Plasmid mediated adaption of bacteria to changing environmental conditions

GND
1119102766
Zugehörigkeit
Julius Kühn-Institute (JKI), Institute for Epidemiology and Pathogen Diagnostics, Germany
Nour Eldeen Hamed Ahmed, Eman

Conjugative plasmids have been frequently reported to carry a considerable variety of bacterial functional genes often coding for traits beneficial to the plasmid host. Transferable plasmids are mobile genetic elements (MGEs) that can transfer by conjugation or mobilization between bacterial cells of the same species or between different taxa. The ability to stably replicate in a host cell determines the replication range. Under rapidly changing environmental conditions plasmids foster the adaptation to stressful environments. The focus of this thesis was to investigate the response of bacterial communities to changing environmental conditions, and the studies showed that IncP-1 plasmid-mediated adaptation to changing environmental conditions is important for biofilter and rhizosphere bacteria. IncP-1 plasmids have a broad host range and transfer efficiently in soils and related habitats. IncP-1 plasmids have the ability to disseminate remarkably diverse functional genes (resistance to antibiotic and/or heavy metal, degradation of xenobiotics) by carrying, or mobilizing plasmids carrying, such genes via a process called horizontal gene transfer (HGT) or the proliferation of IncP-1 carrying bacterial populations. Most of the studied IncP-1 plasmids were isolated from polluted environments. However, the role of IncP-1 plasmids in unpolluted soils was so far not studied. In this context, this thesis is providing insights into the presence, response, type, diversity and potential ecological role of IncP-1 plasmids in two different environments known to be hot spots for HGT. First, on-farm bio-purification systems (BPSs) are an efficient, applicable, simple and low-cost method used by the farmers to treat pesticide-contaminated wastewater through biodegradation and sorption processes. Recently it was shown for on-farm BPSs that during the agricultural season the abundance of IncP-1 plasmids in BPS material increased and the dynamic changes of the composition of different IncP-1 plasmids were revealed by trfA amplicon sequencing. The increase of IncP-1 plasmids seemed to correlate with increasing pesticide concentration. In this thesis microcosm experiments were performed with BPS material where the phenylurea herbicide linuron was added or not. Cultivation-independent methods were used to study the dynamic response of BPS bacterial communities to the addition of linuron. Total community DNA (TC-DNA) extracted from BPS samples taken at different time points after the linuron addition was analyzed by quantitative real-time PCR or by PCR amplification of degradative genes and subsequent Southern blot hybridization. In addition, plasmids were captured in socalled exogenous biparental matings into P. putida as a recipient. The experimental design allowed to demonstrate the link between the presence of linuron and the abundance of IncP-1 plasmids and genes coding for enzymes involved in the degradation of linuron (chapters II and III). In three transconjugants the presence of hylA gene was revealed. Second, the ecology of IncP-1 plasmid in the rhizosphere of three different crops grown under greenhouse conditions in the same soil type was investigated. We could confirm by qPCR the recently reported observation that in the rhizosphere of field-grown lettuce IncP-1 plasmids were enriched. In contrast, the increased relative abundance of IncP-1 plasmids was less pronounced in the rhizosphere of tomato and hardly detectable in potato plants (chapters IV and V). Based on the ability to mobilize IncQ plasmids, numerous IncP-1 plasmids were captured in a so-called triparental mating directly from rhizosphere bacteria. Transconjugants were selected based on the markers of the IncQ plasmid but most of the transconjugants also carried IncP-1 plasmids. The cultivation-independent methods used in this thesis (DGGE, pyrosequencing of 16S rRNA gene fragments, amplicon pyrosequencing of IncP-1 trfA genes, qPCR, PCR and/or Southern blot hybridization) allowed us to explore the dynamics of populations bearing IncP-1 plasmids in the TC-DNA of tested samples. The results presented in this thesis revealed that the IncP-1 plasmids bearing populations are remarkably dynamic in response not only to the introduction of xenobiotic compounds but also to plant root exudates providing evidence of the importance of IncP-1 plasmids in bacterial adaptation to both man-made and root exudates. Almost all IncP-1 subgroups were presented in all tested samples while IncP-1β subgroup showed a notably stronger response. We showed for the first time the high diversity of IncP-1 plasmids (particularly IncP-1β subgroup) regarding the rhizosphere of naturally grown lettuce and tomato plants but not for potato. Additionally, different plants might harbor populations bearing different IncP-1 plasmids even if they were grown in the same soil type. However, these results suggest the plant rhizosphere as a fertile source for novel functional MGEs. Upon author request this thesis is available as printed version only.

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