Molecular procedures were used to identify Thiothrix spp. in biofilms from sulfide-rich waters in two distinct Florida ecosystems. These Thiothrix spp.-containing biofilms at these sites have been consistently observed for over 10 years. Clonal libraries of biofilm 16S rDNA from each site contained rDNA sequences that were 99 to 99.5 percent similar to Thiothrix unzii

Brigmon, R.L.

UNT Digital Library

WSRC-MS-2000-00537R11Identification of Thiothrix unzii in Two Distinct Ecosystems12Robin L. Brigmon,* Michelle Furlong **  and William B. Whitman **3*Environmental Biotechnology Section, Savannah River Technology Center,4Westinghouse Savannah River Company, Aiken, South Carolina, 29808,5**Department of Microbiology, University of Georgia, Athens.67LIST OF KEY WORDS8Thiothrix spp., groundwater, phylogenetic analysis, biofilm.91011*Corresponding Author12Environmental Biotechnology Section13Savannah River Technology Center14Westinghouse Savannah River Company15Bldg. 704-8T (TNX)16Aiken, SC 2980817Phone (803)-557-771918Fax (803)-557-722319EMAIL r03.brigmon@srs.gov20This document was prepared in conjunction with work accomplished under Contract No.DE-AC09-96SR18500 with the U.S. Department of Energy.DISCLAIMERThis report was prepared as an account of work sponsored by an agency of the United States Government.Neither the United States Government nor any agency thereof, nor any of their employees, makes anywarranty, express or implied, or assumes any legal liability or responsibility for the accuracy,completeness, or usefulness of any information, apparatus, product or process disclosed, or represents thatits use would not infringe privately owned rights. Reference herein to any specific commercial product,process or service by trade name, trademark, manufacturer, or otherwise does not necessarily constitute orimply its endorsement, recommendation, or favoring by the United States Government or any agencythereof. The views and opinions of authors expressed herein do not necessarily state or reflect those of theUnited States Government or any agency thereof.This report has been reproduced directly from the best available copy.Available for sale to the public, in paper, from: U.S. Department of Commerce, National TechnicalInformation Service, 5285 Port Royal Road, Springfield, VA 22161, phone: (800)553-6847, fax: (703) 605-6900, email: orders@ntis.fedworld.gov online ordering:http://www.ntis.gov/ordering.htmAvailable electronically at  http://www.doe.gov/bridgeAvailable for a processing fee to U.S. Department of Energy and its contractors, in paper, from: U.S.Department of Energy, Office of Scientific and Technical Information, P.O. Box 62, Oak Ridge, TN37831-0062, phone: (865 ) 576-8401, fax: (865) 576-5728, email: reports@adonis.osti.govWSRC-MS-2000-00537R12Abstract12Molecular procedures were used to identify Thiothrix spp. in biofilms from3sulfide-rich waters in two distinct Florida ecosystems.  These Thiothrix spp.-4containing biofilms at these sites have been consistently observed for over 105years.  Clonal libraries of biofilm 16S rDNA from each site contained rDNA6sequences that were 99 to 99.5% similar to Thiothrix unzii.78910WSRC-MS-2000-00537R13Summary1The genus Thiothrix was created for ensheathed sulfur-oxidizing filamentous2bacteria that deposit sulfur granules internally, attach to substrates, produce3gliding gonidia, and form rosettes (Brigmon & DeRidder, 1998). Thiothrix spp.4have been described as a component of biofilms on a variety of surfaces in5sulfide-containing flowing water in natural systems (Brigmon et al., 1995) and6wastewater treatment plants (Williams & Unz, 1989).  The occurrence of7Thiothrix as an ectosymbiont has been demonstrated for fresh water (Larkin et al.,81990) and salt-water (Polz et al., 1994) organisms. However, little is known about9the molecular ecology of this genus.  In flowing water with sulfide concentrations10of at least 0.1 ppm, oxygen concentrations of less than 10% saturation and neutral11pH, Thiothrix spp. attach to solid surfaces with holdfasts (Brigmon et al., 1995).12In the springs and underwater limestone caves within the Floridan aquifer, visible13white filamentous biofilms containing Thiothrix spp. have been observed as thin14white mats or tufts with scattered distribution patterns on surfaces of rocks,15sediments, and cave floors or filamentous masses in the water column (Brigmon16et al., 1995).17In this study Thiothrix species were identified at two surficial sites with18molecular techniques.  