Cro Gene Research Articles

High-level expression of genes under control of cro translation initiation signals in Escherichia coli

The expression of several genes in Escherichia coli under the control of the lambda p R promoter and translation initiation signals of the lambda cro gene were studied. Fusions were made in frame at the initiation codon and/or with 5′ translated cro fragments. Expression fluctuated strongly when genes were fused directly at the ATG, whereas constructs, which encode hybrid genes that include at least the first nine codons of the cro gene, always directed high-level synthesis. These fusion proteins were mainly intracellularly precipitated. Our results confirm the poor reliability of ATG vectors for the expression of cloned genes. On the other hand, useful levels of expression are obtained when genes are fused to 5′ cro coding sequences, presumably due to an efficient ribosome binding site configuration.

Analysis of nutR, a site required for transcription antitermination in phage lambda.

Deletions extending from the cro gene into boxA and nutR of the Rho-dependent tR1 terminator of bacteriophage lambda have been generated and cloned between promoters and the galK gene of Escherichia coli on a multicopy plasmid. Terminators placed between the promoters and galK restrict transcription and expression of galK on these plasmids. However, when lambda N protein is provided, and if a functional N interaction site, nutR, is intact, transcription antitermination occurs and galK expression increases. Deletions into the nutR region affect the ability to antiterminate. From the results obtained we conclude that: boxA, a site believed to bind host factors (Nus), is not required for transcription antitermination in this system; the host NusA function is required even in the absence of boxA; nutR is required for N antitermination; translation across the nutR sequence prevents N-dependent antitermination.

A ‘phase-shift’ fusion system for the regulation of foreign gene expression by lambda represser in Gram-negative bacteria

A ‘phase-shift’ translation fusion vector was constructed in which mutually compatible restriction sites BamHI, BelI and BglII are positioned in such a manner that the cut point is in a different reading frame, immediately following the ATG start codon and ribosome-binding site of the λ cro gene. The λ cro gene is expressed from promoter p R and controlled by a thermosensitive (cI857) λ repressor. The usefulness of the expression vector was demonstrated using a galϰ gene lacking the ATG start codon and fusing this to the p R promoter and ATG start codon of the λ cro gene, resulting in cI857-regulated expression of galactokinase. The vector is of general use for foreign gene expression in Escherichia coli when the target gene has a compatible cohesive end (5′-GATC-3′) at the N terminus (provided, for example, by a BamHI linker). The A λ cI857- p R - cro-galK cassette was cloned into pJRD215, a wide-host-range plasmid and transferred by conjugation to a variety of Gram-negative bacteria. In all cases, thermosensitive regulation of galactokinase could be demonstrated, though the levels of induction varied considerably. These results show that the powerful λ p R promoter and the efficient A repressor can be used to regulate expression of foreign genes in Gram-negative organisms other than E. coli.

Bacteriophage lambda cro mutations: effects on activity and intracellular degradation.

Following random mutagenesis of the bacteriophage lambda cro gene, we have isolated missense mutations that affect approximately half of the 66 residue positions of Cro. About two-thirds of the mutations change residues involved in the maintenance of Cro structure and stability. The corresponding mutant proteins are severely degraded in the cell but often have specific activities near that of wild-type Cro. The remaining mutations affect residues involved in DNA binding. These mutant proteins are present at moderately reduced intracellular levels, but their specific activities are much lower than that of wild type.

Overproduction of proteins encoded by lambda replicon-integrated plasmid: effect of partial inactivation of cI repressor.

Composite plasmids containing the 7.2-kb fragment of bacteriophage lambda cI857cro27, essential for lambda DNA replication and its control, have been constructed. The plasmids contained, in addition, pBR322, a part of Co1E1, and the Escherichia coli pss gene, coding for phosphatidylserine synthase. When the plasmid-harboring cells were induced at different temperatures, the phosphatidylserine synthase production was maximum at 37 degrees C, and the specific activities at 37 degrees C continuously increased until the culture reached saturation. The results suggest that, when the cro gene is defective, only a transient temperature for inactivation of cI repressor provides both the increase in copy number of the plasmids and cellular activities that allow overproduction of the plasmid-encoded protein.

