Activation of the Inositol (1,4,5)-Triphosphate Calcium Gate Receptor Is Required for HIV-1 Gag Release

The structural precursor polyprotein, Gag, encoded by all retroviruses, including the human immunodeficiency virus type 1 (HIV-1), is necessary and sufficient for the assembly and release of particles that morphologically resemble immature virus particles. Previous studies have shown that the addition of Ca²⁺ to cells expressing Gag enhances virus particle production. However, no specific cellular factor has been implicated as mediator of Ca²⁺ provision. The inositol (1,4,5)-triphosphate receptor (IP3R) gates intracellular Ca²⁺ stores. Following activation by binding of its ligand, IP3, it releases Ca²⁺ from the stores. We demonstrate here that IP3R function is required for efficient release of HIV-1 virus particles. Depletion of IP3R by small interfering RNA, sequestration of its activating ligand by expression of a mutated fragment of IP3R that binds IP3 with very high affinity, or blocking formation of the ligand by inhibiting phospholipase C-mediated hydrolysis of the precursor, phosphatidylinositol-4,5-biphosphate, inhibited Gag particle release. These disruptions, as well as interference with ligand-receptor interaction using antibody targeted to the ligand-binding site on IP3R, blocked plasma membrane accumulation of Gag. These findings identify IP3R as a new determinant in HIV-1 trafficking during Gag assembly and introduce IP3R-regulated Ca²⁺ signaling as a potential novel cofactor in viral particle release.Assembly of the human immunodeficiency virus (HIV) is determined by a single gene that encodes a structural polyprotein precursor, Gag (71), and may occur at the plasma membrane or within late endosomes/multivesicular bodies (LE/MVB) (7, 48, 58; reviewed in reference 9). Irrespective of where assembly occurs, the assembled particle is released from the plasma membrane of the host cell. Release of Gag as virus-like particles (VLPs) requires the C-terminal p6 region of the protein (18, 19), which contains binding sites for Alix (60, 68) and Tsg101 (17, 37, 38, 41, 67, 68). Efficient release of virus particles requires Gag interaction with Alix and Tsg101. Alix and Tsg101 normally function to sort cargo proteins to LE/MVB for lysosomal degradation (5, 15, 29, 52). Previous studies have shown that addition of ionomycin, a calcium ionophore, and CaCl₂ to the culture medium of cells expressing Gag or virus enhances particle production (20, 48). This is an intriguing observation, given the well-documented positive role for Ca²⁺ in exocytotic events (33, 56). It is unclear which cellular factors might regulate calcium availability for the virus release process.Local and global elevations in the cytosolic Ca²⁺ level are achieved by ion release from intracellular stores and by influx from the extracellular milieu (reviewed in reference 3). The major intracellular Ca²⁺ store is the endoplasmic reticulum (ER); stores also exist in MVB and the nucleus. Ca²⁺ release is regulated by transmembrane channels on the Ca²⁺ store membrane that are formed by tetramers of inositol (1,4,5)-triphosphate receptor (IP3R) proteins (reviewed in references 39, 47, and 66). The bulk of IP3R channels mediate release of Ca²⁺ from the ER, the emptying of which signals Ca²⁺ influx (39, 51, 57, 66). The few IP3R channels on the plasma membrane have been shown to be functional as well (13). Through proteomic analysis, we identified IP3R as a cellular protein that was enriched in a previously described membrane fraction (18) which, in subsequent membrane floatation analyses, reproducibly cofractionated with Gag and was enriched in the membrane fraction only when Gag was expressed. That IP3R is a major regulator of cytosolic calcium concentration (Ca²⁺) is well documented (39, 47, 66). An IP3R-mediated rise in cytosolic Ca²⁺ requires activation of the receptor by a ligand, inositol (1,4,5)-triphosphate (IP3), which is produced when phospholipase C (PLC) hydrolyzes phosphatidylinositol-4,5-bisphosphate [PI(4,5)P₂] at the plasma membrane (16, 25, 54). Paradoxically, PI(4,5)P₂ binds to the matrix (MA) domain in Gag (8, 55, 59), and the interaction targets Gag to PI(4,5)P₂-enriched regions on the plasma membrane; these events are required for virus release (45). We hypothesized that PI(4,5)P₂ binding might serve to target Gag to plasma membrane sites of localized Ca²⁺ elevation resulting from PLC-mediated PI(4,5)P₂ hydrolysis and IP3R activation. This idea prompted us to investigate the role of IP3R in Gag function.Here, we show that HIV-1 Gag requires steady-state levels of IP3R for its efficient release. Three isoforms of IP3R, types 1, 2, and 3, are encoded in three independent genes (39, 47). Types 1 and 3 are expressed in a variety of cells and have been studied most extensively (22, 39, 47, 73). Depletion of the major isoforms in HeLa or COS-1 cells by small interfering RNA (siRNA) inhibited viral particle release. Moreover, we show that sequestration of the IP3R activating ligand or blocking ligand formation also inhibited Gag particle release. The above perturbations, as well as interfering with receptor expression or activation, led to reduced Gag accumulation at the cell periphery. The results support the conclusion that IP3R activation is required for efficient HIV-1 viral particle release.     

