Relationship between endoplasmic reticulum- and Golgi-associated calcium homeostasis and 4-NQO-induced DNA repair in Saccharomyces cerevisiae

Calcium (Ca²⁺) is an important ion that is necessary for the activation of different DNA repair mechanisms. However, the mechanism by which DNA repair and Ca²⁺ homeostasis cooperate remains unclear. We undertook a systems biology approach to verify the relationship between proteins associated with Ca²⁺ homeostasis and DNA repair for Saccharomyces cerevisiae. Our data indicate that Pmr1p, a Ca²⁺ transporter of Golgi complex, interacts with Cod1p, which regulates Ca²⁺ levels in the endoplasmic reticulum (ER), and with Rad4p, which is a nucleotide excision repair (NER) protein. This information was used to construct single and double mutants defective for Pmr1p, Cod1p, and Rad4p followed by cytotoxic, cytostatic, and cell cycle arrest analyses after cell exposure to different concentrations of 4-nitroquinoline 1-oxide (4-NQO). The results indicated that cod1Δ, cod1Δrad4Δ, and cod1Δpmr1Δ strains have an elevated sensitivity to 4-NQO when compared to its wild-type (WT) strain. Moreover, both cod1Δpmr1Δ and cod1Δrad4Δ strains have a strong arrest at G₂/M phases of cell cycle after 4-NQO treatment, while pmr1Δrad4Δ have a similar sensitivity and cell cycle arrest profile when compared to rad4Δ after 4-NQO exposure. Taken together, our results indicate that deletion in Golgi- and ER-associated Ca²⁺ transporters affect the repair of 4-NQO-induced DNA damage.     

The Cytosolic Tail Dipeptide Ile-Met of the Pea Receptor BP80 Is Required for Recycling from the Prevacuole and for Endocytosis

Pea (Pisum sativum) BP80 is a vacuolar sorting receptor for soluble proteins and has a cytosolic domain essential for its intracellular trafficking between the trans-Golgi network and the prevacuole. Based on mammalian knowledge, we introduced point mutations in the cytosolic region of the receptor and produced chimeras of green fluorescent protein fused to the transmembrane domain of pea BP80 along with the modified cytosolic tails. By analyzing the subcellular location of these chimera, we found that mutating Glu-604, Asp-616, or Glu-620 had mild effects, whereas mutating the Tyr motif partially redistributed the chimera to the plasma membrane. Replacing both Ile-608 and Met-609 by Ala (IMAA) led to a massive redistribution of fluorescence to the vacuole, indicating that recycling is impaired. When the chimera uses the alternative route, the IMAA mutation led to a massive accumulation at the plasma membrane. Using Arabidopsis thaliana plants expressing a fluorescent reporter with the full-length sequence of At VSR4, we demonstrated that the receptor undergoes brefeldin A–sensitive endocytosis. We conclude that the receptors use two pathways, one leading directly to the lytic vacuole and the other going via the plasma membrane, and that the Ileu-608 Met-609 motif has a role in the retrieval step in both pathways.     

The MMS22L-TONSL complex mediates recovery from replication stress and homologous recombination

Genome integrity is jeopardized each time DNA replication forks stall or collapse. Here we report the identification of a complex composed of MMS22L (C6ORF167) and TONSL (NFKBIL2) that participates in the recovery from replication stress. MMS22L and TONSL are homologous to yeast Mms22 and plant Tonsoku/Brushy1, respectively. MMS22L-TONSL accumulates at regions of ssDNA associated with distressed replication forks or at processed DNA breaks, and its depletion results in high levels of endogenous DNA double-strand breaks caused by an inability to complete DNA synthesis after replication fork collapse. Moreover, cells depleted of MMS22L are highly sensitive to camptothecin,?a topoisomerase I poison that impairs DNA replication progression. Finally, MMS22L and TONSL are necessary for the efficient formation of RAD51 foci after DNA damage, and their depletion impairs homologous recombination. These results indicate that MMS22L and TONSL are genome caretakers that stimulate the recombination-dependent repair of stalled or collapsed replication forks.     

