The Escherichia coli Fis protein prevents initiation of DNA replication from oriC in vitro.

Fis protein participates in the normal control of chromosomal replication in Escherichia coli. However, the mechanism by which it executes its effect is largely unknown. We demonstrate an inhibitory influence of purified Fis protein on replication from oriC in vitro. Fis inhibits DNA synthesis equally well in replication systems either dependent upon or independent of RNA polymerase, even when the latter is stimulated by the presence of HU or IHF. The extent of inhibition by Fis is modulated by the concentrations of DnaA protein and RNA polymerase; the more limiting the amounts of these, the more severe the inhibition by Fis. Thus, the level of inhibition seems to depend on the ease with which the open complex can be formed. Fis-mediated inhibition of DNA replication does not depend on a functional primary Fis binding site between DnaA boxes R2 and R3 in oriC, as mutations that cause reduced binding of Fis to this site do not affect the degree of inhibition. The data presented suggest that Fis prevents formation of an initiation-proficient structure at oriC by forming an alternative, initiation-preventive complex. This indicates a negative role for Fis in the regulation of replication initiation.     

Efficient excision of phage lambda from the Escherichia coli chromosome requires the Fis protein.

The Escherichia coli protein Fis has been shown to bind a single site in the recombination region of phage lambda and to stimulate excisive recombination in vitro (J. F. Thompson, L. Moitoso de Vargas, C. Koch, R. Kahmann, and A. Landy, Cell 50:901-908, 1987). We demonstrate that mutant strains deficient in fis expression show dramatically reduced rates of lambda excision in vivo. Phage yields after induction of a stable lysogen are reduced more than 200-fold in fis cells. The defect observed in phage yield is not due to inefficient phage replication or lytic growth. Direct examination of excisive recombination products reveals a severe defect in the rate of recombination in the absence of Fis. The excision defect observed in fis cells can be fully reproduced in fis+ cells by using phages that lack the Fis binding site on attR, indicating that the entire stimulatory effect of Fis on excisive recombination is due to binding at that site.     

The DnaA box R4 in the minimal oriC is dispensable for initiation of Escherichia coli chromosome replication.

We have developed a genetic system with which to replace oriC+ on the Escherichia coli chromosome with modified oriC sequences constructed on plasmids. Using this system we have demonstrated that chromosomal oriC can tolerate the insertion of a 2 kb fragment at the HindIII site between DnaA boxes R3 and R4, whereas the same insertion completely inactivates cloned oriC. We have further found that although R4 is essential for the origin activity of cloned oriC, cells carrying a deletion of R4 in chromosomal oriC are viable. These results indicate that the oriC sequence necessary for initiation of chromosome replication is different from the so-called minimal oriC that was determined with cloned oriC. Flow cytometric analyses have revealed that these oriC mutations confer the initiation asynchrony phenotype. Introduction of the R4 deletion into a fis::kan mutant, which lacks the DNA bending protein FIS, renders the mutant cells inviable.     

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.     

The stringent response blocks DNA replication outside the ori region in Bacillus subtilis and at the origin in Escherichia coli

When the Bacillus subtilis dnaB37 mutant, defective in initiation, is returned to permissive temperature after growth at 45 degrees C, DNA replication is synchronized. Under these conditions, we have shown previously that DNA replication is inhibited when the Stringent Response is induced by the amino acid analogue, arginine hydroxamate. We have now shown, using DNA-DNA hybridization analysis, that substantial replication of the oriC region nevertheless occurs during the Stringent Response, and that replication inhibition is therefore implemented downstream from the origin. On the left arm, replication continues for at least 190 x 10(3) base-pairs to the gnt gene and for a similar distance on the right arm to the gerD gene. When the Stringent Response is lifted, DNA replication resumed downstream from oriC on both arms, confirming that DNA replication is regulated at a post-initiation level during the Stringent Response in B. subtilis. Resumption of DNA synthesis following the lifting of the Stringent Response did not require protein or RNA synthesis or the initiation protein DnaB. We suggest, therefore, that a specific control region, involving Stringent Control sites, facilitate reversible inhibition of fork movement downstream from the origin via modifications of a replisome component during the Stringent Response. In contrast, in Escherichia coli, induction of the Stringent Response appears to block initiation of DNA replication at oriC itself. No DNA synthesis was detected in the oriC region and, upon lifting the Stringent Response, replication occurred from oriC. Post-initiation control in B. subtilis therefore results in duplication of many key genes involved in growth and sporulation. We discuss the possibility that such a control might be linked to differentiation in this organism.     

Initiation of chromosome replication in dnaA and dnaC mutants of Escherichia coli B/r F.