Bacteriophage P22 Cro protein: sequence, purification, and properties.

The DNA sequence of part of the bacteriophage P22 early regulatory region, including genes cro and c1, was determined. The protein product of the cro gene consists of 61 amino acid residues, and that of c1, 92 amino acid residues. Both genes were placed separately in plasmids from which they are expressed from a controllable promoter in vivo. Induced cells bearing the cro-expressing plasmid were used as a source for purifying and characterizing the Cro protein. The amino-terminal sequence of this protein was found to be as predicted by the DNA sequence; close agreement was also observed between its predicted and experimentally determined amino acid composition and molar extinction coefficient at 280 nm. In gel filtration experiments, Cro protein at concentrations around 10(-5) M appears to have a molecular weight of 8600, which is more consistent with monomers (6800) than with dimers (13 600). Cro protein binds specifically to the three repressor binding sites in the P22 right operator; in order of decreasing affinity, these are OR3, OR1, and OR2.

Synthesis of fused glycoprotein D of Herpes simplex virus type 1 but not type 2 inhibits Escherichia coli hosts

Glycoprotein D from either Herpes simplex virus type 1 (gD-1) or type 2 (gD-2) has been expressed in Escherichia coli as a series of chimeric proteins. The expression vector used in this study, pJS413, was derived from pBR322 and contains several cloning sites between the lacZ promoter-operator and the phage λ cro gene. Plasmids containing fusions between the cro gene, gD-related sequences and lacZ was constructed and shown to direct the synthesis of 160-kDa proteins. The accumulation effusion protein could be visualized as inclusion bodies when the cells were examined by dark phase-contrast or transmission electron microscopy. None of the plasmids that encoded cro : : gD gene fusions yielded significant amounts of material upon induction with isopropyl-β- d-thiogalactopyranoside. In addition, certain plasmids produced a form of Cro-gD-1 fusion protein which resulted in severe growth inhibition of E. coli. These inhibitory effects were attributed to the presence of specific gD-1 sequences i.e., the transmembrane and cytoplasmic anchor region of the protein.

A system for detection of genetic and epigenetic alterations in Escherichia coli induced by DNA-damaging agents.

In order to compare the genetic and epigenetic effects of genotoxic agents, we have constructed Escherichia coli K12 strains that allow the detection of mutagenesis, SOS induction (epigenetic effect) and genetic recombination in the same genetic background. The epigenetic effect was detected in a similar way to any genetic alteration, i.e. by counting altered clones (colonies), using a gene fusion system that responds to a temporary epigenetic effect by a stable, heritable switch. The gene fusion consists of the E. coli gal operon and a partially deleted prophage lambda, resulting in the gal operon coming under the control of the cI and cro genes. It allows the detection of SOS induction and forward mutagenesis in the cI gene. Even a temporary inactivation of the CI repressor in this particular system leads to a stable epigenetic switch transmitted to the cellular progeny, which can be detected as Gal+ (red) colonies. The genetic (mutational inactivation of gene cI) and epigenetic (proteolytic inactivation of the product of gene cI) mechanisms leading to gal expression can be distinguished. Genetic recombination between two heteroallelic lacZ genes, one located in the bacterial chromosome, the other on an F'lac plasmid, can be detected as Lac+ colonies. Radiation and several chemical mutagens show very different capacities in generating mutants, inductants and recombinants; therefore, a dose range of any physical or chemical agent generates a set of relative values for the generation of mutants, inductants and recombinants that are characteristic of the agent.

Dominant negative mutations in the Tn10 tet repressor: evidence for use of the conserved helix-turn-helix motif in DNA binding.

