Structural characterization and assembly of the distal tail structure of the temperate lactococcal bacteriophage TP901-1

Abstract

The tail structures of bacteriophages infecting gram-positive bacteria are largely unexplored, although the phage tail mediates the initial interaction with the host cell. The temperate Lactococcus lactis phage TP901-1 of the Siphoviridae family has a long noncontractile tail with a distal baseplate. In the present study, we investigated the distal tail structures and tail assembly of phage TP901-1 by introducing nonsense mutations into the late transcribed genes dit (orf46), tal(TP901-1) (orf47), bppU (orf48), bppL (orf49), and orf50. Transmission electron microscopy examination of mutant and wild-type TP901-1 phages showed that the baseplate consisted of two different disks and that a central tail fiber is protruding below the baseplate. Evaluation of the mutant tail morphologies with protein profiles and Western blots revealed that the upper and lower baseplate disks consist of the proteins BppU and BppL, respectively. Likewise, Dit and Tal(TP901-1) were shown to be structural tail proteins essential for tail formation, and Tal(TP901-1) was furthermore identified as the tail fiber protein by immunogold labeling experiments. Determination of infection efficiencies of the mutant phages showed that the baseplate is fundamental for host infection and the lower disk protein, BppL, is suggested to interact with the host receptor. In contrast, ORF50 was found to be nonessential for tail assembly and host infection. A model for TP901-1 tail assembly, in which the function of eight specific proteins is considered, is presented.

FIG. 1.

Transmission electron micrographs of TP901-1 phages negatively stained with uranyl acetate. A, TP901-1 wt; B, 48⁻ mutant; C, 49⁻ mutant; D, 46⁻ mutant; E, 47⁻ mutant; F, 50⁻ mutant.

FIG. 2.

Transmission electron micrographs of immunogold-labeled TP901-1 tail fiber protein ORF47. 48⁻ mutant phages were incubated with anti-Tal₂₀₀₉ (C-terminal) antibodies and labeled with secondary antibodies conjugated to 5-nm gold particles. (ORF47_TP901-1 and Tal₂₀₀₉ share 93% identity.) Bars, 50 nm.

FIG. 3.

Silver-stained 10% SDS-PAGE gel with protein profiles of TP901-1 phages. Lane 1, 46⁻ mutant; lane 2, 47⁻ mutant; lane 3, 48⁻ mutant; lane 4, 49⁻ mutant; lane 5, 50⁻ mutant; lane 6, TP901-1 wt. Molecular masses are indicated to the left of the gel. Tail protein bands and estimated molecular masses are indicated with arrows to the right of the gel. *, processed protein.

FIG. 4.

Western blot analysis for detection of ORF46_TP901-1, ORF47_TP901-1, and ORF48_TP901-1 from purified wt and mutant TP901-1 phages. (A) Phage proteins detected with anti-ORF49_Tuc2009 (ORF49_Tuc2009 and ORF46_TP901-1 share 95% identity). (B) Phage proteins detected with anti-Tal₂₀₀₉ (N-terminal) (Tal₂₀₀₉ and ORF47_TP901-1 share 93% identity). (C) Phage proteins detected with anti-ORF51_Tuc2009 (ORF51_Tuc2009 and ORF48_TP901-1 share 80% identity). Lane 1, molecular marker; lane 2, TP901-1 wt; lane 3, 46⁻ mutant; lane 4, 47⁻ mutant; lane 5, 48⁻ mutant; lane 6, 49⁻ mutant; lane 7, 50⁻ mutant. Relevant molecular masses are indicated to the left of the blots. Arrow indicates band of ORF46_TP901-1.

FIG. 5.

TP901-1 tail assembly hypothesis. An initiator complex is assumed to be formed from TMP, ORF46, and the tail fiber protein ORF47. The tail length determining the N-terminal part of TMP is expected to protrude from the complex. The baseplate is assembled onto the conical fiber structure by independent assembly of the upper and lower disks constituted from ORF48 and ORF49, respectively, and the tail tube is formed from polymerization of the major tail protein. It is currently unknown whether the baseplate disks are assembled before, during, or after tail tube formation. Finally, the tail is joined to an independently assembled head structure. ORF43 and ORF43-44 (expected to be formed from a translational frameshift at a slippery sequence in orf43) are putative assisting proteins equivalent to gpG and gpGT in the λ tail assembly process.