Microbial Diversity Dianne Ahmann 1993 I. Search for Degraders of Methylated Purines ABSTRACT An attempt was made to cultivate microorganisms able to utilize one of the methylated purines caffeine, theobromine, or theophylline as a sole source of carbon. Five freshwater and three marine samples were chosen as inocula on the basis of natural proximity to decaying plant matter. Freshwater enrichments were incubated under varying concentrations of sulfate, hydrogen, and formate, while marine enrichments were kept under a single, “standard” marine condition. Each enrichment condition was tested in the presence of caffeine, theobromine, theophylline, and in the absence of any methylated purifies. Analysis of purine concentrations was accomplished by HPLC. Cultures which showed purine loss of 20% or more, as well as greater turbidity than their purine-lacking controls, were transferred to liquid media after twelve days. After four days, transfers which showed turbidity and purine loss of 25% or more were examined for cell morphology, analyzed for acetate concentration by HPLC, and transferred ,— to solid media in an effort to isolate single colonies. Theobromine concentrations were determined for all original enrichment cultures after 18 days. Results indicate that theobromine degraders are ubiquitous but slow acting. Demethylated intermediates, such as xanthine, were not found in any cultures. Certain sources of inocula and certain conditions consistently produced greater purine degradation than others.
Transcript
Microbial Diversity Dianne Ahmann1993
I. Search for Degraders of Methylated Purines
ABSTRACT
An attempt was made to cultivate microorganisms able to utilize oneof the methylated purines caffeine, theobromine, or theophylline as a solesource of carbon. Five freshwater and three marine samples were chosenas inocula on the basis of natural proximity to decaying plant matter.Freshwater enrichments were incubated under varying concentrations ofsulfate, hydrogen, and formate, while marine enrichments were keptunder a single, “standard” marine condition. Each enrichment condition wastested in the presence of caffeine, theobromine, theophylline, and in theabsence of any methylated purifies.
Analysis of purine concentrations was accomplished by HPLC.Cultures which showed purine loss of 20% or more, as well as greater
turbidity than their purine-lacking controls, were transferred to liquidmedia after twelve days. After four days, transfers which showedturbidity and purine loss of 25% or more were examined for cellmorphology, analyzed for acetate concentration by HPLC, and transferred
,— to solid media in an effort to isolate single colonies.Theobromine concentrations were determined for all original
enrichment cultures after 18 days.Results indicate that theobromine degraders are ubiquitous but slow
acting. Demethylated intermediates, such as xanthine, were not found inany cultures. Certain sources of inocula and certain conditions consistentlyproduced greater purine degradation than others.
REPORT
Background:Caffeine, theobromine, and theophylline are methylated derivatives ofxanthine which are produced primarily by tropical plants and are thoughtto function in plant defense systems. As toxic and carcinogenic constituentsof tea leaves, coffee and cacao beans, and cola nuts, they are the drugs
most widely used by humans. Each of the three is resistant to microbialdegradation, and examination of both aerobic and anaerobic purinedegradation pathways suggests that the positioning of the methyl groupsmay interfere with the enzymes which typically begin purine degradationby ring cleavage. Anaerobic degraders of methylated purines have notbeen studied extensively, if at all, and the purpose of this project was todiscover characteristics of such microorganisms.
Enrichment cultures:Samples of compost, garden pond (eutrophic, methanogenic) sediment,
sewage sludge, cedar swamp sediment, and higher termite (Nasutitermes)hindgut were inoculated into freshwater medium under each of thefollowing conditions:
1=1mM sulfate with H2/COz headspace11=1mM sulfate with 2.5mM formate and N2/CO2 (80:20) headspaceffl=lmM sulfate with N2/C02 headspace1V=28mM sulfate with Nz/C02 headspace
Samples of Sippewissett salt marsh phototroph-rich sediment, salt marshphototroph-poor sediment, and ocean sediment were inoculated intomarine medium with N2/CO2 headspace.
Caffeine, theobroniine, and theophylline were added at 5mMconcentrations, and PdC12 and H2 were used as a reducing agent. Allcultures were incubated at 25°C in the dark.
Analytical methods:Caffeine, theobromine, and theophylline were resolved and
quantitated using HPLC. Separation was accomplished by a reverse-phaseCg column, 15cm X 4mm, in 20mM phosphate buffer (pH 2.5) with 25%methanol. Compounds were detected by UV adsorption at 2 lOnm.Sampling error was tested and was found to be approximately 2%.
