Chromosome arrangement and dynamics in the budding bacterium Hyphomonas neptunium

Faithful chromosome replication and segregation are essential for every living cell and must be tightly coordinated with other cell cycle events such as cell division. Our knowledge about prokaryotic chromosome dynamics is based on studies of only a few model organisms that divide by binary fission and are mostly characterized by a rod-like morphology. To broaden our insight into bacterial chromosome segregation, our lab has recently started to analyze chromosome dynamics in the marine alphaproteobacterium Hyphomonas neptunium, which divides by budding at the tip of the stalk and uses its stalk as a reproductive structure. This mode of reproduction distinguishes H. neptunium from so far studied model organisms and renders it an exciting candidate for the study of chromosome dynamics, since the duplicated chromosome must transit the stalk to reach the newly generated daughter cell. Recent work has revealed that the H. neptunium chromosome is segregated in a unique two-step process. At first, one of the duplicated origins is segregated within the mother cell, possibly in a ParABS-dependent manner, and remains at the stalked mother cell pole until a visible bud has formed at the tip of the stalk. In a second step, it is then segregated through the stalk into the bud. Several lines of evidence suggest that the transport through the stalk is mediated by a novel, yet unidentified, segregation mechanism. Commonly, chromosome replication and segregation occur concomitantly in bacteria. However, this two-step segregation mechanism implies a temporal uncoupling of chromosome replication and segregation through the stalk, reminiscent of eukaryotic mitosis. In this work, we analyzed the role of the ParABS system in chromosome segregation of H. neptunium. The ParABS system was shown to be essential for cell viability and chromosome segregation. Impairment of ParA functioning leads to morphological alterations and incomplete origin segregation within the mother cell and, consequently, hampers chromosome segregation through the stalk. This shows that the ParABS system mediates origin segregation within the mother cell. It also implies that chromosome segregation within the mother cell and through the stalk are sequential processes. Furthermore, we analyzed the role of PopZ and SMC in H. neptunium, since these proteins were shown to be involved in chromosome segregation in other bacteria. PopZ localizes to the pole opposite the stalk in the newly generated bud and appears to play only a minor role in the positioning of the ParABS partitioning machinery. SMC seems to be essential in H. neptunium and shows a similar localization pattern as ParB. Determination of the location of seven genomic loci in new-born cells revealed that the chromosome shows a longitudinal arrangement with the origin located at the flagellated pole and the terminus at the opposite cell pole. The other loci are arranged between both cell poles in a linear order that correlates with their position on the genomic map. Moreover, analysis of chromosome dynamics indicates that the ParB/parS complex is the region to be segregated first within the mother cell and also through the stalk, emphasizing its central role in the segregation process. As mentioned above, the observed two-step chromosome segregation mechanism suggested a temporal uncoupling of chromosome replication and its segregation through the stalk. To investigate the coordination between these two processes in more detail, we followed replisome dynamics by fluorescence labeling of different replisome components. The replication machinery shows a dynamic localization within the mother cell: in cells that are most likely at the swarmer-to-stalked cell transition as well as in stalked cells, it assembles at the pole opposite the (future) stalk and moves, via midcell, close to the stalked cell pole, where it disassembles again. This localization pattern is consistent with the observed location of the origin and terminus region. Furthermore, the replisomes appear to track independently along the two chromosome arms. Co-localization of ParB (origin) and DnaN (replisome) revealed that a large part of the chromosome is replicated before its segregation through the stalk commences, indicating that these processes are partially temporally uncoupled. Collectively, these observations expand our insight into chromosome dynamics in H. neptunium and suggest that it combines previously described segregation mechanisms, such as the ParABS system, with a novel segregation mechanism that awaits discovery

PAPC and the Wnt5a/Ror2 pathway control the invagination of the otic placode in Xenopus