Sporicidal Activity of Synthetic Antifungal Undecapeptides and Control of Penicillium Rot of Apples

The antifungal activity of cecropin A(2-8)-melittin(6-9) hybrid undecapeptides, previously reported as active against plant pathogenic bacteria, was studied. A set of 15 sequences was screened in vitro against Fusarium oxysporum, Penicillium expansum, Aspergillus niger, and Rhizopus stolonifer. Most compounds were highly active against F. oxysporum (MIC < 2.5 μM) but were less active against the other fungi. The best peptides were studied for their sporicidal activity and for Sytox green uptake in F. oxysporum microconidia. A significant inverse linear relationship was observed between survival and fluorescence, indicating membrane disruption. Next, we evaluated the in vitro activity against P. expansum of a 125-member peptide library with the general structure R-X¹KLFKKILKX¹⁰L-NH₂, where X¹ and X¹⁰ corresponded to amino acids with various degrees of hydrophobicity and hydrophilicity and R included different N-terminal derivatizations. Fifteen sequences with MICs below 12.5 μM were identified. The most active compounds were BP21 {Ac,F,V} and BP34 {Ac,L,V} (MIC < 6.25 μM), where the braces denote R, X¹, and X¹⁰ positions and where Ac is an acetyl group. The peptides had sporicidal activity against P. expansum conidia. Seven of these peptides were tested in vivo by evaluating their preventative effect of inhibition of P. expansum infection in apple fruits. The peptide Ts-FKLFKKILKVL-NH₂ (BP22), where Ts is a tosyl group, was the most active with an average efficacy of 56% disease reduction, which was slightly lower than that of a commercial formulation of the fungicide imazalil.The discovery of antimicrobial compounds to treat plant diseases of economical importance in agriculture remains a major scientific challenge (1). Antimicrobial peptides are being considered as a good alternative to current fungicides and a great deal of scientific effort has been invested in studying their application in plant disease control (29, 34, 35).Antimicrobial peptides have been reported to display interesting activities against pathogenic microbes that are resistant to conventional antibiotics and to exhibit a broad spectrum of activity against bacteria, fungi, enveloped viruses, parasites, and tumor cells (7-10, 19, 20, 40, 49). The mechanism of action of these peptides against fungi consists of cell lysis by binding to the membrane surface and disrupting its structure, interference with the synthesis of essential cell wall components, or interaction with specific internal targets (12, 13, 15, 23, 29).Despite their good lytic activity, major concerns about the use of antimicrobial peptides as pesticides in plant protection are the high production cost associated with synthetic procedures and their low stability toward protease degradation. Several design strategies have been devised in order to find shorter and more stable peptides, while maintaining or increasing the activity with a low cytotoxicity. These strategies include the juxtaposition of fragments of natural antimicrobial peptides, the modification of natural peptides, and the de novo design of sequences maintaining the crucial features of native antimicrobial peptides (2, 3, 11, 24, 32, 38, 42). However, the process involved in the development of lead candidates is time consuming and limited by the number of individual compounds that can be synthesized. Combinatorial chemistry has allowed the rapid preparation of synthetic libraries and their screening has led to the identification of peptides with high activity against selected phytopathogenic bacteria and fungi (4, 26, 27, 33).During our current research oriented to the development of new antimicrobial agents for use in plant protection, we designed linear undecapeptides (CECMEL11) derived from the cecropin A-melittin hybrid peptide WKLFKKILKVL-NH₂ (Pep3) (5, 17). Using a combinatorial approach, we identified peptides with high activity against plant pathogenic bacteria, such as Erwinia amylovora, Xanthomonas vesicatoria, and Pseudomonas syringae, and with low susceptibility to protease degradation (4, 5).In order to broaden the study, we decided to test the CECMEL11 peptides against the plant pathogenic fungi Fusarium oxysporum, Aspergillus niger, Rhizopus stolonifer, and Penicillium expansum. The fungus F. oxysporum causes Fusarium wilt in more than a hundred species of plants, and it is an important pathogen in horticultural crops (44). Several Rhizopus and Penicillium species cause soft rot and blue mold rot, respectively, which are important postharvest diseases in stone and pome fruits (6, 18, 22, 39). Apart from the economic losses, Aspergillus and Penicillium species are also of interest from a public health point of view due to the production of mycotoxins (45, 47). The importance of Penicillium species in the postharvest of fruits emphasizes the interest to develop antimicrobial peptides to control this fungus.Taking into account the relevance of these pathogens, the aim of the present study was the analysis of the antifungal activity profile of the CECMEL11 peptides in order to identify sporicidal sequences against the above fungi. As a proof of concept, the feasibility of using such peptides to protect fruits from fungal spoilage was evaluated using a P. expansum/apple model.     

