Microbial Diversity Summer Course 1991 MBL, Woods Hole

(D Guenter

1. Characterization of Magnetotactic Bacteria

Introduction: Magnetotactic bacteria were isolated from a glass jar that was set up for theenrichment for magnetotactic bacteria from the sediment of School Street Marsh, WoodsHole, three years ago. Basic properties of their morphology as well as of theft physiologyand theft environment were examined in order to find an appropiate strategy for theircultivation in pure culture.

Isolation: For the isolation of these bacteria advantage was taken of their magnetotacticbehavior: the north pole of a magnet (in form of a stirring bar) was fixed near the wall ofthe enrichment jar. Within a few minutes the magnetotactic bacteria aggregated to a visiblewhite cell pellet on the wall of the jar and were harvested with a pulled out pasteur pipette.The cells were transfered to small glass tubes containing 2m1 PBS (phosphate bufferedsaline: 130 mM sodium chloride, 10 mM sodium phosphate, pH 8.0), again concentratedwith a magnet and the visible pellet sucked off with a pipette (“magnetotactic washing”).Thus magnetotactic bacteria were highly enriched by outdiluting nonmagnetotacticcontaminants.

Environmental conditions (in the enrichment jar):-approximately: 2 1 of sediment (10 cm high), covered by the same volume of water (bothfrom Scholl Street Marsh).-salinity 2ppt (refractometer), pH 8.0, room temperature.-jar was standing in constant orientation of a labeled side to north, but even distribution ofmagnetotactic bacteria all over the cross-section of the jar (“north” = down)

Morphology: According to cell shape and size and kind of magnetosomes (as visible inphase contrast) three major subpopulations could be recognized:A) spherical cells, ca. 2.tm in diameter; magnetosomes = “small dots”, close to cellenvelope; ca. 70% of the cells after the “magnetotactic washing”.B) spherical cells, ca. 2.un two parallel thin chains; ca. 25%.C) almost square cells, ci 3jim; one thick chain; <5%.Besides those single cells of different morphology (rods, “bananas”) containingrnagnetosome-lilce structures could be seen (<<1%).All of the magnetotactic cells were Gram negative.

Reaction to oxygen: Enriched magnetotactic cells were transfered to anaerobic tubes anddifferent amounts of air corresponding to 0.1, 0.5, 1, 3, 6, 12, and 21% oxygen (v/v inheadspace) were added. After different times subsamples were taken and checked formoti]ity/ magnetotaxis under the light microscope.Magnetotaxis could be observed: after 2h in up to 6%, after 6h and 12h in up to 3%, andafter 26h and 48h in up to 1% oxygen.Therefore at least the larger subpopulations of the magnetotactic bacteria aremicroaerotolerant or microaerophi]ic.

Hybridization with probes: After the hybridization with fluorescent rRNA-targetedoligonucleotide probes all magnetotactic bacteria showed a positive signal for the“Eubacterial” probe, the morphological subpopulation “B” also a weak but positive signalfor the “Sulfate Reducer Probe” (within delta-proteobacteria).

Transmission electron microscopy: A pellet of “magnetotacticalLy washed’ cells wasembedded in 2% low-melting agarose (to much cell loss without that), fixed in 4%glutaraldehydeflxPBS and 2% osmium tetroxide/1xPBS, dehydrated, and embedded inplastic. Semi-thin sections (600nm) showed satisfying cell density under thelightmicroscope, but the ultra-thin sections showed relatively poor results after stainingwith 4% uranyl acetate: only one spherical cell (gram-negative cell wall type) without avisible magnetosome and one pair of magnetosomes (two parallel chains of squaresubunits) surrounded by cell debris could be found on several grids.Negative stain of unfixed cells with 1% or 4% uranyl acetate brought no results at all

Cultivation attempt: Agar tube dilution series with cells that were “magnetotacticallywashed” for 4 times yielded only in the growth of one type of contaminant: small, nonmotile, non-magnetotactic cocci.Medium Used: 0.2 gIl Na2SO4, 0,25 gIl NH4C1, 1.0 gIl NaCI, 0.4 gIl MgCl2, 0.5 gIl KC1,0.15 gIl CaCl2 in distilled water. After autoclaving lml of a vitamin solution and lmI ofSL1O mineral solution (cf. course handouts), 3Onil NaHCO3 (84g/l), 15m1 of KHPO, andlOml thioglycolate (0.lg/lOml) were added. The pH was adjusted to 7.4, the medium wasIransfered anaerobically to serum bottles.For the “agar shakes” 5m1 of this medium, lml of sterile filtered water from the enrichmentjar, and 0.2 ml of substrate (1M acetate or 1M lactate or 1M succinate) were added to 3m1of 2.1% Agar, mixed gently and gased with N2/C02 (80/20). Each series of substrateswas parallely incubated strictly anaerobically or after the addition of a volume of aircorresponding to 0.5% oxygen at room temperature in the dark.

Literature: Blackmore, R. P., Maratea, D. and Wolfe, R. S. 1979. Isolation and pureculture of a freshwater magnetic spirillum in chemically defined medium. 3. Bacteriol.140:720-729.

2. Fluorescence Spectrophotometry of Phototrophic Bacteria

fluorescence excitation and emission spectra of suspensions of untreated living cells ofphototrophic bacteria were run on a PTI “ALPHASCAN” fluorescence spectrophotometer.The purpose of these spectra is to find out if there are characteristic peaks (“colors”) ofemitted fluorescence for different groups of phototrophic bacteria, that could be used todifferentiate them at a single cell level in a fluorescence microscope or a flow cytometer.For none of more than ten purple nonsulfur bacteria fluorescence spectra could be obtained,not even after many variations in cell concentration and excitation or emission wavelength(poor energy transfer?).For one green and two purple sulfur bacteria it could be shown that excitation light targetedagainst carotenoid pigments is capable of inducing fluorescence emission in chlorophyllmolecules by energy transfer. The wavelength of the emitted light depends not only on thetype of chlorophyll but also on the association with proteins in the light harvesting systemsor reaction centers. Therefore these are not enough data to see if “signature peaks” fordifferent groups of phototrophic bacteria exist. But at least for part of them it should bepossible to distinguish them from non-phototrophic bacteria, cyanobacteria and algae bytheir infrared fluorescence after excitation with wavelengths that are available from commonlight sources and are absorbed by carotenoid pigments.

Literature: Fetisova, Z. G., Freiberg, A. M. and Timpmann, K. E. 1988. Long-rangemolecular order as an efficient strategy for light harvesting in photosynthesis. Nature.334:633-635.

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