A bacterial RNA polymerase mutant that renders lambda growth independent of the N and cro functions at 42 degrees C.

We describe a bacterial RNA polymerase mutation, rif 501, which confers rifampicin resistance and thermosensitivity to E. coli K 12. The purified RNA polymerase enzyme from rif 501 bacteria shows increased heatsensitivity in vitro at 51 degrees C. However, in vivo, at 42 degrees C the non-permissive temperature, mutant bacteria continue to grow and to synthesize RNA for 90 min. On a lawn of the mutant bacteria, at 40-41 degrees C, phage lambda forms clear plaques (LycA phenotype); this is probably due to an enhancement of cro function; we surmise that at 42 degrees C the transcription originating from the pR (but not from the pL) promoter on the lamdba genome becomes N-independent and less sensitive to the absence of the cro product. We discuss the possibility that both the N and cro proteins of phage lambda interact directly with the bacterial RNA polymerase. These observations indicate that the loss of viability of the rif 501 mutant at the restrictive temperature is not a consequence of an immediate inactivation of RNA polymerase; rather we feel it is due to a modification of the activity of RNA polymerase, leading to a disruption of the cellular regulation.

Induction of a stable, heritable epigenetic change by mutagenic carcinogens: a new test system.

The expression of the E. coli gal operon was set under the direct negative control of phage lambda repressor by a fusion between the gal operon an the active cI cro segment of phage lambda genoma. This system can exist in two stable but reversible epigenetic states: (A) the immune cI+ cro- gal- state (white colonies on McConkey gal plates) and (B) the nonimmune cI- cro+ gal+ state (red colonies). Transcription of this gal operon depends upon activation of the PR promoter; hence requiring the removal of lambda immunity repressor (cI) from the oR operator. Short exposure of this E. coli strain to radiation or chemical mutagens/carcinogens leads to a stable, inherited loss of the cI repressor (due to the take-over by the cro repressor, repressing the cI gene) and subsequent constitutive expression of the gal operon. This switch, from the (A) state to the (B) state, depends upon E. coli recA+ and lambda cro+ genes and is reversible upon supply of lambda cI repressor; this is consistent with the fact that cI and cro proteins are mutual repressors, i.e., only one of them can be expressed at a given time. This E. coli strain provides the easiest, most accurate and sensitive assay for all mutagenic and SOS-inducing agents.

Specificity of polarity suppression in E. coli: correction of defects in gene N, but not in gene Q, of phage lambda.

The bacterial mutation psuA1, known as (suA) a polarity suppressor, partially relieves all N defects in bacteriophage lambda growth. No evidence is found that psuA1 relieves Q defects in lambda growth. Specific mechanisms of action by the N and Q gene products are discussed. The psuA1 mutation was also found to suppress IS1 type but not IS2 type insertion mutations in lambda.

qin101: Promoter mutation which allows the constitutive expression of the late genes.

We describe the isolation and characterization of a mutant (lambda qin101) which renders lambda growth Q independent. We have shown that this mutation creates a new promoter, located between genes P and Q, which results in the constitutive expression of the entire Q late region.

Isolation and heteroduplex mapping of a lambda transducing bacteriophage carrying the structural genes for carbamoylphosphate synthase: regulation of enzyme synthesis in Escherichia coli K-12 lysogens.

A N-lambda bacteriophage transducing the structural genes for Escherichia coli K-12 carbamoylphosphate synthase (glutamine) (CPSase; EC 2.7.2.9) has been isolated and analyzed both genetically and physically. The whole int-N region is substituted for a short chromosomal segment corresponding almost exactly to the car locus. The study of CPSase, ornithine carbamoyltransferase, and aspartate carbamoyltransferase regulation in carriers of lambdadcar confirms the previously reported participation of the argR gene product in the control of CPSase synthesis and points to the existence of a regulatory molecule involved in the control of both CPSase and aspartate carbamoyltransferase synthesis. The general usefulness of using N- lambda transducing bacteriophages for the recovery of large amounts of gene products is discussed.