Gene flux in Streptomyces: conjugative transfer, chromosomal recombination, and impact of biotic factors. – StreptoFlux

Among the mechanisms of evolution, lateral transfer of genetic information (LGT for Lateral Gene Transfer) is the most efficient way to generate genetic variability among bacterial populations on a short evolutionary timescale. LGT provides a constant gene flux allowing the acquisition of highly valuable genes such as polymer degradation genes, secondary metabolite gene clusters or drug resistance determinants. It is presumed to facilitate the adaptation of free-living soil and rhizosphere bacteria, such as our model genus Streptomyces, to changing abiotic and biotic environments in the soil. Up to now, the impact of soil biotic factors on LGT have been very poorly documented in the case of Streptomyces although these Gram-positive bacteria are known to interact with many other bacterial genera as well as different symbiotic, pathogenic and saprotrophic fungi in their natural environment. Streptomyces possesses a large linear chromosome (ca. 9 Mb) showing a very specific genetic organization with a central core region conserved across species (harboring the 'essential genes'), and highly species/strain specific genes ('accessory genes') confined to the terminal regions (up to 20% of the genome). Among the mechanisms of LGT, conjugative transfer is the most likely to be responsible for incoming information in Streptomyces. This hypothesis is supported by the presence in this genus, of numerous conjugative elements (linear or circular plasmids and integrated elements), all able to mobilize chromosomal genes, and by the presence of plasmid-related genes in the chromosomal ends. Moreover, beside the well-known picture of the self-transmissible plasmids encoding a type IV secretion system in Gram negative bacteria, Streptomyces conjugative processes are unusual in several points, i.e. the absence of pilus, the fact that a unique TraG-like coupling protein is sufficient for transfer, and transfer of double stranded DNA. In addition, the mechanisms of mobilization of chromosomal genes are poorly understood, even if both linear and circular conjugative replicons can mediate these events. Recent genomic comparisons also pointed out the unique pattern of recombination along the linear chromosome, and questioned the mechanisms of integration of the transferred DNA. Multiple insertions and deletions (indels) affecting a few genes are detected at the chromosomal ends. At the evolutionary time-scale, these indels progressively erased the gene conservation between species in the terminal regions, resulting in a gradient of recombination events increasing towards the chromosomal ends. Traces of exchanges between plasmids and chromosome were also detected, participating in the evolution of the terminal regions. The main aim of the StreptoFlux project is to characterize the mechanisms of chromosomal gene transfer and chromosomal integration, resulting in the gene flux observed in the extremities of the linear chromosome of Streptomyces, and to test the influence of bacteria and fungi from the soil and the plant rhizosphere on LGT among Streptomyces. The pattern of homologous recombination along the genome will be surveyed in order to test the hypothesis of a recombination gradient. Double strand break (DSB) repair will also be studied in different genetic backgrounds, defective for homologous or illegitimate recombination, to test the involvement of different repair systems in the terminal regions relative to the center. The conjugative transfer of chromosomal markers will be studied using different conjugative elements (circular or linear, integrated or not). The hypothesis of the preferential transfer of chromosomal extremities will be investigated as well as the role of chromosomal sequences related to the plasmid borne clt sequence (clt for cis locus for transfer). The frequencies of transfer as well as the short term maintenance (integration) of the transferred DNA will be assessed by simulating microbiological interactions that exist in soil and plant rhizosphere. Either culture extracts (ectomycorrhizal or pathogenic fungi, soil bacteria), or cell extracts (cell wall, sugars) or bacterial metabolites (e.g. antibiotics) will be added to the conjugation in vitro experiments. The proposed project will involve three teams (UMRs), two INRA-University Nancy teams and a CNRS-Paris Sud University one, internationally recognized in the field of bacterial genetics/genomics and soil microbial ecology. This join pluridisciplinary effort will be performed on S. ambofaciens whose complete genome sequencing is ongoing.