The two-component, ATP-dependent Clp protease of Escherichia coli. Purification, cloning, and mutational analysis of the ATP-binding component

The ATP-binding component (Component II, hereafter referred to as ClpA) of a two-component, ATP-dependent protease from Escherichia coli has been purified to homogeneity. ClpA is a protein with subunit Mr 81,000. It has an intrinsic ATPase activity and activates degradation of protein substrates only in the presence of a second component (Component I, hereafter referred to as ClpP), Mg2+, and ATP. The amount of ClpA varies by less than a factor of 2 in cells grown in different media and at temperatures from 30 to 42 degrees C. ClpA does not appear to be a heat-shock protein since its synthesis is not dependent on htpR. Antibodies against purified ClpA were used to identify lambda transducing phage bearing the clpA gene. The cloned gene contains a DNA sequence expected to code for the first 28 amino acids of ClpA, which were determined by protein sequencing of purified ClpA. The clpA gene in the phage was mutated by insertion of delta kan defective transposons and the mutations were transferred to E. coli by homologous recombination. The clpA gene was mapped to 19 min on the E. coli chromosome. Mutant cells with insertions early in the gene produce no ClpA protein detectable in Western blots, and extracts of such mutant cells have no detectable ClpA activity. clpA- mutants grow well under all conditions tested and are not defective in turnover of proteins during nitrogen starvation nor in the turnover of such highly unstable proteins as the lambda proteins O, N, and cII, or the E. coli proteins SulA, RcsA, and glutamate dehydrogenase. The degradation of abnormal canavanine-containing proteins is defective in clpA mutants especially in cells that also have a lon- mutation. Extracts of clpA- lon- cells have ATP-dependent casein degrading activity.     

The role of the OOP antisense RNA in coliphage λ development

We have made a derivative of bacteriophage lambda that makes no OOP antisense RNA. The mutant phage carries a point mutation that inactivates the OOP promoter, po. The phages lambda + and lambda po- have identical plaque morphologies, one-step growth curves, and frequencies of lysogenization of a sensitive host. OOP RNA synthesis is weakly repressed by the Escherichia coli LexA protein. Consonant with this inducibility of OOP RNA synthesis by ultraviolet light, we find a two-fold greater phage burst following ultraviolet induction of a lambda + than of a lambda po- prophage. In lambda + infections, OOP RNA causes two cleavage events in cll mRNA: one is in the 3'-end of the coding region, and the second is in the intercistronic region between the cll and O genes. The cll gene fragments are subject to additional hydrolytic events, and cll mRNA levels are several-fold lower in lambda + than in lambda po- infections late in the infection cycle. However, O mRNA levels are almost unaffected by the po- mutation.     

An accessory role for Escherichia coli integration host factor: characterization of a lambda mutant dependent upon integration host factor for DNA packaging.

Bacteriophage lambda grows lytically on Escherichia coli defective for integration host factor, a protein involved in lambda site-specific recombination and the regulation of gene expression. We report the characterization of a mutant, lambda cos154, that, unlike wild-type lambda, is defective for growth in integration host factor-defective E. coli. The cis-dominant mutation in lambda cos154 is a single base pair change in a region of hyphenated dyad symmetry close to the lambda left cohesive end; this mutation prevents DNA packaging. We propose the following two alternative roles for this site in lambda DNA packaging: (i) to bind an E. coli accessory protein required in the absence of integration host factor or (ii) to bind the phage-encoded terminase protein that is essential for DNA packaging.     

Instability of bacteriophage lambda initiator O and P proteins in DNA replication

Lambda dv plasmids having an amber mutation in an initiator gene, O or P, were constructed from mutant lambda phages by recombinant DNA techniques and several properties of such derivatives were investigated. These plasmids are perpetuated in suppressor-plus (amber-permissive) cells, but not in non-suppressor cells. The plasmid copy number in the suppressor-plus cells was low as compared to that of the plasmid without the amber mutation. In cells carrying a thermosensitive suppressor 2, raising the temperature is expected to block new production of amber proteins, but should not affect conservation of the protein made prior to heating. It was observed, however, that replication of the plasmids carrying an amber mutation in the O or P gene was abolished soon after raising the temperature, suggesting that neither of the initiator proteins can continue functioning unless replenished. Pulse-chase experiments demonstrated that O protein decays with a half-life of 8 min. Several lines of evidence suggest that this degradation occurs independently of the protein function. On the other hand, P protein was not degraded under the same experimental conditions. These observations are discussed in connection with functional instability of the initiator molecules. It appears that they do not work catalytically.     

