Denitrifying microbial communities provide an important ecosystem function in aquatic systems. Yet, knowledge on predictive and modeling of these complex and changing communities is limited. The emergently challenging question of how the geographical distribution of denitrifiers responds to ongoing and future environmental change is not yet fully understood. In our study we use metadata-based correlative niche modeling to analyze the geographical distribution of selected putative denitrifiers in the genus Sphingomonas, Mycoplana, Shewanella, and Alteromonas at different predicted environmental conditions and future climatic scenarios across the Baltic Sea. Using the predictive power of an ensemble modeling approach and eight different machine-learning algorithms, habitat suitability and the distribution of the selected denitrifiers were evaluated using geophysical and bioclimatic variables, benthic conditions, and four Representative Concentration Pathway (RCP) trajectories of future global warming scenarios. All algorithms provided successful prediction capabilities both for variable importance, and for habitat suitability with Area Under the Curve (AUC) values between 0.89 and 1.00. Model findings revealed that salinity and nitrate concentrations significantly explained the variation in distribution of the selected denitrifiers. Rising temperatures of 0.8 to 1.8 °C at future RCP60–2050 trajectories are predicted to diminish or eliminate the bioclimatic suitable habitats for denitrifier distributions across the Baltic Sea. Multi-collated terrestrial and marine environmental variables contributed to the successful prediction of denitrifier distributions within the study area. The correlative niche modeling approach with high AUC values presented in the study allowed for accurate projections of the future distributions of the selected denitrifiers. The modeling approach can be used to improve our understanding of how ongoing and predicted future environmental changes may affect habitat suitability for organisms with denitrification capacity across the Baltic Sea.

In coastal aphotic sediments, organic matter (OM) input from phytoplankton is the primary food resource for benthic organisms. Current observations from temperate ecosystems like the Baltic Sea report a decline in spring bloom diatoms, while summer cyanobacteria blooms are becoming more frequent and intense. These climate-driven changes in phytoplankton communities may in turn have important consequences for benthic biodiversity and ecosystem functions, but such questions are not yet sufficiently explored experimentally. Here, in a 4-week experiment, we investigated the response of microeukaryotic and bacterial communities to different types of OM inputs comprising five ratios of two common phytoplankton species in the Baltic Sea, the diatom Skeletonema marinoi and filamentous cyanobacterium Nodularia spumigena. Metabarcoding analyses on 16S and 18S ribosomal RNA (rRNA) at the experiment termination revealed subtle but significant changes in diversity and community composition of microeukaryotes in response to settling OM quality. Sediment bacteria were less affected, although we observed a clear effect on denitrification gene expression (nirS and nosZ), which was positively correlated with increasing proportions of cyanobacteria. Altogether, these results suggest that future changes in OM input to the seafloor may have important effects on both the composition and function of microbenthic communities.

The Earth’s permanently cold biosphere is known to harbour abundant microbial biomass and represents a rich resource for the discovery of novel cold-adapted microorganisms, many of which form part of the ‘microbial dark matter’ which cannot be analysed using traditional culture-dependent approaches. The recent development of metagenomics and related multi-omics strategies has provided a means by which entire microbial communities can be studied directly, without the prerequisite of culturing. The advancement of the ‘omic’ methods is directly linked to recent progress in high-throughput sequencing, robust data processing capabilities and the application of cutting-edge analytical tools for high-throughput detection of biomolecules. The combined application of these tools and strategies has provided an unprecedented access to the structure and potential function of microbial communities in cold environments, providing increasingly comprehensive insights into the taxonomic richness and functional capacity of the indigenous microorganisms. Applications of ‘omic’ strategies have enhanced our understanding of psychrophilic adaptation mechanisms, revealing the versatility and adaptability of life in the ‘cryosphere’. In addition to the predicted roles of psychrophiles in biogeochemical cycling, recent multi-omic studies have further emphasised the importance of the ‘cryosphere’ in influencing global atmospheric conditions. Finally, metagenomic bioprospecting of cold environments has yielded a variety of novel bioactive molecules including novel ‘psychrozymes’, with a wide range of potential industrial and biotechnological applications. Here, we have provided an overview of recent developments in metagenomic technologies and their application in the study of the cold biosphere.

BACKGROUND: Through functional screening of a fosmid library, generated from a phytopathogen-suppressive soil metagenome, the novel antifungal chitinase-named Chi18H8 and belonging to family 18 glycosyl hydrolases-was previously discovered. The initial extremely low yield of Chi18H8 recombinant production and purification from Escherichia coli cells (21 μg/g cell) limited its characterization, thus preventing further investigation on its biotechnological potential.

RESULTS: We report on how we succeeded in producing hundreds of milligrams of pure and biologically active Chi18H8 by developing and scaling up to a high-yielding, 30 L bioreactor process, based on a novel method of mild solubilization of E. coli inclusion bodies in lactic acid aqueous solution, coupled with a single step purification by hydrophobic interaction chromatography. Chi18H8 was characterized as a Ca(2+)-dependent mesophilic chitobiosidase, active on chitin substrates at acidic pHs and possessing interesting features, such as solvent tolerance, long-term stability in acidic environment and antifungal activity against the phytopathogens Fusarium graminearum and Rhizoctonia solani. Additionally, Chi18H8 was found to operate according to a non-processive endomode of action on a water-soluble chitin-like substrate.

