The ars Detoxification System Is Advantageous but Not Required for As(V) Respiration by the Genetically Tractable Shewanella Species Strain ANA-3

Arsenate [As(V); HAsO₄²⁻] respiration by bacteria is poorly understood at the molecular level largely due to a paucity of genetically tractable organisms with this metabolic capability. We report here the isolation of a new As(V)-respiring strain (ANA-3) that is phylogenetically related to members of the genus Shewanella and that also provides a useful model system with which to explore the molecular basis of As(V) respiration. This gram-negative strain stoichiometrically couples the oxidation of lactate to acetate with the reduction of As(V) to arsenite [As(III); HAsO₂]. The generation time and lactate molar growth yield (Y_lactate) are 2.8 h and 10.0 g of cells mol of lactate⁻¹, respectively, when it is grown anaerobically on lactate and As(V). ANA-3 uses a wide variety of terminal electron acceptors, including oxygen, soluble ferric iron, oxides of iron and manganese, nitrate, fumarate, the humic acid functional analog 2,6-anthraquinone disulfonate, and thiosulfate. ANA-3 also reduces As(V) to As(III) in the presence of oxygen and resists high concentrations of As(III) (up to 10 mM) when grown under either aerobic or anaerobic conditions. ANA-3 possesses an ars operon (arsDABC) that allows it to resist high levels of As(III); this operon also confers resistance to the As-sensitive strains Shewanella oneidensis MR-1 and Escherichia coli AW3110. When the gene encoding the As(III) efflux pump, arsB, is inactivated in ANA-3 by a polar mutation that also eliminates the expression of arsC, which encodes an As(V) reductase, the resulting As(III)-sensitive strain still respires As(V); however, the generation time and the Y_lactate value are two- and threefold lower, respectively, than those of the wild type. These results suggest that ArsB and ArsC may be useful for As(V)-respiring bacteria in environments where As concentrations are high, but that neither is required for respiration.     

The type IV secretion system of Sinorhizobium meliloti strain 1021 is required for conjugation but not for intracellular symbiosis

The type IV secretion system (T4SS) of the plant intracellular symbiont Sinorhizobium meliloti 1021 is required for conjugal transfer of DNA. However, it is not required for host invasion and persistence, unlike the T4SSs of closely related mammalian intracellular pathogens. A comparison of the requirement for a bacterial T4SS in plant versus animal host invasion suggests an important difference in the intracellular niches occupied by these bacteria.     

Xenopus TACC3/maskin is not required for microtubule stability but is required for anchoring microtubules at the centrosome

Members of the transforming acidic coiled coil (TACC) protein family are emerging as important mitotic spindle assembly proteins in a variety of organisms. The molecular details of how TACC proteins function are unknown, but TACC proteins have been proposed to recruit microtubule-stabilizing proteins of the tumor overexpressed gene (TOG) family to the centrosome and to facilitate their loading onto newly emerging microtubules. Using Xenopus egg extracts and in vitro assays, we show that the Xenopus TACC protein maskin is required for centrosome function beyond recruiting the Xenopus TOG protein XMAP215. The conserved C-terminal TACC domain of maskin is both necessary and sufficient to restore centrosome function in maskin-depleted extracts, and we provide evidence that the N terminus of maskin inhibits the function of the TACC domain. Time-lapse video microscopy reveals that microtubule dynamics in Xenopus egg extracts are unaffected by maskin depletion. Our results provide direct experimental evidence of a role for maskin in centrosome function and suggest that maskin is required for microtubule anchoring at the centrosome.     

Epidermal transglutaminase (TGase 3) is required for proper hair development, but not the formation of the epidermal barrier

Transglutaminases (TGase), a family of cross-linking enzymes present in most cell types, are important in events as diverse as cell-signaling and matrix stabilization. Transglutaminase 1 is crucial in developing the epidermal barrier, however the skin also contains other family members, in particular TGase 3. This isoform is highly expressed in the cornified layer, where it is believed to stabilize the epidermis and its reduction is implicated in psoriasis. To understand the importance of TGase 3 in vivo we have generated and analyzed mice lacking this protein. Surprisingly, these animals display no obvious defect in skin development, no overt changes in barrier function or ability to heal wounds. In contrast, hair lacking TGase 3 is thinner, has major alterations in the cuticle cells and hair protein cross-linking is markedly decreased. Apparently, while TGase 3 is of unique functional importance in hair, in the epidermis loss of TGase 3 can be compensated for by other family members.     

