Characterization of red-shifted phycobilisomes isolated from the chlorophyll f-containing cyanobacterium Halomicronema hongdechloris.

Phycobilisomes are the main light-harvesting protein complexes in cyanobacteria and some algae. It is commonly accepted that these complexes only absorb green and orange light, complementing chlorophyll absorbance. Here, we present a new phycobilisome derived complex that consists only of allophycocyanin core subunits, having red-shifted absorption peaks of 653 and 712 nm. These red-shifted phycobiliprotein complexes were isolated from the chlorophyll f-containing cyanobacterium, Halomicronema hongdechloris, grown under monochromatic 730 nm-wavelength (far-red) light. The 3D model obtained from single particle analysis reveals a double disk assembly of 120-145 Å with two α/ß allophycocyanin trimers fitting into the two separated disks. They are significantly smaller than typical phycobilisomes formed from allophycocyanin subunits and core-membrane linker proteins, which fit well with a reduced distance between thylakoid membranes observed from cells grown under far-red light. Spectral analysis of the dissociated and denatured phycobiliprotein complexes grown under both these light conditions shows that the same bilin chromophore, phycocyanobilin, is exclusively used. Our findings show that red-shifted phycobilisomes are required for assisting efficient far-red light harvesting. Their discovery provides new insights into the molecular mechanisms of light harvesting under extreme conditions for photosynthesis, as well as the strategies involved in flexible chromatic acclimation to diverse light conditions.

Ultrastructural features of the early secretory pathway in Trichoderma reesei.

We have systematically analysed the ultrastructure of the early secretory pathway in the Trichoderma reesei hyphae in the wild-type QM6a, cellulase-overexpressing Rut-C30 strain and a Rut-C30 transformant BV47 overexpressing a recombinant BiP1-VenusYFP fusion protein with an endoplasmic reticulum (ER) retention signal. The hyphae were studied after 24 h of growth using transmission electron microscopy, confocal microscopy and quantitative stereological techniques. All three strains exhibited different spatial organisation of the ER at 24 h in both a cellulase-inducing medium and a minimal medium containing glycerol as a carbon source (non-cellulase-inducing medium). The wild-type displayed a number of ER subdomains including parallel tubular/cisternal ER, ER whorls, ER-isolation membrane complexes with abundant autophagy vacuoles and dense bodies. Rut-C30 and its transformant BV47 overexpressing the BiP1-VenusYFP fusion protein also contained parallel tubular/cisternal ER, but no ER whorls; also, there were very few autophagy vacuoles and an increasing amount of punctate bodies where particularly the recombinant BiP1-VenusYFP fusion protein was localised. The early presence of distinct strain-specific features such as the dominance of ER whorls in the wild type and tub/cis ER in Rut-C30 suggests that these are inherent traits and not solely a result of cellular response mechanisms by the high secreting mutant to protein overload.

Characterization of the interaction between heterodimeric αvß6 integrin and urokinase plasminogen activator receptor (uPAR) using functional proteomics.

Urokinase plasminogen activator receptor (uPAR) and the epithelial integrin αvß6 are thought to individually play critical roles in cancer metastasis. These observations have been highlighted by the recent discovery (by proteomics) of an interaction between these two molecules, which are also both implicated in the epithelial-mesenchymal transition (EMT) that facilitates escape of cells from tissue barriers and is a common signature of cancer metastases. In this study, orthogonal in cellulo and in vitro functional proteomic approaches were used to better characterize the uPAR·αvß6 interaction. Proximity ligation assays (PLA) confirmed the uPAR·αvß6 interaction on OVCA429 (ovarian cancer line) and four different colon cancer cell lines including positive controls in cells with de novo ß6 subunit expression. PLA studies were then validated using peptide arrays, which also identified potential physical sites of uPAR interaction with αvß6, as well as verifying interactions with other known uPAR ligands (e.g., uPA, vitronectin) and individual integrin subunits (i.e., αv, ß1, ß3, and ß6 alone). Our data suggest that interaction with uPAR requires expression of the complete αß heterodimer (e.g., αvß6), not individual subunits (i.e., αv, ß1, ß3, or ß6). Finally, using in silico structural analyses in concert with these functional proteomics studies, we propose and demonstrate that the most likely unique sites of interaction between αvß6 and uPAR are located in uPAR domains II and III.