Septal and lateral wall localization of PBP5, the major D,D‐carboxypeptidase of Escherichia coli,requires substrate recognition and membrane attachment

The distribution of PBP5, the major D,D‐carboxypeptidase in Escherichia coli, was mapped by immunolabelling and by visualization of GFP fusion proteins in wild‐type cells and in mutants lacking one or more D,D‐carboxypeptidases. In addition to being scattered around the lateral envelope, PBP5 was also concentrated at nascent division sites prior to visible constriction. Inhibiting PBP2 activity (which eliminates wall elongation) shifted PBP5 to midcell, whereas inhibiting PBP3 (which aborts divisome invagination) led to the creation of PBP5 rings at positions of preseptal wall formation, implying that PBP5 localizes to areas of ongoing peptidoglycan synthesis. A PBP5(S44G) active site mutant was more evenly dispersed, indicating that localization required enzyme activity and the availability of pentapeptide substrates. Both the membrane bound and soluble forms of PBP5 converted pentapeptides to tetrapeptides in vitro and in vivo, and the enzymes accepted the same range of substrates, including sacculi, Lipid II, muropeptides and artificial substrates. However, only the membrane‐bound form localized to the developing septum and restored wild‐type rod morphology to shape defective mutants, suggesting that the two events are related. The results indicate that PBP5 localization to sites of ongoing peptidoglycan synthesis is substrate dependent and requires membrane attachment.     

Knock-down of the COX3 and COX17 gene expression of cytochrome c oxidase in the unicellular green alga Chlamydomonas reinhardtii

The COX3 gene encodes a core subunit of mitochondrial cytochrome c oxidase (complex IV) whereas the COX17 gene encodes a chaperone delivering copper to the enzyme. Mutants of these two genes were isolated by RNA interference in the microalga Chlamydomonas. The COX3 mRNA was completely lacking in the cox3-RNAi mutant and no activity and assembly of complex IV were detected. The cox17-RNAi mutant presented a reduced level of COX17 mRNA, a reduced activity of the cytochrome c oxidase but no modification of its amount. The cox3-RNAi mutant had only 40% of the wild-type rate of dark respiration which was cyanide-insensitive. The mutant presented a 60% decrease of H₂O₂ production in the dark compared to wild type, which probably accounts for a reduced electron leakage by respiratory complexes III and IV. In contrast, the cox17-RNAi mutant showed no modification of respiration and of H₂O₂ production in the dark but a two to threefold increase of H₂O₂ in the light compared to wild type and the cox3-RNAi mutant. The cox17-RNAi mutant was more sensitive to cadmium than the wild-type and cox3-RNAi strains. This suggested that besides its role in complex IV assembly, Cox17 could have additional functions in the cell such as metal detoxification or Reactive Oxygen Species protection or signaling. Concerning Cox3, its role in Chlamydomonas complex IV is similar to that of other eukaryotes although this subunit is encoded in the nuclear genome in the alga contrary to the situation found in all other organisms.     

Determination of the Diversity of Rhodopirellula Isolates from European Seas by Multilocus Sequence Analysis