CONCLUSIONS: Expression screening of a metagenomic library may allow access to the functional diversity of uncultivable microbiota and to the discovery of novel enzymes useful for biotechnological applications. A persisting bottleneck, however, is the lack of methods for large scale production of metagenome-sourced enzymes from genes of unknown origin in the commonly used microbial hosts. To our knowledge, this is the first report on a novel metagenome-sourced enzyme produced in hundreds-of-milligram amount by recovering the protein in the biologically active form from recombinant E. coli inclusion bodies.

Denitrification in sediments is a key microbial process that removes excess fixed nitrogen, while dissimilatory nitrate reduction to ammonium (DNRA) converts nitrate to ammonium. Although microorganisms are responsible for essential nitrogen (N) cycling, it is not yet fully understood how these microbially mediated processes respond to toxic hydrophobic organic compounds (HOCs) and metals. In this study, we sampled long-term polluted sediment from the outer harbor of Oskarshamn (Baltic Sea), measured denitrification and DNRA rates, and analyzed taxonomic structure and N-cycling genes of microbial communities using metagenomics. Results showed that denitrification and DNRA rates were within the range of a national reference site and other unpolluted sites in the Baltic Sea, indicating that long-term pollution did not significantly affect these processes. Furthermore, our results indicate an adaptation to metal pollution by the N-cycling microbial community. These findings suggest that denitrification and DNRA rates are affected more by eutrophication and organic enrichment than by historic pollution of metals and organic contaminants.

Benthic ecosystems have come under intense pressure, due to eutrophication-driven oxygen decline and industrial metal contamination. One of the most toxic metals is Cadmium (Cd), which is lethal to many aquatic organisms already at low concentrations. Denitrification by facultative anaerobic microorganisms is an essential process to transform, but also to remove, excess nitrate in eutrophied systems. Cd has been shown to decrease denitrification and sequester free sulfide, which is available when oxygen is scarce and generally inhibits complete denitrification (i.e. N2O to N2). In polluted sediments, an interaction between oxygen and Cd may influence denitrification and this relationship has not been studied. For example, in the Baltic Sea some sediments are double exposed to both Cd and hypoxia. In this study, we examined how the double exposure of Cd and fluctuations in oxygen affects denitrification in Baltic Sea sediment. Results show that oxygen largely regulated N2O and N2 production after 21 days of exposure to Cd (ranging from 0 to 500 μg/L, 5 different treatments, measured by the isotope pairing technique (IPT)). In the high Cd treatment (500 μg/L) the variation in N2 production increased compared to the other treatments. Increases in N2 production are suggested to be an effect of 1) enhanced nitrification that increases NO3 − availability thus stimulating denitrification, and 2) Cd successfully sequestrating sulfide (yielding CdS), which allows for full denitrification to N2. The in situ field sediment contained initially high Cd concentrations in the pore water (∼10 μg/L) and microbial communities might already have been adapted to metal stress, making the effect of low Cd levels negligible. Here we show that high levels of cadmium pollution might increase N2 production and influence nitrogen cycling in marine sediments. © 2019 The Authors

The current study assessed the occurrence of the Vibrio cholerae serogroups O1 and O139 in environmental samples along salinity gradients in three selected estuaries of Tanzania both through culture independent methods and by cultured bacteria. Occurrence of V. cholerae was determined by PCR targeting the V. cholerae outer membrane protein gene ompW. Furthermore, the presence of toxigenic strains and serogroups O1 and O139 was determined using multiplex PCR with specific primers targeting the cholera toxin gene subunit A, ctxA, and serotype specific primers, O1-rfb and O139-rfb, respectively. Results showed that V. cholerae occurred in approximately 10% (n = 185) of both the environmental samples and isolated bacteria. Eight of the bacteria isolates (n = 43) were confirmed as serogroup O1 while one belonged to serogroup O139, the first reported identification of this epidemic strain in East African coastal waters. All samples identified as serogroup O1 or O139 and a number of non-O1/O139 strains were ctxA positive. This study provides in situ evidence of the presence of pathogenic V. cholerae O1 and O139 and a number of V. cholerae non-O1/O139 that carry the cholera toxin gene in estuaries along the coast of Tanzania.

