Development of host and vector for high-efficiency transformation and gene disruption in Debaryomyces hansenii

Debaryomyces hansenii is one of the most osmotolerant and halotolerant yeasts. The molecular mechanisms underlying its extreme osmotolerance and halotolerance have drawn considerable attention in the recent past. However, progress in this regard has been limited due to lack of availability of a transformation system and molecular tools to study the functions of the genes in D. hansenii . Here, we have described the development of an efficient transformation system for D. hansenii that is based on a histidine auxotrophic recipient strain and the DhHIS4 gene as the selectable marker. By screening the D. hansenii genomic library, we have isolated several autonomous replication sequences that can be used for constructing a replicating vector. Moreover, our study is the first to demonstrate gene disruption in D. hansenii by homologous recombination.     

Impact of template overhang-binding region of HIV-1 RT on the binding and orientation of the duplex region of the template-primer

Fingers domain of HIV-1 RT is one of the constituents of the dNTP-binding pocket that is involved in binding of both dNTP and the template-primer. In the ternary complex of HIV-1 RT, two residues Trp-24 and Phe-61 located on the β1 and β3, respectively, are seen interacting with N + 1 to N + 3 nucleotides in the template overhang. We generated nonconservative and conservative mutant derivatives of these residues and examined their impact on the template-primer binding and polymerase function of the enzyme. We noted that W24A, F61A, and F61Y and the double mutant (W24A/F61A) were significantly affected in their ability to bind template-primer and also to catalyze the polymerase reaction while W24F remained unaffected. Using a specially designed template-primer with photoactivatable bromo-dU base in the duplex region at the penultimate position to the primer terminus, we demonstrated that F61A, W24A, F61Y as well as the double mutant were also affected in their cross-linking ability with the duplex region of the template-primer. We also isolated the E–TP covalent complexes of these mutants and examined their ability to catalyze single dNTP incorporation onto the immobilized primer terminus. The E–TP covalent complexes from W24F mutant displayed wild-type activity while those from W24A, F61A, F61Y, and the double mutant (W24A/F61A) were significantly impaired in their ability to catalyze dNTP incorporation onto the immobilized primer terminus. This unusual observation indicated that amino acid residues involved in the positioning of the template overhang may also influence the binding and orientation of the duplex region of the template-primer. Molecular modeling studies based on our biochemical results suggested that conformation of both W24 and F61 are interdependent on their interactions with each other, which together are required for proper positioning of the +1 template nucleotide in the binary and ternary complexes.     

Selective isolation,evaluation and characterization of antagonistic actinomycetes against <Emphasis Type="Italic">Rhizoctonia solani</Emphasis>

The baiting bag method was found to be useful for isolating antagonistic actinomycetes from terrestrial habitat. Out of total 110 actinomycetes isolated from rhizospheric and non-rhizospheric soil of Indo Gangetic Plains (IGP) of India, 9 isolates exhibited aggressive antagonism against Rhizoctonia solani, screened through dual culture, well diffusion and sealed plate technique. Maximum growth inhibition was recorded up to 50% under well diffusion (S. toxytricini vh22) and 52.6% in a direct confrontation (Actinomycetales bacterium vh41). Whereas maximum disease suppression (53.33%) under green house condition was achieved on seedling treated with S. tricolor vh85. Scanning electron microscopy of antagonists and pathogen interaction exhibited pore formation and hyphal degradation of test pathogen. Physiological and molecular characterization of selected isolates showed wide diversity and uncommon species has been encountered through the selective isolation technique.     