The Tn10 tet repressor regulates transcription of the tetracycline-resistance determinant in transposon Tn10. Previous DNA sequencing studies identified a region of tet repressor (amino acids 26-47) that is homologous to the helix-turn-helix regions of lambda Cro, lambda repressor, and catabolite gene activator protein that are implicated in sequence-specific DNA binding. Here we report the isolation of dominant tetR mutations that result in tet repressors deficient in tet operator binding but that retain some capacity to form dimers with, and thereby inactivate, wild-type repressor monomers. The mutations were isolated by transforming a tetR+ tetA-lacZ fusion strain with hydroxylamine-mutagenized tetR plasmid DNA and then screening for increased lacZ expression. DNA sequence analysis of 35 independent isolates identified seven different mutations, five of which are in the region of helix-turn-helix sequence homology. In vitro binding studies indicate that the mutations in this region of tet repressor reduce the affinity of tet repressor for tet operator DNA by at least a factor of 1000 but have no significant effect on the affinity of tet repressor for tetracycline. These results provide strong support for the proposal that tet repressor utilizes the conserved helix-turn-helix structural motif in binding to tet operator DNA.

Interaction of rho factor with bacteriophage lambda cro gene transcripts.

Rho protein is responsible for termination of transcription of the cro gene of bacteriophage lambda. Since rho is known to interact with the RNA whose synthesis is being terminated, we measured the specificity and strength of binding of rho to isolated cro transcripts, using a nitrocellulose filter retention assay. The association constant (K alpha) for the binding of rho to a 372-nucleotide cro transcript was determined to be 7 +/- 2 X 10(8) M-1 at 37 degrees C and about 20-fold less at 4 degrees C. Although NTP cleavage is required for rho activity, the presence of ATP did not alter the K alpha. Rho bound less tightly (K alpha less than 10(8) M-1) to partial cro transcripts smaller than 290 nucleotides and had very little affinity (K alpha less than 10(6) M-1) for lambda 4 S RNA, lambda 6 S RNA, and partial cro transcripts smaller than 160 nucleotides. In contrast, cro transcripts as short as 100 nucleotides bound if guanosine residues were replaced with inosine. In addition, rho bound readily to 3' fragments of cro RNA that had 85 or more residues. A common feature of the RNA molecules that bind tightly to rho protein is that they have a stretch of at least 85 nucleotides with relatively few (less than 14%) guanosine residues. Such a segment is thus likely to be largely single-stranded. These results suggest that the binding of rho to lambda cro mRNA is dependent on a 3' terminal segment that has those properties.

Conservation of genome form but not sequence in the transcription antitermination determinants of bacteriophages λ, φ21 and P22

Comparisons are made among DNA sequences upstream from terminators in both leftwards and rightwards early operons of related coliphages λ, φ21 and P22. These sequences include both left and right determinants of response to phage-coded antitermination proteins, “ N”, as well as the N structural genes themselves. Despite almost total disparity of DNA sequence, the three genomes can be discerned to include the same elements in the same order and spacing: downstream from the early left promoter are sequentially a site of recognition for host nusA protein, a dyad symmetry “ nut” essential for N function in λ, overlapping sites for processing of the transcript by RNAase III and then the N structural genes; downstream from the cro gene on the right are sites of nusA recognition and nut dyad symmetries homologous to those on the left. Because the N proteins of λ, φ21 and P22 do not for the most part complement each other, a specific site of N recognition has been postulated for each N-responding operon. The nut dyad symmetry qualifies as such a site, since the loop of the left dyad in λ is marked by mutations that block N function leftwards, and since DNA sequences here show close homology between the loops of left and right dyads for each phage, but less if not little homology for different phages.

Differential expression of influenza N protein and neuraminidase antigenic determinants in Escherichia coli

Two influenza gene products of similar size and codon usage have been expressed in Escherichia coli under control of the phage λ p R promoter. The influenza N protein (NP) was expressed in its entirety after fusion to a short (12 amino acid) segment of the λ cro gene product and constituted about 1–2% of total soluble cell protein after induction. By contrast, constructions using the full length neuraminidase (NA) gene failed to give rise to detectable amounts of NA antigen after fusion to either the 12 amino acid Cro peptide or after fusion to bacterial β-galactosidase (βgal). Rather, expression of NA antigenic determinants was only achieved after deletion of coding sequences at the 3' end of the βgal-NA fusion construct such that the encoded protein precipitated within the cell.

A genetic analysis of defective plasmid formation by N-deficient phage lambda chromosomes.

