ARTICLE IN PRESS Systematic and Applied Microbiology 28 (2005) 242–264 www.elsevier.de/syapm A molecular systematic survey of cultured microbial associates of deep-water marine invertebrates Karen Sfanosa,1, Dedra Harmodya, Phat Dangb, Angela Ledgera, Shirley Pomponia, Peter McCarthya, Jose Lopeza,Ã aDivision of Biomedical Marine Research, Harbor Branch Oceanographic Institution (HBOI), 5600 US Hwy. 1 Fort Pierce, FL 34946, USA bUS Horticultural Research Laboratory, Agricultural Research Service, USDA, Fort Pierce, FL 34945, USA Received 30 August 2004 Abstract A taxonomic survey was conducted to determine the microbial diversity held within the Harbor Branch Oceanographic Marine Microbial Culture Collection (HBMMCC). The collection consists of approximately 17,000 microbial isolates, with 11,000 from a depth of greater than 150 ft seawater. A total of 2273 heterotrophic bacterial isolates were inventoried using the DNA fingerprinting technique amplified rDNA restriction analysis on approximately 750–800 base pairs (bp) encompassing hypervariable regions in the 50 portion of the small subunit (SSU) 16S rRNA gene. Restriction fragment length polymorphism patterns obtained from restriction digests with RsaI, HaeIII, and HhaI were used to infer taxonomic similarity. SSU 16S rDNA fragments were sequenced from a total of 356 isolates for more definitive taxonomic analysis. Sequence results show that this subset of the HBMMCC contains 224 different phylotypes from six major bacterial clades (Proteobacteria (Alpha, Beta, Gamma), Cytophaga, Flavobacteria, and Bacteroides (CFB), Gram+ high GC content, Gram+ low GC content). The 2273 microorganisms surveyed encompass 834 a-Proteobacteria (representing 60 different phylotypes), 25 b-Proteobacteria (3 phylotypes), 767 g-Proteobacteria (77 phylotypes), 122 CFB (17 phylotypes), 327 Gram+ high GC content (43 phylotypes), and 198 Gram+ low GC content isolates (24 phylotypes). Notably, 11 phylotypes were p93% similar to the closest sequence match in the GenBank database even after sequencing a larger portion of the 16S rRNA gene (1400 bp), indicating the likely discovery of novel microbial taxa. Furthermore, previously reported ‘‘uncultured’’ microbes, such as sponge- specific isolates, are part of the HBMMCC. The results of this research will be available online as a searchable taxonomic database (www.hboi.edu/dbmr/dbmr_hbmmd.html). r 2004 Elsevier GmbH. All rights reserved. Keywords: Marine microorganisms; 16S rRNA; Culture collection; Sponge symbiosis Introduction ÃCorresponding author. Tel.: +1772 465 2400; fax: +1772 4612221. Marine invertebrate filter feeders can harbor a great E-mail address: [email protected] (J. Lopez). 1Current address: James Buchanan Brady Urological Institute, abundance of microbial diversity and biomass. For Johns Hopkins University School of Medicine, Baltimore, MD 21287, example, many marine sponges filter 420,000 l of water USA. per day and appear to host microbial communities with 0723-2020/$ - see front matter r 2004 Elsevier GmbH. All rights reserved. doi:10.1016/j.syapm.2004.12.002 ARTICLE IN PRESS K. Sfanos et al. / Systematic and Applied Microbiology 28 (2005) 242–264 243 a wide phylogenetic spectrum [29,30,36,65] that can comprise over 50% of the total sponge biomass [51,67]. Because marine invertebrates can accumulate micro- organisms, samples collected from invertebrates provide a more diverse array of microbes than samples recovered from the water column [30,33,64,67]. In recent years, the deep sea has also proven to be a source of a surprisingly diverse abundance of microorganisms, including cultur- able, newly described species of g-Proteobacteria [4], e-Proteobacteria [7], and actinomycetes [9]. Small subunit (SSU) rRNA has emerged as a reliable tool for phylogenetics because it is present in all living Fig. 1. Flow chart of experimental methods. organisms, functionally constant, and highly conserved [45,59,60]. It therefore serves as the ‘‘backbone’’ for the structuring of the second edition of Bergey’s Manual of Systematic Bacteriology [22,37]. Restriction fragment collected from Aruba, the Bahamas, Barbados, Bonaire, length polymorphism (RFLP) analysis of the 16S SSU Canary Islands, Cape Verde, Curacao, the Galapagos, rRNA gene (also termed amplified rDNA restriction the Gulf of Mexico, Honduras, Jamaica, Madeira, analysis (ARDRA)) has been used to rapidly distinguish Puerto Rico, Turks & Caicos, the US Virgin Islands, microbial species in a variety of applications such as and the USA using Harbor Branch Oceanographic clinical laboratories [14,61,63], industrial wastewater Institution’s underwater submersibles (Johnson-Sea- [8,23], coral diseases [11], agricultural soils [44], lake Link I and II). Bacterial isolation methods involved sediments [12], saline mud volcanoes [69], and microbial the sampling of invertebrate tissues using aseptic communities in the marine environment [1,13,46,58]. technique upon surfacing. Microbial isolates were The Harbor Branch Oceanographic Marine Microbial sampled from a total of 38 invertebrate hosts plus Culture Collection (HBMMCC) has been developed sediment samples (Table 1). The taxonomy of most over the last two decades as a resource for drug invertebrate hosts is resolved to the level of order or discovery [5,47] and is one of the largest collections of family, and ongoing taxonomic identifications will be marine-derived microorganisms. Prior to this survey, continually updated in the online HBMMCC database many of the isolates had not been characterized beyond (www.hboi.edu/dbmr/dbmr_hbmmd.html) [26]. The in- microscopic, morphological, and Gram-stain identifica- vertebrate tissue was ground in sterile seawater and the tions. The objectives of this study were to: (i) develop a subsequent supernatant was diluted in sterile seawater rapid method to taxonomically inventory deep-water before plating onto a series of media designed to recover invertebrate-derived marine microorganisms in the a diverse range of heterotrophic microbes. Media ranged HBMMCC, (ii) compare the relationships between the from extremely nutrient poor (60% seawater, 40% isolates described in this study to previously described deionized water, trace metals, phosphate, agar), to marine bacteria, and (iii) assess the distribution of nutrient rich (Difco Marine Agar 2216) and included a inventoried isolates across various host invertebrate wide variety of carbon sources (e.g. chitin, simple and species, depths, and geographic locales. The present complex sugars, and mucin). Certain isolation media study expands on previous work [42,49] by profiling also included host tissue and other supplements approximately one-fifth of the deep-water (4110 ft designed to increase total microbial recovery [43].In seawater) bacterial isolates in the HBMMCC. some cases, antibiotics were also employed for selective recovery of bacterial populations (e.g. nalidixic acid was used to reduce growth of Gram negative bacteria). The Materials and methods subset of the collection used in this survey was derived from 98 isolation media. The general scheme of the experimental design is depicted in Fig. 1. More detailed methodology is DNA extraction described below. Bacterial cells for DNA extraction were collected with Microbe isolation and selection a sterile 1 ml loop. The cells were added to 125 mlof Chelex-100 (Bio-Rad Inc.) made as a 5% solution in The isolates used in this study were deep-water sterile distilled water. Total genomic DNA was then (4110 ft seawater) invertebrate- or sediment-associated extracted using the standard protocol for Chelex-100 bacteria maintained in the HBMMCC. Samples were [15]. ARTICLE IN PRESS 244 K. Sfanos et al. / Systematic and Applied Microbiology 28 (2005) 242–264 Table 1. Marine invertebrate sources of isolates used in this study Phylum Class Order Family Identified isolates Porifera Demospongiae Astrophorida Ancorinidae (An) 107 Calthropellidae (Ca) 12 Geodiidae (Ge) 65 Pachastrellidae (Pa) 119 Dictyoceratida Irciniidae (Ir) 17 Thorectidae (Tr) 17 Hadromerida Placospongiidae (Pl) 41 Polymastiidae (Pm) 12 Suberitidae (Su) 14 Halichondrida Axinellidae (Ax) 220 Desmoxyidae (Dx) 35 Halichondriidae (Ha) 183 Haplosclerida Phloeodictyidae (Ph) 18 Petrosiidae (Pe) 24 Lithistida Azoricidae (Az) 21 Phymaraphinidae (Py) 8 Scleritodermidae (Sc) 124 Siphonidiidae (Si) 59 Theonellidae (Tn) 138 Vetulinidae (Vt) 9 Poecilolsclerida Acarnidae (Ac) 7 Desmacellidae (Dc) 38 Coelosphaeridae (Co) 244 Mycalidae (My) 8 Raspailiidae (Ra) 66 Verongida Pseudoceratinidae (Ps) 70 Unidentified demospongiae (UD) 317 Unidentified hexactinellida (UH) 65 Cnidaria Anthozoa Alcyonacea Nephtheidae (Ne) 1 Gorgonacea Plexauridae (Px) 88 Isididae (Is) 1 Actinaria (sea 6 anemone) (At) Ectoproctoa Gymnolaemata Ctenostomata Vesiculariidae (Vs) 2 (bryozoans) Mollusca Gastropoda Anaspidea (sea slug) Pleurobranchidae (Pb) 4 Gastropoda Archeogastropoda Pleurotomariidae (Pt) 18 (slit shell) Echinodermata Holothuroidea (sea cucumber) (Ho) 35 Echinoidea Echinothurioidea Echinothuridae (Ec) 16 (sea urchin) Annelida Polychaeta (polychaete worm) (Po) 6 Sediments (Se) 38 Polymerase chain reaction (PCR) [35] ‘‘27F’’ primer) and Loop27rc 50-GACTAC- CAGGGTATCTAATC-30 [36] amplified approximately Universal (consensus) 16S rRNA primers Ecoli9 750–800 base pairs (bp) of the bacterial 16S rRNA gene 50-GAGTTTGATCCTGGCTCAG-30 (equal to Lane (E. coli positions 9–804) as part of a rapid and cost- ARTICLE IN PRESS K. Sfanos et al. / Systematic and Applied Microbiology 28 (2005) 242–264 245 effective method