Production and biochemical characterization of a novel cellulase-poor alkali-thermo-tolerant xylanase from <Emphasis Type="Italic">Coprinellus disseminatus</Emphasis> SW-1 NTCC 1165

Fungi producing xylanases are plentiful but alkali-thermo-tolerant fungi producing cellulase-poor xylanase are rare. Out of 12 fungal strains isolated from various sources, Coprinellus disseminatus SW-1 NTCC 1165 yielded the highest xylanase activity (362.1 IU/ml) with minimal cellulase contamination (0.64 IU/ml). The solid state fermentation was more effective yielding 88.59% higher xylanase activity than that of submerged fermentation. An incubation period of 7 days at 37°C and pH 6.4 accelerated the xylanase production up to the maximum level. Among various inexpensive agro-residues used as carbon source, wheat bran induced the maximum xylanase titres (469.45 IU/ml) while soya bean meal was the best nitrogen source (478.5 IU/ml). A solid substrate to moisture content ratio of 1:3 was suitable for xylanase production while xylanase titre was repressed with the addition of glucose and lactose. The xylanase and laccase activities under optimized conditions were 499.60 and 25.5 IU/ml, respectively along with negligible cellulase contamination (0.86 IU/ml). Biochemical characterization revealed that optimal xylanase activity was observed at pH 6.4 and temperature 55°C and xylanase is active up to pH 9 (40.33 IU/ml) and temperature 85°C (48.81 IU/ml). SDS–PAGE and zymogram analysis indicated that molecular weight of alkali-thermo-tolerant xylanase produced by C. disseminatus SW-1 NTCC 1165 was 43 kDa.     

Genetic polymorphisms of matrix metalloproteinases and their inhibitors in potentially malignant and malignant lesions of the head and neck

Matrix metalloproteinases (MMPs) are a family of zinc-dependent proteinases that are capable of cleaving all extra cellular matrix (ECM) substrates. Degradation of matrix is a key event in progression, invasion and metastasis of potentially malignant and malignant lesions of the head and neck. It might have an important polymorphic association at the promoter regions of several MMPs such as MMP-1 (-1607 1G/2G), MMP-2 (-1306 C/T), MMP-3 (-1171 5A/6A), MMP-9 (-1562 C/T) and TIMP-2 (-418 G/C or C/C). Tissue inhibitors of metalloproteinases (TIMPs) are naturally occurring inhibitors of MMPs, which inhibit the activity of MMPs and control the breakdown of ECM. Currently, many MMP inhibitors (MMPIs) are under development for treating different malignancies. Useful markers associated with molecular aggressiveness might have a role in prognostication of malignancies and to better recognize patient groups that need more antagonistic treatment options. Furthermore, the introduction of novel prognostic markers may also promote exclusively new treatment possibilities, and there is an obvious need to identify markers that could be used as selection criteria for novel therapies. The objective of this review is to discuss the molecular functions and polymorphic association of MMPs and TIMPs and the possible therapeutic aspects of these proteinases in potentially malignant and malignant head and neck lesions. So far, no promising drug target therapy has been developed for MMPs in the lesions of this region. In conclusion, further research is required for the development of their potential diagnostic and therapeutic possibilities.     

Structural Study of the Serratia entomophila Antifeeding Prophage: Three-Dimensional Structure of the Helical Sheath