Nucleotide sequence of the Escherichia coli dnaJ gene and purification of the gene product

The dnaJ and dnaK genes are essential for replication of Escherichia coli DNA, and they constitute an operon, dnaJ being downstream from dnaK. The amount of the dnaJ protein in E. coli is substantially less than that of the dnaK protein, which is produced abundantly. In order to construct a system that over-produces the dnaJ protein, we started our study by determining the DNA sequence of the entire dnaJ gene, and an operon fusion was constructed by inserting the gene downstream of the lambda PL promoter of an expression vector plasmid, pPL-lambda. Cells containing the recombinant plasmid produced dnaJ protein amounting to 2% of the total cellular protein when cells were induced. The overproduced protein was purified, and Edman degradation of the protein indicated that the NH2-terminal methionine was found to be processed. From the DNA sequence of the dnaJ gene, the processed gene product is composed of 375 amino acid residues, and its molecular weight is calculated to be 40,975.     

Analysis of an Escherichia coli dnaB temperature-sensitive insertion mutation and its cold-sensitive extragenic suppressor.

An Escherichia coli mutant, ts121, was isolated following random insertional mutagenesis using phage lambda Mu transposition. The mutant phenotype includes inability to form colonies at temperatures above 38 degrees C and inability to propagate phage lambda at all temperatures. A lambda i434 cI- (ts121)+ transducing phage was isolated on the basis of its ability to form plaques on ts121 mutant bacteria. Using this transducing phage, it was shown through complementation and protein analyses, that the ts121 mutation is located in the dnaB gene. The exact insertion event was identified by polymerase chain reaction amplification of the DNA sequences containing the insertion junction. The mutational insertion event in ts121 was mapped precisely between base pairs 1514 and 1515 of the dnaB gene. This result predicts that the mutant dnaB protein has lost its six terminal amino acids. The reading frame shifts into Mu-specific DNA sequences resulting in an additional 20 amino acid residues. The E. coli wild type dnaB protein participates in host replication and interacts with lambda P protein to initiate phage lambda DNA replication. Our results demonstrate that the extreme carboxyl end of the dnaB protein is required for productive interaction with the lambda P replication protein at all temperatures, and is important for dnaB function at temperatures above 38 degrees C. Cold-sensitive extragenic suppressors of the ts121 mutation were isolated on the basis of their ability to restore colony formation at 42 degrees C. One of these extragenic suppressors was mapped at 54 min on the E. coli genetic map and localized to the suhB gene, whose product may affect the expression of a number of genes at the translational level.     

Protein degradation in E. coli: the lon mutation and bacteriophage lambda N and cII protein stability

The Ion gene of E. coli controls the stability of two bacteriophage lambda proteins. The functional half-life of the phage N gene product, measured by complementation, is increased about 5-fold in Ion mutant strains, from 2 min to 10 min. The chemical half-life of N protein, determined by its disappearance on polyacrylamide gels following pulse-chase labeling, increases about three-fold in Ion cells. In contrast to its effect on the N protein, the Ion mutation produces a 50% decrease in the chemical half-life of cII protein. The decay rate of many other phage proteins, including the unstable gene O product, remains unaffected by a host Ion defect. A Ion mutation alters lambda physiology in two ways. First, upon infection, the phage enters the lytic pathway predominantly. This may result from the deficiency of cII protein caused by its decreased stability, since cII product is required for establishment of lysogeny. Second, brief thermal induction of a Ion (lambda c1857) lysogen leads irreversibly to lysis; repression cannot be restablished and the treated cells are committed to forming infective centers. Although N product is normally required for rapid commitment, Ion lysogens become committed more rapidly than Ion+ lysogens, even in the absence of N function. These results identify for the first time native proteins whose stability is affected by the Lon proteolytic pathway. They also indicate that the Lon system may be important in regulating gene expression in E. coli.     

Stability of coliphage lambda DNA replication initiator, the lambda O protein.

The initiator of coliphage lambda DNA replication, lambda O protein, may be detected among other 35S-labeled phage and bacterial proteins by a method based on immunoprecipitation. This method makes it possible to study lambda O proteolytic degradation in lambda plasmid-harboring or lambda phage-infected cells; it avoids ultraviolet (u.v.)-irradiation of bacteria, used for depression of host protein synthesis, prior to lambda phage infection. We confirm the rapid decay of lambda O protein (half-time of 80 s), but we demonstrate the existence of a stable lambda O fraction. In the standard five minute pulse-chase experiments, 20% of synthesized lambda O is stable. The extension of the [35S]methionine pulse, possible in lambda plasmid-harboring cells, leads to a linear increase of this fraction, as if a part of the synthesized lambda O was constantly made resistant to proteolysis. Less than 5% of lambda O protein synthesized during one minute is transformed into a stable form. We presume that the stable lambda O is identical with lambda O present in the normal replication complex and thus protected from proteases. We cannot find any stable lambda O in Escherichia coli recA+ cells that were irradiated with u.v. light prior to lambda phage infection, but their recA- counterparts behave normally, suggesting that recA function interferes in the assembly of a normal replication complex in u.v.-irradiated bacteria. The stable lambda O found in lambda plasmid-harboring, amino acid-starved relA cells is responsible for the lambda O-dependent lambda plasmid replication that occurs in this system in the absence of lambda O synthesis. The existence of stable lambda O raises doubt concerning its role as the limiting initiator protein in the control of replication. Another significance of lambda O rapid degradation is proposed.     

