Kinetics of bacteriophage lambda repressor synthesis directed by the PRE promoter: influence of temperature, multiplicity of infection, and mutation of PRM or the cro gene

Summary Expression of the P _RE (establishment) pathway for repressor synthesis is regulated both by phage-specific genetic elements and by physiological conditions. Here we describe the effects of temperature, multiplicity of infection, mutations in the cro gene, and a mutation in P _RM on P _RE-directed repressor synthesis. As Reichardt (1975a) has shown, repressor synthesis begins 5–15 min after infection by wildtype phage, and is shut off at 20–30 min after infection, depending on the temperature. At 43°, synthesis starts sooner, shuts off earlier, and leads to lower repressor levels than are attained at lower temperatures. Experiments with the temperature sensitive mutant crots20 demonstrate that, as had been shown previously in experiments at 30° and 37° C, cro protein is responsible for the shut-off of repressor synthesis at 43°. In addition to the effects of temperature, the kinetics of repressor synthesis are strongly affected by multiplicity of infection (moi). At mois greater than 10, repressor synthesis after infection by wildtype at 30° is dramatically inhibited. Unexpectedly, the P _RM mutation prm116, under certain conditions, can alleviate both cro-mediated shutoff and the inhibition of P _RE-directed repressor synthesis at high moi. These effects of prm116 are observed only at low temperature (30°–32° C) and at mois of about 6–10 or greater; they also appear to be cis-specific. Possible mechanisms for the effects of the prm116 mutation are discussed. Finally, these studies demonstrate that crots20, which was isolated as a temperature-sensitive lethal mutation in the cro gene (Herskowitz, unpublished), is temperature-sensitive with respect to the ability to shutoff P _RE-directed repressor synthesis; however, even at low temperature (30° C), the crots20 gene product is only partially active.     

An analysis of repressor overproduction by the lambda cIIIs mutant.

Efficient lysogenization of Escherichia coli K12 by bacteriophage λ requires the high level of synthesis of the phage repressor shortly after infection. This high level of synthesis of repressor requires the action of the λ eII and cIII proteins. Certain mutants of λ (λcIIIs) appear to have excess $\text{c}\text{II}\text{c}\text{III}$ activity and can lysogenize more efficiently than λ⁺. The basis for the enhanced lysogenization is that, while two or more infecting phage are necessary for λ⁺ to lysogenize, a single infecting λcIIIs particle is sufficient for lysogenization. Also, repressor levels in cells infected with λcIIIs are higher than in those infected with λ⁺. I report here that repressor overproduction by λcIIIs (1) is due to a much higher rate of repressor synthesis than that of λ⁺; (2) is most marked at low multiplicities of infection, possibly because λcIIIs produces repressor much more efficiently than λ⁺ as a singly infecting phage.     

Analysis of the reduction in expression of tetracycline resistance determined by transposon Tn10 in the multicopy state

The tet genes of transposon Tn10 have been mapped in a 2,200 bp DNA sequence by analysing deletion and Tn5 insertion mutations. When the tet genes were present on multi-copy plasmids the level of resistance expressed was about ten-fold lower than that determined by a single copy of Tn10 in the E. coli chromosome. The 36K tet protein known to be encoded by R100 in E. coli minicells was not detected when they harboured a multicopy tet plasmid. However, normal high levels of resistance were expressed when the tet genes were recombined into the host chromosome as part of a lambda lysogen, showing that the multicopy effect was phenotypic. Most of the Tn5 insertions and deletions in tet which caused Tc^s mutations also prevented expression of high level Tc^r from a chromosomal Tn10 element present in the same cell. Only those insertions in the promoter-proximal 90–130 bp of a 1,275 bp HindII fragment known to carry the gene encoding the 36K tet protein did not reduce the single copy Tn10 resistance level.A gene fusion system that results in the constitutive synthesis of -galactosidase from a tet promoter has been used to assay tet repressor activity. The basal (uninduced) -galactosidase level in cells carrying multicopy tet plasmids was 10–20 fold lower than those carrying a single copy. The tet:: Tn5 mutants defective in the trans-dominant multicopy effect still made normal amounts of tet repressor showing that repressor overproduction was not responsible for this effect. In addition a repressor-defective constitutive mutant did not exhibit a higher resistance level when located on a multicopy plasmid vector. We postulate that a regulatory mechanism recognises the amino-terminus of the tet structural gene product when attempts are being made to overproduce the protein and prevents further translation.     

