Draft Genomes of Nitrogen-fixing Frankia Strains Ag45/Mut15 and AgPM24 Isolated from Root Nodules of Alnus Glutinosa

Abstract

The genomes of two nitrogen-fixing Frankia strains, Ag45/Mut15 and AgPM24, isolated from root nodules of Alnus glutinosa are described as representatives of a novel candidate species . Phylogenomic and ANI analyses confirmed that both strains are related to cluster 1 frankiae, and that both strains belong to a novel species. At 6.4 - 6.7 Mb, their genomes were smaller than those of other cultivated Alnus -infective cluster 1 strains but larger than that of the non-cultivated Alnus -infective cluster 1 Sp+ strain AgTrS that was their closest neighbor as assessed by ANI. Comparative genomic analyses identified genes essential for nitrogen-fixation, gene composition as regards COGs, secondary metabolites clusters and transcriptional regulators typical of those from Alnus -infective cluster 1 cultivated strains in both genomes. There were 459 genes present in other cultivated Alnus -infective strains lost in the two genomes, spread over the whole of the genome, which indicates genome erosion is taking place in these two strains.

Draft Genomes of Symbiotic Frankia Strains AgB32 and AgKG'84/4 from Root Nodules of Alnus Glutinosa growing under Contrasted Environmental Conditions

“…Several published works on genus Frankia using sub-cluster, OTU, group and genomospecies assignments did provide grounds permitting to affirm that cluster 1 is probably much more diverse than the four species and two candidate species described so far 4 , 11 - 14 . This statement is supported by recent genome analyses of strains Ag45/Mut14 and AgPM24 as representatives of a yet undescribed species 15 , and by comparative sequence analyses of amplicons of an actinobacteria-specific insertion in the 23S rRNA genes of frankiae that identified several strains clustering together but that are distinct from type strains of cluster 1 16 . Strains AgB32 and AgKG'84/4 are two such strains, isolated from root nodules of Alnus glutinosa growing under contrasted environmental conditions at two locations in Germany about 350 km apart.…”

“…Comparative analyses of whole genome sequences between Frankia populations have resulted in the description of twelve species and five candidate species for uncultured populations so far 6 . These numbers were based on the availability of 37 genomes 39 , a number that is increasing regularly 15 , 40 . Comparative sequences analyses of whole genomes and metrics such as ANI 26 or dDDH 41 are now used as foundation for the description of microbial genera, species and subspecies.…”

“…Genomes of Alnus-infective symbiotic strains have initially been found to be quite large at 7.5 Mb with several ancient duplicated genes such as the shc gene coding for the synthesis of hopanoid lipids [48], the hup genes coding for hydrogen uptake for the recycling of hydrogen derived from nitrogenase [33], the cel coding for cellulases [49], the can coding for the carbonic anhydrase necessary for feeding short chain fatty acids (SCFA) into the tricarboxylic acid (TCA) cycle or the kor genes coding for 2-oxoglutarate ferredoxin oxidoreductase that connects the TCA cycle with nitrogenase (with the nitrogen-fixation process) [50,51]. Some of these duplications have been found to be lost in lineages with smaller genome size as is the case for Ag45/Mut15 and AgPM24 [15]. It appears the genomes of strains AgB32 and AgKG'84/4 are also undergoing a parallel process of genome erosion.…”

Draft Genome of Frankia sp. Strain R82, an Isolate from Root Nodules of Myrica gale

“…The strain was isolated from root nodules of Myrica gale from Black Creek National Refuge, Vermont, United States (44°50′N, 73°08′W) in 1980 ( 5 ). This strain was kept at −80°C in 20% glycerol in water (vol/vol) since 2010 and grown in defined propionate medium at 30°C for 15 days ( 6 ). Cells were centrifuged (15,000 × g , 5 min), sonicated (10 s at 20% output in S-450 Sonifier; Branson Ultrasonics, Danbury, CT), and DNA extracted using SurePrep soil DNA isolation kits (Fisher Scientific, Houston, TX).…”

“…The DNA concentration was measured with a Qubit 2.0 fluorometer (Life Technologies, Carlsbad, USA). The DNA was moderately fragmented to 1,000 nucleotides (nt), and the libraries were prepared for sequencing using standard protocols for Illumina tagmentation and a NextSeq 2000 machine (2 × 150 bp) at the Microbial Genomics Sequencing Center (Pittsburgh, PA, USA) ( 6 , 7 ). The total number of reads was 7,362,308, and the average length was 147.8 bp.…”