Report
Characterizing Ancient Bacteria
T
he gemstone amber is formed by the gradual hardening of the resins of conifers and flowering plants through the aging processes of oxidation and polymerization. Amber deposits (Figure 1) occur throughout the world and range in age from more than 590 million years to less than a million years old (copal or young amber). Amber is an excellent source of molecular diversity and ancient organisms because of its preservative properties Thpcp deposits are time capsules of the Earth's biological life and metabolic and chemical diversity In 1991, we isolated a strain of Bacillus sphaericus from the abdominal contents ss a stingless bee in Dominican amber that was 25 million to 40 million years old and showed that this bacterium was of ancient origin based on methodological, phenetic, and molecular studies (1). Since the original report, this finding has been independently verified, and more than 500 microbial isolates have been obtained from ambers dating from 135 million years (Lebanese) to less than 2 million years (Colombian copal). Microorganisms isolated from amber colonized the planet during geological periods characterized by great climactic atmospheric and biological changes (1 2) and it is believed that these microorganisms must have had a rpnprtoire of cpronrlary mptahnlitpQ witVl
chemical structures that were active under a broad spectrum of environmental conditions. These novel chemical structures may provide the basis for the development of chemotherapeutic agents and other biological products. At present, there is no straightforward method for determining whether a bacte0003-2700/96/0368-609 A/S12.00/0 © 1996 American Chemical Society
paring these results with a database of values for a wide range of microorganisms. The first objective is to determine whether the fatty acid distribution in the cell membrane of the ancient bacteria lies within the range of modern populations of the species. Although the fatty acid distribution may be outside the range of modern microorganisms as a result of mutations and evolution, the probability is that it will be sufficiently close for an unequivocal identification, given agreement with other biochemical and morphological assessments. Only after the species is identified is it then practical to undertake a detailed assessment of time-based genetic mutation by comparing selected stretches of DNA sequences in ancient and modern variants.
Amber-entombed pieces of ancient life provide a window on the past and may The geological age of the amber improvide novel chemical plies the age of the bacteria. Evidence supporting the putative age of the ancient structures that could organism is provided by counting the number of base-pair differences between have therapeutic use ancient and modern variants in a stretch rium is ancient or modern—an indirect approach is required. Once tissue or other matrix has been removed from the amber, the microorganism (s) must be separated from the matrix, cultured, and purified. Once colonies are grown, the organisms can be classified by their morphological, staining, and biochemical characteristics. Species are identified by determining the distribution of cellular fatty acids in the isolated, cloned bacteria by GC and com-
Raul Cano California Polytechnic State University
of DNA and calculating the number of mutations per base pair per year over that same stretch. Data from a variety of experiments show that DNA mutation-rate calculations based on differences between modern bacteria and their amber-entombed counterparts agree favorably with results from other sources (1 2) How it's done Many of the bacteria that have been revived have built-in mechanisms for dehydration that allowed them to remain dormant for a long time. These organisms are relatively simple in structure and are therefore apparently better able to withstand the stresses inherent in the dehy-
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Report
Figure 1 . Time capsules of biological life. (a) Fungus gnat in Dominican amber, (b) Sphaerical bacterium isolated from Dominican amber. (Courtesy of Paula Orkland.)
dration process. It is thought that nonreducing sugars such as trehalose, found in the cell walls of many bacteria, help prevent the destruction of the cell during dehydration. Supporting this hypothesis is the observation that inclusion of a natural sugar such as lactose or a trehalose helps prevent a lethal disruption to such organisms preserved by freeze-drying (3). In addition to the organism's own protective mechanisms, encapsulation in amber excludes air and water. To guard against contamination, the amber specimens must be isolated, and all tools and media must be scrupulously sterilized before bacteria are extracted. The amber is opened, and the fossil tissue is teased out using a needle or other mi-
cromanipulations and transferred to a culture medium. In the case of encapsulated plant material or soil, it is sometimes necessary to first cut away extraneous material from the inclusion. Under conditions of asepsis, the inclusions are pulverized in a mortar and pestle or a homogenizer to free the organic material. The resulting material is then introduced into a standardized tripticase soy agar culture medium (4) or a medium specifically designed for the recovery of certain organisms The bacteria-containing samples are resuspended in sterile saline and poured or streaked so as to distribute them over the surface of the culture Dlates Once the individual colonies start to QTOW they are suhnilriired setrregated according to spe-
T a b l e 1 . F a t t y a c i d c o m p o s i t i o n of i s o l a t e s of B.
thuringiensis.
