Characterization of marine temperate phage-host systems isolated from Mamala Bay, Oahu, Hawaii

Abstract

To understand the ecological and genetic role of viruses in the marine environment, it is critical to know the infectivity of viruses and the types of interactions that occur between marine viruses and their hosts. We isolated four marine phages from turbid plaques by using four indigenous bacterial hosts obtained from concentrated water samples from Mamala Bay, Oahu, Hawaii. Two of the rod-shaped bacterial hosts were identified as Sphingomonas paucimobilis and Flavobacterium sp. All of the phage isolates were tailed phages and contained double-stranded DNA. Two of the phage isolates had morphologies typical of the family Siphoviridae, while the other two belonged to the families Myoviridae and Podoviridae. The head diameters of these viruses ranged from 47 to 70.7 nm, and the tail lengths ranged from 12 to 146 nm. The burst sizes ranged from 7.8 to 240 phage/bacterial cell, and the genome sizes, as determined by restriction digestion, ranged from 36 to 112 kb. The members of the Siphoviridae, T-phi HSIC, and T-phi D0, and the member of the Myoviridae, T-phi D1B, were found to form lysogenic associations with their bacterial hosts, which were isolated from the same water samples. Hybridization of phage T-phi HSIC probe with lysogenic host genomic DNA was observed in dot blot hybridization experiments, indicating that prophage T-phi HSIC was integrated within the host genome. These phage-host systems are available for use in studies of marine lysogeny and transduction.

FIG. 1

Morphologies of bacterial isolates from Mamala Bay. (A) HSIC. (B) D0 and phage particles in the cell culture (indicated by arrows). (b) Phage particles found in the D0 culture. Bar = 50 nm. (C) D1B. (D) D2S. (A, B, C, and D) Bars = 0.5 μm.

FIG. 2

Electron photomicrographs of phages isolated from Mamala Bay. (A) T-φD0-3. (B) T-φD2S. (C) T-φD1B. (D) T-φD0. (E) T-φHSIC. (F) T-φD0-3. Bars = 100 nm.

FIG. 3

Phage DNAs digested with restriction endonucleases to determine molecular weights. Lane 1, uncut T-φD0 DNA; lanes 2 through 6, T-φD0 DNA cut with HpaI, HindIII, BglII, AccI, and EcoRV, respectively; lane 7, uncut T-φD1B DNA; lanes 8 through 12, T-φD1B DNA cut with HapI, AccI, BglII, EcoRI, and EcoRV, respectively; lane 13, uncut T-φD2S DNA; lanes 14 through 18, T-φD2S DNA cut with HpaI, HindIII, AccI, EcoRI, and EcoRV, respectively; lane 19, uncut T-φHSIC DNA; lanes 20 through 24, T-φHSIC cut with HpaI, HindIII, AccI, EcoRI and EcoRV, respectively. The positions of molecular weight standards (in kilobases) are indicated on both sides of the gel.

FIG. 4

One-step growth curves for phage isolates from Mamala Bay. (A) T-φHSIC. (B) T-φD0. (C) T-φD1B. (D) T-φD2S.

FIG. 5

Dot blot hybridization preparations probed with the phage T-φHSIC DNA probe. Lane A, 0.4 μg of T-φHSIC DNA; lane B, 2.1 μg of plasmid DNA from HSIC; lane C, 0.5 μg of plasmid DNA from L-HSIC; lane D, 2.1 μg of chromosomal DNA from HSIC; lane E, 1.8 μg of chromosomal DNA from L-HSIC. Rows 1 and 2 are replicates.