Nitai C. Mandal

Studies on polylysogens containing λN−cl− prophages I. control of synthesis and maintenance of a large number of integrated λ genomes

Abstract Study of the control of λ DNA synthesis in polylysogens containing 20–25 copies of integrated λ N − cl − prophages per bacterial genome shows that in absence of the functional products of O and P genes, only 7–8 copies per host genome are maintained. Introduction of constitutive mutations ν1ν3 in the right operator increases the prophage genome copies to 52. In a mixed polylysogen containing both λ N − c I − and λ imm 434 N − c I − prophages, the total number of phage genomes is also increased to 50. The levels of cro activity in all the polylysogens of λ vary almost proportionately with of the number of prophages. All these results indicate that the maintenance of 20–25 copies of integrated phage genome in λ N − c I − polylysogens is the result of both passive and O and P protein-mediated synthesis, and that the latter may be regulated by the cro gene product.

Bacteriophage λ p gene shows host killing which is not dependent on λ DNA replication

Bacteriophage lambda, having a mutation replacing glycine by glutamic acid at the 48th codon of cro, kills the host under N- conditions; we call this the hk mutation. In lambda N-N-cl-hk phage-infected bacteria, the late gene R is expressed to a significant level, phage DNA synthesis occurs with better efficiency, and the Cro activity is around 20% less, all compared to those in lambda N-N-cl-hk(+)-infected bacteria. Segments of lambda DNA from the left of pR to the right of tR2, carrying cro, cII, O, P, and the genes of the nin5 region from the above hk and hk+ phages, were cloned in pBR322. Studies with these plasmids and their derivatives having one or more of the lambda genes deleted indicate that the hk mutation is lethal only when a functional P gene is also present. When expression of P from pR is elevated, due to the deletion of tR1, host killing also occurs without the hk mutation. We conclude that the higher levels of P protein, produced either (1) when cro has the hk mutation or (2) when tR1 is deleted, are lethal to the host. We also show that due to the hk mutation, the Cro protein becomes partially defective in its negative regulation at pR, resulting in the expression of P to a lethal level even in the absence of N protein-mediated antitermination. This P protein-induced host killing depends neither on lambda DNA replication nor on any other gene functions of the phage.

Isolation and preliminary characterization of Escherichia coli mutants resistant to lethal action of the bacteriophage λ P gene

Abstract Both spontaneous and NTG-induced mutants of Escherichia coli 594 insensitive to the lethal action of λ P gene were isolated and called rpl ( r esistant to Pl ethality). These mutants were of two types, showing different phenotypes. On type I rpl mutants, λ c l − and λ v 1 v 3 did not plate, while λ vir , λ c l − c 17, λ imm 434, and λ imm 21 did; plasmid pMR45 carrying the λ P gene could not complement λimm21 P − phage in type I mutants. On the other hand, the type II rpl mutants support the growth of all the above phages including λ c l − . Neither type of rpl mutation affects growth of the bacteria.

Structure and function of the repressor of bacteriophage lambda

SummaryBy mutagenizing a λcIts (λcI857) lysogen, a λ mutant has been isolated with a wild-type phenotype. This mutant phage lysogenizes with low efficiency and produces a low burst. Though the initial rates of repressor synthesis in Escherichia coli after infection with wild-type and mutant λ are the same, the maximum level of repressor that is synthesized in the latter case is only about 30% of that synthesized in the former. Virulent λ plates on the lysogen of mutant λ with slightly less efficiency producing very tiny plaques. Operator-binding studies made in vitro with purified mutant and wild-type repressors show that the binding curve of the former repressor is a rectangular hyperbola while that of the latter is sigmoid. The half-lives of the complexes of mutant and wild-type repressors with right operator are 133 and 27 min, respectively. All these results suggest that the mutant repressor possibly has a higher affinity for the operators. This mutant has been named λcIha (ha=high affinity).

Studies on polylysogens containing λN−cI− prophages II. Role of high multiplicities in lysogen formation by λN−cI− phage

Results of the experiments presented in this paper show that lambda N-cI- phage can lysogenize a nonpermissive host Escherichia coli when it infects at very high multiplicities (around 100), and lambda N-cI-cII- and lambda cIII-N-cI- lysogenize poorly at similar high multiplicities. The latter two phages lysogenize with appreciable frequency when either lambda N-cI- or lambda int-cN-cI-cII- is used as helper. The phages, lambda N-cI-, lambda N-cI-cII-, and lambda cIII-N-cI- can lysogenize also at relatively low m.o.i. of 20 in presence of the above lambda int-c helper, and the lambda int-cN-cI-cII- phage alone forms converted lysogens at an m.o.i. as low as 12. All these results suggest that the establishment of prophage integration by lambda N-cI- is positively regulated, like lambda N+cI+ phage, by the cII/cIII-promoted expression of the int gene of lambda, and under the N- condition, high multiplicities are needed to provide optimum levels of cII and cIII products, especially the latter.

Bacteriophage lambda containing two temperature-sensitive mutations in the cl gene produces clear plaque at 30°

Abstract When λ cIts 2 Psus 3 was crossed with λ Nsus 7 sus 53 c I857, clear plaque formers at 30° were obtained among the λ N + P + recombinants. This clear recombinant of λ was shown to contain the two c I ts mutations, 2 and 857, of the parents. It was also observed that crosses between any two λ c I ts mutants, from a list of eight, produced recombinants forming clear plaques at 30°. These suggest that no pair of ts mutations of the eight tested are compatible with the functioning of the repressor even at 30° when they are present in cis .