Klebsiella michiganensis: a nitrogen-fixing endohyphal bacterium from Ustilago maydis

Abstract

Ustilago maydis is a pathogenic fungus in Basidiomycota causing corn smut disease. A strain of U. maydis YZZF202006 was isolated from the tumor of corn smut collected from Jingzhou city in China. The intracellular bacteria were confirmed inner hyphal of the strain YZZF202006 by PCR amplification and fluorescence in situ hybridization (FISH) and SYTO-9. An endohyphal bacterium YZUMF202001 was isolated from the protoplasts of the strain YZZF202006. It was gram-negative, short rod-shaped with smooth light yellow colony. The endohyphal bacterium was genomic evidenced as Klebsiella michiganensis on the basis of average nucleotide identity (ANI) analysis and the phylogram. Then K. michiganensis was GFP-Labeled and reintroduced into U. maydis, which confirmed the bacterium can live in hyphae of U.maydis. The bacterium can grow on N-free culture media. Its nitrogenase activity was reached av. 646.25 ± 38.61 nmol·mL^{- 1}·h^{- 1} C₂H₄ by acetylene reduction assay. A cluster of nitrogen fixation genes (nifJHDKTXENXUSVWZMFLABQ) was found from its genome. The endohyphal K. michiganensis may play an important role to help nitrogen fixation for fungi in the future.

Keywords: Endohyphal bacterium; Klebsiella michiganensis; Nitrogen fixation; Ustilago maydis.

Fig. 1

Fluorescence in situ hybridization (FISH) assay for Ustilago maydis YZZF202006 and phylogram of endohyphal bacterium YZUMF202001 (a) Cells observed in bright field; (b) Red fluorescent cells hybridized with Cy3; (c) Green fluorescent cells hybridized with FITC; (d) Stains of YZZF202006 with SYTO-9 green-fluorescent nucleic acid stains; FN: Fungal nucleus; BD: Bacterial DNA; (e): Maximum Likelihood tree for endohyphal bacterium Klebsiella sp. YZUMF202001 based on partial 16 S rRNA region

Fig. 2

(a): Colony of endohyphal bacterium strain YZUMF202001 grown on LA meidium. (b): Bacterial cells of strain YZUMF202001 obtained by scanning electron microscope (SEM) observation. (c): Genomic phylogram of strain YZUMF202001 compared with relevant strains. (d): Heatmap of ANI analysis based on the genome sequences of 17 Klebsiella strains

Fig. 3

(a) Preliminary nitrogen fixation capacity test with nitrogen-free culture medium (left: strain YZUMF202001; right: strain DH5α), (b) The value of nitrogenase activity determined by acetylene reduction assay (left: strain DH5α; right: strain YZUMF202001), (c) nitrogen fixation gene cluster of strain YZUMF202001

Fig. 4

GFP-labeled YZUMF202001 colonizing YZZF202006 was observed in fluorescence (a) and white light (b) mode, scale bar represents 25 μm