Rapid conversion of Pseudomonas aeruginosa to a spherical cell morphotype facilitates tolerance to carbapenems and penicillins but increases susceptibility to antimicrobial peptides.

The Gram-negative human pathogen Pseudomonas aeruginosa tolerates high concentrations of ß-lactam antibiotics. Despite inhibiting the growth of the organism, these cell wall-targeting drugs exhibit remarkably little bactericidal activity. However, the mechanisms underlying ß-lactam tolerance are currently unclear. Here, we show that P. aeruginosa undergoes a rapid en masse transition from normal rod-shaped cells to viable cell wall-defective spherical cells when treated with ß-lactams from the widely used carbapenem and penicillin classes. When the antibiotic is removed, the entire population of spherical cells quickly converts back to the normal bacillary form. Our results demonstrate that these rapid population-wide cell morphotype transitions function as a strategy to survive antibiotic exposure. Taking advantage of these findings, we have developed a novel approach to efficiently kill P. aeruginosa by using carbapenem treatment to induce en masse transition to the spherical cell morphotype and then exploiting the relative fragility and sensitivity of these cells to killing by antimicrobial peptides (AMPs) that are relatively inactive against P. aeruginosa bacillary cells. This approach could broaden the repertoire of antimicrobial compounds used to treat P. aeruginosa and serve as a basis for developing new therapeutic agents to combat bacterial infections.

A cyanobacterium that contains chlorophyll f--a red-absorbing photopigment.

A Chl f-containing filamentous cyanobacterium was purified from stromatolites and named as Halomicronema hongdechloris gen., sp. nov. after its phylogenetic classification and the morphological characteristics. Hongdechloris contains four main carotenoids and two chlorophylls, a and f. The ratio of Chl f to Chl a is reversibly changed from 1:8 under red light to an undetectable level of Chl f under white-light culture conditions. Phycobiliproteins were induced under white light growth conditions. A fluorescence emission peak of 748 nm was identified as due to Chl f. The results suggest that Chl f is a red-light inducible chlorophyll.

A microscopic description and ultrastructural characterisation of Dientamoeba fragilis: an emerging cause of human enteric disease.

Dientamoeba fragilis is a pathogenic trichomonad found in the gastrointestinal tract of humans and is implicated as a cause of diarrhoea. Despite its discovery over a century ago, there has been no recent thorough description of this parasite by microscopy. Scanning electron microscopy, transmission electron microscopy, confocal and light microscopy were therefore used to characterise D. fragilis populations growing in xenic culture. Two different populations - smooth and ruffled cells - were identifiable by scanning electron microscopy. No flagella, pelta structures, undulating membrane or pseudocyst-like forms were present. The organelles in D. fragilis were analysed by transmission electron microscopy; like Trichomonas and Histomonas, D. fragilis contains hydrogenosomes that presumably represent the site of anaerobic respiration. The nuclear morphology of D. fragilis trophozoites grown in vitro and trophozoites from clinical isolates were also compared by confocal microscopy and light microscopy. The majority of cells grown in culture were mononucleate while most cells in permanent stained faecal smears were binucleate. The two nuclei of D. fragilis are morphologically indistinguishable and contain equivalent amounts of DNA as determined by DAPI staining. The approximate cell and nuclear volume of four isolates of D. fragilis were measured and shown to be comparable to other trichomonads. In addition, the discovery of a virus-like particle is reported, to our knowledge for the first time in D. fragilis. This study therefore provides extensive and novel details of the ultrastructure of a neglected protozoan parasite that is an emerging cause of human disease.

The fluorescent dyes TO-PRO-3 and TOTO-3 iodide allow detection of microbial cells in soil samples without interference from background fluorescence.

Visualization of microorganisms in soils and sediments using fluorescent dyes is a common method in microbial ecology studies, but is often hampered by strong nonspecific background fluorescence that can mask genuine cellular signals. The cyanine nucleic acid binding dyes TO-PRO-3 and TOTO-3 iodide enabled a clear detection of microbial cells in a mineral soil, while nonspecific background was greatly reduced compared with commonly used dyes. When used as counterstains for fluorescence in situ hybridization (FISH), both cyanine dyes allowed identification of microbial cells despite strong background from nonspecifically bound probes. TO-PRO-3 and TOTO-3 are easy to use and represent superior alternatives for detecting microorganisms in soil environments.

