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1 Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742, USA
2 Department of Microbiology and Cell Biology, University of La Laguna, 38206 La Laguna, Tenerife, Spain
3 Department of Marine Estuarine Environmental Sciences, College Park, MD 20742, USA
Correspondence
Ronald M. Weiner
rw19{at}umail.umd.edu
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Published online ahead of print on 4 March 2005 as DOI 10.1099/ijs.0.63627-0.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain 2-40T is AF055269.
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In 1997, a Gram-negative bacterium that degrades cellulose, xylan and chitin was isolated by Gonzalez et al. (1997)
from a salt marsh in Georgia, USA, and named Microbulbifer hydrolyticus IRE-31T. In 2002, Distel et al. (2002) reported taxonomic criteria for a shipworm symbiont that degrades cellulose and found them to be sufficiently different from the Microbulbifer taxon to be accorded a new genus, Teredinibacter. Within the past 2 years, sequences from six strains related to Microbulbifer have been deposited in the GenBank database.
In 1986, Andrykovitch & Marx (1988) isolated bacteria involved in the degradation of a salt marsh grass, Spartina alterniflora, found in the lower Chesapeake Bay, USA. One of these was designated strain 2-40T. Based upon phenotypic characteristics, it was placed with the alteromonads, where it resides today in the order ‘Alteromonadales’ of the ‘Gammaproteobacteria’, in the family Alteromonadaceae. Later, based on sequence analysis of the 16S rRNA gene, its distinctive cellular morphology and its CP-degradative capabilities, 2-40T was considered to be closely related to the genus Microbulbifer. A more meaningful classification of strain 2-40T had been hampered by the limited availability of 16S rRNA gene sequences from closely related organisms. Now, on the basis of more than 20 available sequences, we propose a new genus, Saccharophagus, to accommodate the most versatile marine carbohydrate degrader yet identified.
Strain 2-40T can degrade at least 10 CPs: agar, alginate, chitin, cellulose, fucoidan, laminarin, pectin, pullulan, starch and xylan (Ensor et al., 1999; Howard et al., 2003). These CPs are derived from numerous sources including algae, land plants, crustaceans, bacteria and fungi. Because of its involvement in ocean and estuarine carbon cycles, the US Department of Energy has recently (February 2005) completed sequencing the genome of strain 2-40T (http://genome.jgi-psf.org/draft_microbes/micde/micde.home.html; named as ‘Microbulbifer degradans’). Genomic analysis has predicted that there are >130 open reading frames that encode enzymes involved in the depolymerization of CPs. Additionally, it is likely that at least another 100–200 genes are involved in signalling, regulation and further metabolism of CPs.
Strain 2-40T clusters with the marine CP-degrading genera. Its 16S rRNA gene sequence is most closely related to those of M. hydrolyticus (90·5 %) and Teredinibacter turnerae (91·5 %); it shares the ability to degrade CPs with both M. hydrolyticus and T. turnerae and it has similar cellular morphology to that of M. hydrolyticus, most notably the copious production of membrane blebs and vesicles when grown with a CP as the sole carbon source (Gonzalez & Weiner, 2000) (Fig. 1). It is proposed that these organisms form a cluster of Gram-negative marine gammaproteobacteria with the ability to degrade CPs. The question remained whether strain 2-40T is sufficiently different from members of these genera to warrant its placement in a new genus.
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), 18 additional 16S rRNA gene sequences of related organisms have become available in GenBank. These permitted clarification of the phylogenetic position of strain 2-40T when considered along with available phenotypic analyses. The 16S rRNA gene sequence of strain 2-40T was obtained from GenBank (accession no. AF055269). The 16S rRNA gene tree (Fig. 2) was generated using CLUSTAL W 1.82 (Chenna et al., 2003) for sequence alignment and the neighbour-joining program in the PHYLIP package (Felsenstein, 1988). Before analysis, a filter was applied to exclude positions with less than 50 % conservation within the sequences being aligned. Only positions 110–1265 (Escherichia coli numbering) were considered. All additional sequences were obtained from the GenBank database. It should also be noted that a metagenomic study of Sargasso Sea prokaryotes revealed putative proteins attributed to isolate Microbulbifer SAR-1 (http://www.ncbi.nlm.nih.gov/Web/Newsltr/Spring04/sargasso.html). However, 16S rRNA gene sequences from the Sargasso Sea shared <90 % similarity with any isolate included in Fig. 2 and so are not included in the proposed Microbulbifer/Teredinibacter/Saccharophagus group.