Abundance of Novel and Diverse tfdA-Like Genes,Encoding Putative Phenoxyalkanoic Acid Herbicide-Degrading Dioxygenases,in Soil

Phenoxyalkanoic acid (PAA) herbicides are widely used in agriculture. Biotic degradation of such herbicides occurs in soils and is initiated by α-ketoglutarate- and Fe²⁺-dependent dioxygenases encoded by tfdA-like genes (i.e., tfdA and tfdAα). Novel primers and quantitative kinetic PCR (qPCR) assays were developed to analyze the diversity and abundance of tfdA-like genes in soil. Five primer sets targeting tfdA-like genes were designed and evaluated. Primer sets 3 to 5 specifically amplified tfdA-like genes from soil, and a total of 437 sequences were retrieved. Coverages of gene libraries were 62 to 100%, up to 122 genotypes were detected, and up to 389 genotypes were predicted to occur in the gene libraries as indicated by the richness estimator Chao1. Phylogenetic analysis of in silico-translated tfdA-like genes indicated that soil tfdA-like genes were related to those of group 2 and 3 Bradyrhizobium spp., Sphingomonas spp., and uncultured soil bacteria. Soil-derived tfdA-like genes were assigned to 11 clusters, 4 of which were composed of novel sequences from this study, indicating that soil harbors novel and diverse tfdA-like genes. Correlation analysis of 16S rRNA and tfdA-like gene similarity indicated that any two bacteria with D > 20% of group 2 tfdA-like gene-derived protein sequences belong to different species. Thus, data indicate that the soil analyzed harbors at least 48 novel bacterial species containing group 2 tfdA-like genes. Novel qPCR assays were established to quantify such new tfdA-like genes. Copy numbers of tfdA-like genes were 1.0 × 10⁶ to 65 × 10⁶ per gram (dry weight) soil in four different soils, indicating that hitherto-unknown, diverse tfdA-like genes are abundant in soils.Phenoxyalkanoic acid (PAA) herbicides such as MCPA (4-chloro-2-methyl-phenoxyacetic acid) and 2,4-D (2,4-dichlorophenoxyacetic acid) are widely used to control broad-leaf weeds in agricultural as well as nonagricultural areas (19, 77). Degradation occurs primarily under oxic conditions in soil, and microorganisms play a key role in the degradation of such herbicides in soil (62, 64). Although relatively rapidly degraded in soil (32, 45), both MCPA and 2,4-D are potential groundwater contaminants (10, 56, 70), accentuating the importance of bacterial PAA herbicide-degrading bacteria in soils (e.g., references 3, 5, 6, 20, 41, 59, and 78).Degradation can occur cometabolically or be associated with energy conservation (15, 54). The first step in the degradation of 2,4-D and MCPA is initiated by the product of cadAB or tfdA-like genes (29, 30, 35, 67), which constitutes an α-ketoglutarate (α-KG)- and Fe²⁺-dependent dioxygenase. TfdA removes the acetate side chain of 2,4-D and MCPA to produce 2,4-dichlorophenol and 4-chloro-2-methylphenol, respectively, and glyoxylate while oxidizing α-ketoglutarate to CO₂ and succinate (16, 17).Organisms capable of PAA herbicide degradation are phylogenetically diverse and belong to the Alpha-, Beta-, and Gammproteobacteria and the Bacteroidetes/Chlorobi group (e.g., references 2, 14, 29-34, 39, 60, 68, and 71). These bacteria harbor tfdA-like genes (i.e., tfdA or tfdAα) and are categorized into three groups on an evolutionary and physiological basis (34). The first group consists of beta- and gammaproteobacteria and can be further divided into three distinct classes based on their tfdA genes (30, 46). Class I tfdA genes are closely related to those of Cupriavidus necator JMP134 (formerly Ralstonia eutropha). Class II tfdA genes consist of those of Burkholderia sp. strain RASC and a few strains that are 76% identical to class I tfdA genes. Class III tfdA genes are 77% identical to class I and 80% identical to class II tfdA genes and linked to MCPA degradation in soil (3). The second group consists of alphaproteobacteria, which are closely related to Bradyrhizobium spp. with tfdAα genes having 60% identity to tfdA of group 1 (18, 29, 34). The third group also harbors the tfdAα genes and consists of Sphingomonas spp. within the alphaproteobacteria (30).Diverse PAA herbicide degraders of all three groups were identified in soil by cultivation-dependent studies (32, 34, 41, 78). Besides CadAB, TfdA and certain TfdAα proteins catalyze the conversion of PAA herbicides (29, 30, 35). All groups of tfdA-like genes are potentially linked to the degradation of PAA herbicides, although alternative primary functions of group 2 and 3 TfdAs have been proposed (30, 35). However, recent cultivation-independent