Ecological Genomics of Marine Picocyanobacteria

Summary: Marine picocyanobacteria of the genera Prochlorococcus and Synechococcus numerically dominate the picophytoplankton of the world ocean, making a key contribution to global primary production. Prochlorococcus was isolated around 20 years ago and is probably the most abundant photosynthetic organism on Earth. The genus comprises specific ecotypes which are phylogenetically distinct and differ markedly in their photophysiology, allowing growth over a broad range of light and nutrient conditions within the 45°N to 40°S latitudinal belt that they occupy. Synechococcus and Prochlorococcus are closely related, together forming a discrete picophytoplankton clade, but are distinguishable by their possession of dissimilar light-harvesting apparatuses and differences in cell size and elemental composition. Synechococcus strains have a ubiquitous oceanic distribution compared to that of Prochlorococcus strains and are characterized by phylogenetically discrete lineages with a wide range of pigmentation. In this review, we put our current knowledge of marine picocyanobacterial genomics into an environmental context and present previously unpublished genomic information arising from extensive genomic comparisons in order to provide insights into the adaptations of these marine microbes to their environment and how they are reflected at the genomic level.     

Whole-genome microarray analyses of Synechococcus–Vibrio interactions

Microbes live in diverse communities yet their physiologies are typically studied in axenic culture. To begin to address this dichotomy, whole-genome microarray analyses were used and revealed that several major metabolic pathways were affected in Synechococcus sp. WH8102, a model phototroph, when grown with Vibrio parahaemolyticus , a model heterotroph. In co-cultures with V. parahaemolyticus , although phosphate was not depleted, Synechococcus sp. WH8102 may have experienced phosphate stress since the expression of phosphate acquisition genes increased and alkaline phosphatase activity was higher than in monocultures. Expression of cell wall synthesis genes and the components of a zinc transporter were also upregulated. In contrast, a ferric uptake regulation (Fur) family gene was downregulated as were genes that encode proteins rich in iron or involved in detoxifying oxygen radicals. Nitrogen use may also have been affected in co-cultures as the gene expression changes share similarities with ammonia-grown Synechococcus . This study demonstrates the multiple impacts that interspecific microbial interactions can have on the physiology of a major primary producer and the importance of investigating microbial physiology from a community perspective.     

Coastal Synechococcus metagenome reveals major roles for horizontal gene transfer and plasmids in population diversity

The extent to which cultured strains represent the genetic diversity of a population of microorganisms is poorly understood. Because they do not require culturing, metagenomic approaches have the potential to reveal the genetic diversity of the microbes actually present in an environment. From coastal California seawater, a complex and diverse environment, the marine cyanobacteria of the genus Synechococcus were enriched by flow cytometry-based sorting and the population metagenome was analysed with 454 sequencing technology. The sequence data were compared with model Synechococcus genomes, including those of two coastal strains, one isolated from the same and one from a very similar environment. The natural population metagenome had high sequence identity to most genes from the coastal model strains but diverged greatly from these genomes in multiple regions of atypical trinucleotide content that encoded diverse functions. These results can be explained by extensive horizontal gene transfer presumably with large differences in horizontally transferred genetic material between different strains. Some assembled contigs showed the presence of novel open reading frames not found in the model genomes, but these could not yet be unambiguously assigned to a Synechococcus clade. At least three distinct mobile DNA elements (plasmids) not found in model strain genomes were detected in the assembled contigs, suggesting for the first time their likely importance in marine cyanobacterial populations and possible role in horizontal gene transfer.     

Structural and functional analysis of the type III secretion system from Pseudomonas fluorescens Q8r1-96