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). The first is a Microbulbifer cluster that contains seven members, including ‘Pelagiobacter variabilis’ (Imamura et al., 1997). This cluster is supported by a high bootstrap score. Six members were isolated from aquatic marine habitats, while ‘Microbulbifer arenaceous’ was isolated within coastal sandstone (Tanaka et al., 2003). The type species of the genus Microbulbifer is M. hydrolyticus (type strain IRE-31T).
The second branch is more diffuse and contains organisms from diverse marine niches, predominantly endosymbionts. Only one of these, T. turnerae, has been given a validly published name (Distel et al., 2002). T. turnerae, LP1, LP2, LP3 and AF102866 are endosymbionts of shipworms from the bivalve family Teredinidae, while ‘Candidatus Endobugula glebosa’ and ‘Candidatus Endobugula sertula’ were isolated from the pallial sinuses of the bivalve Bugula neritina (Haygood & Davidson, 1997; Lim & Haygood, 2004). Strain NKB4 was isolated from deep-sea sediments (Li et al., 1999b). The 16S rRNA gene sequences of strains SCB11 and BD2-13 were derived gene clones from marine environmental samples (Li et al., 1999a). Strain characteristics for these organisms have not been reported.
Strain 2-40T shares a maximum of 91 % 16S rRNA gene sequence similarity with members of the genus Microbulbifer and 93 % similarity with the endosymbiont cluster. It shares 91·9 and 91·3 % similarity with BD2-13 and SCB11, respectively. These data suggest that strain 2-40T is related to the other members included in the 16S rRNA gene tree, but below the species level. This is in agreement with previously published data (Distel et al., 2002; Gonzalez & Weiner, 2000; Tanaka et al., 2003; Yoon et al., 2003a).
The suggestion that strain 2-40T is distinct from the other Microbulbifer/Teredinibacter isolates is also supported by comparison of phenotypic characteristics (Table 1). Most notably, the G+C content of strain 2-40T is significantly lower than that estimated for the other organisms, 46·7 mol% as reported by Gonzalez & Weiner (2000) and 45·8 mol% as determined from the draft genomic sequence, compared with 57·7 mol% for M. hydrolyticus and 49–51 mol% for several T. turnerae strains (Distel et al., 2002). Furthermore, the major fatty acid of strain 2-40T is iso-C16 : 0 (37 %) (Gonzalez & Weiner, 2000), while iso-C15 : 0 is the primary fatty acid of M. hydrolyticus (Gonzalez et al., 1997) and Microbulbifer salipaludis (Yoon et al., 2003a). Strain 2-40T reduces nitrate to nitrite and synthesizes eumelanin via tyrosinase activity (Kelly et al., 1990), both traits reported for only one other strain, ‘M. arenaceous’ (Tanaka et al., 2003), which does not degrade agar and shares only 90·6 % 16S rRNA gene sequence similarity with strain 2-40T.
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). However, because this required additional confirmation, the differences in CP-degradative capabilities within the Microbulbifer/Teredinibacter/Saccharophagus group of bacteria were investigated. M. hydrolyticus, Microbulbifer elongatus and M. salipaludis were tested for their abilities to degrade alginate, cellulose, pectin, pullulan and xylan compared with strain 2-40T. Assays for the depolymerization of cellulose, pullulan and xylan utilized azurine-cross-linked polysaccharides (Megazyme; http://www.megazyme.com/) incorporated into 1/3 strength marine agar (Difco 2216). Degradation of these insoluble substrates by endohydrolases produced soluble dye-labelled fragments that were easily observed as blue haloes around active colonies. M. hydrolyticus was used as a positive control for xylan and cellulose utilization. Gelatin utilization and nitrate reduction were determined by API 20NE test strips supplemented with sea salts medium (2·3 % sea salts, 0·05 % yeast extract).
