Abstract
The repeated failures reported in cultivating some microbial lineages are a major challenge in microbial ecology and probably linked, in the case of Frankia microsymbionts to atypical patterns of auxotrophy. Comparative genomics of the so far uncultured cluster-2 Candidatus Frankia datiscae Dg1, with cultivated Frankiae has revealed genome reduction, but no obvious physiological impairments. A direct physiological assay on nodule tissues from Coriaria myrtifolia infected with a closely-related strain permitted the identification of a requirement for alkaline conditions. A high pH growth medium permitted the recovery of a slow-growing actinobacterium. The strain obtained, called BMG5.1, has short hyphae, produced diazovesicles in nitrogen-free media, and fulfilled Koch’s postulates by inducing effective nodules on axenically grown Coriaria spp. and Datisca glomerata. Analysis of the draft genome confirmed its close proximity to the Candidatus Frankia datiscae Dg1 genome with the absence of 38 genes (trehalose synthase, fumarylacetoacetase, etc) in BMG5.1 and the presence of 77 other genes (CRISPR, lanthionine synthase, glutathione synthetase, catalase, Na+/H+ antiporter, etc) not found in Dg1. A multi-gene phylogeny placed the two cluster-2 strains together at the root of the Frankia radiation.
Highlights
The genome of a cluster-2 strain was determined by directly sequencing DNA isolated from nodule tissues[8] and a comprehensive study of its metabolic pathways permitted to conclude there was no missing function that would impede cluster-2 strains to grow in pure culture[9]
We decided to use this system to study the physiology of cluster-2 Frankia strains and adapt our isolation conditions to fit the endophytic cells
A modified BAP medium[13,14] buffered to pH 9 and supplemented with several organic acids to replace the usual propionate as carbon source and several oxygen scavengers was used, and yielded very slow-growing brownish colonies
Summary
Maher Gtari[1], Faten Ghodhbane-Gtari[1], Imen Nouioui[1], Amir Ktari[1], Karima Hezbri[1], Wajdi Mimouni[1], Imed Sbissi[1], Amani Ayari[1], Takashi Yamanaka[2], Philippe Normand[3], Louis S Tisa4 & Abdellatif Boudabous[1]. Reisolated from these nodules using the same isolation methods, grown in pure culture, and shown by PCR-sequencing to have identical markers (16S rRNA, nifH, gyrB and glnA) It is a stressful situation for a bacterium buffered by host tissues and under oxygen levels kept low by neighboring mitochondria[19] to grow on synthetic medium. Physiological assessment of uncultivated cells can provide reliable physiological data, leading to testable hypotheses for pure culture conditions This new Frankia isolate may form, together with Candidatus Frankia datiscae Dg1 strain, a different species that is mildly alkaliphilic. Status Size in bp Proteins Pseudogenes rRNA tRNA %G+ C Transposons and IS elements Horizontally Transferred Genes Accession
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