We previously observed these biofilms to have similar19physical appearances, physiological characteristics and sulfurous water sources.20While there has been some anthropogenic influence on these aquatic systems, if21left without treatment (i.e. chlorination), the same biofilm formation occurs22WSRC-MS-2000-00537R14(Brigmon et al, 1997).  This is the first reported identification of Thiothrix unzii in1a natural aquatic system.23Materials and Methods45Sampling Sites.67Biofilm samples were obtained  from two groundwater-fed systems in8central and northern Florida including a spring at Orange Springs, Florida and a9municipal water tank at Palatka, Florida.  At both of these sites, visible colonies10of white filamentous biofilms containing Thiothrix spp. have been documented11for over 10 years (Brigmon et al. 1997).12Samples were collected from both sites on the same day with sterile13forceps and immediately placed in sterile 50-ml centrifuge tubes.  The Orange14Springs samples were collected from the white filamentous biofilm covering the15surface of rocks in the spring run at a depth of 0.5 m.   The Palatka sample was16collected from a biofilm-covered aerator pan.  The water chemistry at both17sampling sites was tested in the field for dissolved oxygen, pH, and hydrogen18sulfide as previously described (Brigmon et al., 1997).  Caution was used so that19the tubes were opened under the water, biofilm mats immediately placed into the20tubes, and capped before bringing to the surface. The tubes were stored in a cooler21with ice and returned to the laboratory for processing.22The Thiothrix spp. in the  biofilms were tentatively identified  in situ for23sampling by their typical white, rough, filamentous appearance (Larkin et al.,24WSRC-MS-2000-00537R151990).  Initial microscopic examination of Thiothrix spp. distinguishing1morphological characteristics including rosettes, filaments, gonidia, and sulfur2granules was used for further presumptive identification (Williams and Unz,31989).  Confirmation of Thiothrix spp. in biofilm samples was made by4monoclonal antibodies (MAb) specific for Thiothrix spp. with5immunofluorescence as previously described (Brigmon et al., 1995).67Sample Phylogenetic Analysis8One milliliter of mat material was taken from each sample.  This material9was centrifuged at full speed in a microfuge for 5 min, washed with sterile10distilled water and resuspended in 0.5 ml of sterile distilled water.  The 16S11ribosomal DNA in each sample was PCR-amplified using Ready-To-Go PCR12Beads (Pharmacia Biotech, Piscataway, NJ).  The bacterial primer 27f, 5’-AGA13GTT TGA TCM TGG CTC AG-3’ (Lane, 1991) and the universal primer 1392r,145’-ACG GGC GGT GTG TRC-3’ (Lane, 1991) were used at a concentration of150.8 µM.  For each sample, 2 µg of DNA, was added directly to a PCR reaction16tube.  The reaction mixtures were heated for 5 min at 94 °C, which lysed the cells.17This lysate was used as a source of DNA without further purification.  The18denaturation, elongation, and annealing conditions were 1 min at 94 °C, 2 minutes19at 72 °C and 1 min at 61 °C.  The PCR products were purified by electrophoresis20on a 0.8% agarose gel and eluted from the gel with a using Prep-A-Gene DNA21Purification System (Biorad, Hercules).  The amplified DNA was then ligated22using T4 ligase into vector pCR 2.0 (Invitrogen, Carlsbad) using the vendor’s23protocol and transformed into E. coli Top 10 cells (Ausuvel et al., 1999, Miller et24WSRC-MS-2000-00537R16al., 1988).  After ligation, approximately, 100 colonies containing PCR-amplified1bacterial 16S rDNA inserts from each sample were found.  PCR inserts were2sequenced at the Molecular Genetics Instrumentation Facility at the University of3Georgia.  Primer 27f was used to sequence 3 bacterial clones from each sample.4Approximately 700 base pairs from each clone were sequenced.5For FastA searches, approximately 500 nucleotides of the sequences were6utilized to avoid ambiguous positions in some of the clones.  