Isolation and analysis of Escherichia coli mutants that allow increased replication of bacteriophage lambda.

Escherichia coli mutants were isolated that supported the growth of a lambda Ots and, in at least one case, a lambda Bts phage at the normally nonpermissive temperature of 39 degrees C. In one such strain, Ots and Bts suppression ability appeared to be a function of the guaB gene. Ots suppression by the mutant guaB strain was prevented if high levels of guanine or xanthine were present in the medium. No other base had any effect on Ots suppression in this strain. Other strains carrying spontaneous mutations resulting in guanine or xanthine auxotrophy (guaA or guaB lesions, respectively) all allowed lambda Ots replication at 39 degrees C; Ots suppression in these strains was also abolished by addition of guanine to the medium. Thus, reduced intracellular guanine levels resulting from guaA or guaB mutations appeared to suppress the inability of lambda Ots and, at least in some cases, Bts bacteriophage to form plaques at 39 degrees C. In burst size experiments, a guaB mutant produced a larger phage yield per infected cell of both lambda Ots and lambda O+ phage at 39 degrees C than did a similar guaB+ strain. It appeared that a lower-than-normal level of guanine (or a guanine derivative) in these cells may permit unusually efficient lambda replication. The fact that O+ and lambda Ots bursts in the guaB mutant were reduced significantly by addition of exogenous guanine to the medium is consistent with this suggestion. Another strain that suppresses the Ots allele has no known auxotrophic requirements, and suppression in this strain was unaffected by addition of guanine to the medium; however, addition of cytidine to the medium specifically eliminated Ots suppression in this strain. The mutation responsible for allowing Ots replication in this strain is unknown.     

A bacteriophage T4 gene which functions to inhibit Escherichia coli Lon protease.

A bacteriophage T4 gene which functions to inhibit Escherichia coli Lon protease has been identified. This pin (proteolysis inhibition) gene was selected for its ability to support plaque formation by a lambda Ots vector at 40 degrees C. Southern blot experiments indicated that this T4 gene is included within the 4.9-kilobase XbaI fragment which contains gene 49. Subcloning experiments showed that T4 gene 49.1 (designated pinA) is responsible for the ability of the Ots vector to form plaques at 40 degrees C. Deficiencies in Lon protease activity are the only changes known in E. coli that permit lambda Ots phage to form plaques efficiently at 40 degrees C. lon+ lysogens of the lambda Ots vector containing pinA permitted a lambda Ots phage to form plaques efficiently at 40 degrees C. Furthermore, these lysogens, upon comparison with similar lysogens lacking any T4 DNA, showed reduced levels of degradation of puromycyl polypeptides and of canavanyl proteins. The lon+ lysogens that contained pinA exhibited other phenotypic characteristics common to lon strains, such as filamentation and production of mucoid colonies. Levels of degradation of canavanyl proteins were essentially the same, however, in null lon lysogens which either contained or lacked pinA. We infer from these data that the T4 pinA gene functions to block Lon protease activity; pinA does not, however, appear to block the activity of proteases other than Lon that are involved in the degradation of abnormal proteins.