Plasmid formation by N- derivatives of lambdoid phages has been reinvestigated with transducing phages carrying the trp, lac and gal genes of Escherichia coli. Transduction by lambda N- cI- derivatives was inefficient and short-lived in each case, under both selective and nonselective conditions. Mutant-operators were introduced to relieve possible auto-repression by the cro gene product. Such N-defective phage genomes were able to propagate continually as plasmids, although without selection they were gradually lost from the carrier cells. Plasmid formation remained inefficient, however. The entire chromosome of N- phages can be expressed by transcription that leaks through the serially arranged Rho-dependent terminators. Some functions so expressed are deleterious to the plasmid state and cause the instability of lambda N- plasmids.

Cloning and expression in Escherichia coli of the TL-DNA gene 4 of Agrobacterium tumefaciens under the control of the P r promoter of bacteriophage λ.

A plasmid was constructed that directs expression of the TL-DNA gene 4 protein in E. coli. The different steps of the construction were as follows : i) a region of gene 4 encoding the amino-terminal portion of the protein was fused in frame to DNA encoding an enzymatically active carboxy-terminal fragment of β-galactosidase. The hybrid gene was poorly expressed from the upstream λ P L promoter carried by the vector. ii) in order to generate an efficient procaryotic ribosome binding site, a DNA fragment carrying the λ P R promoter with the nearby Shine-Dalgarno (SD) sequence of gene cro was placed in front of the gene 4- lacZ fusion. A recombinant plasmid, termed pGV793, that expressed efficiently a fused protein 4-β-galactosidase was identified among the Lac + clones. DNA sequencing analysis showed that pGV793 carried a hybrid ribosome binding site composed of the cro SD sequence, a five bp sequence and the ATG codon of gene 4. Plasmid pGV793 directed the synthesis of three polypeptides of molecular weight 132 Kd, 126 Kd and 122 Kd that carried β-galactosidase antigenic determinants. The largest polypeptide had the expected size for the hybrid protein. The fusion proteins which accounted for about 0.5% of the total cellular proteins were purified by immunoadsorption using anti-β-galactosidase antiserum. iii) the complete gene 4 coding sequence was reconstituted, with the λ P R promoter in place. The resulting pGV822 plasmid expressed a polypeptide whose molecular weight 27 Kd corresponded to the expected size for the gene 4 product. The pI was about 7.

Evidence of cell fragility caused by gene kil following λ induction

Escherichia coli cells carrying λ cI857 prophage lyse 40 min after λ thermoinduction; the lysis depends on the λ genes Q, R, and S. If chloramphenicol (CAP) is added within 20 min after λ cI857 induction, an early, unproductive lysis occurs. This lysis is independent of the genes int, rex, O, P, Q, and all late genes. Instead, early lysis depends upon the kil gene. The early lysis is under the positive control of λ gene N and the negative control of gene cro. One or more events specifically connected with λ induction appear to be necessary for the occurrence of early lysis, since early lysis cannot be observed after λ infection. Induced λ kil + lysogens are more sensitive to osmotic shock than induced λ kil- lysogens. CAP-induced early lysis can be prevented in a hypertonic medium. These results suggest that induction of λ causes an osmotic fragility due to a damage of the cell envelope which requires repair; in the absence of protein synthesis the cell envelope is not repaired and cell lysis ensues.

Effect of multiplicity of infection on transcription in Escherichia coli cells infected by bacteriophage lambda.

The effect of multiplicity of infection (m.o.i.) on transcription was studied by infecting Escherichia coli with bacteriophage lambda cI47, lambda cI47O29P3 and lambda cI857cro27P3. DNA-RNA hybridization with lambda cI47 l-strand DNA, phi 80imm lambda r-strand DNA, and lambda imm80 r-strand DNA were used to measure mRNA transcription from the l-strand, the r-strand of the early x-P-Q region and the late A-J-b2 region of lambda bacteriophage respectively. In lambda cI47cro+O-P--infected cells, transcription from the l-strand, r-strand of the early x-P-Q region and the late A-J-b2 region all decreased with increasing m.o.i. The response in the x-P-Q region was less marked than in other regions, but the pattern looked similar to that described above. When phage DNA replication was permitted, as in the case of lambda cI47, the response was similar to that observed in lambda cI47cro+O-P--infected cells, but the level of transcription was increased two- or threefold. In lambda cI857cro-P--infected cells, the leftward transcription and the rightward transcription from the early x-P-Q region and the late A-J-b2 region all increased with increasing m.o.i., but the extent of change was less drastic than with lambda cro+. This result demonstrated clearly that the decrease in transcription from various regions at increasing m.o.i. of lambda cro+ was due to the inhibitory action of the cro gene product. The results obtained with cro- strongly support the view that gene dosage is a significant controlling factor for the extent of gene expression.

