Chemical Markers for Bacteria inExtraterrestrial Samples

ALVIN FOX*Department of Microbiology and Immunology, School of Medicine, University of

South Carolina, Columbia, South Carolina

ABSTRACTInterplanetary missions to collect pristine Martian surface samples for

analysis of organic molecules, and to search for evidence of life, are in theplanning phases. The only extraterrestrial samples currently on Earth arelunar dust and rocks, brought back by the Apollo (U.S.) and Luna (SovietUnion) missions to the moon, and meteorites. Meteorites are contaminatedwhen they pass through the Earth’s atmosphere, and during environmentalexposure on Earth. Lunar fines have been stored on Earth for over 30 yearsunder conditions designed to avoid chemical but not microbiological con-tamination. It has been extremely difficult to draw firm conclusions aboutthe origin of chemicals (including amino acids) in extraterrestrial samples.Of particular concern has been the possibility of bacterial contamination.Recent work using state-of-the-art gas chromatography tandem mass spec-trometry (GC-MS/MS) has dramatically lowered the chemical background,allowing a clear demonstration that lunar fines are remarkably differentfrom terrestrial dust in that they generally lack certain chemical markers(muramic acid and 3-hydroxy fatty acids) characteristic of Earth’s bacteria.Thus, lunar dust might be used as a negative control, in conjunction withGC-MS/MS analyses, in future analytical studies of lunar dust and mete-orites. Such analyses may also be important in studies designed to searchfor the presence of life on Mars. Anat Rec 268:180–185, 2002.© 2002 Wiley-Liss, Inc.

Key words: bacteria; mass spectrometry; exobiology

There is a long history of analyses of meteorites andlunar dust for their organic chemical content. A majorfocus of these studies has been to determine whether thesechemicals were innately derived from the samples or werederived from biological or chemical contamination. A sec-ondary goal has been to determine whether the com-pounds, if indeed they are innate to the extraterrestrialsamples, are biotic or abiotic in origin. The purpose of thecurrent review is to summarize where we are and whatneeds to be done, from the perspective of a bacteriologistexperienced in trace analysis of chemical markers for bac-teria. This perspective is rather unusual, since previouswork has been almost entirely performed by geochemistsand analytical chemists.

EXTRATERRESTRIAL SAMPLESAVAILABLE FOR ANALYSIS

The only extraterrestrial samples currently on Earthare lunar dust and meteorites. The lunar samples werebrought back by the Apollo (U.S.) and Luna (Soviet Union)missions to the moon (Heiken et al., 1991). Many meteor-

ites have landed here naturally. Unfortunately, they wereinvariably contaminated as they passed through theEarth’s atmosphere, and during their long residence here.Viable bacteria were not found in lunar material (collectedon the Apollo missions) when they were tested by classicalmicrobiological culture (Oyama et al., 1970). However,until recently it was unclear whether they remained pris-tine during their storage on Earth (Kozar et al., 2001).

The claim made by McKay et al. (1996) that they iden-tified ancient extraterrestrial life in carbonate formationsin the Martian meteorite ALH84001, believed to have

*Correspondence to: Alvin Fox, Department of Microbiologyand Immunology, School of Medicine, University of South Caro-lina, Columbia, SC 29208. Fax: (803) 733-3275.E-mail: afox@med.sc.edu

Received 5 December 2001; Accepted 21 February 2002DOI 10.1002/ar.10152Published online 00 Month 2002 in Wiley InterScience(www.interscience.wiley.com).

THE ANATOMICAL RECORD 268:180–185 (2002)

© 2002 WILEY-LISS, INC.

arrived on Earth 13,000 years ago, has had a great impacton the field of exobiology. However, it is unclear whetherthese carbonate formations are the result of past terres-trial microbiological activity or they were formed by inor-ganic processes (Buczynsik and Chafetz, 1991; Anders,1996). The presence of polyaromatic hydrocarbons (PAHs)may represent the presence of indigenous fossilized bac-teria. Isoprenoids and chlorophyl (from bacterial andplant sources) can generate such compounds by loss ofoxygen, and PAHs are common in fossil fuels (Volkmanand Maxwell, 1964; Kissin, 1993). ALH84001 could alsohave been naturally contaminated with PAHs duringlong-term exposure on Earth. Magnetite, which may bederived from magnetotactic bacteria (Friedmann et al.,2001), is also present. However, a recent review (Gibson etal., 2001) noted that it is likely that there is terrestrialcontamination in ALH84001, and it may be necessary toawait the return of pristine samples from Mars to deter-mine whether life ever existed there.