The Bloom's syndrome protein (BLM) interacts with MLH1 but is not required for DNA mismatch repair

Bloom's syndrome (BS) is a rare autosomal recessive disorder characterized by pre- and postnatal growth deficiency, immunodeficiency, and a tremendous predisposition to a wide variety of cancers. Cells from BS individuals are characterized by a high incidence of chromosomal gaps and breaks, elevated sister chromatid exchange, quadriradial formations, and locus-specific mutations. BS is the consequence of mutations that lead to loss of function of BLM, a gene encoding a helicase with homology to the RecQ helicase family. To delineate the role of BLM in DNA replication, recombination, and repair we used a yeast two-hybrid screen to identify potential protein partners of the BLM helicase. The C terminus of BLM interacts directly with MLH1 in the yeast-two hybrid assay; far Western analysis and co-immunoprecipitations confirmed the interaction. Cell extracts deficient in BLM were competent for DNA mismatch repair. These data suggest that the BLM helicase and MLH1 function together in replication, recombination, or DNA repair events independent of single base mismatch repair.     

3-Phosphoglycerate dehydrogenase from Corynebacterium glutamicum: the C-terminal domain is not essential for activity but is required for inhibition by L-serine

The serA gene of Corynebacterium glutamicum coding for 3-phosphoglycerate dehydrogenase (PGDH) was isolated and functionally characterized. It encodes a polypeptide of 530 aminoacyl residues (aa), which is substantially longer than the corresponding Escherichia coli polypeptide of 410 aa. The difference is largely due to an additional stretch of aa in the carboxy- (C)-terminal part of the polypeptide. Overexpression of serA in C. glutamicum results in a 16-fold increase in specific PGDH activity to 2.1 U/mg protein, with activity being inhibited by high concentrations of L-serine. A set of muteins that were progressively truncated at the C-terminal end was constructed. When overexpressed, mutein SerADelta197 showed a specific PGDH dehydrogenase activity of 1.3 U/mg protein, with the activity no longer being sensitive to L-serine. Gel filtration experiments showed that wild type PGDH is a homotetramer, whereas mutein SerADelta197 constitutes a dimer. Thus, the specific regulatory features of C. glutamicum PGDH are due to the C-terminal part of the polypeptide, which can be deleted with almost no effect on the catalytic activity of the enzyme.     

Mouse pachytene checkpoint 2 (trip13) is required for completing meiotic recombination but not synapsis

In mammalian meiosis, homologous chromosome synapsis is coupled with recombination. As in most eukaryotes, mammalian meiocytes have checkpoints that monitor the fidelity of these processes. We report that the mouse ortholog (Trip13) of pachytene checkpoint 2 (PCH2), an essential component of the synapsis checkpoint in Saccharomyces cerevisiae and Caenorhabditis elegans, is required for completion of meiosis in both sexes. TRIP13-deficient mice exhibit spermatocyte death in pachynema and loss of oocytes around birth. The chromosomes of mutant spermatocytes synapse fully, yet retain several markers of recombination intermediates, including RAD51, BLM, and RPA. These chromosomes also exhibited the chiasmata markers MLH1 and MLH3, and okadaic acid treatment of mutant spermatocytes caused progression to metaphase I with bivalent chromosomes. Double mutant analysis demonstrated that the recombination and synapsis genes Spo11, Mei1, Rec8, and Dmc1 are all epistatic to Trip13, suggesting that TRIP13 does not have meiotic checkpoint function in mice. Our data indicate that TRIP13 is required after strand invasion for completing a subset of recombination events, but possibly not those destined to be crossovers. To our knowledge, this is the first model to separate recombination defects from asynapsis in mammalian meiosis, and provides the first evidence that unrepaired DNA damage alone can trigger the pachytene checkpoint response in mice.     

