Metagenomic characterization of oyster shell dump reveals predominance of <Emphasis Type="Italic">Firmicutes</Emphasis> bacteria

Metagenomic analyses were conducted to evaluate the biodiversity of oyster shell bacteria, under storage conditions, on the basis of 16s rDNA sequences. Temperature was recorded during a one year storage period, and the highest temperature (about 60°C) was observed after five months of storage. Bacterial diversity was greatest in the initial stage sample, with 33 different phylotypes classified under seven phyla (Proteobacteria, Bacteroidetes, Firmicutes, Actinobacteria, Planctomycetes, Verrucomicrobia and unclassified bacteria), with 42.22% of phylotypes belonging to Proteobacteria. The lowest diversity was found in the high temperature (fermentation) stage sample, with 10 different phylotypes belonging to Firmicutes (78.57%) and Bacteroidetes. In the final stage sample, bacteria were found belonging to Proteobacteria, Bacteroidetes, and Firmicutes, and some were unclassified bacteria. Of the bacteria constituting the final stage metagenome, 69.70% belonged to Firmicutes. Our results show that bacteria belonging to phylum Firmicutes were predominant during fermentation, and during the final stages of oyster shell storage, which suggests that these bacteria supposed to be the key players for oyster shell biodegradation.     

Aggregation of ammonia-oxidizing bacteria in microbial biofilm on oyster shell surface

Summary Formation and activity of bacterial nitrifying biofilms play an important role in the closed seawater systems for shrimp cultivation. The structure of microbial biofilm on empty oyster shells, used as a biofilm carrier in biofiltration of aquacultural water, was studied using fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy. FISH was performed with specific oligonucleotide probes for Bacteria and ammonia-oxidizing Nitrosomonas spp. The bacterial cells were arranged within the biofilm as a layer of vertically elongated aggregates. Aggregates of ammonia-oxidizing bacteria were embedded within the matrix formed by other bacteria. Vertically elongated cell aggregates may be ecologically important in bacterial biofilms because they have a higher surface-to-volume ratio than that of laminated biofilms.     

Metagenomic analysis reveals unexpected subgenomic diversity of magnetotactic bacteria within the phylum Nitrospirae

A targeted metagenomic approach was applied to investigate magnetotactic bacteria (MTB) within the phylum Nitrospirae in Lake Miyun near Beijing, China. Five fosmids containing rRNA operons were identified. Comparative sequence analysis of a total of 172 kb provided new insights into their genome organization and revealed unexpected subgenomic diversity of uncultivated MTB in the phylum Nitrospirae. In addition, affiliation of two novel MTB with the phylum Nitrospirae was verified by fluorescence in situ hybridization. One of them was morphologically similar to "Candidatus Magnetobacterium bavaricum," but the other differed substantially in cell shape and magnetosome organization from all previously described "Ca. Magnetobacterium bavaricum"-like bacteria.     

Transfer of conjugative elements from rumen and human Firmicutes bacteria to Roseburia inulinivorans

Studies on Firmicutes bacteria from the gut are hampered by a lack of gene transfer systems. Here the human colonic anaerobe Roseburia inulinivorans A2-194 was shown to be a transfer recipient for two conjugative transposons, Tn1545 from Eubacterium cellulosolvens and TnK10 from Clostridium saccharolyticum K10.     

Intramembrane-sensing histidine kinases: a new family of cell envelope stress sensors in Firmicutes bacteria

Two-component signal-transducing systems (TCS) consist of a histidine kinase (HK) that senses a specific environmental stimulus, and a cognate response regulator (RR) that mediates the cellular response. Most HK are membrane-anchored proteins harboring two domains: An extracytoplasmic input and a cytoplasmic transmitter (or kinase) domain, separated by transmembrane helices that are crucial for the intramolecular information flow. In contrast to the cytoplasmic domain, the input domain is highly variable, reflecting the plethora of different signals sensed. Intramembrane-sensing HK (IM-HK) are characterized by their short input domain, consisting solely of two putative transmembane helices. They lack an extracytoplasmic domain, indicative for a sensing process at or from within the membrane interface. Most proteins sharing this domain architecture are found in Firmicutes bacteria. Two major groups can be differentiated based on sequence similarity and genomic context: (1) BceS-like IM-HK that are functionally and genetically linked to ABC transporters, and (2) LiaS-like IM-HK, as part of three-component systems. Most IM-HK sense cell envelope stress, and identified target genes are often involved in maintaining cell envelope integrity, mediating antibiotic resistance, or detoxification processes. Therefore, IM-HK seem to constitute an important mechanism of cell envelope stress response in low G+C Gram-positive bacteria.     

