Adaptivity of Archaeal and Bacterial Extremophiles

Microbiology - Extremophilic prokaryotes, inhabitants of hot, cold, acidic, alkaline, saline, and deep-sea ecosystems, are classified as mono- and polyextremophilic or extreme-tolerant. Under...     

Cell Size Distributions of Soil Bacterial and Archaeal Taxa

Cell size is a key ecological trait of soil microorganisms that determines a wide range of life history attributes, including the efficiency of nutrient acquisition. However, because of the methodological issues associated with determining cell sizes in situ, we have a limited understanding of how cell abundances vary across cell size fractions and whether certain microbial taxa have consistently smaller cells than other taxa. In this study, we extracted cells from three distinct soils and fractionated them into seven size ranges (5 μm to 0.2 μm) by filtration. Cell abundances in each size fraction were determined by direct microscopy, with the taxonomic composition of each size fraction determined by high-throughput sequencing of the 16S rRNA gene. Most of the cells were smaller than cells typically grown in culture, with 59 to 67% of cells <1.2 μm in diameter. Furthermore, each size fraction harbored distinct bacterial and archaeal communities in each of the three soils, and many of the taxa exhibited distinct size distribution patterns, with the smaller size fractions having higher relative abundances of taxa that are rare or poorly characterized (including Acidobacteria, Gemmatimonadetes, Crenarchaeota, Verrucomicrobia, and Elusimicrobia). In general, there was a direct relationship between average cell size and culturability, with those soil taxa that are poorly represented in culture collections tending to be smaller. Size fractionation not only provides important insight into the life history strategies of soil microbial taxa but also is a useful tool to enable more focused investigations into those taxa that remain poorly characterized.     

Functional Similarity between Archaeal and Bacterial CorA Magnesium Transporters

The constitutively expressed CorA Mg²⁺ transporter is the major Mg²⁺ influx system of Salmonella typhimurium and Escherichia coli. Genomic sequence data indicated the presence of a homolog in the archaeal organism Methanococcus jannaschii. The putative M. jannaschii CorA was expressed in an Mg²⁺-transport-deficient strain of S. typhimurium to determine its functional characteristics. The archaeal CorA homolog is a functional Mg²⁺ uptake system when expressed in S. typhimurium and has properties which are highly similar to those of the normal CorA transporter of S. typhimurium despite having a low level of sequence identity with the protein and being expressed in a lipid membrane of quite different composition than normal. This implies that the overall function of the proteins is the same and further suggests that their structures are very similar.     

Response of Archaeal and Bacterial Soil Communities to Changes Associated with Outdoor Cattle Overwintering

Archaea and bacteria are important drivers for nutrient transformations in soils and catalyse the production and consumption of important greenhouse gases. In this study, we investigate changes in archaeal and bacterial communities of four Czech grassland soils affected by outdoor cattle husbandry. Two show short-term (3 years; STI) and long-term impact (17 years; LTI), one is regenerating from cattle impact (REG) and a control is unaffected by cattle (CON). Cattle manure (CMN), the source of allochthonous microbes, was collected from the same area. We used pyrosequencing of 16S rRNA genes to assess the composition of archaeal and bacterial communities in each soil type and CMN. Both short- and long- term cattle impact negatively altered archaeal and bacterial diversity, leading to increase of homogenization of microbial communities in overwintering soils over time. Moreover, strong shifts in the prokaryotic communities were observed in response to cattle overwintering, with the greatest impact on archaea. Oligotrophic and acidophilic microorganisms (e.g. Thaumarchaeota, Acidobacteria, and α-Proteobacteria) dominated in CON and expressed strong negative response to increased pH, total C and N. Whereas copiotrophic and alkalophilic microbes (e.g. methanogenic Euryarchaeota, Firmicutes, Chloroflexi, Actinobacteria, and Bacteroidetes) were common in LTI showing opposite trends. Crenarchaeota were also found in LTI, though their trophic interactions remain cryptic. Firmicutes, Bacteroidetes, Methanobacteriaceae, and Methanomicrobiaceae indicated the introduction and establishment of faecal microbes into the impacted soils, while Chloroflexi and Methanosarcinaceae suggested increased abundance of soil-borne microbes under altered environmental conditions. The observed changes in prokaryotic community composition may have driven corresponding changes in soil functioning.     