AMINO ACIDS AND LIFE DETECTIONAmino acids are found in meteorites, but at only slightly

lower levels than in the surrounding terrestrial terrain(Kvenholden et al., 1970; Engel and Nagy, 1982; Bada etal., 1983; Engel et al., 1990; Cronin and Pizzarello, 1997;Engel and Macko, 1997; Glavin et al., 1999). Amino acidsare present at much lower levels in lunar dust. However,lunar samples are readily distinguished from blanks byamino acid levels, which suggests that the source of theamino acids is not the analytical procedure (Ponnampe-ruma et al., 1970; Harada et al., 1971; Nagy et al., 1971;Brinton and Bada, 1996). In any event, the presence ofdetectable levels of amino acids in blanks does complicatedata interpretation.

Whether amino acids in meteorites originate from ex-traterrestrial/terrestrial or biotic/abiotic sources remainscontroversial (Engel and Nagy, 1982; Bada et al., 1983;McKay et al., 1996; Ehrenfreund et al., 2001). As notedabove, meteorites are invariably contaminated on passingthrough the Earth’s atmosphere, and during their longresidence here. Thus it is difficult to rule out contamina-tion by terrestrial life as the source of these amino acids.Several attempts have been made to prove the uniquenessof amino acids found in extraterrestrial samples by assay-ing enantiomeric excesses, by determining the presence ofnonprotein amino acids, and by stable isotope enrichment.However, all of these findings could be explained by bac-terial contamination.

Enantiomeric ExcessMeteorites contain both L- and D-amino acids, with the

former in excess. It has been suggested that amino acidson Earth are generally derived from proteins that are ofthe L- configuration. It has also been postulated that abi-otic (chemical) processes should generate amino acidswith an equal ratio of D- and L-amino acids (Bada, 1997;Cronin and Pizzarello, 1997). Thus a D-amino/L-aminoacid ratio not equal to one was suggested to be evidence ofterrestrial contamination. What was not discussed is thatthere is a major source of D-amino acids in nature: bacte-rial cell walls. Indeed, there are extremely high concen-trations in terrestrial dust; at the one part per 10,000 level(Sonesson et al., 1988). The primary D-amino acids areD-glutamic acid and D-alanine, which are commonly

found in meteorites. Determining the source of D-aminoacids in extraterrestrial samples clearly requires furtherinvestigation.

Presence of Nonprotein Amino AcidsSome unusual amino acids (e.g., 2-amino-2,3-dimeth-

ylpentanoic acid) have been reported to be unique to me-teorites (Cronin and Pizzarello, 1997). However, the dis-tribution and concentration of 2-amino-2,3-dimethyl-pentanoic acid in terrestrial samples has not been exhaus-tively studied. Other nonprotein amino acids, e.g., orni-thine and diaminopimelic acid, are widely found in bacte-rial peptidoglycan (PG) (Schleifer and Kandler, 1972) andare readily detected in organic dust as a consequence ofbacterial contamination (Sonesson et al., 1988; Ueda etal., 1989).

Stable Isotope EnrichmentDifferences in stable isotope concentrations in meteor-

ites relative to their terrestrial counterparts may be anindicator of extraterrestrial origin (Engel and Macko,1997). However, bacterial growth on a meteorite mightincorporate carbon, e.g., from endogenous carbonatepresent in the meteorite (Tull et al., 1998). Thus the aminoacid would appear to have originated from space, but mayactually have been synthesized from simpler precursorson Earth.

In contrast to meteorites, lunar dust samples have beenstored under strict isolation conditions (Allton et al.,1998). Therefore, it is less likely that the amino acidspresent in lunar fines represent terrestrial contamination.However, it is still possible that contamination occurredduring transport to Earth or during curation. The space-ships used in the Apollo missions were documented to becontaminated with bacteria (Puleo et al., 1970, 1973,1977). Furthermore, the lunar curation facility was de-signed to avoid chemical but not microbiological contami-nation. If amino acids in lunar dust are not derived fromterrestrial sources, they must be from extraterrestrialsources (whether biotic or abiotic).

