Genome Analysis of Fimbriiglobus ruber SP5T, a Planctomycete with Confirmed Chitinolytic Capability

Abstract

Members of the bacterial order Planctomycetales have often been observed in associations with Crustacea. The ability to degrade chitin, however, has never been reported for any of the cultured planctomycetes although utilization of N-acetylglucosamine (GlcNAc) as a sole carbon and nitrogen source is well recognized for these bacteria. Here, we demonstrate the chitinolytic capability of a member of the family Gemmataceae, Fimbriiglobus ruber SP5^T, which was isolated from a peat bog. As revealed by metatranscriptomic analysis of chitin-amended peat, the pool of 16S rRNA reads from F. ruber increased in response to chitin availability. Strain SP5^T displayed only weak growth on amorphous chitin as a sole source of carbon but grew well with chitin as a source of nitrogen. The genome of F. ruber SP5^T is 12.364 Mb in size and is the largest among all currently determined planctomycete genomes. It encodes several enzymes putatively involved in chitin degradation, including two chitinases affiliated with the glycoside hydrolase (GH) family GH18, GH20 family β-N-acetylglucosaminidase, and the complete set of enzymes required for utilization of GlcNAc. The gene encoding one of the predicted chitinases was expressed in Escherichia coli, and the endochitinase activity of the recombinant enzyme was confirmed. The genome also contains genes required for the assembly of type IV pili, which may be used to adhere to chitin and possibly other biopolymers. The ability to use chitin as a source of nitrogen is of special importance for planctomycetes that inhabit N-depleted ombrotrophic wetlands.IMPORTANCE Planctomycetes represent an important part of the microbial community in Sphagnum-dominated peatlands, but their potential functions in these ecosystems remain poorly understood. This study reports the presence of chitinolytic potential in one of the recently described peat-inhabiting members of the family Gemmataceae, Fimbriiglobus ruber SP5^T This planctomycete uses chitin, a major constituent of fungal cell walls and exoskeletons of peat-inhabiting arthropods, as a source of nitrogen in N-depleted ombrotrophic Sphagnum-dominated peatlands. This study reports the chitin-degrading capability of representatives of the order Planctomycetales.

Keywords: Fimbriiglobus ruber; Gemmataceae; Planctomycetes; chitinase; chitinolytic ability; genome annotation.

FIG 1

The relative abundance values represent average values calculated by relating the number of reads assigned to the species Fimbriiglobus ruber (blast sequence identity threshold of 97%) to the total number of SSU rRNA reads retrieved from four experimental incubations of peat samples amended with different biopolymers and from the control incubation without added substrate in the study of Ivanova et al. (19). The relative abundance values represent averages of triplicate data sets (pectin, cellulose, and xylan) or duplicate data sets (chitin and control). A significant difference in Ribo-tag abundances between control and biopolymer-amended samples is indicated by an asterisk (P < 0.01).

FIG 2

Specific detection of planctomycete cells on microparticles of amorphous chitin used in growth experiments as a source of carbon (row 1) or nitrogen (row 2). Phase-contrast images (a frames), the respective epifluorescent micrographs of whole-cell hybridizations with Cy3-labeled probes PLA46-PLA886 (b frames), and DAPI staining (c frames) are shown. Bar, 10 μm.

FIG 3

Suggested scheme of chitin degradation in F. ruber SP5^T. Enzymes are given with gene numbers corresponding to those from the genome of F. ruber SP5^T when possible. Abbreviations: OM, outer membrane; PP, periplasm; CP, cytoplasm; GH18, GH18 family chitinase; GH20, GH20 family β-N-acetylglucosaminidase; ABC, ABC-type sugar transporter.

FIG 4

Hydrolytic activities of F. ruber SP5^T crude cell extract. (A) Hydrolysis of different polysaccharides evaluated by measuring the amounts of total reduced sugars released after treatment. The following substrates were tested: lane 1, microcrystalline cellulose; lane 2, starch; lane 3, carboxymethyl cellulose; lane 4, lichenan; lane 5, laminarin; lane 6, xanthan; lane 7, birchwood xylan; lane 8, beechwood xylan. (B) Hydrolysis of aryl glycosides. The following substrates were tested: lane 1, pNPGal; lane 2, pNPXyl; lane 3, pNPMan; lane 4, pNPGlu. Data represent the means of three separate experiments. The activities of the cell extract with carboxymethyl cellulose (A) and pNPGlu (B) were defined as 100%.