Karl-Paul Witzel

Phylogenetic and functional marker genes to study ammonia-oxidizing microorganisms (AOM) in the environment

The oxidation of ammonia plays a significant role in the transformation of fixed nitrogen in the global nitrogen cycle. Autotrophic ammonia oxidation is known in three groups of microorganisms. Aerobic ammonia-oxidizing bacteria and archaea convert ammonia into nitrite during nitrification. Anaerobic ammonia-oxidizing bacteria (anammox) oxidize ammonia using nitrite as electron acceptor and producing atmospheric dinitrogen. The isolation and cultivation of all three groups in the laboratory are quite problematic due to their slow growth rates, poor growth yields, unpredictable lag phases, and sensitivity to certain organic compounds. Culture-independent approaches have contributed importantly to our understanding of the diversity and distribution of these microorganisms in the environment. In this review, we present an overview of approaches that have been used for the molecular study of ammonia oxidizers and discuss their application in different environments.

Widespread distribution of proteorhodopsins in freshwater and brackish ecosystems

Proteorhodopsins (PRs) are light-driven proton pumps that have been found in a variety of marine environments. The goal of this study was to search for PR presence in different freshwater and brackish environments and to explore the diversity of non-marine PR protein. Here, we show that PRs exist in distinctly different aquatic environments, ranging from clear water lakes to peat lakes and in the Baltic Sea. Some of the PRs observed in this study formed unique clades that were not previously observed in marine environments, whereas others were similar to PRs found in non-marine samples of the Global Ocean Sampling (GOS) expedition. Furthermore, the similarity of several PRs isolated from lakes in different parts of the world suggests that these genes are dispersed globally and that they may encode unique functional capabilities enabling successful competition in a wide range of freshwater environments. Phylogenomic analysis of genes found on these GOS scaffolds suggests that some of the freshwater PRs are found in freshwater Flavobacteria and freshwater SAR11-like bacteria.

TRiFLe, a program for in silico terminal restriction fragment length polymorphism analysis with user-defined sequence sets

ABSTRACT We describe TRiFLe, a freely accessible computer program that generates theoretical terminal restriction fragments (T-RFs) from any user-supplied sequence set tailored to a particular group of organisms, sequences from clone libraries, or sequences from specific genes. The program allows a rapid identification of the most polymorphic enzymes, creates a collection of T-RFs for the data set, and can potentially identify specific T-RFs in T-RF length polymorphism (T-RFLP) patterns by comparing theoretical and experimental results. TRiFLE was used for analyzing T-RFLP data generated for the amoA and pmoA genes. The peaks identified in the T-RFLP patterns show an overlap of ammonia- and methane-oxidizing bacteria in the metalimnion of a subtropical lake.

Bacterial Community Composition in Central European Running Waters Examined by Temperature Gradient Gel Electrophoresis and Sequence Analysis of 16S rRNA Genes

ABSTRACT The bacterial community composition in small streams and a river in central Germany was examined by temperature gradient gel electrophoresis (TGGE) with PCR products of 16S rRNA gene fragments and sequence analysis. Complex TGGE band patterns suggested high levels of diversity of bacterial species in all habitats of these environments. Cluster analyses demonstrated distinct differences among the communities in stream and spring water, sandy sediments, biofilms on stones, degrading leaves, and soil. The differences between stream water and sediment were more significant than those between sites within the same habitat along the stretch from the stream source to the mouth. TGGE data from an entire stream course suggest that, in the upper reach of the stream, a special suspended bacterial community is already established and changes only slightly downstream. The bacterial communities in water and sediment in an acidic headwater with a pH below 5 were highly similar to each other but deviated distinctly from the communities at the other sites. As ascertained by nucleotide sequence analysis, stream water communities were dominated by Betaproteobacteria (one-third of the total bacteria), whereas sediment communities were composed mainly of Betaproteobacteria and members of the Fibrobacteres/Acidobacteria group (each accounting for about 25% of bacteria). Sequences obtained from bacteria from water samples indicated the presence of typical cosmopolitan freshwater organisms. TGGE bands shared between stream and soil samples, as well as sequences found in bacteria from stream samples that were related to those of soil bacteria, demonstrated the occurrence of some species in both stream and soil habitats. Changes in bacterial community composition were correlated with geographic distance along a stream, but in comparisons of different streams and rivers, community composition was correlated only with environmental conditions.

