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      Comparing Bacterial Community Composition between Healthy and White Plague-Like Disease States in Orbicella annularis Using PhyloChip™ G3 Microarrays

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          Abstract

          Coral disease is a global problem. Diseases are typically named or described based on macroscopic changes, but broad signs of coral distress such as tissue loss or discoloration are unlikely to be specific to a particular pathogen. For example, there appear to be multiple diseases that manifest the rapid tissue loss that characterizes ‘white plague.’ PhyloChip™ G3 microarrays were used to compare the bacterial community composition of both healthy and white plague-like diseased corals. Samples of lobed star coral ( Orbicella annularis, formerly of the genus Montastraea [1]) were collected from two geographically distinct areas, Dry Tortugas National Park and Virgin Islands National Park, to determine if there were biogeographic differences between the diseases. In fact, all diseased samples clustered together, however there was no consistent link to Aurantimonas coralicida, which has been described as the causative agent of white plague type II. The microarrays revealed a large amount of bacterial heterogeneity within the healthy corals and less diversity in the diseased corals. Gram-positive bacterial groups (Actinobacteria, Firmicutes) comprised a greater proportion of the operational taxonomic units (OTUs) unique to healthy samples. Diseased samples were enriched in OTUs from the families Corynebacteriaceae, Lachnospiraceae, Rhodobacteraceae, and Streptococcaceae. Much previous coral disease work has used clone libraries, which seem to be methodologically biased toward recovery of Gram-negative bacterial sequences and may therefore have missed the importance of Gram-positive groups. The PhyloChip™data presented here provide a broader characterization of the bacterial community changes that occur within Orbicella annularis during the shift from a healthy to diseased state.

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          Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal RNA.

          Using a set of synthetic oligonucleotides homologous to broadly conserved sequences in-vitro amplification via the polymerase chain reaction followed by direct sequencing results in almost complete nucleotide determination of a gene coding for 16S ribosomal RNA. As a model system the nucleotide sequence of the 16S rRNA gene of M.kansasii was determined and found to be 98.7% homologous to that of M.bovis BCG. This is the first report on a contiguous sequence information of an entire amplified gene spanning 1.5 kb without any subcloning procedures.
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            Bacterial diversity and White Plague Disease-associated community changes in the Caribbean coral Montastraea faveolata.

            Increasing evidence confirms the crucial role bacteria and archaea play within the coral holobiont, that is, the coral host and its associated microbial community. The bacterial component constitutes a community of high diversity, which appears to change in structure in response to disease events. In this study, we highlight the limitation of 16S rRNA gene (16S rDNA) clone library sequencing as the sole method to comprehensively describe coral-associated communities. This limitation was addressed by combining a high-density 16S rRNA gene microarray with, clone library sequencing as a novel approach to study bacterial communities in healthy versus diseased corals. We determined an increase in diversity as well as a significant shift in community structure in Montastraea faveolata colonies displaying phenotypic signs of White Plague Disease type II (WPD-II). An accumulation of species that belong to families that include known coral pathogens (Alteromonadaceae, Vibrionaceae), bacteria previously isolated from diseased, stressed or injured marine invertebrates (for example, Rhodobacteraceae), and other species (for example, Campylobacteraceae) was observed. Some of these species were also present in healthy tissue samples, but the putative primary pathogen, Aurantimonas corallicida, was not detected in any sample by either method. Although an ecological succession of bacteria during disease progression after causation by a primary agent represents a possible explanation for our observations, we also discuss the possibility that a disease of yet to be determined etiology may have affected M. faveolata colonies and resulted in (or be a result of) an increase in opportunistic pathogens.
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              Interplay between NS3 protease and human La protein regulates translation-replication switch of Hepatitis C virus

              HCV NS3 protein plays a central role in viral polyprotein processing and RNA replication. We demonstrate that the NS3 protease (NS3pro) domain alone can specifically bind to HCV-IRES RNA, predominantly in the SLIV region. The cleavage activity of the NS3 protease domain is reduced upon HCV-RNA binding. More importantly, NS3pro binding to the SLIV hinders the interaction of La protein, a cellular IRES-trans acting factor required for HCV IRES-mediated translation, resulting in inhibition of HCV-IRES activity. Although overexpression of both NS3pro as well as the full length NS3 protein decreased the level of HCV IRES mediated translation, replication of HCV replicon RNA was enhanced significantly. These observations suggest that the NS3pro binding to HCV IRES reduces translation in favor of RNA replication. The competition between the host factor (La) and the viral protein (NS3) for binding to HCV IRES might regulate the molecular switch from translation to replication of HCV.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2013
                20 November 2013
                : 8
                : 11
                : e79801
                Affiliations
                [1 ]United States Geological Survey, St. Petersburg Coastal and Marine Science Center, St. Petersburg, Florida, United States of America
                [2 ]Lawrence Berkeley National Laboratory, Berkeley, California, United States of America
                [3 ]Second Genome, Inc., San Bruno, California, United States of America
                Pennsylvania State University, United States of America
                Author notes

                Competing Interests: Co-author Christina Kellogg is a PLOS ONE Editorial Board member, and this does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. The authors also agree to declare co-author Todd DeSantis’s affiliation with the company Second Genome, Inc. and confirm that this also does not alter their adherence to all the PLOS ONE policies on sharing data and materials.

                Conceived and designed the experiments: CAK. Performed the experiments: CAK YMP LMT. Analyzed the data: CAK YMP LMT TZD MAG GLA. Contributed reagents/materials/analysis tools: CAK DGZ GLA. Wrote the paper: CAK YMP LMT TZD MAG DGZ GLA.

                Article
                PONE-D-13-34643
                10.1371/journal.pone.0079801
                3835879
                24278181
                0053c8fd-0df9-4e38-8302-b80ecfdac729
                Copyright @ 2013

                This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

                History
                : 21 August 2013
                : 4 October 2013
                Page count
                Pages: 10
                Funding
                This project was supported by the Coral Reef Ecosystems Study (CREST) of the United States Geological Survey’s Coastal and Marine Geology Program. A portion of this work was performed under the auspices of the United States Department of Energy under contract DE-AC02-05CH11231 to Lawrence Berkeley National Laboratory. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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