Unstable Lysogeny and Pseudolysogeny in Vibrio harveyi Siphovirus-Like Phage 1

Abstract

Exposure of Vibrio harveyi (strain VH1114) to V. harveyi siphovirus-like phage 1 (VHS1) resulted in the production of a low percentage of lysogenized clones of variable stability. These were retrieved most easily as small colonies within dot plaques. Analysis revealed that VHS1 prophage was most likely carried by VH1114 as an episome rather than integrated into the host chromosome. In the late exponential growth phase, lysogenized VH1114 continuously produced VHS1 but also gave rise to a large number of cured progeny. The absence of phage DNA in the cured progeny was confirmed by the absence of VHS1 DNA in Southern blot and PCR assays. Curiously, these very stable, cured subclones did not show the parental phenotype of clear plaques with VHS1 but instead showed turbid plaques, both in overlaid lawns and in dot plaque assays. This phenotypic difference from the original parental isolate suggested that transient lysogeny by VHS1 had resulted in a stable genetic change in the cured clones. Such clones may be called pseudolysogens (i.e., false lysogens), since they have undergone transient lysogeny and have retained some resistance to full lytic phage development, despite the loss of viable or detectable prophage.Luminous vibriosis and luminescent bacterial disease are terms that are used commonly to describe mortality caused mostly by pathogenic strains of Vibrio harveyi in hatcheries or rearing ponds of Penaeus species (6, 10). In serious epizootics, moribund shrimp emit a greenish luminescence due to bacterial infection or colonization. There is good evidence that exotoxins are involved in mortality from luminescent vibriosis, since extracellular products of cultured V. harveyi can be toxic when injected into Penaeus monodon (7,8) and since exotoxin production by V. harveyi is involved in shrimp larval disease (5).Upon examining the genetic relationship among virulent isolates of V. harveyi, Pizzutto and Hirst (17) found no discernible associations and thus proposed that virulence factors were transmitted by mobile elements such as transposons, plasmids, or bacteriophages. This led to a search for bacteriophages in Australian isolates of V. harveyi and to the eventual discovery of V. harveyi myo-like phage from a highly virulent V. harveyi isolate derived from diseased shrimp larvae (11-13). Phage conversion leading to toxin production occurs with many human-pathogenic bacteria (1, 2), including Vibrio cholerae (4), but had not previously been proven for V. harveyi.Association of V. harveyi virulence with a bacteriophage has also been reported in Thailand (14,19). However, phage production could not be sustained in culture, and the phage was lost, preventing further study. Later, another bacteriophage was isolated from shrimp culture ponds in Thailand, together with a partner V. harveyi isolate, VH1114 (15). It was an unenveloped virus with a long, noncontractile tail (100 to 120 nm long) and an isometric head (approximately 60 nm in diameter) that morphologically resembled the earlier Thai bacteriophage associa...

A Listeria monocytogenes Mutant Defective in Bacteriophage Attachment Is Attenuated in Orally Inoculated Mice and Impaired in Enterocyte Intracellular Growth

“…We took the precaution of isolating our phage-resistant mutant by screening rather than selection so that the mutant bank would not be subject to artifactual effects, such as lysogeny or pseudolysogeny (20). The phage employed in the isolation of our resistant strain has not been documented to be lysogenic (19).…”

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“…In shrimp ponds, Vibrio harveyi (VH) is a common bacterial species that sometimes causes luminescent disease, although luminescence is not a requirement for shrimp pathogenicity. On the other hand, it has been shown that bacteriophages can sometimes lysogenize VH and alter their pathogenicity for shrimp [1][2][3] . Increased virulence due to lysogeny is well known for human pathogens 4 , including Salmonella enteritica 5 , Vibrio cholerae, 6 and Staphylococcus aureus 7 .…”

Complete Genome Sequence of Virulence-Enhancing Siphophage VHS1 from Vibrio harveyi

“…Since the phage is carried in the lysogens as an episome, cured isolates can be obtained at high rates by isolation of single colonies upon subculture (14). Random clones representing approximately 25% of the VHS1 genome were previously sequenced and deposited in GenBank (26).Vibrio harveyi 1114GL (VH0) that has been lysogenized by VHS1 (VH1) is Ͼ100 times more lethal to the black tiger shrimp than VH0 (13,14). In addition, culture supernatant solutions from VH1 are highly toxic for the black tiger shrimp (Penaeus monodon) but not for the whiteleg shrimp (Penaeus [Litopenaeus] vannamei) (13).…”

“…Vibrio harveyi 1114GL (VH0) that has been lysogenized by VHS1 (VH1) is Ͼ100 times more lethal to the black tiger shrimp than VH0 (13,14). In addition, culture supernatant solutions from VH1 are highly toxic for the black tiger shrimp (Penaeus monodon) but not for the whiteleg shrimp (Penaeus [Litopenaeus] vannamei) (13).…”

“…It produces initially clear lytic plaques on lawns of Vibrio harveyi 1114GL. However, after several days of incubation, colonies of lysogens appear within the formerly clear plaques (14). These lysogens produce VHS1 spontaneously upon subculture, without the need for induction of a lytic cycle.…”