Kolteria novifilia, a novel planctomycetotal strain from the volcanic habitat of Panarea divides by unusual lateral budding

Abstract

Members of the phylum Planctomycetota are ubiquitous bacteria that play important roles in the global carbon and nitrogen cycle. In this study, we sampled the shallow-sea hydrothermal vent system close to Panarea Island, Italy, and analyzed the bacterial diversity in this habitat using a cultivation-independent amplicon sequencing approach. Motivated by the observed abundance of members of the phylum Planctomycetota, we employed cultivation conditions that facilitate the enrichment of planctomycetes and isolated strain Pan216^T. This strain shows a rarely observed type of cell division-lateral budding. Based on 16S rRNA gene- and multi-locus sequence analyses, its phylogenetic position in the phylum Planctomycetota appears vague. Strain Pan216^T clustered between the different described families in the class Planctomycetia. The novel isolate shares the highest sequence identity (85.4%) of the 16S rRNA gene with Thermostilla marina SVX8^T, indicating that this strain belongs to a novel family. In addition to its uncommon cell division mode, Pan216^T cells are pill-shaped and covered by a putative outer surface layer. Genomic analyses of strain Pan216^T revealed many giant genes, putative S-layer protein-encoding genes, and only a limited set of canonical cell division genes. Based on the results of the polyphasic analysis, we conclude that strain Pan216^T constitutes a novel family within the phylum Planctomycetota, for which we propose the name Kolteriaceae fam. nov. The novel species Kolteria novifilia gen. nov., sp. nov. is represented by the type strain Pan216^T (= DSM 100414^T = CECT 9536^T).

Importance: We describe a novel family of the underrepresented bacterial phylum Planctomycetota that divides by unusual lateral budding. Our strain is the only validly described species that uses this mode of cell division. Furthermore, it represents the only planctomycete outside of the anammox bacteria that has an S-layer-like structure. Taken together, the novel family shows a novel mechanism of cell division that could only be studied in this species.

Keywords: 16S rRNA; Panarea; V3 region; binary fission; budding; cell division; planctomycetes; surface layer.

Fig 1

Sampling site and bacterial community composition in the hydrothermal vent system close to Panarea using 16S rRNA (V3) amplicon sequencing. (A) Sampling site in Panarea (Italy). The map was created with material from OpenMapTiles (obtained via https://satellites.pro/). (B) Diving map of the sampling site. The map is a hand-drawn illustration. (C). Bacterial community composition in surface seawater and in the transition zone of a hydrothermal vent was analyzed using fractions obtained by filtration with glass fiber (2.7 µm) and polycarbonate (0.22 µm) filters (n = 2).

Fig 2

Maximum likelihood phylogenetic analysis. Phylogenetic trees showing the position of strain Pan216^T. The 16S rRNA gene sequence- (A) and multilocus sequence analysis (MLSA)-based phylogeny (B) was computed as described in the Materials and Methods section. Bootstrap values after 1,000 re-samplings (16S rRNA gene)/500 re-samplings (MLSA) are given at the nodes (in %). Three 16S rRNA genes of bacterial strains outside of the phylum Planctomycetes, but part of the PVC superphylum, were used as outgroup in the 16S rRNA gene sequence-based tree (NCBI acc. no. AJ229235, CP010904.1 and NR_027571). The genomes of three members of the class Phycisphaerae, Phycisphaera mikurensis FYK2301M01^T (acc. no. AP012338.1), Poriferisphaera corsica KS4^T (acc. no. CP036425.1), and Algisphaera agarilytica 06SJR6-2^T (acc. no. GCA_014207595.1), were used as outgroup in the MLSA-based tree.

Fig 3

Cell morphology and cell size of Pan216^T. The morphology and average cell size of strain Pan216^T were determined by light microscopy with differential interference contrast (DIC, A and C) and phase-contrast (Phaco, B and D) illumination. Cells of strain Pan216^T are of oblong, cylindrical morphology and do not grow in clusters under aerobic conditions. Cell size was determined by measuring 100 individual cells (E). Scale bar 2 µm.

Fig 4

Morphological characteristics of strain Pan216^T. (A) SEM overview of Pan216^T showing an oblong-shaped cell. (B) TEM image of a flagellated (black arrowhead) Pan216^T cell. (C) SEM image of the surface of Pan216^T depicting an unusual cell surface. (D) TEM image of the outer surface of Pan216^T revealing an additional structure (white arrowhead) surrounding the cell. (E) Thin section of a high-pressure frozen and freeze-substituted Pan216^T cell. The inset gives a detailed view of the membrane organization, visualizing the cytoplasm (CP), the inner membrane (IM), the periplasm (PP), the outer membrane (OM), and a putative surface layer (S, white arrowhead). (F) SEM micrograph depicting the novel cell division by lateral budding of Pan216^T. Scale bars: A, B: 1 µm; C, D, and E: 0.1 µm; F: 0.2 µm.

Fig 5

Lateral budding is a novel mechanism of cell division in Planctomycetota. (A) Time series over 3 h (eight representative images) of the polar budding of Planctopirus limnophila. (B) Time series over 6 h (eight representative images) of budding of Gemmata obscuriglobus. (C) Time series over 3 h (eight representative images) of lateral budding of Pan216^T shows the formation of the daughter cell at the midcell of the elongated mother cell. Scale bar A, B: 1 µm. (For details, see Movies S1 to S3).

Fig 6

Circular genome plot of strain Pan216^T. The genome of strain Pan216^T is visualized by six individual circles. The innermost circle depicts the G + C skew in light and dark gray. The second circle shows the color-coded COG (Cluster of Orthologous Groups) categories of the encoded proteins. Circle three illustrates the core genome of strain Pan216^T determined by the method of reciprocal best alignment. The given color code illustrates whether a gene belongs to the general Planctomycetes core genome of all analyzed Planctomycetes (green), to the core genome of Planctomycetes dividing by budding (black), or by binary fission (blue). The fourth circle holds those genes predicted to be in genomic islands. Circle five depicts “giant genes”—a term describing all genes longer than 5 kB. The outermost circle contains genes partaking in cell division, sulfur metabolism, and assimilatory nitrate reduction.