studies focused on 16S rRNA genes or solely on group 1 tfdA sequences in soil (e.g., references 3-5, 13, and 41). Whether group 2 and 3 tfdA-like genes are also quantitatively linked to the degradation of PAA herbicides in soils is unknown. Thus, tools to target a broad range of tfdA-like genes are needed to resolve such an issue. Primers used to assess the diversity of tfdA-like sequences used in previous studies were based on the alignment of approximately 50% or less of available sequences to date (3, 20, 29, 32, 39, 47, 58, 73). Primers specifically targeting all major groups of tfdA-like genes to assess and quantify a broad diversity of potential PAA degraders in soil are unavailable. Thus, the objectives of this study were (i) to develop primers specific for all three groups of tfdA-like genes, (ii) to establish quantitative kinetic PCR (qPCR) assays based on such primers for different soil samples, and (iii) to assess the diversity and abundance of tfdA-like genes in soil.     

Potential Application of N-Carbamoyl-β-Alanine Amidohydrolase from Agrobacterium tumefaciens C58 for β-Amino Acid Production

An N-carbamoyl-β-alanine amidohydrolase of industrial interest from Agrobacterium tumefaciens C58 (βcar_At) has been characterized. βcar_At is most active at 30°C and pH 8.0 with N-carbamoyl-β-alanine as a substrate. The purified enzyme is completely inactivated by the metal-chelating agent 8-hydroxyquinoline-5-sulfonic acid (HQSA), and activity is restored by the addition of divalent metal ions, such as Mn²⁺, Ni²⁺, and Co²⁺. The native enzyme is a homodimer with a molecular mass of 90 kDa from pH 5.5 to 9.0. The enzyme has a broad substrate spectrum and hydrolyzes nonsubstituted N-carbamoyl-α-, -β-, -γ-, and -δ-amino acids, with the greatest catalytic efficiency for N-carbamoyl-β-alanine. βcar_At also recognizes substrate analogues substituted with sulfonic and phosphonic acid groups to produce the β-amino acids taurine and ciliatine, respectively. βcar_At is able to produce monosubstituted β²- and β³-amino acids, showing better catalytic efficiency (k_cat/K_m) for the production of the former. For both types of monosubstituted substrates, the enzyme hydrolyzes N-carbamoyl-β-amino acids with a short aliphatic side chain better than those with aromatic rings. These properties make βcar_At an outstanding candidate for application in the biotechnology industry.N-Carbamoyl-β-alanine amidohydrolase (NCβAA) (EC 3.5.1.6), also known as β-alanine synthase or β-ureidopropionase, catalyzes the third and final step of reductive pyrimidine degradation. In this reaction, N-carbamoyl-β-alanine or N-carbamoyl-β-aminoisobutyric acid is irreversibly hydrolyzed to CO₂, NH₃, and β-alanine or β-aminoisobutyric acid, respectively (43). Eukaryotic NCβAAs have been purified from several sources (10, 25, 33, 39, 42, 44). Nevertheless, only two prokaryotic NCβAAs, belonging to the Clostridium and Pseudomonas genera (4, 29), have been purified to date, although this activity has been inferred for several microorganisms due to the appearance of the reductive pathway of pyrimidine degradation (38, 45). Pseudomonas NCβAA is also able to hydrolyze l-N-carbamoyl-α-amino acids, and indeed, this activity is widespread in the bacterial kingdom (3, 23, 26, 46).β-Amino acids have unique pharmacological properties, and their utility as building blocks of β-peptides, pharmaceutical compounds, and natural products is of growing interest (14). β-Alanine, a natural β-amino acid, is a precursor of coenzyme A and pantothenic acid in bacteria and fungi (vitamin B₅) (7). β-Alanine is widely distributed in the central nervous systems of vertebrates and is a structural analogue of γ-amino-n-butyric acid and glycine, major inhibitory neurotransmitters, suggesting that it may be involved in synaptic transmissions (20). Another important natural β-amino acid is taurine (2-aminoethanesulfonic acid), which plays an important role in several essential processes, such as membrane stabilization, osmoregulation, glucose metabolism, antioxidation, and development of the central nervous system and the retina (9, 28, 33). 