Diazotrophic Actinobacteria of the genus Frankia represent a challenge to classical bacterial taxonomy as they include many unculturable strains. As a consequence, we still have a poor understanding ...of their diversity, evolution and biogeography. In this study, a Multi-Locus Sequence Analysis (MLSA) using atpD, dnaA, ftsZ, pgk, and rpoB loci was done on a large set of cultured and uncultured strains, compared to 16S rRNA and correlated to Average Nucleotide Identity (ANI) from available Frankia genomes. MLSA provided a robust resolution of Frankia genus phylogeny and clarified the status of unresolved species and complex of species.
The robustness of single-gene topologies and their congruence with the MLSA tree were tested. Lateral Gene Transfers (LGT) were few and scattered, suggesting they had no impact on the concatenate topology. The pgk marker – providing the longest sequence, highest mean genetic divergence and least occurrence of LGT – was used to survey an unequalled number of Alnus-infective Frankia — mainly uncultured strains from a broad range of host-species and geographic origins. This marker allowed reliable Single-Locus Strain Typing (SLST) below the species level, revealed an undiscovered taxonomical diversity, and highlighted the effect of cultivation, sporulation phenotype and host plant species on symbiont richness, diversity and phylogeny.
A unique case of microbial symbiont capable of dormancy within its living host cells has been reported in actinorhizal symbioses. Some Frankia strains, named Sp+, are able to sporulate inside plant ...cells, contrarily to Sp- strains. The presence of metabolically slowed-down bacterial structures in host cells alters our understanding of symbiosis based on reciprocal benefits between both partners, and its impact on the symbiotic processes remains unknown. The present work reports a metabolomic study of Sp+ and Sp- nodules (from Alnus glutinosa), in order to highlight variabilities associated with in-planta sporulation. A total of 21 amino acids, 44 sugars and organic acids, and 213 secondary metabolites were detected using UV and mass spectrometric-based profiling. Little change was observed in primary metabolites, suggesting that in-planta sporulation would not strongly affect the primary functionalities of the symbiosis. One secondary metabolite (M27) was detected only in Sp+ nodules. It was identified as gentisic acid 5-O-β-d-xylopyranoside, previously reported as involved in plant defenses against microbial pathogens. This metabolite significantly increased Frankia in-vitro sporulation, unlike another metabolite significantly more abundant in Sp- nodules M168 = (5R)-1,7-bis-(3,4-dihydroxyphenyl)-heptane-5-O-β-d-glucopyranoside. All these results suggest that the plant could play an important role in the Frankia ability to sporulate in planta and allow us to discuss a possible sanction emitted by the host against less cooperative Sp+ symbionts.
Sporulation is a microbial adaptive strategy to resist inhospitable conditions for vegetative growth and to disperse to colonise more favourable environments. This microbial trait is widespread in ...Actinobacteria. Among them, Frankia strains are able to differentiate sporangia in pure culture, while others can sporulate even when in symbiosis with sporulation occurring within host cells. The molecular determinants controlling Frankia sporulation have not been yet described. In order to highlight, for the first time, the molecular players potentially involved in Frankia sporulation, we conducted (i) a comparison of protein contents between Frankia spores and hyphae and (ii) a comparative genomic analysis of Frankia proteomes with sporulating and non-sporulating Actinobacteria. Among the main results, glycogen-metabolism related proteins, as well as oxidative stress response and protease-like proteins were overdetected in hyphae, recalling lytic processes that allow Streptomyces cells to erect sporogenic hyphae. Several genes encoding transcriptional regulators, including GntR-like, appeared up-regulated in spores, as well as tyrosinase, suggesting their potential role in mature spore metabolism. Finally, our results highlighted new proteins potentially involved in Frankia sporulation, including a pyrophosphate-energized proton pump and YaaT, described as involved in the phosphorelay allowing sporulation in Bacillus subtilis, leading us to discuss the role of a phosphorelay in Frankia sporulation.