IncP-1, IncP-7 and IncP-9 plasmids often carry genes encoding enzymes involved in the degradation of man-made and natural contaminants, thus contributing to bacterial survival in polluted environments. However, the lack of suitable molecular tools often limits the detection of these plasmids in the environment. In this study, PCR followed by Southern blot hybridization detected the presence of plasmid-specific sequences in total community (TC-) DNA or fosmid DNA from samples originating from different environments and geographic regions. A novel primer system targeting IncP-9 plasmids was developed and applied along with established primers for IncP-1 and IncP-7. Screening TC-DNA from biopurification systems (BPS) which are used on farms for the purification of pesticide-contaminated water revealed high abundances of IncP-1 plasmids belonging to different subgroups as well as IncP-7 and IncP-9. The novel IncP-9 primer-system targeting the rep gene of nine IncP-9 subgroups allowed the detection of a high diversity of IncP-9 plasmid specific sequences in environments with different sources of pollution. Thus polluted sites are "hot spots" of plasmids potentially carrying catabolic genes.

Community structures of active bacterial populations were investigated along a vertical redox profile in coastal Baltic Sea sediments by terminal-restriction fragment length polymorphism (T-RFLP) and clone library analysis. According to correspondence analysis of T-RFLP results and sequencing of cloned 16S rRNA genes, the microbial community structures at three redox depths (179, -64 and -337 mV) differed significantly. The bacterial communities in the community DNA differed from those in bromodeoxyuridine (BrdU)-labelled DNA, indicating that the growing members of the community that incorporated BrdU were not necessarily the most dominant members. The structures of the actively growing bacterial communities were most strongly correlated to organic carbon followed by total nitrogen and redox potentials. Bacterial identification by sequencing of 16S rRNA genes from clones of BrdU-labelled DNA and DNA from reverse transcription polymerase chain reaction showed that bacterial taxa involved in nitrogen and sulfur cycling were metabolically active along the redox profiles. Several sequences had low similarities to previously detected sequences, indicating that novel lineages of bacteria are present in Baltic Sea sediments. Also, a high number of different 16S rRNA gene sequences representing different phyla were detected at all sampling depths.

Nearly half the seabed of the Baltic Proper is incapable of supporting life of higher organisms as a consequence of oxygen depletion resulting from eutrophication. However, these areas are actually teeming with microbial life. Here we used terminal-restriction fragment length polymorphism (T-RFLP) to investigate the dominant archaeal and bacterial groups, with respect to community structure, in surface layers of bottom sediments of the Baltic Sea along a coastal pollution gradient. Both archaeal and bacterial communities formed distinct clusters along the pollution gradient and the community compositions were different at the polluted sites compared with the relatively clean reference sites. The structures of the bacterial communities were most strongly correlated to water depth, followed by organic carbon, oxygen, salinity and silicate levels. In contrast, the structures of the archaeal communities were most strongly correlated to oxygen, salinity, organic carbon, silicate and nitrate levels. Some members of the microbial communities were identified using a combination of traditional and molecular approaches. Isolates obtained on different culture media were identified by partial sequencing of their 16S rRNA genes and some novel species were found. In addition, we developed a computer program, APLAUS, to elucidate the putative identities of the most dominant community members by T-RFLP.

This report summarizes the events of the 1st International Functional Metagenomics Workshop. The workshop was held on May 7 and 8, 2012, in St. Jacobs, Ontario, Canada and was focused on building an international functional metagenomics community, exploring strategic research areas, and identifying opportunities for future collaboration and funding. The workshop was initiated by researchers at the University of Waterloo with support from the Ontario Genomics Institute (OGI), Natural Sciences and Engineering Research Council of Canada (NSERC) and the University of Waterloo.

The book summarizes our current knowledge in the molecular biology of plant mitochondria. It covers such topics as: - RNA editing - mitochondrial gene organization and expression - protein synthesis and transport - cytoplasmic male sterility Specific emphasis is placed on RNA editing. Different systems known to date in mitochondria and plastids are compared. In addition, their connection with the molecular biology involved with the functional analysis is delineated. Another major topic is the molecular biology of mitochondrial genomes of cytoplasmic male sterile (cms) plants. The similarities observed in different cms systems not only promote our understanding of those processes which lead to male sterile plants but are also helpful for breeding strategies. Concise and timely, this book is a unique collaboration of researchers from different fields.

The mitochondrial general processing peptidase (MPP) in plant mitochondria constitutes an integral part of the cytochromebc ₁ complex of the respiratory chain. Here we present a characterization of this bifunctional complex from spinach leaf mitochondria. The purified MPP/bc ₁ complex has a molecular mass of 550 kDa, which corresponds to a dimer. Increased ionic strength results in partial dissociation of the dimer as well as loss of the processing activity. Micellar concentrations of nonionic and zwitterionic detergents stimulate the activity by decreasing the temperature optimum of the processing reaction, whereas anionic detergents totally suppress the activity. MPP is a metalloendopeptidase. Interestingly, hemin, a potent regulator of mitochondrial and cytosolic biogenesis and inhibitor of proteosomal degradation, inhibits the processing activity. Measurements of the processing activity at different redox states of the bc ₁ complex show that despite bifunctionality of the MPP/bc ₁ complex, there is no correlation between electron transfer and protein processing.