The mutation that makes Escherichia coli resistant to lambda P gene-mediated host lethality is located within the DNA initiator Gene dnaA of the bacterium

Earlier, we reported that the bacteriophage lambda P gene product is lethal to Escherichia coli, and the E. coli rpl mutants are resistant to this lambda P gene-mediated lethality. In this paper, we show that under the lambda P gene-mediated lethal condition, the host DNA synthesis is inhibited at the initiation step. The rpl8 mutation maps around the 83 min position in the E. coli chromosome and is 94 % linked with the dnaA gene. The rpl8 mutant gene has been cloned in a plasmid. This plasmid clone can protect the wild-type E. coli from lambda P gene-mediated killing and complements E. coli dnaAts46 at 42 degrees C. Also, starting with the wild-type dnaA gene in a plasmid, the rpl-like mutations have been isolated by in vitro mutagenesis. DNA sequencing data show that each of the rpl8, rpl12 and rpl14 mutations has changed a single base in the dnaA gene, which translates into the amino acid changes N313T, Y200N, and S246T respectively within the DnaA protein. These results have led us to conclude that the rpl mutations, which make E. coli resistant to lambda P gene-mediated host lethality, are located within the DNA initiator gene dnaA of the host.     

hflB, a new Escherichia coli locus regulating lysogeny and the level of bacteriophage lambda cII protein

The level of the viral cII protein has been proposed to be the crucial determinant in the lysis-lysogeny decision of bacteriophage lambda. A new Escherichia coli locus (hflB) has been identified in which a mutation (hflB29) leads to high frequency of lysogeny by lambda. A double mutant defective in both hflB and the previously identified hflA gene displays a more severe Hfl- phenotype than either single mutant. The hflB locus is at 69 minutes on the E. coli map, 85% co-transducible with argG. The hflB29 mutation results in increased stability of the phage cII protein (increasing its half-life twofold) and is recessive to hflB+. We conclude that the hflB+ locus is a negative regulator of cII, perhaps coding for or regulating a protease that acts on cII. In addition, we observe that the can1 mutation, an alteration of the cII gene that results in enhanced lysogenization, leads to increased stability of cII protein. These observations reinforce the view that the level of cII is a key factor in the lysis-lysogeny decision of lambda.     

Identification and characterization of a gene responsible for inhibiting propagation of methylated DNA sequences in mcrA mcrB1 Escherichia coli strains.

Identifying and eliminating endogenous bacterial enzyme systems can significantly increase the efficiency of propagation of eukaryotic DNA in Escherichia coli. We have recently examined one such system which inhibits the propagation of lambda DNA rescued from transgenic mouse tissues. This rescue procedure utilizes lambda packaging extracts for excision of the lambda DNA from the transgenic mouse genome, as well as E. coli cells for subsequent infection and propagation. This assay, in combination with conjugal mating, P1 transduction, and gene cloning, was used to identify and characterize the E. coli locus responsible for this difference in efficiency. It was determined that the E. coli K-12 mcrB gene when expressed on a high-copy-number plasmid can cause a decrease in rescue efficiency despite the presence of the mcrB1 mutation, which inactivates the classic McrB restriction activity. (This mutation was verified by sequence analysis.) However, this McrB1 activity is not observed when the cloned mcrB1 gene is inserted into the E. coli genome at one copy per chromosome. A second locus was identified which causes a decrease in rescue efficiency both when expressed on a high-copy-number plasmid and when inserted into the genome. The data presented here suggest that this locus is mrr and that the mrr gene product can recognize and restrict cytosine-methylated sequences. Removal of this DNA region including the mrr gene from E. coli K-12 strains allows high rescue efficiencies equal to those of E. coli C strains. These modified E. coli K-12 plating strains and lambda packaging extract strains should also allow a significant improvement in the efficiency and representation of eukaryotic genomic and cDNA libraries.     

Isolation and characterization of a temperature-sensitive dnaK mutant of Escherichia coli B.

