ABSTRACTContractile injection systems (CISs) are a large group of phage tail-like nanostructures conserved among bacteria. Despite their wide distribution, the biological significance of CISs in bacteria remains largely unclear except for a few unicellular bacteria. Here, we show thatStreptomyces lividans—a model organism of filamentous Gram-positive bacteria with highly conserved CIS-related gene clusters—produces intracellular CIS-like nanostructures (Streptomycesphage tail-like particles [SLPs]) that affect phenotypes of this bacterium under hyperosmotic conditions. In contrast to typical CISs released from the cells, SLPs are localized in the cytoplasm ofS. lividans. In addition, loss of SLPs leads to (i) delayed erection of aerial mycelia on hyperosmotic solid medium and (ii) decreased growth during the transition from exponential growth phase to stationary phase in hyperosmotic liquid medium. Localization of fluorescent protein-tagged SLPs showed partial correlation with cell wall synthesis-related proteins, including MreB, an actin-like cytoskeleton protein. Our pulldown assay and subsequent quantitative proteome analysis also suggest that 30S ribosomal proteins and cell wall-related proteins, including MreB, are coeluted with SLPs. Furthermore, an interaction assay using the recombinant proteins revealed a direct interaction between a sheath protein of SLP and ribosomal protein S16. Results of cross-linking experiments show indirect interactions between SLPs and translation elongation factors. These findings collectively suggest that SLPs are directly or indirectly associated with a protein interaction network within the cytoplasm ofS. lividansand that SLP loss ultimately affects the susceptibility of the bacterium to certain stress conditions.IMPORTANCERecent bioinformatic analyses have revealed that CIS-related gene clusters are highly conserved in Gram-positive actinomycetes, especially members of the genusStreptomycesknown for their ability to produce therapeutic antibiotics. While typical CISs are released from the cells and can act as protein translocation systems that inject effector proteins into the target cells, our results indicate the unique intracellular localization of SLPs, CIS-related nanostructures produced byS. lividans. In addition, the direct and indirect interactions of SLPs with cytoplasmic proteins and SLP localization within specific regions of mycelia suggest that the biological significance of SLPs is related to intracellular processes. Further, SLP loss leads to increased susceptibility ofS. lividansto osmotic stress, suggesting that production of these phage tail-like nanostructures ultimately affects the fitness of the bacterium under certain stress conditions. This work will provide new insight into the phage tail-like nanostructures highly conserved inStreptomycesspecies.