Strukturelle und funktionelle Analysen von Bakterienpopulationen mit Hilfe der PCR-SSCP in sechs unterschiedlichen Modellböden

The bacterial diversity of six different model soils was investigated with PCR-SSCP. The eubacterial and actinomycetical community was analysed with a structural and the community of nitrogen fixing bacteria with a functional approach. For the first time it could be shown that it is also possible to analyse bacterial communities using PCR-SSCP with functional genes like the nifH gene. Reference bacteria were used to optimize the SSCP, especially the parameters temperature und gel concentration, with the aim of achieving maximal resolution of the bands on the gel. The method was then tested with samples from garden-, forest-, restored- and Aralsea soil. The PCR, the crucial step of the SSCP, was not reproducible in all cases. Therefore for further studies it is recommended to perform parallel PCRs as a control. Another reason for the lack of reproducibility is due in part to the inability of the software used to analyse the complex band patterns. Despite this restriction most of the parallel samples showed a similarity of more than 90 %. The success of sequencing bands isolated from the gels depends on the diversity of the investigated bacterial group. It was easier to sequence the bands of the actinomycetes and the nitrogen fixing bacteria than of the eubacterial domain, because of their lower diversity, as this resulted in bands which were single products (and therefore easier to sequence). It could be shown, that all used primers were specific and that they have a great potential to detect unculturable and unknown bacteria, especially that the degenerated primers for nitrogen fixing bacteria were very suitable. Furthermore, the sequencing results of this work supports the assumption that Azomonas macrocytogenes has an alternative nitrogenase which is very similar to the nitrogenase 3. The basic difference between the structural and the functional analysis is the fact, that the 16S rRNA gene is more conserved and better investigated than the nifH gene. Therefore, structural analysis comparisons with the database show more agreement and higher homologies than the functional analysis comparisons. The method PCR-SSCP can visualise differences of the complex substrate soil, where every particle is colonized by different bacteria. When the diversity is high, the bands on the SSCP-gel blend with the background. In this case, the computer analysis is problematic. The six model soils depicted very different diversities of the investigated bacteria groups. The bandpatterns of the actinomycetes and the nitrogen fixing bacteria were less complex than the ones of the eubacteria.

Grantor dc:publisher

Publikationsserver der RWTH Aachen University

Year dc:date

2002

Author dc:creator

• Seibold, Anja

Contributors dc:contributor

• Priefer, Ursula B.

• info:eu-repo/classification/ddc/570
• Biowissenschaften, Biologie
• PCR-SSCP
• Diversität
• Boden
• Aktinomyceten
• stickstoffixierende Bakterien
• Eubakterien

Statement dc:rights

• info:eu-repo/semantics/openAccess

Language dc:language

ger

Identifier

https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-120736%22

OAI identifier oai:identifier

oai:publications.rwth-aachen.de:58910