A temperature-sensitive dnaK mutant (strain MT112) was isolated from Escherichia coli B strain H/r30RT by thymineless death selection at 43 degrees C. By genetic mapping, the mutation [dnaK7(Ts)] was located near the thr gene (approximately 0.2 min on the may). E. coli K-12 transductants of the mutation to temperature sensitivity were assayed for their susceptibility to transducing phage lambda carrying the dnaK and/or the dnaJ gene. All of the transductants were able to propagate phage lambda carrying the dnaK gene. When macromolecular synthesis of the mutant was assayed at 43 degrees C, it was observed that both deoxyribonucleic acid and ribonucleic acid syntheses were severely inhibited. Thus, it was suggested that the conditionally defective dnaK mutation affects both cellular deoxyribonucleic acid and ribonucleic acid syntheses at the nonpermissive temperature in addition to inability to propagate phage lambda at permissive temperature.     

The ClpX heat-shock protein of Escherichia coli, the ATP-dependent substrate specificity component of the ClpP-ClpX protease, is a novel molecular chaperone.

All major classes of protein chaperones, including DnaK (the Hsp70 eukaryotic equivalent) and GroEL (the Hsp60 eukaryotic equivalent) have been found in Escherichia coli. Molecular chaperones enhance the yields of correctly folded polypeptides by preventing aggregation and even by disaggregating certain protein aggregates. Previously, we identified the ClpX heat-shock protein of E. coli because it enables the ClpP catalytic protease to degrade the bacteriophage lambda O replication protein. Here we report that ClpX alone possesses all the properties expected of a molecular chaperone protein. Specifically, it can protect the lambda O protein from heat-induced aggregation, disaggregate preformed lambda O aggregates, and even promote efficient binding of lambda O to its DNA recognition sequence. A lambda O-ClpX specific protein-protein interaction can be detected either by a modified ELISA assay or through the stimulation of ClpX's weak ATPase activity by lambda O. Unlike the behaviour of the major DnaK and GroEL chaperones, ClpX requires the presence of ATP or its non-hydrolysable analogue ATP-gamma-S for efficient interaction with other proteins including the protection of lambda O from aggregation. However, ClpX's ability to disaggregate lambda O aggregates requires hydrolysable ATP. We propose that the ClpX protein is a bona fide chaperone, whose biological role includes the maintenance of certain polypeptides in a form competent for proteolysis by the ClpP protease. Furthermore, our results suggest that the ClpX protein also performs typical chaperone protein functions independent of ClpP.     

Saturation and specificity of the Lon protease of Escherichia coli.

Lon is an ATP-dependent protease of Escherichia coli. The lon mutation has a pleiotropic phenotype: UV sensitivity, mucoidy, deficiency for lysogenization by bacteriophage lambda and P1, and lower efficiency in the degradation of abnormal proteins. All of these phenotypes are correlated with the loss of protease activity. Here we examine the effects of overproduction of one Lon substrate, SulA, and show that it protects two other substrates from degradation. To better understand this protection, we mutagenized the sulA gene and selected for mutants that have partially or totally lost their ability to saturate the Lon protease and thus can no longer protect another substrate. Some of the SulA mutants lost their ability to protect RcsA from degradation but could still protect the O thermosensitive mutant protein (Ots). All of the mutants retained their capacity to induce cell division inhibition. It was also found that deletion of the C-terminal end of SulA affected its activity but did not affect its susceptibility to Lon. We propose that Lon may have more than one specificity for peptide cleavage.     

Cloning, nucleotide sequence, and mutagenesis of a gene (irpA) involved in iron-deficient growth of the cyanobacterium Synechococcus sp. strain PCC7942.

We describe the cloning and sequencing of a gene from the cyanobacterium Synechococcus sp. strain PCC7942, designated irpA (iron-regulated protein A), that encodes for a protein involved in iron acquisition or storage. Polyclonal antibodies raised against proteins which accumulate during iron-deficient growth were used as probes to isolate immunopositive clones from a lambda gt11 genomic expression library. The clone, designated lambda gtAN26, carried a 1.7-kilobase (kb) chromosomal DNA insert and was detected by cross-reactivity with antibody against a 36-kilodalton protein. It was possible to map a 20-kb portion of the chromosome with various DNA probes from lambda gt11 and lambda EMBL-3 clones, and Southern blot analysis revealed that the irpA gene was present in a single copy and localized within a 1.7-kb PstI fragment. DNA sequencing revealed an open reading frame of 1,068 nucleotides capable of encoding 356 amino acids which yields a protein with a molecular weight of 38,584. The hydropathy profile of the polypeptide indicated a putative N-terminal signal sequence of 44 amino acid residues. IrpA is a cytoplasmic membrane protein as determined by biochemistry and electron microscopy immunocytochemistry. The upstream region of the irpA gene contained a consensus sequence similar to the aerobactin operator in Escherichia coli. This fact, plus a mutant with a mutation in irpA that is unable to grow under iron-deficient conditions, led us to suggest that irpA is regulated by iron and that the gene product is involved in iron acquisition or storage.