DNA double strand break repair in Escherichia coli perturbs cell division and chromosome dynamics.

To prevent the transmission of damaged genomic material between generations, cells require a system for accommodating DNA repair within their cell cycles. We have previously shown that Escherichia coli cells subject to a single, repairable site-specific DNA double-strand break (DSB) per DNA replication cycle reach a new average cell length, with a negligible effect on population growth rate. We show here that this new cell size distribution is caused by a DSB repair-dependent delay in completion of cell division. This delay occurs despite unperturbed cell size regulated initiation of both chromosomal DNA replication and cell division. Furthermore, despite DSB repair altering the profile of DNA replication across the genome, the time required to complete chromosomal duplication is invariant. The delay in completion of cell division is accompanied by a DSB repair-dependent delay in individualization of sister nucleoids. We suggest that DSB repair events create inter-sister connections that persist until those chromosomes are separated by a closing septum.

RecBCD coordinates repair of two ends at a DNA double-strand break, preventing aberrant chromosome amplification.

DNA double-strand break (DSB) repair is critical for cell survival. A diverse range of organisms from bacteria to humans rely on homologous recombination for accurate DSB repair. This requires both coordinate action of the two ends of a DSB and stringent control of the resultant DNA replication to prevent unwarranted DNA amplification and aneuploidy. In Escherichia coli, RecBCD enzyme is responsible for the initial steps of homologous recombination. Previous work has revealed recD mutants to be nuclease defective but recombination proficient. Despite this proficiency, we show here that a recD null mutant is defective for the repair of a two-ended DSB and that this defect is associated with unregulated chromosome amplification and defective chromosome segregation. Our results demonstrate that RecBCD plays an important role in avoiding this amplification by coordinating the two recombining ends in a manner that prevents divergent replication forks progressing away from the DSB site.

Profiling antibiotic resistance and electrotransformation potential of Ensifer adhaerens OV14; a non-Agrobacterium species capable of efficient rates of plant transformation.

Ensifer adhaerens OV14 underpins the successful crop transformation protocol termed Ensifer-mediated transformation but issues exist in regard to addressing the pleomorphic tendency of the bacterium in suboptimal conditions, identifying the optimal parameters for electrotransformation and defining the strain's antibiotic profile. Here, modifications made to growth medium composition addressed the pleomorphic trait of E. adhaerens OV14, delivering uniform E. adhaerens OV14 growth to ensure efficient rates of electroporation with plasmids up to 42.2 kb in size. Separately, 63 putative antibiotic resistance coding sequences were identified across the E. adhaerens OV14 genome, with testing confirming the strain's susceptibility to gentamicin (≥10 mg L(-1)), tetracycline (≥10 mg L(-1)), chloramphenicol (≥100 mg L(-1)) and cefotaxime (≥500 mg L(-1)) and resistance to ampicillin, paramomycin, streptomycin, spectinomycin, ticarcillin-clavulanate and kanamycin. Partial resistance against carbenicillin, rifampicin, hygromycin-B and neomycin was also recorded. Resistance to kanamycin was supported by seven independent nptII-like homologs located within the E. adhaerens OV14 genome. Transcriptional analysis of these targets highlighted two homologs (AHK42288 and AHK42618) whose transcription was significantly elevated within 2 h exposure to kanamycin and which in the case of AHK42288 was maintained out to 6 h post-exposure. In conclusion, our results have identified optimal conditions for the handling of E. adhaerens and have identified a future genome editing target (AHK42288) to negate the kanamycin-resistant phenotype of E. adhaerens.

Repair on the go: E. coli maintains a high proliferation rate while repairing a chronic DNA double-strand break.

