Mohan C. Joshi

Escherichia coli sister chromosome separation includes an abrupt global transition with concomitant release of late-splitting intersister snaps

The basis for segregation of sister chromosomes in bacteria is not established. We show here that two discrete ~150-kb regions, both located early in the right replichore, exhibit prolonged juxtaposition of sister loci, for 20 and 30 min, respectively, after replication. Flanking regions, meanwhile, separate. Thus, the two identified regions comprise specialized late-splitting intersister connections or snaps. Sister snap loci separate simultaneously in both snap regions, concomitant with a major global nucleoid reorganization that results in emergence of a bilobed nucleoid morphology. Split snap loci move rapidly apart to a separation distance comparable with one-half the length of the nucleoid. Concomitantly, at already split positions, sister loci undergo further separation to a comparable distance. The overall consequence of these and other effects is that thus far replicated sister chromosomes become spatially separated (individualized) into the two nucleoid lobes, while the terminus region (and likely, all unreplicated portions of the chromosome) moves to midcell. These and other findings imply that segregation of Escherichia coli sister chromosomes is not a smooth continuous process but involves at least one and likely, two major global transition(s). The presented patterns further suggest that accumulation of internal intranucleoid forces and constraining of these forces by snaps play central roles in global chromosome dynamics. They are consistent with and supportive of our previous proposals that individualization of sisters in E. coli is driven primarily by internally generated pushing forces and is directly analogous to sister individualization at the prophase to prometaphase transition of the eukaryotic cell cycle.

Engineered proteins detect spontaneous DNA breakage in human and bacterial cells

Spontaneous DNA breaks instigate genomic changes that fuel cancer and evolution, yet direct quantification of double-strand breaks (DSBs) has been limited. Predominant sources of spontaneous DSBs remain elusive. We report synthetic technology for quantifying DSBs using fluorescent-protein fusions of double-strand DNA end-binding protein, Gam of bacteriophage Mu. In Escherichia coli GamGFP forms foci at chromosomal DSBs and pinpoints their subgenomic locations. Spontaneous DSBs occur mostly one per cell, and correspond with generations, supporting replicative models for spontaneous breakage, and providing the first true breakage rates. In mammalian cells GamGFP—labels laser-induced DSBs antagonized by end-binding protein Ku; co-localizes incompletely with DSB marker 53BP1 suggesting superior DSB-specificity; blocks resection; and demonstrates DNA breakage via APOBEC3A cytosine deaminase. We demonstrate directly that some spontaneous DSBs occur outside of S phase. The data illuminate spontaneous DNA breakage in E. coli and human cells and illustrate the versatility of fluorescent-Gam for interrogation of DSBs in living cells. DOI: http://dx.doi.org/10.7554/eLife.01222.001

Regulation of Sister Chromosome Cohesion by the Replication Fork Tracking Protein SeqA

Analogously to chromosome cohesion in eukaryotes, newly replicated DNA in E. coli is held together by inter-sister linkages before partitioning into daughter nucleoids. In both cases, initial joining is apparently mediated by DNA catenation, in which replication-induced positive supercoils diffuse behind the fork, causing newly replicated duplexes to twist around each other. Type-II topoisomerase-catalyzed sister separation is delayed by the well-characterized cohesin complex in eukaryotes, but cohesion control in E. coli is not currently understood. We report that the abundant fork tracking protein SeqA is a strong positive regulator of cohesion, and is responsible for markedly prolonged cohesion observed at “snap” loci. Epistasis analysis suggests that SeqA stabilizes cohesion by antagonizing Topo IV-mediated sister resolution, and possibly also by a direct bridging mechanism. We show that variable cohesion observed along the E. coli chromosome is caused by differential SeqA binding, with oriC and snap loci binding disproportionally more SeqA. We propose that SeqA binding results in loose inter-duplex junctions that are resistant to Topo IV cleavage. Lastly, reducing cohesion by genetic manipulation of Topo IV or SeqA resulted in dramatically slowed sister locus separation and poor nucleoid partitioning, indicating that cohesion has a prominent role in chromosome segregation.

An Insecticidal GroEL Protein with Chitin Binding Activity from Xenorhabdus nematophila

Xenorhabdus nematophila secretes insecticidal proteins to kill its larval prey. We have isolated an ∼58-kDa GroEL homolog, secreted in the culture medium through outer membrane vesicles. The protein was orally insecticidal to the major crop pest Helicoverpa armigera with an LC50 of ∼3.6 μg/g diet. For optimal insecticidal activity all three domains of the protein, apical, intermediate, and equatorial, were necessary. The apical domain alone was able to bind to the larval gut membranes and manifest low level insecticidal activity. At equimolar concentrations, the apical domain contained approximately one-third and the apical-intermediate domain approximately one-half bioactivity of that of the full-length protein. Interaction of the protein with the larval gut membrane was specifically inhibited by N-acetylglucosamine and chito-oligosaccharides. Treatment of the larval gut membranes with chitinase abolished protein binding. Based on the three-dimensional structural model, mutational analysis demonstrated that surface-exposed residues Thr-347 and Ser-356 in the apical domain were crucial for both binding to the gut epithelium and insecticidal activity. Double mutant T347A,S356A was 80% less toxic (p < 0.001) than the wild type protein. The GroEL homolog showed α-chitin binding activity with Kd ∼ 0.64 μm and Bmax ∼ 4.68 μmol/g chitin. The variation in chitin binding activity of the mutant proteins was in good agreement with membrane binding characteristics and insecticidal activity. The less toxic double mutant XnGroEL showed an ∼8-fold increase of Kd in chitin binding assay. Our results demonstrate that X. nematophila secretes an insecticidal GroEL protein with chitin binding activity.