Summary
Two major types of Frankia strains are usually recognized, based on the ability to sporulate in‐planta: spore‐positive (Sp+) and spore‐negative (Sp−). We carried out a study of Sp+ and Sp− ...Frankia strains based on nodules collected on Alnus glutinosa, Alnus incana and Alnus viridis. The nodules were phenotyped using improved histology methods, and endophytic Frankia strain genotype was determined using a multilocus sequence analysis approach. An additional sampling was done to assess the relation between Sp+ phenotype frequency and genetic diversity of Frankia strains at the alder stand scale. Our results revealed that (i) Sp+ and Sp− Alnus‐infective Frankia strains are genetically different even when sampled from the same alder stand and the same host–plant species; (ii) there are at least two distinct phylogenetic lineages of Sp+ Frankia that cluster according to the host–plant species and without regard of geographic distance and (iii) genetic diversity of Sp+ strains is very low at the alder stand scale compared with Sp− strains. Difference in evolutionary history and genetic diversity between Sp+ and Sp− Frankia allows us to discuss the possible ecological role of in‐planta sporulation.
Rhizosphere bacterial community and endophytes are now known to influence plant health and response to environmental stress. Very few studies have reported the diversity of endophytic bacterial ...communities of Vanilla planifolia and their potential roles in promoting plant growth or contributing to aromatic quality. In this study, the composition and diversity of the Vanilla rhizosphere bacterial community were explored by analyzing rhizosphere soil and root tissue samples as well as green pods of three accessions of Vanilla planifolia grown on different types of substrates (compost and leaf litter). In addition, the endophytic bacterial diversity of roots and green pods as well as the evolution of endophytic bacteria after the curing process of vanilla green pods were analyzed based on a metabarcoding approach. The results showed that bacterial species richness and diversity were higher in the compost. The analysis of the soil bacterial composition displayed that Halomonas, Pseudoalteromonas, Enterobacter and Bradyrhizobium were the most abundant genera. Moreover, the results indicated that the soil bacterial community structure was linked to the host plant genotype. Regarding the roots endophytic bacteria composition, the genera Halomonas, Pseudoalteromonas, Bacillus and Carboxydocella genera were present in all samples, independently from the substrate nature. Several genera including Bacillus, Bradyrhizobium, Burkholderia and Halomonas were transmitted internally from the roots to the green pods. The curing process reduced the bacterial richness and bacterial diversity associated with the green pods. Halomonas, Pseudoalteromonas, Bacillus, and Carboxydocella are the dominant genera in the pods after the curing process. This study provides an overview of changes of the bacterial communities dynamics especially endophytic in the roots and the green pods. It highlighted bacterial genera (Halomonas, Pseudoalteromonas, Bacillus, and Carboxydocella) potentially implicated in the formation of aroma compounds of vanilla beans.
Frankia Sp+ strains maintain their ability to sporulate in symbiosis with actinorhizal plants, producing abundant sporangia inside host plant cells, in contrast to Sp− strains, which are unable to ...perform in-planta sporulation. We herein examined the role of in-planta sporulation in Frankia infectivity and competitiveness for root infection. Fifteen strains belonging to different Sp+ and Sp− phylogenetic lineages were inoculated on seedlings of Alnus glutinosa (Ag) and A. incana (Ai). Strain competitiveness was investigated by performing Sp−/Sp+ co-inoculations. Plant inoculations were standardized using crushed nodules obtained under laboratory-controlled conditions (same plant species, age, and environmental factors). Specific oligonucleotide primers were developed to identify Frankia Sp+ and/or Sp− strains in the resulting nodules. Single inoculation experiments showed that (i) infectivity by Sp+ strains was significantly greater than that by Sp− strains, (ii) genetically divergent Sp+ strains exhibited different infective abilities, and (iii) Sp+ and Sp− strains showed different host preferences according to the origin (host species) of the inocula. Co-inoculations of Sp+ and Sp− strains revealed the greater competitiveness of Sp+ strains (98.3 to 100% of Sp+ nodules, with up to 15.6% nodules containing both Sp+ and Sp− strains). The results of the present study highlight differences in Sp+/Sp− strain ecological behaviors and provide new insights to strengthen the obligate symbiont hypothesis for Sp+ strains.
The present study aimed to use comparative genomics to explore the relationships between
and actinorhizal plants using a data set made of 33
genomes. The determinants of host specificity were first ...explored for "
-infective strains" (i.e.,
strains belonging to Cluster Ia). Several genes were specifically found in these strains, including an agmatine deiminase which could possibly be involved in various functions as access to nitrogen sources, nodule organogenesis or plant defense. Within "
-infective strains", Sp+
genomes were compared to Sp- genomes in order to elucidate the narrower host specificity of Sp+ strains (i.e., Sp+ strains being capable of
sporulation, unlike Sp- strains). A total of 88 protein families were lost in the Sp+ genomes. The lost genes were related to saprophytic life (transcriptional factors, transmembrane and secreted proteins), reinforcing the proposed status of Sp+ as obligatory symbiont. The Sp+ genomes were also characterized by a loss of genetic and functional paralogs, highlighting a reduction in functional redundancy (e.g.,
genes) or a possible loss of function related to a saprophytic lifestyle (e.g., genes involved in gas vesicle formation or recycling of nutrients).
