Genome plasticity in soil bacteria is predicted to be evolutionarily advantageous, allowing bacteria to sample genetic variation for adaptation to local soil ecology. In the field population of mesorhizobia where the symbiosis island (ICEMlSym[R7A]; an I̲ntegrative C̲onjugative E̲lement) was first identified, individual members were found to have significant chromosomal variation downstream of the phe-tRNA gene or phe-tRNA integrated ICEMlSym[R7A]. However, the nature of this genetic variation and whether it contributed to the adaptation of the indigenous mesorhizobia to their field environment were unknown.
This work focused on a nodule isolate, Mesorhizobium sp. strain R88B, a member of the indigenous mesorhizobial population that received ICEMlSym[R7A] from strain R7A. The region downstream of ICEMlSym[R7A] was sequenced, revealing three distinct regions of non-conserved DNA, totalling 34.5 kb. Integrated directly downstream of ICEMlSym[R7A] was IMEMlAdh[R88B], a 24.3-kb novel I̲ntegrative M̲obilisable E̲lement. Using a PCR-based assay, it was shown that the IMEMlAdh[R88B] integrase could excise not only IMEMlAdh[R88B], but also a dual-IMEMlAdh[R88B]/ICEMlSym[R7A] hybrid, indicating the potential mobility of IMEMlAdh[R88B], and a likely evolutionary intermediate of a novel ICE. However, a functional role for MadA, (a putative adhesin and the sole adaptive trait encoded on IMEMlAdh[R88B]) was not discovered. Southern hybridisations with the mesorhizobial population provided evidence for the existence of a novel family of IMEs in the mesorhizobia, which, by diversifying their internal sequences, provide allele-specific variation to the population.
The two other regions downstream of IMEMlAdh[R88B] possessed no obvious mobile genetic element structures, and only the region adjacent to the core-chromosome encoded ORFs with putative functions. Mutation of two of these ORFs, fhuD1 and fhuB1, identified their function as two of the four components of a ferrichrome ABC-uptake (Fhu) system. Using genetic screens, the remaining components of this transporter were mapped to two separate loci. Thus, the functional transporter in R88B was a composite of at least two independently-acquired Fhu systems. The genetic screens also revealed that ferrichrome utilisation was dependent on a TonB energy-transduction system encoded downstream of the Fhu ATPase gene, fhuC.
Expression studies on the three fhu loci demonstrated that, despite their separate acquisition, their expression was coordinately up-regulated in response to low-iron conditions. Bioinformatics on the predicted promoter regions of the fhu genes identified the binding site of the rhizobial Fur analogue, RirA, which is likely to be responsible for this expression profile.
Southern hybridisations of DNA isolated from members of the mesorhizobial population revealed the three fhu loci were not conserved in the mesorhizobial population. The presence of FhuA was the best predictive marker for the trait. It is proposed that multiple rounds of acquisitions and recombinations, both illegitimate and legitimate, formed this transporter, with the constant need for iron offset by the negative selection pressure of FhuA being a target for phage. None of the Fhu-specific genes was present in the sequenced M. loti strain MAFF303099 though flanking sequences were, further emphasizing the role of genome microevolution in forming the Fhu phenotype.
| Identifer | oai:union.ndltd.org:ADTP/217471 |
| Date | January 2006 |
| Creators | Carlton, Timothy M., n/a |
| Publisher | University of Otago. Department of Microbiology & Immunology |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | http://policy01.otago.ac.nz/policies/FMPro?-db=policies.fm&-format=viewpolicy.html&-lay=viewpolicy&-sortfield=Title&Type=Academic&-recid=33025&-find), Copyright Timothy M. Carlton |
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