Daughter cells as an important factor in determining the physiological state of yeast populations

Cells of Candida utilis grown in a single-stage chemostat at D = 0.05, 0.1, 0.25, and 0.35 hr^?l were separated into a fraction of scar-bearing mother cells and a fraction of scar-free daughter cells. The scar-free cells were transferred into small batch cultures where the length of the maturation phase, changes in length and width of cells, specific growth rate, and specific rate of RNA and protein synthesis were examined for 5 hr. The daughter cells grown at D = 0.05 hr^?1 were very small at the moment of separation from the mother cells (about one-third of the mother cell). Their maturation phase (in a batch culture), at the beginning of which they attain the specific growth rate approaching the μ_max of the strain used, lasts for 3 hr. On the other hand, daughter cells grown at D = 0.35 hr^?1 are almost the same size as the mother cells at the moment of separation. After transfer to a batch culture they begin to bud almost immediately. Similarly, in their other morphological and physiological parameters they differ strikingly from immature daughter cells which are formed at low specific growth rates. The importance of these differences from the point of view of mathematical modeling of growth processes is discussed.     

Kinetics of c2-repressor synthesis in a regulatory defective P22 mutant

Summary Phage P22 defective in gene 24 and harbouring the o^c mutation k5 in O_R exhibits a strongly increased c2-repressor synthesis after infection of non-lysogenic S. typhimurium. The repressor synthesis depends strictly on an intact c1 gene. The kinetics of its synthesis, as monitored by polyacrylamid gel electrophoresis, is the same as with P22 c ⁺, namely a turn off 8–10 min after infection. — After infection of P22-lysogenic bacteria with either P22 24 ^– k5 or P22 24 ^– k5 cl, much lower amounts of repressor are synthesized but again with the same kinetics. These results suggest a cro-like function acting at P_RE and P_RM of P22. The possible reason for the c2 overproduction is discussed.     

Lysogenization by bacteriophage lambda: II. - Identification of genes involved in the multiplicity dependent processes

We previously showed that, under conditions of rapid exponential growth, lysogenization of E. coli cells by phage λ requires that the cell is infected by at least 2 phages able to replicate their DNA, or 3 or 4 phages unable to replicate their DNA [ref. 4]. Since genes dealing with prophage integration appear not to be involved in these multiplicity dependent processes, a determination was made as to whether more than one copy of the genes involved in repressor synthesis or its activation are needed for lysogenization. The complementation patterns which we obtained indicate multiplicity effects involving gene cII (and, perhaps, cIII) in lysogenization by both phage able or unable to replicate. In the former case, we propose that cII protein (and, perhaps, cIII) both induces repressor synthesis and inhibits phage DNA replication. In lysogenization by phage unable to replicate, the data suggest that the expression of early phage genes and repressor synthesis in the course of lysogenization are mutually exclusive processes which do not take place on the same phage chromosome.     

Inhibition of peptide chain initiation by a nonhemin-regulated translational repressor from Friend leukemia cells.

A nonhemin-regulated translational repressor protein has been purified partially from the postribosomal supernatant fraction of Friend leukemia cells grown in the absence of dimethylsulfoxide. This repressor inhibits protein synthesis in lysates from rabbit reticulocytes or Friend leukemia cells and in a fractionated system using Artemia salina ribosomes, reticulocyte mRNA, and soluble components from reticulocytes. In contrast, the hemin-controlled repressor from reticulocytes does not inhibit protein synthesis in lysates from Friend leukemia cells. The repressor from Friend leukemia cells has no effect on poly(U)-directed synthesis of polyphenylalanine using reticulocyte ribosomes nor on the extension and release of nascent globin chains that were initiated in intact reticulocytes. It does not block completion of peptides on ribosomes isolated from reticulocytes incubated with NaF nor does it inhibit initiation factor-dependent formation of methionylpuromycin, but it inhibits globin mRNA-dependent methionylvaline synthesis. The Friend leukemia cell repressor promotes peptide synthesis-dependent breakdown of polysomes in reticulocyte lysates that appears to involve inhibition of ribosome reattachment to mRNA during peptide chain initiation. It is concluded that the Friend leukemia cell repressor blocks peptide initiation at a point between the addition of methionyl-tRNA^fMet to the ribosomal initiation complex and the NaF-sensitive reaction.     

Identification of protein X of Escherichia coli as the recA+/tif+ gene product.