From Simple Bacterial and Archaeal Replicons to Replication N/U-Domains

The Replicon Theory proposed 50 years ago has proven to apply for replicons of the three domains of life. Here, we review our knowledge of genome organization into single and multiple replicons in bacteria, archaea and eukarya. Bacterial and archaeal replicator/initiator systems are quite specific and efficient, whereas eukaryotic replicons show degenerate specificity and efficiency, allowing for complex regulation of origin firing time. We expand on recent evidence that ~ 50% of the human genome is organized as ~ 1,500 megabase-sized replication domains with a characteristic parabolic (U-shaped) replication timing profile and linear (N-shaped) gradient of replication fork polarity. These N/U-domains correspond to self-interacting segments of the chromatin fiber bordered by open chromatin zones and replicate by cascades of origin firing initiating at their borders and propagating to their center, possibly by fork-stimulated initiation. The conserved occurrence of this replication pattern in the germline of mammals has resulted over evolutionary times in the formation of megabase-sized domains with an N-shaped nucleotide compositional skew profile due to replication-associated mutational asymmetries. Overall, these results reveal an evolutionarily conserved but developmentally plastic organization of replication that is driving mammalian genome evolution.     

Phylogeny of Bacterial and Archaeal Genomes Using Conserved Genes: Supertrees and Supermatrices

Over 3000 microbial (bacterial and archaeal) genomes have been made publically available to date, providing an unprecedented opportunity to examine evolutionary genomic trends and offering valuable reference data for a variety of other studies such as metagenomics. The utility of these genome sequences is greatly enhanced when we have an understanding of how they are phylogenetically related to each other. Therefore, we here describe our efforts to reconstruct the phylogeny of all available bacterial and archaeal genomes. We identified 24, single-copy, ubiquitous genes suitable for this phylogenetic analysis. We used two approaches to combine the data for the 24 genes. First, we concatenated alignments of all genes into a single alignment from which a Maximum Likelihood (ML) tree was inferred using RAxML. Second, we used a relatively new approach to combining gene data, Bayesian Concordance Analysis (BCA), as implemented in the BUCKy software, in which the results of 24 single-gene phylogenetic analyses are used to generate a “primary concordance” tree. A comparison of the concatenated ML tree and the primary concordance (BUCKy) tree reveals that the two approaches give similar results, relative to a phylogenetic tree inferred from the 16S rRNA gene. After comparing the results and the methods used, we conclude that the current best approach for generating a single phylogenetic tree, suitable for use as a reference phylogeny for comparative analyses, is to perform a maximum likelihood analysis of a concatenated alignment of conserved, single-copy genes.     

Distribution and Diversity of Archaeal and Bacterial Ammonia Oxidizers in Salt Marsh Sediments