Diversity of Bacteroidetes in high-altitude saline evaporitic basins in northern Chile

The phylum Bacteroidetes represents one of the most abundant bacterial groups of marine and freshwater bacterioplankton. We investigated the diversity of Bacteroidetes in water and sediment samples from three evaporitic basins located in the highlands of northern Chile. We used both 16S rRNA gene clone libraries created with targeted Bacteroidetes-specific primers and separation of specifically amplified gene fragments by denaturing gradient gel electrophoresis (DGGE). DGGE analysis revealed a reduced richness of these organisms in samples from Salar de Huasco (two to four DGGE bands) increasing in Salar de Ascotan (two to seven DGGE bands) and Laguna Tebenquiche at Salar de Atacama (four to eight DGGE bands). Cluster analysis (WPGMA) of DGGE bands showed that bands from Salar de Huasco and Salar de Ascotan grouped together and samples from Salar de Atacama formed separate clusters in water and sediment samples, reflecting different Bacteroidetes communities between sites. Most of the sequences analyzed belonged to the family Flavobacteriaceae and clustered with the genera Psychroflexus, Gillisia, Maribacter, Muricauda, Flavobacterium, and Salegentibacter. The most abundant phylotype was highly related to Psychroflexus spp. and was recovered from all three study sites. The similarity of the analyzed sequences with their closest relatives in GenBank was typically <97% and notably lower when compared with type strains, demonstrating the unique character of these sequences. Culture efforts will be necessary to get a better description of the diversity of this group in saline evaporitic basins of northern Chile.

Cyanobacterial diversity in Salar de Huasco, a high altitude saline wetland in Northern Chile, are highly similar to Antarctic cyanobacteria

The diversity of Cyanobacteria in water and sediment samples from four representative sites of the Salar de Huasco was examined using denaturing gradient gel electrophoresis and analysis of clone libraries of 16S rRNA gene PCR products. Salar de Huasco is a high altitude (3800 m altitude) saline wetland located in the Chilean Altiplano. We analyzed samples from a tributary stream (H0) and three shallow lagoons (H1, H4, H6) that contrasted in their physicochemical conditions and associated biota. Seventy-eight phylotypes were identified in a total of 268 clonal sequences deriving from seven clone libraries of water and sediment samples. Oscillatoriales were frequently found in water samples from sites H0, H1 and H4 and in sediment samples from sites H1 and H4. Pleurocapsales were found only at site H0, while Chroococcales were recovered from sediment samples of sites H0 and H1, and from water samples of site H1. Nostocales were found in sediment samples from sites H1 and H4, and water samples from site H1 and were largely represented by sequences highly similar to Nodularia spumigena. We suggest that cyanobacterial communities from Salar de Huasco are unique - they include sequences related to others previously described from the Antarctic, along with others from diverse, but less extreme environments.

Unique clusters of Archaea in Salar de Huasco, an athalassohaline evaporitic basin of the Chilean Altiplano

Analyses of clone libraries from water and sediments of different sites from Salar de Huasco, a high-altitude athalassohaline wetland in the Chilean Altiplano, revealed the presence of five unique clusters of uncultured Archaea that have not been previously reported or specifically assigned. These sequences were distantly related (83-96% sequence identity) to a limited number of other clone sequences and revealed no identity to cultured Archaea. The abundance of Archaea and Bacteria was estimated using qPCR and community composition was examined through the construction of clone libraries of archaeal 16S rRNA gene. Archaea were found to be dominant over Bacteria in sediments from two saline sites (sites H4: 6.31 x 10(4) and site H6: 1.37 x 10(4) microS cm(-1)) and in one of the water samples (freshwater from site H0: 607 muS cm(-1)). Euryarchaeotal sequences were more abundant than crenarchaeotal sequences. Many of the clone sequences (52%) were similar to uncultured archaeal groups found in marine ecosystems having identity values between 99% and 97%. A major fraction of the sequences (40%) were members of Methanobacteria, while others were included in the Marine Benthic Groups B and D, the Miscellaneous Crenarchaeotic Group, the Terrestrial Miscellaneous Euryarchaeotal Group, Marine Group I and Halobacteria. The presence of uncultured archaeal groups in Salar de Huasco extends their known distribution in inland waters, providing new clues about their possible function in the environment.

