ABSTRACT Bacterial type VIIb secretion systems (T7SSb) are multi-subunit integral membrane protein complexes found in Firmicutes that play a role in both bacterial competition and virulence by secreting toxic effector proteins. The majority of characterized T7SSb effectors adopt a polymorphic domain architecture consisting of a conserved N-terminal Leu-X-Gly (LXG) domain and a variable C-terminal toxin domain. Recent work has started to reveal the diversity of toxic activities exhibited by LXG effectors; however, little is known about how these proteins are recruited to the T7SSb apparatus. In this work, we sought to characterize genes encoding domains of unknown function (DUFs) 3130 and 3958, which frequently co-occur with LXG effector-encoding genes. Using coimmunoprecipitation-mass spectrometry analyses, in vitro copurification experiments and T7SSb secretion assays, we find that representative members of these protein families form heteromeric complexes with their cognate LXG domain and in doing so, function as targeting factors that promote effector export. Additionally, an X-ray crystal structure of a representative DUF3958 protein, combined with predictive modelling of DUF3130 using AlphaFold2, reveals structural similarity between these protein families and the ubiquitous WXG100 family of T7SS effectors. Interestingly, we identify a conserved FxxxD motif within DUF3130 that is reminiscent of the YxxxD/E “export arm” found in Mycobacterial T7SSa substrates and mutation of this motif abrogates LXG effector secretion. Overall, our data experimentally link previously uncharacterized bacterial DUFs to type VIIb secretion and reveal a molecular signature required for LXG effector export. Significance statement Type VIIb secretion systems (T7SSb) are protein secretion machines used by an array of Gram-positive bacterial genera including Staphylococcus, Streptococcus, Bacillus , and Enterococcus . These bacteria use the T7SSb to facilitate interbacterial killing and pathogenesis through the secretion of toxins. Although the modes of toxicity for a number of these toxins have been investigated, the mechanisms by which they are recognized and secreted by T7SSb remains poorly understood. The significance of this work is the discovery of two new protein families, termed Lap1 and Lap2, that directly interact with these toxins and are required for their secretion. Overall, Lap1 and Lap2 represent two widespread families of proteins that function as targeting factors that participate in T7SSb-dependent toxin release from Gram-positive bacteria.

Dual targeting factors are required for LXG toxin export by the bacterial type VIIb secretion system