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Reverse transcription of 16S rRNA to monitor ribosome-synthesizing bacterial populations in the environment.

ABSTRACT: Identification and quantification of phylogenetically defined bacterial populations in the environment are often performed using molecular tools targeting 16S rRNA. Fluorescence in situ hybridization has been used to monitor the expression and processing of rRNA by targeting the 3' tail of precursor 16S rRNA. To expand this approach, we employed reverse transcription of total RNA using primer S-D-Bact-0338-a-A-18. Length heterogeneity detected by slab gel analysis, denaturing high-performance liquid chromatography (DHPLC) was used to differentiate the 5' tail of the precursor from mature 16S rRNA, and the relative abundance of the precursor compared to the abundance of mature 16S rRNA was shown to be a sensitive indicator of the physiologic state of Acinetobacter calcoaceticus ATCC 23055(T). Our results demonstrate that this is a sensitive and reliable method with a detection limit of 10 ng of single-stranded DNA. The assay was also used to differentiate among precursor 16S rRNA levels with mixed pure cultures, as well as to examine the response of a mixed activated sludge culture exposed to fresh growth medium and the antibiotic chloramphenicol. The results of this study demonstrate that this assay is a novel reverse transcription assay that simultaneously measures the mature and precursor 16S rRNA pools for mixed bacterial populations in an engineered environment. Furthermore, collection of the reverse transcription products derived from activated sludge samples by the DHPLC approach enabled identification of the active bacterial genera. Comparison of 16S and precursor 16S rRNA clone library results indicated that the precursor 16S rRNA library is a more sensitive indicator for active bacteria in engineered environmental samples.

SUBMITTER: Lu T

PROVIDER: S-EPMC2704851 | biostudies-literature | 2009 Jul

REPOSITORIES: biostudies-literature

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Reverse transcription of 16S rRNA to monitor ribosome-synthesizing bacterial populations in the environment.

Lu Ting T Stroot Peter G PG Oerther Daniel B DB

Applied and environmental microbiology 20090424 13

Identification and quantification of phylogenetically defined bacterial populations in the environment are often performed using molecular tools targeting 16S rRNA. Fluorescence in situ hybridization has been used to monitor the expression and processing of rRNA by targeting the 3' tail of precursor 16S rRNA. To expand this approach, we employed reverse transcription of total RNA using primer S-D-Bact-0338-a-A-18. Length heterogeneity detected by slab gel analysis, denaturing high-performance li ...[more]

PMID: 19395563

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Project description:Microbiomes of full-scale seawater reverse osmosis membranes are complex and subject to variation within and between membrane units. The pre-existing bacterial communities of unused membranes before operation have been largely ignored in biofouling studies. This study is novel as unused membranes were used as a critical benchmark for comparison. Fouled seawater reverse osmosis membrane biofilm communities from an array of autopsied membrane samples, following a 7-year operational life-span in a full-scale desalination plant in Western Australia, were characterised by 16S rRNA gene metabarcoding using the bacterial primers 515F and 806R. Communities were then compared based on fouling severity and sampling location. Microbiomes of proteobacterial predominance were detected on control unused membranes. However, fouled membrane communities differed significantly from those on unused membranes, reflecting that operational conditions select specific bacteria on the membrane surface. On fouled membranes, Proteobacteria were also predominant but families differed from those on unused membranes, followed by Bacteriodetes and Firmicutes. Betaproteobacteria correlated with stable, mature and thick biofilms such as those in severely fouled membranes or samples from the feed end of the membrane unit, while Alpha and Gammaproteobacteria were predominantly found in biofilms on fouled but visually clean, and moderately fouled samples or those from reject ends of membrane units. Gammaproteobacteria predominated the thin, compact biofilms at the mid-feed end of membrane units. The study also supported the importance of Caulobacterales and glycosphingolipid-producing bacteria, namely Sphingomonadales, Rhizobiales and Sphingobacteriia, in primary attachment and biofilm recalcitrance. Nitrate-and-nitrite-reducing bacteria such as Rhizobiales, Burkholderiales and some Pseudomonadales were also prevalent across all fouled membranes and appeared to be critical for ecological balance and biofilm maturation.

Dataset Information

Reverse transcription of 16S rRNA to monitor ribosome-synthesizing bacterial populations in the environment.

Publications

Reverse transcription of 16S rRNA to monitor ribosome-synthesizing bacterial populations in the environment.

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