In this work, we analyzed the community structure and metabolic potential of sediment microbial communities in high-latitude coastal environments subjected to low to moderate levels of chronic pollution. Subtidal sediments from four low-energy inlets located in polar and subpolar regions from both Hemispheres were analyzed using large-scale 16S rRNA gene and metagenomic sequencing. Communities showed high diversity (Shannon's index 6.8 to 10.2), with distinct phylogenetic structures (<40% shared taxa at the Phylum level among regions) but similar metabolic potential in terms of sequences assigned to KOs. Environmental factors (mainly salinity, temperature, and in less extent organic pollution) were drivers of both phylogenetic and functional traits. Bacterial taxa correlating with hydrocarbon pollution included families of anaerobic or facultative anaerobic lifestyle, such as Desulfuromonadaceae, Geobacteraceae, and Rhodocyclaceae. In accordance, biomarker genes for anaerobic hydrocarbon degradation (bamA, ebdA, bcrA, and bssA) were prevalent, only outnumbered by alkB, and their sequences were taxonomically binned to the same bacterial groups. BssA-assigned metagenomic sequences showed an extremely wide diversity distributed all along the phylogeny known for this gene, including bssA sensu stricto, nmsA, assA, and other clusters from poorly or not yet described variants. This work increases our understanding of microbial community patterns in cold coastal sediments, and highlights the relevance of anaerobic hydrocarbon degradation processes in subtidal environments.

Abstract Nuclear  encoded  mitochondrial  precursor  proteins  are  cleaved  to  mature  size products  by the  general  mitochondrial  processing  peptidase  (MPP). In  contrast  to  non-plant  sources  where  MPP  is  a matrix  enzyme,  the  plant  mitochondrial  MPP  is localised  in  the  inner  membrane  and  constitutes an  integral  part  of  the  bc,  complex  of  the  respiratory  chain.  Core  proteins  of  the  complex  are  immunologically  related  and  show  high  sequence similarity  to the MPP  subunits  from  non-plant  sources.  The bc,  complex  in plants  is thus  bifunctional,  being  involved  both  in respiration  and  in protein processing

Several hundreds of mitochondrial proteins are nuclear encoded and are synthesised on cytosolic polyribosomes as precursor proteins. Most of these precursors contain an N-terminal extension called presequence which functions as targeting signal and which is cleaved off after import. Despite the fact that there are no sequence similarities and no consensus for the cleavage site in mitochondrial presequences, cleavage of almost all presequences is catalysed by a single, highly specific metalloendopeptidase, called general mitochondrial processing peptidase (MPP). MPP in plants is integrated into the bc₁, complex of the respiratory chain and both subunits, α-MPP and β-MPP, are identical to the core proteins of the complex. Despite the fact that the bc₁ complex in plants is bifunctional, catalysing bothelectron transport and protein processing, these two functions are distinct. MPP belongs to the Pitrilysin family of peptidases, characterised by a zinc binding motif, HXXEH_74–76E, involved in catalysis. Both the membrane-bound integrated MPP/bc₁ complex of plants and the soluble mammalian MPP recognise similar higher-order structural elements upstream from the cleavage site that are important for processing. The secondary structure with flexibility and stabilising elements, hydrofobicity, charge and length seem to influence the interaction with MPP. The newly imported non-assembled precursor inside mitochondria is degraded by a proteinase that is distinct from MPP or any other previously characterised proteinases, a novel ATP-dependent, membrane-associated serine-type proteinase.

Mitochondrial biogenesis requires a coordinated expression of both the nuclear and the organellar genomes and specific intracellular protein trafficking, processing and assembly machinery. Mostmitochondrial proteins are synthesised as precursor proteins containing an N-terminal extension which functions as a targeting signal, which is proteolytically cleaved off after import into mitochondria. We review our present knowledge on components and mechanisms involved in the mitochondrial proteinimport process in plants. This encompasses properties of targeting peptides, sorting of precursor proteinsbetween mitochondria and chloroplasts, signal recognition, mechanism of translocation across the mitochondrial membranes and the role of cytosolic and organellar molecular chaperones in this process. The mitochondrial protein processing in plants is catalysed by the mitochondrial processing peptidase (MPP), which in contrast to other sources, is integrated into the bc₁ complex of the respiratory chain. This is the most studied component of the plant import machinery characterised to date. What are the biochemical consequences of the integration of the MPP into an oligomeric protein complex and how are several hundred presequences of precursor proteins with no sequence similarities and no consensus for cleavage, specifically cleaved off by MPP? Finally we will address the emerging area of the control of protein import into mitochondria.