2-Aminoethylphosphonate, the most common naturally occurring phosphonate, also known as ciliatine, is an important precursor used in the biosynthesis of phosphonolipids, phosphonoproteins, and phosphonoglycans (5). β-Homoalanine (β-aminobutyric acid) has been used successfully for the design of nonnatural ligands for therapeutic application against autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, or autoimmune uveitis (30). Substituted β-amino acids can be denominated β², β³, and β^2,3, depending on the position of the side chain(s) (R) on the amino acid skeleton (18). β²-Amino acids are not yet as readily available as their β³-counterparts, as they must be prepared using multistep procedures (17).We decided to characterize NCβAA (β-carbamoylase) from Agrobacterium tumefaciens C58 (βcar_At) after showing that some dihydropyrimidinases belonging to the Arthrobacter and Sinorhizobium genera are able to hydrolyze different 5- or 6-substituted dihydrouracils to the corresponding N-carbamoyl-β-amino acids (18, 22). If βcar_At could decarbamoylate the reaction products of dihydrouracils, different β-amino acids would be obtained enzymatically in the same way that α-amino acids are produced via the hydantoinase process (6, 21). We therefore describe the physical, biochemical, kinetic, and substrate specificity properties of recombinant βcar_At.     

Environmental Factors Shape Sediment Anammox Bacterial Communities in Hypernutrified Jiaozhou Bay,China

Bacterial anaerobic ammonium oxidation (anammox) is an important process in the marine nitrogen cycle. Because ongoing eutrophication of coastal bays contributes significantly to the formation of low-oxygen zones, monitoring of the anammox bacterial community offers a unique opportunity for assessment of anthropogenic perturbations in these environments. The current study used targeting of 16S rRNA and hzo genes to characterize the composition and structure of the anammox bacterial community in the sediments of the eutrophic Jiaozhou Bay, thereby unraveling their diversity, abundance, and distribution. Abundance and distribution of hzo genes revealed a greater taxonomic diversity in Jiaozhou Bay, including several novel clades of anammox bacteria. In contrast, the targeting of 16S rRNA genes verified the presence of only “Candidatus Scalindua,” albeit with a high microdiversity. The genus “Ca. Scalindua” comprised the apparent majority of active sediment anammox bacteria. Multivariate statistical analyses indicated a heterogeneous distribution of the anammox bacterial assemblages in Jiaozhou Bay. Of all environmental parameters investigated, sediment organic C/organic N (OrgC/OrgN), nitrite concentration, and sediment median grain size were found to impact the composition, structure, and distribution of the sediment anammox bacterial community. Analysis of Pearson correlations between environmental factors and abundance of 16S rRNA and hzo genes as determined by fluorescent real-time PCR suggests that the local nitrite concentration is the key regulator of the abundance of anammox bacteria in Jiaozhou Bay sediments.Anaerobic ammonium oxidation (anammox, NH₄⁺ + NO₂⁻ → N₂ + 2H₂O) was proposed as a missing N transformation pathway decades ago. It was found 20 years later to be mediated by bacteria in artificial environments, such as anaerobic wastewater processing systems (see reference 32 and references therein). Anammox in natural environments was found even more recently, mainly in O₂-limited environments such as marine sediments (28, 51, 54, 67, 69) and hypoxic or anoxic waters (10, 25, 39-42). Because anammox may remove as much as 30 to 70% of fixed N from the oceans (3, 9, 64), this process is potentially as important as denitrification for N loss and bioremediation (41, 42, 73). These findings have significantly changed our understanding of the budget of the marine and global N cycles as well as involved pathways and their evolution (24, 32, 35, 72). Studies indicate variable anammox contributions to local or regional N loss (41, 42, 73), probably due to distinct environmental conditions that may influence the composition, abundance, and distribution of the anammox bacteria. However, the interactions of anammox bacteria with their environment are still poorly understood.The chemolithoautotrophic anammox bacteria (64, 66) comprise the new Brocadiaceae family in the Planctomycetales, for which five Candidatus genera have been described (see references 32 and 37 and references therein): “Candidatus Kuenenia,” “Candidatus Brocadia,” “Candidatus Scalindua,” “Candidatus Anammoxoglobus,” and “Candidatus Jettenia.” Due to the difficulty of cultivation and isolation, anammox bacteria are not yet in pure culture. Molecular detection by using DNA probes or PCR primers targeting the anammox bacterial 16S rRNA genes has thus been the main approach for the detection of anammox bacteria and community analyses (58). However, these studies revealed unexpected target sequence diversity and led to the realization that due to biased coverage and specificity of most of the PCR primers (2, 