Strains CN4
, CN6, CN7 and CNm7 were isolated from root nodules of
from Murree in Pakistan. They do not form root nodules on
nor on
although they deformed root hairs of
. The colonies are bright ...red-pigmented, the strains form hyphae and sporangia but no N
-fixing vesicles and do not fix nitrogen
. The peptidoglycan of strain CN4
contains
-diaminopimelic acid; whole cell sugars consist of ribose, mannose, glucose, galactose and rhamnose. Diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and two unknown lipids represent the major polar lipids; MK-9(H
) and MK-9(H
) are the predominant menaquinones (>15 %), and iso-C
and C
ω8
are the major fatty acids (>15 %). The results of comparative 16S rRNA gene sequence analyses indicated that strain CN4
is most closely related to
CN 3
. An MLSA phylogeny using amino acids sequences of AtpD, DnaA, FtsZ, Pgk and RpoB, assigned the strain to cluster 4 non-nodulating species, close to
CN 3
M16386
and
EuI1c
with 0.04 substitutions per site, while that value was 0.075 with other strains. Digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values between CN4
and all species of the genus
with validly published names were below the defined threshold for prokaryotic species demarcation, with dDDH and ANI values at or below 27.8 and 83.7 %, respectively. The four strains CN4
, CN6, CN7 and CNm7 had dDDH (98.6-99.6 %) and ANI values that grouped them as representing a single species. CN4
has a 10.76 Mb genome. CN4
was different from its close phylogenetic neighbours with validly published names in being red-pigmented, in having several lantibiotic-coding clusters, a carbon monoxide dehydrogenase cluster and a clustered regularly interspaced short palindromic repeats (CRISPR) cluster. The results of phenotypic, physiological and phylogenomic analyses confirmed the assignment of strain CN4
(=DSM 114740
= LMG 32595
) to a novel species, with CN4
as type strain, for which the name
sp. nov. is proposed.
strain Ag45/Mut15
was isolated from a root nodule of
growing in a swamp at lake Grossensee, Germany. The strain forms root nodules on
, in which it produces hyphae and clusters of N
-fixing vesicles. ...N
-fixing vesicles are also produced in nitrogen-free growth medium, in addition to hyphae and sporangia. The whole-cell hydrolysates of strain Ag45/Mut15
contained
-diaminopimelic acid in the peptidoglycan and ribose, xylose, mannose, glucose, galactose and a trace of rhamnose as cell-wall sugars. The major polar lipids were phosphatidylglycerol, phosphatidylinositol, diphosphatidylglycerol and glyco-phospholipid. The predominant (>20 %) menaquinones were MK-9(H
) and MK-9(H
). The major fatty acid profile (>10 %) consisted of iso-C
, C
ω8
and C
. Pairwise 16S rRNA gene distances showed that strain Ag45/Mut15
was most closely related to
CpI1
and
Frankia nodulisporulans with 16S rRNA gene similarity values of 0.001335 substitutions per site. An multilocus sequence analysis phylogeny based on
,
,
,
and
amino acid sequences positioned the strain within cluster 1 of
- and
-nodulating species, close to
F. nodulisporulans AgTrS
and
ARgP5
. The digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values between the studied strain Ag45/Mut15
and all validly named
species were below the defined threshold for prokaryotic species demarcation.
F. nodulisporulans AgTrS
, which cannot be cultivated
, was found to be the closest phylogenetic neighbour to strain strain Ag45/Mut15
with dDDH and ANI values of 61.8 and 97 %, respectively. Strain Ag45/Mut15
was not able to sporulate in nodule tissues like strain AgTrS
.Phenotypic, physiological and phylogenomic analyses confirmed the assignment of strain Ag45/Mut15
(=DSM 114737
=LMG 326O1
) to a novel species, with Ag45/Mut15
as type strain, for which the name
sp. nov. is proposed.
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