Substance P, tyrosine hydroxylase and serotonin terminals in the rat caudal nucleus ambiguus.

Substance P (SP), tyrosine hydroxylase (TH) and serotonin inputs onto laryngeal motoneurons (LMNs) are known to exist, but the distribution of their terminals in the caudal nucleus ambiguus (NA), remains unclear. Using immunofluorescence and confocal microscopy, we assessed simultaneously the distribution of SP, TH, serotonin and synaptophysin immunoreactive (ir) terminals in the caudal NA. SP, TH and serotonin-ir varicosities were considered to represent immunoreactive synapses if, using confocal microscopy, they were co-localized with the presynaptic protein, synaptophysin. Relative to the total number of synapses, we found only a modest number of SP, TH or serotonin-ir synaptic terminals in the caudal NA. The density of SP-ir synaptic terminals was higher than that of TH-ir and serotonin-ir synaptic terminals. Our results suggest that SP, TH, and serotonin-ir inputs may play only a modest role in regulating the activity of LMN. We conclude that SP, TH and serotonin are not always co-localized in terminals forming inputs with LMN and that they arise from separate subpopulations of neurons.

Ultrastructural localization of highly variable 185/333 immune response proteins in the coelomocytes of the sea urchin, Heliocidaris erythrogramma.

The 185/333 proteins of sea urchins represent a family of highly variable immune response molecules with unknown functions. In this study, we show that 185/333 proteins are expressed by three cell types: amoebocytes, colourless spherule cells and gut-associated amoebocytes. A sub-population of amoebocytes express 185/333 proteins on the membranes of vesicles emanating from the trans-Golgi and which later fuse with the plasma membranes of the cells. The previously uncharacterized gut-associated amoebocytes also show a high level of 185/333 protein expression on their internal vesicles and plasma membranes. Colourless spherule cells contain 185/333 proteins within large spherules (specialized intracellular vesicles). In the presence of bacteria and yeast, the ultrastucture of colourless spherule cells changes and 185/333 proteins disappear. In contrast, 185/333 proteins were not found in the phagosomes of coelomocytes. The 185/333-positive gut amoebocytes were often associated with anuclear bodies, which appeared to incorporate material of microbial origin that was surrounded by 185/333 proteins. The association between 185/333 proteins on gut amoebocytes and anuclear bodies suggests that these proteins may be involved in the phagocytosis of microbes in the gut epithelium.

Iron uptake and toxin synthesis in the bloom-forming Microcystis aeruginosa under iron limitation.

Toxin production during cyanobacterial blooms poses a significant public health threat in water bodies globally and requires the development of effective bloom management strategies. Previously, synthesis of the hepatotoxin microcystin has been proposed to be regulated by iron availability, but the contribution of the toxin to the adaptation of cyanobacteria to environmental stresses, such as changing light intensity and nutrient limitation, remains unclear. The aim of this study was to compare the iron stress response in toxic and non-toxic strains of Microcystis aeruginosa subjected to moderate and severe iron limitation. The transcription of a number of genes involved in iron uptake, oxidative stress response, toxin synthesis and transcriptional control of these processes was accessed by quantitative real-time PCR (qRT-PCR). The process of adaptation of M. aeruginosa to iron stress was found to be highly dynamic and strain-specific. Toxin production in PCC 7806 increased in an iron-dependent manner and appeared to be regulated by FurA. The inability to produce microcystin, either due to natural mutations in the mcy gene cluster or due to insertional inactivation of mcyH, affected the remodelling of the photosynthetic machinery in iron-stressed cells, the transport of Fe(II) and transcription of the Fur family of transcriptional regulators. The presence of the toxin appears to give an advantage to microcystin-producing cyanobacteria in the early stages of exposure to severe iron stress and may protect the cell from reactive oxygen species-induced damage.

Characterisation of an immunodominant, high molecular weight glycoprotein on the surface of infectious Neoparamoeba spp., causative agent of amoebic gill disease (AGD) in Atlantic salmon.