Studies on shell formation. VII. The submicroscopic structure of the shell of the oyster Crassostrea virginica

The submicroscopic structure of the growing surface of the shell of the oyster, Crassostrea virginica, was studied by means of shadowed replicas. The outer edge of the prismatic region consists of a fine grained matrix enclosing crystals, the surfaces of which show a finely pebbled structure. Crystal size varies continously from 0.01 micro to 8 micro. The matrix surface shows no evidence of fibrous structure. The outer portions of the prismatic region exhibit a tile-like arrangement of large crystals separated by granular matrix 0.02 to 0.08 micro in thickness. The exposed crystal surfaces have indentations of varying form which appear as roughly parallel grooves spaced at intervals of approximately 0.3 micro. The final form of this region is believed to result from the random distribution of crystal seeds, which grow without orientation and through coalescence and growth come into contact, producing polygonal areas. The crystal arrangement of the nacreous region is one of overlapping rows of crystals in side to side contact, and with one end of each crystal free, permitting continued increase in length. Crystal angles and plane indices are presented.     

Characterization of pathogenic bacteria of the cupped oyster Crassostrea gigas

The French mollusc production is mainly based on the Pacific cupped oyster, Crassostrea gigas. Since 1991, outbreaks of mass mortality of juveniles are reported during the summer period. These outbreaks are a major concern of oyster industry. Several studies have established given bacterial strains to be pathogenic for bivalve species, including oysters. Here we present a study of mortality outbreaks of C. gigas, as initiated in 1995. In a first step, bacterial strains were isolated during mass mortality outbreak and were biochemically characterised. Among the isolated strains, some strains of Vibrio splendidus biovar II were found to be pathogenic by means of experimental challenge of oyster juveniles. In the second step, a genotypical identification of the pathogenic strain was undertaken, based on 16S RNA sequences and phylogenetic analysis. It confirmed that the pathogenetic strain belonged to Vibrio splendidus biovar II.     

Transesterification of soybean oil using combusted oyster shell waste as a catalyst

Transesterification of soybean oil catalyzed by combusted oyster shell, which is waste material from shellfish farms, was examined. Powdered oyster shell combusted at a temperature above 700 degrees C, at which point the calcium carbonate of oyster shell transformed to calcium oxide, acted as a catalyst in the transesterification of soybean oil. On the basis of factorial design, the reaction conditions of catalyst concentration and reaction time were optimized in terms of the fatty acid methyl ester concentration expressed as biodiesel purity. Under the optimized reaction conditions of a catalyst concentration and reaction time of 25wt.%. and 5h, respectively, the biodiesel yield, expressed relative to the amount of soybean oil poured into the reaction vial, was more than 70% with high biodiesel purity. These results indicate oyster shell waste combusted at high temperature can be reused in biodiesel production as a catalyst.     

Nucleation and growth of calcite on native versus pyrolyzed oyster shell folia

The thin sheets of calcite, termed folia, that make up much of the shell of an oyster are covered by a layer of discrete globules that has been proposed to consist of agglomerations of protein and mineral. Foliar fragments, treated at 475 degrees C for 36 h to remove organic matter, were imaged by atomic force microscopy (AFM) as crystals grew on the foliar surfaces in artificial seawater at calcite supersaturations up to 52-fold. Crystals were also viewed later by scanning electron microscopy. After pyrolysis, the foliar globules persisted only as fragile remnants that were quickly washed away during AFM imaging, revealing an underlying morphology on the foliar laths of a tightly packed continuum of nanometer-scale protrusions. At intermediate supersaturations, crystal formation was seen immediately almost everywhere on these surfaces, each crystal having the same distinctive shape and orientation, even at the outset with crystals as small as a few nanometers. In contrast, nucleation did not occur readily on non-pyrolyzed foliar surfaces, and the crystals that did grow, although slowly at intermediate supersaturations, had irregular shapes. Possible crystallographic features of foliar laths are considered on the basis of the morphology of ectopic crystals and the atomic patterns of various surfaces. A model for foliar lath formation is presented that includes cycles of pulsed secretion of shell protein, removal of the protein from the mineralizing solution upon binding to mineral, and mineral growth at relatively high supersaturation over a time frame of about 1 h for each turn of the cycle.     

