Archaea, Bacteria, and Algal Plastids Associated with the Reef-Building Corals Siderastrea stellata and Mussismilia hispida from Búzios, South Atlantic Ocean, Brazil

Reef-building corals may be seen as holobiont organisms, presenting diverse associated microbial communities. Best known is the symbiotic relationship with zooxanthellae, but Archaea, Bacteria, fungi, viruses, and algal plastids are also abundant. Until now, there is little information concerning microbial communities associated with Brazilian corals. The present study aims to describe the diversity of Archaea, Bacteria, and eukaryotic algal plastid communities associated with two sympatric species, Siderastrea stellata and Mussismilia hispida, from Southeastern Brazil, using 16S rRNA gene libraries. Since corals present a high number of other associated invertebrates, coral barcoding (COI) was performed to confirm the exclusive occurrence of coral DNA in our samples. Our analysis yielded 354 distinct microbial OTUs, represented mainly by novel phylotypes. Richness (Chao1 and ACE) and diversity (H') estimations of the microbial communities associated with both species were high and comparable to other studies. Rarefaction analyses showed that microbial diversity of S. stellata is higher than that of M. hispida. Libshuff comparative analyses showed that the highest microbial community similarity between the two coral species occurred in the bacterial libraries, while archaeal and plastidial communities were significantly different. Crenarchaeota dominated archaeal communities, while Proteobacteria was the most abundant bacterial phylum, dominated by alpha-Proteobacteria. Plastids were also represented by novel phylotypes and did not match with any 16S rRNA sequences of Cyanobacteria and zooxanthellae from GenBank. Our data improves the pool of available information on Brazilian coral microbes and shows corals as sources of diverse prokaryotic and picoeukaryotic communities.     

In vitro Synthesis of Native,Fibrous Long Spacing and Segmental Long Spacing Collagen

Collagen fibrils are present in the extracellular matrix of animal tissue to provide structural scaffolding and mechanical strength. These native collagen fibrils have a characteristic banding periodicity of ~67 nm and are formed in vivo through the hierarchical assembly of Type I collagen monomers, which are 300 nm in length and 1.4 nm in diameter. In vitro, by varying the conditions to which the monomer building blocks are exposed, unique structures ranging in length scales up to 50 microns can be constructed, including not only native type fibrils, but also fibrous long spacing and segmental long spacing collagen. Herein, we present procedures for forming the three different collagen structures from a common commercially available collagen monomer. Using the protocols that we and others have published in the past to make these three types typically lead to mixtures of structures. In particular, unbanded fibrils were commonly found when making native collagen, and native fibrils were often present when making fibrous long spacing collagen. These new procedures have the advantage of producing the desired collagen fibril type almost exclusively. The formation of the desired structures is verified by imaging using an atomic force microscope.     

Spectroscopic properties of the Chlorophyll a–Chlorophyll c 2–Peridinin-Protein-Complex (acpPC) from the coral symbiotic dinoflagellate Symbiodinium

Femtosecond time-resolved transient absorption spectroscopy was performed on the chlorophyll a–chlorophyll c ₂–peridinin-protein-complex (acpPC), a major light-harvesting complex of the coral symbiotic dinoflagellate Symbiodinium. The measurements were carried out on the protein as well on the isolated pigments in the visible and the near-infrared region at 77 K. The data were globally fit to establish inter-pigment energy transfer paths within the scaffold of the complex. In addition, microsecond flash photolysis analysis was applied to reveal photoprotective capabilities of carotenoids (peridinin and diadinoxanthin) in the complex, especially the ability to quench chlorophyll a triplet states. The results demonstrate that the majority of carotenoids and other accessory light absorbers such as chlorophyll c ₂ are very well suited to support chlorophyll a in light harvesting. However, their performance in photoprotection in the acpPC is questionable. This is unusual among carotenoid-containing light-harvesting proteins and may explain the low resistance of the acpPC complex against photoinduced damage under even moderate light conditions.     

