Raju Sekar

Bio: Raju Sekar is an academic researcher from Xi'an Jiaotong-Liverpool University. The author has contributed to research in topics: Black band disease & Water quality. The author has an hindex of 21, co-authored 59 publications receiving 2253 citations. Previous affiliations of Raju Sekar include University of Sheffield & University of Liverpool.

An improved protocol for quantification of freshwater Actinobacteria by fluorescence in situ hybridization.

Abstract: We tested a previously described protocol for fluorescence in situ hybridization of marine bacterioplankton with horseradish peroxidase-labeled rRNA-targeted oligonucleotide probes and catalyzed reporter deposition (CARD-FISH) in plankton samples from different lakes The fraction of Bacteria detected by CARD-FISH was significantly lower than after FISH with fluorescently monolabeled probes In particular, the abundances of aquatic Actinobacteria were significantly underestimated We thus developed a combined fixation and permeabilization protocol for CARD-FISH of freshwater samples Enzymatic pretreatment of fixed cells was optimized for the controlled digestion of gram-positive cell walls without causing overall cell loss Incubations with high concentrations of lysozyme (10 mg ml 1 ) followed by achromopeptidase (60 U ml 1 ) successfully permeabilized cell walls of Actinobacteria for subsequent CARD-FISH both in enrichment cultures and environmental samples Between 72 and >99% (mean, 86%) of all Bacteria could be visualized with the improved assay in surface waters of four lakes For freshwater samples, our method is thus superior to the CARD-FISH protocol for marine Bacteria (mean, 55%) and to FISH with directly fluorochrome labeled probes (mean, 67%) Actinobacterial abundances in the studied systems, as detected by the optimized protocol, ranged from 32 to >55% (mean, 45%) Our findings confirm that members of this lineage are among the numerically most important Bacteria of freshwater picoplankton The taxonomic composition of picoplankton communities profoundly differs in freshwater and marine systems Cultivation-independent molecular approaches have demonstrated that bacteria from large phylogenetic lineages of typical freshwater microbes, eg, the -subdivision of the proteobacteria, are virtually absent in the marine picoplankton (15, 23) For other bacterial lineages the evidence is less conclusive Comparative analysis of 16S rDNA genes indicate that uncultured members of the class Actinobacteria are ubiquitous in lakes of various trophic state, size or geographic location (16, 36), but actinobacterial sequence types are also known from marine systems (31)

Abundances, Identity, and Growth State of Actinobacteria in Mountain Lakes of Different UV Transparency

Abstract: Members of the class Actinobacteria (44) are regarded as typical inhabitants of soil environments (14). Evidence is accumulating that these bacteria are also present in a variety of freshwater habitats (7, 10, 17, 48, 49). Bacteria from this group may in fact constitute a large fraction of the bacterioplankton in oligo- to mesotrophic lakes or in humic lakes (8, 13, 36). In order to understand the role of Actinobacteria in different freshwater habitats, it is important to distinguish between the various phylogenetic groups within this lineage (13, 47, 48) and to determine their population sizes in situ. This can be achieved by fluorescence in situ hybridization (FISH) with specific rRNA-targeted oligonucleotide probes (36). However, the occurrence of particular phylogenetic groups of Actinobacteria in the plankton of lakes does not necessarily imply that these bacteria are growing in this environment. Many cultivated Actinobacteria perform a life cycle which may involve a vegetative and a resting stage (i.e., spores) (11). Spores are highly resistant to unfavorable environmental conditions and can survive for extended periods of time. This life strategy promotes the survival and dispersal of Actinobacteria in soils. Thus, actinobacterial phylotypes that are found in freshwater might potentially be resting stages of predominently soil-dwelling allochthonous microorganisms. In order to prove that this phylogenetic group is an indigenous component of freshwater planktonic microbial assemblages, it is necessary to demonstrate cell growth in situ, e.g., by visualization of DNA de novo synthesis at the single-cell level (31). Our knowledge on the environmental conditions that favor the occurrence of Actinobacteria in lakes is scarce. The first sequence types from this group originate from acidified lakes in the Adirondack Mountains (17). Actinobacteria were conspicuously abundant in the water column of an alpine lake that is characterized by high levels of incident solar UV radiation and by high water transparency (13, 42). Solar UV radiation is harmful to various aquatic microorganisms, including viruses, bacteria, and unicellular eukaryotes (19, 40, 45). Furthermore, UV radiation is known to inhibit bacteria-mediated processes (41), and picoplankton organisms from clear-water ecosystems accumulate significant UV-induced DNA damage (6). DNA rich in A+T content can be particularly susceptible due to the higher probability of production of cyclobutane thymine dimers. One typical feature of the Actinobacteria is their high genomic G+C content (5). It is thus conceivable that such bacteria might be favored in the bacterioplankton assemblages of UV-transparent lakes. In order to test this hypothesis, the relative abundances of Actinobacteria were determined in the bacterial assemblages of 10 mountain lakes. These lakes are located along an altitude gradient, and they cover a wide range of water column UV transparencies. The composition of the microbial assemblages in these lakes was analyzed using a set of available and newly designed FISH probes. Specifically, we attempted to identify the numerically important taxa of freshwater Actinobacteria. In addition, the proportions of growing Actinobacteria were determined by pulse-labeling with bromodeoxyuridine (31).