Ancient
Modern
Fatty acid type
AG4
AG187
AG262B
AG567
25414
8249
12:0i 12:0 13:01 13:0a 14:0i 14:0 15:01 15:0a 15:0 16:1w/7C-OH 16:0i 16:1 w/11 C 16:0 15:0 w/2 OH 17:1iw/10C 17:1iw/5C 17:11 A 17:0i 17:0a
1.46% 0.48 11.42 1.24 7.13 3.09 27.92 3.98 0.77 1.75 7.56 0.67 4.60 0.87 3.25 3.24 0.61 5.13 0.83
2.05% 0.86 13.11 1.92 5.30 3.81 25.17 3.59 0.82 1.34 7.20 0.95 5.34 0.00 1.63 2.39 0.53 6.58 1.04
1.56% 0.43 13.57 1.97 4.70 3.58 26.86 4.17 0.56 1.39 6.59 1.65 5.70 0.00 2.71 2.18 0.00 7.94 1.30
1.54% 0.41 10.65 1.80 6.86 3.58 26.63 5.45 0.73 1.74 7.64 0.76 5.31 0.64 3.03 2.44 0.85 4.82 1.27
0.67% 0.00 10.29 0.67 3.67 3.13 35.83 3.12 0.00 0.74 0.00 4.96 3.52 0.81 3.38 5.72 0.85 7.23 0.72
0.76% 0.00 9.69 1.13 5.07 2.75 32.46 4.38 0.00 1.00 0.00 7.03 5.59 0.00 3.22 3.82 0.00 8.40 1.46
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Analytical Chemistry News & Features, October 1, 1996
cies, and frozen for subsequent study. In most cases, an amber sample yields only a few species of bacteria. For fatty acid analysis, the cloned colony is grown overnight under standardized conditions. The cell mass is then extracted from the culture tube, saponified with sodium hydroxide and methanol, methylated with hydrochloric acid and methanol, and extracted in hexane and methyl tertiary butyl ether. Once cleaned with sodium hydroxide, the solution is analyzed for fatty acids by GC on a phenyl-methyl-silicon fused-silica column using flame ionization detection (5) Currently under study are four representative samples of Bacillus thuringiensis from ancient ambers 2 million to 40 million years old. These bacteria produce a characteristic crystalline protein (parasporal body), strong evidence that they all belong to the same species. Table 1 shows the eesults of a representative fatty acid methyl ester analysis of B. thuringiensis from amber and from modern sources. Fatty acid composition alone, however, is not sufficient to assess the degree of similarity (or diversity) among populations. Multivariate statistical analyses are done to better assess the distribution of fatty acid variation in populations of similar bacteria. We have extensively used principal component analysis (PCA) and discriminant analysis to graphically assess the degree variation among populations of bacteria classified in a single species. Figure 2 illustrates the PCA performed with the fattv acid data from Reference 1 on B sbhaericus Notice that most strains of B sbhaericus cluster totrether and that the ancient isolate BCA16 although related morpholoffirallv and biochemically to the rest of the strains stands alone in the outskirts of the cluster. This kind of information, consistently replicated, can be used to validate claims of antiquity. Applications
In addition to the wealth of knowledge that can be gained by manipulating and assessing living ancient organisms, the potential exists for the discovery of valuable new products. Although DNA coding for vital functions tends to be conserved, genes coding for secondary metabolic processes of ancient organisms are more likely to have mutated over time and thus
able for further characterization by MS, NMR, and other techniques. In the future The ability to recover ancient microorganisms and study them using a broad range of modern analytical tools will undoubtedly provide valuable informational benchmarks and deepen our understanding of many biochemical mechanisms and cellular processes. It also offers opportunities to discover novel products witfi wide-ranging applications. At this writing, numerous fractions of cloned ancient material and many diverse samples of amber have yet to be investigated
Figure 2. Graphical representation of PCA of the fatty acid composition of B. sphaericus.
