Peter L. Kuempel

The cytoplasmic domain of FtsK protein is required for resolution of chromosome dimers

Chromosome dimers, formed by homologous recombination between sister chromosomes, normally require cell division to be resolved into monomers by site‐specific recombination at the dif locus of Escherichia coli. We report here that it is not in fact cell division per se that is required for dimer resolution but the action of the cytoplasmic domain of FtsK, which is a bifunctional protein required both for cell division and for chromosome partition.

Plant factors induce expression of nodulation and host-range genes in Rhizobium trifolii

SummaryIn Rhizobium trifolii ANU843, host specific nodulation capability is encoded within a 14kb HindIII fragment of the symbiosis plasmid. To gain a better understanding of the regulation of the nodulation (Nod) genes, we have isolated lac operon transcriptional fusions to several genes within this fragment, using the mini-Mu-lac bacteriophage transposon MudI1734. Using a broad-host-range vector, fragments containing MudI1734 insertions were introduced into R. trifolii ANU845, a derivative of ANU843 which lacks the symbiosis plasmid. Four distinct regions were identified within the Nod fragment, insertions in which resulted in nodulation phenotypes similar to those found previously for Tn5. Region I mutants were Nod- and defective in root hair curling (Hac-) and corresponded to the nodABC and D genes identified by sequence analysis. Region II mutants showed an exaggerated root hair curling (Hac++) response on clover plants and a greatly reduced nodulation ability. Region III mutants were affected in host-range properties, as they gained the ability to nodulate Pisum sativum (peas), but showed only poor nodulation ability on the normal host plant, Trifolium repens (white clover). Region IV mutants showed a delay in the nodulation of Trifolium repens, but only when plants were grown under high light-regimes. When ANU845 strains carrying the above MudI1734 insertions were grown in standard laboratory media, only insertions in nodD expressed β-galactosidase at high levels. However, when cells were placed in medium in which Trifolium repens was growing, insertions in nodA, nodB, region II, region III, and region IV were all induced from 5–10 times above basal levels. This allowed us to determine the directions of transcription in these regions.

Identification of the DNA sequence from the E. coli terminus region that halts replication forks

The terminus region of the E. coli chromosome contains two loci, T1 and T2, that inhibit the progress of replication forks and require the trans-acting factor tus. We have identified a 23 bp terminator signal at T1 and T2 that is within 100 bp of the sites of replication arrest. When an oligodeoxyribonucleotide containing the terminator signal was inserted into a plasmid, replication was halted only in a tus+ strain and when the terminator signal was oriented properly. We also found this terminator sequence in the terminus region of the plasmid R6K and in the origin region of RepFIIA class plasmids. In addition, we found striking similarities between the E. coli terminator signal and the terminator sequence of B. subtilis.

Cell division, guillotining of dimer chromosomes and SOS induction in resolution mutants (dif, xerC and xerD) of Escherichia coli

We have studied the growth and division of xerC, xerD and dif mutants of Escherichia coli, which are unable to resolve dimer chromosomes. These mutants express the Dif phenotype, which includes reduced viability, SOS induction and filamentation, and abnormal nucleoid morphology. Growth was studied in synchronous cultures and in microcolonies derived from single cells. SOS induction and filamentation commenced after an apparently normal cell division, which sheared unresolved dimer chromosomes. This has been called guillotining. Microcolony analysis demonstrated that cell division in the two daughter cells was inhibited after guillotining, and microcolonies formed that consisted of two filaments lying side by side. Growth of these filaments was severely reduced in hipA+ strains. We propose that guillotining at dif destroys the expression of the adjacent hipBA genes and, in the absence of continued formation of HipB, HipA inhibits growth. The length of the filaments was also affected by SfiA: sfiA dif hipA mutants initially formed filaments, but cell division at the ends of the filaments ultimately produced a number of DNA‐negative cells. If SOS induction was blocked by lexA3 (Ind−), filaments did not form, and cell division was not inhibited. However, pedigree analysis of cells in microcolonies demonstrated that lethal sectoring occurred as a result of limited growth and division of dead cells produced by guillotining.

A possible function of DNA polymerase in chromosome replication

Abstract The Escherichia coli DNA polymerase mutant isolated by de Lucia and Cairns is impaired in its ability to convert low molecular weight, newly synthesized DNA into high molecular weight material. The mutation affects the joining of the low molecular weight strands and not their synthesis, but the strands are eventually incorporated into high molecular weight DNA. The in vitro DNA ligase activity of the mutant is normal. It is proposed both DNA polymerase and DNA ligase are required to close the breaks in newly synthesized DNA in vivo , and either the low levels of polymerase activity or interference with DNA ligase by an altered DNA polymerase reduce the rate of closing in the mutant.

