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

• Published in: Gene 1998-11, Vol.223 (1-2), p.115-128
• Main Authors: Hayes, S, Hayes, C, Bull, HJ, Pelcher, LA, Slavcev, R A
• Format: Article
• Language: English
• Subjects: Escherichia coli; Phage lambda
• ISSN: 0378-1119

The majority of bacteria, which carry the N super(+)-cI857[Ts]-cro super(-)-O super(+) -P super(+) fragment of lambda genome, are killed when derepressed by shifting from 30 degree C to 42 degree C. Among rare survivors, we observed a proportion of colony-forming units (cfu) that retained the typical imm super( lambda )-immunity phenotype when grown at 30 degree C; however, when shifted from 30 degree C to 42 degree C, they lost lambda immunity and acquired a non-immune exclusion-state (Nie phenotype). We also found that the imm super( lambda ) survivor cfu quickly lost their Rex super(+) exclusion phenotype (as measured by T4rII plating inhibition) when shifted from 30 degree C to 42 degree C, even though they produced CII, which stimulates p sub(E)-cI-rexA-rexB transcription. The Nie phenotype was characterized by an inhibition of plating of the homoimmune phage, lambda wt, and the heteroimmune phage, lambda imm super(434). However, lambda vir and spontaneous mutants of lambda wt ( lambda se mutations localized within o sub(R)) escaped the Nie exclusion-state and plated efficiently on lawns of Nie cfu at 42 degree C. Thus, we examined the scope of the Nie exclusion-state toward lambda mutants blocked for lysogeny, and lambda hybrids substituted for immunity or replication genes. Phage like lambda wt, competent for lysogeny, were severely excluded compared to some mutants of lambda defective for lysogeny. Among this latter type, there was high variance in the Nie exclusion of various cI mutants; some of which were not excluded. The Nie exclusion-state was attributed to the constitutive expression of the defective lambda fragment in the survivor cfu, made possible by the acquired replication defect(s). We characterized, both genetically and physically, the mutations in the defective integrated lambda prophage that permitted growth of the survivor cfu at 42 degree C. In five of seven survivor cfu, we identified IS2 insertions within lambda genes O and P that can block replication initiation from the lambda fragment. The remaining survivor cfu had multiple base 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 ochre nonsense mutation or a single-base frameshift deletion inactivated P.