Both GenBank7and EMBL databases were searched.  Sequence comparisons were performed8with PHYLIP 3.5 (Felsenstein, 1993).  Evolutionary distances were calculated9using the Jukes-Cantor formula, and the Neighbor Joining algorithm was used10to construct the phylogenetic trees.  Bootstrap analysis was performed with11100 replicates.1213Results and Discussion1415The clonal library from each sample site contained one clone that was1699%-99.5% similar to Thiothrix unzii.  These clones, TX1 and PL3, were 99.8%17similar to each other, suggesting that they both represented the same or very18similar species.  These two clones branched from the clade of Thiothrix spp. and19clustered most closely with Thiothrix unzii with bootstrap value of 100  (Figure 120& 2,data not shown)  T. unzii has previously been found associated with21wastewater systems (Howarth et al., 1999), and this is the first report of this taxon22in a natural spring. This result suggests that this group may be more widely23WSRC-MS-2000-00537R17distributed than previously known.  The four other clones sequenced from the1libraries were not related to the Thiothrix group and were less than 85% similar to2each other.  Presumably, they represent heterotrophic bacteria associated with3these biofilms.  The Thiothrix species was abundant enough in both mats to be4detected in the Tx and PL clone libraries after sequencing only 3 clones.5Moreover, this result is consistent with the previous observation that  Thiothrix6spp. comprised 18% w/w of the biofilm in the Palatka municipal water storage7tanks (Brigmon et al., 1997).  Previous work at these two sites indicated8morphological and immunological similarity of the biofilms at these two sites9(Brigmon et al.,1995).  The water chemistry of the groundwater analyzed in the10field at sampling time were quite similar.  For Orange Spring and Palatka the11groundwater constituents were to determined to be respectively 1 and 1.5 ppm12sulfide,  2 and 5 % dissolved oxygen, and 7.2 and 7.3 pH.  These conditions are13conducive to growth of Thiothrix spp.14Knowledge of the microbial ecology in these aquifers and associated15biofilms is limited.  By increasing our understanding of the geomicrobial ecology16of these environments, valuable information will be gained on our aquatic17resources. This is important to document as anthropogenic pressure on these18aquifers increases. Molecular procedures were applied here successfully to19identify Thiothrix spp. in biofilms in two distinct ecosystems.  These Thiothrix20spp.-containing biofilms have been documented for over 10 years by microscopic,21microbiological, and immunologic techniques.  This confirmation by phylogenetic22methods verifies the ecological distribution of related organisms in surficial23WSRC-MS-2000-00537R18systems supplied by geochemically similar groundwater (Brigmon et al., 1994,11997).  This is the first report of T. unzii in a natural system.23This paper was prepared in connection with work done under Contract No. DE-4AC09-96SR18500 with the U. S. Department of Energy.  We thank the Oak5Ridge Institute for Science and Education (ORISE) for support of Ms. Furlong.6WSRC-MS-2000-00537R19REFERENCES1Ausuvel, F.M., R. Brent, R.E. Kingston, D.D. Moore, J. G. Seidman, J.A. Smith,2and K. Struhl (eds.) 1999. Short Protocols in Molecular Biology. 4th ed. John-3Wiley and Sons, New York.45Benson D.A., Boguski M.S., Lipman D.J., Ostell J., Ouellette B.F., Rapp B.A.,6Wheeler D.L.  (1999)  Genbank.  Nucleic Acids Research 27: 12-17.78Brigmon, R.L., G. Bitton, S.G. Zam, and B. O'Brien. (1995) Development and9application of a monoclonal antibody against Thiothrix spp. Applied10Environmental Microbiology. 61: 13-20.1112Brigmon R. L. & C. De Ridder. (1998) Symbiotic Relationship of Thiothrix spp.13with Echinoderms. Applied Environmental Microbiology 64:3491-3495.1415Brigmon, R.L., H.W. Martin, and H. Aldrich. 