Tus and the terminators: The arrest of replication in prokaryotes

Bidirectional replication of prokaryotic circular chromosomes concludes when the two replication forks converge opposite the origin of replication in a region called the terminus. The terminus regions of several replicons contain sites referred to as terminators that arrest DNA replication and prevent replication forks from exiting the terminus. Function of the terminators is absolutely dependent upon a DNA binding protein that specifically binds the terminator sites and impedes replication forks. Studies on prokaryotic termination systems have now progressed to the point where termination sites have been sequenced and DNA binding proteins have been purified to near homogeneity. Very recently, several laboratories have completed the initial biochemical characterization of the mechanism of replication fork arrest, and one of these papers appears in this issue of cell. The studies that elucidated the components of the termination systems in Escherichia coli, Bacillus subtiiis, and the R6K plasmid will be reviewed here, as well as the more recent mechanistic studies. Tsrmlnator Sites The terminus of the E. coli chromosome is a large region (~360 kb) flanked on both sides by terminator sites, WA, Ter8, TerC, and TerD, that arrest replication forks (see figure; nomenclature for these sites is presented in the footnote). Before proceeding to the details of this inhibition of replication, the role of these sites in the replication cycle should be considered. inhibition is polar, and the important concept was developed that the terminus region functions as a replication-fork trap (Hill et al., 1987; deMassy et al., 1987). Specifically, replication forks from either direction are not prevented from entering the terminus region, but they are inhibited when they progress across the terminus and reach the other side. With respect to bidirectional replication of the chromosome, this means that if the two forks

Norfloxacin‐induced DNA cleavage occurs at the dif resolvase locus in Escherichia coli and is the result of interaction with topoisomerase IV

The dif locus is a site‐specific recombination site located within the terminus region of the chromosome of Escherichia coli. Recombination at dif resolves circular dimer chromosomes to monomers, and this recombination requires the XerC, XerD and FtsK proteins, as well as cell division. In order to characterize other enzymes that interact at dif, we tested whether quinolone‐induced cleavage occurs at this site. Quinolone drugs, such as norfloxacin, inhibit the type 2 topoisomerases, DNA gyrase and topoisomerase IV, and can cleave DNA at sites where these enzymes interact with the chromosome. Using strains in which either DNA gyrase or topoisomerase IV, or both, were resistant to norfloxacin, we determined that specific interactions between dif and topoisomerase IV caused cleavage at that site. This interaction required XerC and XerD, but did not require the C‐terminal region of FtsK or cell division.

In vivo characterization of tus gene expression in Escherichia coli

The tus gene encodes a DNA‐binding protein (Tus) that inhibits replication forks at specific block‐sites within the terminus region of the Escherichia coli chromosome. One of these block‐sites, TerB, is adjacent to the tus gene. Using primer extension and a promoter fusion to characterize in vivo expression, we have demonstrated that the tus transcription start site is within TerB, and that Tus protein autoregulates expression at this weak promoter We have also demonstrated that a minority of tus transcripts are initiated from an upstream region that contains two additional open reading frames. This readthrough transcription into tus is reduced in the presence of Tus protein.

Loss of rac locus DNA in merozygotes of Escherichia coli K12.

SummaryDNA-DNA hybridization was used to demonstrate that the substituted DNA in the bacteriophage λrecE (formerly called λ reverse) is homologous to DNA at the rac locus in Escherichia coli. Strains that are rac- do not contain appreciable amounts of this DNA, and it is lost from a rac+ episome (F′ 123) after transmission to a rac- recipient. This is consistent with the proposal that the rac locus contains a cryptic prophage (Low, 1973).

DNA degradation in the terminus region of resolvase mutants of Escherichia coli, and suppression of this degradation and the Dif phenotype by recD.

We recently proposed that guillotining of dimer chromosomes occurs at cell division in resolvase mutants of Escherichia coli. This was based on the abnormal pattern of cell division observed in 10-14% of the cells in microcolonies of xerC, xerD and dif mutants. A prediction of this guillotining is that DNA degradation should occur in the terminus region, in the vicinity of the dif locus. We have tested this by DNA-DNA hybridization and have observed that dif was absent in about 22% of the chromosomes in exponentially growing xerC mutants. A locus 206 kb from dif was not affected by this degradation. We have also observed that degradation did not occur in xerC recD mutants, and that the low efficiency of plating associated with the Dif phenotype was suppressed in this strain. A model is proposed in which rapid degradation of the terminus region does not occur in recD mutants following guillotining, and that this permits the initiation of repair of broken dimer chromosomes prior to completion of cell division.