PHENOTYPIC CONVERSIONS AS A RESULT OF PSEUDOLYSOGENY

Julie J. Shaffer, John O. Schrader, and Tyler A. Kokjohn

School of Biological Sciences, E151 Beadle Center, University of Nebraska-Lincoln, Lincoln, NE 68588-0666

SUMMARY

Although two of the life-styles of bacteriophages, lysis and lysogeny, have been studied intensively, pseudolysogeny or the unstable carrier-state has been virtually unstudied since it was first recognized. The characterization of pseudolysogeny has made so little progress that researchers have not even been able to agree on a definition of this unusual state which is known to occur in many of the lytic bacteriophage. This lack of research may be due to the highly unstable nature of the pseudolysogen.

The goal of our research is to characterize the interactions of the bacterium and the pseudolysogenic bacteriophage and to elucidate the role of the pseudolysogen in nature. Our research has revealed several phenotypic changes that occur in the host cell after infection. In some instances, the cells' pigment changes dramatically from normal pyocyanin blue to a brown with varying intensities. The pseudolysogens are more resistant to ultraviolet light and hydrogen peroxide. These new phenotypes could play an important role in the survival of the bacteria and phage in the natural ecosystem.

Key words: Pseudolysogeny, carrier state, Pseuomonas aeruginosa, bacteriophage, UV

INTRODUCTION

Substantial numbers of bacteriophage can be found in freshwater, marine, and other ecological systems (Torrella and Morita, 1979; Bergh et al., 1989; Proctor and Fuhrman, 1990; Paul et al., 1991; 1993; Wommack et al., 1992, Marsh and Wellington, 1994). A substantial fraction of bacterial mortality in these systems is attributed to the lytic action of bacteriophages (Proctor and Fuhrman, 1990, 1993, Heldal and Bratbak, 1991). Based on these observations of large numbers and known facts regarding bacteriophage, it is believed they play an important role in the ecological balance of nature.

Three life-styles are exhibited by bacteriophage, i. lysis, ii. lysogeny, and iii. pseudolysogeny or the carrier-state. The lytic cycle is characterized by the initial infection of the virus particle, replication of the viral genome, synthesis of viral components, and lysis of the host cell, releasing the virions. Lysogeny is the indefinite persistence of the phage genome in the host cell. Pseudolysogeny is defined as an unstable coexistence of a bacteriophage in a host bacterium without a constant inheritance of the phage genome. This leads to both infected and phage sensitive progeny in the same culture.

Research has shown that the large numbers of bacteriophage in marine water may be due to lytic interactions (Wilcox and Fuhrman, 1994) and not to lysogenic induction as previously thought (Bratbak et al., 1990, 1992, Thingstad et al., 1993). Yet, it has also been shown that there are few virus per milliliter of seawater (Spencer, 1960; Moebus, 1987) and the decay rate of free-phage is rapid (Suttle and Chen, 1992). Pseudolysogeny may account for these conflicting observations. Pseudolysogenic interaction may account for the relatively low numbers of free bacteriophage in the seawater with the bacteria acting as a safe-haven for the phage. This unstable interaction of normally lytic bacteriophage with bacteria may have a significant role in the marine system.

Pseudolysogeny is difficult to study due on the unstable nature of the interaction. Little research has been done on this topic, but the results available seem to have a recurring theme: phenotypic changes (Jones et al., 1961; Koibong et al., 1961; Nida and Ferretti, 1982; Thompson et al., 1980). Some of the pseudolysogeny-associated changes are pigment production, nutritional changes, and toxin production.

Based on the potential role of pseudolysogens in nature, we have taken initiated investigations of the interactions between bacterium and phage that result in pseudolysogenic interactions.

MATERIALS AND METHODS

Bacterial and Bacteriophage strains. Bacterial strains used were Pseudomonas aeruginosa PAO1 and PAC5 Nal^R. Bacteriophage were UT1 (Ogunseitan et al., 1990) and Phage S, a newly isolated P. aeruginosa bacteriophage.

Lysogeny Establishment Methods. Pseudolysogens were established by modifications to Kokjohn and Miller (1988).

UV radiation resistance. Survival of bacteria after exposure to ultraviolet radiation was quantified using a modification of the methods of Simonson et al. (1990). The source of UV-C radiation was a GE germicidal lamp. Ultraviolet radiation A and B was provided by an Oriel Corp (Stratford, CT) Model 66002 Solar simulator.

Hydrogen peroxide resistance. Survival of bacteria after exposure to hydrogen peroxide was quantified using modifications to Sammartano et al. (1986).

RESULTS

During the course of studies to quantify host cell survival after infection with bacteriophages, we noticed low frequency of clones with novel phenotypes. Some of the survivors of infection with both UT1 and Phage S produced a brown pigment. The pigment can be seen after a couple of days of incubation at room temperature. The brown pigment-producing pseudolysogens were included in the test of the resistance of these cells to ultraviolet radiation.

Enhanced UV-C resistance was found to be present in both the brown and pseudolysogens with normal pigmentation of PAO1-UT1. (Figure 1) Both cell types, pigmented and unpigmented, exhibited at least a ten fold higher survival than the non-pseudolysogens at longer exposures. Preliminary experiments suggest that a similar resistance is also present to solar radiation.

Phage S pseudolysogens were examined for resistance to hydrogen peroxide. These pseudolysogens are more resistant to the lethal effects of hydrogen peroxide than isogenic controls (Figure 2).

DISCUSSION

The survivors of infection with the lytic bacteriophage UT1 appear to be unstable pseudolysogens. The interaction we have observed is similar to the "facultative lysogeny" of bacteriophage T3 described by Kruger and Schroeder (1981). It is very difficult to grow clones to midlog phase without induction of lysis although the cultures may be grown at 30C with some success. Clones are unstable so that the same cell line may not show the same results the next time the experiment is done.

A second general characteristic of the pseudolysogens is that they may not release phage consistently. After probing by colony hybridization we know at least part of the viral genome is still present. A similar set of observations have been made for Azotobacter vinelandii after infection by Phage A21 (Thompson et al., 1980). Researchers found what they describe as "permanently converted" cells. They believe part of the viral DNA is still present in the cell, but not a complete viral genome. This viral genome still present is responsible for the persistence of the unusual phenotype.

Although the exact nature of the brown pigment is still unknown, a similar brown color has been reported previously in P. aeruginosa (Ogunnariwo and Hamilton-Miller, 1975). The brown pigment may be a pyomelanin that is chemically unrelated to animal melanin. It has been suggested that pyomelanin is a catechol melanin which can be found in plants.

The phenotypic changes that a cell undergoes after the establishment of pseudolysogeny may aid in an increased survivability for both host and bacteriophage. In nature those cells that can withstand high amounts of ultraviolet radiation and oxygen radicals may out-compete or persist longer than those that are more sensitive. Pseudolysogeny may be an important factor in the ecology of bacteriophages and their hosts.

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Figure 1. UV-C sensitivity of P. aeruginosa PAO1 with and without bacteriophage UT1. Survivors were quantified by viable plate counts. P. aeruginosa PAO1 control (n), P. aeruginosa PAO-UT1 without pigment (l), and P. aeruginosa PAO1-UT1 with brown pigment (s).

Figure 2. Hydrogen peroxide sensitivity of P. aeruginosa PAC5 Nal^R with and without phage S. Survivors were quantified by viable plate counts. P. aeruginosa PAC5 Nal^R control (n), P. aeruginosa PAC5 Nal^R-phage S (l).