Soil that is suppressive to disease caused by fungal pathogens is an interesting source to target for novel chitinases that might be contributing towards disease suppression. In this study, we screened for chitinase genes, in a phytopathogen-suppressive soil in three ways: (1) from a metagenomic library constructed from microbial cells extracted from soil, (2) from directly extracted DNA and (3) from bacterial isolates with antifungal and chitinase activities. Terminal restriction fragment length polymorphism (T-RFLP) of chitinase genes revealed differences in amplified chitinase genes from the metagenomic library and the directly extracted DNA, but approximately 40% of the identified chitinase terminal restriction fragments (TRFs) were found in both sources. All of the chitinase TRFs from the isolates were matched to TRFs in the directly extracted DNA and the metagenomic library. The most abundant chitinase TRF in the soil DNA and the metagenomic library corresponded to the TRF103 of the isolate Streptomyces mutomycini and/or Streptomyces clavifer. There were good matches between T-RFLP profiles of chitinase gene fragments obtained from different sources of DNA. However, there were also differences in both the chitinase and the 16S rRNA gene T-RFLP patterns depending on the source of DNA, emphasizing the lack of complete coverage of the gene diversity by any of the approaches used.

Plant disease caused by fungal pathogens results in vast crop damage globally. Microbial communities of soil that is suppressive to fungal crop disease provide a source for the identification of novel enzymes functioning as bioshields against plant pathogens. In this study, we targeted chitin-degrading enzymes of the uncultured bacterial community through a functional metagenomics approach, using a fosmid library of a suppressive soil metagenome. We identified a novel bacterial chitinase, Chi18H8, with antifungal activity against several important crop pathogens. Sequence analyses show that the chi18H8 gene encodes a 425-amino acid protein of 46 kDa with an N-terminal signal peptide, a catalytic domain with the conserved active site F¹⁷⁵DGIDIDWE¹⁸³, and a chitinase insertion domain. Chi18H8 was expressed (pGEX-6P-3 vector) in Escherichia coli and purified. Enzyme characterization shows that Chi18H8 has a prevalent chitobiosidase activity with a maximum activity at 35 °C at pH lower than 6, suggesting a role as exochitinase on native chitin. To our knowledge, Chi18H8 is the first chitinase isolated from a metagenome library obtained in pure form and which has the potential to be used as a candidate agent for controlling fungal crop diseases. Furthermore, Chi18H8 may also answer to the demand for novel chitin-degrading enzymes for a broad range of other industrial processes and medical purposes.

A novel lipase was isolated from a metagenomic library of Baltic Sea sediment bacteria. Prokaryotic DNA was extracted and cloned into a copy control fosmid vector (pCC1FOS) generating a library of > 7000 clones with inserts of 24-39 kb. Screening for clones expressing lipolytic activity based on the hydrolysis of tributyrin and p-nitrophenyl esters, identified 1% of the fosmids as positive. An insert of 29 kb was fragmented and subcloned. Subclones with lipolytic activity were sequenced and an open reading frame of 978 bp encoding a 35.4-kDa putative lipase/esterase h1Lip1 (DQ118648) with 54% amino acid similarity to a Pseudomomas putida esterase (BAD07370) was identified. Conserved regions, including the putative active site, GDSAG, a catalytic triad (Ser148, Glu242 and His272) and a HGG motif, were identified. The h1Lip1 lipase was over expressed, (pGEX-6P-3 vector), purified and shown to hydrolyse p-nitrophenyl esters of fatty acids with chain lengths up to C-14. Hydrolysis of the triglyceride derivative 1,2-di-O-lauryl-rac-glycero-3-glutaric acid 6'-methylresorufin ester (DGGR) confirmed that h1Lip1 was a lipase. The apparent optimal temperature for h1Lip1, by hydrolysis of p-nitrophenyl butyrate, was 35 degrees C. Thermal stability analysis showed that h1Lip1 was unstable at 25 degrees C and inactivated at 40 degrees C with t(1/2) < 5 min.

In this study we report the first comparison of the mitochondrial protein import and processing events in two different tissues from the same organism. Both spinach leaf and root mitochondria were able to import and process the in vitro transcribed and translated Neurospora crassa F₁ subunit of ATP synthase to the mature size product. Temperature optimum for protein import, 20 °C, was considerably lower than that found in other systems. In spinach leaf mitochondria, the processing peptidase has been shown to constitute an integral part of the bc₁ complex of the respiratory chain. In accordance with these results, the majority of the processing activity in root mitochondria was also localized in the membrane. However, although the same amount of the processing peptidase was present per mg of membrane protein in both leaf and root mitochondria, as determined immunologically, the specific processing activity was several-fold higher in roots. Furthermore, in contrast to the processing enzyme in leaf, a portion of the processing activity could be disassociated from the root membrane with relatively weak salt treatment. The processing event in both the leaf and root membranes was always accompanied by a degradation of the F₁ precursor. The degradation activity was found to be several-fold higher in roots than in leaves and was also partially dissociated from the membrane after salt treatment. Both the processing and degradation activities were inhibited by orthophenanthroline, a known metalloprotease inhibitor. These results show tissue-specific differencies of the processing event catalyzed by the bc₁ complex and indicate the presence of two populations of the processing peptidase in root mitochondria.

