WSRC-MS-2000-00537R1 1
Identification of Thiothrix unzii in Two Distinct Ecosystems
2 Robin L. Brigmon, * Michelle Furlong ** and William B. Whitman **
3 *
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Environmental Biotechnology Section, Savannah River Technology Center, Westinghouse Savannah River Company, Aiken, South Carolina, 29808, **
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Department of Microbiology, University of Georgia, Athens.
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LIST OF KEY WORDS
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Thiothrix spp., groundwater, phylogenetic analysis, biofilm.
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*
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Environmental Biotechnology Section
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Savannah River Technology Center
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Westinghouse Savannah River Company
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Bldg. 704-8T (TNX)
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Aiken, SC 29808
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Phone (803)-557-7719
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Fax (803)-557-7223
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EMAIL
[email protected]
Corresponding Author
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This document was prepared in conjunction with work accomplished under Contract No. DE-AC09-96SR18500 with the U.S. Department of Energy.
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[email protected]
WSRC-MS-2000-00537R1 1
Abstract
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Molecular procedures were used to identify Thiothrix spp. in biofilms from
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sulfide-rich waters in two distinct Florida ecosystems. These Thiothrix spp.-
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containing biofilms at these sites have been consistently observed for over 10
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years. Clonal libraries of biofilm 16S rDNA from each site contained rDNA
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sequences that were 99 to 99.5% similar to Thiothrix unzii.
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WSRC-MS-2000-00537R1 1
Summary
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The genus Thiothrix was created for ensheathed sulfur-oxidizing filamentous
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bacteria that deposit sulfur granules internally, attach to substrates, produce
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gliding gonidia, and form rosettes (Brigmon & DeRidder, 1998). Thiothrix spp.
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have been described as a component of biofilms on a variety of surfaces in
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sulfide-containing flowing water in natural systems (Brigmon et al., 1995) and
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wastewater treatment plants (Williams & Unz, 1989). The occurrence of
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Thiothrix as an ectosymbiont has been demonstrated for fresh water (Larkin et al.,
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1990) and salt-water (Polz et al., 1994) organisms. However, little is known about
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the molecular ecology of this genus. In flowing water with sulfide concentrations
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of at least 0.1 ppm, oxygen concentrations of less than 10% saturation and neutral
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pH, Thiothrix spp. attach to solid surfaces with holdfasts (Brigmon et al., 1995).
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In the springs and underwater limestone caves within the Floridan aquifer, visible
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white filamentous biofilms containing Thiothrix spp. have been observed as thin
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white mats or tufts with scattered distribution patterns on surfaces of rocks,
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sediments, and cave floors or filamentous masses in the water column (Brigmon
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et al., 1995).
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In this study Thiothrix species were identified at two surficial sites with
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molecular techniques. We previously observed these biofilms to have similar
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physical appearances, physiological characteristics and sulfurous water sources.
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While there has been some anthropogenic influence on these aquatic systems, if
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left without treatment (i.e. chlorination), the same biofilm formation occurs
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WSRC-MS-2000-00537R1 1
(Brigmon et al, 1997). This is the first reported identification of Thiothrix unzii in
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a natural aquatic system.
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Materials and Methods
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Sampling Sites.
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Biofilm samples were obtained from two groundwater-fed systems in
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central and northern Florida including a spring at Orange Springs, Florida and a
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municipal water tank at Palatka, Florida. At both of these sites, visible colonies
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of white filamentous biofilms containing Thiothrix spp. have been documented
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for over 10 years (Brigmon et al. 1997).
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Samples were collected from both sites on the same day with sterile
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forceps and immediately placed in sterile 50-ml centrifuge tubes. The Orange
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Springs samples were collected from the white filamentous biofilm covering the
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surface of rocks in the spring run at a depth of 0.5 m. The Palatka sample was
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collected from a biofilm-covered aerator pan. The water chemistry at both
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sampling sites was tested in the field for dissolved oxygen, pH, and hydrogen
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sulfide as previously described (Brigmon et al., 1997). Caution was used so that
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the tubes were opened under the water, biofilm mats immediately placed into the
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tubes, and capped before bringing to the surface. The tubes were stored in a cooler
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with ice and returned to the laboratory for processing.
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The Thiothrix spp. in the biofilms were tentatively identified in situ for sampling by their typical white, rough, filamentous appearance (Larkin et al.,
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WSRC-MS-2000-00537R1
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1990). Initial microscopic examination of Thiothrix spp. distinguishing
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morphological characteristics including rosettes, filaments, gonidia, and sulfur
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granules was used for further presumptive identification (Williams and Unz,
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1989). Confirmation of Thiothrix spp. in biofilm samples was made by
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monoclonal antibodies (MAb) specific for Thiothrix spp. with
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immunofluorescence as previously described (Brigmon et al., 1995).