PYROLYSIS GAS CHROMATOGRAPHY-MASSSPECTROMETRY STUDIES

Experiments employing pyrolysis (pyr) gas chromatog-raphy-mass spectrometry (GC-MS), one of the first ana-lytical chemical techniques used for studies of lunar dust,have demonstrated the presence of trace levels of organiccarbon in the lunar samples (Burlingame et al., 1970).However, it proved difficult to categorically state thatthese compounds are of lunar origin as opposed to beingformed by terrestrial contamination (e.g., from microbes).Pyr GC-MS involves heating a sample in the range of400-1,000°C in the absence of oxygen. Complex organicmonomers, oligomers, and polymers are converted intosimpler molecules (e.g., CO and CH4 ) that are separatedby gas chromatography and structurally identified bymass spectrometry (Nagy et al., 1971). Unfortunately, pyrresults in a complex series of reactions, including scission,rearrangement, and dehydration of chemical bonds. Thusit is often difficult to relate the structure of pyr products tothe parent molecules present in bacteria and other lifeforms (Simmonds, 1970; Eudy et al., 1985; Smith et al.,1987; Watt et al., 1991; Knabner-Kogel, 2000).

It has proven tremendously difficult to perform pyr-GC-MS studies on the surface of Mars (Biemann et al.,

181CHEMICAL MARKERS FOR BACTERIA

1977). Indeed, the initial results were widely interpretedas indicating the absence of organic carbon or life. How-ever, a recent reexamination of these data suggests thatbacteria could have been present and missed (Glavin etal., 2001).

CHEMICAL MARKERS FOR BACTERIAStandard culture techniques are able to detect bacteria

when the nutritional requirements and culture conditionsare known. However, most environmental organisms arenon-culturable and remain undetected. Analytical micro-biology techniques (primarily GC-MS) detect not only vi-able bacteria, but also their nonviable cell envelope rem-nants when present in complex biological matrices(Maitra et al., 1978; Fox et al., 1980; Findlay et al., 1983;).More recently, gas chromatography tandem mass spec-trometry (GC-MS/MS) has been proven to be capable ofdetecting terrestrial bacterial markers at minute concen-trations in environmental and clinical samples (Fox et al.,1995, 1996; Saraf and Larsson, 1996; Saraf et al., 1999;Bal and Larsson, 2000; Kozar et al., 2000). GC-MS/MSwas only recently used for the first time in studies ofmaterial of extraterrestrial origin (Kozar and Fox, 2001).

There are a number of substances present in the cellenvelope of bacteria that are of limited distribution innature, including muramic acid (3-O-lactyl, 2-deoxy-2-amino glucose) and certain 3-hydroxy fatty acids (3-OHFAs). Muramic acid (Mur) is both an amino acid and anamino sugar, and is a constituent of the cell wall PGbackbone of Gram-positive (Gm�) and Gram-negative(Gm–) bacteria. PG consists of a backbone of repeatingalternating subunits of Mur and glucosamine. There areattached tetra- or penta peptide side chains andcrosslinks. Unlike proteins (which contain exclusively L-amino acids) there are both D- and L-amino acids in PG.The most common amino acids in PG are D-glutamic acid,L-alanine, and D-alanine (Schleifer and Kandler, 1972),which are among the major amino acids found in meteor-ites (Engel and Nagy, 1982). Since PG is the primarysource of both Mur and D-amino acids in terrestrial sam-ples, they generally are present in similar concentrations(at the 10–100 parts per million (ppm) level) (Sonesson etal., 1988; Fox et al., 1993).

Lipopolysaccharide (LPS) is a component of the Gm–

bacterial cell envelope. LPS is found in the surface layer ofthe outer membrane, but not in the cytoplasmic mem-brane. The structure of LPS is similar among all Gm–

bacteria and is composed of an outer O antigen, a middlecore region covalently bound to a glycolipid termed lipid A.The lipid A portion of LPS is composed of a disaccharide ofglucosamine with covalently bound 2- and 3-OH FAs (Ri-etschel, 1984).

In a recent study using GC-MS/MS, levels of Mur and3-OH FAs were assayed in lunar samples that have beenstored at the Johnson Space Center (JSC) under strictisolation conditions. This was the first study of chemicalmarkers, specific for bacteria, in lunar material (or indeedany other sample of extraterrestrial origin). It was notedthat lunar and terrestrial samples are strikingly differentin that chemical markers are absent in the former. Theability to measure trace levels of chemical markers forEarth bacteria in extraterrestrial samples would greatlyaid future studies designed to confirm the extraterrestrialorigin of amino acids. The absence of chemical markers forEarth bacteria would indicate that samples are not con-

taminated, and, consequently, if organic chemicals arepresent, they could not be from this source (Kozar et al.,2001).