Summary Protein X, molecular weight 40,000, has been separated from the other proteins of E. coliby a two-dimensional gel electrophoretic technique which separates proteins according to isoelectric point (pI) in the firstdimension and according to molecular weight in the second. When protein X is induced in wild-type cells by mitomycin C treatmentit has a pI6.0. However, when protein X is induced in a tif-1 mutant, either by temperatureshift-up to 42° or by mitomycin C treatment at 30°, it has a pI6.2. The low level of protein X which is present inuninduced tif mutants at 30° also has a pI6.2. These results suggest thattif-1 is a mis-sense mutation in the gene coding for protein X. Since transduction andcomplementation studies indicate that tif-1 is a mutation of therecA ⁺ gene (Castellazzi, Morand, George and Buttin, 1977) it follows that protein X is the recA ⁺ gene product.A model has been formulated to account for the relationship between protein X synthesis and the recA ⁺ and lexA ⁺ genes. In this model, a repressor coded by lexA ⁺ binds to the operator of the recA ⁺ gene from whence it can normally only be removed by the combined action of an inducer and protein X, the recA ⁺ product. Thus, protein X controls its own synthesis. The tif-1 mutation leads to a temperature sensitive form of protein X which, at 42°, can spontaneously remove the repressor without the intervention of the inducer.     

Deletions fusing the i and lac regions of the chromosome in E. coli: isolation and mapping

Summary Three hundred and fifty deletions ending within the i gene (which codes for the lac repressor) at one terminus and within the z gene at the other terminus have been isolated. Twenty-nine of these have been mapped in detail and have endpoints which are distributed throughout the i gene. Two deletions which remove only the very end of the i gene still permit the synthesis of altered repressor molecules retaining some properties of the wildtype lac repressor. In strains carrying these deletions, the synthesis of lac permease is under the control of the i promoter. An additional 40 deletions have been found which apparently fuse the lac permease to an untranslated portion of the terminus of the i-message.     

Mutational analysis of the operators of bacteriophage lambda

Summary O ^c mutations in the operators of bacteriophage lambda have been used to analyze the functional organization of the operators. In each operator, repressor binding sites 1 and 2, as identified biochemically, were found to be primarily responsible for the repressor affinity of the operators in vitro and for the repression of lytic functions in vivo. In addition, both sites were shown to be involved in the action of cro product at the operators. The data obtained have been used to estimate the repressor affinities of the individual binding sites. These affinities suggest that repressor bound at O _R1 and O _R2 interacts cooperatively. The results obtained support a model for repression of the early lambda operons where repressor bound at binding sites 1 and 2 interferes with RNA polymerase binding to the promoter sites.     

Alteration of the rate of cell division independent of the rate of DNA synthesis in a mutant of Salmonella typhimurium

Summary Salmonella typhimurium strain IIG has a temperature—sensitive DNA synthesis initiation apparatus and completes rounds of DNA replication when shifted to 38°. At this temperature there is a period of apparently normal division followed by a second phase in which DNA-less cells are produced. The rate of division in this second phase can be markedly increased if a culture growing in MM is shifted to nutrient broth at the time of the temperature shift. The extra divisions induced by the nutritional shift are not due to extra replication forks being introduced by this process nor to the rapid growth of ts ⁺ revertants. It is concluded that in this strain at 38°, the rate of division can be increased without altering the rate of DNA synthesis. The extra divisions induced by the shift-up do not take place for about 90 min. The possible occurrence of such a period between the triggering of division and the division event in normal cells is discussed.     

Direct identification of the amino acid changes in two mutant lac repressors.

Deletions extending into the trp operon at one terminus and the lacI control region at the other terminus have been examined. One of these, B116, ends within the trp leader sequence and eliminates the trp attenuator site, placing the synthesis of lac repressor under trp control. We have isolated and characterized the B116 repressor. The protein sequence of the aminoterminus of B116 shows that an additional 16 residues are added to the amino-terminal end of wild-type repressor. Moreover, a valine residue appears in place of methionine at position 17 (the original amino-terminal residue of the wild-type repressor). A comparison of the messenger RNA sequence of the trp leader region and of the I leader region demonstrates that the translation of the B116 repressor is initiated at an AUG codon within the trp leader sequence. The GUG initiation codon at the start point for translation of wild-type repressor is now read as valine, since it appears at an internal position (residue 17 of the altered repressor). The B116 repressor accumulates at levels as high as 1% of the soluble cell protein in trpR^? strains. The efficiency of the trp leader initiation codon in translation suggests that in wild-type strains this AUG is also active in directing protein synthesis, which would result in a polypeptide consisting of 14 amino acids. We have examined the physical properties of the B116 repressor, which shows a marked tendency to form higher aggregates. Other characteristics of B116 are also described.     

