Specific Glucoside Transporters Influence Septal Structure and Function in the Filamentous, Heterocyst-Forming Cyanobacterium Anabaena sp Strain PCC 7120

T When deprived of combined nitrogen, some filamentous cyanobacteria contain two cell types: vegetative cells that fix CO2 through oxygenic photosynthesis and heterocysts that are specialized in N2 fixation. In the diazotrophic filament, the vegetative cells provide the heterocysts with reduced carbon (mainly in the form of sucrose) and heterocysts provide the vegetative cells with combined nitrogen. Septal junctions traverse peptidoglycan through structures known as nanopores and appear to mediate intercellular molecular transfer that can be traced with fluorescent markers, including the sucrose analog esculin (a coumarin glucoside) that is incorporated into the cells. Uptake of esculin by the model heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 was inhibited by the -glucosides sucrose and maltose. Analysis of Anabaena mutants identified components of three glucoside transporters that move esculin into the cells: GlsC (Alr4781) and GlsP (All0261) are an ATP-binding subunit and a permease subunit of two different ABC transporters, respectively, and HepP (All1711) is a major facilitator superfamily (MFS) protein that was shown previously to be involved in formation of the heterocyst envelope. Transfer of fluorescent markers (especially calcein) between vegetative cells of Anabaena was impaired by mutation of glucoside transporter genes. GlsP and HepP interact in bacterial two-hybrid assays with the septal junction-related protein SepJ, and GlsC was found to be necessary for the formation of a normal number of septal peptidoglycan nanopores and for normal subcellular localization of SepJ. Therefore, beyond their possible role in nutrient uptake in Anabaena, glucoside transporters influence the structure and function of septal junctions.Peer reviewe

Diabetic fetopathy associated with bilateral adrenal hyperplasia and ambiguous genitalia: a case report

<p>Abstract</p> <p>Introduction</p> <p>Many fetal malformations can occur because of maternal diabetes. However, ambiguous genital organs have never been reported as an associated finding in the literature. This is the first report of associated ambiguous genital organ and bilateral adrenal hyperplasia in a case of diabetic fetopathy.</p> <p>Case presentation</p> <p>A 19-year-old Thai primigravida with familial history of diabetes mellitus (DM) was diagnosed as having gestational DM type 2, based on 100 g oral glucose tolerance test, and was poorly controlled with insulin injections. Delayed targeted ultrasonography at 28 weeks gestation revealed multiple fetal anomalies. The woman underwent low transverse cesarean section at 30 weeks gestation due to preterm labor and transverse lie. The newborn with ambiguous genitalia was delivered but expired after birth. Autopsy findings revealed alobar holoprosencephaly, a prominent forehead, hypotelorism, an absent nose, absent bilateral ears, median cleft lip and palate, preaxial polydactyly of the right hand, accessory spleens, single umbilical artery, markedly enlarged adrenal glands and ambiguous external genitalia The subsequent fetal chromosomal study revealed 46,XX.</p> <p>Conclusion</p> <p>We describe a case of diabetic fetopathy with classic facial malformation and preaxial hallucal polydactyly which has been proposed as a marker of diabetic embryopathy. Bilateral adrenal hyperplasia with ambiguous genitalia, an uncommon associated anomaly, was also identified. It is controversial whether adrenal hyperplasia can be a novel feature of diabetic fetopathy or just a coincidental finding. Further observation and adequate investigation are needed in such cases.</p

Characterisation of the bacterial and fungal communities associated with different lesion sizes of Dark Spot Syndrome occurring in the Coral Stephanocoenia intersepta

