Limited differential mRNA inactivation in the atp (unc) operon of Escherichia coli.

Individual subunits of ATP synthase, encoded by the eight genes of the atp operon (atpA through atpH), have been found to be synthesized at a 10-fold range in molar amounts (D.L. Foster and R.H. Fillingame, J. Biol. Chem. 257:2009-2015, 1982; K. von Meyenburg, B.B. Jorgensen, J. Nielsen, F.G. Hansen, and O. Michelsen. Tokai J. Exp. Clin. Med. 7:23-31, 1982). We have determined the functional half-lives at 30 degrees C of mRNAs transcribed from these genes either during constitutive expression in a partial diploid strain or after induced expression from a plasmid. Accurate decay kinetics of the relative mRNA levels were determined by monitoring the rates of synthesis of the individual ATP synthase subunits by radioactive pulse labeling at different times after blocking transcription initiation with rifampin. The mRNA transcribed from the atp operon was found to be inactivated about twice as fast as the bulk mRNA in E. coli. Exceptions are the mRNA from the promoter-proximal atpB gene, which was inactivated about three times as fast as the bulk mRNA, and atpC mRNA, the inactivation rate of which was comparable to that of the bulk mRNA. These moderate differences in the kinetics of functional decay explain only a minor part of the differences in expression levels of the atp genes. We conclude, therefore, that the individual atp mRNAs must be translated with widely different efficiencies. The present analysis further revealed that mRNA degradation is sensitive to heat shock; i.e., after incubation at 39 degrees C for 5 min followed by a shift back to 30 degrees C, the decay rate of the bulk mRNA was decreased by 30%.

Initiation of DNA replication in Escherichia coli after overproduction of the DnaA protein.

Flow cytometry was used to study initiation of DNA replication in Escherichia coli K12 after induced expression of a plasmid-borne dnaA+ gene. When the dnaA gene was induced from either the plac or the lambda pL promoter initiation was stimulated, as evidenced by an increase in the number of origins and in DNA content per mass unit. During prolonged growth under inducing conditions the origin and DNA content per mass unit were stabilized at levels significantly higher than those found before induction or in similarly treated control cells. The largest increase was observed when using the stronger promoter lambda pL compared to plac. Synchrony of initiation was reasonably well maintained with elevated DnaA protein concentrations, indicating that simultaneous initiation of all origins was still preferred under these conditions. A reduced rate of replication fork movement was found in the presence of rifampin when the DnaA protein was overproduced. We conclude that increased synthesis levels or increased concentrations of the DnaA protein stimulate initiation of DNA replication. The data suggest that the DnaA protein may be the limiting factor for initiation under normal physiological conditions.

The DnaA protein determines the initiation mass of Escherichia coli K-12.

DNA replication was studied in a dnaA(Ts) strain containing a plasmid with the dnaA+ gene under plac control. At 42 degrees C, initiation of DNA replication was totally dependent upon the gratuitous inducer isopropyl beta-D-thiogalactopyranoside (IPTG). Flow cytometric measurements showed that at 13% induction of the lac promoter the growth rate, cell size, DNA content, and timing of initiation of DNA replication were indistinguishable from those observed in a wild-type control cell. Higher levels of induction resulted in initiations earlier in the cell cycle and a corresponding increase in the time from initiation to termination. We conclude that the concentration of DnaA protein determines the time of initiation and thereby the initiation mass. With an induction level equal to or above 13%, the synchrony of multiple initiations within one cell was close to that found in a wild-type control cell, showing that a cyclic variation in DnaA content is not necessary for a high degree of synchrony.

Coordination of chromosome replication initiation in Escherichia coli: effects of different dnaA alleles.

The synchrony of initiation of chromosomal replication in single cells was determined in ten different dnaA(Ts) mutants. After inhibiting the initiation of replication but allowing initiated rounds of replication to terminate, we measured the number of fully replicated chromosomes per individual cell by flow cytometry. Synchronous initiation at the several independent origins (oriC) in single rapidly growing cells would give 2'' (n = 0,1,2,3,...) chromosomes per cell, whereas asynchronous initiation was indicated by the presence of a different number of chromosomes. Mutations mapping in the central part of the dnaA gene (dnaA5, dnaA46, dnaA601, dnaA602, and dnaA604) lead to a high degree of asynchrony (class I mutants), whereas mutations mapping in either of the distal parts of the gene (dnaA508, dnaA167, dnaA203, and dnaA204) yielded a low degree of asynchrony at the permissive temperature (class 2 mutants). The dnaA205 mutant exhibited an intermediate degree of asynchrony. Mutants dnaA203 and dnaA204 (promoter distal) differed from the other class 2 mutants (dnaA167, dnaA508; promoter proximal) in that asynchrony increased no more than twofold between 25 and 37 degrees C compared with the more-than-fourfold increase in the latter. The high degree of asynchrony in class 1 mutants was independent of temperature and was not due to insufficient functional DnaA protein, because overproduction of DnaA46 protein did not decrease the asynchrony. The data demonstrate that the DnaA protein has functions in addition to acting positively in the initiation process and negatively as its own repressor, namely in coordinating initiations at all oriC sites within a single cell.