Pseudomonas fluorescens Q8r1-96 represents a group of rhizosphere strains responsible for the suppressiveness of agricultural soils to take-all disease of wheat. It produces the antibiotic 2,4-diacetylphloroglucinol and aggressively colonizes the roots of cereal crops. In this study, we analyzed the genome of Q8r1-96 and identified a type III protein secretion system (T3SS) gene cluster that has overall organization similar to that of the T3SS gene cluster of the plant pathogen Pseudomonas syringae. We also screened a collection of 30 closely related P. fluorescens strains and detected the T3SS genes in all but one of them. The Q8r1-96 genome contained ropAA and ropM type III effector genes, which are orthologs of the P. syringae effector genes hopAA1-1 and hopM1, as well as a novel type III effector gene designated ropB. These type III effector genes encoded proteins that were secreted in culture and injected into plant cells by both P. syringae and Q8r1-96 T3SSs. The Q8r1-96 T3SS was expressed in the rhizosphere, but mutants lacking a functional T3SS were not altered in their rhizosphere competence. The Q8r1-96 type III effectors RopAA, RopB, and RopM were capable of suppressing the hypersensitive response and production of reactive oxygen species, two plant immune responses.     

Adherence and motility characteristics of clinical Acinetobacter baumannii isolates

Acinetobacter baumannii continues to be a major health problem especially in hospital settings. Herein, features that may play a role in persistence and disease potential were investigated in a collection of clinical A. baumannii strains from Australia. Twitching motility was found to be a common trait in A. baumannii international clone I strains and in abundant biofilm formers, whereas swarming motility was only observed in isolates not classified within the international clone lineages. Bioinformatic analysis of the type IV fimbriae revealed a correlation between PilA sequence homology and motility. A high level of variability in adherence to both abiotic surfaces and epithelial cells was found. We report for the first time the motility characteristics of a large number of A. baumannii isolates and present a direct comparison of A. baumannii binding to nasopharyngeal and lung epithelial cells.     

Insights into the molecular regulation of FasL (CD178) biology

Fas ligand (FasL, CD95L, APO-1L, CD178, TNFSF6, APT1LG1) is the key death factor of receptor-triggered programmed cell death in immune cells. FasL/Fas-dependent apoptosis plays a pivotal role in activation-induced cell death, termination of immune responses, elimination of autoreactive cells, cytotoxic effector function of T and NK cells, and the establishment of immune privilege. Deregulation or functional impairment of FasL threatens the maintenance of immune homeostasis and defense and results in severe autoimmunity. In addition, FasL has been implicated as an accessory or costimulatory receptor in T cell activation. The molecular mechanisms underlying this reverse signaling capacity are, however, poorly understood and still controversially discussed. Many aspects of FasL biology have been ascribed to selective protein-protein interactions mediated by a unique polyproline region located in the membrane-proximal intracellular part of FasL. Over the past decade, we and others identified a large number of putative FasL-interacting molecules that bind to this polyproline stretch via Src homology 3 or WW domains. Individual interactions were analyzed in more detail and turned out to be crucial for the lysosomal storage, the transport and the surface appearance of the death factor and potentially also for reverse signaling. This review summarizes the work in the framework of the Collaborative Research Consortium 415 (CRC 415) and provides facts and hypotheses about FasL-interacting proteins and their potential role in FasL biology.     

A statistical model of ChIA-PET data for accurate detection of chromatin 3D interactions

Identification of three-dimensional (3D) interactions between regulatory elements across the genome is crucial to unravel the complex regulatory machinery that orchestrates proliferation and differentiation of cells. ChIA-PET is a novel method to identify such interactions, where physical contacts between regions bound by a specific protein are quantified using next-generation sequencing. However, determining the significance of the observed interaction frequencies in such datasets is challenging, and few methods have been proposed. Despite the fact that regions that are close in linear genomic distance have a much higher tendency to interact by chance, no methods to date are capable of taking such dependency into account. Here, we propose a statistical model taking into account the genomic distance relationship, as well as the general propensity of anchors to be involved in contacts overall. Using both real and simulated data, we show that the previously proposed statistical test, based on Fisher''s exact test, leads to invalid results when data are dependent on genomic distance. We also evaluate our method on previously validated cell-line specific and constitutive 3D interactions, and show that relevant interactions are significant, while avoiding over-estimating the significance of short nearby interactions.     

