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1.
The composition of the methanogenic archaeal community in the foregut contents of Tammar wallabies (Macropus eugenii) was studied using 16S rRNA and methyl coenzyme reductase subunit A (mcrA) gene clone libraries. Methanogens belonging to the Methanobacteriales and a well-supported cluster of uncultivated archaeon sequences previously observed in the ovine and bovine rumens were found. Methanogen densities ranged from 7.0 × 105 and 3.9 × 106 cells per gram of wet weight.Kangaroos and wallabies belong to the marsupial family Macropodidae and are native to Australia. Because of their geographical isolation, macropod marsupials have evolved separately from other herbivorous animals, such as ruminants, but like ruminants, macropods have a complex gut microbiome that includes fungi, archaea, bacteria, and protozoa to coordinate plant biomass breakdown (11). The macropod foregut is functionally analogous to the rumen, yet for reasons unknown, macropod species produce relatively low levels of methane compared to ruminants (5, 13, 30).New species of bacteria (21) and protozoa (2-4) have been indentified in the macropod foregut, and the presence of fungi has also been reported (5). Preliminary studies have shown that methanogens are present in the kangaroo foregut (1) but can be absent or at levels below detection limits (22). This study represents the first attempt to describe the diversity of methanogens residing in the macropod foregut by using 16S rRNA and methyl coenzyme reductase A (mcrA) clone libraries in combination with quantitative real-time PCR.  相似文献   

2.
Although the level of diversity of root-associated fungi can be quite high, the effect of plant distribution and soil environment on root-associated fungal communities at fine spatial scales has received little attention. Here, we examine how soil environment and plant distribution affect the occurrence, diversity, and community structure of root-associated fungi at local patch scales within a mature forest. We used terminal restriction fragment length polymorphism and sequence analysis to detect 63 fungal species representing 28 different genera colonizing tree root tips. At least 32 species matched previously identified mycorrhizal fungi, with the remaining fungi including both saprotrophic and parasitic species. Root fungal communities were significantly different between June and September, suggesting a rapid temporal change in root fungal communities. Plant distribution affected root fungal communities, with some root fungi positively correlated with tree diameter and herbaceous-plant coverage. Some aspects of the soil environment were correlated with root fungal community structure, with the abundance of some root fungi positively correlated with soil pH and moisture content in June and with soil phosphorous (P) in September. Fungal distribution and community structure may be governed by plant-soil interactions at fine spatial scales within a mature forest. Soil P may play a role in structuring root fungal communities at certain times of the year.In temperate forests, most trees form relationships with ectomycorrhizal (ECM) fungi, and the diversity of this fungal group alone can approach 100 species within a forest stand (17, 20, 60). The ECM mutualism may be necessary for the success of some native plant species, as approximately 90% of roots of some tree species are colonized by ECM fungi (65). Nevertheless, we still know surprisingly little about what controls the community structure and distribution of root-associated fungi in forest systems (44, 46). The occurrence of root-associated fungi may broadly reflect soil environmental conditions and the presence of preferred plant hosts (28, 61), but how these factors interact to influence the diversity, distribution, and community structure of these fungi within forest habitat patches at a local scale is uncertain.The distribution of root-associated fungi may be primarily a species response to local soil environmental conditions. For example, both the quality (i.e., nutrient content) and the quantity of soil organic matter are known to influence the diversity of ECM communities (18, 20, 32). ECM fungi also vary in drought tolerance (14, 36), resistance to fire (61, 65), and tolerance to soil acidity (19) and temperature (56). Changes in soil chemistry, especially as they relate to pH and the availability of nitrogen (N) and phosphorous (P), might favor selection of fungi most capable of tolerating environmental extremes (2, 28, 29).Plant distribution and identity may, however, play the strongest role in structuring the below-ground diversity of root-associated fungi. Many ECM fungi can colonize a wide range of plant species, and plant species can be host to a large number of ECM fungi (63), especially those in the families Russulaceae and Thelephoraceae (34, 35, 62). Moreover, some ECM fungi are also specific to certain tree species (e.g., Suillus and Rhizopogon species are specific to species in the family Pinaceae [38, 39]). At the local scale, fungal distribution and richness might be influenced by differences in root growth and architecture (30, 42), by the distance to the bole of the tree (11, 42, 49), or by the presence of neighboring trees (29, 64). Temporal changes in ECM communities could be associated with seasonal changes in plant physiology and phenology (3, 8, 17).An often overlooked factor influencing root-associated fungi of tree roots is the occurrence of herbaceous plant species within forest stands. Many species of parasitic, achlorophyllous angiosperms obtain carbon (C) from ECM fungi that colonize tree roots (43), and some autotrophic plants could also obtain C from ECM fungi during certain times of the year (58). Herbaceous plants also influence the cycling of nutrients, including N, P, and K (potassium) (31, 50), within forests, which could affect the distribution of root-associated fungi. Herbaceous plants can also produce secondary compounds that inhibit colonization of tree roots (68).In this study, we examine the effect of soil environment and plant distribution on root-associated fungi of tree roots in a mature beech-maple forest at two points in the growing season. We predict that plant distribution, both the distribution of host trees and that of herbaceous plants, influences fungi associated with tree roots in terms of both community structure and diversity. Molecular typing protocols, including a site-specific database of fungal sequences and fingerprints, were used to identify fungi on tree roots (i.e., beech or maple trees) to the species level.  相似文献   

3.