Amoebic gill disease can be experimentally induced by the exposure of salmonids to Neoparamoeba spp. freshly isolated from infected fish, while cultured amoebae are non-infective. Results from our previous work suggested that one key difference between infectious and non-infectious Neoparamoeba were the highly glycosylated molecules in the glycocalyx. To characterise these surface glycans or glycoproteins we used a monoclonal antibody (mAb 44C12) specific to a surface molecule unique to infective parasites. This mAb recognised a carbohydrate epitope on a high molecular weight antigen (HMWA) that make up 15-19% of the total protein in a soluble extract of infectious parasites. The HMWA consisted of at least four glycoprotein subunits of molecular weight (MW) greater than 150 kDa that form disulfide-linked complexes of MW greater than 600 kDa. Chemical deglycosylation yielded at least four protein bands of approximate MW 46, 34, 28 and 18 kDA. While a similar HMWA complex was present in non-infective parasites, the glycoprotein subunits were of lower MW and exhibited differences in glycosylation. The four glycoproteins subunits recognised by mAb 44C12 were resistant to degradation by PNGase F, PNGase A, O-glycosidase plus ß-1, 4-galactosidase, ß-N-acetylglucosaminidase and neuraminidase. The major monosaccharides in the HMWA from infectious parasites were rhamnose, fucose, galactose, and mannose while sialic acids were absent. The carbohydrate portion constituted more than 90% of the total weight of the HMWA from infectious Neoparamoeba spp. Preliminary results indicate that immunisation of salmon with HMWA does not lead to protection against challenge infection; rather it may even have an immunosuppressive effect.

Haemocyte morphology and function in the Akoya pearl oyster, Pinctada imbricata.

The morphology and cytochemistry of Pinctada imbricata haemocytes were studied in vitro. Three distinct blood cell types were identified; hyalinocytes, granulocytes, and serous cells. Haemocytes were classified based on the presence/absence of granules, and nucleus to cytoplasm ratio. Granulocytes were the most common cell type (62+/-2.81%), followed by hyalinocytes (36+/-2.35%), and serous cells (2+/-0.90%). Granulocytes, and hyalinocytes were found to be immunologically active, with the ability to phagocytose Congo red stained yeast. Of the cells involved in phagocytosis, granulocytes were the most active with 88.8+/-3.9% of these haemocytes engulfing yeast. Cytochemical stains (phenoloxidase, peroxidase, superoxide, melanin, neutral red) showed that enzymes associated with phagocytic activity were localised in granules within granulocytes. Based on their affinities for Giemsa/May-Grünwald stain, haemocytes were also defined as either acidic, basic or neutral. Hyalinocytes and serous cells were found to be eosinophilic, whilst granulocytes were either basophilic (large granulocytes), eosinophilic (small granulocytes) or a combination of the two (combination granulocytes). Light, differential interference contrast and epi-fluorescence microscopy identified three sub-populations of granulocytes based on size and granularity; small (4.00-5.00 microm in diameter, with small granules (0.05-0.5 microm in diameter), large (5.00-9.00 microm in diameter, with large granules (0.50-2.50 microm in diameter) and combination (5.00-9.00 microm in diameter, with both large and small granules). These observations demonstrate that P. imbricata have a variety of morphologically and functionally specialized haemocytes, many of which maybe associated with immunological functions.

Phagocytosis of the protozoan parasite, Marteilia sydneyi, by Sydney rock oyster (Saccostrea glomerata) hemocytes.

QX disease is a fatal disease in Sydney rock oysters caused by the protozoan parasite Marteilia sydneyi. The current study investigates the phagocytosis of M. sydneyi by Sydney rock oyster hemocytes. It also compares the in vitro phagocytic activities of hemocytes from oysters bred for QX disease resistance (QXR) with those of wild-type oysters. After ingestion of M. sydneyi, hemocyte granules fused with phagosome membranes and the pH of phagosomes decreased. Significantly (p=<0.05) more phagosomes in QXR hemocytes showed obvious changes in pH within 40 min of phagocytosis, when compared with wild-type hemocytes. Phenoloxidase deposition was also evident in phagosomes after in vitro phagocytosis. Most importantly, ingested and melanised M. sydneyi were detected in vivo among hemocytes from infected oysters. Overall, the data suggest that Sydney rock oyster hemocytes can recognise and phagocytose M. sydneyi, and that resistance against QX disease may be associated with enhanced phagolysosomal activity in QXR oysters.

Rapid purification method for the 26S proteasome from the filamentous fungus Trichoderma reesei.