XRCC4's interaction with XLF is required for coding (but not signal) end joining

XRCC4 and XLF are structurally related proteins important for DNA Ligase IV function. XRCC4 forms a tight complex with DNA Ligase IV while XLF interacts directly with XRCC4. Both XRCC4 and XLF form homodimers that can polymerize as heterotypic filaments independently of DNA Ligase IV. Emerging structural and in vitro biochemical data suggest that XRCC4 and XLF together generate a filamentous structure that promotes bridging between DNA molecules. Here, we show that ablating XRCC4's affinity for XLF results in DNA repair deficits including a surprising deficit in VDJ coding, but not signal end joining. These data are consistent with a model whereby XRCC4/XLF complexes hold DNA ends together--stringently required for coding end joining, but dispensable for signal end joining. Finally, DNA-PK phosphorylation of XRCC4/XLF complexes disrupt DNA bridging in vitro, suggesting a regulatory role for DNA-PK's phosphorylation of XRCC4/XLF complexes.     

(Arg)3 within the N-terminal domain of glucosidase I contains ER targeting information but is not required absolutely for ER localization

Glucosidase I is an endoplasmic reticulum (ER) type II membrane enzyme that cleaves the distal alpha1,2-glucose of the asparagine-linked GlcNAc2-Man9-Glc3 precursor. To identify sequence motifs responsible for ER localization, we prepared a protein chimera by transferring the cytosolic and transmembrane domain of glucosidase I to the luminal domain of Golgi-Man9-mannosidase. The GIM9 hybrid was overexpressed in COS 1 cells as an ER-resident protein that displayed alpha1,2-mannosidase activity, excluding the possibility that the glucosidase I-specific domains interfere with folding of the Man9-mannosidase catalytic domain. After substitution of the Args in position 7, 8, or 9 relative to the N-terminus by leucine, the GIM9 mutants were transported to the cell surface indicating that the (Arg)3 sequence functions as an ER-targeting motif. Cell surface expression was also observed after substitution of Arg-7 or Arg-8 but not Arg-9 in GIM9 by either lysine or histidine. Thus the side chain structure, including its positive charge, appears to be essential for signal function. Analysis of the N-linked glycans suggests that the (Arg)3 sequence mediates ER localization through Golgi-to-ER retrograde transport. Glucosidase I remained localized in the ER after truncation or mutation of the N-terminal (Arg)3 signal, in contrast to comparable GIM9 mutants. ER localization was also observed with an M9GI chimera consisting of the cytosolic and transmembrane domain of Man9-mannosidase and the glucosidase I catalytic domain. ER-specific targeting information must therefore be provided by sequence motifs contained within the glucosidase I luminal domain. This structural information appears to direct ER localization by retention rather than by retrieval, as concluded from N-linked Man9-GlcNAc2 being the major glycan released from the wild-type enzyme.     

Precipitation of alacranite (As8S9) by a novel As(V)‐respiring anaerobe strain MPA‐C3

Strain MPA‐C3 was isolated by incubating arsenic‐bearing sediments under anaerobic, mesophilic conditions in minimal media with acetate as the sole source of energy and carbon, and As(V) as the sole electron acceptor. Following growth and the respiratory reduction of As(V) to As(III), a yellow precipitate formed in active cultures, while no precipitate was observed in autoclaved controls, or in uninoculated media supplemented with As(III). The precipitate was identified by X‐ray diffraction as alacranite, As₈S₉, a mineral previously only identified in hydrothermal environments. Sequencing of the 16S rRNA gene indicated that strain MPA‐C3 is a member of the Deferribacteres family, with relatively low (90%) identity to Denitrovibrio acetiphilus DSM 12809. The arsenate respiratory reductase gene, arrA, was sequenced, showing high homology to the arrA gene of Desulfitobacterium halfniense. In addition to As(V), strain MPA‐C3 utilizes NO₃^?, Se(VI), Se(IV), fumarate and Fe(III) as electron acceptors, and acetate, pyruvate, fructose and benzoate as sources of carbon and energy. Analysis of a draft genome sequence revealed multiple pathways for respiration and carbon utilization. The results of this work demonstrate that alacranite, a mineral previously thought to be formed only chemically under hydrothermal conditions, is precipitated under mesophilic conditions by the metabolically versatile strain MPA‐C3.     

