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1.  Molecular analysis of bacterial diversity in mudflats along the salinity gradient of an acidified tropical Bornean estuary (South East Asia) 
Aquatic Biosystems  2014;10:10.
The Brunei River and Bay estuarine system (BES) in the northwest of Borneo is acidic and highly turbid. The system supports extensive intertidal mudflats and presents a potentially steep salinity and pH gradient along its length (45 km). Temporal variation in physical parameters is observed diurnally due to seawater flux during tidal forcing, and stochastically due to elevated freshwater inflow after rains, resulting in a salinity range between 0 and 34 psu. High velocity freshwater run-off from acid sulphate formations during monsoon seasons results in highly variable and acidic conditions (pH 4) at the upper reaches of the BES, whereas the pH is relatively stable (pH 8) at the seaward extremes, due to mixing with seawater from the South China Sea. At their surfaces, the BES mudflats present microbial ecosystems driven by oxygenic phototrophs. To study the effect of various physical parameters on the bacterial diversity of the BES mudflats, surface samples were collected from six sites stretching over 40 km for molecular and phylogentic analysis.
The bacterial diversity at these sites was compared by community fingerprinting analysis using 16S rRNA gene based denaturing gradient gel electrophoresis and by 16S rRNA gene sequencing and phylogenetic analyses. Results revealed functionally conserved, diatom-driven microbial mudflat communities composed of mainly novel, uncultured species. Species composition was evaluated as 50-70% unique for each site along the BES. Clustering of the sequences commonly occurred and revealed that proteobacterial diversity was related to the salinity gradient. When considering all phyla, the diversity varied consistently with physical parameters (including anthropogenic) that are expected to influence microbial composition.
The BES mudflats were found to comprise the typical functional groups of microorganisms associated with photosynthetic carbon flux, sulfur cycling (Gamma- and Deltaproteobacteria), and decomposition (Bacteroidetes). From a structural perspective, however, the mudflats constituted discretely distributed communities along the physical gradient of the BES, composed of largely novel species of Bacteria. This study provides first insights into patterns of bacterial community structure in tropical South East Asian coastal ecosystems that are potentially threatened by increasing variability in pH and salinity, in line with predicted future environmental change.
PMCID: PMC4229359  PMID: 25392733
Marine Environmental Research; Borneo; Brunei; Mudflats; Acid sulfide; 16S rRNA; Bacterial diversity; Microbial mats
2.  Chemoautotrophic Carbon Fixation Rates and Active Bacterial Communities in Intertidal Marine Sediments 
PLoS ONE  2014;9(7):e101443.
Chemoautotrophy has been little studied in typical coastal marine sediments, but may be an important component of carbon recycling as intense anaerobic mineralization processes in these sediments lead to accumulation of high amounts of reduced compounds, such as sulfides and ammonium. We studied chemoautotrophy by measuring dark-fixation of 13C-bicarbonate into phospholipid derived fatty acid (PLFA) biomarkers at two coastal sediment sites with contrasting sulfur chemistry in the Eastern Scheldt estuary, the Netherlands. At one site where free sulfide accumulated in the pore water right to the top of the sediment, PLFA labeling was restricted to compounds typically found in sulfur and ammonium oxidizing bacteria. At the other site, with no detectable free sulfide in the pore water, a very different PLFA labeling pattern was found with high amounts of label in branched i- and a-PLFA besides the typical compounds for sulfur and ammonium oxidizing bacteria. This suggests that other types of chemoautotrophic bacteria were also active, most likely Deltaproteobacteria related to sulfate reducers. Maximum rates of chemoautotrophy were detected in first 1 to 2 centimeters of both sediments and chemosynthetic biomass production was high ranging from 3 to 36 mmol C m−2 d−1. Average dark carbon fixation to sediment oxygen uptake ratios were 0.22±0.07 mol C (mol O2)−1, which is in the range of the maximum growth yields reported for sulfur oxidizing bacteria indicating highly efficient growth. Chemoautotrophic biomass production was similar to carbon mineralization rates in the top of the free sulfide site, suggesting that chemoautotrophic bacteria could play a crucial role in the microbial food web and labeling in eukaryotic poly-unsaturated PLFA was indeed detectable. Our study shows that dark carbon fixation by chemoautotrophic bacteria is a major process in the carbon cycle of coastal sediments, and should therefore receive more attention in future studies on sediment biogeochemistry and microbial ecology.
PMCID: PMC4086895  PMID: 25003508
3.  Coastal Microbial Mat Diversity along a Natural Salinity Gradient 
PLoS ONE  2013;8(5):e63166.
