David M. Ward

Ribosomal RNA Analysis of Microorganisms as They Occur in Nature

Advances in molecular biology are now providing the means for solving long-standing problems in microbiology. One of the best examples is the development of a rational approach to the phylogenetic classification of microorganisms, based on comparative analysis of slowly evolving molecular components, most notably ribosomal RNAs (Woese, 1987). Molecular biologists and microbiologists have been quick to recognize how rRNA sequence variation could be used to answer major questions limiting progress in microbial ecology. Only a few years after the initial rRNA-based phylogenetic observations were published (Woese and Fox, 1977), the 16S rRNA molecule was used to characterize Prochloron, an uncultivated symbiont of marine invertebrates (Seewaldt and Stackebrandt, 1982), and the smallest ribosomal RNA molecule, 5S rRNA, was used to analyze the composition of a few simple microbial communities (Stahl et al., 1984, 1985; Lane et al., 1985b). Some further ecologic work with 5S rRNA has appeared (Colwell et al., 1989), but extensive community analysis with this molecule is complicated by the difficulty of physically separating 5S rRNAs, and by the relatively small size and thus limited information content of this molecule. In the last few years, considerable emphasis has been given in both microbial phylogeny and microbial ecology to the development of methods for studying the larger and more informative rRNAs. Most of the work has been with small ribosomal subunit rRNA (SSU rRNA, 16S in prokaryotes and 18S in eukaryotes), though a limited amount of work has been done with the larger rRNAs of large ribosomal subunits (here termed LSU rRNA, 23S in prokaryotes and 28S in eukaryotes) and with internal transcribed spacer (ITS) regions separating rRNA genes.

Identifying the fundamental units of bacterial diversity: A paradigm shift to incorporate ecology into bacterial systematics

The central questions of bacterial ecology and evolution require a method to consistently demarcate, from the vast and diverse set of bacterial cells within a natural community, the groups playing ecologically distinct roles (ecotypes). Because of a lack of theory-based guidelines, current methods in bacterial systematics fail to divide the bacterial domain of life into meaningful units of ecology and evolution. We introduce a sequence-based approach (“ecotype simulation”) to model the evolutionary dynamics of bacterial populations and to identify ecotypes within a natural community, focusing here on two Bacillus clades surveyed from the “Evolution Canyons” of Israel. This approach has identified multiple ecotypes within traditional species, with each predicted to be an ecologically distinct lineage; many such ecotypes were confirmed to be ecologically distinct, with specialization to different canyon slopes with different solar exposures. Ecotype simulation provides a long-needed natural foundation for microbial ecology and systematics.

A natural species concept for prokaryotes

Direct molecular analyses of natural microbial populations reveal patterns that should compel microbiologists to adopt a more natural species concept that has been known to biologists for decades. The species debate can be exploited to address a larger issue - microbiologists need, in general, to take a more natural view of the organisms they study.

Microbial population dynamics associated with crude-oil biodegradation in diverse soils.

ABSTRACT Soil bacterial population dynamics were examined in several crude-oil-contaminated soils to identify those organisms associated with alkane degradation and to assess patterns in microbial response across disparate soils. Seven soil types obtained from six geographically distinct areas of the United States (Arizona, Oregon, Indiana, Virginia, Oklahoma, and Montana) were used in controlled contamination experiments containing 2% (wt/wt) crude oil spiked with [1-14C]hexadecane. Microbial populations present during hydrocarbon degradation were analyzed using both 16S rRNA gene sequence analysis and by traditional methods for cultivating hydrocarbon-oxidizing bacteria. After a 50-day incubation, all seven soils showed comparable hydrocarbon depletion, where >80% of added crude oil was depleted and approximately 40 to 70% of added [14C]hexadecane was converted to 14CO2. However, the initial rates of hydrocarbon depletion differed up to 10-fold, and preferential utilization of shorter-chain-length n-alkanes relative to longer-chain-length n-alkanes was observed in some soils. Distinct microbial populations developed, concomitant with crude-oil depletion. Phylogenetically diverse bacterial populations were selected across different soils, many of which were identical to hydrocarbon-degrading isolates obtained from the same systems (e.g., Nocardioides albus, Collimonas sp., and Rhodococcus coprophilus). In several cases, soil type was shown to be an important determinant, defining specific microorganisms responding to hydrocarbon contamination. However, similar Rhodococcus erythropolis-like populations were observed in four of the seven soils and were the most common hydrocarbon-degrading organisms identified via cultivation.

Mid-chain branched mono- and dimethyl alkanes in hot spring cyanobacterial mats: A direct biogenic source for branched alkanes in ancient sediments?

Hot spring cyanobacterial mats from Yellowstone National Park, U.S.A. and the Orakei Korako thermal area, New Zealand have been found to contain suites of monomethyl alkanes, compounds frequently reported in cultured and natural populations of cyanobacteria. The complete range of possible structural isomers for mid-chain branching for C17/C18 and C16/C18 alkanes occurs in the two mats, respectively. Several C19 dimethyl and C20 multibranched alkanes were also observed as major hydrocarbons of the Orakei Korako mat. The recognition of series of mid-chain branched alkanes in modern cyanobacterial mats suggests that their occurrence in ancient sediments may reflect direct biogenic contributions, rather than formation by diagenetic processes.

Population level functional diversity in a microbial community revealed by comparative genomic and metagenomic analyses.