Bacterial and archaeal diversity in high altitude wetlands of the Chilean Altiplano

The microbial diversity of five unconnected high altitude (≥ 3800 m a.s.l.) wetlands from the Chilean Altiplano was analyzed by a culture-independent approach, using 16 S rRNA gene sequences of different microbial groups. The wetlands (Chungara Lake, Parinacota wetland, Piacota Lake, Salar de Huasco and Salar de Ascotan) differed in terms of habitat type and physicochemical properties. The bacterial communities of these systems were dominated by Bacteroidetes (24-94 % of the clones) and Proteobacteria (Alpha, Beta, Gamma and Delta subgroups) with smaller contributions by the Firmicutes, Actinobacteria, Planctomycetes, Verrucomicrobia, Chloroflexi, Cyanobacteria, Acidobacteria, Deinococcus-Thermus and Candidate Division WS3. Fourteen phylotypes matching Alphaproteobacteria were part of the marine Roseobacter clade, representing new clusters of this group. Archaeal diversity was much lower than that seen for bacteria, and was dominated by Euryarchaeota; however Crenarchaeota were also present. Considering the large differences in microbial community composition between sites and samples, the presence of eleven phylotypes common to two or more habitats is highlighted. The frequent presence of new taxa in different phylogenetic groups in the altiplanic wetlands studied here revealed the unique characteristics of Bacteria and Archaea in these fragile Andean ecosystems.

Significance for Extracellular Enzymes for Organic Matter Degradation and Nutrient Regeneration in Small Streams

Extracellular enzymatic hydrolysis is the first step in the microbial degradation of macromolecular organic matter. Investigations into this process in lotic systems are scarce (e.g., Duddridge and Wainwright, 1982; Boon, 1989; Marxsen and Witzel, 1990), even though it may limit microbial substrate uptake and production. The first publications on extracellular enzymes in aquatic systems appeared more than 20 years ago (Overbeck, 1961; Reichardt et al., 1967; Chapter 1), but for a long time afterwards there were few investigations into this subject. One reason was probably the lack of an appropriate method. However, in recent years artificial substrates that release colored (Meyer-Reil, 1981; 1983; 1984; Hoppe et al., 1983) or fluorescent (Pettersson and Jansson, 1978; Hoppe, 1983; Somville and Billen, 1983; Rego et al., 1985; Chrost, 1990) compounds after enzymatic hydrolysis have been introduced into the field of aquatic microbial ecology. In particular, the fluorogenic model substrates have made sensitive, simple, and rapid measurements of enzyme activity possible.

Comparative in silico analysis of PCR primers suited for diagnostics and cloning of ammonia monooxygenase genes from ammonia-oxidizing bacteria

Over recent years, several PCR primers have been described to amplify genes encoding the structural subunits of ammonia monooxygenase (AMO) from ammonia-oxidizing bacteria (AOB). Most of them target amoA, while amoB and amoC have been neglected so far. This study compared the nucleotide sequence of 33 primers that have been used to amplify different regions of the amoCAB operon with alignments of all available sequences in public databases. The advantages and disadvantages of these primers are discussed based on the original description and the spectrum of matching sequences obtained. Additionally, new primers to amplify the almost complete amoCAB operon of AOB belonging to Betaproteobacteria (betaproteobacterial AOB), a primer pair for DGGE analysis of amoA and specific primers for gammaproteobacterial AOB, are also described. The specificity of these new primers was also evaluated using the databases of the sequences created during this study.