Raúl Riesco

Endophytic Actinobacteria and the Interaction of Micromonospora and Nitrogen Fixing Plants.

For a long time, it was believed that a healthy plant did not harbor any microorganisms within its tissues, as these were often considered detrimental for the plant. In the last three decades, the numbers of studies on plant microbe-interactions has led to a change in our view and we now know that many of these invisible partners are essential for the overall welfare of the plant. The application of Next Generation Sequencing techniques is a powerful tool that has permitted the detection and identification of microbial communities in healthy plants. Among the new plant microbe interactions recently reported several actinobacteria such as Micromonospora are included. Micromonospora is a Gram-positive bacterium with a wide geographical distribution; it can be found in the soil, mangrove sediments, and freshwater and marine ecosistems. In the last years our group has focused on the isolation of Micromonospora strains from nitrogen fixing nodules of both leguminous and actinorhizal plants and reported for the first time its wide distribution in nitrogen fixing nodules of both types of plants. These studies have shown how this microoganism had been largely overlooked in this niche due to its slow growth. Surprisingly, the genetic diversity of Micromonospora strains isolated from nodules is very high and several new species have been described. The current data indicate that Micromonospora saelicesensis is the most frequently isolated species from the nodular tissues of both leguminous and actinorhizal plants. Further studies have also been carried out to confirm the presence of Micromonospora inside the nodule tissues, mainly by specific in situ hybridization. The information derived from the genome of the model strain, Micromonospora lupini, Lupac 08, has provided useful information as to how this bacterium may relate with its host plant. Several strategies potentially necessary for Micromonospora to thrive in the soil, a highly competitive, and rough environment, and as an endophytic bacterium with the capacity to colonize the internal plant tissues which are protected from the invasion of other soil microbes were identified. The genome data also revealed the potential of M. lupini Lupac 08 as a plant growth promoting bacterium. Several loci involved in plant growth promotion features such as the production of siderophores, phytohormones, and the degradation of chitin (biocontrol) were also located on the genome and the functionality of these genes was confirmed in the laboratory. In addition, when several host plants species were inoculated with Micromonospora strains, the plant growth enhancing effect was evident under greenhouse conditions. Unexpectedly, a high number of plant-cell wall degrading enzymes were also detected, a trait usually found only in pathogenic bacteria. Thus, Micromonospora can be added to the list of new plant-microbe interactions. The current data indicate that this microorganism may have an important application in agriculture and other biotechnological processes. The available information is promising but limited, much research is still needed to determine which is the ecological function of Micromonospora in interaction with nitrogen fixing plants.

Genome Features of the Endophytic Actinobacterium Micromonospora lupini Strain Lupac 08: On the Process of Adaptation to an Endophytic Life Style?

Endophytic microorganisms live inside plants for at least part of their life cycle. According to their life strategies, bacterial endophytes can be classified as “obligate” or “facultative”. Reports that members of the genus Micromonospora, Gram-positive Actinobacteria, are normal occupants of nitrogen-fixing nodules has opened up a question as to what is the ecological role of these bacteria in interactions with nitrogen-fixing plants and whether it is in a process of adaptation from a terrestrial to a facultative endophytic life. The aim of this work was to analyse the genome sequence of Micromonospora lupini Lupac 08 isolated from a nitrogen fixing nodule of the legume Lupinus angustifolius and to identify genomic traits that provide information on this new plant-microbe interaction. The genome of M. lupini contains a diverse array of genes that may help its survival in soil or in plant tissues, while the high number of putative plant degrading enzyme genes identified is quite surprising since this bacterium is not considered a plant-pathogen. Functionality of several of these genes was demonstrated in vitro, showing that Lupac 08 degraded carboxymethylcellulose, starch and xylan. In addition, the production of chitinases detected in vitro, indicates that strain Lupac 08 may also confer protection to the plant. Micromonospora species appears as new candidates in plant-microbe interactions with an important potential in agriculture and biotechnology. The current data strongly suggests that a beneficial effect is produced on the host-plant.

Modestobacter caceresii sp. nov., novel actinobacteria with an insight into their adaptive mechanisms for survival in extreme hyper-arid Atacama Desert soils

