Canonical T7SS substrate EsxA is conserved across GBS T7SS subtypes I—III.

a) ClustalW alignments and percent identity matrix of EsxA amino acid sequences from GBS T7SS subtypes I-III that are encoded upstream of the putative GBS T7SS loci. Subtype I example strain CJB111 (accession: NZ_CP063198.2) encodes EsxA1 and EsxA2. Subtype II example strain 2603V/R (accession: NC_004116.1) encodes EsxA2. Subtype III example strain CNCTC 10/84 (accession: NZ_CP006910.1) encodes EsxA1 and a truncated EsxA2. In the above matrix, the purple shading corresponds to the level of identity between two strains (on a spectrum of 0 to 100% identity), with darker shading indicative of higher percent identity. b) un-cropped, full picture of the Western blot shown in Fig 4B. The only other band (CJB111 supernatant) may indicate oligomerization of monomeric EsxA over time, but this needs to be further investigated. c) Coomassie-stained SDS PAGE gel indicating that wells were equally loaded (see blue arrows) for the Western blot shown in Fig 4B.

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