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A cyanophage PaV-LD, previously isolated from harmful filamentous cyanobacterium Planktothrix agardhii, was sequenced, and co-expression of its two ORFs in tandem, ORF123 and ORF124, inhibited growth on the model cyanobacterium Synechocystis sp. PCC6803 cells. However, the mechanism of action of ORF123 and ORF124 alone remains to be elucidated. In this study, we aimed to study the individual function of ORF123 or ORF124 from PaV-LD. Our data showed that the ORF123 encoded an endopeptidase, which harbored an M23 family peptidase domain and a transmembrane region. The expression of the endopeptidase in Escherichia coli alone revealed that the protein exhibited remarkable bacteriostatic activity, as evidenced by observation of growth inhibition, membrane damage, and leakage of the intracellular enzyme. Similarly, the holin, a membrane-associated protein encoded by the ORF124, showed weak bacteriostatic activity on E. coli. Moreover, deletion mutations indicated that the transmembrane domains of endopeptidase and holin were indispensable for their bacteriostatic activity. Meanwhile, the bacteriostatic functions of endopeptidase and holin on cyanobacteria cells were confirmed by expressing them in the cyanobacterium Synechocystis sp. PCC6803. Collectively, our study revealed the individual role of endopeptidase or holin and their synergistic bacteriolytic effect, which would contribute to a better understanding of the lytic mechanism of cyanophage PaV-LD.

Additional file 1: Fig. S1. Visualization of DNA labeled by EdU in RGV infections at 6 and 12 hpi. GSTC cells were infected with RGV at 1 MOI. The cells were incubated for 1 h with 10 ��M EdU at the indicated time points and then fixed, permeabilized, and reacted with Alexa Fluor 488 azide. Cellular DNA was stained with Hoechst 33342 (blue). The EdU-labeled DNA is shown in green. The visible Hoechst-labeled cytoplasmic viral factories are indicated with arrows. Fig. S2. EdU labeling of ADRV at different times post infection. ADRV-infected GSTC cells were labeled with EdU at the indicated time points for 1 h. EdU-labeled nascent DNA is shown in green. Hoechst 33342-stained DNA is shown in blue. Normal GSTC cells were used as a control under the same processing. With lasting infection, the number of EdU-labeled nuclei decreased, while the number, size, and intensity of the cytoplasmic foci increased. The green color was located completely in the cytoplasm at 6���7 hpi. Fig. S3. EdU labeling of ADRV-infected cells at different treatment times. Infected GSTC cells were labeled with EdU for different periods (10 min, 20 min, and 30 min) at 12 hpi. The nuclei were stained with Hoechst 33342 (blue). EdU-labeled nascent DNA is presented in green. The 30 min labeling resulted in strong signals. Fig. S4. Characterization of RGV-27R. A. Prokaryotic expression and purification of recombinant RGV-27R. The protein markers, bacteria without induction (Uninduced), bacteria with induction (Induced), and purified proteins (Purified) are labeled at the top. The recombinant proteins with molecular weights of approximately 50 kDa are indicated with asterisks. B. Electrophoretic mobility shift analysis of the DNA���protein complexes. The ��X174 DNA���protein complexes migrated more slowly with increasing protein amounts (0���10 ��g). C. Temporal expression of RGV-27R in virus infected GSTC cells by Western blot analysis. D. Subcellular localization of RGV-27R in virus infected GSTC cells by immunofluorescence. Viral nascent DNA was labeled with EdU as described above (green). RGV-27R was detected with anti-vSSB antibody (red). Cell nuclei were stained with Hoechst 33342 (blue). The visible Hoechst-labeled cytoplasmic viral factories are indicated with arrows. Fig. S5. Domain organization of core replisome components ADRV-88L/RGV-24R and ADRV-47L/RGV-63R. The domains (gray box) were searched using MOTIF Search in the Pfam and PROSITE databases. ADRV-88L/RGV-24R has a length of 975 aa and contains Primase_C, D5_N, and SF3 helicase domains. ADRV-47L/RGV-63R has a length of 1013 aa and contains exonuclease and DNA polymerase domains. Numbers at the top indicate the amino acid sites of the searched domains. Fig. S6. Temporal expression and localization of RGV-24R and RGV-91R. A. Western blot analysis of vHelicase/primase (RGV-24R) and vPCNA (RGV-91R). GSTC cells were infected by RGV at 0.5 MOI and collected at indicated time points. B. Localization of RGV-24R by immunofluorescence in GSTC cells. RGV-24R (green) colocalized with the vSSB (red). Representative overlaps are indicated by white arrows. C. Localization of RGV-91R by immunofluorescence. RGV-91R (green) colocalized with the vSSB (red). Representative overlaps are indicated by white arrows. The visible Hoechst-labeled cytoplasmic viral factories are indicated with yellow arrows. Fig. S7. Localization of Rpb3, Rpb6, and Rpb11 in RGV infected BHK-21 cells. The cells were infected with RGV at an MOI of 0.5 and fixed at the indicated time points. Rpb3, Rpb6, and Rpb11 (red) were stained with commercial antibodies. Replicating DNA was stained with EdU (green) as described above. Cell nuclei were stained with DAPI (blue). Bar = 5 ��M. Rpb3, Rpb6, and Rpb11 were located in viral factories during RGV infection. Fig. S8. Gel electrophoresis of the deproteinized DNA. In the iPOND assay, cross-links were reversed in lysates collected after DNA sonication. The bound proteins were digested, and the DNA fragments were separated on a 1% agarose gel. The main molecular weights of the DNA fragments in the present study are from 250 to 500 bp.

Additional file 2: Table S1. Primers and constructs used in the study.

Additional file 3: Table S2. Sequences of the siRNAs used in the study.