Protein modeling and molecular dynamics simulation of the two novel surfactant proteins SP-G and SP-H

The prospects of a control for a novel gallium nitride pseudo-halide vapor phase epitaxy (PHVPE) with HCN were thoroughly analyzed for hydrocarbons–NH₃–Ga gas phase on the basis of quantum chemical investigation with DFT (B3LYP, B3LYP with D3 empirical correction on dispersion interaction) and ab-initio (CASSCF, coupled clusters, and multireference configuration interaction including MRCI+Q) methods. The computational screening of reactions for different hydrocarbons (CH₄, C₂H₆, C₃H₈, C₂H₄, and C₂H₂) as readily available carbon precursors for HCN formation, potential chemical transport agents, and for controlled carbon doping of deposited GaN was carried out with the B3LYP method in conjunction with basis sets up to aug-cc-pVTZ. The gas phase intermediates for the reactions in the Ga-hydrocarbon systems were predicted at different theory levels. The located π-complexes Ga…C₂H₂ and Ga…C₂H₄ were studied to determine a probable catalytic activity in reactions with NH₃. A limited influence of the carbon-containing atmosphere was exhibited for the carbon doping of GaN crystal in the conventional GaN chemical vapor deposition (CVD) process with hydrocarbons injected in the gas phase. Our results provide a basis for experimental studies of GaN crystal growth with C₂H₄ and C₂H₂ as auxiliary carbon reagents for the Ga-NH₃ and Ga-C-NH₃ CVD systems and prerequisites for reactor design to enhance and control the PHVPE process through the HCN synthesis.     

Articular cartilage chondrocytes express aromatase and use enzymes involved in estrogen metabolism

Introduction

Sex hormones, especially estrogens, have been implicated in articular cartilage metabolism and the pathogenesis of postmenopausal osteoarthritis. The conversion by aromatase (CYP19A1) of androstenedione into estrone (E1) and of testosterone into 17β-estradiol (E2) plays a key role in the endogenous synthesis of estrogens in tissue.

Methods

We analyzed the expression of aromatase (CYP19A1) in immortalized C-28/I2 and T/C-28a2 chondrocytes, as well as in cultured primary human articular chondrocytes and human articular cartilage tissue, by means of RT-PCR, Western blotting and immunohistochemistry. By means of quantitative RT-PCR and enzyme-linked immunosorbent assay, we also determined whether the aromatase inhibitor letrozole influences estrogen metabolism of cultured chondrocytes in immortalized C-28/I2 chondrocytes.

Results

Aromatase mRNA was detected in both immortalized chondrocyte cell lines, in cultured primary human chondrocytes, and in human articular cartilage tissue. By means of Western blot analysis, aromatase was detected at the protein level in articular cartilage taken from various patients of both sexes and different ages. Cultured primary human articular chondrocytes, C-28/I2 and T/C-28a2, and human articular cartilage tissue reacted with antibodies for aromatase. Incubation of C-28/I2 chondrocytes with 10⁻¹¹ M to 10⁻⁷ M letrozole as an aromatase inhibitor revealed significantly increased amounts of the mRNAs of the enzyme cytochrome P4501A1 (CYP1A1), which is involved in the catagen estrogen metabolism, and of the estrogen receptors ER-α and ER-β. Concomitantly, synthesis of estrone (E1) was significantly downregulated after incubation with letrozole.

Conclusions

We demonstrate that human articular cartilage expresses aromatase at the mRNA and protein levels. Blocking of estrone synthesis by the aromatase inhibitor letrozole is counteracted by an increase in ER-α and ER-β. In addition, CYP1A1, an enzyme involved in catabolic estrogen metabolism, is upregulated. This suggests that articular chondrocytes use ERs functionally. The role of endogenous synthesized estrogens in articular cartilage health remains to be elucidated.     

A review of the primary types of the Hawaiian stag beetle genus Apterocyclus Waterhouse (Coleoptera,Lucanidae, Lucaninae), with the description of a new species

The species of the Hawaiian stag beetle genus Apterocyclus Waterhouse (Coleoptera: Lucanidae) are reviewed following an examination of all primary types. Although the continued existence of the species is unknown and some possibly are extinct there are five recently extant species, including one species that is described here as new. The holotypes for all available names are pictured, and synonymies discussed and updated. Lectotypes are designated for Apterocyclus honoluluensis Waterhouse and A. munroi Sharp. A key to species and a revised catalog for the genus are provided.