1997. Biofouling in aquatic systems16by Thiothrix spp. Current Microbiology 35:169-174.1718Felsenstein, J. (1993) PHYLIP: phylogeny inference package. University of19Washington, Seattle.2021Howarth, R., R. F. Unz, E. M. Seviour, R. J. Seviour, L. L. Blacjkall, R.W.22Pickup, J.G. Jones, J. Yaguchi, and I. M. Head. (1999) Phylogenetic relationships23WSRC-MS-2000-00537R110between filamentous sulfur bacteria (Thiothrix spp. and Eikelboom type 021N1bacteria) isolated from wastewater-treatment plants and description of Thiothrix2eikelboomi sp. Nov., Thiothrix unzii sp. Nov., Thiothrix fructosivorans sp. Nov.3and Thiothrix defluvii sp. Nov.  International Journal of Systematic Bacteriology449, 1817-1827.56Larkin, J.M., M.C. Henk, and S.D. Burton. (1990) Occurrence of Thiothrix sp.7attached to Mayfly larva and presence of a parasitic bacteria in the Thiothrix sp.8Applied Environmental Microbiology 56, 357-361.910Lane, D. J. (1991) 16S/23S rRNA sequencing, p. 115-175. In E. Stackebrandt and11M. Goodfellow (ed.), Nucleic Acid Technology in Bacterial Systematics.  John12Wiley and Sons, New York.1314Miller, J. F., W.J. Dower, and L.S. Tompkins. 1988. High-voltage electroporation15of bacteria: Genetic Transformation of Campylobacter jejuni with Plasmid DNA.16Proceedings of the National Academy of Sciences 85, 856-860.1718Polz, M.F., D.L. Distel, B. Zarda, R. Amann, H. Felbeck, J.A. Ott, and C.M.19Cavanaugh. (1994) Phylogenetic analysis of a highly specific association between20ectosymbiotic, sulfur-oxidizing bacteria and a marine nematode. Applied21Environmental Microbiology 60, 4461-4467.2223WSRC-MS-2000-00537R111Williams, T.M., and R.F. Unz. (1989) The nutrition of Thiothrix, type 021N,1Beggiatoa, and Leucothrix strains. Water Research 2,15-22.23Figure 1.  Phylogenetic tree generated by the neighbor-joining method from an4alignment of 400 nucleotide positions showing the relationships between well PL5and TX clones and described bacteria. PL and TX represent the samples from6Palatka and Orange Springs, respectively.  The scale bar indicates the Jukes7Cantor distance. The number in parentheses indicates the accession number for8the sequence. This tree was rooted using a Methanococcus maripaludis 16S9rDNA sequence.  Bootstrap values greater than 50 are reported adjacent to each10node.1112Figure 2.  Phylogenetic tree generated by the neighbor-joining method from an13alignment of 659 nucleotide positions showing the relationships between the14Thiothrix  clones and described bacteria in the Thiothrix group. PL and TX15represent the samples from Palatka and Orange Springs respectively.  The scale16bar indicates the Jukes Cantor distance.  The number in parentheses indicates the17accession number for the sequence.  This tree was rooted using a Burkholderia18caryophylli 16S rDNA sequence.  Bootstrap values greater than 50 are reported19adjacent to each node.20210.16Thiothrix unzii (L79961)Thiothrix nivea Enterobacter amnigenus (AB004749)(Z76667)Pseudomonas putida (Z93449)Acinetobacter sp.Cycloclasticus pugetii(AF016046)Thiomicrospira thyasirae(AF011346)Azoarcus sp.(D88008)Alcaligenes faecalis(AB003627)Variovorax sp. Rhodocyclus purpureus(AJ012231) Pseudoxanthomonas broegbernensis Xanthomonas campestrisMethanococcus maripaludis(D25306)Methylobacterium sp.Agrobacterium tumefaciensRhodospirillum rubrumHolophaga foetidaGeothrix fermentansChlorobium vibrioformeHelicobacter pylori(AF162266)Flavobacterium xylanivorumBenzene mineralizing clone (AF029041)Bacteroides acidofaciens (AB021165)5091100996610052 6810054616179746910010051Clone PL1 (AF298766)Clone TX2 (AF298764)Clone TX3 (AF298765)Clone TX1 (AF298763)Clone PL2 (AF298767)Clone PL3 (AF298768)(M34132)(X95917)(L40993)(L34955)(X67223)(X87278)(X77215)(U41563)(Y10648)(U00679)(AF005049)Thiothrix niveaThiothrix unzii0.06100100729867Clone TX1 (AF298763)Clone PL3 (AF298768)Thiothrix ramosa62781001001007061Thiothrix eikelboomiiThiothrix fructosivoransThiomicrospira thyasirae (AF016046)Thiomicrospira crunogena (AF064545 )Methylophaga marina (X87338)Methylophaga sulfidovorans (X95461 )Burkholderia caryophylli (AB021423)(L40993)(L79961)(L79963)(U32940)(AB042819)Thiothrix I ( AF127020)Cycloclasticus pugetii (U12624)Cycloclasticus oligotrophus (AF148215)

Identification of Thiothrix unzii in Two Distinct Ecosystems

Abstract

Similar works

Full text

Available Versions

UNT Digital Library