Induction of the inhibitor of RecBCD enzyme inEscherichia coli is alexA-independent SOS response

Exonuclease V (ExoV), an enzyme involved in the RecBCD pathway of recombination, was inhibited in cells induced for SOS functions. In vitro experiments showed that an ExoV inhibitor (Exi) induced after SOS induction was responsible for the inhibition of ExoV. Unlike other SOS functions, Exi protein was induced even inlexA(Ind^–) mutants. Phage Mud(amp^r,lac) was fused to the promoter of theexi gene in alexA(Ind^–) strain, and in these fusion strains-galactosidase was inducible five- to six-fold after DNA damage. The Exi protein, in addition to the inhibition of ATP-dependent DNase activity of ExoV, appeared to repress the synthesis of polypeptide subunits of ExoV as well. Further, Exi protein appeared to be an inducible repressor of a number of other genes in SOS-induced cells.     

The DNA sequence change resulting from the IQ1mutation, which greatly increases promoter strength

Summary DNA sequencing shows that the mutational alteration resulting from I ^Q1is a deletion of 15 base pairs in the lacI promoter. The deletion creates a greatly improved — 35 homology region, explaining the 50–100-fold increase in lac repressor synthesis.     

Evidence supporting a physiological role for the hemin-controlled translational repressor of globin synthesis in reticulocytes.

The synthesis of heme and globin in rabbit reticulocytes was compared at 35 and 25 degrees C. The lower temperature decreased heme synthesis significantly more than globin synthesis and resulted in a much greater accumulation of globin dimers. After 16 h of incubation in the absence of iron, globin synthesis in reticulocytes which were at 35 degrees C could not be stimulated by iron, whereas cells which were at 25 degrees C responded with nearly control levels of globin synthesis. Since the formation of the hemin-controlled translational repressor in reticulocyte lysates is also decreased much more than protein synthesis at reduced temperature the results provide evidence for a physiological role for the translational repressor in controlling globin synthesis in reticulocytes.     

Characterization of mutations in the penicillinase operon of Staphylococcus aureus

Summary Mutant penicillinase plasmids, in which penicillinase synthesis is not inducible by penicillin or a penicillin analogue, were examined by biochemical and genetic analyses. In five of the six mutants tested, penicillinase synthesis could be induced by growth in the presence of 5-methyltryptophan. It is known that the tryptophan analogue 5-methyltryptophan is readily incorporated into protein by S. aureus and that staphylococcal penicillinase lacks tryptophan. 5-methyltryptophan seems to induce penicillinase synthesis in wild-type plasmids by becoming incorporated into the repressor and thereby inactivating the operator binding function of the penicillinase repressor. Therefore, induction of penicillinase synthesis in the mutant plasmids by 5-methyltryptophan strongly suggests that the noninducible phenotype of these five plasmids is due to a mutation that inactivates the effector binding site of the penicillinase repressor (i.e., the five mutant plasmids carry an i^s genotype for the penicillinase repressor). This conclusion was supported by heterodiploid analysis. The mutant plasmid that did not respond to 5-methyltryptophan either produces an exceedingly low basal level of penicillinase or does not produce active enzyme. This plasmid seems to carry a mutation in the penicillinase structural gene or in the promoter for the structural gene. Thus, a genetic characterization of many mutations in the penicillinase operon can be accomplished easily and rapidly by biochemical analysis.Journal Paper No. J-7994 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project No. 2029     

ComGA‐RelA interaction and persistence in the Bacillus subtilis K‐state

The bistably expressed K‐state of Bacillus subtilis is characterized by two distinct features; transformability and arrested growth when K‐state cells are exposed to fresh medium. The arrest is manifested by a failure to assemble replisomes and by decreased rates of cell growth and rRNA synthesis. These phenotypes are all partially explained by the presence of the AAA⁺ protein ComGA, which is also required for the binding of transforming DNA to the cell surface and for the assembly of the transformation pilus that mediates DNA transport. We have discovered that ComGA interacts with RelA and that the ComGA‐dependent inhibition of rRNA synthesis is largely bypassed in strains that cannot synthesize the alarmone (p)ppGpp. We propose that the interaction of ComGA with RelA prevents the hydrolysis of (p)ppGpp in K‐state cells, which are thus trapped in a non‐growing state until ComGA is degraded. We show that some K‐state cells exhibit tolerance to antibiotics, a form of type 1 persistence, and we propose that the bistable expression of both transformability and the growth arrest are bet‐hedging adaptations that improve fitness in the face of varying environments, such as those presumably encountered by B. subtilis in the soil.