The bacterium causing cholera, Vibrio cholerae, is essentially a marine organism and its ecological dynamics have been linked to oceanographic conditions and climate. We used autoregressive models with external inputs to identify potential relationships between the number of cholera cases in the coastal regions of mainland Tanzania with climatic and environmental indices (maximum air temperature, sea surface temperature, wind speed and chlorophyll a). Results revealed that, between 2004 and 2010, coastal regions of mainland Tanzania inhabited by approximately 21% of the total population accounted for approximately 50% of the cholera cases and 40% of the total mortality. Significant co-variations were found between seasonally adjusted cholera cases and coastal ocean chlorophyll a and, to some degree, sea surface temperature, the outbreaks lagging behind by one to four months. Cholera cases in Dar es Salaam were also weakly related to the Indian Ocean Dipole Mode Index, lagging by five months, suggesting that it may be possible to predict cholera outbreaks for Dar es Salaam this period ahead. The results also suggest that the severity of cholera in coastal regions can be predicted by ocean conditions and that longer-term environmental and climate parameters may be used to predict cholera outbreaks along the coastal regions.

Rhodopsins are broadly distributed. In this work we analyzed 23 metagenomes corresponding to marine sediment samples from four regions which share cold climate conditions (Norway; Sweden; Argentina and Antarctica). In order to investigate the genes evolution of viral-rodopsins, an initial set of 6224 bacterial rhodopsins sequences according COG5524 were retrieved from the 23 metagenomes. After selection by the presence of transmembrane domains and alignment 123 viral (51) and non-viral (72) sequences (>50 aminoacids) were finally included in further analysis. Viral rhodopsin genes were homologues of Phaeocystis globosa virus and Organic lake Phycodnavirus Non-viral microbial rhodopsin genes were ascribed to Bacteroidetes, Planctomycetes, Firmicutes, Actinobacteria, Cyanobacteria, Proteobacteria, Deinococcus-Thermus as well as Cryptophyta and Fungi. A re-screening using Blastp, using as queries the viral sequences previously described, retrieved 30 sequences (>100 aminoacids). Phylogeographic analysis revealed a geographycal clustering of the sequences affiliated to the viral group. This clustering was not observed for the microbial non-viral sequences. The phylogenetic reconstruction allowed us to propose the existence of a putative ancestor of viral rhodopsins (PAVR) genes related to Actinobacteria and Chloroflexi This is the first report about the existence of a phylogeographic association of the viral rhodopsins sequences from marine sediments.

Alginates are abundant polysaccharides in brown algae that constitute an important energy source for marine heterotrophic bacteria. Despite the key role of alginate degradation processes in the marine carbon cycle, little information is available on the bacterial populations involved in these processes. The aim of this work was to gain a better understanding of alginate utilization capabilities in cold coastal environments. Sediment metagenomes from four high-latitude regions of both Hemispheres were interrogated for alginate lyase gene homologue sequences and their genomic context. Sediments contained highly abundant and diverse bacterial assemblages with alginolytic potential, including members of Bacteroidetes and Proteobacteria, as well as several poorly characterized taxa. The microbial communities in Arctic and Antarctic sediments exhibited the most similar alginolytic profiles, whereas brackish sediments showed distinct structures with a higher proportion of novel genes. Examination of the gene neighbourhood of the alginate lyase homologues revealed distinct patterns depending on the potential lineage of the scaffolds, with evidence of evolutionary relationships among alginolytic gene clusters from Bacteroidetes and Proteobacteria. This information is relevant for understanding carbon fluxes in cold coastal environments and provides valuable information for the development of biotechnological applications from brown algae biomass.

The goal of this work was to identify sequences encoding monooxygenase biocatalysts with novel features by in silico mining an assembled metagenomic dataset of polar and subpolar marine sediments. The targeted enzyme sequences were Baeyer-Villiger and bacterial cytochrome P450 monooxygenases (CYP153). These enzymes have wide-ranging applications, from the synthesis of steroids, antibiotics, mycotoxins and pheromones to the synthesis of monomers for polymerization and anticancer precursors, due to their extraordinary enantio-, regio-, and chemo- selectivity that are valuable features for organic synthesis. Phylogenetic analyses were used to select the most divergent sequences affiliated to these enzyme families among the 264 putative monooxygenases recovered from the ~14 million protein-coding sequences in the assembled metagenome dataset. Three-dimensional structure modeling and docking analysis suggested features useful in biotechnological applications in five metagenomic sequences, such as wide substrate range, novel substrate specificity or regioselectivity. Further analysis revealed structural features associated with psychrophilic enzymes, such as broader substrate accessibility, larger catalytic pockets or low domain interactions, suggesting that they could be applied in biooxidations at room or low temperatures, saving costs inherent to energy consumption. This work allowed the identification of putative enzyme candidates with promising features from metagenomes, providing a suitable starting point for further developments.