Analysis of nutR: A region of phage lambda required for antitermination of transcription

The N gene product of coliphage lambda acts with host factors (Nus) through sites (nut) to render subsequent downstream transcription resistant to a variety of termination signals. These sites, nutR and nutL, are downstream, respectively, from the early promoters PR and PL. Thus a complicated set of molecular interactions are likely to occur at the nut sites. We have selected mutations in the nutR region that reduce the effectiveness of pN in altering transcription initiating at the PR promoter. DNA sequence analysis of three independently selected mutations revealed, in each case, a deletion of a single base pair in the cro gene. Consideration of the effect of such mutations on the extension of translation of cro message into the adjacent downstream nut region led to the identification of a consensus sequence CGCTCT(T)TAA that appears to play a role in the recognition of a host factor, possibly the NusA protein.

An IS4 transposition causes a 13-bp duplication of phage λ DNA and results in the constitutive expression of the cI and cro gene products

We have examined the nature of the additional DNA present in λ hyp $ ̄ mutants (Eisen et al., 1982). This DNA is an IS4 element in orientation I, in the y region of bacteriophage λ at nucleotide position 39 139 (see Moore et al., 1979). Our assignment is based on (i) the similarity in size derived from the PstI, AvaI, and HindII restriction pattern and (ii) the DNA sequence of both the left and right λ-lS4 DNA junctions in phage λ hyp15rev4. The IS 4 integration event resulted in the duplication of 13 bp of X DNA in contrast to the 11- and 12-bp duplications previously observed at the sites of IS 4 integrations elsewhere (Klaer et al., 1981).

Mutants in the y region of bacteriophage X constitutive for represser synthesis: Their isolation and the characterization of the Hyp phenotype

Bacteriophage λ hyp mutants have been isolated as survivors of Escherichia coli K-12 bacteria lysogenic for λ Nam7am53cI857. The hyp mutants are characterized by (i) their localization in the y region very close to the immλ/ imm 434 boundary, (ii) polarity on O gene expression, (iii) immediate recovery of X immunity at 30°C after prolonged growth of λ Nfam7am53cI857 hyp lysogens at 42°C even in the presence of an active cro gene product, (iv) ability of phage λ v 2 v 3 v s326 but not λ v 1 v 2 v 3to propagate on gl cI + hyp lysogens, (v) inability to express X 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 ell-independent constitutive cl-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 λ cI + hyp lysogens. A model is presented which is consistent with all the experimental observations.

Enhanced expression of cro-beta-galactosidase fusion proteins under the control of the PR promoter of bacteriophage lambda.

Hybrid plasmids carrying cro-lacZ gene fusions have been constructed by joining DNA segments carrying the PR promoter and the start of the cro gene of bacteriophage lambda to the lacZ gene fragment carried by plasmid pLG400 . Plasmids in which the translational reading frames of the cro and lacZ genes are joined in-register (type I) direct the synthesis of elevated levels of cro-beta-galactosidase fusion protein amounting to 30% of the total cellular protein, while plasmids in which the genes are fused out-of-register (type II) produce a low level of beta-galactosidase protein. Sequence rearrangements downstream of the cro initiator AUG were found to influence the efficiency of translation, and have been correlated with alterations in the RNA secondary structure of the ribosome-binding site. Plasmids which direct the synthesis of high levels of beta-galactosidase are conditionally lethal and can only be propagated when the PR promoter is repressed. Deletion of sequences downstream of the lacZ gene restored viability, indicating that this region of the plasmid encodes a function which inhibits the growth of the cells. The different applications of these plasmids for expression of cloned genes are discussed.