Regulatory aspects of chromosome replication were investigated in dnaA5 and dnaC2 mutants of the Escherichia coli B/r F. When cultures growing at 25 degrees C were shifted to 41 degrees C for extended periods and then returned to 25 degrees C, the subsequent synchronous initiations of chromosome replication were spaced at fixed intervals. When chloramphenicol was added coincident with the temperature downshift, the extend of chromosome replication in the dnaA mutant was greater than that in the dnaC mutant, but the time intervals between initiations were the same in both mutants. Furthermore, the time interval between the first two initiation events was unaffected by alterations in the rate of rifampin-sensitive RNA synthesis or cell mass increase. In the dnaC2 mutant, the capacities for both initiations were achieved in the absence of extensive DNA replication at 25 degrees C as long as protein synthesis was permitted, but the cells did not progress toward the second initiation at 25 degrees C when both protein synthesis and DNA replication were prevented. Cells of the dnaA5 mutant did not achieve the capacity for the second initiation event in the absence of extensive chromosome replication, although delayed initiation may have taken place. A plausible hypothesis to explain the data is that the minimum interval is determined by the time required for formation of a supercoiled, membrane-attached structure in the vicinity of oriC which is required for initiation of DNA synthesis.     

Initiation of heat-induced replication requires DnaA and the L-13-mer of oriC

An upshift of 10 degrees C or more in the growth temperature of an Escherichia coli culture causes induction of extra rounds of chromosome replication. This stress replication initiates at oriC but has functional requirements different from those of cyclic replication. We named this phenomenon heat-induced replication (HIR). Analysis of HIR in bacterial strains that had complete or partial oriC deletions and were suppressed by F integration showed that no sequence outside oriC is used for HIR. Analysis of a number of oriC mutants showed that deletion of the L-13-mer, which makes oriC inactive for cyclic replication, was the only mutation studied that inactivated HIR. The requirement for this sequence was strictly correlated with Benham's theoretical stress-induced DNA duplex destabilization. oriC mutations at DnaA, FIS, or IHF binding sites showed normal HIR activation, but DnaA was required for HIR. We suggest that strand opening for HIR initiation occurs due to heat-induced destabilization of the L-13-mer, and the stable oligomeric DnaA-single-stranded oriC complex might be required only to load the replicative helicase DnaB.     

Drunken-cell footprints: nuclease treatment of ethanol-permeabilized bacteria reveals an initiation-like nucleoprotein complex in stationary phase replication origins.

The nucleoprotein complex formed on oriC, the Escherichia coli replication origin, is dynamic. During the cell cycle, high levels of the initiator DnaA and a bending protein, IHF, bind to oriC at the time of initiation of DNA replication, while binding of Fis, another bending protein, is reduced. In order to probe the structure of nucleoprotein complexes at oriC in more detail, we have developed an in situ footprinting method, termed drunken-cell footprinting, that allows enzymatic DNA modifying reagents access to intracellular nucleoprotein complexes in E.coli, after a brief exposure to ethanol. With this method, we observed in situ binding of Fis to oriC in exponentially growing cells, and binding of IHF to oriC in stationary cells, using DNase I and Bst NI endonuclease, respectively. Increased binding of DnaA to oriC in stationary phase was also noted. Because binding of DnaA and IHF results in unwinding of oriC in vitro, P1 endonuclease was used to probe for intracellular unwinding of oriC. P1 cleavage sites, localized within the 13mer unwinding region of oriC ', were dramatically enhanced in stationary phase on wild-type origins, but not on mutant versions of oriC unable to unwind. These observations suggest that most oriC copies become unwound during stationary phase, forming an initiation-like nucleoprotein complex.     

Protein synthesis is required for in vivo activation of polysialic acid capsule synthesis in Escherichia coli K1.

The kinetics of in vivo expression of the polysialosyl (K1) capsular antigen in Escherichia coli has been studied. Growth of E. coli K1 strains at 15 degrees C prevents K1 polysaccharide synthesis (F. A. Troy and M. A. McCloskey, J. Biol. Chem. 254:7377-7387, 1979). Synthesis is reactivated in cells grown at 15 degrees C after upshift to 37 degrees C. The early expression and resultant morphology of K1 capsular antigen was monitored in temperature upshift experiments by using electron microscopy. Morphological stabilization of the capsule was achieved by treatment of cells with an antiserum specific for the alpha, 2-8-linked polysialosyl antigen. The kinetics of K1 capsule expression in growing cells was measured by bacteriophage adsorption with phage K1F, which required the K1 capsule for binding. The results of temperature upshift experiments showed that capsule first appeared on the cell surface after 10 min. Subsequent bacteriophage binding increased linearly with time until a fully encapsulated state was reached 45 min after upshift. The initiation of K1 capsule appearance was dependent on protein synthesis and the addition of chloramphenicol before temperature upshift prevented any expression of the K1 antigen. Chloramphenicol reduced the rate of K1 synthesis when added after temperature upshift. We conclude from these results that protein synthesis is a prerequisite for activation of capsule expression in vivo, but not for subsequent elongation of polysialosyl chains.     