从18S rDNA序列初探散胞盘菌亚科属间亲缘关系

散胞盘菌亚科Encoelioideae建于1932年,为盘菌纲Discomycetes锤舌菌目Leotiales锤舌菌科（广义）Leotiaceae的成员,它包括腐生、植物寄生、真菌上生的盘状子囊菌,现含18属,其中若干为单种属。传统分类从形态学角度将外囊盘表层细胞松散结合或者表面覆盖短的菌丝延伸物视为此亚科的主要特征,宏观上,其成员的共性表现为外囊盘被表面糠皮状、鳞屑状或霜状。迄今,学者们从未对散孢盘菌亚科这一性状是否反映属间亲缘关系进行过任何探讨,国际基因库中亦无此亚科的可利用序列。为了探明此性状在系统演化过程中的作用以及散胞盘菌亚科的属间亲缘关系,我们对此亚科中绿散胞盘菌Chlorencoelia、复柄盘菌属Cordierites、散胞盘菌属Encoelia、霍氏盘菌属Holwaya、拟爪毛盘菌属 Unguiculariopsis、以及丝绒盘菌属Velutarina代表种的18SrDNA片段进行了序列测定,受实验材料所限,每属只获得了一个种的18SrDNA序列。为了分析和比较,还对同一科内锤舌菌亚科Leotioideae中绿杯菌属Chlorociboria一个种的相关序列进行了测定。在构建系统树的过程中,核盘菌科的核盘菌Sclerotiniasclerotiorum被选为外群;从基因库中调入属于同一个目的地舌菌科和锤舌菌亚科几个属的代表即胶鼓菌属Bulgaria、锤舌菌属Leotia、小舌菌属Microglossum、新胶鼓菌属Neobulgaria和地勺菌属Spathularia的序列;利用ClustalW和TreeView软件,构建出Neighbour-joining系统进化树。初步的研究结果表明,散孢盘菌亚科不是单源的,该亚科的成员与其他亚科或者其他科的物种一起形成四个系统演化分支。散胞盘菌亚科的Holwaya与另一亚科的Bulgaria（Leotiaceae,Leotioideae）聚在一起,具有95%的序列相似性和97.4%支持率（bootstrapvalue）;此二属与Leotia（Leotiaceae,Leotioideae）和Microglossum（Geoglossaceae）组成姐妹群。散胞盘菌亚科Cordierites和Encoelia接近,支持率为93.6%;它们是Leotioideae中Chlorociboria和Neobulgaria两属的姐妹群。散胞盘菌亚科的Unguiculariopsis与Velutarina在一起,独立成为一个分支,支持率高达99.3%。Chlorencoelia与本研究所涉及的散胞盘菌亚科其他成员的关系都比较远。以上研究结果表明,散胞盘菌亚科很可能是多源的;但是,由于该亚科大多数属缺乏分子生物学证据,本研究结果只能作为推断。     

Juxtaposition of signal-peptide charge and core region hydrophobicity is critical for functional signal peptides

In Escherichia coli, exported proteins are synthesized as precursors containing an amino-terminal signal peptide which directs transport through the translocase to the proper destination. We have constructed a series of signal peptide mutants, incorporating linker sequences of varying lengths between the amino-terminal charge and core region hydrophobicity, to examine the requirement for the juxtaposition of these two structural features in promoting protein transport. In vivo and in vitro analyses indicated that high transport efficiency via signal peptides with core regions of marginal hydrophobicity absolutely requires the proximity of sufficient charge.     

Abnormalities and reduced reproductive potential of sperm from Tnp1- and Tnp2-null double mutant mice

Previous studies have demonstrated the importance of transition nuclear proteins, TP1 and TP2, in spermatogenesis and male fertility. However, importance of the overall level of transition proteins and their level of redundancy in the production of normal sperm is not clear. Epididymal sperm from the nine possible Tnp1 and Tnp2 null genotypes demonstrated a general decrease in normal morphology, motility, chromatin condensation, and degree of protamine 2 processing with decreasing levels of transition proteins in mutant sperm. Nuclei of some mutant epididymal sperm stained poorly with hematoxylin and DNA fluorochromes, suggesting that the DNA of these sperm underwent degradation during epididymal transport. When epididymal sperm were injected directly into oocytes, fertilization and embryonic development were reduced only in the two most severely affected genotypes. These phenotypes indicated some functional redundancy of transition proteins; however, redundancy of transition protein function was not complete, as, for example, sperm from double heterozygous males had fewer abnormalities than sperm from males homozygous for a single Tnp null mutation. Our study suggests that each TP fulfills some unique function during spermiogenesis even though sperm phenotypes strongly indicate defects are largely attributable to an overall gene dosage effect. Similarities between sperm defects found in Tnp mutants and infertile patients make the Tnp mutants a valuable tool with which to study outcomes following fertilization using sperm with compromised DNA integrity.     