The Cytotoxic Fimbrial Structural Subunit of Xenorhabdus nematophila Is a Pore-Forming Toxin

We have purified a fimbrial shaft protein (MrxA) of Xenorhabdus nematophila. The soluble monomeric protein lysed larval hemocytes of Helicoverpa armigera. Osmotic protection of the cells with polyethylene glycol suggested that the 17-kDa MrxA subunit makes pores in the target cell membrane. The internal diameter of the pores was estimated to be >2.9 nm. Electron microscopy confirmed the formation of pores by the fimbrial subunit. MrxA protein oligomerized in the presence of liposomes. Electrophysiological studies demonstrated that MrxA formed large, voltage-gated passive-diffusion channels in lipid bilayers.

DNA Replication Initiation Is Blocked by a Distant Chromosome–Membrane Attachment

Although it has been recognized for several decades that chromosome structure regulates the capacity of replication origins to initiate, very little is known about how or if cells actively regulate structure to direct initiation. We report that a localized inducible protein tether between the chromosome and cell membrane in E. coli cells imparts a rapid and complete block to replication initiation. Tethers, composed of a trans-membrane and transcription repressor fusion protein bound to an array of operator sequences, can be placed up to 1 Mb from the origin with no loss of penetrance. Tether-induced initiation blocking has no effect on elongation at pre-existing replication forks and does not cause cell or DNA damage. Whole-genome and site-specific fluorescent DNA labeling in tethered cells indicates that global nucleoid structure and chromosome organization are disrupted. Gene expression patterns, assayed by RNA sequencing, show that tethering induces global supercoiling changes, which are likely incompatible with replication initiation. Parallels between tether-induced initiation blocking and rifampicin treatment and the role of programmed changes in chromosome structure in replication control are discussed.

MioC and GidA proteins promote cell division in E. coli

The well-conserved genes surrounding the E. coli replication origin, mioC and gidA, do not normally affect chromosome replication and have little known function. We report that mioC and gidA mutants exhibit a moderate cell division inhibition phenotype. Cell elongation is exacerbated by a fis deletion, likely owing to delayed replication and subsequent cell cycle stress. Measurements of replication initiation frequency and origin segregation indicate that mioC and gidA do not inhibit cell division through any effect on oriC function. Division inhibition is also independent of the two known replication/cell division checkpoints, SOS and nucleoid occlusion. Complementation analysis indicates that mioC and gidA affect cell division in trans, indicating their effect is at the protein level. Transcriptome analysis by RNA sequencing showed that expression of a cell division septum component, YmgF, is significantly altered in mioC and gidA mutants. Our data reveal new roles for the gene products of gidA and mioC in the division apparatus, and we propose that their expression, cyclically regulated by chromatin remodeling at oriC, is part of a cell cycle regulatory program coordinating replication initiation and cell division.

Bacteria-to-human protein networks reveal origins of endogenous DNA damage

DNA damage provokes mutations and cancer, and results from external carcinogens or endogenous cellular processes. Yet, the intrinsic instigators of DNA damage are poorly understood. Here we identify proteins that promote endogenous DNA damage when overproduced: the DNA-damaging proteins (DDPs). We discover a large network of DDPs in Escherichia coli and deconvolute them into six DNA-damage-causing function clusters, demonstrating DDP mechanisms in three: reactive-oxygen increase by transmembrane transporters, chromosome loss by replisome binding, and replication stalling by transcription factors. Their 284 human homologs are over-represented among known cancer drivers, and their expression in tumors predicts heavy mutagenesis and poor prognosis. Half of tested human homologs, when overproduced in human cells, promote DNA damage and mutation, with DNA-damaging mechanisms like those in E. coli. Together, our work reveals DDP networks that provoke endogenous DNA damage and may indicate functions of many human known and newly implicated cancer-promoting proteins.

Multilocus Imaging of the E. coli Chromosome by Fluorescent In Situ Hybridization

Fluorescence in situ hybridization (FISH) is a widely used technique to detect and localize specific DNA or RNA sequences in cells. Although supplanted in many ways by fluorescently labeled DNA binding proteins, FISH remains the only cytological method to examine many genetic loci at once (up to six), and can be performed in any cell type and genotype. These advantages have proved invaluable in studying the spatial relationships between chromosome regions and the dynamics of chromosome segregation in bacteria. A detailed protocol for DNA FISH in E. coli is described.