Initiator (DnaA) protein concentration as a function of growth rate in Escherichia coli and Salmonella typhimurium

Hansen, Flemming; Atlung, Tove; Braun, Robert E.; Wright, Andrew; Hughes, Patrick; Kohiyama, Masamichi

doi:10.1128/jb.173.16.5194-5199.1991

Cited by 107 publications

(121 citation statements)

References 35 publications

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“…FH1496 is a wild-type E. coli strain that carries a plasmid with lacP in front of the S. typhimurium dnaA (dnaAs5y) gene and the E. coli dnaA (dnaAECO)'-'lacZ fusion phage XRB1 located in the Xatt site. By using this strain, we can measure the amounts of DnaA protein produced from the dnaA ,y gene on the plasmid and the dnaAECO gene on the chromosome independently, as the DnaAsty protein and the DnaAECO protein show different migration behaviors in SDSpolyacrylamide gel electrophoresis (SDS-PAGE) (19). repression of the chromosomal dnaA gene, as well as the dnaA'-'lacZ fusion gene, when the total DnaA protein concentration was increased more than 1.5-fold.…”

Section: Resultsmentioning

confidence: 99%

Three distinct chromosome replication states are induced by increasing concentrations of DnaA protein in Escherichia coli

1993

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The DnaA protein concentration in Escherichia coli was increased above the wild-type level by inducing a lacP-controlled dnaA gene located on a plasmid. In these cells with different DnaA protein levels, we measured several parameters: dnaA gene expression; cell size, amount of DNA per cell, and number of origins per cell by flow cytometry; and origin-to-terminus ratio and the frequencies of five other markers on the chromosome by Southern hybridization. The response of the cells to higher levels of DnaA protein could be divided into three states. From the normal level to a level 1.5-fold higher, DnaA protein had little effect on dnaA gene expression and the rate of DNA replication but led to nearly proportional increases in DNA and origin concentrations. Between 1.5- and 3-fold, the normal DnaA protein concentration, dnaA gene expression was gradually decreased. In this interval, the origin concentration increased significantly; however, the replication rate was severely affected, becoming slower--especially near the origin--the higher the DnaA protein concentration, and as a result, the DNA concentration was constant. Further increases in the DnaA protein concentration did not lead to an increased origin concentration. Thus, the initiation mass was set by the DnaA protein from the normal level to an at least twofold-increased level, but the increased initiation did not lead to a large increase in the amount of DNA per unit of mass because of the inhibition of replication fork velocity.

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Section: Resultsmentioning

confidence: 99%

Three distinct chromosome replication states are induced by increasing concentrations of DnaA protein in Escherichia coli

1993

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“…The amount of DnaA protein in rapidly growing mycelia from S. lividans was estimated to be 0.013 to 0.040% of total cellular protein; this corresponds to the quantities found in E. coli (13) and B. subtilis (39).…”

Section: Discussionmentioning

confidence: 99%

Minimal requirements of the Streptomyces lividans 66 oriC region and its transcriptional and translational activities

