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27

562

Table 1. Genome relatedness indexes between strain SS37A-ReT and Methylocystis and Methylosinus species

563

ANI and dDDH values are shown in the upper and lower triangles, respectively. Accession number are presented in parentheses.

Strain

10

1. SS37A-ReT (GCA_027925385.1)

100

82.3

82.5

80.0

79.2

80.0

78.6

80.0

79.2

79.4

2. Methylocystis parva OBBPT (GCA_000283235.1)

24.8

100

82.2

79.7

78.9

79.8

78.7

79.8

78.7

78.9

24.9

24.3

100

80.0

79.0

80.1

78.9

79.9

79.1

78.7

4. Methylocystis rosea SV97T (GCA_000372845.1)

22.3

21.6

21.9

100

78.4

91.9

78.4

92.4

78.7

78.1

5. Methylocystis heyeri H2T (GCA_004802635.2)

22.5

21.2

21.6

20.8

100

78.7

78.7

78.3

78.5

78.2

6. Methylocystis hirsuta CSC1T (GCA_003722355.1)

22.0

21.7

22.2

47.2

21.9

100

78.3

94.1

78.6

78.3

7. Methylocystis bryophila H2sT (GCA_027925445.1)

21.7

21.3

21.9

21.5

21.4

21.5

100

78.6

78.7

78.4

8. Methylocystis silviterrae FST (GCA_013350005.1)

21.8

21.7

21.8

49.1

21.1

56.0

21.0

100

78.7

78.5

9. Methylosinus trichosporium OB3bT (GCA_002752655.1)

21.7

21.4

21.6

21.4

21.3

21.5

21.0

21.6

100

83.1

10. Methylosinus sporium 5T (GCA_009811675.1)

22.0

21.3

21.2

20.6

21.2

20.6

20.9

20.5

25.4

100

3. Methylocystis echinoides LMG27198T

(BSEC01000001–BSEC01000011)

564

28

565

566

567

568

569

570

Table 2. Cellular fatty acids composition of SS37A-ReT and type strains of

Methylocystis and Methylosinus

Strains: 1, strain SS37A-ReT; 2, Methylocystis parva OBBPT [25]; 3, Methylocystis echinoides IMET10491T [60]; 4, Methylocystis

rosea SV97T [26]; 5, Methylocystis heyeri H2T [23]; 6, Methylocystis hirsuta CSC1T [26]; 7, Methylocystis bryophila H2sT [25]; 8,

Methylocystis silviterrae FST [26]; 9, Methylosinus trichosporium OB3bT [61]; 10, Methylosinus sporium 5T [61].Values are

percentages of the total fatty acids. −, Not detected; tr, Trace component.

Fatty acid

10

iso-C14:0

0.1

iso-C15:0

0.2

0.5

anteiso-C15:0

3.5

C15:0

tr

0.7

iso-C16:0

1.4

10-Methyl C16:0

3.5

C16:1ω9c

0.6

C16:1ω9t

2.3

C16:1ω8c

29.0

C16:1ω7c

1.2*

0.5

0.8

3.4

15.4

14.2

9.3

C16:1ω6c

0.5

2.8

C16:1ω5t

C16:0

4.7

0.2

0.3

0.9

1.2

0.3

2.4

0.7

2.2

iso-C17:0

0.2

0.2

0.1

0.6

0.2

0.2

0.3

0.2

C17:0

0.4

tr

0.3

iso-C18:0

3.6

anteiso-C17:0

C17:1ω8c

C17:0 cyclo

571

572

573

C18:2ω7, 12c

8.0

22.7

7.8

C18:2ω6, 12c

22.1

4.4

(tr)†

C18:2ω6, 9c

9.0

(tr)†

C18:1ω9c

0.2

C18:1ω9t

1.5

14.7

C18:1ω8c

27.3‡

45.5

51.9

74.8

32.0

71.1

53.1

74.5

67.5

C18:1ω8t

51.8‡

0.8

C18:1ω7c

23.7

18.1

23.7

10.9

26.1

19.3

24.7

13.1

78.7

C18:1ω7t

4.6

C18:1ω5c

0.3

C18:0 2-OH

0.3

C18:0

1.4

0.2

0.7

0.3

0.8

1.2

C19:0 branched

0.8

C19:0 cyclo

0.2

C20:0

0.1

* The fatty acid was assigned to C16:1ω7c/ C16:1ω9t as “summed feature” in the report of the MIDI System.

† Bowman et al. [61] showed the fatty acid as “C18:2ω6”.