The North Sea coast of the Dutch barrier island of Schiermonnikoog is covered by microbial mats that initiate a succession of plant communities that eventually results in the development of a densely vegetated salt marsh. The North Sea beach has a natural elevation running from the low water mark to the dunes resulting in gradients of environmental factors perpendicular to the beach. These gradients are due to the input of seawater at the low water mark and of freshwater from upwelling groundwater at the dunes and rainfall. The result is a natural and dynamic salinity gradient depending on the tide, rainfall and wind. We studied the microbial community composition in thirty three samples taken every ten meters along this natural salinity gradient by using denaturing gradient gel electrophoresis (DGGE) of rRNA gene fragments. We looked at representatives from each Domain of life (Bacteria, Archaea and Eukarya) and with a particular emphasis on Cyanobacteria. Analysis of the DGGE fingerprints together with pigment composition revealed three distinct microbial mat communities, a marine community dominated by diatoms as primary producers, an intermediate brackish community dominated by Cyanobacteria as primary producers and a freshwater community with Cyanobacteria and freshwater green algae.
PMCID: PMC3660559  PMID: 23704895
4.  Insights into the Physiology and Ecology of the Brackish-Water-Adapted Cyanobacterium Nodularia spumigena CCY9414 Based on a Genome-Transcriptome Analysis 
PLoS ONE  2013;8(3):e60224.
Nodularia spumigena is a filamentous diazotrophic cyanobacterium that dominates the annual late summer cyanobacterial blooms in the Baltic Sea. But N. spumigena also is common in brackish water bodies worldwide, suggesting special adaptation allowing it to thrive at moderate salinities. A draft genome analysis of N. spumigena sp. CCY9414 yielded a single scaffold of 5,462,271 nucleotides in length on which genes for 5,294 proteins were annotated. A subsequent strand-specific transcriptome analysis identified more than 6,000 putative transcriptional start sites (TSS). Orphan TSSs located in intergenic regions led us to predict 764 non-coding RNAs, among them 70 copies of a possible retrotransposon and several potential RNA regulators, some of which are also present in other N2-fixing cyanobacteria. Approximately 4% of the total coding capacity is devoted to the production of secondary metabolites, among them the potent hepatotoxin nodularin, the linear spumigin and the cyclic nodulapeptin. The transcriptional complexity associated with genes involved in nitrogen fixation and heterocyst differentiation is considerably smaller compared to other Nostocales. In contrast, sophisticated systems exist for the uptake and assimilation of iron and phosphorus compounds, for the synthesis of compatible solutes, and for the formation of gas vesicles, required for the active control of buoyancy. Hence, the annotation and interpretation of this sequence provides a vast array of clues into the genomic underpinnings of the physiology of this cyanobacterium and indicates in particular a competitive edge of N. spumigena in nutrient-limited brackish water ecosystems.
PMCID: PMC3610870  PMID: 23555932
5.  Analysis of bacterial and archaeal diversity in coastal microbial mats using massive parallel 16S rRNA gene tag sequencing 
The ISME Journal  2011;5(11):1701-1712.
Coastal microbial mats are small-scale and largely closed ecosystems in which a plethora of different functional groups of microorganisms are responsible for the biogeochemical cycling of the elements. Coastal microbial mats play an important role in coastal protection and morphodynamics through stabilization of the sediments and by initiating the development of salt-marshes. Little is known about the bacterial and especially archaeal diversity and how it contributes to the ecological functioning of coastal microbial mats. Here, we analyzed three different types of coastal microbial mats that are located along a tidal gradient and can be characterized as marine (ST2), brackish (ST3) and freshwater (ST3) systems. The mats were sampled during three different seasons and subjected to massive parallel tag sequencing of the V6 region of the 16S rRNA genes of Bacteria and Archaea. Sequence analysis revealed that the mats are among the most diverse marine ecosystems studied so far and consist of several novel taxonomic levels ranging from classes to species. The diversity between the different mat types was far more pronounced than the changes between the different seasons at one location. The archaeal community for these mats have not been studied before and revealed a strong reaction on a short period of draught during summer resulting in a massive increase in halobacterial sequences, whereas the bacterial community was barely affected. We concluded that the community composition and the microbial diversity were intrinsic of the mat type and depend on the location along the tidal gradient indicating a relation with salinity.
PMCID: PMC3197164  PMID: 21544102
microbial diversity; microbial mats; 16S tag sequencing
6.  Actinomycete integrative and conjugative elements 
Antonie Van Leeuwenhoek  2008;94(1):127-143.
