Science - USA (2022-01-14)

SCIENCEscience.org 14 JANUARY 2022¥VOL 375 ISSUE 6577 223

Fig. 2. bGSDMs are associated with proteases,
defend from phages, and execute cell death.
(A) Representative instances of bGSDMs and
associated proteases in their genomic neighborhoods.
Genes known to be involved in antiphage defense
are shown in yellow. TA, toxin-antitoxin; Abi, abortive
infection; ATPase, adenosine triphosphatase.
(B) Types of proteases found adjacent to bGSDMs
(n= 59). Some bGSDMs appear with more than
one adjacent protease. Caspase-like proteases include
peptidase C14 (n= 15) and CHAT (n= 23). Cases
in which the protease gene also encodes an additional
domain are indicated. TPR, tetratricopeptide repeat;
LRR, leucine-rich repeat. (C) A bGSDM-containing
operon protects against phages. The efficiency
of plating of phages onE. coliMG1655 cells
expressing theLysobacterbGSDM WT or mutated
operon is shown. Data represent plaque-forming units
(PFU) per milliliter and are the averages of three
independent replicates, with individual data points
overlaid. GFP represents a control strain. Statistical
significance was determined by a one-way analysis of
variance (ANOVA) and Tukey multiple comparison
test. Not significant (n.s.)≥0.05; **P= 0.001 to 0.01.
(D) Growth of liquid cultures ofE. coliexpressing
the WT and mutatedLysobacterbGSDM operons.
Cells were infected with phage T6. For each experi-
ment, data represent one out of three biological
replicates (replicates are shown in fig. S6). OD 600 ,
optical density at 600 nm. (E) TheRunellabGSDM
operon causes cell death.E. coliDH5acells expressing
theRunellaprotease and WT or C3A-mutated bGSDM were examined by time-lapse microscopy. Overlay images from PI (red) and phase contrast of cells captured at the
start of the experiment and after 120 min of incubation are shown. Scale bar, 2mm. (F) bGSDM operons are toxic. Cells encoding protease and WT or mutated
bGDSM were plated in 10-fold serial dilution on LB-agar in conditions that repress operon expression (1% glucose) or induce expression (0.2% arabinose).

110k 120k 130k

20k 10k 1k

5k 15k 25k

Runella zeae

DSM 19591

G563DRAFT_02010

Bradyrhizobium tropiciagri

SEMIA 6148

Ga0098714_109513

Desulfuromonadales

Unclassified Bin 1

Ga0182885_104519

A

CRISPR-cas type I

Transposase Abi bGSDM

Caspase-like

Trypsin-like

Caspase-like

TA Abi

Runella

Desulfuromonadales

1% glucose 0.2% arabinose

Runella (C3A)

Bradyrhizobium

C

E

Trypsin-like

Subtilase

Peptidase

U49

Peptidase

C1-like

7

6

6 2

NACHT LRR

TPR WD40 repeat

B D

pBAD

WT C3A

Runella bGSDM + protease

0 min

120 min

[

Caspase

-like

38

GFP

full systemΔ

bGSDM

PFU / mL (log)

102

105

108

103

104

106

107

109

T5 T4 T6

0 100 200 300

Time (min)

0.2

0.4

0.6

OD

600

0.3

0.5

0.1

MOI T6 2 2×10-3 2×10-4 uninfected

full system

ΔbGSDM

5481K 5491K 5501K

AT Pa s e TA

Lysobacter enzymogenes

YC36

Ga0399710_4913

Trypsin-like

5471K

GFP

full sy

stem

ΔbG

SDMGF

P

full systemΔ

bGSDM

pBAD

F

Lysobacter system

n.s.

n.s.

** n.s.

**

****

****

Fig. 3. bGSDMs are activated by proteolytic
cleavage.(A) Toxicity ofRunellabGSDM
in vivo requires the associated protease. Bacteria
expressing WT and mutated versions of the
RunellabGSDM–protease operon were grown on
LB-agar in conditions that repress or induce
expression. Data represent colony-forming units
(CFU) per milliliter, and bar graphs represent an
average of three independent replicates, with
individual data points overlaid. Asterisks indicate
statistically significant differences compared
with the respective noninduced control using two-
sidedttest. n.s.≥0.05; *P= 0.0001 to 0.001;
**P< 0.0001. (B)RunellabGSDM cleavage
by its associated protease is dependent on
catalytic histidine and cysteine residues in vitro.
Fifteen percent SDS–polyacrylamide gel electropho-
resis (SDS-PAGE) gels were run after cleavage at
room temperature for 18 hours and visualized by
Coomassie staining. (C) TheRunellabGSDM crystal
structure and protease cleavage site. TheRunella
bGSDM structure is shown in lavender with the last
21 amino acids highlighted as gray spheres.
(D) Close-up view of theRunellabGSDM cleavage
site wherein cleavage occurs after the P1 L247 residue. (E) Structural overview of theBradyrhizobiumCTD and autoinhibitory interactions. The bGSDM is colored
purple except for its last 16 residues, which are colored yellow. Insets show interactions of F245 and F247 adjacent to D21 of the N-terminalbsheet (top) and F253
and the palmitoyl modification at C3 (bottom). The 2FO−FC(contoured at 1.5s) map is shown as gray mesh fit to the last 16 residues.

cleaved

full-length

bGSDM

AC

kDa

17

26

34

43

95

250

+ bGSDM

WTnoneWTH796AC840A

B

protease:

Runella

bGSDM

D E

N244

R245

V246

L247

G248

E249

N250

M251

Runella bGSDM loop

cleavage

site

C N

2FO−FC map

90°

F253 C3

F247

A246

F245

N21

Bradyrhizobium bGSDM

P244

Runella bGSDM

non-induced

induced

bGSDM+protease

(separate plasmids)

103

106

109

protease (alone)bGSDM (alone)bGSDM+proteaseprotease (H796A)protease (C840A)

104

105

107

108

n.s. n.s. **** n.s. n.s. ***

CFU / mL (log)

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