Diversity and abundance of ammonia-oxidizing Betaproteobacteria (β-AOB) and archaea (AOA) were investigated in a New England salt marsh at sites dominated by short or tall Spartina alterniflora (SAS and SAT sites, respectively) or Spartina patens (SP site). AOA amoA gene richness was higher than β-AOB amoA richness at SAT and SP, but AOA and β-AOB richness were similar at SAS. β-AOB amoA clone libraries were composed exclusively of Nitrosospira-like amoA genes. AOA amoA genes at SAT and SP were equally distributed between the water column/sediment and soil/sediment clades, while AOA amoA sequences at SAS were primarily affiliated with the water column/sediment clade. At all three site types, AOA were always more abundant than β-AOB based on quantitative PCR of amoA genes. At some sites, we detected 10⁹ AOA amoA gene copies g of sediment⁻¹. Ratios of AOA to β-AOB varied over 2 orders of magnitude among sites and sampling dates. Nevertheless, abundances of AOA and β-AOB amoA genes were highly correlated. Abundance of 16S rRNA genes affiliated with Nitrosopumilus maritimus, Crenarchaeota group I.1b, and pSL12 were positively correlated with AOA amoA abundance, but ratios of amoA to 16S rRNA genes varied among sites. We also observed a significant effect of pH on AOA abundance and a significant salinity effect on both AOA and β-ΑΟΒ abundance. Our results expand the distribution of AOA to salt marshes, and the high numbers of AOA at some sites suggest that salt marsh sediments serve as an important habitat for AOA.Nitrification, the sequential oxidation of ammonia to nitrite and nitrate, is a critical step in the nitrogen cycle and is mediated by a suite of phylogenetically and physiologically distinct microorganisms. The recent discovery of ammonia oxidation among Archaea (17, 38) has led to a dramatic shift in the current model of nitrification and to new questions of niche differentiation between putative ammonia-oxidizing Archaea (AOA) and the more-well-studied ammonia-oxidizing Betaproteobacteria (β-AOB). Based on surveys of 16S rRNA genes and archaeal amoA genes, it is evident that AOA occupy a wide range of niches (10), suggesting a physiologically diverse group of Archaea. Additionally, in studies where AOA and β-AOB were both targeted, AOA were typically more abundant than their bacterial counterparts (19, 21, 42). However, there are reports of β-AOB outnumbering AOA in estuarine systems (6, 33), suggesting a possible shift in competitive dominance under certain conditions.Patterns of β-AOB diversity in estuaries have been well characterized and appear to be regulated by similar mechanisms within geographically disparate systems (4, 11, 32). However, AOA distribution and their role in nitrification relative to β-AOB remain to be determined. A few studies have begun to address this question in different estuaries, but no unifying patterns or mechanisms have emerged. Although β-AOB have been well studied along estuarine salinity gradients (1, 3, 4, 7, 11, 13, 22, 33, 39) and recent studies have begun to address AOA in estuaries (1, 6, 22, 32, 33), few have investigated β-AOB in salt marshes (9), and none has included AOA.In this study, we investigated the distribution and abundance of AOA and β-AOB based on the distribution and abundance of amoA genes in salt marsh sediments dominated by different types of vegetation. Although we equate the presence of archaeal amoA genes with the genetic potential to oxidize ammonia, we acknowledge the possibility that all Archaea that have amoA genes may not all represent functional ammonia oxidizers. Vegetation patterns of New England salt marshes are strongly correlated with marsh elevation and are controlled by a combination of interspecific competition and tolerance to physico-chemical stress (28). The dominant grasses of New England salt marshes are Spartina alterniflora and Spartina patens, which typically grow as pure stands. S. alterniflora is found in two phenotypically distinct but genetically identical forms, a tall and a short growth form (34). The tall S. alterniflora grows to heights of 1 to 2 m and is typically found at the edges of the marsh and along creek banks (SAT sites), while the short-form S. alterniflora may reach heights of only 30 cm and is found in sites (SAS sites) slightly higher on the marsh where soil drainage is limited and conditions are more reduced compared to SAT sites (14). Conversely, S. patens, due to its lower tolerance of salt and more reduced conditions, is found in sites (SP sites) highest on the marsh, in areas that receive less flooding (5). Because the marsh is subjected to daily tidal fluctuations, most sites experience periods of anoxia, the degree of which depends on the marsh elevation. We hypothesized that ammonia-oxidizing communities in areas dominated by different marsh grasses would reflect the different edaphic conditions associated with each type of grass, due to differences in vertical zonation in the marsh.     

Potential Interactions of Particle-Associated Anammox Bacteria with Bacterial and Archaeal Partners in the Namibian Upwelling System

