Analysis of microbial metabolism

(©Max Planck Institute for Marine Microbiology, O. Lemaire)
What resembles here an origami butterfly is a crystallized enzyme that can produce methane and is found in all methanogens. The yellow-greenish color of the crystal is coming from the F430-cofactor containing a nickel. It is the cofactor, deeply buried in the enzyme core, which orchestrates the magical reaction of methane generation. (©Max Planck Institute for Marine Microbiology, O. Lemaire)

Why is relevant to study microbial metabolism?

The Mi­cro­bial Meta­bol­ism Group aims to un­der­stand, at the mo­lecu­lar level, how meth­ano­gens are sur­viv­ing and grow­ing in ex­treme en­vir­on­ments. How do they gen­er­ate meth­ane from dif­fer­ent sources of car­bon so ef­fi­ciently? How do they con­vert min­er­als into the ele­ment­ary bricks of life? And how do they pro­tect them­selves against stresses from their nat­ural en­vir­on­ment?

How does the analysis of microbial metabolism work?

To find an an­swer to these ques­tions, we must cul­tiv­ate these mi­croor­gan­isms and study the dif­fer­ent chem­ical re­ac­tions oc­cur­ring in­side them. The en­zymes in­volved in the con­ver­sion of min­er­als and gases are pro­teins that or­ches­trate strange re­ac­tions highly chal­len­ging for chem­ists. We have to ex­tract these en­zymes, and sort them out from other pro­teins by us­ing the nat­ive puri­fic­a­tion. We then pierce the mo­lecu­lar secret of their re­ac­tion by look­ing at them with X-ray crys­tal­lo­graphy, which means that we have to crys­tal­lize the en­zymes, and use X-ray to get their pic­tures.

The most crit­ical part of his groups’ study is meth­ano­gen cul­tiv­a­tion: we need spe­cial gases, equip­ments and most im­port­antly the know­ledge to take care of them.

Which instruments are important for the analysis?

Autoclave

Autoclaves are used to sterilize solutions and materials, or even waste prior to disposal. You might have heard of autoclaves from the medical sector, for instance to sterilize surgical tools. At the institute we have a hospital-size autoclave, which allows us to even sterilize large devices (fermenter).

Manual Gas-exchanger

Many of our organisms grow anaerobically, so without any oxygen. Therefore, we require the manual Gas-exchanger to make solutions and cultures anaerobic, using the gases nitrogen or a mixture of hydrogen and carbon dioxide.

Automatic Gas-exchanger

This device runs automatically and also removes oxygen from solutions. In this case, the oxygen can only be replaced by nitrogen gas.

Incubator

Our lab has special incubators, which can maintain temperatures up to 80 °C and shake up to 220 rotation per minute. These incubators allow us to provide the perfect growth conditions for many microorganisms cultivated in erlenmeyer or pressure-resistant bottles.

Fermenter

Often, small and pressure protected bottles are used to grow anaerobic microorganisms. The use of fermenter allows us to increase the volume of culture – up to 10 liter and provides a constant amount of gas(es) to stimulate the growth of microorganisms.

Fermenter (©Max Planck Institute for Marine Microbiology, T. Wagner)

Sonication

To extract the proteins within the microorganisms the cells need to be broken. We often break the cells via ultra-sonication. Since this device is rather small, one Sonicator is placed inside an anaerobic chamber (see below).

French Press

The French Press is another way to break the cells but with high pressure, which can be manually adjusted.

Aekta Avant

The Aekta Avant is used to aerobically purify proteins (liquid chromatography) – so in presence of oxygen. However, Aekta systems are also placed inside anaerobic chambers to guarantee a protein purification without oxygen.

Anaerobic chambers

The MicroMet team has five anaerobic chambers.

- The first tent has a nitrogen/carbon-dioxide atmosphere. Anaerobic organisms are “harvested” in this tent and cells can be broken anaerobically by a sonication device.

- Three tents with a hydrogen/nitrogen atmosphere stand in a room which can be kept under 18 °C and yellow-light conditions. This is important as many proteins are light sensitive and could be damaged by normal room light. Two tents comprise all devices required to perform anaerobic protein purifications. Here, we can fractionate and separate pro­teins from each other. One tent is solely used for anaerobic protein-crystallization. Crystallization is a delicate process, for which reason the tent stands on a anti-vibration table and the atmosphere is kept dust-free via a filter system.