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Sample Phylogenetic Analysis
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One milliliter of mat material was taken from each sample. This material
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was centrifuged at full speed in a microfuge for 5 min, washed with sterile
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distilled water and resuspended in 0.5 ml of sterile distilled water. The 16S
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ribosomal DNA in each sample was PCR-amplified using Ready-To-Go PCR
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Beads (Pharmacia Biotech, Piscataway, NJ). The bacterial primer 27f, 5’-AGA
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GTT TGA TCM TGG CTC AG-3’ (Lane, 1991) and the universal primer 1392r,
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5’-ACG GGC GGT GTG TRC-3’ (Lane, 1991) were used at a concentration of
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0.8 µM. For each sample, 2 µg of DNA, was added directly to a PCR reaction
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tube. The reaction mixtures were heated for 5 min at 94 °C, which lysed the cells.
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This lysate was used as a source of DNA without further purification. The
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denaturation, elongation, and annealing conditions were 1 min at 94 °C, 2 minutes
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at 72 °C and 1 min at 61 °C. The PCR products were purified by electrophoresis
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on a 0.8% agarose gel and eluted from the gel with a using Prep-A-Gene DNA
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Purification System (Biorad, Hercules). The amplified DNA was then ligated
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using T4 ligase into vector pCR 2.0 (Invitrogen, Carlsbad) using the vendor’s
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protocol and transformed into E. coli Top 10 cells (Ausuvel et al., 1999, Miller et
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WSRC-MS-2000-00537R1 1
al., 1988). After ligation, approximately, 100 colonies containing PCR-amplified
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bacterial 16S rDNA inserts from each sample were found. PCR inserts were
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sequenced at the Molecular Genetics Instrumentation Facility at the University of
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Georgia. Primer 27f was used to sequence 3 bacterial clones from each sample.
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Approximately 700 base pairs from each clone were sequenced.
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For FastA searches, approximately 500 nucleotides of the sequences were
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utilized to avoid ambiguous positions in some of the clones. Both GenBank
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and EMBL databases were searched. Sequence comparisons were performed
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with PHYLIP 3.5 (Felsenstein, 1993). Evolutionary distances were calculated
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using the Jukes-Cantor formula, and the Neighbor Joining algorithm was used
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to construct the phylogenetic trees. Bootstrap analysis was performed with
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100 replicates.
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Results and Discussion
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The clonal library from each sample site contained one clone that was
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99%-99.5% similar to Thiothrix unzii. These clones, TX1 and PL3, were 99.8%
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similar to each other, suggesting that they both represented the same or very
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similar species. These two clones branched from the clade of Thiothrix spp. and
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clustered most closely with Thiothrix unzii with bootstrap value of 100 (Figure 1
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& 2,data not shown) T. unzii has previously been found associated with
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wastewater systems (Howarth et al., 1999), and this is the first report of this taxon
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in a natural spring. This result suggests that this group may be more widely
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WSRC-MS-2000-00537R1 1
distributed than previously known. The four other clones sequenced from the
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libraries were not related to the Thiothrix group and were less than 85% similar to
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each other. Presumably, they represent heterotrophic bacteria associated with
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these biofilms. The Thiothrix species was abundant enough in both mats to be
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detected in the Tx and PL clone libraries after sequencing only 3 clones.
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Moreover, this result is consistent with the previous observation that Thiothrix
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spp. comprised 18% w/w of the biofilm in the Palatka municipal water storage
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tanks (Brigmon et al., 1997). Previous work at these two sites indicated
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morphological and immunological similarity of the biofilms at these two sites
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(Brigmon et al.,1995). The water chemistry of the groundwater analyzed in the
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field at sampling time were quite similar. For Orange Spring and Palatka the
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groundwater constituents were to determined to be respectively 1 and 1.5 ppm
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sulfide, 2 and 5 % dissolved oxygen, and 7.2 and 7.3 pH. These conditions are
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conducive to growth of Thiothrix spp.
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Knowledge of the microbial ecology in these aquifers and associated
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biofilms is limited. By increasing our understanding of the geomicrobial ecology
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of these environments, valuable information will be gained on our aquatic
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resources. This is important to document as anthropogenic pressure on these
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aquifers increases. Molecular procedures were applied here successfully to
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identify Thiothrix spp. in biofilms in two distinct ecosystems. These Thiothrix
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spp.-containing biofilms have been documented for over 10 years by microscopic,
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microbiological, and immunologic techniques. This confirmation by phylogenetic
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methods verifies the ecological distribution of related organisms in surficial
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WSRC-MS-2000-00537R1 1
systems supplied by geochemically similar groundwater (Brigmon et al., 1994,
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1997). This is the first report of T. unzii in a natural system.
