Polysaccharides promote the deep-sea Lentisphaerae strain WC36 growth and stimulate the expression of phage-associated genes.

(A) Maximum likelihood phylogenetic tree of 16S rRNA gene sequences from strain WC36, strain zth2, and some Planctomycetes-Verrucomicrobia-Chlamydia (PVC) group bacteria. Bacillus cereus ATCC 14579 was used as the outgroup. Bootstrap values (%) > 80 are indicated at the base of each node with the gray dots (expressed as percentages of 1000 replications). (B) Growth assays of strain WC36 cultivated in rich medium either without supplementation, with 5 g/L or 10 g/L laminarin or with 5 g/L or 10 g/L starch. (C) Transcriptomics-based heat map showing all up-regulated genes encoding phage-associated proteins in strain WC36 cultured in rich medium supplemented with 10 g/L laminarin. “Rich” indicates strain WC36 cultivated in rich medium; “Lam” indicates strain WC36 cultivated in rich medium supplemented with 10 g/L laminarin. (D) RT-qPCR detection of the expression of some genes encoding phage-associated proteins shown in panel C. The heat map was generated by Heml 1.0.3.3. The numbers in panel C represent multiple differences in gene expression (by taking log2 values).

Polysaccharides induce the production of bacteriophages in Lentisphaerae strain WC36.

(A) TEM observation of phages extracted from the supernatant of WC36 cells cultured in rich medium supplemented with or without polysaccharide. Panel I shows the absence of phages in the supernatant of WC36 cells cultivated in rich medium. Panels II-IV show the morphology of phages present in the supernatant of WC36 cells cultivated in rich medium supplemented with 10 g/L laminarin. Yellow hollow arrows indicate the typical granular structure of filamentous phages, and green arrows indicate phage-like particles with other shapes. Panels V-VIII show the morphology of phages present in the supernatant of strain WC36 cells cultivated in rich medium supplemented with 10 g/L starch. Typical filamentous phages are indicated with yellow hollow arrows, and two other kinds of phage-like particles with different shapes are indicated by orange and green arrows, respectively. (B) TEM observation of strain WC36 cultured in rich medium supplemented with or without 10 g/L laminarin or 10 g/L starch. Panel I shows representative morphology of strain WC36 cultivated in rich medium. Panel II shows the morphology of strain WC36 cultivated in rich medium supplemented with 10 g/L laminarin. Panels III-IV show the morphology of strain WC36 cultivated in rich medium supplemented with 10 g/L starch. Red arrows indicate filamentous phages associated with bacterial cells. (C) TEM of an ultrathin section of strain WC36 cultured in rich medium supplemented with or without 10 g/L laminarin or 10 g/L starch. Panel I shows an ultrathin section of strain WC36 cultivated in rich medium; Panels II-III show ultrathin sections of strain WC36 cultivated in rich medium supplemented with 10 g/L laminarin; Panel IV shows an observation of the ultrathin section of strain WC36 cultivated in rich medium supplemented with 10 g/L starch. Red arrows indicate filamentous phages being released from or entering bacterial cells.

Bacteriophages induced from Lentisphaerae strain WC36 by polysaccharides are released via chronic infections.

(A) Growth curve of strain WC36 cultivated in either rich medium alone or rich medium supplemented with 5 g/L or 10 g/L laminarin for 30 days. The number of filamentous phages (B) and hexagonal phages (C) extracted from 1 μL of the supernatant of strain WC36 cell cultured in rich medium supplemented with or without 5 g/L or 10 g/L laminarin (for 5, 10, and 30 days). The average numbers are shown at the top of the bar charts.

Polysaccharides promote the growth of deep-sea Lentisphaerae strain zth2 and induce the production of bacteriophages.

(A) Growth assays of strain zth2 cultivated in rich medium supplemented with or without 3 g/L laminarin for four days. (B) Transcriptomics-based heat map showing all up-regulated genes encoding phage-associated proteins in strain zth2 cultured in rich medium supplemented with 3 g/L laminarin. (C) RT-qPCR detection of the expressions of some genes encoding phage-associated proteins shown in panel B. The numbers in panel C represent multiple differences in gene expression (by taking log2 values). (D) TEM observation of phages extracted from the supernatant of a cell suspension of strain zth2 cultured in the rich medium supplemented with or without polysaccharides. Panels I-III show the morphology of phages in the cell supernatant of strain zth2 cultivated in rich medium supplemented with 3 g/L laminarin. Typical filamentous phage is indicated with yellow hollow arrows, and two other kinds of phage-like particles with different shapes are indicated by orange and green arrows, respectively. Panels IV-V show the morphology of phages present in the cell supernatant of strain zth2 cultivated in rich medium supplemented with 3 g/L starch. Typical filamentous phages are indicated with red arrows, and other phage-like particles with different shapes are indicated by green arrows. Panel VI shows that no phages were observed in the cell supernatant of strain zth2 cultivated in rich medium. “Rich” indicates strain zth2 cultivated in rich medium; “Lam” indicates strain zth2 cultivated in rich medium supplemented with 3 g/L laminarin.

Genomic organization of bacteriophages released from Lentisphaerae strains WC36 and zth2 cultured in rich medium supplemented with polysaccharide.

(A) Schematic of the genomic composition of Phage-WC36-1 and Phage-zth2-1. (B) A diagram showing the genomic composition of Phage-WC36-2. (C) The genomic composition of Phage-zth2-2. Arrows represent different ORFs and the direction of transcription. The main putative gene products of the phages are shown, and the numbers in brackets indicate the numbers of amino acids within each ORF. Hypothetical proteins are indicated with gray arrows, structural modules are indicated by green arrows, the replication module is indicated by blue-grey arrows, the regulatory module is indicated by golden arrows, the assembly and secretion modules are indicated by blue arrows and auxiliary metabolic genes (AMGs) encoding proteins associated with polysaccharide transport and degradation are indicated by red arrows. The size of the phage genomes is shown behind each gene cluster.

Bacteriophages released from Lentisphaerae strain WC36 promote the growth and polysaccharide metabolism of a marine bacterium Pseudomonas stutzeri 273.

(A) Growth curve and status of P. stutzeri 273 cultivated in basal medium, basal medium supplemented with 20 μl/mL Phages-WC36, basal medium supplemented with 5 g/L starch, basal medium supplemented with 5 g/L starch and 20 μl/mL Phages-WC36. (B) Number of filamentous phages and hexagonal phages extracted from 1 μL of P. stutzeri 273 cell supernatant after culture in four different types of media as in panel A. The average numbers are shown at the top of the bar charts. (C) Continuous culture model of Phages-WC36 infected strain 273. The number of filamentous phages (D) and hexagonal phages (E) extracted from 1 μL of P. stutzeri 273 cell supernatant after three successive cultures. The average numbers are shown at the top of the bar charts.