We have developed a fast and simple two column chromatographic method for the purification of the 26S proteasome from the filamentous fungus Trichoderma reesei that simplifies the overall procedure and reduces the purification time from 5 to 2.5 days. The combination of only the anionic exchange POROS HQ column (Applied Biosystems) together with a size exclusion column has not been used previously for proteasome purification. The purified complex was analysed further by two-dimensional electrophoresis (2DE) and examined by transmission electron microscopy (TEM). A total of 102 spots separated by 2DE were identified by mass spectrometry using cross-species identification (CSI) or an in-house custom-made protein database derived from the T. reesei sequencing project. Fifty-one spots out of 102 represented unique proteins. Among them, 30 were from the 20S particle and eight were from the 19S particle. In addition, seven proteasome-interacting proteins as well as several non-proteasome related proteins were identified. Co-purification of the 19S regulatory particle was confirmed by TEM and Western blotting. The rapidity of the purification procedure and largely intact nature of the complex suggest that similar procedure may be applicable to the isolation and purification of the other protein complexes.

Sydney rock oyster (Saccostrea glomerata) hemocytes: morphology and function.

In this study, three major hemocyte types were identified in the Sydney rock oyster. They were characterized primarily by light and electron microscopy based on the presence or absence of granules and nucleus to cytoplasm ratios. Hemoblast-like cells were the smallest cell type 4.0+/-0.4microm and comprised 15+/-3% of the hemocyte population. They had large nuclei and scanty basic cytoplasm. This cell type also had some endoplasmic reticuli and mitochondria. The second major type were hyalinocytes. Hyalinocytes represented 46+/-6% of all hemocytes. They were large cells (7.1+/-1.0microm) that had low nucleus:cytoplasm ratios and agranular basic or acidic cytoplasm. Hyalinocytes had the ability to phagocytose yeast cells and formed the core of hemocyte aggregates associated with agglutination. Four discrete sub-populations of hyalinocytes were identified. The third major cell type were the granulocytes, comprising 38+/-1% of the hemocyte population. These cells were large (9.3+/-0.3microm) and were characterized by cytoplasm containing many acidic or basic granules. Granulocytes were more phagocytic than hyalinocytes and they formed the inner layer of hemocytes during the encapsulation of fungal hyphae. Five discrete sub-populations of granulocytes were identified based on the types of granules in their cytoplasm. Flow cytometry showed that the hemocytes of rock oysters could be divided into between two and four major cell types based on their light scattering properties. The most common of the cell types identified by flow cytometry corresponded to hyalinocytes and granulocytes. Cytochemical assays showed that most enzymes associated with immunological activity were localized in granulocytes. Their granules contained acid phosphatase, peroxidase, phenoloxidase, superoxide and melanin. Hyalinocytes were positive only for acid phosphatase. All of these observations suggest that Sydney rock oysters have a broad variety of functionally specialized hemocytes, many of which are involved in host defense.

Embryonic gonadal and sexual organ development in a small viviparous skink, Niveoscincus ocellatus.

The majority of research into the timing of gonad differentiation (and sex determination) in reptiles has focused on oviparous species. This is largely because: (1) most reptiles are oviparous; (2) it is easier to manipulate embryonic developmental conditions (e.g., temperature) of eggs than oviductal embryos and (3) modes of sex determination in oviparous taxa were thought to be more diverse since viviparity and environmental sex determination (ESD)/temperature-dependent sex determination (TSD) were considered incompatible. However, recent evidence suggests the two may well be compatible biological attributes, opening potential new lines of enquiry into the evolution and maintenance of sex determination. Unfortunately, the baseline information on embryonic development in viviparous species is lacking and information on gonad differentiation and sexual organ development is almost non-existent. Here we present an embryonic morphological development table (10 stages), the sequence of gonad differentiation and sexual organ development for the viviparous spotted snow skink (Niveoscincus ocellatus). Gonad differentiation in this species is similar to other reptilian species. Initially, the gonads are indifferent and both male and female accessory ducts are present. During stage 2, in the middle third of development, differentiation begins as the inner medulla regresses and the cortex thickens signaling ovary development, while the opposite occurs in testis formation. At this point, the Müllerian (female reproductive) duct regresses in males until it is lost (stage 6), while females retain both ducts until after birth. In the later stages of testis development, interstitial tissue forms in the medulla corresponding to maximum development of the hemipenes in males and the corresponding regression in the females.