The sheath of the Serratia entomophila antifeeding prophage, which is pathogenic to the New Zealand grass grub Costelytra zealandica, is a 3-fold helix formed by a 4-fold symmetric repeating motif disposed around a helical inner tube. This structure, determined by electron microscopy and image processing, is distinct from that of the other known morphologically similar bacteriophage sheaths.The antifeeding prophage (Afp) of Serratia entomophila and Serratia proteamaculans is a naturally occurring virus tail-like structure which delivers a putative toxin molecule that leads to starvation of the New Zealand grass grub Costelytra zealandica (5). Afp is composed of 18 different gene products (molecular masses of 6.5 to 263 kDa). The first 16 open reading frames have orthologues (Photorhabdus virulence cassettes [PVC]) in the insecticidal bacterium Photorhabdus luminescens TTO1 genome (5). Afp and PVCs morphologically resemble a typical R-type bacteriocin (6, 12, 16) However, Afp is the only known phage tail-like protein complex that is not a bacteriocin-protein complex of considerable medical relevance that targets the same or closely related bacterial strains (1, 3, 8, 12). The major component of Afp is a contractile cylindrical outer sheath encasing an inner tube speculated to house the toxin molecule (6). A dome-shaped “head” defines one extremity of the tube, while the other end is attached to a “bell-shaped” structure with a base morphologically similar to the base plate of the T4 bacteriophage tail (9).Transmission electron micrographs of two-dimensional (2D) projections of negatively stained (Fig. ​(Fig.11 A) or frozen-hydrated and vitrified (Fig. ​(Fig.1B)1B) recombinant Afp particles (see Fig. S1 in the supplemental material) were used for computational image analysis. A globally averaged image of the Afp particle in the major configuration (called E here) (Fig. ​(Fig.1C),1C), generated using negatively stained specimens, clearly distinguished the morphologies of the various constituent structural parts. Thus, the cylindrical sheath appears to be formed by a periodic structure harboring a distinctive, inverted-V-shaped feature. A minor population of Afp particles displays an alternate configuration (called C here) where, concomitant with contraction of the sheath (averaged axial compression of ∼52% [see Table S1 in the supplemental material]), the inner tube, shorn off the bell-shaped structure, is revealed (Fig. ​(Fig.1A)1A) (6). Several other bacteriocins undergo such a high degree of compression, which has been characterized in detail for the tail sheath of bacteriophage T4 (9). We also generated individual global averages for the periodic sheath structure, for the bell-shaped structure, and for the inner tube (Fig. ​(Fig.1C)1C) which provide more accurate dimensions of these different sections (see Table S1 in the supplemental material) than those reported earlier (6).Open in a separate windowFIG. 1.(A) Electron micrograph of a negatively stained preparation of partially purified recombinant Afp particles. The gray, white, and black arrows point to an Afp particle in the extended (E) configuration, to an Afp particle with the sheath contracted, exposing the inner tube in the contracted (C) configuration, and to an inner tube with the bell-shaped structure attached at one end, respectively. (B) Cryoelectron micrograph recorded from a preparation similar to that shown in panel A. (C) Globally averaged images of Afp particles (3,026 images) in the E state and the three distinguishable parts visualized by negative staining. Bars, 200 Å. (D) Averaged power spectrum of the sheath of vitrified Afp particles. The black and white arrows indicate reflections delineating the axial rise (1/78.5 Å) and the helical pitch (1/118 Å), respectively. In panels A and C, lighter regions represent protein and the contrast is reversed in panel B. Global averages were created using classalign2 of the EMAN suite (10) and visualized in bshow (4).For a better insight, we determined the 3D structure of the central periodic section of the Afp particle in the E state. A global power spectrum derived from the cryoimages established the structure to be helical with a clear first meridional reflection at 1/78.5 Å and the first, strongest nonmeridional reflection at 1/118 Å (Fig. ​(Fig.1D).1D). These reflections correspond to the axial rise (Δx) and the pitch of the helix, respectively, and reflect a turn angle (Δψ) of about ±240° (3₂ helix) for the repeating motif. The correct sign, i.e., the hand, of the helix remains to be determined. Computationally excised overlapping segments of this helical section from images of vitrified and negatively stained Afp particles were subjected to 3D reconstruction using the iterative helical real-space reconstruction (IHRSR) algorithm (2) using the determined helical parameters (see Fig. S2 in the supplemental material). After a few iterations, the presence of an in-plane 4-fold symmetry (C4) was apparent in the density map (see Fig. S2 in the supplemental material), which was then imposed in the subsequent reconstruction exercises. However, no stable solution was forthcoming, even after many (e.g., 30) iterative cycles. This is generally indicative of the presence of heterogeneity in the form of variations in helix translation and/or twist angle (15) in the structure. As a first step, we focused our attention on the pitch value, and following classification (see Fig. S3 in the supplemental material), we found that the majority of the image segments correspond to a pitch of 120 Å. These segments were then selected out of the full data set and led to a stable and refined 3D reconstruction. We also obtained very comparable results for the helical section when images of negatively stained Afp sheath sections were used, thus supporting our computational approach (see Fig. S4 in the supplemental material) and general conclusions about the E state described below.Figure ​Figure22 A is an isosurface representation of the density map of the helical Afp sheath in the E state calculated at ∼21.5-Å resolution (see Fig. S5 in the supplemental material). To the best of our knowledge, a 4-fold rotational symmetry has not been seen for any other contractile T4 bacteriophage taillike structure, which points to the unique architecture of the Afp sheath. A power spectrum generated using the 2D projection from the final density map, compared to the experimental global power spectrum (Fig. ​(Fig.2D),2D), showed strong agreement, confirming the fidelity of the computational image analysis. The density map displays protein layers, ∼80 Å thick, that are stacked on each other in a periodic fashion. The uneven outer surface of the sheath is perforated and decorated with ∼35-Å protrusions. When rendered with a raised threshold, a characteristic feature of the map is a contiguous, high-electron-density region having an inverted-Y-shaped structure (Fig. 2C and E; see Fig. S4 in the supplemental material). At the modest ∼21.5-Å resolution, the boundary of the repeating subunit cannot be defined. A 25 ± 3-Å-wide central lumen is seen clearly when viewed along the helix axis (Fig. ​(Fig.2B)2B) and likely represents the pore of the inner tube (see also below). Using scanning transmission electron microscopy (STEM) (see Fig. S6 in the supplemental material), we estimated the averaged molecular mass of the central helical section of an Afp particle to be 9.8 ± 0.4 kDa/Å (Fig. ​(Fig.3)3) (14) and that of only the inner tube to be ∼2.5 kDa/Å, based on a relatively small pool of such images. These values translate to a mass contribution of approximately 145 kDa of the subunit whose periodic arrangement forms the outer component of the sheath (i.e., excluding the inner tube) (see Fig. S6 in the supplemental material). This value is not very different from the cumulative mass of the different proteins, i.e., homologous afp2, afp3, and afp4, thought to be involved in Afp sheath formation (5) (see Fig. S7 in the supplemental material).Open in a separate windowFIG. 2.Orthogonal isosurface rendering, at 1 σ (standard deviation) of the computed ∼21.5-Å density map of the helical sheath of the vitrified Afp particle viewed normal (A) and parallel (B) to the helix. The images were generated using the software package CHIMERA (13). The arrow indicates a surface perforation. (C) The Afp density map rendered at 3.5 σ to highlight the largest contiguous high-electron-density regions; one circumscribed by a black ellipse is computationally extracted and shown in panel E. (D) Comparison of the experimental, averaged power spectrum of the helical sheath of Afp (left) with that computed (right) from the 2D projection of the calculated density map. (F) Global average of the inner tube of the Afp particle and a plot of the surface density variation (scaled from 0 to 1) along the helical (y) axis. The dimension along the tube is plotted on the x axis.Open in a separate windowFIG. 3.(A) Dark-field micrograph of a freeze-dried, unstained preparation of Afp particles used in STEM measurements. An Afp particle in the E state, an Afp inner tube with the attached bell-shaped structure, and a tobacco mosaic virus particle, used as a calibration standard, are marked by the arrowhead and the gray and white arrows, respectively. (B) Histogram plot of the measured distributions of mass per unit length corresponding to the uniform periodic section of the Afp particles overlaid with a fitted Gaussian curve produced by using the ORIGIN6 software package.A paucity of images of the C state (∼5% of the complete data set) precluded a full, refined 3D reconstruction, but based on the available 2,774 overlapping image segments of the isolated inner tube, a global average was calculated. A plot of contrast variation (Fig. ​(Fig.2F)2F) indicates that the surface is characterized by ∼40-Å spaced elevated crests and invaginated grooves, in agreement with the calculated axial rise of ∼39 Å for the subunit (see Fig. S8 in the supplemental material) comprising the tube. Based on these preliminary results, it appears that the helical symmetry of the inner tube is markedly different from that of the sheath.Our observation that the pitch of the helix in the E state can vary by as much as ∼50 Å attests to the flexible nature of the sheath, which is required for compressibility and may be facilitated by the somewhat porous nature of the sheath (Fig. ​(Fig.2).2). Preliminary deductions (data not shown) based on a small pool of images of the C state suggest profound rearrangement of the elements of the sheath. How that translates to extrusion of the toxin remains to be revealed.      