Zakrzewska‐Czerwińska

Majka

Schrempf

1995

J Bacteriol

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Deletion analysis of a previously constructed minichromosome revealed that a stretch of DNA which is longer than 623 bp but shorter than 837 bp is required for autonomous replication of the Streptomyces lividans chromosome. Each of the dnaA and dnaN genes flanking the oriC region is individually transcribed from two promoters. Within the intergenic, nontranslatable region between the dnaA and dnaN genes, five main transcripts and several less abundant transcripts of various lengths as well as one of the promoters were identified. The introduction of additional DnaA boxes in S. lividans led to a significant increase in dnaA gene transcripts and to an enhanced level of the DnaA (73-kDa) protein. In summary, the data suggest that dnaA gene transcription is autoregulated and that initiation of the S. lividans chromosome is tightly controlled.The replication of several bacterial chromosomes was found to be initiated at the origin of replication, oriC. The features of oriC and, with a few exceptions, the arrangement of genes in its vicinity are remarkably similar among eubacteria (25, 38). Sequence analyses revealed that the origins of various eubacteria contain highly conserved segments which are separated by variable spacer regions. The DnaA box motifs (TTATC/AC AC/AA) (36) constituting the binding sites for the initiator protein DnaA are prominent in the conserved parts of oriC regions from various bacteria. The only relevant variation is an A-to-G exchange at the third position of the DnaA box motif within GC-rich DNA present in gram-positive bacteria such as Micrococcus luteus (10) and Streptomyces lividans (42). Most eubacteria contain a block of genes, dnaA-dnaN-recF-gyrB (10, 38), encoding the initiator protein DnaA, the ␤-subunit of DNA polymerase III holoenzyme, a product for recombination, and the ␤-subunit of DNA gyrase, respectively. Within most bacteria, oriC is situated close to the dnaA gene. In Escherichia coli, the arrangement of the dnaA region matches that in other bacteria; however, this region is located about 42 kb away from oriC (35).Procaryotic chromosomal replication has been best studied in E. coli. Its DnaA protein is a basic protein of 52 kDa to which ATP binds. About 10 to 40 ATP-DnaA molecules interact with four DnaA boxes within oriC (19,36). According to a current model (14), the newly synthesized DnaA protein is titrated by these DnaA boxes. As soon as the number of DnaA molecules exceeds the number of boxes, initiation is supposed to occur. A high number of GATC sites which can be methylated by the Dam methyltransferase have been found in the oriC region of E. coli, as well as in other members of the family Enterobacteriaceae (44), and GANTC sites are present in the Caulobacter crescentus oriC (43).Contrary to many bacteria, Streptomyces species grow as substrate mycelia which differentiate to aerial mycelia and spores upon depletion of nutrients. Depending on the species, the size of the GC-rich Streptomyces chromosome ranges between 6.6 and 8 Mb (18,32). Recently, we identified the...

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“…Western immunoblot analysis of total cellular DnaA protein concentration was carried out as described by Hansen et al. (11). dnaA gene expression was analyzed in strains SPE83 (rpoC907) and SPE84 (rpoC+), carrying the XRB1 phage (4).…”

Section: Methodsmentioning

confidence: 99%

A missense mutation in the rpoC gene affects chromosomal replication control in Escherichia coli

Petersen

Hansen

1991

J Bacteriol

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An RNA polymerase mutant with a single-base-pair change in the rpoC gene affects chromosome initiation control. The mutation, which is recessive, is a G to A transition leading to the substitution of aspartate for glycine at amino acid residue 1033 in the RNA polymerase beta' subunit. The chromosome copy number is increased twofold in the mutant at semipermissive growth temperatures (39 degrees C). In a delta oriC strain, in which chromosome initiation is governed by an F replicon, chromosome copy number is not affected. Plasmid pBR322 copy number is also increased in the mutant at 39 degrees C. The mutation causes a more than fivefold increased expression of the dnaA gene at 39 degrees C. It is conceivable that it is this high DnaA concentration which causes the high chromosome copy number and that the mutant RNA polymerase beta' subunit exerts its effect by altering the expression of the dnaA gene. However, other factors must be affected as well to explain why the RNA polymerase mutant can grow in a balanced fashion with a high chromosome concentration. This is in contrast to wild-type cells, which exhibit higher origin concentrations when DnaA protein is overproduced, but in which the overall DNA concentration is only moderately affected.

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Initiator (DnaA) protein concentration as a function of growth rate in Escherichia coli and Salmonella typhimurium

Cited by 107 publications

References 35 publications

Three distinct chromosome replication states are induced by increasing concentrations of DnaA protein in Escherichia coli

Three distinct chromosome replication states are induced by increasing concentrations of DnaA protein in Escherichia coli

Minimal requirements of the Streptomyces lividans 66 oriC region and its transcriptional and translational activities

A missense mutation in the rpoC gene affects chromosomal replication control in Escherichia coli

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