ANALYTICAL MICROBIOLOGICALCONSIDERATIONS

There have been extensive chemical, and some biologi-cal, characterizations of lunar samples and meteorites.For example, as noted above, amino acids have been de-tected at trace levels. In recent years GC-MS and high-performance liquid chromatography with fluorescence de-tection (LC-fluor) have primarily been used for identifyingamino acids in lunar dust and meteorites. GC-MS in totalion mode allows identification when compounds arepresent at relatively high concentrations. However, co-eluting peaks are often present which may confound iden-tification (Pizarello and Cronin, 2000). Alternatively, se-lected ion monitoring (SIM) can home in on characteristicfeatures in a mass spectrum, thereby eliminating much ofthis background. This dramatically lowers the detectionlimit for trace analysis. Unfortunately, definitive identifi-cation is lost in SIM mode.

In LC-fluor analysis, amino groups, which are presentin amino acids and other basic organic materials, areconverted to fluorescent derivatives prior to analysis. Thistechnique is the most sensitive approach available forscreening for such organic materials. Unfortunately, anycompound with an amino group is labeled, and thus back-ground interferences are high. As noted by Glavin et al.(1999), for definitive identification it is vital that suchresults be confirmed by mass spectrometry. Unfortu-nately, the mass spectrometric procedures used in thatwork were too insensitive to corroborate LC-fluor analy-ses.

GC-MS/MS for trace analysis of chemical markers forbacteria in environmental matrices has been used in theU.S. and Sweden for the past 6 years. GC-MS/MS, likeGC-MS, employs a high-resolution GC separation. How-ever, the exquisite selectivity of MS/MS detection dramat-ically reduces background, thereby lowering detection lim-its approximately two orders of magnitude over GC-MS(employing SIM). Furthermore, the product ion spectrum(MS/MS chemical fingerprint) allows for categorical iden-tification of chemical identity at parts per billion (ppb)levels (Kozar et al., 2001).

LC-fluor and GC-MS/MS are complementary tech-niques. LC-fluor will detect all compounds that haveamino groups, including amino acids, with high sensitiv-ity. GC-MS/MS (which has comparable sensitivity butmuch greater specificity) allows definitive analysis, butmust target specific chemicals that are likely to bepresent. Thus, LC-fluor is a useful screening method totarget more definitive analysis by GC-MS/MS.

Classical microbiology techniques focus on the detectionof live bacteria. The LC-fluor, GC-MS, and GC-MS/MSmethods alternatively detect both viable bacteria andtheir nonviable cell wall remnants. The most unconven-tional technique, GC-MS/MS, has proven capable of de-tecting terrestrial bacterial markers at minute concentra-tions (�100 ppb) in a variety of complex environmentaland clinical matrices (Fox et al., 1996; Krahmer et al.,1998, 1999; Saraf et al., 1999; Kozar et al., 2000; Liu et al.,2000). However, GC-MS/MS has been used in only oneextraterrestrial study to date (Kozar et al., 2001). In thatwork it was demonstrated that pristine lunar dust does

182 FOX

not contain chemical markers for bacteria, and therefore itcan be used as a negative control in studies of othersamples of extraterrestrial origin. In view of the longhistory of difficulties in interpreting results due to chem-ical and biological contamination (Tasch, 1964), this resultcould be a highly significant observation.

PLANS TO VISIT MARS TO COLLECTSAMPLES FOR LIFE DETECTION STUDIESIn order to directly address the issue of whether life

exists beyond the Earth, NASA is planning a series ofmissions to retrieve Martian surface samples for analysis.Contamination of these samples with terrestrial materialduring passage to Earth, or upon subsequent curation,would be a catastrophic loss for the scientific community.These studies will be particularly important since exper-iments conducted by the Viking expeditions to Mars weregenerally interpreted as negative for organic matter anddetection of life (Biemann et al., 1977). This does not ruleout the possibility of organic matter and/or life existingelsewhere on Mars, especially below the ultraviolet-irra-diated surface of the planet (Weiss et al., 2000). Recentreports of detection of organic molecules in meteorites(Glavin et al., 1999), simple sugars in interstellar clouds(Hollis et al., 2000), and the probability of liquid water onJupiter’s moon, Europa (Kivelson et al., 2000), serve tointensify the search for extraterrestrial life. The discoveryof planets revolving around stars has also intensified thedebate about the possibility of life on other planets(Lunine, 1999).

