Bacterial genome-encoded ParMs

Abstract

ParMs generally exist on low-copy number plasmids where they contribute to plasmid segregation and stable inheritance. We carried out bioinformatics analysis, which indicated that ParM genes are not only confined to plasmids but are also occasionally found on genomes. Here we report the discovery and characterization of two chromosome-encoded ParMs (cParMs) from the genomes of Desulfitobacterium hafniense (Dh-cParM1) and Clostridium botulinum (Cb-cParM). Both cParMs form filaments, exhibit nucleotide hydrolysis, and possess characteristic ParM subunit structures. Dh-cParM1 forms single and tightly coupled double filaments and is highly conserved on the chromosomes of five of six Desulfitobacterium species. Interestingly, these bacteria have not been reported to harbor plasmids. Cb-cParM possesses unique properties. Its filaments were stable after nucleotide hydrolysis and Pi release, and its ParR (Cb-cParR) did not affect the initial phase of Cb-cParM polymerization but displayed properties of a depolymerization factor for mature filaments. These results indicate functional, polymerizing ParMs can be encoded on genomes, suggesting that ParM roles may extend to other functions beyond plasmid segregation.

Keywords: DNA segregation; ParCMR system; ParM; nucleotide hydrolysis; plasmid.

Figure 1

Putative cParMR cassettes from different bacterial species. Color coding: cParMs (blue), cParRs (red), and unrelated genes (green). Operons that contain cParMs or cParR are boxed in orange. The directions of the arrows show the direction of transcription. Three putative cParMR systems are encoded on the Desulfitobacterium hafniense Y51 chromosome (accession numbers: AP008230.1 or NC_007907.1, 5,727,534 bp): Dh-cParMR-1 (Dh-cParM1, WP_011460071.1; Dh-cParR1, WP_011460070.1); Dh-ParMR-2 (Dh-cParM2, WP_011461902.1; Dh-cParR2, WP_041272685.1); and Dh-cParMR-3 (Dh-cParM3, WP_011461997.1, Dh-cParR3, WP_011461998.1). Natranaerobius thermophilus strain JW/NM-WN-LF (accession number: NZ_CP144221.1, 3,137,840 bp) also encodes 3 cParMR systems - Nt-cParMR-1 (Nt-cParM1, WP_148206872.1; Nt-cParR1, WP_012449064.1), Nt-cParMR-2 (Nt-cParM2, WP_012448769.1) and Nt-cParMR-3 (Nt-cParM3, WP_012446843.1; Nt-cParR3, WP_012446842.1). This Natranaerobius thermophilus strain has two associated plasmids, pNTHE01 and pNTHE02 (accession numbers and sizes: NC_010715.1, 17,207 bp and NC_010724.1, 8689 bp, respectively). Bacillus tropicus strain FDAARGOS_920 (accession number: NZ_CP065739.1, 5,298,747 bp) encodes two putative cParMR systems Bt-cParMR-1 (Bt-cParM1, WP_001968526.1; Bt-cParR1, WP_129075283.1) and Bt-cParMR-2 (Bt-cParM2, WP_000025611.1; Bt-cParR2a, WP_001978111.1; Bt-cParR2b, WP_001257752.1). Other bacterial genomes have single cParMR cassettes, such as Caldicellulosiruptor saccharolyticus DSM 8903 (accession number: NC_009437.1, 2,970,275 bp): Cs-cParMR cassette (Cs-cParM, WP_011916932.1; Cs-cParR, WP_011916933.1), Burkholderia multivorans ATCC 17616 chromosome 3 (accession number: CP000870.1, 919,806 bp): Bm-cParMR (Bm-cParM, ABX19247.1; Bm-cParR, ABX19246.1) and Moorella thermoacetica strain 39073-HH (accession number: CP031054.1, 2,645,661 bp): Mt-cParMR cassette (Mt-cParM, WP_011391888.1; Mt-cParR, WP_053104303.1) and no associated plasmids (29, 76).

Figure 2

Variations in helical parameters of the two types of Dh-cParM1 filaments.A, electron micrograph of a negatively stained sample displaying the two types of Dh-cParM1 filament morphologies – single and coupled filaments. B, extraction of two distinct filament types from the same micrograph. The single and coupled filaments were extracted and straightened for helical parameter determinations. Top: single filament and Lower: coupled filament. C, the averaged diffraction pattern of single filament morphology from negatively stained micrographs (left image) and from reconstructed 3D structure (right image). D, 2D classification of Dh-cParM1 extracted particles of single filaments from cryoEM micrographs. E, 2D class averages of the coupled filament morphology from cryoEM micrographs. Detailed analysis showed these structures comprise two single filaments lying side by side, which sometimes taper at the ends (Fig. S6).

Figure 3

Dh-cParM1 shows a double-helical single filament structure.A, protomers fit into the 4 Å resolution density map of Dh-cParM1 single filament. B, a short segment of the Dh-cParM1 filament model. Protomer structures are shown with different colors in the filament. C, a dimer of Dh-cParM1 protomer showing the longitudinal contacts D, Dh-cParM1 monomer displaying its four subdomains (numbered) similar to actin and other ParMs. The nucleotide which is likely to be ADP from hydrolysis of ATP is indicated in magenta and the magnesium ion in red.