The unusual intersubunit ferroxidase center of Listeria innocua Dps is required for hydrogen peroxide detoxification but not for iron uptake. A study with site-specific mutants

The role of the ferroxidase center in iron uptake and hydrogen peroxide detoxification was investigated in Listeria innocua Dps by substituting the iron ligands His31, His43, and Asp58 with glycine or alanine residues either individually or in combination. The X-ray crystal structures of the variants reveal only small alterations in the ferroxidase center region compared to the native protein. Quenching of the protein fluorescence was exploited to assess stoichiometry and affinity of metal binding. Substitution of either His31 or His43 decreases Fe(II) affinity significantly with respect to wt L. innocua Dps (K approximately 10(5) vs approximately 10(7) M(-)(1)) but does not alter the binding stoichiometry [12 Fe(II)/dodecamer]. In the H31G-H43G and H31G-H43G-D58A variants, binding of Fe(II) does not take place with measurable affinity. Oxidation of protein-bound Fe(II) increases the binding stoichiometry to 24 Fe(III)/dodecamer. However, the extent of fluorescence quenching upon Fe(III) binding decreases, and the end point near 24 Fe(III)/dodecamer becomes less distinct with increase in the number of mutated residues. In the presence of dioxygen, the mutations have little or no effect on the kinetics of iron uptake and in the formation of micelles inside the protein shell. In contrast, in the presence of hydrogen peroxide, with increase in the number of substitutions the rate of iron oxidation and the capacity to inhibit Fenton chemistry, thereby protecting DNA from oxidative damage, appear increasingly compromised, a further indication of the role of ferroxidation in conferring peroxide tolerance to the bacterium.     

Pyoverdine-mediated iron uptake in Pseudomonas aeruginosa: the Tat system is required for PvdN but not for FpvA transport

Under iron-limiting conditions, Pseudomonas aeruginosa PAO1 secretes a fluorescent siderophore called pyoverdine (Pvd). After chelating iron, this ferric siderophore is transported back into the cells via the outer membrane receptor FpvA. The Pvd-dependent iron uptake pathway requires several essential genes involved in both the synthesis of Pvd and the uptake of ferric Pvd inside the cell. A previous study describing the global phenotype of a tat-deficient P. aeruginosa strain showed that the defect in Pvd-mediated iron uptake was due to the Tat-dependent export of proteins involved in Pvd biogenesis and ferric Pvd uptake (U. Ochsner, A. Snyder, A. I. Vasil, and M. L. Vasil, Proc. Natl. Acad. Sci. USA 99:8312-8317, 2002). Using biochemical and biophysical tools, we showed that despite its predicted Tat signal sequence, FpvA is correctly located in the outer membrane of a tat mutant and is fully functional for all steps of the iron uptake process (ferric Pvd uptake and recycling of Pvd on FpvA after iron release). However, in the tat mutant, no Pvd was produced. This suggested that a key element in the Pvd biogenesis pathway must be exported to the periplasm by the Tat pathway. We located PvdN, a still unknown but essential component in Pvd biogenesis, at the periplasmic side of the cytoplasmic membrane and showed that its export is Tat dependent. Our results further support the idea that a critical step of the Pvd biogenesis pathway involving PvdN occurs at the periplasmic side of the cytoplasmic membrane.     