‡ The fatty acids were identified by the analysis of the DMDS derivatives (Fig. S9)

29

574

Table 3. Phenotypic characters of type strains of Methylocystis and Methylosinus species

575

576

577

Strains: 1, strain SS37A-ReT; 2, Methylocystis parva OBBPT [20, 57, 60]; 3, Methylocystis echinoides IMET10491T [20, 60, 62, this study]; 4, Methylocystis rosea SV97T [22, 26]; 5,

Methylocystis heyeri H2T [23]; 6, Methylocystis hirsuta CSC1T [24, 26]; 7, Methylocystis bryophila H2sT [25, this study]; 8, Methylocystis silviterrae FST [26]; 9, Methylosinus

trichosporium OB3bT [20, 57]; 10, Methylosinus sporium 5T [20, 57, 61]. +, Positive; -, negative; NR, Not reported.

Characteristic

10

Cell shape

Rods

Reniform,

coccobacilli

Straight,

Reniform,

coccobacilli,

Rods

rods

polymorphic or

regularly curved

Small curved

Dumbbell

coccoids,

short rods

rods, ovoids

Small curved

Pear

Vibrioids,

coccoids/rods

(pyriform)

rods

Cell width (µm)

1.1

0.3–0.5

0.6

0.8–1.1

0.8–1.2

0.3–0.6

0.9–1.4

0.5–0.7

0.5–1.5

0.5–1.0

Cell length (µm)

2.7

0.5–1.5

0.8–1.2

1.1–2.5

1.4–4.0

0.7–1.0

1.8–3.4

1.7–3.4

2.0–3.0

1.5–3.0

Motility

Single polar

NR

NR

NR

NR

Polar tuft

Polar tuft

Lipid cyst formation

NR

NR

Exospore formation

NR

NR

Rosette formation

NR

Temperature (ºC)

25–30

30

30

27

25

30

25–30

25–30

30

30

pH

7.0–8.0

7.0

7.0

5.5–9.0

5.8–6.2

7.0

6.0–6.5

6.0–6.5

6.5–7.0

6.5–7.0

Growth at 37 ºC

sMMO

Predominant fatty acids

C18:1ω8t

C18:1ω8c

C18:1ω8c

C18:1ω8c

C18:1ω8c

C18:1ω8c

C18:1ω8c

C18:1ω8c

C18:1ω7c

G+C content (mol%)*

63.2

63.9

63.9

62.5

62.4

63.2

62.6

65.9

65.2

Flagellum

Optimum growth

condition:

578

C18:1ω8c

C16:1ω8c

63.0

* Determined by genome analysis.

30

579

Figure legends

580

Fig. 1. Maximum-likelihood phylogenetic tree of 16S rRNA gene sequences showing

581

the relationships between strain SS37A-ReT and related bacteria. Bar represents 0.02

582

substitutions per nucleotide sequence position. Closed circles indicate internal nodes

583

with at least 50% bootstrap support from 1000 data resampling. The tree was rooted

584

using Rhodoplanes elegans AS130T as the outgroup. GenBank accession numbers are

585

given in parentheses.

586

587

Fig. 2. Maximum-likelihood phylogenetic tree of pmoA gene sequences showing the

588

relationships between strain SS37A-ReT and related bacteria. Bar represents 0.1

589

substitutions per nucleotide sequence position. Closed circles indicate internal nodes

590

with at least 50% bootstrap support from 1000 data resampling. The tree was rooted

591

using Methylomonas koyamae Fw12E-YT as the outgroup. GenBank accession numbers

592

are given in parentheses.

593

594

Fig. 3. Phylogenomic tree based on the concatenated nucleotide sequence of core genes

595

of Methylocystis and Methylosinus species by the distance method. The bar shows

596

nucleotide substitutions per site. Closed circles indicate internal nodes with at least 70%

597

bootstrap support from 1000 data resamplings. The tree was rooted using Methylomonas

598

koyamae Fw12E-YT as the outgroup. GenBank accession numbers are given in

599

parentheses.

600

601

Fig. 4. (a) Transmission electron micrograph of negatively stained cells of strain

602

SS37A-ReT. Bar, 1 µm. (b) Phase-contrast micrograph of strain SS37A-ReT cells. Bar, 5

603

µm. (c) Phase-contrast micrograph of rosettes of strain SS37A-ReT cells. Bar, 5 µm. (d)

604

Transmission electron micrograph of ultrathin sections of strain SS37A-ReT cells. Bar,

605

200 nm. Arrow shows the intracytoplasmic membrane structure.

31

606

607

Supplementary figures

608

609

Fig. S1. Phylogenetic trees of 16S rRNA gene sequences showing the relationships

610

between strain SS37A-ReT and related bacteria by the parsimony (a) and distance (b)

611

methods. Bars represent 0.01 (a) and 0.005 (b) substitutions per nucleotide sequence

612

position. Closed circles indicate internal nodes with at least 50% bootstrap support from

613

1000 data resamplings. The tree was rooted using Rhodoplanes elegans AS130T as the

614

outgroup. GenBank accession numbers are given in parentheses.

615

616

Fig. S2. Phylogenetic trees of pmoA gene sequences showing the relationships between

617

strain SS37A-ReT and related bacteria by the parsimony (a) and distance (b) methods.

