Section 3 update: Microbial community analysis by PCR-single-strand conformation polymorphism (PCR-SSCP)

Dohrmann, Anja B.; Tebbe, Christoph C.

doi:10.1007/978-1-4020-2177-0_316

Cited by 26 publications

(42 citation statements)

References 48 publications

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“…Isopycnic centrifugation and fractionation of density gradients. 12 C-and 13 Clabeled RNA were separated by density gradient centrifugation using cesium trifluoroacetate (CsTFA) gradients (33). Density gradient centrifugation was performed in 4.9-ml OptiSeal polyallomer centrifuge tubes (Beckman Coulter, Krefeld, Germany) in a VTi 65.2 rotor (Beckman Coulter) in an ultracentrifuge (L8-70 M; Beckman Coulter).…”

Section: Methodsmentioning

confidence: 99%

“…In order to determine the source of the bacteria, the bacterial diversity associated with the food (tobacco leaves) and with M. sexta eggs deposited on the leaf surfaces were also determined. To allow the distinction between abundant and metabolically active bacteria, comparisons were made between genetic profiles with the single-strand conformation polymorphism (SSCP) technique (12,48) of the partial 16S rRNA genes PCR amplified from the total community DNA and 16S rRNA itself using reverse transcriptase PCR (RT-PCR). In addition, it was explored whether stable isotope probing (SIP) of 16S rRNA sequences (34) would also be capable of and possibly more sensitive in detecting metabolically active gut bacteria.…”

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confidence: 99%

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Origin and Diversity of Metabolically Active Gut Bacteria from Laboratory-Bred Larvae of Manduca sexta (Sphingidae, Lepidoptera, Insecta)

Brinkmann

Martens

Tebbe

2008

Appl Environ Microbiol

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Cultivation-independent analyses based on genetic profiling of partial bacterial 16S rRNA genes by PCRsingle-strand conformation polymorphism (PCR-SSCP), reverse transcriptase (RT)-PCR-SSCP of the 16S rRNA itself, and stable isotope probing (SIP), followed by RT-PCR-SSCP, were applied to characterize the diversity of metabolically active bacteria in the larval gut of Manduca sexta bred on tobacco leaves under greenhouse conditions. For SIP, hatching larvae were fed with leaves from tobacco plants grown in a 13 CO 2 -enriched atmosphere. Dominant SSCP bands were sequenced and phylogenetically analyzed. Only one major gut colonizer, an Enterococcus relative, was detected; it occurred in the heavy RNA fraction, demonstrating its metabolic activity, and it originated from eggs, where its metabolic activity was also indicated by rRNA-based SSCP profiles. In contrast, a Citrobacter sedlakii relative was detected on eggs by DNA-SSCP, but rRNA-SSCP and SIP-rRNA-SSCP were negative, suggesting that these bacterial cells were inactive. A Burkholderia relative was dominant and metabolically active on the tobacco leaves but inactive inside the gut, where it was also quantitatively reduced, as suggested by lower band intensities in the DNA-based SSCP profiles. SIP-RNA-SSCP detected another metabolically active gut bacterium (Enterobacter sp.) and more bacteria in the light RNA fraction, indicating low or no metabolic activity of the latter inside the gut. We conclude that the larval gut supported only a low diversity of metabolically active bacteria.Insects represent one of the largest reservoirs of bacterial diversity on Earth, and more than 250 million years of coevolution have resulted in manifold interactions between insects and bacteria, ranging from pathogenicity to highly sophisticated symbiotic relationships (13, 56). One of the most striking interactions is that bacteria have extended the nutritional range of insects by supplying nutrients as endosymbionts (14) and by accessing otherwise indigestible substrates, such as lingo-cellulose-derived organic matter from soils, with the help of gut bacteria (7).Manduca sexta (Sphingidae) represents a lepidopteran species adapted to extreme habitat conditions because their larvae can feed exclusively on the leaves of tobacco plants (43). Tobacco plants typically contain high amounts of alkaloids which are highly toxic to other insects and vertebrates but not to M. sexta (21,49,57). Once hatched from eggs deposited on tobacco leaves, the M. sexta larvae consume large amounts of such leaves and pass through five larval stages (instars) within 2 to 3 weeks, increasing their individual body weight from approximately 1 mg to 10 g (22, 43). While this simple feeding strategy is a large problem for tobacco growers (23), it is, under controlled conditions, an excellent system to generate larvae for biological research, which explains the broad use of M. sexta in laboratories worldwide (for examples, see references 26, 29, and 52).Despite their wide laboratory dissemination and their ...

