Lactococcal abortive infection protein AbiV interacts directly with the phage protein SaV and prevents translation of phage proteins

Abstract

AbiV is an abortive infection protein that inhibits the lytic cycle of several virulent phages infecting Lactococcus lactis, while a mutation in the phage gene sav confers insensitivity to AbiV. In this study, we have further characterized the effects of the bacterial AbiV and its interaction with the phage p2 protein SaV. First, we showed that during phage infection of lactococcal AbiV(+) cells, AbiV rapidly inhibited protein synthesis. Among early phage transcripts, sav gene transcription was slightly inhibited while the SaV protein could not be detected. Analyses of other phage p2 mRNAs and proteins suggested that AbiV blocks the activation of late gene transcription, probably by a general inhibition of translation. Using size exclusion chromatography coupled with on-line static light scattering and refractometry, as well as fluorescence quenching experiments, we also demonstrated that both AbiV and SaV formed homodimers and that they strongly and specifically interact with each other to form a stable protein complex.

FIG. 1.

Total incorporation of [¹⁴C]uridine and [³⁵S]methionine in L. lactis during phage infection. The vertical arrow represents the time of lysis of the sensitive AbiV⁻ culture. Open symbols represent AbiV⁻, while closed symbols represent AbiV⁺.

FIG. 2.

Detection of early (A), middle (B), and late (C) phage p2 transcripts during infection of AbiV⁺ (closed symbols) and AbiV⁻ (open symbols) L. lactis cells.

FIG. 3.

Western blotting for detection of ORF26 (SaV), ORF11, and ORF16 during phage infection. (A) Temporal phage protein production in AbiV⁻ (left) and AbiV⁺ (right) L. lactis cells. Numbers represent minutes in relation to time of infection (time zero). Lanes NI, noninfected cells; lane SaV, purified SaV; lanes p2, CsCl-purified p2 virions. (B) Graphical representation of the expression levels shown in panel A. Open symbols represent AbiV⁻, while closed symbols represent AbiV⁺.

FIG. 4.

SEC-MALS/UV/RI analysis and fluorescence quenching assay. (A) SEC-MALS/UV/RI analysis. The abscissa indicates the time scale for the HPLC injections. AU, absorbance units. (B) Fluorescence quenching assay fitting curve, determined using nonlinear regression with a double binding site equation. The initial concentration of SaV was 0.5 μM, followed by progressive addition of different concentrations of AbiV (calculated for the monomeric forms). Error bars represent standard deviations. (C) Fluorescence spectra obtained for SaV-AbiV, SaV-SSB, and Sak3-AbiV. The Δ values obtained were 17%, 3.9%, and 3.5%, respectively.