Concepción de los recursos naturales desde los estudiantes:

For determination of the amino acids, cells were grown (growth conditions described in subsubsection 2.2.1.2) in synthetic medium with defined composition (Koch, 2005), with and without glycerine. The cell culture was harvested at stationary phase (OD600

= 1.5) by centrifugation for 20 min at 8000 rpm (10°C in a Sorvall RC5C centrifuge with a GS3 rotor).

Afterwards the pellet was suspended in basal salt (like complex medium forHbt. sa- linarum (section 2.2.2.1) but without peptone) till an OD600 of 4.5 was reached and

35 ml of the cell suspension were given in a klett-flask. Arginine, ornithine or both (0.5 %) were added to the concentrated cell suspension and again incubated at 37◦_C,

aerobically or anaerobically in the dark or in the light.

For cells cultured under anaerobic growth conditions, 1 ml of a 1 M sodiumcarbon- ate solution was added into the sidearm of the klett-flask. The cell suspension was flushed with nitrogen for 5 min and finally the flask made airtightly closure with a plug containing a septum inside. At various times, 0.5 ml samples were taken (in case of anaerobic conditions, samples were taken with a syringe through the septum), and the cells were removed by centrifugation (14 000 rpm, 5 min, Eppendorf desk centrifuge). Aliquots of the supernatant (400µl) were frozen and later analyzed for arginine or

ornithine on an amino acid analyzer (Biotronik LC3000).

2.5.1.1 Calculations of the intracellular change of concentration of ornithine

(∆orn) and arginine (∆arg) per cell

Parameters (calculated by Ricardo Del Rosario from the internal Cell Database): • cell density: 450 Klett/ml = 4,59 OD/ml

• 1 OD600 ml = 1,36*109 cells

• cell water volume : 0.59 fl

First, the absolute number of the measured amino acid molecules was converted into a number per 1 ml. After calculating the number of cells in 1 ml using the optical density, the absorption or secretion of the amino acid per cell could be determined.

2.5.2 Stable Isotope Labeling in

Nmn. pharaonis

with

C carbon

sources

Instead of12_{C carbon sources,}13_{C labeled acetate or/and}13_{C labeled amino acids and} 13C labeled sodium carbonate were added to the minimal medium for_{Nmn. pharaonis}

(subsubsection 2.2.2.2). All isotopomeres and deuterated solvents were ordered from Euriso-Top. Nmn. pharaoniscells were inoculated twice consecutively in 35 ml minimal

medium containing the respective labeled carbon source before inoculation into 350 ml medium.

2.5.3 Preparation of lipids

Before extracting lipids it was necessary to separate the membrane fraction of the harvested cells from the remaining cell components.

2.5.3.1 Cell harvest and disruption

For labeling experiments, lipids yielded from at least 350 ml cell culture in stationary phase were required. Upon reaching the stationary phase, the cells were harvested by centrifugation in a Sorvall RC 5c plus centrifuge (4◦_{C, 10500 rpm, Rotor SS34) for}

30 min. The pellet was resuspended in basal salts (NaCl 3.4 M, KCl 13 mM, KH2PO4

7.3 mM, pH 9) and then homogenized for 2 h by adding a small quantity of DNase I and mixing it with a magnetic stir bar. Finally the cells were broken up by osmotic shock (Oesterhelt & Stoeckenius, 1971): the cell suspension was dialyzed against 30 l water in dialysis cassettes (Pierce Slide-A-Lyzer, 10 000 MWCO) over night at 4◦_{C. After}

dialysis, lipids were separated from proteins, DNA and RNA by ultra-centrifugation at 40 000 rpm for 2.5 h at 4◦_{C (Beckman Coulter LE-80K, Ti50 rotor).}

2.5.3.2 Lipid extraction

Lipids were extracted according to Bligh and Dyer (Bligh & Dyer, 1959). The polar fraction of lipids was obtained by extraction with chloroform, methanol and citric acid (2:2:1.8; v/v/v). The pellet was dissolved in 40 ml citric acid and transferred into a separatory funnel. 50 ml chloroform and 50 ml methanol were added and mixed by shaking the funnel. 50 ml of both, chloroform and citric acid were added again and

the solution allowed to separate into two layers after shaking. After collecting the chloroform layer in a round flask, additional chloroform was added into the funnel. Extractions were repeated for as long as this layer stayed colored. To exclude the water in the solution, some Na2SO4 was added to the chloroform layer and finally

filtered through a fluted filter. The solvent was removed by rotary evaporation.

2.5.3.3 Extraction of polar lipids by acetone precipitation

Total lipid extracts were dissolved by shaking in 600µl CHCl3 and 100µl MeOH (6:1).

The solutions were transferred into precooled (-20◦_{C) glass centrifuge tubes (COREX)}

after prior flushing of the tubes twice with 300µl of solvents. Then, 4 ml of ice-cold

acetone was added and the solution incubated at -20◦_{C over night. The solution was}

centrifuged (Sorvall centrifuge, SS 34 rotor) at 2900 g, 30 min at 5◦_{C and the lipid}

precipitate was removed with a metal spatula and resuspended again in 2 ml ice-cold acetone. The suspension was sonicated in a water bath. After centrifugation, acetone precipitation was repeated twice and the sample incubated at -20◦_{C for 1 h after every}

repetition.

2.5.3.4 Cleavage of ether lipids with boron trichloride

For analyzing the alkyl chains of extracted ether lipids, cleavage with BCl3 (dissolved

in methylene chloride) was performed (Nishihara & Koga, 1988; Kates et al., 1965;

Gerrard & Lappert, 1952). After drying the lipids in glass vials (Pierce) under stream of nitrogen, 1 ml of BCl3 in CH2Cl2 was added and the vial closed by an appropriate

screw top. The reaction was allowed to proceed for 30 min with occasional shaking of the tube. Afterwards, the solution was evaporated and the BCl3 and borate removed

by two rounds of suspension in MeOH and drying. Cleaved lipids were dissolved in 1.5 ml CHCl3/MeOH/H2O (1:1:0.9; v:v:v) and then the upper phase, which contained

the polar headgroups, was removed. The phytanoylchloride (attached chloride to the isoprenoid chain) which was dissolved in CHCl3 was evaporated again before 1 ml

petroleum ether was added. The solution was shaken well and sonified in a water bath. To avoid taking up precipitates, the solution was transferred into a new glass vial and washed with 1 ml H2O. The upper phase containing the phytanoylchloride in

petroleum ether was again transferred into a new vial and finally dried off under a nitrogen stream.