Cloning, sequencing, and mutation of a gene for azurin in Methylobacillus flagellatum KT

The gene cluster for methylamine utilization (mau genes) has been cloned from the obligate methylotrophic bacterium Methylobacillus flagellatum KT. Partial sequence data showed that the organization of these genes was similar to that found in Methylophilus methylotrophus W3A1-NS, including the lack of a gene for amicyanin, which had been thought to be the electron acceptor for methylamine dehydrogenase in M. flagellatum KT. However, a gene encoding azurin was discovered at the 3' end of the mau gene cluster, transcribed in the opposite orientation. A mutant with a defect in this gene showed impaired growth on methylamine, suggesting that azurin is involved in methylamine oxidation in M. flagellatum KT

Draft Genome Sequence of the Putative Marine Pathogen Thalassobius sp. I31.1

Thalassobius sp. I31.1 is a putative pathogen involved in epizootic shell disease in the American lobster (Homarus americanus). We report here the draft genome sequence for Thalassobius sp. I31.1 and provide insight into its metabolism and links to environmental pollutant degradation

The diversity of sulfide oxidation and sulfate reduction genes expressed by the bacterial communities of the Cariaco Basin, Venezuela

© The Author(s), 2016. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Open Microbiology Journal 10 (2016): 140-149, doi:10.2174/1874285801610010140.Qualitative expression of dissimilative sulfite reductase (dsrA), a key gene in sulfate reduction, and sulfide:quinone oxidoreductase (sqr), a key gene in sulfide oxidation was investigated. Neither of the two could be amplified from mRNA retrieved with Niskin bottles but were amplified from mRNA retrieved by the Deep SID. The sqr and sqr-like genes retrieved from the Cariaco Basin were related to the sqr genes from a Bradyrhizobium sp., Methylomicrobium alcaliphilum, Sulfurovum sp. NBC37-1, Sulfurimonas autotrophica, Thiorhodospira sibirica and Chlorobium tepidum. The dsrA gene sequences obtained from the redoxcline of the Cariaco Basin belonged to chemoorganotrophic and chemoautotrophic sulfate and sulfur reducers belonging to the class Deltaproteobacteria (phylum Proteobacteria) and the order Clostridiales (phylum Firmicutes).Support for this work came from NSF grant MCB03-47811 to AYC, MIS, and GTT and NSF grant OCE-1061774 to VPE and CT

Massively parallel tag sequencing reveals the complexity of anaerobic marine protistan communities

© 2009 The Authors. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in BMC Biology 7 (2009): 72, doi:10.1186/1741-7007-7-72.Recent advances in sequencing strategies make possible unprecedented depth and scale of sampling for molecular detection of microbial diversity. Two major paradigm-shifting discoveries include the detection of bacterial diversity that is one to two orders of magnitude greater than previous estimates, and the discovery of an exciting 'rare biosphere' of molecular signatures ('species') of poorly understood ecological significance. We applied a high-throughput parallel tag sequencing (454 sequencing) protocol adopted for eukaryotes to investigate protistan community complexity in two contrasting anoxic marine ecosystems (Framvaren Fjord, Norway; Cariaco deep-sea basin, Venezuela). Both sampling sites have previously been scrutinized for protistan diversity by traditional clone library construction and Sanger sequencing. By comparing these clone library data with 454 amplicon library data, we assess the efficiency of high-throughput tag sequencing strategies. We here present a novel, highly conservative bioinformatic analysis pipeline for the processing of large tag sequence data sets.The analyses of ca. 250,000 sequence reads revealed that the number of detected Operational Taxonomic Units (OTUs) far exceeded previous richness estimates from the same sites based on clone libraries and Sanger sequencing. More than 90% of this diversity was represented by OTUs with less than 10 sequence tags. We detected a substantial number of taxonomic groups like Apusozoa, Chrysomerophytes, Centroheliozoa, Eustigmatophytes, hyphochytriomycetes, Ichthyosporea, Oikomonads, Phaeothamniophytes, and rhodophytes which remained undetected by previous clone library-based diversity surveys of the sampling sites. The most important innovations in our newly developed bioinformatics pipeline employ (i) BLASTN with query parameters adjusted for highly variable domains and a complete database of public ribosomal RNA (rRNA) gene sequences for taxonomic assignments of tags; (ii) a clustering of tags at k differences (Levenshtein distance) with a newly developed algorithm enabling very fast OTU clustering for large tag sequence data sets; and (iii) a novel parsing procedure to combine the data from individual analyses. Our data highlight the magnitude of the under-sampled 'protistan gap' in the eukaryotic tree of life. This study illustrates that our current understanding of the ecological complexity of protist communities, and of the global species richness and genome diversity of protists, is severely limited. Even though 454 pyrosequencing is not a panacea, it allows for more comprehensive insights into the diversity of protistan communities, and combined with appropriate statistical tools, enables improved ecological interpretations of the data and projections of global diversity.The International Census of Marine Microbes and the W.M. Keck Foundation award to the Marine Biological Laboratory at Woods Hole (MA) supported the pyrosequencing part of this study. Further financial support came from a grant from the Deutsche Forschungsgemeinschaft to TS (STO414/3-1). Support for the unpublished work on Cariaco Basin protists came from NSF MCB-0348407 to VE (collaborative project with S Epstein at Northeastern University, Boston, MA, USA). Financial support to AC was provided by NSF MCB-0348045. Financial support to RC was provided by the ANR-Biodiversité project Aquaparadox