Mode of initiation of constitutive stable DNA replication in RNase H-defective mutants of Escherichia coli K-12.

The alternative pathway of DNA replication in rnh mutants of Escherichia coli can be continuously initiated in the presence of chloramphenicol, giving rise to constitutive stable DNA replication (cSDR). We conducted a physiological analysis of cSDR in rnh-224 mutants in the presence or absence of the normal DNA replication system. The following results were obtained. cSDR allowed the cells to grow in the absence of the normal replication system at a 30 to 40% reduced growth rate and with an approximately twofold-decreased DNA content. cSDR initiation was random with respect to time in the cell cycle as well as choice of origins. cSDR initiation continued to increase exponentially for more than one doubling time when protein synthesis was inhibited by chloramphenicol. cSDR initiation was inhibited during amino acid starvation in stringent (relA+) but not in relaxed (relA1) strains, indicating its sensitivity to ppGpp. cSDR initiation was rifampin sensitive, demonstrating that RNA polymerase was involved. cSDR functioned in dnaA+ rnh-224 strains parallel to the normal oriC+ dnaA+-dependent chromosome replication system.

Nonrandom minichromosome replication in Escherichia coli K-12.

The intervals between rounds of chromosome and minichromosome replication were measured by density shift experiments and found to be similar. Thus the minichromosome, a lambda asnA oriC bacteriophage, mostly replicates once each division cycle rather than randomly, despite its high copy number. Slight differences between the chromosome and the oriC plasmid are explained.

Mechanism of postsegregational killing by the hok gene product of the parB system of plasmid R1 and its homology with the relF gene product of the E. coli relB operon.

The parB region of plasmid R1 encodes two genes, hok and sok, which are required for the plasmid-stabilizing activity exerted by parB. The hok gene encodes a potent cell-killing factor, and it is regulated by the sok gene product such that cells losing a parB-carrying plasmid during cell division are rapidly killed. Coinciding with death of the host cell, a characteristic change in morphology is observed. Here we show that the killing factor encoded by the hok gene is a membrane-associated polypeptide of 52 amino acids. A gene located in the Escherichia coli relB operon, designated relF, is shown to be homologous to the hok gene. The relF gene codes for a polypeptide of 51 amino acids, which is 40% homologous to the hok gene product. Induced overexpression of the hok and relF gene products results in the same phenomena: loss of cell membrane potential, arrest of respiration, death of the host cell and change in cell morphology. The parB region and the relB genes were cloned into unstably inherited oriC minichromosomes. Whereas the parB region also conferred a high degree of genetic stability to an oriC minichromosome, the relB operon (with relF) did not; therefore the latter does not appear to 'stabilize' its replicon (the chromosome). The function of the relF gene is not known.

Conservation of genes and their organization in the chromosomal replication origin region of Bacillus subtilis and Escherichia coli.

The organization of six open reading frames which were deduced from the nucleotide sequence of some 10 kb from the replication origin region of Bacillus subtilis resembles the organization of the genes in the rnpA-dnaA-gyrB region of the Escherichia coli chromosome. Based on the detection of homology with the E. coli genes the open reading frames were found to represent the Bacillus 'rnpA', 'rpmH', 'dnaA', 'dnaN', recF and gyrB genes. Only the latter two have also been defined by genetic analysis. Two regulatory regions containing nine and four copies of a repeating sequence, DnaA-box, which is identical with the DnaA protein-binding sequence repeated four times in the E. coli origin of replication, flank the 'dnaA' gene of B. subtilis. One or both of them are proposed to function as origins in the initiation of chromosomal replication. Transcription of the 'dnaA' gene of Bacillus starts in one of these regions and appears to be coupled to initiation of chromosomal replication. We propose that the conserved gene organization in the 'dnaA'-'gyrB' region of B. subtilis is representative of the replication origin region of a primordial replicon. The oriC sequence of E. coli has either been translocated to its present location 44 kb away from the primordial origin or has independently evolved there.

Proton conduction by subunit a of the membrane-bound ATP synthase of Escherichia coli revealed after induced overproduction.