The reconstruction of bacterial and archaeal genomes from shotgun metagenomes has enabled insights into the ecology and evolution of environmental and host-associated microbiomes. Here we applied this approach to >10,000 metagenomes collected from diverse habitats covering all of Earth’s continents and oceans, including metagenomes from human and animal hosts, engineered environments, and natural and agricultural soils, to capture extant microbial, metabolic and functional potential. This comprehensive catalog includes 52,515 metagenome-assembled genomes representing 12,556 novel candidate species-level operational taxonomic units spanning 135 phyla. The catalog expands the known phylogenetic diversity of bacteria and archaea by 44% and is broadly available for streamlined comparative analyses, interactive exploration, metabolic modeling and bulk download. We demonstrate the utility of this collection for understanding secondary-metabolite biosynthetic potential and for resolving thousands of new host linkages to uncultivated viruses. This resource underscores the value of genome-centric approaches for revealing genomic properties of uncultivated microorganisms that affect ecosystem processes.

Variations in sediment biochemistry and abundance of meiofauna were investigated in three tropical habitats: mangrove forest, intertidal lagoon, and subtidal reef area in eastern Africa. Both the biochemical components of the pore water and the meiofauna varied substantially over small distances in all three habitats. In the mangrove area, the color of the pore water was found to be positively correlated with the major meiofaunal taxa, especially the nematodes. In the coastal lagoon, polychaetes showed negative correlation with particulate organic matter while other groups showed no correlation with any of the chemical components analyzed. In the subtidal reef area no chemical components were found to correlate with the meiofauna. When data from the three habitats are taken together two clear associations emerge. First, grain size shows a highly significant relationship with both total fauna and nematode numbers. Second, there is clear association between the amount of particulate organic carbon and particulate organic nitrogen in the pore water.

Coastal areas in East Africa are experiencing rapid economic, resource management, demographic and technological shifts. In response diverse Community-based Natural Resource Management (CBNRM) applications have been embraced to provide mutual conservation and use benefits. These initiatives have met with mixed success in practice. Reflecting on the limitations of past research using common pool resources theory theory to study CBNRM we use insights from actor oriented theory combined with satellite image analysis to describe and discuss the forces dynamically influencing institutional and mangrove forest cover change at Kisakasaka, Zanzibar focussing on the formal CBNRM project period between 1996 and 2001, but also considering the period before and after this. We examine the shifting social relations that affected the performance and viability of the formal CBNRM arrangements. An integrated approach was taken to the presentation and discussion of results where it was possible to enrich and expand explanations of socio-environmental change, which was driven by a lack of government support, the undermining effects of party political divisions, and the lack of institutional adaptive capacity. We conclude that this was a useful approach to explain CBNRM intervention events at Kisakasaka.

This study employs insights largely derived from critical reflections on the common pool resources (CPR) theory to examine the current governance arrangements in place to manage the mangrove forest at Kisakasaka, in Zanzibar, Tanzania. Kisakasaka was used as a site for a community-based management pilot project of forest resources in Zanzibar. After some initial success in setting up a local management structure and regulating access to the mangrove for mainly charcoal production, there are now clear indications that forest conditions have deteriorated dramatically with concomitant ongoing resource use problems for local villagers who have relied heavily on forest resources as a source of cash income. Extra-local factors, such as urban population increases and associated market pressures for charcoal, are also conjectured to overlay and interact with the institutional problems at Kisakasaka. As a result, over concern about the deterioration in the condition of the mangrove forest, the responsible government authority decided not to renew the community-based governance arrangements after an initial five-year pilot period. While revealing the inadequacies of existing governance arrangements and of its relationship to deteriorating forest conditions at Kisakasaka, this study concludes by suggesting an approach to more fully understand forces driving local resource management and use. 

Cleavage sites in nuclear-encoded mitochondrial protein targeting peptides (mTPs) from mammals, yeast, and plants have been analysed for characteristic physicochemical features using statistical methods, perceptrons, multilayer neural networks, and self-organizing feature maps, Three different sequence motifs were found, revealing loosely defined arginine motifs with Arg in positions -10, -3, and -2. A self-organizing feature map was able to cluster these three types of endopeptidase target sites but did not identify any species-specific characteristics in mTPs, Neural networks were used to define local sequence features around precursor cleavage sites.

The microbial diversity in maritime meltwater pond sediments from Bratina Island, Ross Sea, Antarctica was investigated by 16S rDNA-dependent molecular phylogeny. Investigations of the vertical distribution, phylogenetic composition, and spatial variability of Bacteria and Archaea in the sediment were carried out. Results revealed the presence of a highly diverse bacterial population and a significantly depth-related composition. Assessment of 173 partial 16S rDNA clones analyzed by amplified rDNA restriction analysis (ARDRA) using tetrameric restriction enzymes (HinP1I 5'GdelCGC3'and Msp I. 5'CdelCGG3', BioLabs) revealed 153 different bacterial OTUs (operational taxonomic units). However, only seven archaeal OTUs were detected, indicating low archaeal diversity. Based on ARDRA results, 30 bacterial clones were selected for sequencing and the sequenced clones fell into seven major lineages of the domain Bacteria; the alpha, gamma, and delta subdivisions of Proteobacteria, the Cytophaga-Flavobacterium-Bacteroides, the Spirochaetaceae, and the Actinobacteria. All of the archaeal clones sequenced belonged to the group Crenarchaeota and phylogenetic analysis revealed close relationships with members of the deep-branching Group 1 Marine Archaea.