Acetate analysis was also accomplished by HPLC, using a Waterscolumn designed for separation of volatile fatty acids with a refractometerdetector and .1% H3P04 buffer.
,Th Materials:U Caffeine, theobromine, and theophylline were purchased from Sigma.
pn-i-L
C Results and Conclusions:The enclosed tables indicate transferred cultures, results of HPLC
analyses, and cell morphologies.Theobromine appears to be the most readily degradable of the three
methylated purines, as the only transferred cultures showing purine lossafter four days were those grown in the presence of theobromine. Inaddition, all original enrichment cultures showed at least 18% loss oftheobromine by day 18, with several degrading as much as 50%. Noneshowed loss of more than 52%, however, indicating that while theobrorninedegraders may be ubiquitous, they are nevertheless slow in theirdegradation.
No demethylated intermediates, such as xanthine, were detected inpurine-degrading cultures at any time. Degradation by initialdemethylation was hypothesized as a potential pathway, but thishypothesis was not supported. If initial demethylation is, in fact, occurringto transform the methylated compounds into xanthine, which is thencleaved by the known purine catabolic enzymes, then such demethylationmust be the rate-limiting step in the process.
Of the inocula chosen, some produced consistently greater purine lossunder all conditions. In the case of theobromine degradation, garden pondsediment was most effective, with purine disappearance averaging 45-50%. Sippewissett marsh and ocean sediments were also effective,degrading 40-45% of the theobrontine. Sewage sludge and swampsediments were the least effective, with an average purine loss of onlyabout 30%.
Of the freshwater conditions tested, enrichments under condition I(methanogenic) consistently degraded slightly more theobromine thanthose under other conditions, while sulfidogenic enrichments consistentlyproduced below-average losses of theobromine.
As no autoclaved controls were examined for comparison with liveenrichment cultures, microbiological activity cannot be assumed to havecaused all observed purine loss. Some purines may have bound firmly tosolid particles or may have been destroyed by chemical means, thusescaping detection by HPLC. These possibilities should be checked in futureexperiments.
Recommendations:A future attempt to isolate degraders of methylated purines would
benefit from narrowing the enrichment panel considerably and followingenrichment and transfer cultures more closely. Conditions II and lv, as
Owell as sewage sludge and cedar swamp inocula, should be the firstparameters to be eliminated.
Data from transferred cultures
O inoculum turbidity acetate (+1-) purine loss cell morphology
loss (%) loss (%) loss (%) loss (%) loss (%)day4 day6 day8 dayl2 dayla
termite I C 0hindgut Tb 0 41
Tp
II CTb 34Tp
Ill C 0 100C transfer
Tb 0 35 41Tb transfer 32
Tp
IV CTb 39Tp
termite Ill Cmicroaerophiles
termite A (1% oxygen) 14 15termite B (5% oxygen) 21 23termite C (anaerobic) 1 6 8termite D (5% oxygen) 1 0 1 3termite E (1% oxygen) 8 8
salt marsh V Csediment 1 Tb 45
Tp
salt marsh V Csediment 2 Tb 36
Tp
ocean V C 42sediment Tb
Tp
0
r
-N Microbial Diversity Dianne Ahmann1993
II. Investigation of chemotaxis in sulfate-reducing bacteria
ABSTRACT
A motile culture of sulfate-reducing bacteria was examined forchemotactic behavior toward sulfate. Simple anaerobic chemotaxischambers were constructed from glass slides, cover slips, and flat capillarytubes for observation of cell behavior without use of a glovebox.Chemotactic migration of bacteria toward sulfate and away from air wereobserved.
REPORT
Background:Many motile bacteria have been observed to use their motility for
travel from less favorable toward more favorable conditions: Rbizobiuznmeliloci and Agrobacteriuxn turnefaciens travel toward specific plant
N compounds, Aquaspfrillum snagnetotacticum travels north (in the northerni hemisphere) and usually to lower redox potentials as a result, B. coil moves
toward a variety of sugars, and many aerobes are known to seek oxygen.Oxygen is apparently the only terminal electron acceptor which has beenstudied as a chemoattractant, however, and in addition, very little isknown about chemotaxis of anaerobes. Chemotaxis of sulfate-reducingbacteria, for example, appears to be an entirely unexplored area. Sincemany sulfate-reducing bacteria are highly motile, and because theiroptimal growth requires a terminal electron acceptor such as sulfate ornitrate, the question was posed: Do motile sulfate-reducing bacteria exhibitchemotaxis toward sulfate?