- The last tent has a nitrogen atmosphere and is used for anaerobic robotic crystallization and protein characterization.

 

Gas chromatographs

The gas chro­ma­to­graphs are used to meas­ure gases and to measure their concentrations, for ex­ample, dur­ing the growth of cell cul­tures. Among oth­ers, the fol­low­ing gases can be meas­ured: Meth­ane, car­bon monox­ide, car­bon di­ox­ide, hy­dro­gen and oxy­gen.

Photometer

The photometer measures enzyme activities and records spectra (wavelength scan from 200-1100 nm). A maximum of 18 cuvettes can be measured simultaneously, the smallest sample volume is 5 - 1000 µl. The temperature can be set up to 90 degree celsius.

Research Examples

CODH/ACS crystals obtained without oxygen. The brown color is coming from the natural metals harboured by the proteins. (© Max Planck Institute for Marine Microbiology/T. Wagner)
CODH/ACS crystals obtained without oxygen. The brown color is coming from the natural metals harboured by the proteins. (© Max Planck Institute for Marine Microbiology/T. Wagner)

Cel­lu­lar power­plant re­cycles waste gases

Carbon monoxide is a very poisonous gas. Humans die within minutes when they inhale it. However, some microorganisms tolerate carbon monoxide and even use it to breathe and replicate. Knowledge about how these bacteria survive opens a window into the primeval times of the earth and the origin of life. At the same time, they might be very useful for the future as they can be used to clean waste gases and produce biofuels.

In this context, scientists from the Max Planck Institute for Marine Microbiology in Bremen have made a surprising discovery. The two sci­ent­ists used the crys­tal­liz­a­tion method to ob­tain crys­tals of the en­zyme CODH/​ACS and de­term­ine the pro­tein 3D-struc­ture by X-ray crys­tal­lo­graphy. The surprising discovery: The CODH-ACS in­ter­face from one bacteria drastic­ally dif­fers from another, even though it was the same en­zyme and sim­ilar bac­teria.

 

Read more in the press release "Cellular powerplant recycles waste gases"

 

Original publication

Oli­vier N. Le­mai­re and Tris­tan Wag­ner: Gas channel rerouting in a primordial enzyme: Structural insights of the carbon-monoxide dehydrogenase/acetyl-CoA synthase complex from the acetogen Clostridium autoethanogenum. BBA – Bio­en­er­ge­tics, 2020

DOI: 10.1016/j.bbabio.2020.148330

Pictures of F420H2-oxidase crystals obtained aerobically with a typical size of 0.1 mm. With oxygen the Fe and flavin inside the enzyme give the natural yellow color to the crystals. (© Max Planck Institute for Marine Microbiology/T. Wagner)
Pictures of F420H2-oxidase crystals obtained aerobically with a typical size of 0.1 mm. With oxygen the Fe and flavin inside the enzyme give the natural yellow color to the crystals. (© Max Planck Institute for Marine Microbiology/T. Wagner)

Wa­ter at the end of the tun­nel

We humans need oxygen to breath – for a lot of microbes it is a lethal poison. That is why microorganisms have developed ways to render oxygen molecules harmless. Microbiologists from Bremen, Marburg and Grenoble have now succeeded in decrypting such a mechanism. They show, how methane-generating microbes transform oxygen into water without causing any damage to the cell. These findings are relevant for future bio-inspired processes.

 

Read more in the press release "Water at the end of the tunnel"

 

Original publication

Sylvain En­gil­berge#, Tristan Wag­ner#, Phil­ippe Car­pen­tier, Eric Gir­ard, Seigo Shima: Krypton-derivatization highlights O2-channeling in a four-electron reducing oxidase. Chem­ical Com­mu­nic­a­tion, Septem­ber 2020

DOI: 10.1039/d0cc04557h

# both au­thors con­trib­uted equally to this work

Contact

Head of Group

Dr. Tristan Wagner

MPI for Marine Microbiology
Celsiusstr. 1
D-28359 Bremen
Germany

Phone: 

+49 421 2028-7440

Dr. Tristan Wagner
 
 
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