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Origin and Diversity of Metabolically Active Gut Bacteria from Laboratory-Bred Larvae of Manduca sexta (Sphingidae, Lepidoptera, Insecta)

Brinkmann

Martens

Tebbe

2008

Appl Environ Microbiol

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“…Triplicate amplifications, each in a 50 ml volume, were performed for each sample and then pooled. After digestion of the phosphorylated DNA-strand with lambda exoncuclease (New England Biolabs, Frankfurt, Germany), DNA was analyzed by SSCP as described before (Dohrmann and Tebbe, 2004).…”

Section: Dna Analyses Of Cscl-gradient Fractionsmentioning

confidence: 99%

Importance of soil organic matter for the diversity of microorganisms involved in the degradation of organic pollutants

et al. 2014

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Many organic pollutants are readily degradable by microorganisms in soil, but the importance of soil organic matter for their transformation by specific microbial taxa is unknown. In this study, sorption and microbial degradation of phenol and 2,4-dichlorophenol (DCP) were characterized in three soil variants, generated by different long-term fertilization regimes. Compared with a non-fertilized control (NIL), a mineral-fertilized NPK variant showed 19% and a farmyard manure treated FYM variant 46% more soil organic carbon (SOC). Phenol sorption declined with overall increasing SOC because of altered affinities to the clay fraction (soil particles o2 mm in diameter). In contrast, DCP sorption correlated positively with particulate soil organic matter (present in the soil particle fractions of 63-2000 lm). Stable isotope probing identified Rhodococcus, Arthrobacter (both Actinobacteria) and Cryptococcus (Basidiomycota) as the main degraders of phenol. Rhodococcus and Cryptococcus were not affected by SOC, but the participation of Arthrobacter declined in NPK and even more in FYM. 14 C-DCP was hardly metabolized in the NIL variant, more efficiently in FYM and most in NPK. In NPK, Burkholderia was the main degrader and in FYM Variovorax. This study demonstrates a strong effect of SOC on the partitioning of organic pollutants to soil particle size fractions and indicates the profound consequences that this process could have for the diversity of bacteria involved in their degradation.

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“…Characterization of bacterial communities in fluid and filter samples was done by AMODIA Bioservice GmbH (Braunschweig, Germany), including filtration of 1 liter fluid on a 0.22 µm cellulose acetate filter (Sartorius, Goettingen, Germany), and using single strand conformation polymorphism (SSCP) fingerprinting of PCR-amplified 16S rRNA gene fragments according to Schwieger and Tebbe (1998) and Dohrmann and Tebbe (2004). Portions of the filter sample taken at the topside facility were processed in parallel with the fluid samples.…”

Section: Genetic Fingerprintingmentioning

confidence: 99%

Thermal effects on microbial composition and microbiologically induced corrosion and mineral precipitation affecting operation of a geothermal plant in a deep saline aquifer

et al. 2013

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The microbial diversity of a deep saline aquifer used for geothermal heat storage in the North German Basin was investigated. Genetic fingerprinting analyses revealed distinct microbial communities in fluids produced from the cold and warm side of the aquifer. Direct cell counting and quantification of 16S rRNA genes and dissimilatory sulfite reductase (dsrA) genes by real-time PCR proved different population sizes in fluids, showing higher abundance of bacteria and sulfate reducing bacteria (SRB) in cold fluids compared with warm fluids. The operation-dependent temperature increase at the warm well probably enhanced organic matter availability, favoring the growth of fermentative bacteria and SRB in the topside facility after the reduction of fluid temperature. In the cold well, SRB predominated and probably accounted for corrosion damage to the submersible well pump, and iron sulfide precipitates in the near wellbore area and topside facility filters. This corresponded to lower sulfate content in fluids produced from the cold well as well as higher content of hydrogen gas that was probably released from corrosion, and maybe favored growth of hydrogenotrophic SRB. This study reflects the high influence of microbial populations for geothermal plant operation, because microbiologically induced precipitative and corrosive processes adversely affect plant reliability.2

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Section 3 update: Microbial community analysis by PCR-single-strand conformation polymorphism (PCR-SSCP)

Cited by 26 publications

References 48 publications

Origin and Diversity of Metabolically Active Gut Bacteria from Laboratory-Bred Larvae of Manduca sexta (Sphingidae, Lepidoptera, Insecta)

Origin and Diversity of Metabolically Active Gut Bacteria from Laboratory-Bred Larvae of Manduca sexta (Sphingidae, Lepidoptera, Insecta)

Importance of soil organic matter for the diversity of microorganisms involved in the degradation of organic pollutants

Thermal effects on microbial composition and microbiologically induced corrosion and mineral precipitation affecting operation of a geothermal plant in a deep saline aquifer

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