The number and prevalence of coral diseases/syndromes are increasing worldwide. Dark Spot Syndrome (DSS) afflicts numerous coral species and is widespread throughout the Caribbean, yet there are no known causal agents. In this study we aimed to characterise the microbial communities (bacteria and fungi) associated with DSS lesions affecting the coral Stephanocoenia intersepta using nonculture molecular techniques. Bacterial diversity of healthy tissues (H), those in advance of the lesion interface (apparently healthy AH), and three sizes of disease lesions (small, medium, and large) varied significantly (ANOSIM R = 0.052 p,0.001), apart from the medium and large lesions, which were similar in their community profile. Four bacteria fitted into the pattern expected from potential pathogens; namely absent from H, increasing in abundance within AH, and dominant in the lesions themselves. These included ribotypes related to Corynebacterium (KC190237), Acinetobacter (KC190251), Parvularculaceae (KC19027), and Oscillatoria (KC190271). Furthermore, two Vibrio species, a genus including many proposed coral pathogens, dominated the disease lesion and were absent from H and AH tissues, making them candidates as potential pathogens for DSS. In contrast, other members of bacteria from the same genus, such as V. harveyii were present throughout all sample types, supporting previous studies where potential coral pathogens exist in healthy tissues. Fungal diversity varied significantly as well, however the main difference between diseased and healthy tissues was the dominance of one ribotype, closely related to the plant pathogen, Rhytisma acerinum, a known causal agent of tar spot on tree leaves. As the corals’ symbiotic algae have been shown to turn to a darker pigmented state in DSS (giving rise to the syndromes name), the two most likely pathogens are R. acerinum and the bacterium Oscillatoria, which has been identified as the causal agent of the colouration in Black Band Disease, another widespread coral disease

Metagenomic and Metatranscriptomic Analysis of Microbial Community Structure and Gene Expression of Activated Sludge

The present study applied both metagenomic and metatranscriptomic approaches to characterize microbial structure and gene expression of an activated sludge community from a municipal wastewater treatment plant in Hong Kong. DNA and cDNA were sequenced by Illumina Hi-seq2000 at a depth of 2.4 Gbp. Taxonomic analysis by MG-RAST showed bacteria were dominant in both DNA and cDNA datasets. The taxonomic profile obtained by BLAST against SILVA SSUref database and annotation by MEGAN showed that activated sludge was dominated by Proteobacteria, Actinobacteria, Bacteroidetes, Firmicutes and Verrucomicrobia phyla in both DNA and cDNA datasets. Global gene expression annotation based on KEGG metabolism pathway displayed slight disagreement between the DNA and cDNA datasets. Further gene expression annotation focusing on nitrogen removal revealed that denitrification-related genes sequences dominated in both DNA and cDNA datasets, while nitrifying genes were also expressed in relative high levels. Specially, ammonia monooxygenase and hydroxylamine oxidase demonstrated the high cDNA/DNA ratios in the present study, indicating strong nitrification activity. Enzyme subunits gene sequences annotation discovered that subunits of ammonia monooxygenase (amoA, amoB, amoC) and hydroxylamine oxygenase had higher expression levels compared with subunits of the other enzymes genes. Taxonomic profiles of selected enzymes (ammonia monooxygenase and hydroxylamine oxygenase) showed that ammonia-oxidizing bacteria present mainly belonged to Nitrosomonas and Nitrosospira species and no ammonia-oxidizing Archaea sequences were detected in both DNA and cDNA datasets

Short clones or long clones? A simulation study on the use of paired reads in metagenomics

<p>Abstract</p> <p>Background</p> <p>Metagenomics is the study of environmental samples using sequencing. Rapid advances in sequencing technology are fueling a vast increase in the number and scope of metagenomics projects. Most metagenome sequencing projects so far have been based on Sanger or Roche-454 sequencing, as only these technologies provide long enough reads, while Illumina sequencing has not been considered suitable for metagenomic studies due to a short read length of only 35 bp. However, now that reads of length 75 bp can be sequenced in pairs, Illumina sequencing has become a viable option for metagenome studies.</p> <p>Results</p> <p>This paper addresses the problem of taxonomical analysis of paired reads. We describe a new feature of our metagenome analysis software MEGAN that allows one to process sequencing reads in pairs and makes assignments of such reads based on the combined bit scores of their matches to reference sequences. Using this new software in a simulation study, we investigate the use of Illumina paired-sequencing in taxonomical analysis and compare the performance of single reads, short clones and long clones. In addition, we also compare against simulated Roche-454 sequencing runs.</p> <p>Conclusion</p> <p>This work shows that paired reads perform better than single reads, as expected, but also, perhaps slightly less obviously, that long clones allow more specific assignments than short ones. A new version of the program MEGAN that explicitly takes paired reads into account is available from our website.</p

Genetic characterization of Erve virus, a European Nairovirus distantly related to Crimean-Congo hemorrhagic fever virus