Recent studies have shown that the anaerobic oxidation of ammonium by anammox bacteria plays an important role in catalyzing the loss of nitrogen from marine oxygen minimum zones (OMZ). However, in situ oxygen concentrations of up to 25 μM and ammonium concentrations close to or below the detection limit in the layer of anammox activity are hard to reconcile with the current knowledge of the physiology of anammox bacteria. We therefore investigated samples from the Namibian OMZ by comparative 16S rRNA gene analysis and fluorescence in situ hybridization. Our results showed that “Candidatus Scalindua” spp., the typical marine anammox bacteria, colonized microscopic particles that were likely the remains of either macroscopic marine snow particles or resuspended particles. These particles were slightly but significantly (P < 0.01) enriched in Gammaproteobacteria (11.8% ± 5.0%) compared to the free-water phase (8.1% ± 1.8%). No preference for the attachment to particles could be observed for members of the Alphaproteobacteria and Bacteroidetes, which were abundant (12 to 17%) in both habitats. The alphaproteobacterial SAR11 clade, the Euryarchaeota, and group I Crenarchaeota, were all significantly depleted in particles compared to their presence in the free-water phase (16.5% ± 3.5% versus 2.6% ± 1.7%, 2.7% ± 1.9% versus <1%, and 14.9% ± 4.6% versus 2.2% ± 1.8%, respectively, all P < 0.001). Sequence analysis of the crenarchaeotal 16S rRNA genes showed a 99% sequence identity to the nitrifying “Nitrosopumilus maritimus.” Even though we could not observe conspicuous consortium-like structures of anammox bacteria with particle-enriched bacterioplankton groups, we hypothesize that members of Gammaproteobacteria, Alphaproteobacteria, and Bacteroidetes play a critical role in extending the anammox reaction to nutrient-depleted suboxic water layers in the Namibian upwelling system by creating anoxic, nutrient-enriched microniches.     

Bacterial,Archaeal and Fungal Succession in the Forefield of a Receding Glacier

Glacier forefield chronosequences, initially composed of barren substrate after glacier retreat, are ideal locations to study primary microbial colonization and succession in a natural environment. We characterized the structure and composition of bacterial, archaeal and fungal communities in exposed rock substrates along the Damma glacier forefield in central Switzerland. Soil samples were taken along the forefield from sites ranging from fine granite sand devoid of vegetation near the glacier terminus to well-developed soils covered with vegetation. The microbial communities were studied with genetic profiling (T-RFLP) and sequencing of clone libraries. According to the T-RFLP profiles, bacteria showed a high Shannon diversity index (H) (ranging from 2.3 to 3.4) with no trend along the forefield. The major bacterial lineages were Proteobacteria, Actinobacteria, Acidobacteria, Firmicutes and Cyanobacteria. An interesting finding was that Euryarchaeota were predominantly colonizing young soils and Crenarchaeota mainly mature soils. Fungi shifted from an Ascomycota-dominated community in young soils to a more Basidiomycota-dominated community in old soils. Redundancy analysis indicated that base saturation, pH, soil C and N contents and plant coverage, all related to soil age, correlated with the microbial succession along the forefield.     

Analysis of the Sulfate-Reducing Bacterial and Methanogenic Archaeal Populations in Contrasting Antarctic Sediments

The distribution and activity of communities of sulfate-reducing bacteria (SRB) and methanogenic archaea in two contrasting Antarctic sediments were investigated. Methanogenesis dominated in freshwater Lake Heywood, while sulfate reduction dominated in marine Shallow Bay. Slurry experiments indicated that 90% of the methanogenesis in Lake Heywood was acetoclastic. This finding was supported by the limited diversity of clones detected in a Lake Heywood archaeal clone library, in which most clones were closely related to the obligate acetate-utilizing Methanosaeta concilii. The Shallow Bay archaeal clone library contained clones related to the C₁-utilizing Methanolobus and Methanococcoides and the H₂-utilizing Methanogenium. Oligonucleotide probing of RNA extracted directly from sediment indicated that archaea represented 34% of the total prokaryotic signal in Lake Heywood and that Methanosaeta was a major component (13.2%) of this signal. Archaea represented only 0.2% of the total prokaryotic signal in RNA extracted from Shallow Bay sediments. In the Shallow Bay bacterial clone library, 10.3% of the clones were SRB-like, related to Desulfotalea/Desulforhopalus, Desulfofaba, Desulfosarcina, and Desulfobacter as well as to the sulfur and metal oxidizers comprising the Desulfuromonas cluster. Oligonucleotide probes for specific SRB clusters indicated that SRB represented 14.7% of the total prokaryotic signal, with Desulfotalea/Desulforhopalus being the dominant SRB group (10.7% of the total prokaryotic signal) in the Shallow Bay sediments; these results support previous results obtained for Arctic sediments. Methanosaeta and Desulfotalea/Desulforhopalus appear to be important in Lake Heywood and Shallow Bay, respectively, and may be globally important in permanently low-temperature sediments.     

