Bacteroides species are Gram‐negative, obligate anaerobic bacteria. They are the predominant indigenous bacterial species in the human intestinal tract, where they play an important role in the normal physiology of the host, but they can also be significant opportunistic pathogens. The fact that these are obligate anaerobes is the prevailing feature that affects the methodology used for their cultivation. Several techniques are described for anaerobic culturing; the anaerobic glove box and anaerobic jars are the two techniques most adaptable to a range of research needs. Straightforward methods are presented for propagation on solid media and in broth cultures, and for the long‐term storage and maintenance of stock cultures. The Bacteroides species are saccharolytic, which is the second feature of their physiology that impacts cultivation methodology. Several flexible media formulations, including a defined minimal media, are provided that allow the researcher to choose the carbon source best suited for his or her work. Curr. Protoc. Microbiol. 9:13C.1.1‐13C.1.21. © 2008 by John Wiley & Sons, Inc.

The genetic structure and functional organization of a Bacteroides conjugative transposon (CTn), CTn341, were determined. CTn341 was originally isolated from a tetracycline-resistant clinical isolate of Bacteroides vulgatus. The element was 51,993 bp long, which included a 5-bp coupling sequence that linked the transposon ends in the circular form. There were 46 genes, and the corresponding gene products fell into three major functional groups: DNA metabolism, regulation and antibiotic resistance, and conjugation. The G؉C content and codon usage observed in the functional groups suggested that the groups belong to different genetic lineages, indicating that CTn341 is a composite, modular element. Mutational analysis of genes representing the different functional groups provided evidence for the gene assignments and showed that the basic conjugation and excision genes are conserved among Bacteroides spp. A group IIA1 intron, designated B.f.I1, was found to be inserted into the bmhA methylase gene. Reverse transcriptase PCR analysis of CTn341 RNA showed that B.fr.I1 was functional and was spliced out of the bmhA gene. Six related CTn-like elements were found in the genome sequences of Bacteroides fragilis NCTC9343 and Bacteroides thetaiotaomicron VPI5482. The putative elements were similar to CTn341 primarily in the tra and mob regions and in the exc gene, and several appeared to contain intron elements. Our data provide the first reported sequence for a complete Bacteroides CTn, and they should be of considerable benefit to further functional and genetic analyses of antibiotic resistance elements and genome evolution in Bacteroides.

The range of types of microbes with dimethylsulfoniopropionate (DMSP) lyase capability (enzymatic release of dimethylsulfide [DMS] from DMSP) has recently been expanded from bacteria and eukaryotic algae to include fungi (a species of the genus Fusarium[M. K. Bacic and D. C. Yoch, Appl. Environ. Microbiol. 64:106–111, 1998]). Fungi (especially ascomycetes) are the predominant decomposers of shoots of smooth cordgrass, the principal grass of Atlantic salt marshes of the United States. Since the high rates of release of DMS from smooth cordgrass marshes have a temporal peak that coincides with peak shoot death, we hypothesized that cordgrass fungi were involved in this DMS release. We tested seven species of the known smooth cordgrass ascomycetes and discovered that six of them exhibited DMSP lyase activity. We also tested two species of ascomycetes from other DMSP-containing plants, and both were DMSP lyase competent. For comparison, we tested 11 species of ascomycetes and mitosporic fungi from halophytes that do not contain DMSP; of these 11, only 3 were positive for DMSP lyase. A third group tested, marine oomycotes (four species of the genera Halophytophthora andPythium, mostly from mangroves), showed no DMSP lyase activity. Two of the strains of fungi found to be positive for DMSP lyase also exhibited uptake of DMS, an apparently rare combination of capabilities. In conclusion, a strong correlation exists between a fungal decomposer’s ability to catabolize DMSP via the DMSP lyase pathway and the host plant’s production of DMSP as a secondary product.

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A fungus, Fusarium lateritium, with dimethylsulfoniopropionate (DMSP) lyase activity was isolated from both seawater and a salt marsh due to its ability to grow on DMSP (with the evolution of dimethyl sulfide) as the sole source of carbon. This is the first reported case of DMSP lyase activity in a fungus. Several other common fungal genera tested did not have DMSP lyase activity. DMSP was taken up more rapidly by F. lateritium than it was utilized, leading to its intracellular accumulation. Inhibitor studies with nystatin and cyanide indicated that DMSP uptake was an energy-dependent process. The lyase was inducible by its substrate, DMSP (Km , 1.2 mM), and by the substrate analogs choline and glycine betaine. During induction, DMSP lyase activity increased with time and then dropped rapidly. This loss of activity could be prevented by spiking the culture with fresh DMSP or choline. The V max for DMSP lyase was 34.7 mU · mg of protein−1. The inhibitory effects of nystatin, andp-chloromercuriphenylsulfonate on DMSP lyase activity suggested that the enzyme is cytosolic. Because plants likeSpartina (a marsh grass) and marine algae contain high concentrations of DMSP, we speculate that DMSP-utilizing fungi may be involved in their decay.