Erve virus (ERVEV) is a European Nairovirus that is suspected to cause severe headache (thunderclap headache) and intracerebral hemorrhage. The mode of transmission to humans (ticks or mosquitoes) is still unknown. Currently, no standardized testing method for ERVEV exists and only a small partial sequence of the polymerase gene is available. Here, we present the first complete genome sequence of ERVEV S, M, and L segments. Phylogenetic comparison of the amino acid sequence of the L-protein (RNA-dependent RNA polymerase) revealed only 48 % homology to available L-protein sequences of other Nairoviruses like Crimean-Congo hemorrhagic fever virus, Nairobi sheep disease virus, Hazara virus, Kupe virus, and Dugbe virus. Among themselves, these Nairoviruses show 62-89 % homology in the L-protein sequences. Therefore, ERVEV seems to be only distantly related to other Nairoviruses. The new sequence data can be used for the development of diagnostic methods and the identification of the natural vector

Bunyaviridae RNA Polymerases (L-Protein) Have an N-Terminal, Influenza-Like Endonuclease Domain, Essential for Viral Cap-Dependent Transcription

Bunyaviruses are a large family of segmented RNA viruses which, like influenza virus, use a cap-snatching mechanism for transcription whereby short capped primers derived by endonucleolytic cleavage of host mRNAs are used by the viral RNA-dependent RNA polymerase (L-protein) to transcribe viral mRNAs. It was recently shown that the cap-snatching endonuclease of influenza virus resides in a discrete N-terminal domain of the PA polymerase subunit. Here we structurally and functionally characterize a similar endonuclease in La Crosse orthobunyavirus (LACV) L-protein. We expressed N-terminal fragments of the LACV L-protein and found that residues 1-180 have metal binding and divalent cation dependent nuclease activity analogous to that of influenza virus endonuclease. The 2.2 Å resolution X-ray crystal structure of the domain confirms that LACV and influenza endonucleases have similar overall folds and identical two metal binding active sites. The in vitro activity of the LACV endonuclease could be abolished by point mutations in the active site or by binding 2,4-dioxo-4-phenylbutanoic acid (DPBA), a known influenza virus endonuclease inhibitor. A crystal structure with bound DPBA shows the inhibitor chelating two active site manganese ions. The essential role of this endonuclease in cap-dependent transcription was demonstrated by the loss of transcriptional activity in a RNP reconstitution system in cells upon making the same point mutations in the context of the full-length LACV L-protein. Using structure based sequence alignments we show that a similar endonuclease almost certainly exists at the N-terminus of L-proteins or PA polymerase subunits of essentially all known negative strand and cap-snatching segmented RNA viruses including arenaviruses (2 segments), bunyaviruses (3 segments), tenuiviruses (4–6 segments), and orthomyxoviruses (6–8 segments). This correspondence, together with the well-known mapping of the conserved polymerase motifs to the central regions of the L-protein and influenza PB1 subunit, suggests that L-proteins might be architecturally, and functionally equivalent to a concatemer of the three orthomyxovirus polymerase subunits in the order PA-PB1-PB2. Furthermore, our structure of a known influenza endonuclease inhibitor bound to LACV endonuclease suggests that compounds targeting a potentially broad spectrum of segmented RNA viruses, several of which are serious or emerging human, animal and plant pathogens, could be developed using structure-based optimisation

Inhibition of Interferon Induction and Action by the Nairovirus Nairobi Sheep Disease Virus/Ganjam Virus

The Nairoviruses are an important group of tick-borne viruses that includes pathogens of man (Crimean Congo hemorrhagic fever virus) and livestock animals (Dugbe virus, Nairobi sheep disease virus (NSDV)). NSDV is found in large parts of East Africa and the Indian subcontinent (where it is known as Ganjam virus). We have investigated the ability of NSDV to antagonise the induction and actions of interferon. Both pathogenic and apathogenic isolates could actively inhibit the induction of type 1 interferon, and also blocked the signalling pathways of both type 1 and type 2 interferons. Using transient expression of viral proteins or sections of viral proteins, these activities all mapped to the ovarian tumour-like protease domain (OTU) found in the viral RNA polymerase. Virus infection, or expression of this OTU domain in transfected cells, led to a great reduction in the incorporation of ubiquitin or ISG15 protein into host cell proteins. Point mutations in the OTU that inhibited the protease activity also prevented it from antagonising interferon induction and action. Interestingly, a mutation at a peripheral site, which had little apparent effect on the ability of the OTU to inhibit ubiquitination and ISG15ylation, removed the ability of the OTU to block the induction of type 1 and the action of type 2 interferons, but had a lesser effect on the ability to block type 1 interferon action, suggesting that targets other than ubiquitin and ISG15 may be involved in the actions of the viral OTU