Genomic and physiological characterization of Novosphingobium terrae sp. nov., an alphaproteobacterium isolated from Cerrado soil containing a mega-sized chromid

Abstract

A novel bacterial strain, designated GeG2^T, was isolated from soils of the native Cerrado, a highly biodiverse savanna-like Brazilian biome. 16S rRNA gene analysis of GeG2^T revealed high sequence identity (100%) to the alphaproteobacterium Novosphingobium rosa; however, comparisons with N. rosa DSM 7285^T showed several distinctive features, prompting a full characterization of the new strain in terms of physiology, morphology, and, ultimately, its genome. GeG2^T cells were Gram-stain-negative bacilli, facultatively anaerobic, motile, positive for catalase and oxidase activities, and starch hydrolysis. Strain GeG2^T presented planktonic-sessile dimorphism and cell aggregates surrounded by extracellular matrix and nanometric spherical structures were observed, suggesting the production of exopolysaccharides (EPS) and outer membrane vesicles (OMVs). Despite high 16S rDNA identity, strain GeG2^T showed 90.38% average nucleotide identity and 42.60% digital DNA-DNA hybridization identity with N. rosa, below species threshold. Whole-genome assembly revealed four circular replicons: a 4.1 Mb chromosome, a 2.7 Mb extrachromosomal megareplicon, and two plasmids (212.7 and 68.6 kb). The megareplicon contains a few core genes and plasmid-type replication/maintenance systems, consistent with its classification as a chromid. Genome annotation shows a vast repertoire of carbohydrate-active enzymes and genes involved in the degradation of aromatic compounds, highlighting the biotechnological potential of the new isolate. Chemotaxonomic features, including polar lipid and fatty acid profiles, as well as physiological, molecular, and whole-genome comparisons showed significant differences between strain GeG2^T and N. rosa, indicating that it represents a novel species, for which the name Novosphingobium terrae is proposed. The type strain is GeG2^T (= CBMAI 2313^T = CBAS 753 ^T).

Keywords: Cerrado; Chromid; Cultivation; Novosphingobium; Soils; Sphingomonadales.

Fig. 1

Transmission electron micrograph of strain GeG2^T cells showing intracytoplasmic electron-dense granules (A and B) and scanning electron micrographs of strain GeG2^T cells surrounded by amorphous polymeric matrix, with the characteristic aspect of exopolysaccharides (EPS) (C) and presenting smooth or granular surfaces (D). Magnifications and scale bars are indicated under each micrograph. IG, intracytoplasmic granule. CM, cytoplasmic membrane. OM, outer membrane. P, peptidoglycan layer

Fig. 2

Scanning electron micrographs of strain GeG2^T cultures presenting macroscopic flocks grown in MM for 14 days. Cells presenting granular surfaces are indicated by white arrows and spherical structures resembling large vesicles are indicated by yellow arrows. Magnifications and scale bars are indicated under each micrograph

Fig. 3

Circular maps and genetic features of the chromosome, chromid, and plasmids of strain GeG2^T. From outside to center: forward CDS (green), reverse CDS (light blue), genomic islands (brown), transposases (pink), tRNAs (purple), GC content, and GC skew (red and dark blue). Replicons are not shown to scale

Fig. 4

Circular visualization of whole-genome alignment between strain GeG2^T (chromosome in grey and chromid in orange) and N. rosa NBRC 15208^T reference genome (in blue). Only genome alignment blocks with at least 1,000 nucleotides and 90% identity are shown

Fig. 5

Comparison of phylogenetic trees of 40 Novosphingobium species based on core genes (left) and 16S rRNA genes (right). The trees show phylogenetic positions of strain GeG2^T and the 39 Novosphingobium genomes selected based on the highest ANI values to GeG2^T. A total of 178 core genes were identified and used to construct a maximum-likelihood phylogenetic tree using the M1CR0B1AL1Z3R web server [41]. The 16S rRNA gene-based tree was inferred from Global Blast Distance Phylogeny (GBDP) distances by the TYGS server [38]. Branch lengths are displayed in the bottom scales and only the bootstrap support values below 100% are indicated in the core genes tree. Sphingomonas paucimobilis NCTC 11030^T was used as an outgroup

Fig. 6

COG functional categories (A) and carbohydrate-active enzymes (CAZymes) (B) predicted in each replicon of strain GeG2^T genome. COG categories with significantly different proportions as shown by the two-proportions Z-test are indicated with *. D: cell cycle control, cell division, chromosome partitioning; M: cell wall/membrane/envelop biogenesis; N: cell motility; O: post-translational modification, protein turnover, chaperone functions; T: signal transduction mechanisms; U: intracellular trafficking, secretion, and vesicular transport; V: defense mechanisms; Z: cytoskeleton; A: RNA processing and modification; B: chromatin structure and dynamics; J: translation, ribosomal structure, and biogenesis; K: transcription; L: replication and repair; C: energy production and conversion; E: amino acid metabolism and transport; F: nucleotide metabolism and transport; G: carbohydrate metabolism and transport; H: coenzyme metabolism and transport; I: lipid metabolism and transport; P: inorganic ion transport and metabolism; Q: secondary metabolites biosynthesis, transport, and catabolism; S: function unknown. GT: glycosyl transferases; GH: glycosyl hydrolases; CE: carbohydrate esterases; CBM: carbohydrate-binding module; AA: enzymes for the auxiliary activities; PL: polysaccharide lyases

Fig. 7

Schematic representation of sulfur metabolism genes identified in strain GeG2^T chromosome (in blue) and chromid (in orange). Genes associated with the assimilation of environmental sulfur compounds that were not detected in the genome of strain GeG2^T but can be found in other Novosphingobium species [101] are represented in grey. APS, adenosine phosphosulfate; PAPS, phosphoadenosine phosphosulfate

Fig. 8

Pathways associated with the degradation of aromatic compounds, obtained with the KEGG Mapper tool, indicating genes detected in the chromosome (in blue), the chromid (in orange), or in both replicons (in pink) of strain GeG2^T genome. 1.1.1.90: aryl-alcohol dehydrogenase; 1.2.1.28: benzaldehyde dehydrogenase (xylC); 1.14.12.10: benzoate/toluate 1,2-dioxygenase subunit alpha (benA-xylX); 1.3.1.25: dihydroxycyclohexadiene carboxylate dehydrogenase (benD-xylL); 1.13.11.1: catechol 1,2-dioxygenase (catA); 5.5.1.1: muconate cycloisomerase (catB); 5.3.3.4: muconolactone D-isomerase (catC); 3.1.1.24: 3-oxoadipate enol-lactonase (pcaD); 1.13.11.2: catechol 2,3-dioxygenase (dmpB-xylE); 5.3.2.6: 4-oxalocrotonate tautomerase (praC-xylH); 4.2.1.80: 2-keto-4-pentenoate hydratase (mhpD)