Flow sorting of marine bacterioplankton after fluorescence in situ hybridization.

Abstract: We describe an approach to sort cells from coastal North Sea bacterioplankton by flow cytometry after in situ hybridization with rRNA-targeted horseradish peroxidase-labeled oligonucleotide probes and catalyzed fluorescent reporter deposition (CARD-FISH). In a sample from spring 2003 >90% of the cells were detected by CARD-FISH with a bacterial probe (EUB338). Approximately 30% of the microbial assemblage was affiliated with the Cytophaga-Flavobacterium lineage of the Bacteroidetes (CFB group) (probe CF319a), and almost 10% was targeted by a probe for the β-proteobacteria (probe BET42a). A protocol was optimized to detach cells hybridized with EUB338, BET42a, and CF319a from membrane filters (recovery rate, 70%) and to sort the cells by flow cytometry. The purity of sorted cells was >95%. 16S rRNA gene clone libraries were constructed from hybridized and sorted cells (S-EUB, S-BET, and S-CF libraries) and from unhybridized and unsorted cells (UNHYB library). Sequences related to the CFB group were significantly more frequent in the S-CF library (66%) than in the UNHYB library (13%). No enrichment of β-proteobacterial sequence types was found in the S-BET library, but novel sequences related to Nitrosospira were found exclusively in this library. These bacteria, together with members of marine clade OM43, represented >90% of the β-proteobacteria in the water sample, as determined by CARD-FISH with specific probes. This illustrates that a combination of CARD-FISH and flow sorting might be a powerful approach to study the diversity and potentially the activity and the genomes of different bacterial populations in aquatic habitats.

Microbial Communities in the Surface Mucopolysaccharide Layer and the Black Band Microbial Mat of Black Band-Diseased Siderastrea siderea

Abstract: Microbial community profiles and species composition associated with two black band-diseased colonies of the coral Siderastrea siderea were studied by 16S rRNA-targeted gene cloning, sequencing, and amplicon-length heterogeneity PCR (LH-PCR). Bacterial communities associated with the surface mucopolysaccharide layer (SML) of apparently healthy tissues of the infected colonies, together with samples of the black band disease (BBD) infections, were analyzed using the same techniques for comparison. Gene sequences, ranging from 424 to 1,537 bp, were retrieved from all positive clones (n = 43 to 48) in each of the four clone libraries generated and used for comparative sequence analysis. In addition to LH-PCR community profiling, all of the clone sequences were aligned with LH-PCR primer sequences, and the theoretical lengths of the amplicons were determined. Results revealed that the community profiles were significantly different between BBD and SML samples. The SML samples were dominated by γ-proteobacteria (53 to 64%), followed by β-proteobacteria (18 to 21%) and α-proteobacteria (5 to 11%). In contrast, both BBD clone libraries were dominated by α-proteobacteria (58 to 87%), followed by verrucomicrobia (2 to 10%) and 0 to 6% each of δ-proteobacteria, bacteroidetes, firmicutes, and cyanobacteria. Alphaproteobacterial sequence types related to the bacteria associated with toxin-producing dinoflagellates were observed in BBD clone libraries but were not found in the SML libraries. Similarly, sequences affiliated with the family Desulfobacteraceae and toxin-producing cyanobacteria, both believed to be involved in BBD pathogenesis, were found only in BBD libraries. These data provide evidence for an association of numerous toxin-producing heterotrophic microorganisms with BBD of corals.

The role of microorganisms in coral health, disease and evolution.

Abstract: Coral microbiology is an emerging field, driven largely by a desire to understand, and ultimately prevent, the worldwide destruction of coral reefs. The mucus layer, skeleton and tissues of healthy corals all contain large populations of eukaryotic algae, bacteria and archaea. These microorganisms confer benefits to their host by various mechanisms, including photosynthesis, nitrogen fixation, the provision of nutrients and infection prevention. Conversely, in conditions of environmental stress, certain microorganisms cause coral bleaching and other diseases. Recent research indicates that corals can develop resistance to specific pathogens and adapt to higher environmental temperatures. To explain these findings the coral probiotic hypothesis proposes the occurrence of a dynamic relationship between symbiotic microorganisms and corals that selects for the coral holobiont that is best suited for the prevailing environmental conditions. Generalization of the coral probiotic hypothesis has led us to propose the hologenome theory of evolution.

