Broken replication forks trigger heritable DNA breaks in the terminus of a circular chromosome

<p><u>(A) Circular map of the <i>E</i>. <i>coli</i> chromosome</u>: <i>oriC</i>, <i>dif</i> and <i>terD</i> to <i>terB</i> sites are indicated. Numbers refer to the chromosome coordinates (in kb) of MG1655. (<u>B) Linear map of the terminus region:</u> chromosome coordinates are shown increasing from left to right, as in the marker frequency panels (see Figure 1C for example), therefore in the opposite direction to the circular map. In addition to <i>dif</i> and <i>ter</i> sites, the positions of the <i>parS</i>_pMT1 sites used for microscopy experiments are indicated. (<u>C) MFA analysis of terminus DNA loss in the <i>recB</i> mutant</u>: sequence read frequencies of exponential phase cells normalized to the total number of reads were calculated for each strain. Ratios of normalized reads in isogenic wild-type and <i>recB</i> mutant are plotted against chromosomal coordinates (in kb). The profile ratio of the terminus region is enlarged and the profile of the corresponding entire chromosomes is shown in inset. Original normalized profiles used to calculate ratios are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007256#pgen.1007256.s005" target="_blank">S1 Fig</a>. The position of <i>dif</i> is indicated by a red arrow. The <i>ter</i> sites that arrest clockwise forks (<i>terC</i>, <i>terB</i>, green arrow) and counter-clockwise forks (<i>terA</i>, <i>terD</i>, blue arrow) are shown. <u>(D) Schematic representation of focus loss in the <i>recB</i> mutant:</u> Time-lapse microscopy experiments showed that loss of a focus in the <i>recB</i> mutant occurs concomitantly with cell division in one of two daughter cells, and that the cell that keeps the focus then generates a focus-less cell at each generation. The percentage of initial events was calculated as the percentage of cell divisions that generate a focus-less cell, not counting the following generations. In this schematic representation, two initial events occurred (generations #2 and #7) out of 9 generations, and focus loss at generation #2 is heritable. Panels shown in this figure were previously published in [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007256#pgen.1007256.ref019" target="_blank">19</a>] and are reproduced here to introduce the phenomenon.</p

The Stringent Response and Cell Cycle Arrest in Escherichia coli

The bacterial stringent response, triggered by nutritional deprivation, causes an accumulation of the signaling nucleotides pppGpp and ppGpp. We characterize the replication arrest that occurs during the stringent response in Escherichia coli. Wild type cells undergo a RelA-dependent arrest after treatment with serine hydroxamate to contain an integer number of chromosomes and a replication origin-to-terminus ratio of 1. The growth rate prior to starvation determines the number of chromosomes upon arrest. Nucleoids of these cells are decondensed; in the absence of the ability to synthesize ppGpp, nucleoids become highly condensed, similar to that seen after treatment with the translational inhibitor chloramphenicol. After induction of the stringent response, while regions corresponding to the origins of replication segregate, the termini remain colocalized in wild-type cells. In contrast, cells arrested by rifampicin and cephalexin do not show colocalized termini, suggesting that the stringent response arrests chromosome segregation at a specific point. Release from starvation causes rapid nucleoid reorganization, chromosome segregation, and resumption of replication. Arrest of replication and inhibition of colony formation by ppGpp accumulation is relieved in seqA and dam mutants, although other aspects of the stringent response appear to be intact. We propose that DNA methylation and SeqA binding to non-origin loci is necessary to enforce a full stringent arrest, affecting both initiation of replication and chromosome segregation. This is the first indication that bacterial chromosome segregation, whose mechanism is not understood, is a step that may be regulated in response to environmental conditions

A naturally occurring point mutation in the 13-mer R repeat affects the oriCfunction of the large chromosome of Vibrio choleraeO1 classical biotype

The genome of Vibrio cholerae consists of two circular chromosomes of di.erent sizes. Here, a comparative analysis of the replication origins of the large chromosomes (oriCIVC) of classical and El Tor biotypes of the pathogen is reported. Extensive nucleotide sequence analyses revealed that the oriCIVC region has six DnaA boxes instead of the .ve found in Escherichia coli oriC. The additional DnaA box, designated RV, was unique in V. cholerae as well as in other members of the family Vibrionaceae. However, RV was not found to be essential for the autonomous replication function of the 307-bp oriCIVC minimal region. In contrast to El Tor and the recently evolved V. cholerae O139 strains, the oriCIVC region of the classical biotype showed only a single base transition (T . G) in a highly conserved AT-rich 13-mer R repeat region. From the minichromosome copy number and its transformational e.ciency analyses, it appears that the single base substitution in the oriCIVC of the classical biotype has a signi.cant e.ect on its replication initiation