobes and for the recovery of anaerobic organisms from clinical specimens, and compared its performance to that of an anaerobic chamber and the GasPak®System. Of the 54 stock strains tested, the Anoxomat®, the chamber, and the GasPak®recovered 95%, 95% and 93% at 24 h, respectively. On 29 occasions (51%), the colonies on the Anoxomat®plates were slightly larger than those in the chamber and on 17 (30%) occasions larger than the colonies on the GasPak®jar plates. At 48 h, the Anoxomat®, the chamber, and the GasPak®recovered 93.5%, 94.4% and 88.9%, respectively; of 108 anaerobes isolated from 31 clinical specimens. Methylene blue indicators became decolorized (average of 10 tests) within 2 h inside the Anoxomat®jars, 2 h 10 min inside the anaerobic chamber, and 2 h 30 min inside the GasPak®jars.
Metronidazole is active in vitro against obligate anaerobes such as Bacteroides, Fusobacterium, Veillonella, Peptococcus and Peptostreptococcus, but has no activity against facultative anaerobes. ...However it is generally less active against non-sporing Gram-positive bacilli such as Actinomyces and Propionibacterium, it is also less active against some species of Peptostreptococcus, but less sensitive strains are usually not truly anaerobic. Metronidazole may, therefore, be a useful tool in differentiation of obligate anaerobic bacteria. And so paper strips each containing 5μg of metronidazole were prepared to differentiate single colonies of obligate from facultative anaerobes on primary plate cultures of mixed bacterial suspensions containing obligate and facultative anaerobes. This suspension was inoculated onto a GAM agar plate to obtain single colonies and a metronidazole strip was placed at right angles to the well of inoculum, so that it extended into the area where single colonies would be expected. After 48 hours of anaerobic incubation, in the area uniformly inoculated, a zone of inhibition around the metronidazole strip with intrazonal growth revealed the presence of an obligate anaerobe together with a facultative anaerobe. The area where this phenomenon is recognized is called “sparser zone”. Complete inhibition of growth around the metronidazole strip indicated that obligate anaerobes only were present. In the area where single colonies were obtained, colonies of facultative anaerobes were recognized even right up to the edge of the strip and colonies of obligate anaerobes were recognized at a distance of 5 to 15 mm at least from the edge of the strip. Therefore it seems to be able to differentiate single colonies of obligate from facultative anaerobes without examining the plate which was incubated aerobically. Facultative anaerobes have little influence on the metronidazole strip method. Although metronidazole was inactivated by certain facultative anaerobes such as Streptococcus faecalis, their presence in mixed culture with obligate anaerobes did not inhibit the activity of metronidazole. This was because of the more rapid bactericidal effect of metronidazole before any significant inactivation by the facultative anaerobes. Therefore the metronidazole strip method is able to detect obligate anaerobes on primary plate cultures of clinical specimens containing 102 to 103 times as many facultative anaerobes as obligate anaerobes so that it is difficult to obtain single colonies of the obligate anaerobes. In this case multiple anaerobic selective media are recommended for the isolation of the obligate anaerobes of all types.
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