Community analysis of biofilters using fluorescence in situ hybridization including a new probe for the Xanthomonas branch of the class Proteobacteria - PubMed (original) (raw)

Community analysis of biofilters using fluorescence in situ hybridization including a new probe for the Xanthomonas branch of the class Proteobacteria

U Friedrich et al. Appl Environ Microbiol. 1999 Aug.

Abstract

Domain-, class-, and subclass-specific rRNA-targeted probes were applied to investigate the microbial communities of three industrial and three laboratory-scale biofilters. The set of probes also included a new probe (named XAN818) specific for the Xanthomonas branch of the class Proteobacteria; this probe is described in this study. The members of the Xanthomonas branch do not hybridize with previously developed rRNA-targeted oligonucleotide probes for the alpha-, beta-, and gamma-Proteobacteria. Bacteria of the Xanthomonas branch accounted for up to 4.5% of total direct counts obtained with 4',6-diamidino-2-phenylindole. In biofilter samples, the relative abundance of these bacteria was similar to that of the gamma-Proteobacteria. Actinobacteria (gram-positive bacteria with a high G+C DNA content) and alpha-Proteobacteria were the most dominant groups. Detection rates obtained with probe EUB338 varied between about 40 and 70%. For samples with high contents of gram-positive bacteria, these percentages were substantially improved when the calculations were corrected for the reduced permeability of gram-positive bacteria when formaldehyde was used as a fixative. The set of applied bacterial class- and subclass-specific probes yielded, on average, 58.5% (+/- a standard deviation of 23.0%) of the corrected eubacterial detection rates, thus indicating the necessity of additional probes for studies of biofilter communities. The Xanthomonas-specific probe presented here may serve as an efficient tool for identifying potential phytopathogens. In situ hybridization proved to be a practical tool for microbiological studies of biofiltration systems.

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Figures

FIG. 1

FIG. 1

Quantification of binding of probe XAN818 relative to that of probe EUB338 measured as fluorescence intensity. Different stringencies were obtained by varying the concentration of formamide in the hybridization buffer. The target sequence of probe XAN818 and base changes in nontarget organisms are shown in Table 3. Symbols: ●, S. nitritireducens DSM 12575T; ◊, B. alba DSM 1416; □, A. acidophilum DSM 700T; ▴, G. oxydans subsp. oxydans DSM 3503T. Error bars show standard deviations.

FIG. 2

FIG. 2

Probe-specific counts, relative to direct enumeration with DAPI (percentages) (■). Percentages of EUB338 positive bacteria corrected for samples with high proportions of members of the Firmicutes (▨). Percentages of LGC obtained when PFA fixation was more efficient than ethanol fixation (□) (see Materials and Methods and Table 5). Percentages given are corrected for negative control NON338. Probe names are abbreviations of those shown in Table 2. Error bars indicate standard deviations for two to four replicates.

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