Transcriptional fusions between the phage lambda promotor pR and ATP synthase genes, atp, on plasmid pBR322 were constructed in order to study the effects upon growth and physiology of Escherichia coli of induced overproduction of H+-ATPase subunits. Constitutive overproduction of the complete enzyme had earlier been found to result in decreased growth rate and cytological defects. When a 15-fold overproduction of subunit a alone, or together with subunit c, or with all other ATP synthase subunits was suddenly induced, the following effects were observed. Inhibition of growth and protein synthesis within 10 min of induction, which effect was suppressed by N,N'-dicyclohexylcarbodiimide, also when the chromosomal atp genes coding for the Fo subunits a, b and c were deleted. Partial collapse of the membrane potential delta psi at 4-6 min after induction paralleled by inhibition of thiomethylgalactoside and guanosine transport. Respiration and alpha-methylglucoside transport was not affected. The partial collapse of delta psi, and the specific inhibition of proton-driven transport systems is taken to show that the subunit a has--when suddenly overproduced and inserted into the membrane--a protonophoric activity. It is suggested that this protonophoric activity of subunit a is related to the function of this subunit in the Fo sector in H+-ATPases.

RecA protein acts at the initiation of stable DNA replication in rnh mutants of Escherichia coli K-12.

Escherichia coli rnh mutants lacking RNase H activity are capable of recA+-dependent DNA replication in the absence of concomitant protein synthesis (stable DNA replication). In rnh dnaA::Tn10 and rnh delta oriC double mutants in which the dnaA+-dependent initiation of DNA replication at oriC is completely blocked, the recA200 mutation encoding a thermolabile RecA protein renders both colony formation and DNA synthesis of these mutants temperature sensitive. To determine which stage of DNA replication (initiation, elongation, or termination) was blocked, we analyzed populations of these mutant cells incubated at 30 or 42 degrees C in the presence or absence of chloramphenicol (CM) by dual-parameter (DNA-light scatter) flow cytometry. Incubation at 30 degrees C in the presence of CM resulted in cells with a continuum of DNA content up to seven or more chromosome equivalents per cell. The cultures which had been incubated at 42 degrees C in the absence or presence of CM consisted of cells with integral numbers of chromosomes per cell. It is concluded that active RecA protein is required specifically for the initiation of stable DNA replication.

Quantitation with monoclonal antibodies of Escherichia coli H protein suggests histone function.

The abundance of the histonelike H protein of Escherichia coli (U. Hübscher, H. Lutz, and A. Kornberg, Proc. Natl. Acad. Sci. U.S.A. 77:5097-5101, 1980) was determined by using monoclonal antibodies against H protein, immunoblotting, and homogeneous H protein as a standard. H protein was found to be present at approximately 120,000 monomeric molecules per fast-growing E. coli cell. This amount of H protein corresponds to a ratio of one H protein molecule to approximately 200 base pairs of the bacterial chromosome. Together with previous results, these findings suggest that H protein has histonelike function similar to that of histone protein H2A, its counterpart in the eucaryotic cell.

Function of ribonuclease H in initiation of DNA replication in Escherichia coli K-12.

Escherichia coli rnh mutants lacking ribonuclease H (RNase H) activity can tolerate deletion of the origin of DNA Replication (delta oriC) and transposon-insertional inactivation of an initiator gene (dnaA::Tn10). Introduction of the recA200 allele encoding a thermolabile RecA protein into rnh- dnaA::Tn10 and rnh- delta oriC mutants strains rendered DNA synthesis and colony formation of these mutants temperature sensitive. The temperature sensitivity and the broth sensitivity (Srm-) of the rnh- dnaA::Tn10 recA200 strain was suppressed by the presence of plasmids (pBR322 derivatives) carrying dnaA+ only when the intact oriC site was present on the chromosome. Lack of RNase H activity neither promoted replication of minichromosomes (pOC24 and p lambda asn20) in the absence of required DnaA+ protein nor inhibited dnaA+-dependent minichromosome replication. These results led to the conclusion that RNase H is not directly involved in the events leading to initiation of DNA replication at oriC. Rather, it functions as a specificity factor by eliminating certain forms of RNA-DNA hybrids which could otherwise be used to prime DNA replication at sites other than oriC.

Physiological and morphological effects of overproduction of membrane-bound ATP synthase in Escherichia coli K-12.

Effects of increased biosynthesis of the membrane-bound ATP synthase of Escherichia coli K-12 were analysed at the physiological and morphological level. Overproduction of the enzyme complex was achieved by molecular cloning of the structural genes into plasmid pBR322. A series of plasmids resulting in 2-fold, 4- to 5-fold and 10- to 12-fold overproduction, respectively, was constructed. The ATP synthase was calculated to represent 3%, 6-7% and 18-23%, respectively, of total protein in cells with these plasmids. In wild-type cells ATP synthase represents 1.5-2% of total protein equivalent to approximately 3000 enzyme complexes per average cell. While 2- or 4- to 5-fold wild-type levels of the ATP synthase had only minor effects it was found that 10- to 12-fold overproduction resulted in pronounced inhibition of cell division and growth and in formation of membrane cisterns and vesicles within the cells. Inclusion bodies, probably representing deposits of excess ATP synthase, were also observed in these cells.

