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SUMMARYHighly passaged defective-interfering (DI) particle preparations of equine herpesvirus type 1 (EHV-1) were found to mediate the co-establishment of persistent infection and oncogenic transformation of permissive hamster embryo cells. Four cell lines, designated DI-1 to DI-4, were shown to possess biological properties typical of transformed cells and to induce the rapid formation of metastatic fibrosarcomas when injected into syngeneic LSH hamsters. Corresponding DI tumour cell lines, designated DI-1T to DI-4T, were found to be virus non-producing, to be transplantable in the hamster, and, like the four parent DI cell lines, to express EHV-1-coded antigens and to be resistant to superinfection with EHV-1 but not with a heterologous virus, vesicular stomatitis virus. All transformed cell lines, but not the tumour cell lines, contained a population of cells (2.6 to 9%) that continued to release infectious virus after 100 passages in culture. The production of interferon and selection of temperature-sensitive mutants did not appear to play a role in the maintenance of persistent infection. However, it was demonstrated that these persistently infected cells continued to release not only infectious virus but also DI particles after more than 2 years in culture. DI particles were shown to be present in released virus by: (i) detection of the defective virus DNA species (density 1-724 g/ml; standard EHV-1, density 1.716 g/ml) by CsCI analytical ultracentrifugation techniques; (ii) measurement of interference activity of virus released from DI-I to DI-4 cells against standard EHV-1 replication; (iii) the presence of the 35 megadalton BgllI fragment unique to the DI particle genome in DNA of released virus. These studies indicate that herpesvirus DI particles may function both in the initiation and maintenance of persistent infection and alter the cytocidal effects of infection to allow the establishment of oncogenic transformation and persistent infection.

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Electron microscopic studies of equine herpesvirus DNA revealed that single strands that were allowed to reanneal formed single-stranded loops with double-stranded stems only at one end of the molecule. These observations support restriction enzyme analyses which indicate that the 92-megadalton DNA molecule exists as a long region of unique sequences covalently linked to a short region. The short region is comprised of an internal unique sequence, which forms the loop during reannealing of single strands, and two terminal inverted repeat sequences that bracket the unique sequence and form the double-stranded stem structure observed upon reannealing of single strands. Measurements of the unique sequence and terminal inverted repeat subgenomic sequences indicate a size of 6.4 megadaltons for each and thus fix the size of the short region at approximately 19.2 megadaltons.

Prodigiosin, the bright red pigment produced by many strains of Serratia marcescens, is synthesized by a bifurcated pathway that terminates in the enzymatic condensation of the two final products, a monopyrrole and a bipyrrole, Sau3A fragments of S. marcescens (Nima) DNA were introduced into a strain of Escherichia coli K-12 by use of the cosmid vector pHC79, and transformed clones were selected based on resistance to ampicillin. Among 879 transformants screened, 2 could be induced to synthesize prodigiosin when supplied with either one or both terminal products of the bifurcated pathway. Data are presented to support the idea that production of prodigiosin is not usually mediated by a plasmid.Many strains of Serratia marcescens produce pigment via a bifurcated pathway in which 2-methyl-3-amylpyrrole (MAP) and 4-methoxy-2,2'-bipyrrole-5-carboxyaldehyde (MBC) are enzymatically condensed into 2-methyl-3-amyl-6methoxypyrodigiosene, or prodigiosin ( Fig. 1). Several mutants of S. marcescens have been identified as being blocked in either the MAP or MBC pathway. However, little is known about the precursors accumulated by these mutants, and nothing is known about the enzymes or gene products involved (8).Prodigiosin is an easily assayed secondary metabolite of S. marcescens and may be useful as a model system to study the mechanism of expression of secondary metabolites in bacteria. Isolation of recombinant molecules encoding the prodigiosin biosynthetic pathway would provide an approach to identifying gene products and understanding the enzymology and the genetics of prodigiosin biosynthesis.In this paper, we describe the isolation of DNA sequences encoding part of the prodigiosin biosynthetic pathway by use of a cosmid vector-Escherichia coli cloning system. In addition, we present a novel method for the screening of bacterial clones for prodigiosin genes, using mutant strains of S. marcescens that are defective in pigment production. MATERIALS AND METHODSBacteria and conditions of growth. Wild-type strains of S. marcescens, Nima and Hy, were grown routinely at 37°C on peptone-glycerol agar (PGA; 0.5% Bacto-Peptone [Difco Laboratories, Detroit, Mich.], 1% glycerol, and 1.5% agar) and were maintained on Trypticase soy agar (BBL Microbiology Systems, Cockeysville, Md.). Mutant strains of S. marcescens, 933 and WF, were cultured and maintained under the conditions indicated above. E. coli HB101 (pro leu thi lacY hsdR endA recA rpsL20 ara-4 galK2 xyl-5 mtl-i supE44) and PK243 (HB101[pHC79]) (2) were cultivated at 37°C. E. coli NS428 (N205[X Aamll b2 red3 cIts857 Sam7]) and NS433 (N205[A Eam4 b2 red3 cIts857 Sam7]) were grown at 30°C on L agar (1% tryptone, 0.5% yeast extract, 0.5% NaCl, 1.5% agar) unless otherwise indicated. * Corresponding author. t Present address: Louisiana Reference Laboratories, International Clinical Laboratories, Baton Rouge, LA 70806.Purification of DNA from S. marcescens. Two milliliters of an overnight broth culture of S. marcescens (Nima) was transferred to 100 ml of fresh tryptic soy broth (...