In microbial mat communities of Yellowstone hot springs, ribosomal RNA (rRNA) sequence diversity patterns indicate the presence of closely related bacterial populations along environmental gradients of temperature and light. To identify the functional bases for adaptation, we sequenced the genomes of two cyanobacterial (Synechococcus OS-A and OS-B′) isolates representing ecologically distinct populations that dominate at different temperatures and are major primary producers in the mat. There was a marked lack of conserved large-scale gene order between the two Synechococcus genomes, indicative of extensive genomic rearrangements. Comparative genomic analyses showed that the isolates shared a large fraction of their gene content at high identity, yet, differences in phosphate and nitrogen utilization pathways indicated that they have adapted differentially to nutrient fluxes, possibly by the acquisition of genes by lateral gene transfer or their loss in certain populations. Comparisons of the Synechococcus genomes to metagenomic sequences derived from mats where these Synechococcus stains were originally isolated, revealed new facets of microbial diversity. First, Synechococcus populations at the lower temperature regions of the mat showed greater sequence diversity than those at high temperatures, consistent with a greater number of ecologically distinct populations at the lower temperature. Second, we found evidence of a specialized population that is apparently very closely related to Synechococcus OS-B′, but contains genes that function in the uptake of reduced ferrous iron. In situ expression studies demonstrated that these genes are differentially expressed over the diel cycle, with highest expression when the mats are anoxic and iron may be in the reduced state. Genomic information from these mat-specific isolates and metagenomic information can be coupled to detect naturally occurring populations that are associated with different functionalities, not always represented by isolates, but which may nevertheless be important for niche partitioning and the establishment of microbial community structure.

The importance of physical isolation to microbial diversification.

The importance of physical isolation, defined as the spatial separation of two or more populations, to the evolution of organisms has been well studied in plants and animals yet its significance regarding microbial evolution has not been fully appreciated. Here we review the theoretical paradigm of physical isolation for the diversification of organisms in general and then provide a variety of evidence indicating that microbial populations also fit into a similar evolutionary framework.

Distinctive hydrocarbon biomarkers from fossiliferous sediment of the Late Proterozoic Walcott Member, Chuar Group, Grand Canyon, Arizona

Abundant extractable hydrocarbons, in association with well preserved kerogen, have been recognised in a dolostone from the Chuar Group (approx. 850 Ma). The biomarkers present include C15-C30, acyclic isoprenoids, C26-C28 steranes, C27-C35 hopanes and extended tricyclic terpanes. Among the striking characteristics of the distributions are the dominance of steranes and methyl steranes lacking side-chain alkylation and the presence of putative C29-C35 neohopanes and gammacerane. The high abundance of steranes is consistent with the presumed eukaryotic affinities of several types of microfossils, including Chuaria, present in associated mudstones. This stratum is the oldest in which gammacerane has been found, and suggests contributions from protozoa to the depositional environment which appears to have been hypersaline. There is a strong correlation between the carbon isotopic composition of the kerogen and that of the various polarity fractions of the bitumens. Chemical degradation and pyrolysis of the kerogens yielded n-alkanes with similar characteristics to the bitumens, but there are differences in the patterns of distribution of the acyclic isoprenoid, sterane and triterpane biomarkers.

The Fate of Amoco Cadiz Oil

The Amoco Cadiz oil spill (223,000 metric tons) of March 1978 is the largest and best studied tanker spill in history. Of the total oil lost, 30,000 tons (13.5 percent) rapidly became incorporated into the water column, 18,000 tons (8 percent) were deposited in subtidal sediments, 62,000 tons (28 percent) washed into the intertidal zone, and 67,000 tons (30 percent) evaporated. While still at sea, approximately 10,000 tons of oil were degraded microbiologically. After 3 years, the most obvious effects of the spill have passed, although hydrocarbon concentrations remain elevated in those estuaries and marshes that were initially most heavily oiled.

Archaeal and bacterial glycerol dialkyl glycerol tetraether lipids in hot springs of yellowstone national park.

ABSTRACT Glycerol dialkyl glycerol tetraethers (GDGTs) are core membrane lipids originally thought to be produced mainly by (hyper)thermophilic archaea. Environmental screening of low-temperature environments showed, however, the abundant presence of structurally diverse GDGTs from both bacterial and archaeal sources. In this study, we examined the occurrences and distribution of GDGTs in hot spring environments in Yellowstone National Park with high temperatures (47 to 83°C) and mostly neutral to alkaline pHs. GDGTs with 0 to 4 cyclopentane moieties were dominant in all samples and are likely derived from both (hyper)thermophilic Crenarchaeota and Euryarchaeota. GDGTs with 4 to 8 cyclopentane moieties, likely derived from the crenarchaeotal order Sulfolobales and the euryarchaeotal order Thermoplasmatales, are usually present in much lower abundance, consistent with the relatively high pH values of the hot springs. The relative abundances of cyclopentane-containing GDGTs did not correlate with in situ temperature and pH, suggesting that other environmental and possibly genetic factors play a role as well. Crenarchaeol, a biomarker thought to be specific for nonthermophilic group I Crenarchaeota, was also found in most hot springs, though in relatively low concentrations, i.e., <5% of total GDGTs. Its abundance did not correlate with temperature, as has been reported previously. Instead, the cooccurrence of relatively abundant nonisoprenoid GDGTs thought to be derived from soil bacteria suggests a predominantly allochthonous source for crenarchaeol in these hot spring environments. Finally, the distribution of bacterial branched GDGTs suggests that they may be derived from the geothermally heated soils surrounding the hot springs.