Identification of Acidic pH-dependent Ligands of Pentameric C-reactive Protein

C-reactive protein (CRP) is a phylogenetically conserved protein; in humans, it is present in the plasma and at sites of inflammation. At physiological pH, native pentameric CRP exhibits calcium-dependent binding specificity for phosphocholine. In this study, we determined the binding specificities of CRP at acidic pH, a characteristic of inflammatory sites. We investigated the binding of fluid-phase CRP to six immobilized proteins: complement factor H, oxidized low-density lipoprotein, complement C3b, IgG, amyloid β, and BSA immobilized on microtiter plates. At pH 7.0, CRP did not bind to any of these proteins, but, at pH ranging from 5.2 to 4.6, CRP bound to all six proteins. Acidic pH did not monomerize CRP but modified the pentameric structure, as determined by gel filtration, 1-anilinonaphthalene-8-sulfonic acid-binding fluorescence, and phosphocholine-binding assays. Some modifications in CRP were reversible at pH 7.0, for example, the phosphocholine-binding activity of CRP, which was reduced at acidic pH, was restored after pH neutralization. For efficient binding of acidic pH-treated CRP to immobilized proteins, it was necessary that the immobilized proteins, except factor H, were also exposed to acidic pH. Because immobilization of proteins on microtiter plates and exposure of immobilized proteins to acidic pH alter the conformation of immobilized proteins, our findings suggest that conformationally altered proteins form a CRP-ligand in acidic environment, regardless of the identity of the protein. This ligand binding specificity of CRP in its acidic pH-induced pentameric state has implications for toxic conditions involving protein misfolding in acidic environments and favors the conservation of CRP throughout evolution.     

Identification and Partial Characterization of a Dynamin-Like Protein,EhDLP1, from the Protist Parasite Entamoeba histolytica