<p>Abstract</p> <p>Background</p> <p>Paraxial protocadherin (PAPC) plays a crucial role in morphogenetic movements during gastrulation and somitogenesis in mouse, zebrafish and Xenopus. PAPC influences cell-cell adhesion mediated by C-Cadherin. A putative direct adhesion activity of PAPC is discussed. PAPC also promotes cell elongation, tissue separation and coordinates cell mass movements. In these processes the signaling function of PAPC in activating RhoA/JNK and supporting Wnt-11/PCP by binding to frizzled 7 (fz7) is important.</p> <p>Results</p> <p>Here we demonstrate by loss of function experiments in Xenopus embryos that PAPC regulates another type of morphogenetic movement, the invagination of the ear placode. Knockdown of PAPC by antisense morpholinos results in deformation of the otic vesicle without altering otocyst marker expression. Depletion of PAPC could be rescued by full-length PAPC, constitutive active RhoA and by the closely related PCNS but not by classical cadherins. Also the cytoplasmic deletion mutant M-PAPC, which influences cell adhesion, does not rescue the PAPC knockdown. Interestingly, depletion of Wnt5a or Ror2 which are also expressed in the otocyst phenocopies the PAPC morphant phenotype.</p> <p>Conclusions</p> <p>PAPC signaling via RhoA and Wnt5a/Ror2 activity are required to keep cells aligned in apical-basal orientation during invagination of the ear placode. Since neither the cytoplasmic deletion mutant M-PAPC nor a classical cadherin is able to rescue loss of PAPC we suggest that the signaling function of the protocadherin rather than its role as modulator of cell-cell adhesion is required during invagination of the ear placode.</p

Detection and function of an intramolecular disulfide bond in the pH-responsive CadC of Escherichia coli

Background: In an acidic and lysine-rich environment Escherichia coli induces expression of the cadBA operon which encodes CadA, the lysine decarboxylase, and CadB, the lysine/cadaverine antiporter. cadBA expression is dependent on CadC, a membrane-integrated transcriptional activator which belongs to the ToxR-like protein family. Activation of CadC requires two stimuli, lysine and low pH. Whereas lysine is detected by an interplay between CadC and the lysine-specific transporter LysP, pH alterations are sensed by CadC directly. Crystal structural analyses revealed a close proximity between two periplasmic cysteines, Cys208 and Cys272. Results: Substitution of Cys208 and/or Cys272 by alanine resulted in CadC derivatives that were active in response to only one stimulus, either lysine or pH 5.8. Differential in vivo thiol trapping revealed a disulfide bond between these two residues at pH 7.6, but not at pH 5.8. When Cys208 and Cys272 were replaced by aspartate and lysine, respectively, virtually wild-type behavior was restored indicating that the disulfide bond could be mimicked by a salt bridge. Conclusion: A disulfide bond was found in the periplasmic domain of CadC that supports an inactive state of CadC at pH 7.6. At pH 5.8 disulfide bond formation is prevented which transforms CadC into a semi-active state. These results provide new insights into the function of a pH sensor

CSD-grown Y1−xGdxBa2Cu3O7−δ-BaHfO3 nanocomposite films on Ni5W and IBAD technical substrates

Chemical solution deposition (CSD) was used to grow Y1-xGdxBa2Cu3O7-delta-BaHfO3 (YGBCO-BHO) nanocomposite films containing 12 mol% BHO nanoparticles and various amounts of Gd, x, on two kinds of buffered metallic tapes: Ni5W and IBAD. The influence of the rare-earth stoichiometry on structure, morphology and superconducting properties of these films was studied. The growth process was carefully studied in order to find the most appropriate growth conditions for each composition and substrate. This led to a clear improvement in film quality, probably due to the reduction of BaCeO3 formation. In general, the superconducting properties of the films on Ni5W are significantly better. For x > 0.5, epitaxial 270 nm thick YGBCO-BHO films with T-c > 93 K and self-field J(c) at 77 K 2 MA/cm(2) were obtained on Ni5W. These results highlight the potential of this approach for the fabrication of high-quality coated conductors