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candidate system we selected source organisms Altogether, we attempted to heterologously possible, we verified system consistency by test-
from which the system was taken and heterolo- clone 61 representative instances of the 28 can- ing for phage resistance in systems where indi-
gously cloned into one of the model organisms. didate new systems, and successful cloning was vidual genes were deleted (Figs. 3 to 5 and figs.
To increase the probability that the cloned system verified by whole-genome sequencing (table S4). S4 and S5). We found between several hundred
would be compatible and functionally expressed For 27 of these 28 systems, there was at least and several thousand representations of each
within the receiving bacterium, we selected sys- one candidate locus for which cloning was suc- of the defense systems in sequenced microbial
tems from mesophilic organisms as close phylo- cessful, and RNA sequencing (RNA-seq) of the genomes, usually with broad phylogenetic dis-
genetically as possible to E. coli or to B. subtilis transformants showed that, for 26 of the sys- tribution (fig. S6 and tables S6 to S15). Most
and included the upstream and downstream in- tems, at least one of the candidate loci was ex- systems were detected in >10 taxonomic phyla,
tergenic regions so that promoters, terminators, pressed in the receiving E. coli or B. subtilis strain. and 7 of them appear in archaea (fig. S6). Some
or other regulatory sequences would be preserved. The engineered bacteria were then challenged of the systems seem to target a specific family
Where possible, we took at least two instances by an array of phages consisting of 10 B. subtilis of phages (e.g., the Thoeris system appears to
of each system (from two different source ge- and six E. coli phages, spanning the three major specifically protect from myophages), whereas
nomes), to account for the possibility that some families of tailed double-stranded DNA (dsDNA) others, such as the Hachiman system, provide
systems may not be active in their source orga- phages (myo-, sipho-, and podophages), as well as broader defense (Fig. 2B). The genes comprising
nism (21, 22). The DNA of each system, spanning one single-stranded DNA (ssDNA) phage infecting the new systems encode many protein domains
the predicted genes and the intergenic spaces, E. coli (Fig. 2, B and C). Measuring phage effi- that are commonly present in antiviral systems
was synthesized or amplified from the source ciency of plating (EOP) on system-containing bac- such as CRISPR-Cas and RNA interference (RNAi),
genome and cloned into the phylogenetically teria versus control cells, we found that 9 of the including helicases, nucleases, and nucleic acid
closest model organism—either to E. coli (on a 26 tested systems (35%) showed protection from binding domains, in addition to many domains
plasmid) or to B. subtilis (genomically integrated). infection by at least onephage (Fig.2,BandC of unknown function and also atypical domains
As a control, we repeated the procedure with five andfigs. S2 andS3).In comparison, threeofthe as described below. Three of the systems contain
known defense systems (instances of types I, II, six positive control systems showed defense, with membrane-associated proteins, as predicted by
and III R-M systems, a type III toxin/antitoxin the remaining three showing no protection against thepresenceofmultipletransmembrane helices.
system, and an abortive infection gene of the the 10 B. subtilis phages tested (see Discussion). Below, we focus on further functional analyses Downloaded from
AbiH family) for which source organisms were We named the nine verified new systems after for a selected set of systems.
similarly selected and cloning was performed protective deities from various world mytholo-
into B. subtilis,aswellasasixthcontrol com- gies. These defense systems comprise between The Zorya defense system
posed of the recently discovered DISARM de- 1 and 5 genes and span between 2 and 12 kb of The Zorya system (named after a deity from
fense system (9) (table S4). genomic DNA (Table 1 and table S5). Where Slavic mythology) was identified based on the http://science.sciencemag.org/

on March 1, 2018

Fig. 1. Discovery of new antiphage defense systems in defense islands. in the vicinity (10 genes on each side) of one or more known defense genes
(A) Illustration of the computational analysis employed for each pfam found is recorded. Pink, a set of 123 pfams known to participate in antiphage
to be enriched in defense islands. Pfams that are enriched in the vicinity of defense (“positive set”); blue, the remaining 13,960 pfams analyzed in this
known defense genes are identified, and their neighboring genes are clustered study. (C) Neighborhood variability score for the analyzed pfams. Score
based on sequence homology to identify conserved cassettes that represent represents the fraction of pfam members occurring in different defense
putative defense systems. (B) Tendency of protein families to occur neighborhoods out of total occurrences of pfam members (see Methods).
next to defense genes. The genomic neighborhood for each member gene Pink, the 123 positive pfams; blue, a set of 576 pfams that passed the 65%
in each pfam is examined, and the fraction of member genes occurring threshold for fraction of members occurring with defense genes in proximity.

Doron et al., Science 359, eaar4120 (2018) 2 March 2018 2of11