In the biogeography of microorganisms, the habitat size of an attached-living bacterium has never been investigated. We approached this theme with a multilocus sequence analysis (MLSA) study of new strains of Rhodopirellula sp., an attached-living planctomycete. The development of an MLSA for Rhodopirellula baltica enabled the characterization of the genetic diversity at the species level, beyond the resolution of the 16S rRNA gene. The alleles of the nine housekeeping genes acsA, guaA, trpE, purH, glpF, fumC, icd, glyA, and mdh indicated the presence of 13 genetically defined operational taxonomic units (OTUs) in our culture collection. The MLSA-based OTUs coincided with the taxonomic units defined by DNA-DNA hybridization experiments. BOX-PCR supported the MLSA-based differentiation of two closely related OTUs. This study established a taxon-area relationship of cultivable Rhodopirellula species. In European seas, three closely related species covered the Baltic Sea and the eastern North Sea, the North Atlantic region, and the southern North Sea to the Mediterranean. The last had regional genotypes, as revealed by BOX-PCR. This suggests a limited habitat size of attached-living Rhodopirellula species.The biogeography of microorganisms describes the habitat size of the species and the distribution of microorganisms on Earth. The experimental approaches depend on the focus of the studies. Habitats are often analyzed by environmental microbiologists with genetic-fingerprinting techniques, with up to 200 bands or fragments representing the whole community. Although the taxonomic resolution of these operational taxonomic units (OTUs) is limited, the studies revealed a community biogeography (22). Medical microbiologists analyze the alleles of housekeeping genes of microorganisms to gain insight into the epidemiology of pathogens, the population biogeography (2). This strain-specific, fine-scale taxonomic resolution within a species is well suited to observance of recent dispersal events. At the species level, multilocus sequence typing (MLST) and analysis (MLSA), which were developed for intraspecies and intragenus specific studies, respectively, consist of the sequences of several (at least seven) housekeeping gene fragments concatenated to an approximately 5-kilobase alignment (17). Recent MLSA studies revealed its applicability to marine isolates and the analysis of biogeographic patterns: Alteromonas macleodii isolates could be grouped in an epipelagic and an abyssal clade (6), and strains of Pseudomonas aeruginosa were genetically well separated into groups of coastal and oceanic origin (8). However, for Salinibacter ruber strains, biogeographical distinctness was not resolved in an MLSA study but showed allopatry in a metabolic analysis (31). Several studies used MLSA together with DNA-DNA hybridization (DDH) for the delineation of new species, e.g., for Vibrio and Ensifer spp. (20, 36).In the biogeography of microorganisms, the experimental proof of a local genetic evolution was first revealed at sample sites that were physically separated by over 18,000 km (39). Large populations and the small size of microbes have been considered as facilitators for dispersal over long distances, eventually establishing cosmopolitan microbial populations. On the other hand, the smallest spatial scale of a microbial species in an open system has not been investigated. Attached-living bacteria disperse only during a distinct, short time span in their lives. This limitation of the dispersal time stimulated this study of the biogeography of Rhodopirellula baltica in European seas.R. baltica is a planctomycete with typical morphological features. The peptidoglycanless bacteria have an intracellular compartmentation: the riboplasm with the nucleoid is separated by a membrane from the surrounding paryphoplasm. Cells attach with a holdfast substance to surfaces or, in culture, to themselves, forming typical rosettes. Proliferation occurs by budding, and offspring cells live free in the water column: they are motile with a flagellum until they settle on the sediment (4).Seventy recently isolated strains affiliated according to the 16S rRNA gene analysis with R. baltica SH1^T as the closest validly described species (40). The 16S rRNA gene sequences do not offer sufficient information at the species level. A dissimilarity of the 16S rRNA genes of more than 3%, recently reduced to 1.3% (34, 35), indicates that the strains under consideration belong to two species. These thresholds yielded in our strain collection, according to an ARB-based calculation, five or eight operational taxonomic units besides the species R. baltica (40). For strains with highly identical sequences, whole-genome DDH experiments have to be performed to identify the affiliation to established species. Recently, multilocus sequence analyses have emerged as a possible alternative method. Our strain collection comprised many strains with a 16S rRNA gene sequence very closely related to that of R. baltica SH1^T. To gain insight into the genetic identity of the isolates on the species level and the habitat sizes of the species, we developed a multilocus sequence analysis and applied it to the strain collection. The MLSA results were calibrated with a DDH study. The closely related strains were additionally characterized by BOX-PCR, a fingerprinting method (15). Transmission electron microscopy (EM) was performed on some isolates to support the identification as Planctomycetes and to visualize morphological differences between strains.     

Homologous expression of the nrdF gene of Corynebacterium ammoniagenes strain ATCC 6872 generates a manganese-metallocofactor (R2F) and a stable tyrosyl radical (Y˙) involved in ribonucleotide reduction

