Development and application of 16S rRNA-targeted probes for detection of iron- and manganese-oxidizing sheathed bacteria in environmental samples

• Published in: Applied and Environmental Microbiology Vol. 63; no. 2; pp. 644 - 651
• Main Authors: Siering, P.L. (Cornell University, Ithaca, NY.), Ghiorse, W.C
• Format: Journal Article
• Language: English
• Published: Washington, DC American Society for Microbiology 01-02-1997
• Subjects: BACTERIA; Bacteria - cytology; Bacteria - genetics; Bacteria - isolation & purification; Bacteriology; Biological and medical sciences; Biology of microorganisms of confirmed or potential industrial interest; Biotechnology; DNA, Ribosomal - genetics; Environmental Microbiology; Freshwater; Fundamental and applied biological sciences. Psychology; Genes; Genetics; In Situ Hybridization, Fluorescence - methods; Iron - metabolism; Leptothrix; MANGANESE; Manganese - metabolism; MANGANESO; Marine; Metals, Heavy - metabolism; Microbiological Techniques; Microbiology; Mission oriented research; Oligonucleotide Probes; Oxidation-Reduction; Ribonucleic acid; RNA; RNA, Ribosomal, 16S - genetics; Sensitivity and Specificity; Sequence Analysis, DNA; Soil Microbiology; SOL DE TERRES HUMIDES; Sphaerotilus natans; SUELO DE TIERRAS HUMEDAS; Water Microbiology; Molecular hybridization; In situ; Sequence alignment; Ribosomal DNA; Homology; Method; Specificity; Fluorescent labelling; 16S-RNA; Bacteria; Leptothrix; Molecular probe; Detection; Comparative study; Fluorescence microscopy
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/aem.63.2.644-651.1997

Comparative sequence analysis of the 16S rRNA genes from several Leptothrix and Sphaerotilus strains led to the design of an oligonucleotide probe (PS-1) based on a sequence within the hypervariable region 1 specific for four Leptothrix strains and for one of the four Sphaerotilus natans strains examined. Another probe (PSP-6) was based on a sequence within the hypervariable region 2. PSP-6 was specific for one of the two evolutionary lineages previously described for Leptothrix spp. (P. L. Siering and W. C. Ghiorse, Int. J. Syst. Bacteriol. 46:173-182, 1996). Fluorescein-labeled oligonucleotide probes were synthesized, and their specificity for fluorescence in situ hybridization identification was confirmed by a laser scanning microscopy technique (W. C. Ghiorse, D. N. Miller, R. L. Sandoli, and P. L. Siering, Microsc. Res. Tech. 33:73-86, 1996) to compare whole-cell hybridizations of closely related bacteria. Probe specificity was also tested in dot blots against total RNA isolated from four Leptothrix strains, four Sphaerotilus strains, and 15 other members of the class Proteobacteria. When the probes were tested on samples from the Sapsucker Woods wetland habitat where Leptothrix spp. are thought to play a role in manganese and iron oxidation, positive signals were obtained from several sheathed filamentous bacteria including some that were morphologically similar to previously isolated strains of "Leptothrix discophora." Other unknown filamentous sheathed bacteria also gave strong positive signals. This work provides a foundation for future studies correlating the presence of members of the Leptothrix-Sphaerotilus group of sheathed bacteria with manganese and iron oxidation activity in habitats where biological iron and manganese oxidation are important environmental processes