Isolation, morphology, and phylogenetic analysis of P. heterotrophicis ZRK32.

(A) Diagram showing the strategy used to isolate the Planctomycetes bacteria. (B, C) TEM observation of strain ZRK32. (D) TEM observation of ultrathin sections of cells from strain ZRK32. Abbreviations: CM, outer membrane; Pi, cytoplasm; R, ribosome; N, nucleoid; ICM, cytoplasmic membrane; Py, periplasm; V, vesicle-like organelles. (E) Phylogenetic analysis of strain ZRK32. Phylogenetic placement of strain ZRK32 within the phylum Planctomycetes, based on almost complete 16S rRNA gene sequences. The NCBI accession number for each 16S rRNA gene is indicated after each corresponding strain’s name. The tree was inferred and reconstructed using the maximum likelihood criterion, with bootstrap values (%) > 80; these are indicated at the base of each node with a gray dot (expressed as a percentage from 1,000 replications). The 16S rRNA gene sequence of Actinoplanes derwentensis LA107T was used as the outgroup. Bar, 0.1 substitutions per nucleotide position.

Growth assay and transcriptomic analysis of P. heterotrophicis ZRK32 strains cultivated in basal medium and rich medium.

(A) Growth curves of ZRK32 strains cultivated in basal medium and rich medium. (B) Diagram of the TCA cycle. The gene numbers shown in this schemeatic are the same as those shown in panel C. Transcriptomics-based heat map showing the relative expression levels of genes associated with the TCA cycle (C), NADH-quinone oxidoreductase (D), and flagellar assembly (E) of strain ZRK32 cultivated in rich medium (Rich) compared with strain cutivated in basal medium (Basal). The numbers in panels C, D, and E represent the fold change of gene expression (by using the log2 value).

The mode of cell division utilized by P. heterotrophicis ZRK32.

(A) Ultrathin TEM sections showing the process of polar budding division (panels 1-8) in strain ZRK32. Images representing the different phases of cell division are shown. Scale bars are 200 nm in panels A and B. (B) The proposed model of cell division of strain ZRK32 based on the TEM observation shown in panel B. The numbers in panels A and B correspond to the same phase of division. (C) Transcriptomics-based heat map showing the differentially expressed genes that encode different key proteins associated with cell division in strain ZRK32. The numbers in panel A represent the fold change of gene expression (by using the log2 value).

Nitrogen metabolism assays of P. heterotrophicis ZRK32.

(A) Growth curves of ZRK32 strains cultivated in the rich medium alone and cultivated in rich medium supplemented with either 20 mM NO3-, 20 mM NH4+, or 20 mM NO -. (B) The dynamics of concentrations of NO3-, NH4+, and NO2- in strains of ZRK32 cultivated in the rich medium supplemented with 20 mM NO3-. (C) The dynamics of concentrations of NO3-, NH4+, and NO2- in strains of ZRK32 cultivated in the rich medium supplemented with 20 mM NH +. (D) The predicted nitrogen metabolism pathway of strain ZRK32. Abbreviations: NapA, periplasmic nitrate reductase; NapB, periplasmic nitrate reductase, electron transfer subunit; NirB, nitrite reductase (NADH) large subunit; NirD, nitrite reductase (NADH) small subunit; GlnA, glutamine synthetase; GLT1, glutamate synthase. Transcriptomics-based heat map showing the relative expression levels of genes associated with nitrogen metabolism (E), the TCA cycle (F), NADH-quinone oxidoreductase (G), and flagellar assembly (H) in strains of ZRK32 cultivated in the rich medium supplemented with different inorganic nitrogen sources (20 mM NO -, 20 mM NH + or 20 mM NO -) compared with strains cultivated in the rich medium alone. “Rich” indicates rich medium. “NO3-, NH4+, and NO -” indicate rich medium supplemented with 20 mM NO -, 20 mM NH +, and 20 mM NO -, respectively. The numbers in panels E, F, G, and H represent the fold change of gene expression (by using the log2 value).

Observation and functional assay of the chronic bacteriophage induced by NO3- or NH4+ from P. heterotrophicis ZRK32.

(A) TEM observation of phages extracted from the cell suspensions of ZRK32 strains that cultured in either the rich medium alone, or rich medium supplemented with 20 mM of either NO3- or NH4+. (A, Panel I) No phage-like particles were observed in the cell suspensions from the ZRK32 strain cultured in the rich medium. (A, Panels II and III) Hexagonal phages (indicated with yellow arrows) observed in the cell suspensions from the ZRK32 strains cultured in the rich medium supplemented with 20 mM of either NO3- or NH4+. The scale bar is 200 nm. (B) A diagram showing the genomic composition of Phage-ZRK32. The arrows represent different ORFs and the direction of transcription. The main putative gene products of this phage are shown, and the numbers in brackets indicate the numbers of amino acids. Hypothetical proteins are indicated by gray arrows, structural modules are indicated by green arrows, nucleotide metabolism is indicated by blue-gray arrows, the replication module is indicated by gold arrows, and AMGs are indicated by red arrows. The size of the phage genome is shown beside the gene cluster. (C) Bacterial growth curve showing the growth rate of strains of Pseudomonas stutzeri 273 cultivated in either oligotrophic medium, oligotrophic medium supplemented with Phage-ZRK32, oligotrophic medium supplemented with 20 mM NO3-, or oligotrophic medium supplemented with 20 mM NO3- and Phage-ZRK32. (D) Bacterial growth curve showing the growth rate of strains of Pseudomonas stutzeri 273 cultivated in either oligotrophic medium, oligotrophic medium supplemented with Phage-ZRK32, oligotrophic medium supplemented with 20 mM NH4+, or oligotrophic medium supplemented with 20 mM NH4+ and Phage-ZRK32. “Oligo” indicates oligotrophic medium.