Acquired mutations in phage lambda genes O or P that enable constitutive expression of a cryptic lambda N(+)cI[Ts]cro(-) prophage in E-coli cells shifted from 30 degrees C to 42 degrees C, accompanied by loss of imm(lambda) and Rex(+) phenotypes and emergence of a non-immune exclusion-state

The majority of bacteria, which carry the N+-cI857[Ts]-cro(-)-O+-P+ fragmen t of lambda genome, are killed when derepressed by shifting from 30 degrees C to 42 degrees C. Among rare survivors, we observed a proportion of colon y-forming units (cfu) that retained the typical imm(lambda)-immunity phenot ype when grown at 30 degrees C; however, when shifted from 30"C to 42 degre es C, they lost lambda immunity and acquired a non-immune exclusion-state ( Nie phenotype). We also found that the imm(lambda) survivor cfu quickly los t their Rex(+) exclusion phenotype (as measured by T4rII plating inhibition ) when shifted from 30 degrees C to 42 degrees C, even though they produced CII, which stimulates p(E)-cI-rexA-rexB transcription. The Nie phenotype w as characterized by an inhibition of plating of the homoimmune phage, lambd a wt, and the heteroimmune phage, lambda imm(434). However, lambda vir and spontaneous mutants of lambda wt (lambda se mutations localized within o(R) ) escaped the Nie exclusion-state and plated efficiently on lawns of Nie cf u at 42 degrees C. Thus, we examined the scope of the Nie exclusion-state t oward lambda mutants blocked for lysogeny, and lambda hybrids substituted f or immunity or replication genes. Phage like lambda wt, competent for lysog eny, were severely excluded compared to some mutants of lambda defective fo r lysogeny. Among this latter type, there was high variance in the Nie excl usion of various cI mutants; some of which were not excluded. The Nie exclu sion-state was attributed to the constitutive expression of the defective l ambda fragment in the survivor cfu, made possible by the acquired replicati on defect(s). We characterized, both genetically and physically, the mutati ons in the defective integrated lambda prophage that permitted growth of th e survivor cfu at 42 degrees C. In five of seven survivor cfu, we identifie d IS2 insertions within lambda genes O and P that can block replication ini tiation from the lambda fragment. The remaining survivor cfu had multiple b ase substitutions within the C-terminal end of O and N-terminal half of P, the majority of which were silent. In some of these mutants, either an ochr e nonsense mutation or a single-base frameshift deletion inactivated P. (C) 1998 Elsevier Science B. V. All rights reserved.