Bacteria and culture conditions:The culture of sulfate-reducing bacteria used in these experiments
was originally obtained as an enrichment of freshwater sediment undersulfidogenic conditions. The culture has been propagated in freshwatermedium supplemented with lactate and sulfate, and it appears to be amonoculture of vibrioid forms. Since the bacteria have never been isolatedfrom single colonies, however, the culture cannot be considered pure. Thecells are highly motile, with nearly 100% swimming rapidly inexponentially growing cultures.
Before chemotaxis experiments, cultures were maintained in anoxicC freshwater medium with low (5mM) sodium sulfate and 10mM lactate sothat cells would be limited by available sulfate. Cultures were transferredevery two days to sustain rapid growth and motility. PdCl2 and H2 wereused as the reductant in all culture media.
Chemoattractant solutions:Experimental chemoattractants consisted of anoxic, reduced
freshwater medium with 10mM lactate and 0, 1, or 10mM sodium sulfate.Solutions were reduced with PdC12 and H2.
Capillary tubes:Flat capillary cubes known as “Microslides” (part number 5010; path
length .1mm) were obtained from Vitro Dynamics Inc., Rockaway, NJ.
Chemo taxis chamber:Each chemotaxis chamber was assembled from a standard glass slide,
a cover slip, two flat capifiary tubes, and a molten mixture of Vaseline andparaffin known as Vaspar. At one end of the slide, two small fragments ofthe flat capillary tube were positioned to support the edges of the coverslip, and the cover slip was placed over them. This structure was then
Osealed by a liberal application of Vaspar, extending from the edges of thecover slip to the edges of the slide on three sides. The open end of thecover slip-chamber faced the length of the slide.
Anoxic samples of the cell culture and the chemoattractant solutionwere taken in intl plastic syringes, and within the syringes they remainedreduced for several minutes. The chamber was filled with the cellsuspension by holding the slide vertically, inserting the tip of the needleinto the space between the slide and the cover slip, and slowly ejecting thefluid so that no air bubbles were trapped. When the chamber was filled toslightly overflowing, a flat capillary was grasped with forceps, clipped intothe tip of the syringe in which the attractant solution was stored, andslipped under the cover slip so that it protruded about 5mm into the cellsuspension. The chamber was then thoroughly sealed with Vaspar,including the sides and distant end of the attractant-bearing capillary. Thecomplete assembly was then placed into a large tube, gassed with N2/COz,stoppered and parafilmed, and stored in the dark for several (10-45)minutes.
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Results and Conclusions:After 45 minutes of anaerobic incubation, cells were observed to have
migrated several millimeters into capifiaries bearing medium with 1 or10mM sulfate, but not in those carrying sulfate-free medium. Fan-likeaccumulations at capifiary openings were first observed in peculiar bandformations, in which individual bacteria appeared to be stuck to concentricarcs radiating into the tubes. These bands were subsequently observed inempty capillaries, however, and consequently pattern formations wereregarded purely as an artifact. Further examination revealed that thecapifiary tubes were apparently banded only at the ends touching thebottom of their package cylinder, and repeat experiments using the non-banded ends again showed very high cell accumulation in sulfate-containing capillaries relative to controls.
Initial experiments used chemotaxis chambers with less effective gasexclusion and were incubated in air; as a result, tactic behavior toward airbubbles was also observed. In response to an air bubble emerging beneaththe cover slip, cells retreated, leaving a clear zone near the bubble with adense band of cells just beyond.
These results indicate that the cells in the sediment enrichment ofmotile sulfate-reducing bacteria are capable of tactic behavior. Oxygenappears to serve as a repellent, while sulfate apparently attracts the cells.
The results also show that chemotaxis of anaerobes can bequalitatively studied outside a glovebox with the use of flat capifiary tubesand a sealed chemotaxis chamber. Although quantitation was notattempted, capifiaries used in an assay such as the one described abovecould be emptied into molten agar and diluted serially for estimation ofbacterial numbers.