8), the in situ diversity of anammox bacteria was likely missed. Thus, the use of additional marker genes for phylogenetic analysis was suggested in hopes of better capturing the diversity of this environmentally important group of bacteria. By analogy to molecular ecological studies of aerobic ammonia oxidizers, most recent studies have attempted to include anammox bacterium-specific functional genes. All anammox bacteria employ hydrazine oxidoreductase (HZO) (= [Hzo]₃) to oxidize hydrazine to N₂ as the main source for a useable reductant, which enables them to generate proton-motive force for energy production (32, 36, 65). Phylogenetic analyses of Hzo protein sequences revealed three sequence clusters, of which the cladistic structure of cluster 1 is in agreement with the anammox bacterial 16S rRNA gene phylogeny (57). The hzo genes have emerged as an alternative phylogenetic and functional marker for characterization of anammox bacterial communities (43, 44, 57), allowing the 16S rRNA gene-based investigation methods to be corroborated and improved.The contribution of anammox to the removal of fixed N is highly variable in estuarine and coastal sediments (50). For instance, anammox may be an important pathway for the removal of excess N (23) or nearly negligible (48, 54, 67, 68). This difference may be attributable to a difference in the structure and composition of anammox bacterial communities, in particular how the abundance of individual cohorts depends on particular environmental conditions. Anthropogenic disturbance with variable source and intensity of eutrophication and pollution may further complicate the anammox bacterium-environment relationship.Jiaozhou Bay is a large semienclosed water body of the temperate Yellow Sea in China. Eutrophication has become its most serious environmental problem, along with red tides (harmful algal blooms), species loss, and contamination with toxic chemicals and harmful microbes (14, 15, 21, 61, 71). Due to different sources of pollution and various levels of eutrophication across Jiaozhou Bay (mariculture, municipal and industrial wastewater, crude oil shipyard, etc.), a wide spectrum of environmental conditions may contribute to a widely varying community structure of anammox bacteria. This study used both 16S rRNA and hzo genes as targets to measure their abundance, diversity, and spatial distribution and assess the response of the resident anammox bacterial community to different environmental conditions. Environmental factors with potential for regulating the sediment anammox microbiota are discussed.     

A Role for DNA Polymerase μ in the Emerging DJH Rearrangements of the Postgastrulation Mouse Embryo

The molecular complexes involved in the nonhomologous end-joining process that resolves recombination-activating gene (RAG)-induced double-strand breaks and results in V(D)J gene rearrangements vary during mammalian ontogeny. In the mouse, the first immunoglobulin gene rearrangements emerge during midgestation periods, but their repertoires have not been analyzed in detail. We decided to study the postgastrulation DJ_H joints and compare them with those present in later life. The embryo DJ_H joints differed from those observed in perinatal life by the presence of short stretches of nontemplated (N) nucleotides. Whereas most adult N nucleotides are introduced by terminal deoxynucleotidyl transferase (TdT), the embryo N nucleotides were due to the activity of the homologous DNA polymerase μ (Polμ), which was widely expressed in the early ontogeny, as shown by analysis of Polμ^−/− embryos. Based on its DNA-dependent polymerization ability, which TdT lacks, Polμ also filled in small sequence gaps at the coding ends and contributed to the ligation of highly processed ends, frequently found in the embryo, by pairing to internal microhomology sites. These findings show that Polμ participates in the repair of early-embryo, RAG-induced double-strand breaks and subsequently may contribute to preserve the genomic stability and cellular homeostasis of lymphohematopoietic precursors during development.The adaptive immune system is characterized by the great diversity of its antigen receptors, which result from the activities of enzymatic complexes that cut and paste the genomic DNA of antigen receptor loci. The nonhomologous end-joining (NHEJ) machinery is then recruited to repair the double-strand DNA breaks (DSBs) inflicted by the products of the recombination-activating genes (RAGs) (45, 65). Within B cells, each immunoglobulin (Ig) receptor represents a singular shuffling of two heavy (H) and two light (L) chains, which are derived from the recombination of V, D, and J gene segments of the IgH locus and of V and J for IgL (71). Besides these combinatorial possibilities, most Ig variability derives from extensive processing of the coding ends, including exonucleolytic trimming of DNA ends, together with the addition of palindromic (P) nucleotides templated by the adjacent germ line sequence and of nontemplated (N) nucleotides secondary to the activity of the terminal deoxynucleotidyl transferase (TdT), a lymphoid-specific member of family X of DNA polymerases (reviewed in reference 56). During B-lineage differentiation, IgH rearrangements occur before those of the IgL locus, and D-to-J_H rearrangements precede V-to-DJ_H rearrangements (62). DJ_H joints are formed in any of the three open reading frames (ORFs). ORF1 is predominantly used in mature Igs, ORF2 is transcribed as a Dμ protein that provides negative signals to the B-cell precursors, and ORF3 frequently leads to stop codons (32, 33, 37). Germ line V, D, and J gene segments display short stretches of mutually homologous nucleotides (SSH), which are frequently used in gene rearrangements during perinatal periods, when N additions are absent (27, 32, 55, 57). The actual Ig V-region repertoires represent both the results of the NHEJ process associated with genomic VDJ recombination and those of antigen-independent and -dependent selection events. Although the core NHEJ components (Ku-Artemis-DNA-PK and XLF-XRCC4-DNA ligase IV) are by themselves able to join RAG-induced, incompatible DNA ends, family X DNA polymerases can be recruited to fill gaps created by imprecise coding ends with 3′ overhangs (DNA polymerase μ [Polμ] and Polλ) and/or to promote diversity through the addition of N nucleotides (TdT) (34, 56).The lymphoid differentiation pathways and clonotypic repertoires are developmentally regulated and differ between the embryo-fetal and adult periods (2, 44, 68). The perinatal B cells result from a wave of B lymphopoiesis occurring during the last third of mouse gestation (13, 14, 21, 70). Perinatal V_H gene usage differs from that predominating in the adult (1, 69), and the former VDJ joints rarely display N additions, leading to V-region repertoires enriched in multi- and self-reactive specificities (36, 40). The program of B-cell differentiation starts at embryonic days 10 to 11 (E10 to E11) in embryo hematopoietic sites, after the emergence of multipotent progenitors (at E8.5 to E9.5) (18, 19, 23, 31, 51, 73). DJ_H rearrangements were detected in these early embryos, whereas full VDJ_H sequences were not observed before E14 (14, 18, 51, 66), when VJκ rearrangements were also found (63). The earliest mouse DJ_H/VDJ_H Ig sequences analyzed to date corresponded to late fetuses (E16) (14, 53). We reasoned that the true baseline of the Ig rearrangement process occurs in midgestation embryos, when the first DJ_Hs are not yet transcribed and, consequently, not subjected to selection and are conditioned only for the evolutionarily established and developmentally regulated usage of distinct NHEJ machineries.We report here the sequence profiles of the earliest embryo E10 to E12 DJ_H joints. Unexpected frequencies of embryonic DJ_H joints bearing N nucleotides, in the absence of detectable TdT expression, were found. Moreover, the embryo DJ_H joints lacking N nucleotides (N⁻) used fewer SSH to recombine than newborn DJ_Hs, and these SSH were widely dispersed along the embryo D sequences, in contrast to the most joint-proximal ones, which predominated in newborn DJ_Hs. Considering that Polμ is the closest relative of TdT (42% amino acid identity) (22), which is able to introduce N nucleotides in vitro (4, 22, 34, 39, 49) and to join DNA ends with minimal or even null complementarity (17, 58), and that it is expressed in early-embryo organs, we decided to investigate its putative contribution to the first embryo DJ_H joints. The DJ_H joints obtained from Polμ^−/− embryos (48) showed a significant reduction of N nucleotides compared to wild-type (WT) embryos. Moreover, highly preserved DJ_H joints (with <3 deleted nucleotides) were selectively depleted in the Polμ^−/− mouse embryos, while the remaining DJ_Hs preferentially relied upon longer stretches of homology for end ligation. These findings support the idea that Polμ is active during early-embryo DJ_H rearrangements and that both its template-dependent and -independent ambivalent functions may be used to fill in small nucleotide gaps generated after asymmetric hairpin nicking and also to extend coding ends via a limited TdT-like activity.     

