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Two special genes carried in pairs by the native prophages y and psi of Serratia marcescens HY, with functionally rather similar counterparts each, were assigned to restriction fragments and tested for homology. The y genes any and sky, as well as the psi genes anp and skp, are specifically activated by infection of HY cells with kappa phage. The kappa genes exerting this effect are tay for y and tap for psi. By means of tay and tap mutants, insertions of phage Mu DNA in the relevant parts of y and psi prophage, respectively, could be discovered. These insertions and subsequent deletions of Mu were the basis of our studies. The use of Mu was made possible by the isolation of an HY variant giving appropriate plaques with Mu particles of the G(-) type. In the case of another HY variant, Mu plaque formation depended on the presence of a host range mutation isolated by its ability to allow plaque formation on E. coli C, an indicator only for G(-) particles. Unexpectedly, this HY strain was an indicator of both G(-) and G(+) particles, but unfortunately had become adsorption resistant to y and psi. As an accessory result, it provided evidence for a second restriction/modification system. In both cases the O-specific polysaccharides were reduced. The main results of our paper concerning the two pairs of y and psi genes are as follows: the orders any-sky and anp-skp correspond with each other in y and psi; the sky gene, just as the skp gene, lies near one prophage end. However, despite the similarity in function and order, these genes are not homologous, in contrast to any and anp, which are at least partially homologous. The homologous regions of y and psi amount to only about 0.5 kbp. Another observation was that bacteria with a Mu insertion near the skp end of psi prophage were no longer cured of psi when infected with kappa tay-l, in contrast to the efficient curing observed with an ordinary psi prophage.

Temperate phage kappa originated from the defectively lysogenic Serratia marcescens strain K, from where it was liberated after uv irradiation with low efficiency. The phage is usually indicated on strain HY that can be easily lysogenized by it and rather efficiently uv-induced. Comparing the Eco RI restriction profiles of the kappa prophage in HY revealed a DNA rearrangement, by which the precursor structure in K is converted into the non-defective form. Apparently the fragment containing the pac sequence is concerned since a phage DNA probe prepared from the assumed initiation fragments of the first particles of the packaging series gave two signals instead of one with genomic K DNA. Since several independent new kappa isolates showed the same Eco RI restriction pattern as the original phage of ELLMAUER and KAPLAN (1959), the generation of kappa is a reproducible event.

Prophage psi carried along with prophage y by Serratia marcescens HY is subject of moderate curing at heteroimmune superinfection of cells from stationary phase with phage kappa. Curing becomes considerably more frequent when the bacteria are non-lysogenic for y. Both psi, y-double-lysogenic and psi-single-lysogenic cells with a mutation in the ink gene are very efficiently cured of psi if infected by kappa tay, although this mutant was characterized as being deficient in transactivation of certain genes in prophage y. On the other hand to get efficiently cured after kappa wild-type infection these cells too must be devoid of a y prophage. Thus a y function turned on by tay+ seems to counteract the elimination of psi. However, interestingly enough psi curing is boosted by a further y function under special circumstances. Efficient curing depends on an intact kappa tap gene, a gene reported to cause transactivation of certain psi genes. Curing at kappa tay infection is specifically accompanied by induction of the psi prophage in a part of the infected cells. However, there is no such induction at kappa wild-type infection, either in the absence or presence of a y prophage. An explanation of these findings is suggested which includes an antirepressive effect exerted on psi and a hypothetical interaction between the products of genes tap and tay.