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This paper was prepared in connection with work done under Contract No. DE-
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AC09-96SR18500 with the U. S. Department of Energy. We thank the Oak
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Ridge Institute for Science and Education (ORISE) for support of Ms. Furlong.
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WSRC-MS-2000-00537R1 1
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Felsenstein, J. (1993) PHYLIP: phylogeny inference package. University of
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Washington, Seattle.
21 22
Howarth, R., R. F. Unz, E. M. Seviour, R. J. Seviour, L. L. Blacjkall, R.W.
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Pickup, J.G. Jones, J. Yaguchi, and I. M. Head. (1999) Phylogenetic relationships
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WSRC-MS-2000-00537R1 1
between filamentous sulfur bacteria (Thiothrix spp. and Eikelboom type 021N
2
bacteria) isolated from wastewater-treatment plants and description of Thiothrix
3
eikelboomi sp. Nov., Thiothrix unzii sp. Nov., Thiothrix fructosivorans sp. Nov.
4
and Thiothrix defluvii sp. Nov. International Journal of Systematic Bacteriology
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Larkin, J.M., M.C. Henk, and S.D. Burton. (1990) Occurrence of Thiothrix sp.
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attached to Mayfly larva and presence of a parasitic bacteria in the Thiothrix sp.
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Applied Environmental Microbiology 56, 357-361.
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M. Goodfellow (ed.), Nucleic Acid Technology in Bacterial Systematics. John
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of bacteria: Genetic Transformation of Campylobacter jejuni with Plasmid DNA.
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Polz, M.F., D.L. Distel, B. Zarda, R. Amann, H. Felbeck, J.A. Ott, and C.M.
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Williams, T.M., and R.F. Unz. (1989) The nutrition of Thiothrix, type 021N,
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Figure 1. Phylogenetic tree generated by the neighbor-joining method from an
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alignment of 400 nucleotide positions showing the relationships between well PL
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and TX clones and described bacteria. PL and TX represent the samples from
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Palatka and Orange Springs, respectively. The scale bar indicates the Jukes
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Cantor distance. The number in parentheses indicates the accession number for
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the sequence. This tree was rooted using a Methanococcus maripaludis 16S
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rDNA sequence. Bootstrap values greater than 50 are reported adjacent to each
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node.
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Figure 2. Phylogenetic tree generated by the neighbor-joining method from an
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alignment of 659 nucleotide positions showing the relationships between the
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Thiothrix clones and described bacteria in the Thiothrix group. PL and TX
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represent the samples from Palatka and Orange Springs respectively. The scale
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bar indicates the Jukes Cantor distance. The number in parentheses indicates the
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accession number for the sequence. This tree was rooted using a Burkholderia
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caryophylli 16S rDNA sequence. Bootstrap values greater than 50 are reported
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adjacent to each node.
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100
Xanthomonas campestris (X95917) Pseudoxanthomonas broegbernensis (AJ012231) Rhodocyclus purpureus (M34132) Azoarcus sp. (AF011346)
54
61
79
Clone PL2 (AF298767) Variovorax sp. (AB003627)
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Alcaligenes faecalis (D88008) Clone PL3 (AF298768) Clone TX1 (AF298763) 100 51 Thiothrix unzii (L79961) Thiothrix nivea (L40993) Thiomicrospira thyasirae (AF016046) Cycloclasticus pugetii (L34955) 100
69
74
68
52
50
Enterobacter amnigenus (AB004749) Pseudomonas putida (Z76667) Acinetobacter sp. (Z93449) Agrobacterium tumefaciens (X67223) Methylobacterium sp. (D25306) Rhodospirillum rubrum (X87278) Holophaga foetida (X77215) 100 Geothrix fermentans (U41563) Chlorobium vibrioforme (Y10648)
100
Helicobacter pylori (U00679) Clone TX3 (AF298765) Flavobacterium xylanivorum (AF162266)
99 91
66
Clone TX2 (AF298764) Benzene mineralizing clone (AF029041) Clone PL1 (AF298766) Bacteroides acidofaciens (AB021165) Methanococcus maripaludis (AF005049) 0.16
Thiothrix nivea (L40993) 67 Clone TX1 (AF298763) 100 Clone PL3 (AF298768) 98
62
72
Thiothrix unzii (L79961) Thiothrix fructosivorans (L79963)
100 Thiothrix ramosa (U32940)
78 Thiothrix eikelboomii (AB042819) Thiothrix I ( AF127020)
100
Thiomicrospira crunogena (AF064545 ) Thiomicrospira thyasirae (AF016046)
61
Cycloclasticus oligotrophus (AF148215) 100 Cycloclasticus pugetii (U12624) 70 100
Methylophaga marina (X87338) Methylophaga sulfidovorans (X95461 ) Burkholderia caryophylli (AB021423)
0.06
