Characterization of universal small-subunit rRNA hybridization probes for quantitative molecular microbial ecology studies

Based on comparative analysis of 16S rRNA sequences and the recently established phylogeny of the genus Desulfotomaculum, a set of phylogenetically nested hybridization probes was developed and characterized. A genus-specific probe targets all known Desulfotomaculum species (with the exception of Desulfotomaculum acetoxidans), and five specific probes target subclusters within the Desulfotomaculum genus. The dissociation temperature of each probe was determined experimentally. Probe specificities were verified through hybridizations with pure culture rRNA isolated from a wide variety of target and non-target organisms and through an evaluation of probe 'nesting' using samples obtained from four different environments. Fixation and hybridization conditions for fluorescence in situ hybridizations were also optimized. The probes were used in quantitative membrane hybridizations to determine the abundance of Desulfotomaculum species in thermophilic anaerobic digesters, in soil, in human faeces and in pig colon samples. Desulfotomaculum rRNA accounted for 0.3-2.1% of the total rRNA in the digesters, 2.6-6.6% in soil, 1.5-3.3% in human faeces and 2.5-6.2% in pig colon samples.

Section: Methodsmentioning

confidence: 99%

Section: Optimization Of Wash Temperaturesmentioning

confidence: 99%

See 1 more Smart Citation

Desulfotomaculum genus‐ and subgenus‐specific 16S rRNA hybridization probes for environmental studies

Hristova¹,

Mau²,

Zheng³

et al. 2000

“…Oligonucleotide probe sequences complementary to ampli-fied rRNA sequences were either obtained from the literature or were designed specifically for the new organisms identified in the nitrate-reducing culture. The universal probe (5¢-CTG CCC GCC ACA CAT GTT-3¢) was taken from Zheng et al (1996). The probe targeting Cart-N1 (5¢-GAC ATC GGC CGC TCC TAT AAC A-3¢) was based on a previously published probe TH15 (Hurek et al, 1993), with four nucleotides changed (in bold) to match our cloned sequence.…”

Section: Oligonucleotide Probe Sequencesmentioning

confidence: 99%

Physiological and molecular characterization of anaerobic benzene‐degrading mixed cultures

Ulrich

Edwards

2003

142

169

Nine distinct anaerobic benzene-degrading cultures were enriched from sediment samples from four different sites. These cultures used nitrate, sulphate or CO2 as electron acceptors. The shortest doubling times were observed in nitrate-reducing cultures, although cell yield was lowest in these cultures. The highest substrate concentration utilized and maximum absolute rates of benzene degraded (in micro M day-1) were observed in methanogenic cultures. The microbial compositions of a methanogenic and nitrate-reducing culture were determined from a clone library of 16S rRNA genes. Five Bacterial 16S rRNA sequences, one of which resembled a clone previously found in a sulphate-reducing, benzene-degrading culture and four Archaeal 16S rRNA sequences were identified in a methanogenic culture. Four Bacterial and no Archaeal 16S rRNA sequences were identified in a nitrate-reducing culture. The relative abundance of the four nitrate-reducing putative species was determined by slot blot hybridization. Two green sulphur bacteria together formed 52% of the clone library, but were found to be less than 4% of the culture by slot blot analysis. One of the cloned 16S rRNA gene sequences comprised 70% of the culture and was phylogenetically 93% similar to both Azoarcus and Dechloromonas species, which have been shown to degrade aromatic compounds, including benzene, under nitrate-reducing conditions.

“…b. All primers used were designed in the study, except Bact-008F (Hicks et al, 1992) and Univ1390R ( Zheng et al, 1996). Annealing temperature for all primer sets was 57°C, except for set 7 (53°C).…”

Section: The Extent and Diversity Of Wwe3 Representativesmentioning

confidence: 99%

Discovery and characterization of a new bacterial candidate division by an anaerobic sludge digester metagenomic approach

Guermazi

Daegelen²,

Dauga

et al. 2008

We have constructed a large fosmid library from a mesophilic anaerobic digester and explored its 16S rDNA diversity using a high-density filter DNA–DNA hybridization procedure. We identified a group of 16S rDNA sequences forming a new bacterial lineage named WWE3 (Waste Water of Evry 3). Only one sequence from the public databases shares a sequence identity above 80% with the WWE3 group which hence cannot be affiliated to any known or candidate prokaryotic division. Despite representing a non-negligible fraction (5% of the 16S rDNA sequences) of the bacterial population of this digester, the WWE3 bacteria could not have been retrieved using the conventional 16S rDNA amplification procedure due to their unusual 16S rDNA gene sequence. WWE3 bacteria were detected by polymerase chain reaction (PCR) in various environments (anaerobic digesters, swine lagoon slurries and freshwater biofilms) using newly designed specific PCR primer sets. Fluorescence in situ hybridization (FISH) analysis of sludge samples showed that WWE3 microorganisms are oval-shaped and located deep inside sludge flocs. Detailed phylogenetic analysis showed that WWE3 bacteria form a distinct monophyletic group deeply branching apart from all known bacterial divisions. A new bacterial candidate division status is proposed for this group.