In temperate phage kappa of Serratia marcescens several special features of different phages are combined. The unessential genes lI, iny, cII and, at least to some extent, even the integrase gene int are not subject to negative control by the repressor, the product of gene cIII. A genetic map of the prophage was established using defective, heat-induced lysates of int- lysogens both in vegetative crosses with sus mutants of essential genes and in transduction of the four unessential genes to lysogenic recipients. Results from reciprocal four factor-crosses concerning the order of the four genes had to be included. The four genes are located near the right end of the prophage, whereas cIII lies near its left end. In vegetative phage all five genes lie in an interval between the essential genes T and U, comprising 10% of kappa's genetic map. The right prophage end appears to face at least two trp cistrons, among them the gene encoding anthranilate synthetase. lI encodes a product that masks the phage receptors in the cell wall. The gene product of iny interferes with the growth of infecting phage y. The natural function of cII is still unknown, but some of its mutants display a cold-sensitive phenotype, their plaques being clear at 30 degrees C and turbid at 37 degrees C. Bacteria with such prophages stop producing viable progeny when the cultures are shifted from 37 degrees C to 30 degrees C. These cold-sensitive mutants are partly dominant and partly recessive. Analysing a virulent mutant, a gene ant encoding an antirepressor was discovered, but so far there is no evidence that it is regulated by an extra repressor. The gene is located relatively near the left prophage end. Evidence is presented that the exogenotes in transduction with the defective lysates continue to exist for some time after a first recombinational event.

Plaque formation of phage kappa on Serratia marcescens strain HY normally depends on the presence of either a psi or y prophage in the indicator bacteria. Bacterial ink mutants allowing kappa growth in the absence of either prophage were isolated from the doubly cured strain HY (psi, y)--. By means of a kappa mutant, named gdy, an active participation of kappa in antagonizing inhibition of its own growth on HY (psi, y)-- was demonstrated. The gdy mutation is closely linked to gene cIII coding for the kappa repressor. The prophages psi and y enable kappa to grow undisturbed probably by modifying the kappa DNA during replication in such a way that it is not susceptible to the ink+ effect. Whereas kappa grown on HY (psi, y)--ink-- gave only rare productive infections of HY (kappa, y)--, psi and y grown on the same strain were fully infective. The interference exerted by a kappa prophage on vegetative propagation of y is based upon a multi-component mechanism, the interference being removed, or diminished by mutations residing either inside or outside of the kappa prophage. The responsive phage gene iny+ is dominant over its mutant allele iny--; hence it codes for a diffusible product. Both on the vegetative and the prophage genome iny is located near gene lI responsible for lysogenic conversion of bacteria to non-adsorption of kappa. The restriction-modification system of HY is not involved in the growth inhibition of kappa by HY (psi, y)--. Contrary to the other phages used in this work y is refractory to restriction.

Serratia marcescens HY bacteria must be lysogenic with either prophage y or psi to make it possible for phage kappa to form plaques unless they carry a so-called ink mutation. Genes in y and psi termed any and anp were identified that after infection of ink+ cells are necessary for an effective propagation of these phages as well as of coinfecting kappa phage. When kappa infects y and/or psi-lysogenic cells it transactivates the respective prophage genes by means of two early genes termed tay and tap. It appears that on infection of nonlysogenic ink+ cells kappa damps its own development, provided the regulatory region of the responsible gene is undermethylated. After kappa infection duly to achieve the special methylation of this region seems to be the function of any and anp. There are some more genes in y and psi prophage under the control of tay and tap, concerning in both cases a Dam methylation (recognition sequence GATC) of kappa DNA, a recombination proneness under restricting conditions of kappa DNA not modified by the modification enzyme of HY, and the kappa plaque size. By hybridization studies a region of homology common to y and psi was demonstrated which from its size might comprise all the transactivated genes. The view is supported by genetic data indicating an affinity among the any and anp genes. Investigation of various any mutants were indicative of DNA inversions in this region of the y genome. Surprisingly some of the any mutants had become sensitive in their plaque forming ability to an inhibitory activity exerted by prophage psi. Mutants of psi unable to interfere but still able to lysogenize were isolated. A model is presented accounting for the formation of pleiotropic and nonpleiotropic mutations with Any phenotype and their reversion types. Possible functions of the y genes and their counterparts in psi are discussed.