produce novel products when compared with modern variants of the same species. Thus, antibiotics created from ancient organisms are likely to be sufficiently different from their modern counterparts that modern pathogens will not yet have evolved resistance mechanisms against them. For example, the Bacillus and Streptomyces genera, of which ancient variants are preserved and clonable, have produced antibiotics, some of which are commercially available. Because it is straightforward and relatively rapid, determination of the cellular fatty acid distribution also provides a useful method for screening organisms for the potential to produce novel metabolic products. If the objective is to search for active products among a group of organisms, ancient or modern, the likelihood of finding organisms rich in such products is greater in populations with diverse fatty acid profiles. Such an approach may be used to assess diversity at the microbial level and can be incorporated into a routine, well-defined, natural-product discovery program. In addition to antibiotics, other microbial metabolic products of interest include enzymes for laundry detergents, fabric treatment, and bioremediation; insecticidal and fungicidal proteins and peptides; and polymers for the cosmetics industry.
Isolating and characterizing metabolic products Although GC is the method of choice for bacterial characterization, LC is the technique primarily used to analyze, purify, and recover metabolic products from cloned colonies of ancient bacteria. Typical examples include nucleic acids, short peptides, polyketides, and fatty acids. After culturing, the broth is combined with an equal part of 90% aqueous acetone, centrifuged, and filtered. The resulting solution is evaporated to dryness, and the solids are dissolved in 10% dimethylsulfoxide and injected into a liquid chromatograph with a reversed-phase column and diode array UV-vis detection. In many cases, the presence of active metabolic products can be verified before chromatographic analysis. For example, antimicrobial, antifungal, or even antineoplastic activity can be determined by culturing an ancient organism of interest and testing the culture medium against a panel of bacteria, fungi, or tumor cells for growth inhibition. If a positive result occurs, the fermentation is scaled up to produce adequate biomass for purification and further assays. The LC separation is run on a preparative column to collect larger amounts of each fraction, each of which is tested against the same panel of cells. In this way, the active component is isolated and avail-
Searching out ancient microorganisms as a source for new metabolic products represents only the tip of the discovery iceberg. It is estimated that less than 5% of the microbial diversity of the planet has been retrieved (6). Currently, we are seeking to develop DNA sequencing and GC methods to assess diversity without culturing the organism—for example, by examining fatty acid methyl ester chromatograms for the presence of a predominant peak that correlates with species characteristics I wish to express my gratitude to Julius Meisel for his help with the fatty acid analysis of ancient isolates and for providing much of the data from the modern isolate of B. sphaericus and to Jorge Galazzo for his help with the LC analysis of secondary metabolites of ancient isolates. References (1) Johnsonbaugh, J.; Dodge, D.; Cano, R. AbstractttfPapers; American Society for Microbiology: New Orleans, LA, 1996; Abstract R18. (2) Cano, R. J.; Borucki, M. Science 1995, 268,1060. (3) Potts, M. Microbiol. Rev. 1994,58, 755. (4) Atlas, R. Parks, L. C; Handbook of Microbiological Media; CRC Press: Boca Raton, FL, 1993. (5) DeBoer, S. H.; Sasser, M. Can. J. Microbiol. .986,32, 786. (6) Embley, T. M.; Hirp, R. P.. Williamss D. M. Philos. Trans. R. Soc. Land. B. Biol. Sci. 1994,345, 21. Raul J. Cano, professor of biological sciences and director of the Environmental Biotechnology Institute at California Polytechnic State University, is also a microbiology and forensic biology yonsultant. Address correspondence tt him at Biological Sciences Dept, California Polytechnic ctate University, San Luis Obispo, CA A3407 (rcano@calpoly. edu).
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