Evidence of Intense Archaeal and Bacterial Methanotrophic Activity in the Black Sea Water Column

In the northwestern Black Sea, methane oxidation rates reveal that above shallow and deep gas seeps methane is removed from the water column as efficiently as it is at sites located off seeps. Hence, seeps should not have a significant impact on the estimated annual flux of ~4.1 × 10⁹ mol methane to the atmosphere [W. S. Reeburgh, B. B. Ward, S. C. Wahlen, K. A. Sandbeck, K. A. Kilatrick, and L. J. Kerkhof, Deep-Sea Res. 38(Suppl. 2):S1189-S1210, 1991]. Both the stable carbon isotopic composition of dissolved methane and the microbial community structure analyzed by fluorescent in situ hybridization provide strong evidence that microbially mediated methane oxidation occurs. At the shelf, strong isotope fractionation was observed above high-intensity seeps. This effect was attributed to bacterial type I and II methanotrophs, which on average accounted for 2.5% of the DAPI (4′,6′-diamidino-2-phenylindole)-stained cells in the whole oxic water column. At deep sites, in the oxic-anoxic transition zone, strong isotopic fractionation of methane overlapped with an increased abundance of Archaea and Bacteria, indicating that these organisms are involved in the oxidation of methane. In underlying anoxic water, we successfully identified the archaeal methanotrophs ANME-1 and ANME-2, eachof which accounted for 3 to 4% of the total cell counts. ANME-1 and ANME-2 appear as single cells in anoxicwater, compared to the sediment, where they may form cell aggregates with sulfate-reducing bacteria (A. Boetius, K. Ravenschlag, C. J. Schubert, D. Rickert, F. Widdel, A. Giesecke, R. Amann, B. B. Jørgensen, U. Witte, and O. Pfannkuche, Nature 407:623-626, 2000; V. J. Orphan, C. H. House, K.-U. Hinrichs, K. D. McKeegan, and E. F. DeLong, Proc. Natl. Acad. Sci. USA 99:7663-7668, 2002).     

Responses of Ammonia-Oxidizing Bacterial and Archaeal Populations to Organic Nitrogen Amendments in Low-Nutrient Groundwater

