Selective retention of recombinant plasmids coding for human insulin

Plasmids may be lost from Escherichia coli K-12 hosts that are cultured without selection for plasmid retention. This is particularly true for chimeric plasmids that incorporate genes for human insulin into vectors derived from pBR322. The cIts857 gene of bacteriophage lambda was inserted into the bla gene of the human-insulin-coding plasmids, pIA7 delta 4 delta 1, pIB7 delta 4 delta 1 and pHI7 delta 4 delta 1, generating the new plasmids pPR17, pPR18 and pPR19, respectively, which produced the thermosensitive lambda repressor. The cI gene was downstream from the pM and pbla promoters, so that it may have been expressed from either or both promoters. Separate E. coli K-12 RV308 host strains containing the new recombinants were lysogenized with the repressor-defective bacteriophage lambda cI90. Loss of the plasmid from the lysogens causes concomitant loss of the lambda repressor and cell death, because the prophage is induced to enter the lytic growth cycle. The system effectively forces retention of the plasmid in all viable cells in the culture.     

Induction of lambdoid prophages by amino acid deprivation: Differential inducibility; Role of recA

Summary Lambda prophage in auxotrophic lysogens can be induced by omission of one or combinations of the required amino acids from the culture medium. Such amino acid deprivation can result in nearly as effective induction of lambda as thymine deprivation. Prophage 424 is also induced equally effectively under both conditions although to a lesser extent than lambda. By contrast prophage 21 and i²¹ are differentially induced effectively by thymine deprivation and virtually not at all during amino acid deprivation. The same differential induction of 21 and equivalent induction of and 424 occur when all three prophages are present in the same lysogen. Increasing the levels of repressor with a cI carrying-plasmid prevented amino acidless induction of as did the ind ^– mutation. A recA, but not a recB, mutation in the host prevented induction by amino acid deprivation. A recC mutant host showed increased spontaneous induction of and 21 prophages. The findings reported are used as an argument that the recA protease probably is not itself acting as the inducing protease and that a likely source of the observed specificity is an effector molecule. Different effector molecules may be produced in response to different exigent situations, to which the phage repressors may have evolved sensitivity. i⁸⁰ was inducible both by amino acid and thymine deprivation.     

Mutants in the y region of bacteriophage lambda constitutive for repressor synthesis: their isolation and the characterization of the Hyp phenotype

Bacteriophage lambdahyp mutants have been isolated as survivors of Escherichia coli K-12 bacteria lysogenic for lambda Nam7am53cI857. The hyp mutants are characterized by (i) their localization in the y region very close to the imm lambda/imm434 boundary, (ii) polarity on O gene expression, (iii) immediate recovery of lambda immunity at 30 degrees C after prolonged growth of lambda Nam7am53cI857 hyp lysogens at 42 degrees C even in the presence of an active cro gene product, (iv) ability of phage lambda v2v3vs326 but not lambda v1v2v3 to propagate on lambda cI+hyp lysogens, (v) inability to express lambda exonuclease activity after prophage induction, and (vi) inviability at any temperature of phage carrying the hyp mutation. All these properties are referred to collectively as the Hyp phenotype. We show that the Hyp phenotype is due to cII-independent constitutive cI-gene-product synthesis originating in the y region, which results in the synthesis of anti-cro RNA species, and constitutive levels of cro gene product present even in lambda cI+hyp lysogens. A model is presented which is consistent with all the experimental observations.     

Correlation between UV dose requirement for lambda bacteriophage induction and lambda repressor concentration.

Escherichia coli K-12 wild type and a uvrA mutant derivative were used to construct isogenic strains bearing one, two, three, or more phage lambda cI genomes and containing increasing concentration of lambda repressor as measured by in vitro operator DNA-binding assays. The survival and phage induction in response to UV irradiation were determined. In both strains, dose-response relationships were obtained as a function of the cellular repressor concentration. The uvrA lysogens required one-tenth the UV fluence of the wild-type counterparts for induction. Lysogenic strains containing plasmids that overproduce the lambdaind+ repressor and the same lysogens with plasmids overproducing the lambdaind- repressor displayed the same survival curves as the nonlysogenic parental strain; however, only the former produced infectious centers (at a frequency of 2 x 10(-3) to 5 x 10(-4) in response to radiation.     

Bacteriophage lambda repressor allelic modulation of the Rex exclusion phenotype

The sensitivity of delta red-gam delta ren mutants of bacteriophage lambda to Rex exclusion by lambda rexA+ rexB+ lysogens is modulated by the prophage cI repressor allele. We show the following: (i) lambda spi156 delta nin5 forms plaques on a cI+-rexA+-rexB+ lysogen with 10(5)-fold higher efficiency than on cI[Ts]-rexA+-rexB+ derivatives. (ii) The cI[Ts]857 allele augmentation of Rex exclusion is recessive to cI+. (iii) The cI857-mediated increase in Rex exclusion activity involves the participation of a genetic element mapping outside of cI-rexA-rexB.     

Efficiency of induction of prophage lambda mutants as a function of recA alleles.

