Microvirga sesbaniae sp. nov. and Microvirga yunnanensis sp. nov., Pink-Pigmented Bacteria Isolated from Root Nodules of Sesbania cannabina (Retz.) Poir

Abstract

Four pigment-producing rhizobial strains nodulating Sesbania cannabina (Retz.) Poir. formed a unique group in genus Microvirga in the phylogeny of a 16S rRNA gene and five housekeeping genes (gyrB, recA, dnaK, glnA, and atpD) in a genome analysis, phenotypic characteristics analysis, and chemotaxonomic analysis. These four strains shared as high as 99.3% similarity with Microvirga tunisiensis LmiM8^T in the 16S rRNA gene sequence and, in an MLSA, were subdivided into two clusters, ANI (genome average nucleotide) and dDDH (digital DNA-DNA hybridization) which shared sequence similarities lower than the species thresholds with each other and with the reference strains for related Microvirga species. The polar lipids elucidated that phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and cardiolipin were the main components for strain SWF67558^T and for strain HBU65207^T, with the exception of PC. SWF67558^T and HBU65207^T strains had similar predominant cellular fatty acids, including C16:0, C18:0, summed feature 2, and summed feature8, but with different contents. In addition, all the four novel strains produced pink-pigment, and the main coloring material extract from strain SWF67558^T was identified as zeaxanthin, which presented antioxidant ability and reduction power. With all the phylogenetic and phenotypic divergency, we proposed these pink-pigmented symbiotic bacteria as two novel species, named Microvirga sesbaniae sp. nov. and Microvirga yunnanensis sp. nov., with SWF67558^T (=KCTC82331^T=GDMCC1.2024^T) and HBU65207^T (=KCTC92125^T=GDMCC1.2023^T) as the type strains, respectively.

Keywords: Microvirga; Sesbania cannabina; carotenoids; symbiotic nodules.

Figure 1

Phylogenetic tree based on 16S rDNA sequences (all the aligned sequences trimmed to 1400 bp) showing the relationships of the rhizobial strains isolated from Sesbania and the defined Microvirga species. The reference strains marked in gray letter were not validly published. The tree was constructed by using the maximum likelihood method. The bootstrap values > 50% are indicated in the main nodes in a bootstrap analysis of 1000 replicates. The scale bar represents 1% nucleotide substitutions per site.

Figure 2

Phylogenetic tree based on the 5 concatenated housekeeping genes (atpD, recA, dnaK, gyrB, and glnA) sequences (3944 bp) showing the groups of rhizobial strains isolated from Sesbania species. The reference strains marked in gray letter were not validly published. The phylogeny was constructed using the maximum likelihood method. The bootstrap values greater than 50% are indicated on the main nodes with a bootstrap analysis of 1000 replicates. The scale bar represents 5% substitutions per site.

Figure 3

The heatmap of the distribution of the functional classes of the predicted genes in the genomes from Microvirga sesbaniae SWF66558^T, Microvierga yunnanensis HBU65207^T, Microvirga tunisiensis LmiM8^T, Microvirga calopongonii CCBAU65841^T, and Microvirga lotononidis WSM3557^T, according to the COG category.

Figure 4

Phylogenetic tree based on 120 core genes extracted from genomes showing the relationships of the rhizobial strains isolated from Sesbania with the defined Microvirga species. The reference strains marked in gray letters were not validly published. The tree was constructed with the UBCG software. Bootstrap values (>50) are indicated in the main nodes in a bootstrap analysis of 1000 replicates. The scale bar represents 2% substitutions per site.

Figure 5

Phylogenetic tree based on nodA gene sequences (428 bp) showing the relationships of the Microvirga strains isolated in this study and the other rhizobia. The reference strains marked in gray letter were not validly published. The tree was constructed by using maximum likelihood method. Bootstrap values > 50% are indicated in the main nodes in a bootstrap analysis of 1000 replicates. The scale bar represents 5% nucleotide substitutions per site.

Figure 6

Transmission electron micrograph of the strain Microvirga sesbaniae SWF67558^T cell with its subpolar flagellum its of strain Microvirga sesbaniae SWF67558^T (A) and Microvirga yunnanensis HBU65207^T (B) grown on TY plate.