Metagenomic study of red biofilms from Diamante Lake reveals ancient arsenic bioenergetics in haloarchaea

Arsenic metabolism is proposed to be an ancient mechanism in microbial life. Different bacteria and archaea use detoxification processes to grow under high arsenic concentration. Some of them are also able to use arsenic as a bioenergetic substrate in either anaerobic arsenate respiration or chemolithotrophic growth on arsenite. However, among the archaea, bioenergetic arsenic metabolism has only been found in the Crenarchaeota phylum. Here we report the discovery of haloarchaea (Euryarchaeota phylum) biofilms forming under the extreme environmental conditions such as high salinity, pH and arsenic concentration at 4589 m above sea level inside a volcano crater in Diamante Lake, Argentina. Metagenomic analyses revealed a surprisingly high abundance of genes used for arsenite oxidation (aioBA) and respiratory arsenate reduction (arrCBA) suggesting that these haloarchaea use arsenic compounds as bioenergetics substrates. We showed that several haloarchaea species, not only from this study, have all genes required for these bioenergetic processes. The phylogenetic analysis of aioA showed that haloarchaea sequences cluster in a novel and monophyletic group, suggesting that the origin of arsenic metabolism in haloarchaea is ancient. Our results also suggest that arsenite chemolithotrophy likely emerged within the archaeal lineage. Our results give a broad new perspective on the haloarchaea metabolism and shed light on the evolutionary history of arsenic bioenergetics.     

Metagenomic characterization of airborne viral DNA diversity in the near-surface atmosphere

Airborne viruses are expected to be ubiquitous in the atmosphere but they still remain poorly understood. This study investigated the temporal and spatial dynamics of airborne viruses and their genotypic characteristics in air samples collected from three distinct land use types (a residential district [RD], a forest [FR], and an industrial complex [IC]) and from rainwater samples freshly precipitated at the RD site (RD-rain). Viral abundance exhibited a seasonal fluctuation in the range between 1.7 × 10(6) and 4.0 × 10(7) viruses m(-3), which increased from autumn to winter and decreased toward spring, but no significant spatial differences were observed. Temporal variations in viral abundance were inversely correlated with seasonal changes in temperature and absolute humidity. Metagenomic analysis of air viromes amplified by rolling-circle phi29 polymerase-based random hexamer priming indicated the dominance of plant-associated single-stranded DNA (ssDNA) geminivirus-related viruses, followed by animal-infecting circovirus-related sequences, with low numbers of nanoviruses and microphages-related genomes. Particularly, the majority of the geminivirus-related viruses were closely related to ssDNA mycoviruses that infect plant-pathogenic fungi. Phylogenetic analysis based on the replication initiator protein sequence indicated that the airborne ssDNA viruses were distantly related to known ssDNA viruses, suggesting that a high diversity of viruses were newly discovered. This research is the first to report the seasonality of airborne viruses and their genetic diversity, which enhances our understanding of viral ecology in temperate regions.     

Microtexture of larval shell of oyster,Crassostrea nippona: A FIB-TEM study

The initial formation and subsequent development of larval shells in marine bivalve, Crassostrea nippona were investigated using the FIB-TEM technique. Fourteen hours after fertilization (the trochophore stage), larvae form an incipient shell of 100–150 nm thick with a columnar contrast. Selected-area electron diffraction analysis showed a single-crystal aragonite pattern with the c-axis perpendicular to the shell surface. Plan-view TEM analysis suggested that the shell contains high density of {110} twins, which are the origin of the columnar contrast in the cross-sectional images. 72 h after fertilization (the veliger stage), the shell grows up to 1.2–1.4 μm thick accompanying an additional granular layer between the preexisting layer and embryo to form a distinctive two-layer structure. The granular layer is also composed of aragonite crystals sharing their c-axes perpendicular to the shell surface, but the crystals are arranged with a flexible rotation around the c-axes and not restricted solely to the {110} twin relation. No evidence to suggest the existence of amorphous calcium carbonate (ACC) was found through the observation. The well-regulated crystallographic properties found in the present sample imply initial shell formation probably via a direct deposition of crystalline aragonite.     

Location, synthesis and function of glycolipids and polyglycerolphosphate lipoteichoic acid in Gram-positive bacteria of the phylum Firmicutes

Lipoteichoic acid (LTA) is a zwitterionic polymer found in the cell wall of many Gram-positive bacteria. A widespread and one of the best-studied forms of LTA consists of a polyglycerolphosphate (PGP) chain that is tethered to the membrane via a glycolipid anchor. In this review, we will summarize our current understanding of the enzymes involved in glycolipid and PGP backbone synthesis in a variety of different Gram-positive bacteria. The recent identification of key LTA synthesis proteins allowed the construction and analysis of mutant strains with defined defects in glycolipid or backbone synthesis. Using these strains, new information on the functions of LTA for bacterial growth, physiology and during developmental processes was gained and will be discussed. Furthermore, we will reintroduce the idea that LTA remains in close proximity to the bacterial membrane for its function during bacterial growth rather than as a surface-exposed structure.     