Culture-independent analysis of bacterial communities in hemolymph of American lobsters with epizootic shell disease

Epizootic shell disease (ESD) of the American lobster Homarus americanus H. Milne Edwards, 1837 is a disease of the carapace that presents grossly as large, melanized, irregularly shaped lesions, making the lobsters virtually unmarketable because of their grotesque appearance. We analyzed the bacterial communities present in the hemolymph of lobsters with and without ESD using nested-PCR of the 16S rRNA genes followed by denaturing gradient gel electrophoresis. All lobsters tested (n = 42) had bacterial communities in their hemolymph, and the community profiles were highly similar regardless of the sampling location or disease state. A number of bacteria were detected in a high proportion of samples and from numerous locations, including a Sediminibacterium sp. closely related to a symbiont of Tetraponera ants (38/42) and a Ralstonia sp. (27/42). Other bacteria commonly encountered included various Bacteroidetes, Pelomonas aquatica, and a Novosphingobium sp. One bacterium, a different Sediminibacterium sp., was detected in 20% of diseased animals (n = 29), but not in the lobsters without signs of ESD (n = 13). The bacteria in hemolymph were not the same as those known to be present in lesion communities except for the detection of a Thalassobius sp. in 1 individual. This work demonstrates that hemolymph bacteremia and the particular bacterial species present do not correlate with the incidence of ESD, providing further evidence that microbiologically, ESD is a strictly cuticular disease. Furthermore, the high incidence of the same species of bacteria in hemolymph of lobsters may indicate that they have a positive role in lobster fitness, rather than in disease, and further investigation of the role of bacteria in lobster hemolymph is required

Real-time PCR assay for Aquimarina macrocephali subsp. homaria and its distribution in shell disease lesions of Homarus americanus, Milne-Edwards, 1837, and environmental samples

Epizootic shell disease (ESD) is causing major losses to the lobster fishery in southern New England. Potential pathogens have been identified in lesion communities, but there are currently no efficient means of detecting and quantifying their presence. A qPCR assay was developed for a key potential pathogen, Aquimarina macrocephali subsp. homaria found to be ubiquitous in ESD lesions but not the unaffected integument. Application of the assay to various samples demonstrated that A. macrocephali subsp. homaria is ubiquitous and abundant in lobster lesions, commonly associated with healthy surfaces of crabs and is scarce in water and sediment samples from southern New England suggesting the affinity of this microorganism to the Arthropod integument. The qPCR assay developed here can be applied in future in vivo and in vitro studies to better understand the ecology and role of A. macrocephali subsp.homaria. in shell disease

Diversity of Diazotrophic Unicellular Cyanobacteria in the Tropical North Atlantic Ocean

We present data on the genetic diversity and phylogenetic affinities of N(2)-fixing unicellular cyanobacteria in the plankton of the tropical North Atlantic Ocean. Our dinitrogenase gene (nifH) sequences grouped together with a group of cyanobacteria from the subtropical North Pacific; another subtropical North Pacific group was only distantly related. Most of the 16S ribosomal DNA sequences from our tropical North Atlantic samples were closely allied with sequences from a symbiont of the diatom Climacodium frauenfeldianum. These findings suggest a complex pattern of evolutionary and ecological divergence among unicellular cyanobacteria within and between ocean basins

Bacterial Communities Associated with Lesions of Shell Disease in the American Lobster,Homarus americanusMilne-Edwards

Shell disease in Crustacea is a widely recognized syndrome having a polymicrobial etiology, and manifesting itself as lesions of the shell with a variable structure and shell location. We characterized major members of bacterial communities in epizootic shell disease lesions of the American lobster (Homarus americanus, Milne Edwards) and compared these communities with the ones found in study cases of impoundment and enzootic shell disease. Bacteria belonging to several Flavobacteriaceae genera (Aquimarina, Tenacibaculum, Polaribacter, Maribacter, Cellulophaga) within the phylum Bacteroidetes appear to have particular attraction to lobster lesions. The most prominent Bacteroidetes in lobster lesions were representatives of the genus Aquimarina sp., but only Aquimarina ‘homaria’ was detected in all analyzed lesions of epizootic, impoundment, and enzootic shell disease. It was found on 45% of surfaces unaffected by shell disease, but in smaller numbers compared with lesions. Alphaproteobacteria represent the most diverse class of proteobacteria found in both lesions and on unaffected surfaces. Three bacteria of this class appear to be ubiquitous in shell disease lesions, but only one specific alphaproteobacterium tentatively assigned to the genus Thalassobius (herein designated as ‘Thalassobius’ sp.) was present in all analyzed lesions of epizootic, impoundment, and enzootic shell disease. A ubiquitous gammaproteobacterium called ‘Candidatus Homarophilus dermatus’ was also prevalent in lesions, but just as commonly it was associated with surfaces unaffected by shell disease. The bacteria A. ‘homaria’ and ‘Thalassobius’ sp. are dominant and appear obligatory in lobster shell lesions, and are only occasionally detected on unaffected surfaces, which serve as intermediate reservoirs for the two potential pathogens. Therefore, these two bacteria stand out as potential shell-disease pathogens