To assay for growth on alginate or pectin, strains were grown in minimal sea salts liquid medium incorporating the polysaccharide of interest (0·2 % final concentration). The disappearance of the polysaccharides and a reduction of broth viscosities indicated the degradation of alginate and pectin. Negative controls included media without polysaccharide. Strain 2-40T was used as a positive control. The results are summarized in Table 1.
Strain 2-40T depolymerized all the tested substrates shown. Each of the other related strains was negative for at least one of the substrates, even considering the fact that not every strain was available. The versatility of strain 2-40T CP degradation is unique among the other strains of the Microbulbifer/Teredinibacter/Saccharophagus group and even among any other known bacteria. We believe that the phylogenetic and phenotypic analyses presented here, together with other published recommendations (Distel et al., 2002; Solano & Sanchez-Amat, 1999), support the classification of strain 2-40T as the type strain of Saccharophagus degradans gen. nov., sp. nov.
Description of Saccharophagus gen. nov.
Saccharophagus (Sac'cha.ro.pha'gus. Gr. n. saccharon sugar; N.L. masc. n. phagus from Gr. masc. n. phagos glutton; N.L. masc. n. Saccharophagus sugar-devourer).
Gram-negative, motile, heterotrophic, pleomorphic, rod-shaped, aerobic, catalase-positive and oxidase-positive. Numerous cell surface blebs and vesicles are produced. Degrades numerous complex polysaccharides. Requires sea salts for growth. The type species is Saccharophagus degradans.
Description of Saccharophagus degradans sp. nov.
Saccharophagus degradans (de.gra'dans L. part. adj. degradans bringing back into the former order, used to refer to the ability of the type strain to degrade several complex carbohydrates).
Cells are pleomorphic rods, averaging 1·5–3·0 µm long and 0·5 µm wide during the exponential phase of growth in glucose. In media containing complex polysaccharides as sole carbon sources, cells can be pleomorphic and produce surface protuberances and vesicles. Cells form coils and filaments when grown at high salinity. Colonies are cream in colour and then turn black upon eumelanin production. Colonies rapidly pit agar plates. Capable of utilizing the following complex carbohydrates as sole carbon sources: agar, alginate, chitin, cellulose, fucoidan, laminarin, pectin, pullulan, starch and xylan. Hydrolyses tyrosine. Temperature range for growth is 4–37 °C with an optimum of 30 °C. Optimum pH for growth is 7·5 with a range of 4·5–10. Requires sea salts for growth in the range 1–10 % with an optimum of 3·5 %. Secretes proteases. The G+C content of the type strain is 45·8 mol% as determined by genomic sequencing.
The type strain (and only strain to date) is 2-40T (=ATCC 43961T=DSM 17024T). It was isolated from the surface of degrading salt marsh cord grass, Spartina alterniflora, in the lower Chesapeake Bay, Mathews County, VA, USA.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Haygood, M. G. & Davidson, S. K. (1997). Small-subunit rRNA genes and in situ hybridization with oligonucleotides specific for the bacterial symbionts in the larvae of the bryozoan Bugula neritina and proposal of "Candidatus Endobugula sertula". Appl Environ Microbiol 63, 4612–4616.[Abstract]
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Solano, F. & Sanchez-Amat, A. (1999). Studies on the phylogenetic relationships of melanogenic marine bacteria: proposal of Marinomonas mediterranea sp. nov. Int J Syst Bacteriol 49, 1241–1246.
Tanaka, T., Yan, L. & Burgess, J. G. (2003). Microbulbifer arenaceous sp. nov., a new endolithic bacterium isolated from the inside of red sandstone. Curr Microbiol 47, 412–416.[Medline]
Yoon, J. H., Kim, I. G., Shin, D. Y., Kang, K. H. & Park, Y. H. (2003a). Microbulbifer salipaludis sp. nov., a moderate halophile isolated from a Korean salt marsh. Int J Syst Evol Microbiol 53, 53–57.
Yoon, J. H., Kim, H., Kang, K. H., Oh, T. K. & Park, Y. H. (2003b). Transfer of Pseudomonas elongata Humm 1946 to the genus Microbulbifer as Microbulbifer elongatus comb. nov. Int J Syst Evol Microbiol 53, 1357–1361.
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