Biomineralization: functions of calmodulin-like protein in the shell formation of pearl oyster

Calmodulin-like protein (CaLP) was believed to be involved in the shell formation of pearl oyster. However, no further study of this protein was ever performed. In this study, the in vitro crystallization experiment showed that CaLP can modify the morphology of calcite. In addition, aragonite crystals can be induced in the mixture of CaLP and a nacre protein (at 16 kDa), which was detected and purified from the EDTA-soluble matrix of nacre. These results agreed with that of immunohistological staining in which CaLP was detected not only in the organic layer sandwiched between nacre (aragonite) and the prismatic layer (calcite), but also around the prisms of the prismatic layer. Take together, we concluded that (1) CaLP, as a component of the organic layer, can induce the nucleation of aragonite through binding with the 16-kDa protein, and (2) CaLP may regulate the growth of calcite in the prismatic layer.     

Use of extracts from oyster shell and soil for cultivation of Spirulina maxima

Calcium ion and trace metals play important roles in various metabolisms of photosynthetic organisms. In this study, simple methods were developed to extract calcium ion and micronutrients from oyster shell and common soil, and the prepared extracts were tested as a replacement of the corresponding chemicals that are essential for growth of microalgae. The oyster shell and soil were treated with 0.1 M sodium hydroxide or with 10 % hydrogen peroxide, respectively. The potential application of these natural sources to cultivation was investigated with Spirulina maxima. When compared to standard Zarrouk medium, the Spirulina maxima cultivated in a modified Zarrouk media with elements from oyster shell and soil extract exhibited increases in biomass, chlorophyll, and phycocyanin by 17, 16, and 64 %, respectively. These results indicate that the extracts of oyster shell and soil provide sufficient amounts of calcium and trace metals for successful cultivation of Spirulina maxima.     

A novel extracellular EF-hand protein involved in the shell formation of pearl oyster

Mollusk shell formation is a complicated and highly controlled calcium metabolism process. Previous studies revealed that several EF-hand calcium-binding proteins actively participate in the regulation of shell mineralization. In this study, we cloned a full-length cDNA encoding a novel extracellular EF-hand calcium-binding protein (named EFCBP) from the pearl oyster, Pinctada fucata, according to the EF-hand motifs of calmodulin. Although it shares high similarity with the calmodulin family in its EF-hand signatures, EFCBP just has two EF-hand motifs and belongs to a new separate group from the other EF-hand proteins according to a phylogenetic analysis. EFCBP is specifically expressed in shell mineralization-related tissues, viz. the mantle, the gill, and the hemocytes. Moreover, its expression responds quickly only to the shell damage, but not to the damage of other tissues and the infection of the lipopolysaccharides from Escherichia coli. These results suggest that EFCBP might be an important regulator of shell formation. This finding may help better understand the functions of EF-hand proteins on the regulation of mollusk shell formation.     

A novel phosphorylated glycoprotein in the shell matrix of the oyster Crassostrea nippona

We found a novel 52 kDa matrix glycoprotein MPP1 in the shell of Crassostrea nippona that was unusually acidic and heavily phosphorylated. Deduced from the nucleotide sequence of 1.9 kb cDNA, which is likely to encode MPP1 with high probability, the primary structure of this protein shows a modular structure characterized by repeat sequences rich in Asp, Ser and Gly. The most remarkable of these is the DE-rich sequence, in which continuous repeats of Asp are interrupted by a single Cys residue. Disulfide-dependent MPP1 polymers occurring in the form of multimeric insoluble gels are estimated to contain repetitive locations of the anionic molecules of phosphates and acidic amino acids, particularly Asp. Thus, MPP1 and its polymers possess characteristic features of a charged molecule for oyster biomineralization, namely accumulation and trapping of Ca2+. In addition, MPP1 is the first organic matrix component considered to be expressed in both the foliated and prismatic layers of the molluscan shell microstructure. In vitro crystallization assays demonstrate the induction of tabular crystals with a completely different morphology from those formed spontaneously, indicating that MPP1 and its polymers are potentially the agent that controls crystal growth and shell microstructure.     

A novel silk-like shell matrix gene is expressed in the mantle edge of the Pacific oyster prior to shell regeneration

During shell formation, little is known about the functions of organic matrices, especially about the biomineralization of shell prismatic layer. We identified a novel gene, shelk2, from the Pacific oyster presumed to be involved in the shell biosynthesis. The Pacific oyster has multiple copies of shelk2. Shelk2 mRNA is specifically expressed on the mantle edge and is induced during shell regeneration, thereby suggesting that Shelk2 is involved in shell biosynthesis. To our surprise, the database search revealed that it encodes a spider silk-like alanine-rich protein. Interestingly, most of the Shelk2 primary structure is composed of two kinds of poly-alanine motifs-GXNA(n)(S) and GSA(n)(S)-where X denotes Gln, Arg or no amino acid. Occurrence of common motifs of Shelk2 and spider silk led us to the assumption that shell and silk are constructed under similar strategies despite of their living environments.