A Guide to the Natural History of Freshwater Lake Bacteria

Abstract: Freshwater bacteria are at the hub of biogeochemical cycles and control water quality in lakes. Despite this, little is known about the identity and ecology of functionally significant lake bacteria. Molecular studies have identified many abundant lake bacteria, but there is a large variation in the taxonomic or phylogenetic breadths among the methods used for this exploration. Because of this, an inconsistent and overlapping naming structure has developed for freshwater bacteria, creating a significant obstacle to identifying coherent ecological traits among these groups. A discourse that unites the field is sorely needed. Here we present a new freshwater lake phylogeny constructed from all published 16S rRNA gene sequences from lake epilimnia and propose a unifying vocabulary to discuss freshwater taxa. With this new vocabulary in place, we review the current information on the ecology, ecophysiology, and distribution of lake bacteria and highlight newly identified phylotypes. In the second part of our review, we conduct meta-analyses on the compiled data, identifying distribution patterns for bacterial phylotypes among biomes and across environmental gradients in lakes. We conclude by emphasizing the role that this review can play in providing a coherent framework for future studies.

Archaeal nitrification in the ocean

Abstract: Marine Crenarchaeota are the most abundant single group of prokaryotes in the ocean, but their physiology and role in marine biogeochemical cycles are unknown. Recently, a member of this clade was isolated from a sea aquarium and shown to be capable of nitrification, tentatively suggesting that Crenarchaeota may play a role in the oceanic nitrogen cycle. We enriched a crenarchaeote from North Sea water and showed that its abundance, and not that of bacteria, correlates with ammonium oxidation to nitrite. A time series study in the North Sea revealed that the abundance of the gene encoding for the archaeal ammonia monooxygenase alfa subunit (amoA) is correlated with a decline in ammonium concentrations and with the abundance of Crenarchaeota. Remarkably, the archaeal amoA abundance was 1–2 orders of magnitude higher than those of bacterial nitrifiers, which are commonly thought to mediate the oxidation of ammonium to nitrite in marine environments. Analysis of Atlantic waters of the upper 1,000 m, where most of the ammonium regeneration and oxidation takes place, showed that crenarchaeotal amoA copy numbers are also 1–3 orders of magnitude higher than those of bacterial amoA. Our data thus suggest a major role for Archaea in oceanic nitrification.

The riverine ecosystem synthesis : Biocomplexity in river networks across space and time

Abstract: We propose an integrated, heuristic model of lotic biocomplexity across spatiotemporal scales from headwaters to large rivers. This riverine ecosystem synthesis (RES) provides a framework for understanding both broad, often discontinuous patterns along longitudinal and lateral dimensions of river networks and local ecological patterns across various temporal and smaller spatial scales. Rather than posing a completely new model, we arrange a conceptual marriage of eco-geomorphology (ecological aspects of fluvial geomorphology) with a terrestrial landscape model describing hierarchical patch dynamics. We modify five components of this terrestrial model for lotic ecosystems: (1) nested, discontinuous hierarchies of patch mosaics; (2) ecosystem dynamics as a composite of intra- and inter-patch dynamics; (3) linked patterns and processes; (4) dominance of non-equilibrial and stochastic processes; and (5) formation of a quasi-equilibrial, metastable state. Our conceptual model blends our perspectives on biocomplexity with aspects of aquatic models proposed from 1980–2004. Contrasting with a common view of rivers as continuous, longitudinal gradients in physical conditions, the RES portrays rivers as downstream arrays of large hydrogeomorphic patches (e.g. constricted, braided and floodplain channel areas) formed by catchment geomorphology and climate. The longitudinal distribution of these patches, which are identifiable using standard geomorphic techniques, varies amongst rivers and is difficult to forecast above ecoregional scales. Some types of hydrogeomorphic patches may reoccur along this downstream passage. Unique ecological ‘functional process zones’ are formed by individual types of hydrogeomorphic patches because of physiochemical habitat differences which affect ecosystem structure and function. The RES currently includes 14 tenets predicting how patterns of individual species distributions, community regulation, lotic ecosystem processes, and floodplain interactions will vary over spatiotemporal scales, especially as they relate to the functional process zones formed by hydrogeomorphic differences in the river network. Copyright © 2006 John Wiley & Sons, Ltd.