Genetic analysis of determinants for spike glycoprotein assembly into murine coronavirus virions: distinct roles for charge-rich and cysteine-rich regions of the endodomain

The coronavirus spike protein (S) forms the distinctive virion surface structures that are characteristic of this viral family, appearing in negatively stained electron microscopy as stems capped with spherical bulbs. These structures are essential for the initiation of infection through attachment of the virus to cellular receptors followed by fusion to host cell membranes. The S protein can also mediate the formation of syncytia in infected cells. The S protein is a type I transmembrane protein that is very large compared to other viral fusion proteins, and all except a short carboxy-terminal segment of the S molecule constitutes the ectodomain. For the prototype coronavirus mouse hepatitis virus (MHV), it has previously been established that S protein assembly into virions is specified by the carboxy-terminal segment, which comprises the transmembrane domain and the endodomain. We have genetically dissected these domains in the MHV S protein to localize the determinants of S incorporation into virions. Our results establish that assembly competence maps to the endodomain of S, which was shown to be sufficient to target a heterologous integral membrane protein for incorporation into MHV virions. In particular, mutational analysis indicated a major role for the charge-rich carboxy-terminal region of the endodomain. Additionally, we found that the adjacent cysteine-rich region of the endodomain is critical for fusion of infected cells, confirming results previously obtained with S protein expression systems.     

The soft-bottom macrobenthos of Gray's Reef National Marine Sanctuary and nearby shelf waters off the coast of Georgia, USA

As part of an ongoing ecological assessment of the Gray's Reef National Marine Sanctuary (GRNMS), a 58-km² marine protected area 32 km off the coast of Georgia, USA, surveys of benthic macroinfaunal communities, contaminant levels in sediments and biota, and general habitat conditions were conducted during 2000-2002 at 20 stations within the sanctuary and along three cross-shelf transects in nearby shelf waters. Macroinfaunal community structure and composition exhibited distinct cross-shelf patterns associated with sediment granulometry, depth and possibly other factors related to shoreline proximity (e.g., erosional effects, recruitment of estuarine species). Finer-scale spatial patterns of benthic fauna among stations within the sanctuary appear to be related to proximity to live-bottom habitat and other features of seafloor structure (e.g., rippled vs. flat sand). Population densities of dominant fauna within the sanctuary also varied considerably among years, resulting in shifts in the ranking of dominants at most stations. Chemical contaminants generally were at low background concentrations below probable bioeffect levels and thus are not a likely cause of the observed spatial patterns of benthic fauna. However, trace concentrations of pesticides, PCBs, and PAHs were detectable in sediments and biota throughout the study area, demonstrating that chemicals originating from human activities are capable of reaching the offshore sanctuary environment, possibly from atmospheric deposition or cross-shelf transport of materials outwelled through coastal sounds. Highly diverse infaunal assemblages also were observed within the sanctuary and nearby sites of similar depth, suggesting that the sanctuary is an important reservoir of marine biodiversity. Results of this study should be useful in addressing long-term science and management needs of the GRNMS and in furthering our understanding of broader ecological patterns and dynamics of the surrounding South Atlantic Bight (SAB) ecosystem.     

Amplification and sequence analysis of the full length toxR gene in Vibrio harveyi

This study was focused on obtaining the complete gene sequence of the toxR gene in V. harveyi by using toxR-targeted PCR to amplify 5' and 3' regions flanking the 576-bp Vibrio harveyi (NBRC 15634) toxR gene fragment previously amplified using degenerate PCR. To obtain the 5' flanking sequences, a forward PCR primer (VhtoxRpv) was designed based on known sequences upstream of toxR in V. parahaemolyticus and V. vulnificus. The reverse primer (VctoxR2R) was based on the sequence of the 576-bp Vibrio harveyi toxR fragment. The resulting 750-bp amplicon was sequenced, providing the 5' sequences of the V. harveyi (NBRC 15634) toxR gene. The 3' flanking region was amplified using a primer pair toxRS1 and toxRS2 based on V. parahaemolyticus and V. vulnificus toxR and toxS, resulting in a 900-bp amplicon that contained the remaining 3' sequences of the V. harveyi NBRC 15634 toxR. This paper reports, for the first time, a complete 882-bp nucleotide sequence for toxR in Vibrio harveyi. Sequence analysis and alignment revealed that the complete toxR gene in V. harveyi shares 87% sequence similarity with toxR of V. parahaemolyticus, 84% similarity with V. fluvialis, 83% with V. vulnificus and partial sequence of V. campbellii. The phylogenetic trees revealed wider divergence in toxR compared to 16S rRNA genes, so that V. harveyi could easily be distinguished from V. campbellii and V. parahaemolyticus.