Significance: Many pathogenic bacteria utilize dynamic appendages called pili to facilitate important functions, such as gene transfer and motility. These processes contribute to the spread of antibiotic resistance genes and support persistence in bacterial infections, making them prime targets for therapeutic purposes. The single-stranded (ss) RNA bacteriophages all use dynamic pili to facilitate infection; however, key mechanistic details describing how the pilus promotes viral entry into the host cell have remained elusive. Here we used fluorescence microscopy to uncover a telling phenomenon associated with ssRNA phage infection: initial penetration of the viral payload causes breakage of host pili. This provides a selective advantage for the infecting phage, and thus this phenomenon may be widespread among other pilus-specific phage systems.

The retractile type IV pilus (T4P) is important for virulence of the opportunistic human pathogen Pseudomonas aeruginosa . The single-stranded RNA (ssRNA) phage PP7 binds to T4P and is brought to the cell surface through pilus retraction. Using fluorescence microscopy, we discovered that PP7 detaches T4P, which impairs cell motility and restricts the pathogen’s virulence. Using cryo–electron microscopy, mutagenesis, optical trapping, and Langevin dynamics simulation, we resolved the structure of PP7, T4P, and the PP7/T4P complex and showed that T4P detachment is driven by the affinity between the phage maturation protein and its bound pilin, plus the pilus retraction force and speed, and pilus bending. Pilus detachment may be widespread among other ssRNA phages and their retractile pilus systems and offers new prospects for antibacterial prophylaxis and therapeutics.

Three Bacillus bacteriophage-derived endolysins, designated PlyP56, PlyN74, and PlyTB40, were identified, cloned, purified, and characterized for their antimicrobial properties. Sequence alignment reveals these endolysins have an N-terminal enzymatically active domain (EAD) linked to a C-terminal cell wall binding domain (CBD). PlyP56 has a Peptidase_M15_4/VanY superfamily EAD with a conserved metal binding motif and displays biological dependence on divalent ions for activity. In contrast, PlyN74 and PlyTB40 have T7 lysozyme-type Amidase_2 and carboxypeptidase T-type Amidase_3 EADs, respectively, which are members of the MurNAc-LAA superfamily, but are not homologs and thus do not have a shared protein fold. All three endolysins contain similar SH3-family CBDs. Although minor host range differences were noted, all three endolysins show relatively broad antimicrobial activity against members of the Bacillus cereus sensu lato group with the highest lytic activity against B. cereus ATCC 4342. Characterization studies determined the optimal lytic activity for these enzymes was at physiological pH (pH 7.0–8.0), over a broad temperature range (4–55 °C), and at low concentrations of NaCl (<50 mM). Direct comparison of lytic activity shows the PlyP56 enzyme to be twice as effective at lysing the cell wall peptidoglycan as PlyN74 or PlyTB40, suggesting PlyP56 is a good candidate for further antimicrobial development as well as bioengineering studies.

Here, we describe the complete genome sequence of the T4-like Klebsiella pneumoniae myophage Marfa. In its 168,532-bp genome, Marfa has 289 genes, for which 122 gene functions were predicted. Many similar proteins are shared between Marfa and phage T4, as well as its closest phage relatives.

Soil from George Town, Grand Cayman Island, yielded the bacteriophage Belinda, isolated on Bacillus thuringiensis DSM 350. We present here the analysis of the complete genome sequence of 162,308 bp, with 298 predicted genes. The genome also contains three tRNA genes. Belinda belongs to the C1 cluster of Bacillus phages.

Proteus mirabilis is a Gram-negative enteric bacterium associated with complicated human urinary tract infections. Here, we present the complete genome annotation for P. mirabilis siphophage Saba. With a 60,056-bp genome and 75 predicted genes, Saba is most similar at the nucleotide and protein levels to phage Chi and Chi-like viruses.