618

Bars represent 0.02 (a) and 0.01 (b) substitutions per nucleotide sequence position.

619

Closed circles indicate internal nodes with at least 50% bootstrap support from 1000

620

data resamplings. The tree was rooted using Methylomonas koyamae Fw12E-YT as the

621

outgroup. GenBank accession numbers are given in parentheses.

622

623

Fig. S3. Phylogenetic trees of mxaF gene sequences showing the relationships between

624

strain SS37A-ReT and related bacteria by the maximum-likelihood (a), parsimony (b),

625

and distance (c) methods. Bars represent 0.05 (a) and 0.02 (b and c) substitutions per

626

nucleotide sequence position. Closed circles indicate internal nodes with at least 50%

627

bootstrap support from 1000 data resamplings. The tree was rooted using Methylomonas

628

koyamae Fw12E-YT as the outgroup. GenBank accession numbers are given in

629

parentheses.

630

631

Fig. S4. Phylogenetic trees of mmoX gene sequences showing the relationships between

632

strain SS37A-ReT and related bacteria by the maximum-likelihood (a), parsimony (b)

32

633

and distance (c) methods. Bars represent 0.1 (a) and 0.02 (b and c) substitutions per

634

nucleotide sequence position. Closed circles indicate internal nodes with at least 50%

635

bootstrap support from 1000 data resamplings. The tree was rooted using

636

Methylomagnum ishizawai RS11D-PrT as the outgroup. GenBank accession numbers

637

are given in parentheses.

638

639

Fig. S5. Phylogenetic trees of 16S rRNA gene sequences showing the relationships

640

between strain SS37A-ReT and related Methylocystis and Methylosinus strains isolated

641

from various environments [41] by the maximum-likelihood (a), parsimony (b), and

642

distance (c) methods. Bars represent 0.02 (a), 0.01 (b) and 0.002 (c) substitutions per

643

nucleotide sequence position. Closed circles indicate internal nodes with at least 50%

644

bootstrap support from 1000 (a and c) or 100 (b) data resamplings. The tree was rooted

645

using Methylocapsa palsarum NE2T as the outgroup. GenBank accession numbers are

646

given in parentheses.

647

648

Fig. S6. Phylogenetic trees of pmoA gene sequences showing the relationships between

649

strain SS37A-ReT and related Methylocystis and Methylosinus strains isolated from

650

various environments [41] by the maximum-likelihood (a), parsimony (b), and distance

651

(c) methods. Bars represent 0.01 (a) and 0.02 (b and c) substitutions per nucleotide

652

sequence position. Closed circles indicate internal nodes with at least 50% bootstrap

653

support from 1000 (a and c) or 100 (b) data resamplings. The tree was rooted using

654

Methylomonas koyamae Fw12E-YT as the outgroup. GenBank accession numbers are

655

given in parentheses.

656

657

Fig. S7. Phylogenetic trees of mxaF gene sequences showing the relationships between

658

strain SS37A-ReT and related Methylocystis and Methylosinus strains isolated from

659

various environments [41] by the maximum-likelihood (a), parsimony (b), and distance

33

660

(c) methods. Bars represent 0.05 (a and b) and 0.02 (c) substitutions per nucleotide

661

sequence position. Closed circles indicate internal nodes with at least 50% bootstrap

662

support from 1000 (a and c) or 100 (b) data resamplings. The tree was rooted using

663

Methylomonas koyamae Fw12E-YT as the outgroup. GenBank accession numbers are

664

given in parentheses.

665

666

Fig. S8. Phylogenetic trees of mmoX gene sequences showing the relationships between

667

strain SS37A-ReT and related Methylocystis and Methylosinus strains isolated from

668

various environments [41] by the maximum-likelihood (a), parsimony (b), and distance

669

(c) methods. Bars represent 0.1 (a), 0.02 (b) and 0.01 (c) substitutions per nucleotide

670

sequence position. Closed circles indicate internal nodes with at least 50% bootstrap

671

support from 1000 (a and c) or 100 (b) data resamplings. The tree was rooted using

672

Methylomagnum ishizawai RS11D-PrT as the outgroup. GenBank accession numbers

673

are given in parentheses.

674

675

Fig. S9. (a) Total ion current chromatogram of dimethyl disulphide adducts from strain

676

SS37A-ReT monounsaturated fatty acids. Mass spectra of dimethyl disulphide adducts

677

of C18:1 fatty acids eluted at the retention times 10.23 min (b) and 10.44 min (c),

678

respectively, on the chromatogram (a). Ions at m/z 159, 231, and 390 correspond to ω-

679

fragment, D-fragment, and M+ of the dimethyl disulphide adduct of C18:1ω8 [54].

680

34

681

682

Figure 1

683

35

684

685

Figure 2

686

36

687

688

Figure 3

689

37

690

691

Figure 4

38

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