The dynamin superfamily of proteins includes a large repertoire of evolutionarily conserved GTPases that interact with different subcellular organelle membranes in eukaryotes. Dynamins are thought to participate in a number of cellular processes involving membrane remodeling and scission. Dynamin-like proteins (DLPs) form a subfamily of this vast class and play important roles in cellular processes, such as mitochondrial fission, cytokinesis, and endocytosis. In the present study, a gene encoding a dynamin-like protein (EhDLP1) from the protist parasite Entamoeba histolytica was identified and the protein was partially characterized using a combination of in silico, biochemical, and imaging methods. The protein was capable of GTP binding and hydrolysis, lipid binding, and oligomerization. Immunofluorescence studies showed the protein to be associated with the nuclear membrane. A mutant of EhDLP1 lacking GTP binding and hydrolyzing activities did not associate with the nuclear membrane. The results suggest a nucleus-associated function for EhDLP1.Dynamins are a vast family of GTPases implicated in myriad processes, some of which lead to alteration of membrane structure (22). Classical dynamins, such as mammalian dynamins 1 to 3 (5) and the shibire protein from Drosophila melanogaster (29), are required mainly for scission of vesicles, acting as mechanoenzymes or molecular switches (12). In addition, several dynamin-like proteins (DLPs) have been identified in different organisms ranging from yeast to mammals. DLPs play a key role in the division of organelles such as chloroplasts, mitochondria, and peroxisomes (15, 22). For example, Candida albicans Vps1 has been shown to be associated with virulence-related phenotypes like filamentation and biofilm formation (2). DLPs have also been identified in protists. Downregulation or ablation of the gene products in protists by RNA interference or other methods has helped to decipher the multiple functions carried out by these proteins. These include mitochondrial division and endocytosis in Trypanosoma brucei (6, 20), cytokinesis in Dictyostelium discoideum (31), phagocytosis in Paramecium species (30), endocytic transport in Giardia lamblia (11), and biogenesis of secretory vesicles in Toxoplasma gondii (4). Apart from cellular membranes, some DLPs may also associate with nuclear membranes. Recently, a study on Tetrahymena thermophila reported the requirement of Drp6 for macronuclear development (23). The human DLP MxB has been shown previously to localize to the cytoplasmic face of the nuclear envelope and is involved in regulation of nuclear import (14). Dynamins and DLPs share a minimal domain architecture which includes an N-terminal GTPase domain, a middle domain, and a GTPase effector domain (GED). The GED is involved in enzyme oligomerization and the regulation of the GTPase activity. The GTPase domain contains a well-conserved GTP binding motif required for guanine-nucleotide binding and hydrolysis (22). DLPs lack a pleckstrin homology (PH) domain and a proline-rich domain (PRD), normally associated with protein-lipid and protein-protein interaction.The endocytic, secretory, and adhesion pathways of the parasite Entamoeba histolytica play crucial roles in nutrient uptake, host cell destruction, and the endocytosis of gut resident bacteria, erythrocytes, and cell debris (21). The trophozoites of E. histolytica are known to have robust endocytic capabilities, turning over approximately a third of their cellular volume every hour (1, 19). The presence of a classical receptor-mediated pathway has not yet been clearly demonstrated, though some of the molecules involved in this pathway, such as clathrin, have been identified in E. histolytica (28). Typical eukaryotic cytoplasmic organelles have not been observed in this organism. However, the functional equivalents of a Golgi network and an endoplasmic reticulum are reported to be present (3, 26). Entamoeba also contains a genomeless variant of mitochondria, termed mitosomes (17). The division or biogenesis of these organelles during cell division is not understood. Nuclear division in E. histolytica occurs without nuclear membrane dissolution and reassembly. Since dynamins and DLPs are known to be involved in endocytosis and organelle division, it is likely that these proteins may be performing similar functions in this organism. Although the E. histolytica genome encodes putative dynamins and DLPs, none of these have been characterized. In order to understand the roles of these molecules in amebic biology, we have initiated studies to characterize these proteins from E. histolytica. Here, we report the basic characterization of E. histolytica dynamin-like protein 1 (EhDLP1).     

Anchor-Based Whole Genome Phylogeny (ABWGP): A Tool for Inferring Evolutionary Relationship among Closely Related Microorganims

Phenotypic behavior of a group of organisms can be studied using a range of molecular evolutionary tools that help to determine evolutionary relationships. Traditionally a gene or a set of gene sequences was used for generating phylogenetic trees. Incomplete evolutionary information in few selected genes causes problems in phylogenetic tree construction. Whole genomes are used as remedy. Now, the task is to identify the suitable parameters to extract the hidden information from whole genome sequences that truly represent evolutionary information. In this study we explored a random anchor (a stretch of 100 nucleotides) based approach (ABWGP) for finding distance between any two genomes, and used the distance estimates to compute evolutionary trees. A number of strains and species of Mycobacteria were used for this study. Anchor-derived parameters, such as cumulative normalized score, anchor order and indels were computed in a pair-wise manner, and the scores were used to compute distance/phylogenetic trees. The strength of branching was determined by bootstrap analysis. The terminal branches are clearly discernable using the distance estimates described here. In general, different measures gave similar trees except the trees based on indels. Overall the tree topology reflected the known biology of the organisms. This was also true for different strains of Escherichia coli. A new whole genome-based approach has been described here for studying evolutionary relationships among bacterial strains and species.