This paper reviews current knowledge on actinomycete integrative and conjugative elements (AICEs). The best characterised AICEs, pSAM2 of Streptomyces ambofaciens (10.9 kb), SLP1 (17.3 kb) of Streptomyces coelicolor and pMEA300 of Amycolatopsis methanolica (13.3 kb), are present as integrative elements in specific tRNA genes, and are capable of conjugative transfer. These AICEs have a highly conserved structural organisation, with functional modules for excision/integration, replication, conjugative transfer, and regulation. Recently, it has been shown that pMEA300 and the related elements pMEA100 of Amycolatopsis mediterranei and pSE211 of Saccharopolyspora erythraea form a novel group of AICEs, the pMEA-elements, based on the unique characteristics of their replication initiator protein RepAM. Evaluation of a large collection of Amycolatopsis isolates has allowed identification of multiple pMEA-like elements. Our data show that, as AICEs, they mainly coevolved with their natural host in an integrated form, rather than being dispersed via horizontal gene transfer. The pMEA-like elements could be separated into two distinct populations from different geographical origins. One group was most closely related to pMEA300 and was found in isolates from Australia and Asia and pMEA100-related sequences were present in European isolates. Genome sequence data have enormously contributed to the recent insight that AICEs are present in many actinomycete genera. The sequence data also provide more insight into their evolutionary relationships, revealing their modular composition and their likely combined descent from bacterial plasmids and bacteriophages. Evidence is accumulating that AICEs act as modulators of host genome diversity and are also involved in the acquisition of secondary metabolite clusters and foreign DNA via horizontal gene transfer. Although still speculative, these AICEs may play a role in the spread of antibiotic resistance factors into pathogenic bacteria. The novel insights on AICE characteristics presented in this review may be used for the effective construction of new vectors that allows us to engineer and optimise strains for the production of commercially and medically interesting secondary metabolites, and bioactive proteins.
PMCID: PMC2440964  PMID: 18523858
Actinomycete integrative and conjugative element; Genome plasticity; Geographical distribution; Horizontal gene transfer; Modular evolution; Replication
7.  Environmental genomics of "Haloquadratum walsbyi" in a saltern crystallizer indicates a large pool of accessory genes in an otherwise coherent species 
BMC Genomics  2006;7:171.
Mature saturated brine (crystallizers) communities are largely dominated (>80% of cells) by the square halophilic archaeon "Haloquadratum walsbyi". The recent cultivation of the strain HBSQ001 and thesequencing of its genome allows comparison with the metagenome of this taxonomically simplified environment. Similar studies carried out in other extreme environments have revealed very little diversity in gene content among the cell lineages present.
The metagenome of the microbial community of a crystallizer pond has been analyzed by end sequencing a 2000 clone fosmid library and comparing the sequences obtained with the genome sequence of "Haloquadratum walsbyi". The genome of the sequenced strain was retrieved nearly complete within this environmental DNA library. However, many ORF's that could be ascribed to the "Haloquadratum" metapopulation by common genome characteristics or scaffolding to the strain genome were not present in the specific sequenced isolate. Particularly, three regions of the sequenced genome were associated with multiple rearrangements and the presence of different genes from the metapopulation. Many transposition and phage related genes were found within this pool which, together with the associated atypical GC content in these areas, supports lateral gene transfer mediated by these elements as the most probable genetic cause of this variability. Additionally, these sequences were highly enriched in putative regulatory and signal transduction functions.
These results point to a large pan-genome (total gene repertoire of the genus/species) even in this highly specialized extremophile and at a single geographic location. The extensive gene repertoire is what might be expected of a population that exploits a diverse nutrient pool, resulting from the degradation of biomass produced at lower salinities.
PMCID: PMC1560387  PMID: 16820057
8.  The genome of the square archaeon Haloquadratum walsbyi : life at the limits of water activity 
BMC Genomics  2006;7:169.
The square halophilic archaeon Haloquadratum walsbyi dominates NaCl-saturated and MgCl2 enriched aquatic ecosystems, which imposes a serious desiccation stress, caused by the extremely low water activity. The genome sequence was analyzed and physiological and physical experiments were carried out in order to reveal how H. walsbyi has specialized into its narrow and hostile ecological niche and found ways to cope with the desiccation stress.
A rich repertoire of proteins involved in phosphate metabolism, phototrophic growth and extracellular protective polymers, including the largest archaeal protein (9159 amino acids), a homolog to eukaryotic mucins, are amongst the most outstanding features. A relatively low GC content (47.9%), 15–20% less than in other halophilic archaea, and one of the lowest coding densities (76.5%) known for prokaryotes might be an indication for the specialization in its unique environment
Although no direct genetic indication was found that can explain how this peculiar organism retains its square shape, the genome revealed several unique adaptive traits that allow this organism to thrive in its specific and extreme niche.
PMCID: PMC1544339  PMID: 16820047

Results 1-8 (8)