The presence of non-indigenous microbial contaminants resulting from human faecal contamination of old and currently occupied base and field camp sites in South Victoria Land, Antarctica, was assessed by PCR amplification of extracted soil DNA using species-specific PCR primers. Positive controls (samples recovered from the environs of Scott Base, including the sewage outfall) gave strong signals with Escherichia coli primers whereas Clostridium clostridiiforme primers yielded a signal only with the sewage outfall sample. A comparison was made of PCR amplification results from samples from the abandoned Canada Glacier camp site, the Lake Fryxell summer camp site, the Cape Bird Adelie penguin colony and pristine sites from relatively inaccessible regions of the Taylor Valley. Results indicated a possible residual level of E. coli contamination in the abandoned Canada Glacier camp site, but no significant contamination of the currently occupied Lake Fryxell camp site. These data may provide indirect evidence for improved awareness and standards of waste handling and disposal over the past two decades of Dry Valley field research.

The rapidly expanding field of metagenomics has revolutionized the ability to analyzemicrobial communities by providing access to ‘true’ microbial diversity. This field has a broad range of applications in the areas of biodiversity, systems biology and biotechnology (Handelsman et al. 2002). As most microbial communities in soil, sediment or aquatic environments are highly complex, consisting of hundreds oreven thousands of species of which only a few have been cultured, the approaches collectively described as metagenomics, community genomics or environmental genomics have been developed to help to unlock this thus far hidden diversity. Metagenomics is therefore the application of modern genomic tools used to analyze the collective genomes of whole microbial communities (the metagenome) in an environmental sample, thereby bypassing the need for isolation or cultivation (Béjà et al. 2000; Riesenfeld et al. 2004b). Even though the tip of the microbial ice-berg has barely been scratched, this approach is rapidly increasing our knowledge of microbial genetic and functional diversity through gene/pathway discovery either by sequencing or activity-based screening strategies (Handelsman 2005).

The mitochondrial processing peptidase (MPP) in lower eucaryots and mammals is a matrix enzyme, whereas MPP in plants constitutes an integral part of the bc1 complex of the respiratory chain. The isolated spinach leaf bc1 complex catalyzes cleavage of the precursor of Nicotiana plumbaginifolia F1 beta subunit of the ATP synthase, resulting in a production of mature protein and a presequence that consists of 54 amino acids. A synthetic peptide derived from the C-terminal part of the presequence, containing 17 amino acids with a helical structural element, p-F1 beta(38-54), was an efficient inhibitor of the processing, whereas a peptide derived from the N-terminal part of the presequence, p-F1 beta(1-18), was much less effective. ATIII, a helical peptide derived from antithrombin III, was not recognized by MPP. Synthetic peptides corresponding to 4, 6, and 11 amino acids of the C terminus of the presequence, p-F1 beta(51-54), p-F1 beta(49-54), and p-F1 beta(44-54) were almost completely inert. Competition studies show that MPP recognizes the C-terminal domain of the presequence rather than the N-terminal domain. Furthermore, the alpha-helical element of the C-terminal domain is shown to be required for the recognition event.

Proteins function at distinct sites in the cell, yet most are primarily synthesized in the cytosol and must therefore be transported intracellularly to their final destination. Mitochondrial targeting peptides are localized at the N-terminus of the precursor proteins and most are cleaved off after import into mitochondria. Cleavage is catalysed by the general mitochondrial processing peptidase, which, in plants, is integrated into the bc₁ complex of the respiratory chain. This peptidase recognizes the cleavage sites of nearly 1000 precursor proteins that have no sequence similarity.

The bacterial diversity and nutrient dynamics of mangrove sediments in Kisakasaka, Tanzania, was investigated in order to evaluate potential changes associated with deforestation of mangroves. Study sites included relatively undisturbed, recently protected mangroves and clear-cut mangrove areas that were sampled during both the wet and dry seasons. Physicochemical parameters, nitrogenase activity, pore water nutrient concentrations and bacterial diversity were measured in sediment depth profiles using both molecular and chemical techniques. Results show that there are significant differences in sediment pore water nutrient concentrations and bacterial diversity in sediments of mangrove areas which have been deforested compared to those which have been protected. Average measured values for protected and deforested areas, respectively, were: sulphide (S-2-),S- < 42 +/- 10 mu M and > 1.9 +/- 0.5 mM at 30 cm depth; ammonium (NH4+), 58 +/- 2 mu M and 113 +/- 12 mu M at 4-5 cm depth; soluble reactive phosphate, 40.2 +/- 11 mu M and 18.4 +/- 1.2 at 4-5 cm depth. Nitrogen fixation rates were lower in deforested areas during day and night, organic content was higher in protected areas (20 +/- 5%) compared to deforested areas (12 +/- 3%). The bacterial diversity was lower in deforested areas as determined by Shannon index using 16S rRNA gene analysis with terminal restriction fragment length polymorphism.