Role of architectural elements in combinatorial regulation of initiation of DNA replication in Escherichia coli

Bending of DNA is a prerequisite of site-specific recombination and gene expression in many regulatory systems involving the assembly of specific nucleoprotein complexes. We have investigated how the uniquely clustered Dam methylase sites, GATCs, in the origin of Escherichia coli replication ( oriC  ) and their methylation status modulate the geometry of oriC and its interaction with architectural proteins, such as integration host factor (IHF), factor for inversion stimulation (Fis) and DnaA initiator protein. We note that 3 of the 11 GATC sites at oriC are strategically positioned within the IHF protected region. Methylation of the GATCs enhances IHF binding and alters the IHF-induced bend at oriC . GATC motifs also contribute to intrinsic DNA curvature at oriC and the degree of bending is modulated by methylation. The IHF-induced bend at oriC is further modified by Fis protein and IHF affinity for its binding site may be impaired by protein(s) binding to GATCs within the IHF site. Thus, GATC sites at oriC affect the DNA conformation and GATCs, in conjunction with the protein-induced bends, are critical cis -acting elements in specifying proper juxtapositioning of initiation factors in the early steps of DNA replication.     

Fis binding in the dnaA operon promoter region.

The region between the rpmH and dnaA genes contains five promoters that divergently express the ribosomal protein L34 and the proteins of the dnaA operon, including DnaA, the beta clamp of DNA polymerase III holoenzyme, and RecF. The DNA-binding protein Fis was shown by the band shift assay to bind near the rpmHp2 and dnaAp2 promoters and by DNase I footprinting to bind to a single site in the dnaAp2 promoter overlapping the -35 and spacer sequences. There were no observable differences in Fis affinity or the angle of bending induced by Fis between methylated and unmethylated DNA fragments containing the Fis binding site in the dnaAp2 promoter. Fis directly or indirectly represses the expression of DnaA protein and the beta clamp of DNA polymerase III. A fis null mutant containing a dnaA-lacZ in-frame fusion had twofold greater beta-galactosidase activity than a fis wild-type strain, and induced expression of Fis eliminated the increase in activity of the fusion protein. A two- to threefold increase in the levels of DnaA and beta clamp proteins was found in a fis null mutant by immunoblot gel analysis.     

Escherichia coli prereplication complex assembly is regulated by dynamic interplay among Fis, IHF and DnaA

Initiator DnaA and DNA bending proteins, Fis and IHF, comprise prereplication complexes (pre-RC) that unwind the Escherichia coli chromosome's origin of replication, oriC. Loss of either Fis or IHF perturbs synchronous initiation from oriC copies in rapidly growing E. coli. Based on dimethylsulphate (DMS) footprinting of purified proteins, we observed a dynamic interplay among Fis, IHF and DnaA on supercoiled oriC templates. Low levels of Fis inhibited oriC unwinding by blocking both IHF and DnaA binding to low affinity sites. As the concentration of DnaA was increased, Fis repression was relieved and IHF rapidly redistributed DnaA to all unfilled binding sites on oriC. This behaviour in vitro is analogous to observed assembly of pre-RC in synchronized E. coli. We propose that as new DnaA is synthesized in E. coli, opposing activities of Fis and IHF ensure an abrupt transition from a repressed complex with unfilled weak affinity DnaA binding sites to a completely loaded unwound complex, increasing both the precision of DNA replication timing and initiation synchrony.     

Elevated Fis expression enhances recombinant protein production in Escherichia coli

A genetic strategy to enhance recombinant protein production is discussed. A small DNA bending protein, Fis, which has been shown to activate rRNA synthesis upon a nutrient upshift, was overexpressed in E. coli strain W3110 carrying vector pUCR1. Overexpression of Fis during exponential growth was shown to activate rrn promoters to different extents. A 5-fold improvement in chloramphenicol acetyltransferase (CAT) production in cultures with elevated Fis level was observed in shake-flask cultivations. A similar improvement in the culture performance was also observed during fed-batch fermentation; the specific CAT activity increased by more than 50% during the fed-batch phase for cultures with elevated Fis expression. In contrast, no increase in specific CAT activity was detected for cultures carrying pUCR2, expressing a frame-shift Fis mutant. Expression of Fis from a complementary vector, pKFIS, restored CAT production from W3110:pUCR2 to approximately the same level as cultures carrying pUCR1, indicating that the enhancement in CAT production was indeed Fis-dependent. The framework presented here suggests that differential activation in recombinant protein production may be achieved with differential Fis overexpression. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 138-144, 1997.