Figure 4

Gene clusters of Clostridium species containing cParMR system. Genes within 5000 bp of cParM are depicted using clinker & cluster map (55). Clostridium species containing homologous cParM sequences (identity cutoff = 50%) are aligned. Genes are depicted as arrows. Conserved genes, cParM (light green), cParR (lime green), putative replication initiation factor (magenta), sporulation-specific N-acetylmuramoyl-L-alanine amidase (orange), dihydrodipicolinate reductase (purple), and Cro/CI family transcriptional regulator-like protein (blue), are colored. Annotations are from GenBank records. cparC is not displayed, since a conserved cparC sequence was not identified among strains.

Figure 5

Characterization of the Cb-cParCMR system.A, cParCMR system present on the genome of C. botulinum strain Bf. Cb-cParC includes three palindromic repeats. The red arrows above cParM and cParR indicate the direction of the transcription based on the operon analysis (Fig. 1). cparC is positioned after cParR sequence. HP represents hypothetical proteins on both sides of the cParCMR operon. B, electrophoretic mobility shift assay of Cb-cParC with 10 × to 1000 × molar excess of cParR. C, light scattering time courses of Cb-cParM polymerization. 10 μM Cb-cParM green, ATP; black, GTP; red, ADP; blue, GDP. D, Pi release from 10 μM Cb-cParM, green with ATP and black with GTP. Three measurements for each nucleotide are superimposed. E, light scattering time courses of Cb-cParM polymerization in the presence of Cb-cParR, blue, with ATP; black, with GTP; Corresponding time courses without Cb-cParR, red, with ATP; green, with GTP.

Figure 6

Cryo-EM images of Cb-cParM filaments. A–C, cryo-EM images of Cb-cParM filament formed with ATP, with GTP, or with GTP and short incubation time, respectively. Scale bars = 100 nm. D–F, averaged images after a 2D classification. The images with GTP were classified into two classes: class 1, similar to those with ATP; and class 2, similar to those with GTP and short incubation time.

Figure 7

Maps and Cb-cParM structural states with bound nucleotides. The nucleotides are indicated in red and blue colors. A and E, with ATP. B and F, class 1 with GTP. C and G. class 2 with GTP. D and H. with GTP and short incubation time. The structures with ATP (A and E) were almost identical to those of class 1 with GTP (B and F). The gamma phosphate could not be observed in A and B (insets), showing that the binding nucleotides were ADP and GDP, respectively. The model for class 2 with GTP (C and G) and the model with GTP and a short incubation time (D and H) were similar to each other.

Figure 8

Identification of rigid bodies in Cb-cParM.A, the crystal structure is without nucleotide. The side chains of R204D, K230D, and N234D are shown in red and are labeled. B, Cryo-EM structure with bound GDP (Fig. 7B). Two rigid bodies were identified by comparing these structures (ID rigid body, black, and OD rigid body, magenta). C, the bound GDP and Mg²⁺ connected the two rigid bodies (ID rigid body in black, OD rigid body in magenta, and the rest of the protein in green). Possible hydrogen bonds corresponding to GDP were determined using UCSF chimera [78] and presented as gray lines and possible salt bridges with the Mg²⁺ presented by red dotted lines, although additional hydrogen bonds via water molecules may exist. D, Models for the class 2 state with GTP (orange) and the short incubation time with GTP (brown) were aligned by the ID rigid body and superposed on the model with GDP (green, black, and magenta). The nucleotide-binding cleft was more open in the models for the class 2 with GTP and short incubation time with GTP relative to the GDP state. The orange arrow indicates the direction of domain movement in the open conformation.

Figure 9

Structural shift in Cb-cParM filament strands.A and B, two subunits in one strand (A: front view and B: top view). The ID rigid bodies of the lower subunit were aligned with each other. GDP state: green and black, class 2 with GTP: orange and yellow, short incubation with GTP: brown and pink. The interactions between the subunits appear similar in the three states except for the upper subunit position, which was slightly shifted in the GDP state compared to the other two states. The closure of the cleft in the GDP state may explain this difference because the closure of the cleft can push the upper subunit leftward. A red arrow indicates the direction of the shift of the upper subunit. C–E, relative positions of the two strands. C, the model with GDP is presented as a surface model in green and light gray. Ribbon models for class 2 with GTP (orange and yellow) and the short incubation time with GTP (brown and pink) are superposed. The ID rigid bodies of the center subunits are aligned with each other. D, 180° rotated view of C. The opposite strand position in the GDP state was completely different from that in the other two states (compare the gray surface to the cartoon), indicating a substantial strand movement after phosphate release. E, top view of two adjacent subunits in the different strands. GDP state: green and black, class 2 with GTP: orange and yellow, short incubation with GTP: brown and pink. The ID rigid bodies of the lower subunit (green, orange, and brown) were aligned with each other. A red arrow indicates the direction of the shift in D and E from the class 2 state with GTP to the GDP state. F, inter-strand interactions in the Cb-cParM filament model with GDP. Atoms from the center molecule (green) in contact with the other strand (black) are presented in space-filling representation. 176 contacts are observed. G, inter-strand interactions in the model for class 2 with GTP. 36 contacts were observed. H, the strands of the model with GDP (light gray) were replaced by the strands of the model for class 2 with GTP (orange and yellow). The strands were aligned by the molecules indicated by black arrows. The clashing atoms were presented in space-filling representation. The contacts and clashes were identified by ChimeraX (73).