As noted above, the only extraterrestrial samples cur-rently on the Earth are lunar dust, collected on the Apolloand Luna missions to the Moon, and meteorites. Unfortu-nately, meteorites are contaminated on passing throughthe Earth’s atmosphere and during environmental expo-sure on earth. Thus, it has been difficult to interpret thebiological or chemical origin of compounds found in mete-orites (Kvenholden et al., 1970, 1971; Engel and Nagy,1982; Bada et al., 1983; Engel et al., 1990; McKay et al.,1996; Cronin and Pizzarello, 1997; Engel and Macko,1997; Bada et al., 1998; Glavin et al., 1999; Ehrenfreundet al., 2001;). Organic substances have also been detectedin lunar dust, but at much lower (trace) levels (Burlin-game et al., 1970; Ponnamperuma et al., 1970; Harada etal., 1971; Nagy et al., 1971; Brinton and Bada, 1996).

The spacecraft used in the Apollo missions were con-taminated with terrestrial bacteria (Puleo et al., 1977).However, viable bacteria were not detected (using classi-cal microbiological culture) in the lunar material when itwas first brought by the Apollo missions (Oyama et al.,1970). Unfortunately, chemical methods that detect deadbacterial remnants, or non-culturable bacteria, were in aprimitive stage of development at that time (Maitra et al.,1978; Fox et al., 1980, 1993, 1995, 1996; Findlay et al.,1983; Saraf and Larsson, 1996; Saraf et al., 1999; Bal andLarsson, 2000; Kozar et al., 2000).

The lunar samples have been stored under strict isola-tion conditions for the past 30 years (Allton et al., 1998). Itis possible that terrestrial contamination could have oc-curred during this extended curation period. Although itwas hypothesized that “pristine” lunar dust lacks chemi-cal markers for terrestrial bacteria, this remained to bedemonstrated experimentally until only recently. The ex-perimental test of this hypothesis was the primary subjectof a recent study (Kozar et al., 2001). That work also

provided an important negative baseline for future studiesof extraterrestrial samples (e.g., from Mars).

CONCLUSIONSDespite a great deal of research, the origin of organic

chemicals (including amino acids) in samples of extrater-restrial origin is still far from clear. Part of the problemstems from the difficulty of obtaining definitive analyses oftrace organics in complex environmental matrices. An-other problem is the ubiquitous contamination of meteor-ites with terrestrial bacteria.

Kozar et al. (2001) proposed a null hypothesis, as fol-lows: Pristine lunar samples, since they come from a sat-ellite believed to be void of life, should not contain markersfor terrestrial bacteria. This has been confirmed experi-mentally (Kozar et al., 2001). As noted above, to ourknowledge this is the first study of the levels of specificchemical markers (e.g., Mur and 3-OH FAs) for terrestrialbacteria in a curated lunar sample (or any other sample ofextraterrestrial origin). State-of-the art GC-MS/MS meth-odology was employed to eliminate the background thatoften complicates interpretation of data at trace levels.

If, in future studies, amino acids are detected in pristinelunar samples (as shown by a lack of Mur and 3-OH FAs),this would suggest that they are not of terrestrial origin.Interpretation of the amino acid content of meteorites willbe more difficult. Finding that D-amino acid levels aresimilar to those of 3-OH FAs and Mur would indicate thatall are primarily of bacterial origin. This will reinforce theneed to collect uncontaminated samples from Mars.

Other difficulties stem from limited communication be-tween geochemists and microbiologists. The former have agreat deal of knowledge about space science, but less ex-perience with microbial chemistry. The classical microbi-ologist, on the other hand, has not spent a great deal oftime speculating on the origin of life or how to study itexperimentally. It is hoped that this review may play apart in encouraging further interdisciplinary interactions,which will be extremely important as we begin to drawconclusions about the presence of life in a previously un-studied world.

Contamination of samples can occur during their trans-port and/or long-term curation. In future interplanetarymissions, assessing the levels of chemical markers, bothpre- and post-mission, would be advisable in life-detectionstudies. Current protocols for sample storage in the LunarCuration Facility at JSC appear to have been successful atsafeguarding the lunar sample collection from terrestrialbacterial contamination. However, this facility has beenprimarily concerned with keeping the collection cleanfrom chemical contamination. It may be necessary in fu-ture planetary missions to store subsets of samples duringboth the return mission and curation to minimize biolog-ical contamination, using standard microbiological ap-proaches (e.g., storage at –20 to 70°C).

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