Mutants of the cI gene of prophage lambda have been defined phenotypically in a recA+ host as noninducible (Ind-), inducible (Ind+), or induction sensitive (Inds). We showed that a phage lambda cI+ carrying operator mutations v2 and v3 displays an Inds phenotype, as does lambda cI inds-1. We characterized a fourth induction phenotype called induction resistant (Indr). Using these four prophage types, we tested the influence of bacterial recA mutations on prophage induction. Indr prophages were fully induced in recA441 bacteria whose RecA441 protein is activated constitutively. Indr prophages were not induced in a mutant overproducing RecA+ protein, confirming that RecA+ protein must be activated to promote prophage induction. Inds prophages were induced in recA142 and recA453-441 lysogens, previously described as deficient in prophage induction.     

Spontaneous Mutation at a 5-Methylcytosine Hotspot Is Prevented by Very Short Patch (Vsp) Mismatch Repair

In many strains of Escherichia coli, the product of gene dcm methylates the internal cytosines in the sequence 5'CC(A or T)GG. Spontaneous deamination of 5-methylcytosine produces thymine which, if not corrected, can result in a transition mutation. 5-Methylcytosines in the lacI gene are hotspots for spontaneous C to T mutations. dcm is linked to vsr, a gene required for very short patch (VSP) repair. VSP repair corrects T.G mispairs in the following contexts:CTAAGGGGTCC, CTTGGGGACC, TAGGGTCC and CTAGGGTC. I have investigated the relationships between cytosine methylation, mutation, and VSP repair. Spontaneous mutations in the repressor (cI) gene of lambda prophage were isolated in wild-type and mutant lysogens. A hotspot for spontaneous mutation that corresponds with a 5-methylcytosine was observed in wild-type lysogens but was not present in bacteria lacking both methylase and VSP repair activity. Introduction of a plasmid containing dcm+ and vsr+ restored the mutation hotspot. If the added plasmid carried only dcm+, the frequency of spontaneous mutations at the 5-methylcytosine was over 10-fold higher than in Dcm+Vsr+ lysogens. The addition of vsr on a plasmid to a wild-type lysogen resulted in a 4-fold reduction in mutation at the hotspot. These findings support the previously untested hypothesis that VSP repair prevents mutations resulting from deamination of 5-methylcytosine.     

Altered temperature induction sensitivity of the lambda pR/cI857 system for controlled gene E expression in Escherichia coli

Cell lysis of Gram-negative bacteria can be efficiently achieved by expression of the cloned lysis gene E of bacteriophage PhiX174. Gene E expression is tightly controlled by the rightward lambda pR promoter and the temperature-sensitive repressor cI857 on lysis plasmid pAW12. The resulting empty bacterial cell envelopes, called bacterial ghosts, are currently under investigation as candidate vaccines. Expression of gene E is stringently repressed at temperatures up to 30 degrees C, whereas gene E expression, and thus cell lysis, is induced at temperatures higher than 30 degrees C due to thermal inactivation of the cI857 repressor. As a consequence, the production of ghosts requires that bacteria have to be grown at 28 degrees C before the lysis process is induced. In order to reflect the growth temperature of pathogenic bacteria in vivo, it seemed favorable to extend the heat stability of the lambda pR promoter/cI857 repressor system, allowing pathogens to grow at 37 degrees C before induction of lysis. In this study we describe a mutation in the lambda pR promoter, which allows stringent repression of gene E expression at temperatures up to 36 degrees C, but still permits induction of cell lysis at 42 degrees C.     

The OR control system of bacteriophage lambda. A physical-chemical model for gene regulation

A quantitative model has been developed for processes in the bacteriophage lambda that control the switchover from lysogenic to lytic modes of growth. These processes include the interactions of cI repressor and cro proteins at the three DNA sites of the right operator, OR, the binding of RNA polymerase at promoters PR and PRM, the synthesis of cI repressor and cro proteins, and the degradative action of recA during induction of lysis. The model is comprised of two major physical-chemical components: a statistical thermodynamic theory for relative probabilities of the various molecular configurations of the control system; and a kinetic model for the coupling of these probabilities to functional events, including synthesis of regulatory proteins cI and cro. Using independently evaluated interaction constants and rate parameters, the model was found capable of predicting essential physiological characteristics of the system over an extended time. Sufficiency of the model to predict known physiological properties lends credence to the physical-chemical assumptions used in its construction. Several major physiological characteristics were found to arise as "system properties" through the non-linear, time-dependent, feedback-modulated combinations of molecular interactions prescribed by the model. These include: maintenance of the lysogenic state in the absence of recA-mediated cI repressor degradation; induction of lysis and the phenomenon of subinduction; and autogenous negative control of cro. We have used the model to determine the roles, within the composite system, of several key molecular processes previously characterized by studies in vitro. These include: co-operativity in cI repressor binding to DNA; interactions between repressors and RNA polymerase (positive control); and the monomer-dimer association of cI repressor molecules. A major role of cI repressor co-operativity is found to be that of guaranteeing stability of the lysogenic state against minor changes in cI repressor levels within the cell. The role of positive control seems to be that of providing for a peaked, rather than monotonic, dependence of PRM activity on cI repressor level, while permitting PR activity to be a step function. The model correlates an immense body of studies in vivo and in vitro, and it makes testable predictions about molecular phenomena as well as physiological characteristics of bacteriophage lambda. The approach developed in this study can be extended to include more features of the lambda system and to treat other systems of gene regulation.