DNA damage checkpoints exist to promote cell survival and the faithful inheritance of genetic information. It is thought that one function of such checkpoints is to ensure that cell division does not occur before DNA damage is repaired. However, in unicellular organisms, rapid cell multiplication confers a powerful selective advantage, leading to a dilemma. Is the activation of a DNA damage checkpoint compatible with rapid cell multiplication? By uncoupling the initiation of DNA replication from cell division, the Escherichia coli cell cycle offers a solution to this dilemma. Here, we show that a DNA double-strand break, which occurs once per replication cycle, induces the SOS response. This SOS induction is needed for cell survival due to a requirement for an elevated level of expression of the RecA protein. Cell division is delayed, leading to an increase in average cell length but with no detectable consequence on mutagenesis and little effect on growth rate and viability. The increase in cell length caused by chronic DNA double-strand break repair comprises three components: two types of increase in the unit cell size, one independent of SfiA and SlmA, the other dependent of the presence of SfiA and the absence of SlmA, and a filamentation component that is dependent on the presence of either SfiA or SlmA. These results imply that chronic checkpoint induction in E. coli is compatible with rapid cell multiplication. Therefore, under conditions of chronic low-level DNA damage, the SOS checkpoint operates seamlessly in a cell cycle where the initiation of DNA replication is uncoupled from cell division.

Histone deacetylase complex1 expression level titrates plant growth and abscisic acid sensitivity in Arabidopsis.

Histone deacetylation regulates gene expression during plant stress responses and is therefore an interesting target for epigenetic manipulation of stress sensitivity in plants. Unfortunately, overexpression of the core enzymes (histone deacetylases [HDACs]) has either been ineffective or has caused pleiotropic morphological abnormalities. In yeast and mammals, HDACs operate within multiprotein complexes. Searching for putative components of plant HDAC complexes, we identified a gene with partial homology to a functionally uncharacterized member of the yeast complex, which we called Histone Deacetylation Complex1 (HDC1). HDC1 is encoded by a single-copy gene in the genomes of model plants and crops and therefore presents an attractive target for biotechnology. Here, we present a functional characterization of HDC1 in Arabidopsis thaliana. We show that HDC1 is a ubiquitously expressed nuclear protein that interacts with at least two deacetylases (HDA6 and HDA19), promotes histone deacetylation, and attenuates derepression of genes under water stress. The fast-growing HDC1-overexpressing plants outperformed wild-type plants not only on well-watered soil but also when water supply was reduced. Our findings identify HDC1 as a rate-limiting component of the histone deacetylation machinery and as an attractive tool for increasing germination rate and biomass production of plants.

WITHDRAWN: Symmetries and Asymmetries Associated with Non-Random Segregation of Sister DNA Strands in Escherichia coli.

The Publisher regrets that this article is an accidental duplication of an article that has already been published, http://dx.doi.org/10.1016/j.semcdb.2013.05.010. The duplicate article has therefore been withdrawn.

Symmetries and asymmetries associated with non-random segregation of sister DNA strands in Escherichia coli.

The successful inheritance of genetic information across generations is a complex process requiring replication of the genome and its faithful segregation into two daughter cells. At each replication cycle there is a risk that new DNA strands incorporate genetic changes caused by miscopying of parental information. By contrast the parental strands retain the original information. This raises the intriguing possibility that specific cell lineages might inherit "immortal" parental DNA strands via non-random segregation. If so, this requires an understanding of the mechanisms of non-random segregation. Here, we review several aspects of asymmetry in the very symmetrical cell, Escherichia coli, in the interest of exploring the potential basis for non-random segregation of leading- and lagging-strand replicated chromosome arms. These considerations lead us to propose a model for DNA replication that integrates chromosome segregation and genomic localisation with non-random strand segregation.

Non-random segregation of sister chromosomes in Escherichia coli.

It has long been known that the 5' to 3' polarity of DNA synthesis results in both a leading and lagging strand at all replication forks. Until now, however, there has been no evidence that leading or lagging strands are spatially organized in any way within a cell. Here we show that chromosome segregation in Escherichia coli is not random but is driven in a manner that results in the leading and lagging strands being addressed to particular cellular destinations. These destinations are consistent with the known patterns of chromosome segregation. Our work demonstrates a new level of organization relating to the replication and segregation of the E. coli chromosome.

SbcCD regulation and localization in Escherichia coli.

The SbcCD complex and its homologues play important roles in DNA repair and in the maintenance of genome stability. In Escherichia coli, the in vitro functions of SbcCD have been well characterized, but its exact cellular role remains elusive. This work investigates the regulation of the sbcDC operon and the cellular localization of the SbcC and SbcD proteins. Transcription of the sbcDC operon is shown to be dependent on starvation and RpoS protein. Overexpressed SbcC protein forms foci that colocalize with the replication factory, while overexpressed SbcD protein is distributed through the cytoplasm.