Cellobiose Dehydrogenase from the Ligninolytic Basidiomycete Ceriporiopsis subvermispora

Cellobiose dehydrogenase (CDH), an extracellular flavocytochrome produced by several wood-degrading fungi, was detected in cultures of the selective delignifier Ceriporiopsis subvermispora when grown on a cellulose- and yeast extract-based liquid medium. CDH amounted to up to 2.5% of total extracellular protein during latter phases of the cultivation and thus suggested an important function for the fungus under the given conditions. The enzyme was purified 44-fold to apparent homogeneity. It was found to be present in two glycoforms of 98 kDa and 87 kDa with carbohydrate contents of 16 and 4%, respectively. The isoelectric point of both glycoforms is around 3.0, differing by 0.1 units, which is the most acidic value so far reported for a CDH. By using degenerated primers of known CDH sequences, one cdh gene was found in the genomic DNA, cloned, and sequenced. Alignment of the 774-amino-acid protein sequence revealed a high similarity to CDH from other white rot fungi. One notable difference was found in the longer interdomain peptide linker, which might affect the interdomain electron transfer at higher temperatures. The preferred substrate of C. subvermispora CDH is cellobiose, while glucose conversion is strongly discriminated by a 155,000-fold-lower catalytic efficiency. This is a typical feature of a basidiomycete CDH, as are the acidic pH optima for all tested electron acceptors in the range from 2.5 to 4.5.White rot fungi are the most efficient lignocellulose degraders in our ecosystem, and several species, e.g., Phanerochaete chrysosporium, Trametes versicolor, and Ceriporiopsis subvermispora, have been studied in great detail as model organisms for this complex process. The ability to degrade phenolic and nonphenolic lignin structures in wood has made these strains attractive for biotechnological applications mainly in the pulp and paper industry, where C. subvermispora exhibits a substantial advantage over P. chrysosporium and T. versicolor through its ability for selective removal of a large fraction of lignin without attacking the valuable cellulose (16, 38). The lignin-degrading system of these fungi is composed of extracellular enzymes together with low-molecular-mass cofactors (21, 46). Typically found ligninolytic enzymes are lignin peroxidase, manganese peroxidase (MnP), and laccase. The secretion pattern of these enzymes varies greatly in white rot fungi (22) and is influenced by culture conditions and medium composition. Whereas P. chrysoporium secretes high lignin and manganese peroxidase activities but no laccase activity (32, 33), C. subvermispora produces several MnP and laccase isoforms but no lignin peroxidase. T. versicolor is the only one of these model organisms known so far to express all three of these ligninolytic enzymes efficiently (5). Together with the cellulolytic enzyme system, these patterns of enzyme activities cause varied degrees of lignin and cellulose breakdown at different cultivation stages. The simultaneous attack of cellulose and lignin is the preferred strategy of T. versicolor, whereas C. subvermispora is a selective delignifier in the first stages of biotreatment, secreting only low activities of cellulolytic enzymes at a late culture stage (12, 23), and apparently lacks cellobiohydrolase activity (23).Cellobiose dehydrogenase (CDH; EC1.1.99.18; cellobiose (acceptor) 1-oxidoreductase) is an extracellular flavocytochrome secreted by some white rot and brown rot plant pathogenic and saprotrophic fungi from the dicaryotic phyla Basidiomycota and Ascomycota (50). It shows a strong preference for cellobiose and cello-oligosaccharides, which are oxidized to the corresponding lactones during the reductive half-reaction of the FAD cofactor, and further hydrolyze to aldonic acids in the bulk water. In the oxidative half-reaction FAD transfers two reduction equivalents to either one two-electron acceptor, e.g., various quinones, or to two one-electron acceptors, like complexed Fe(III) or Mn(II) ions. At low pH values (usually below 5.5), the heme cofactor can be involved in the electron transfer to one-electron acceptors. Even though CDH has been studied for a considerable time, the exact role and function of the two prosthetic groups are not fully understood. The pH optima with most electron acceptors are rather acidic, but oxygen, although a poor electron acceptor, is also reduced to H₂O₂ under neutral and alkaline conditions (30).In recent years CDH was shown to participate in the ligninolytic or cellulolytic metabolism of white rot fungi (3, 10, 24, 26, 50). The currently favored mechanism is the production of hydroxyl radicals through Fenton reaction chemistry by the ability of CDH to reduce Fe³⁺ to Fe²⁺ and to produce H₂O₂ (28, 31, 36, 37). CDH is believed to be involved in early stages of cellulose breakdown: knocking out the cdh gene in T. versicolor did not considerably affect its ability to grow on amorphous cellulosic substrates, while it could not grow on crystalline cellulose or recalcitrant substrates such as birch wood (13).Interestingly, no CDH activity has been reported so far from cultures of C. subvermispora, even though it is closely related to other white rotters producing this enzyme, e.g., Trametes spp. (35, 41) or Pycnoporus cinnabarinus (45). It has been speculated that the lack of CDH might contribute to the selectivity of C. subvermispora in degrading lignin while growing on wood. It was therefore the aim of our study to show unequivocally whether C. subvermispora carries a cdh gene and can produce the enzyme under certain growth conditions.