Cdc42 is required for PIP(2)-induced actin polymerization and early development but not for cell viability

BACKGROUND: Cdc42 and other Rho GTPases are conserved from yeast to humans and are thought to regulate multiple cellular functions by inducing coordinated changes in actin reorganization and by activating signaling pathways leading to specific gene expression. Direct evidence implicating upstream signals and components that regulate Cdc42 activity or for required roles of Cdc42 in activation of downstream protein kinase signaling cascades is minimal, however. Also, whereas genetic analyses have shown that Cdc42 is essential for cell viability in yeast, its potential roles in the growth and development of mammalian cells have not been directly assessed. RESULTS: To elucidate potential functions of Cdc42 mammalian cells, we used gene-targeted mutation to inactivate Cdc42 in mouse embryonic stem (ES) cells and in the mouse germline. Surprisingly, Cdc42-deficient ES cells exhibited normal proliferation and phosphorylation of mitogen- and stress-activated protein kinases. Yet Cdc42 deficiency caused very early embryonic lethality in mice and led to aberrant actin cytoskeletal organization in ES cells. Moreover, extracts from Cdc42-deficient cells failed to support phosphatidylinositol 4,5-bisphosphate (PIP(2))-induced actin polymerization. CONCLUSIONS: Our studies clearly demonstrate that Cdc42 mediates PIP(2)-induced actin assembly, and document a critical and unique role for Cdc42 in this process. Moreover, we conclude that, unexpectedly, Cdc42 is not necessary for viability or proliferation of mammalian early embryonic cells. Cdc42 is, however, absolutely required for early mammalian development.     

Poliovirus cre(2C)-dependent synthesis of VPgpUpU is required for positive- but not negative-strand RNA synthesis

The cre(2C) hairpin is a cis-acting replication element in poliovirus RNA and serves as a template for the synthesis of VPgpUpU. We investigated the role of the cre(2C) hairpin on VPgpUpU synthesis and viral RNA replication in preinitiation RNA replication complexes isolated from HeLa S10 translation-RNA replication reactions. cre(2C) hairpin mutations that block VPgpUpU synthesis in reconstituted assays with purified VPg and poliovirus polymerase were also found to completely inhibit VPgpUpU synthesis in preinitiation replication complexes. Surprisingly, blocking VPgpUpU synthesis by mutating the cre(2C) hairpin had no significant effect on negative-strand synthesis but completely inhibited positive-strand synthesis. Negative-strand RNA synthesized in these reactions immunoprecipitated with anti-VPg antibody and demonstrated that it was covalently linked to VPg. This indicated that VPg was used to initiate negative-strand RNA synthesis, although the cre(2C)-dependent synthesis of VPgpUpU was inhibited. Based on these results, we concluded that the cre(2C)-dependent synthesis of VPgpUpU was required for positive- but not negative-strand RNA synthesis. These findings suggest a replication model in which negative-strand synthesis initiates with VPg uridylylated in the 3' poly(A) tail in virion RNA and positive-strand synthesis initiates with VPgpUpU synthesized on the cre(2C) hairpin. The pool of excess VPgpUpU synthesized on the cre(2C) hairpin should support high levels of positive-strand synthesis and thereby promote the asymmetric replication of poliovirus RNA.     

The assembly of proline-rich membrane anchor (PRiMA)-linked acetylcholinesterase enzyme: glycosylation is required for enzymatic activity but not for oligomerization

Acetylcholinesterase (AChE) anchors onto cell membranes by a transmembrane protein PRiMA (proline-rich membrane anchor) as a tetrameric form in vertebrate brain. The assembly of AChE tetramer with PRiMA requires the C-terminal "t-peptide" in AChE catalytic subunit (AChE(T)). Although mature AChE is well known N-glycosylated, the role of glycosylation in forming the physiologically active PRiMA-linked AChE tetramer has not been studied. Here, several lines of evidence indicate that the N-linked glycosylation of AChE(T) plays a major role for acquisition of AChE full enzymatic activity but does not affect its oligomerization. The expression of the AChE(T) mutant, in which all N-glycosylation sites were deleted, together with PRiMA in HEK293T cells produced a glycan-depleted PRiMA-linked AChE tetramer but with a much higher K(m) value as compared with the wild type. This glycan-depleted enzyme was assembled in endoplasmic reticulum but was not transported to Golgi apparatus or plasma membrane.