A polyphasic study was designed to determine the taxonomic provenance of three Modestobacter strains isolated from an extreme hyper-arid Atacama Desert soil. The strains, isolates KNN 45-1a, KNN 45-2b(T) and KNN 45-3b, were shown to have chemotaxonomic and morphological properties in line with their classification in the genus Modestobacter. The isolates had identical 16S rRNA gene sequences and formed a branch in the Modestobacter gene tree that was most closely related to the type strain of Modestobacter marinus (99.6% similarity). All three isolates were distinguished readily from Modestobacter type strains by a broad range of phenotypic properties, by qualitative and quantitative differences in fatty acid profiles and by BOX fingerprint patterns. The whole genome sequence of isolate KNN 45-2b(T) showed 89.3% average nucleotide identity, 90.1% (SD: 10.97%) average amino acid identity and a digital DNA-DNA hybridization value of 42.4±3.1 against the genome sequence of M. marinus DSM 45201(T), values consistent with its assignment to a separate species. On the basis of all of these data, it is proposed that the isolates be assigned to the genus Modestobacter as Modestobacter caceresii sp. nov. with isolate KNN 45-2b(T) (CECT 9023(T)=DSM 101691(T)) as the type strain. Analysis of the whole-genome sequence of M. caceresii KNN 45-2b(T), with 4683 open reading frames and a genome size of ∽4.96Mb, revealed the presence of genes and gene-clusters that encode for properties relevant to its adaptability to harsh environmental conditions prevalent in extreme hyper arid Atacama Desert soils.

Microbacterium diaminobutyricum sp. nov., isolated from Halimione portulacoides, which contains diaminobutyric acid in its cell wall, and emended description of the genus Microbacterium.

Three actinobacterial strains were isolated from roots of the salt-marsh plant Halimione portulacoides collected in Ria de Aveiro, Portugal. Molecular typing using enterobacterial repetitive intergenic consensus ERIC-PCR fingerprinting showed the strains to be highly similar. Phylogenetic analyses based on the 16S rRNA gene sequence and multilocus sequence analysis (MLSA) using gyrB, rpoB, recA and ppk and 16S rRNA genes sequences showed that the strains represented a member of the genus Microbacterium, with Microbacterium lacus DSM 18910T as the closest phylogenetic relative. DNA-DNA hybridization between strain RZ63T and its closest relative was below 70 %, supporting the hypothesis that it represented a distinct genomic species. Chemotaxonomic analyses of the novel strains and their DNA G+C contents confirmed their affiliation to the genus Microbacterium, however, the peptidoglycan of RZ63T contained diaminobutyric acid as the diagnostic diamino acid. In addition, physiological and fatty acid analyses revealed differences between these strains and their phylogenetic relatives, reinforcing their status as a distinct species. Based on the physiological, genetic and chemotaxonomic characterisation it is proposed that the strains studied represent a novel species of the genus Microbacterium for which the name Microbacterium diaminobutyricum sp. nov. is proposed (type strain RZ63T=DSM 27101T=CECT 8355T).

Micromonospora ureilytica sp. nov., Micromonospora noduli sp. nov. and Micromonospora vinacea sp. nov., isolated from Pisum sativum nodules.

A diversity study on the presence of strains representing the genus Micromonospora in Pisum sativum nodules collected from Cañizal (Spain) has provided evidence of the high number of isolates that might represent novel species. In the present work, we have characterized three of these isolates: GUI23T, GUI43T and GUI63T. Phenotypic and genotypic analyses confirmed that all strains represent novel species of the genus Micromonospora with the following proposed names: Micromonospora ureilytica sp. nov., type strain GUI23T (=CECT 9022T=DSM 101692T), Micromonospora noduli sp. nov., type strain GUI43T (=CECT 9020T=DSM 101694T), and Micromonospora vinacea sp. nov., type strain GUI63T (=CECT 9019T=DSM 101695T).

Microbacterium proteolyticum sp. nov. isolated from roots of Halimione portulacoides.

An endophytic actinobacterial strain RZ36T, isolated from roots of the salt-marsh plant Halimione portulacoides, was subjected to a polyphasic taxonomic characterization. 16S rRNA gene sequence analysis revealed that this strain belonged to the genus Microbacterium. The closest phylogenetic relative was Microbacterium hominis DSM 12509T, with a pairwise 16S rRNA gene sequence similarity of 98.8 %. The DNA-DNA hybridization value between strain RZ36T and M. hominis DSM 12509T was 16 %. The affiliation to the genus Microbacterium was corroborated by phenotypic and chemotaxonomic characteristics. The cell-wall peptidoglycan type was B2β and the diagnostic diamino acid was ornithine. Whole-cell sugars detected were galactose, glucose, rhamnose, ribose and xylose. The major fatty acids were anteiso-C15 : 0 and iso-C16 : 0 and the major menaquinone was MK-11 (64 %). Main polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol and two unknown glycolipids. The genomic DNA G+C content was 69.7 mol%. Thus, on the basis of phenotypic, genotypic and chemotaxonomic data, strain RZ36T is considered to represent a novel species of the genus Microbacterium, for which the name Microbacterium proteolyticum sp. nov. (type strain RZ36T = DSM 27100T = CECT 8356T) is proposed.