To evaluate the potential for organic nitrogen addition to stimulate the in situ growth of ammonia oxidizers during a field scale bioremediation trial, samples collected from the Eastern Snake River Plain Aquifer in Idaho before, during, and after the addition of molasses and urea were subjected to PCR analysis of ammonia monooxygenase subunit A (amoA) genes. Ammonia-oxidizing bacteria (AOB) and archaea (AOA) were present in all of the samples tested, with AOA amoA genes outnumbering AOB amoA genes in all of the samples. Following urea addition, nitrate levels rose and bacterial amoA copy numbers increased dramatically, suggesting that urea hydrolysis stimulated nitrification. Bacterial amoA diversity was limited to two Nitrosomonas phylotypes, whereas archaeal amoA analyses revealed 20 distinct operational taxonomic units, including several that were markedly different from all previously reported sequences. Results from this study demonstrate the likelihood of stimulating ammonia-oxidizing communities during field scale manipulation of groundwater conditions to promote urea hydrolysis.Subsurface calcite precipitation driven by microbial urea hydrolysis has been proposed as a means of remediating trace metal or radionuclide contaminants (e.g., strontium-90) that can be coprecipitated and retained in the solid phase (11, 12, 42). Urea hydrolysis generates carbonate alkalinity and raises pH, both of which promote calcite precipitation. However, another product of urea hydrolysis is ammonium, as shown in the following equation: In low-nutrient groundwater, the ammonium resulting from urea hydrolysis can have a number of fates, including uptake by nitrogen-limited microorganisms or transformation to nitrite by ammonia-oxidizing microorganisms. Microbial oxidation of ammonia is a net acid-yielding process. The resultant acidity from this reaction could inhibit calcite precipitation or promote destabilization of preexisting calcite, potentially liberating contaminants from the solid phase. In addition, the further transformation of nitrite by nitrite-oxidizing bacteria leads to the formation of nitrate, a regulated contaminant of drinking water.The first step of bacterial ammonia oxidation, the conversion of ammonia to hydroxylamine, is catalyzed by the membrane-bound enzyme ammonia monooxygenase. The gene coding for the catalytic α subunit of this enzyme, amoA, has proven to be an effective molecular marker for ammonia-oxidizing bacteria (AOB) (20, 34). All of the currently known chemoautotrophic AOB are associated with the Nitrosomonas and Nitrosospira genera within the Betaproteobacteria or the genus Nitrosococcus within the Gammaproteobacteria (15, 32). Although ammonia oxidation was long believed to be carried out exclusively by members of the domain Bacteria, considerable evidence now suggests that recently discovered ammonia-oxidizing archaea (AOA) (18) are key players in this critical step of the microbial nitrogen cycle (8).The archaeal amoA gene has been found in a wide range of environments (9; reviewed in references 8 and 31), and its expression has been documented in enrichment cultures (35) and soil microcosms (40), as well as in marine and terrestrial environments (21, 23). Reported quantitative PCR (qPCR) analyses of amoA in marine and terrestrial environments suggest that AOA typically outnumber AOB by orders of magnitude (23, 26, 44), and AOA abundance has also recently been shown to be highly correlated with water column ¹⁵NH₄⁺ oxidation rates (1). However, some recent studies have reported that AOB are more abundant under certain conditions (6, 27, 35, 43, 45).In an effort to better understand the fate of ammonium generated from urea hydrolysis, we monitored the abundance and diversity of bacterial and archaeal amoA genes during a field experiment designed to test stimulation of urea hydrolysis in groundwater. Dilute molasses and urea were sequentially introduced into a well in the Eastern Snake River Plain Aquifer (ESRPA) in Idaho (13). Previous laboratory experiments indicated that molasses, an inexpensive and commonly used bioremediation amendment (14), was effective in increasing overall microbial populations, as well as total ureolytic activity (13, 39). The ESRPA is a deep basalt aquifer and is considered oligotrophic (4, 22, 29); however, previous work has demonstrated the presence of ureolytic microbes in this environment (11, 13). Erwin et al. also reported evidence of AOB during the analysis of methane monooxygenase clone libraries from ESRPA samples (7), but in general, the structure and function of ammonia-oxidizing microbial communities (and especially AOA) in deep aquifers like the ESRPA have been relatively unexplored.     

Layered Structure of Bacterial and Archaeal Communities and Their In Situ Activities in Anaerobic Granules

The microbial community structure and spatial distribution of microorganisms and their in situ activities in anaerobic granules were investigated by 16S rRNA gene-based molecular techniques and microsensors for CH₄, H₂, pH, and the oxidation-reduction potential (ORP). The 16S rRNA gene-cloning analysis revealed that the clones related to the phyla Alphaproteobacteria (detection frequency, 51%), Firmicutes (20%), Chloroflexi (9%), and Betaproteobacteria (8%) dominated the bacterial clone library, and the predominant clones in the archaeal clone library were affiliated with Methanosaeta (73%). In situ hybridization with oligonucleotide probes at the phylum level revealed that these microorganisms were numerically abundant in the granule. A layered structure of microorganisms was found in the granule, where Chloroflexi and Betaproteobacteria were present in the outer shell of the granule, Firmicutes were found in the middle layer, and aceticlastic Archaea were restricted to the inner layer. Microsensor measurements for CH₄, H₂, pH, and ORP revealed that acid and H₂ production occurred in the upper part of the granule, below which H₂ consumption and CH₄ production were detected. Direct comparison of the in situ activity distribution with the spatial distribution of the microorganisms implied that Chloroflexi contributed to the degradation of complex organic compounds in the outermost layer, H₂ was produced mainly by Firmicutes in the middle layer, and Methanosaeta produced CH₄ in the inner layer. We determined the effective diffusion coefficient for H₂ in the anaerobic granules to be 2.66 × 10⁻⁵ cm² s⁻¹, which was 57% in water.