The Viral Gene ORF79 Encodes a Repressor Regulating Induction of the Lytic Life Cycle in the Haloalkaliphilic Virus ϕCh1

ABSTRACT In this study, we describe the construction of the first genetically modified mutant of a halovirus infecting haloalkaliphilic Archaea. By random choice, we targeted ORF79, a currently uncharacterized viral gene of the haloalkaliphilic virus ϕCh1. We used a polyethylene glycol (PEG)-mediated transformation method to deliver a disruption cassette into a lysogenic strain of the haloalkaliphilic archaeon Natrialba magadii bearing ϕCh1 as a provirus. This approach yielded mutant virus particles carrying a disrupted version of ORF79. Disruption of ORF79 did not influence morphology of the mature virions. The mutant virus was able to infect cured strains of N. magadii, resulting in a lysogenic, ORF79-disrupted strain. Analysis of this strain carrying the mutant virus revealed a repressor function of ORF79. In the absence of gp79, onset of lysis and expression of viral proteins occurred prematurely compared to their timing in the wild-type strain. Constitutive expression of ORF79 in a cured strain of N. magadii reduced the plating efficiency of ϕCh1 by seven orders of magnitude. Overexpression of ORF79 in a lysogenic strain of N. magadii resulted in an inhibition of lysis and total absence of viral proteins as well as viral progeny. In further experiments, gp79 directly regulated the expression of the tail fiber protein ORF34 but did not influence the methyltransferase gene ORF94. Further, we describe the establishment of an inducible promoter for in vivo studies in N. magadii. IMPORTANCE Genetic analyses of haloalkaliphilic Archaea or haloviruses are only rarely reported. Therefore, only little insight into the in vivo roles of proteins and their functions has been gained so far. We used a reverse genetics approach to identify the function of a yet undescribed gene of ϕCh1. We provide evidence that gp79, a currently unknown protein of ϕCh1, acts as a repressor protein of the viral life cycle, affecting the transition from the lysogenic to the lytic state of the virus. Thus, repressor genes in other haloviruses could be identified by sequence homologies to gp79 in the future. Moreover, we describe the use of an inducible promoter of N. magadii. Our work provides valuable tools for the identification of other unknown viral genes by our approach as well as for functional studies of proteins by inducible expression.

Prophage-mediated modulation of interaction of Streptococcus thermophilus J34 with human intestinal epithelial cells and its competition against human pathogens

The human intestinal microbiota plays an important role in human health. While adhesion to gastrointestinal mucosa is a prerequisite for colonisation, inhibition of adhesion is a property which may prevent or reduce infections by food borne pathogens. Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus represent the two lactic bacteria constituting the yoghurt culture. These starter cultures have been claimed to be probiotic. In our study we compared two S. thermophilus strains (i.e. lysogenic strain J34 and corresponding non-lysogenic [prophage-cured] strain J34-6), with respect to (1) their in vitro adhesion properties to HT29 cells and (2) their cell surface hydrophobicities. Effects of the two strains on inhibition of adhesion of the pathogens Listeria monocytogenes Scott A, Staphylococcus aureus 6732 and Salmonella enteritidis S489 were studied in vitro with HT29 cell cultures. Lysogenic strain J34 was shown to be considerably more effective than the non-lysogenic derivative strain J34-6.

Assessment of genotoxicity and cytotoxicity of "lixeira" (Curatella americanaL.) λ using the prophage induction test (SOS inductest)

Curatella americana L., commonly known as "lixeira" in Brazil, has been used in folk medicine to treat ulcers and inflammations. The purpose of the present work was to evaluate the cytotoxic and genotoxic potential of the ethanolic extract of C. americana stem bark using the prophage λ induction test (SOS inductest). To evaluate the cytotoxicity of this plant, after treatment with different concentrations of the extract, Escherichia coli WP2s(λ) cultures were diluted in M9 buffer, inoculated into LB plates, and incubated for 24 h at 37 °C. To assess genotoxicity, the lysogenic strain E. coli WP2s(λ) was treated with different concentrations of the extract. Then, the lysogenic strain was added to the indicator strain (RJF013), LB(1/2)(malt/amp), seeded into plates with the matches, and incubated for 24 h at 37 °C. After this period, the total number of colonies and the number of plaques were counted to evaluate C. americana cytotoxicity and genotoxicity, respectively. Our results showed that although the extract of "lixeira" did not modify the survival of bacteria (p > 0.05), it caused a significant increase in prophage λ induction, especially at the higher concentrations (p<0.05). Therefore, we conclude that the ethanolic extract of C. americana stem bark did not present cytotoxic effect, but some genotoxic potential was observed.