Fine structure genetic map and complementation analysis of mutations in the dnaA gene of Escherichia coli.

A fine structure genetic map of several mutations in the dnaA gene of Escherichia coli was constructed by the use of recombinant lambda and M13 phages. The dnaA508 mutation was found to be the mutation most proximal to the promoter, while the dnaA203 mutation was found to be the most distal one. The order of mutations established in this analysis was: dnaA508, dnaA167, (dnaA5, dnaA46, dnaA211), dnaA205, dnaA204, dnaA203. The mutations dnaA601, dnaA602, dnaA603, dnaA604 and dnaA606 were found to map very close to each other and close to dnaA205 in the middle third of the dnaA gene. In analysing the dominance relationship all 13 dnaA mutations were found to be recessive to the wild type. Characteristic phenotypes of the dnaA(Ts) mutants, like reversibility of the temperature inactivation of the dnaA protein, cold sensitivity of haploid or of merodiploid strains and suppressibility by rpoB mutations, are found to correlate with clusters of mutations within the gene.

The topology of the proton translocating F0 component of the ATP synthase from E. coli K12: studies with proteases.

The accessibility of the three F0 subunits a, b and c from the Escherichia coli K12 ATP synthase to various proteases was studied in F1-depleted inverted membrane vesicles. Subunit b was very sensitive to all applied proteases. Chymotrypsin produced a defined fragment of mol. wt. 15,000 which remained tightly bound to the membrane. The cleavage site was located at the C-terminal region of subunit b. Larger amounts of proteases were necessary to attack subunit a (mol. wt. 30,000). There was no detectable cleavage of subunit c. It is suggested that the major hydrophilic part of subunit b extends from the membrane into the cytoplasm and is in contact with the F1 sector. The F1 sector was found to afford some protection against proteolysis of the b subunit in vitro and in vivo. Protease digestion had no influence on the electro-impelled H+ conduction via F0 but ATP-dependent H+ translocation could not be reconstituted upon binding of F1. A possible role for subunit b as a linker between catalytic events on the F1 component and the proton pathway across the membrane is discussed.

Membrane integration and function of the three F0 subunits of the ATP synthase of Escherichia coli K12.

Integration into the cytoplasmic membrane and function of the three F0 subunits, a, b and c, of the membrane-bound ATP synthase of Escherichia coli K12 were analysed in situations where synthesis of only one or two types of subunits was possible. This was achieved by combined use of atp mutations and plasmids carrying and expressing one or two of the atp genes coding for ATP synthase subunits. AU three F0 subunits were found to be required for the establishment of efficient H+ conduction. Subunits a and b individually as well as together were found to bind F1 ATPase to the membrane while subunit c did not. The ATPase activity bound to either of these single subunits, or in pairwise combinations, was not inhibited by N,N'-dicyclohexylcarbodiimide. Also ATP-dependent H+ translocation was not catalysed unless all three F0 subunits were present in the membrane. The integration into the membrane of the subunits a and b was independent of the presence of other ATP synthase subunits.

The origin of replication, oriC, and the dnaA protein are dispensable in stable DNA replication (sdrA) mutants of Escherichia coli K-12.

The sdrA224 mutants of Escherichia coli K-12, capable of continued DNA replication in the absence of protein synthesis (stable DNA replication), tolerate inactivation of the dnaA gene by insertion of transposon Tn10. Furthermore, oriC, the origin of E. coli chromosome replication, can be deleted from the chromosome of sdrA mutants without loss of viability. The results suggest the presence of a second, normally repressed, initiation system for chromosome replication alternative to the 'normal' dnaA+ oriC+-dependent initiation mechanism.

The nucleotide sequence of the dnaA gene and the first part of the dnaN gene of Escherichia coli K-12.

The nucleotide sequence of the dnaA gene and the first 10% of the dnaN gene was determined. From the nucleotide sequence the amino acid sequence of the dnaA gene product was derived. It is a basic protein of 467 amino acid residues with a molecular weight of 52.5 kD. The expression of the dnaA gene is in the counterclockwise direction like the one of the dnaN gene, for which potential startsites were found.

The membrane bound ATP synthase of Escherichia coli: a review of structural and functional analyses of the atp operon.

The structure of the atp operon, which contains the genes for the eight subunits alpha, beta, gamma, delta, epsilon, a, b and c of the membrane bound ATP synthase of Escherichia coli as determined by genetic experiments and DNA sequencing, is reviewed. The localization of transcription signals, namely of one major and two minor promoters, as well as the determination of the stoichiometry of the subunits (alpha:beta:gamma:delta:epsilon:a:b:c = 3:3:1:1:1:1:2:12-15) is summarized.