Pseudonocardia nigra sp. nov., isolated from Atacama Desert rock

Eleven actinobacterial strains were isolated from a rock sample collected in the Atacama Desert. Molecular typing by BOX-PCR divided the strains into three clusters and showed that, although very similar, they were not clones. Three strains, ATK01, ATK03T and ATK17, each representing one of the defined BOX clusters, were chosen for further characterization. Phylogenetic analysis indicated that the strains were related to the genus Pseudonocardia and were recovered in a cluster together with Pseudonocardia bannensis YIM 63101T and Pseudonocardia xinjiangensis AS 4.1538T. Chemotaxonomic analyses confirmed their affiliation to the genus Pseudonocardia but differences were found between the new strains and their closest phylogenetic relatives. Physiological and fatty acid analyses also revealed differences between these strains and their phylogenetic neighbours supporting their status as a distinct species. Based on the overall data, it is proposed that strains ATK01, ATK03T and ATK17 represent a novel species of the genus Pseudonocardia for which the name Pseudonocardia nigra sp. nov. is proposed (type strain ATK03T=DSM 104088T=CECT 9183T).

Micromonospora phytophila sp. nov. and Micromonospora luteiviridis sp. nov., isolated as natural inhabitants of plant nodules

Two actinobacterial isolates, strains SG15T and SGB14T, were recovered through a microbial diversity study of nitrogen fixing nodules from Pisum sativum plants collected in Salamanca (Spain). The taxonomic status of these isolates was determined using a polyphasic approach and both presented chemotaxonomic and morphological properties consistent with their classification in the genus Micromonospora. For strains SG15T and SGB14T, the highest 16S rRNA gene sequence similarities were observed with Micromonospora coxensis JCM 13248T (99.2 %) and Micromonospora purpureochromogenes DSM 43821T (99.4 %), respectively. However, strains SG15T and SGB14T were readily distinguished from their phylogenetic neighbours both genetically and phenotypically indicating that they represent two new Micromonospora species. The following names are proposed for these species: Micromonosporaphytophila sp. nov. type strain SG15T (=CECT 9369T; =DSM 105363T), and Micromonosporaluteiviridis sp. nov. type strain SGB14T (=CECT 9370T; =DSM 105362T).

Defining the species Micromonospora saelicesensis and Micromonospora noduli under the framework of genomics

The type isolates of species Micromonospora saelicesensis and Micromonospora noduli are Gram-stain positive actinobacteria that were originally isolated from nitrogen fixing nodules of the legumes Lupinus angustifolius and Pisum sativum, respectively. These two species are very closely related and questions arise as to whether they should be merged into a single species. To better delineate the relationship of M. saelicesensis and M. noduli, 10 strains isolated from plant tissue (nodules and leaves) and identified by their 16S rRNA gene sequences as either M. saelicensesis or M. noduli, based on a cut-off value of ≥99.5% were selected for whole-genome sequencing and compared with the type strains of M. saelicesensis Lupac 09T and M. noduli GUI43T using overall genome relatedness indices (OGRI) which included ANI, OrthoANI and digital DNA-DNA hybridization. Whole- and core-genome phylogenomic analyses were also carried out. These results were compared with the topologies of the 16S rRNA and gyrB gene phylogenies. Good correlation was found between all trees except for the 16S rRNA gene. Overall results also supported the current classification of M. saelicesensis and M. noduli as separate species. Especially useful was the core-genome phylogenetic analyses based on 92 genes and the dDDH results which were highly correlated. The importance of using more than one strain for a better definition of a species was also shown. A series of in vitro phenotypic assays performed at different times were compared with in silico predictions based on genomic data. In vitro phenotypic tests showed discrepancies among the independent studies, confirming the lack of reproducibility even when tests were performed in the same laboratory. On the other hand, the use of in silico predictions proved useful for defining a stable phenotype profile among the strains analyzed. These results provide a working framework for defining Micromonospora species at the genomic and phenotypic level.

Blastococcus atacamensis sp. nov., a novel strain adapted to life in the Yungay core region of the Atacama Desert

A polyphasic study was undertaken to establish the taxonomic status of a Blastococcus strain isolated from an extreme hyper-arid Atacama Desert soil. The isolate, strain P6T, was found to have chemotaxonomic and morphological properties consistent with its classification in the genus Blastococcus. It was shown to form a well-supported branch in the Blastococcus 16S rRNA gene tree together with the type strains of Blastococcus capsensis and Blastococcus saxobsidens and was distinguished from the latter, its close phylogenetic neighbour, by a broad range of phenotypic properties. The draft genome sequence of isolate P6T showed 84.6 % average nucleotide identity, 83.0 % average amino acid identity and a digital DNA-DNA hybridisation value of 27.8 % in comparison with the genome sequence of B. saxobsidens DSM 44509T, values consistent with its assignment to a separate species. Based on these data it is proposed that isolate P6T (NCIMB 15090T=NRRL B-65468T) be assigned to the genus Blastococcus as Blastococcus atacamensis sp. nov. Analysis of the whole genome sequence of B. atacamensis P6T, with 3778 open reading frames and a genome size of 3.9 Mb showed the presence of genes and gene clusters that encode for properties that reflect its adaptation to the extreme environmental conditions that prevail in Atacama Desert soils.