Ribonucleotide reduction, the unique step in the pathway to DNA synthesis, is catalyzed by enzymes via radical-dependent redox chemistry involving an array of diverse metallocofactors. The nucleotide reduction gene (nrdF) encoding the metallocofactor containing small subunit (R2F) of the Corynebacterium ammoniagenes ribonucleotide reductase was reintroduced into strain C. ammoniagenes ATCC 6872. Efficient homologous expression from plasmid pOCA2 using the tac-promotor enabled purification of R2F to homogeneity. The chromatographic protocol provided native R2F with a high ratio of manganese to iron (30:1), high activity (69 μmol 2'-deoxyribonucleotide·mg?1 ·min?1) and distinct absorption at 408 nm, characteristic of a tyrosyl radical (Y˙), which is sensitive to the radical scavenger hydroxyurea. A novel enzyme assay revealed the direct involvement of Y˙ in ribonucleotide reduction because 0.2 nmol 2'-deoxyribonucleotide was formed, driven by 0.4 nmol Y˙ located on R2F. X-band electron paramagnetic resonance spectroscopy demonstrated a tyrosyl radical at an effective g-value of 2.004. Temperature dependent X/Q-band EPR studies revealed that this radical is coupled to a metallocofactor. Similarities of the native C. ammoniagenes ribonucleotide reductase to the in vitro activated Escherichia coli class Ib enzyme containing a dimanganese(III)-tyrosyl metallocofactor are discussed.     

Identification of human-pathogenic strains of Shiga toxin-producing Escherichia coli from food by a combination of serotyping and molecular typing of Shiga toxin genes

We examined 219 Shiga toxin-producing Escherichia coli (STEC) strains from meat, milk, and cheese samples collected in Germany between 2005 and 2006. All strains were investigated for their serotypes and for genetic variants of Shiga toxins 1 and 2 (Stx1 and Stx2). stx(1) or variant genes were detected in 88 (40.2%) strains and stx(2) and variants in 177 (80.8%) strains. Typing of stx genes was performed by stx-specific PCRs and by analysis of restriction fragment length polymorphisms (RFLP) of PCR products. Major genotypes of the Stx1 (stx(1), stx(1c), and stx(1d)) and the Stx2 (stx(2), stx(2d), stx(2-O118), stx(2e), and stx(2g)) families were detected, and multiple types of stx genes coexisted frequently in STEC strains. Only 1.8% of the STEC strains from food belonged to the classical enterohemorrhagic E. coli (EHEC) types O26:H11, O103:H2, and O157:H7, and only 5.0% of the STEC strains from food were positive for the eae gene, which is a virulence trait of classical EHEC. In contrast, 95 (43.4%) of the food-borne STEC strains carried stx(2) and/or mucus-activatable stx(2d) genes, an indicator for potential high virulence of STEC for humans. Most of these strains belonged to serotypes associated with severe illness in humans, such as O22:H8, O91:H21, O113:H21, O174:H2, and O174:H21. stx(2) and stx(2d) STEC strains were found frequently in milk and beef products. Other stx types were associated more frequently with pork (stx(2e)), lamb, and wildlife meat (stx(1c)). The combination of serotyping and stx genotyping was found useful for identification and for assignment of food-borne STEC to groups with potential lower and higher levels of virulence for humans.     

Identification of NCAM-binding peptides promoting neurite outgrowth via a heterotrimeric G-protein-coupled pathway

A combinatorial library of undecapeptides was produced and utilized for the isolation of peptide binding to the fibronectin type 3 modules (F3I–F3II) of the neural cell adhesion molecule (NCAM). The isolated peptides were sequenced and produced as dendrimers. Two of the peptides (denoted ENFIN2 and ENFIN11) were confirmed to bind to F3I–F3II of NCAM by surface plasmon resonance. The peptides induced neurite outgrowth in primary cerebellar neurons and PC12E2 cells, but had no apparent neuroprotective properties. NCAM is known to activate different intracellular pathways, including signaling through the fibroblast growth factor receptor, the Src-related non-receptor tyrosine kinase Fyn, and heterotrimeric G-proteins. Interestingly, neurite outgrowth stimulated by ENFIN2 and ENFIN11 was independent of signaling through fibroblast growth factor receptor and Fyn, but could be inhibited with pertussis toxin, an inhibitor of certain heterotrimeric G-proteins. Neurite outgrowth induced by trans- homophilic NCAM was unaffected by the peptides, whereas knockdown of NCAM completely abrogated ENFIN2- and ENFIN11-induced neuritogenesis. These observations suggest that ENFIN2 and ENFIN11 induce neurite outgrowth in an NCAM-dependent manner through G-protein-coupled signal transduction pathways. Thus, ENFIN2 and ENFIN11 may be valuable for exploring this particular type of NCAM-mediated signaling.