Bacteria and fungi are ubiquitous in the atmosphere. The diversity and abundance of airborne microbes may be strongly influenced by atmospheric conditions or even influence atmospheric conditions themselves by acting as ice nucleators. However, few comprehensive studies have described the diversity and dynamics of airborne bacteria and fungi based on culture-independent techniques. We document atmospheric microbial abundance, community composition, and ice nucleation at a high-elevation site in northwestern Colorado. We used a standard small-subunit rRNA gene Sanger sequencing approach for total microbial community analysis and a bacteria-specific 16S rRNA bar-coded pyrosequencing approach (4,864 sequences total). During the 2-week collection period, total microbial abundances were relatively constant, ranging from 9.6 × 105 to 6.6 × 106 cells m−3 of air, and the diversity and composition of the airborne microbial communities were also relatively static. Bacteria and fungi were nearly equivalent, and members of the proteobacterial groups Burkholderiales and Moraxellaceae (particularly the genus Psychrobacter) were dominant. These taxa were not always the most abundant in freshly fallen snow samples collected at this site. Although there was minimal variability in microbial abundances and composition within the atmosphere, the number of biological ice nuclei increased significantly during periods of high relative humidity. However, these changes in ice nuclei numbers were not associated with changes in the relative abundances of the most commonly studied ice-nucleating bacteria.Microbes are abundant in the atmosphere, with both cultivation-dependent and molecular approaches showing that the atmosphere harbors a diverse assemblage of bacteria and fungi, including taxa also commonly found on leaf surfaces (5, 49) and in soil habitats (30). The abundance and composition of airborne microbial communities are variable across time and space (14, 24, 27, 33, 47, 48, 69). However, the atmospheric conditions responsible for driving the observed changes in microbial abundances are unknown. The diversity of airborne microorganisms, and the factors influencing diversity levels, also remains poorly characterized. One reason for these limitations in knowledge is that until recently, culture-based microbiological methods have been the standard, and it is well-recognized that such methods capture only a small portion of the total microbial diversity (59). As demonstrated in a number of recent studies (6, 13, 22, 23, 33, 52, 59, 63, 73), advances in culture-independent techniques allow far more of the microbial diversity present in the atmosphere to be surveyed and the spatiotemporal variability in microbial communities to be examined.Microbes are often considered passive inhabitants of the atmosphere, dispersing via airborne dust particles. However, recent studies suggest that many atmospheric microbes may be metabolically active (3, 4, 64), even up to altitudes of 20,000 m (34). Some airborne microbes may alter atmospheric conditions directly by acting as cloud condensation nuclei (7, 25, 56) and/or ice nuclei (IN) (19, 41, 56, 57, 61); this hypothesis is supported by the observation that most ice nuclei in snow samples are inactivated by a 95°C heat treatment (16, 17). However, the overall contribution of airborne microbes to atmospheric processes such as ice nucleation remains unclear.The best-studied ice-nucleating microbes are gram-negative bacteria that have also been isolated from leaf surfaces, including Pseudomonas syringae, Pseudomonas fluorescens, Erwinia herbicola, Xanthomonas campestri, and Sphingomonas spp. (45). These bacteria have been cultured extensively, and their ice-nucleating activity has been traced to a membrane-bound glycoprotein (40, 42, 70). However, their specific influence on atmospheric processes remains, at this point, largely anecdotal. Less is known about the ice-nucleating activities of fungi, but a few studies have shown that fungi can be effective ice nucleators, capable of initiating ice nucleation at temperatures as high as −2°C (41, 61). At this point, all known ice-nucleating microorganisms are amenable to culture-based studies, but given that the vast majority of microorganisms have yet to be cultured, it is likely that other ice-nucleating microbes remain undiscovered.The work presented here addresses three overarching questions. (i) Are microbial abundances altered by changes in atmospheric conditions? (ii) How is the diversity and composition of airborne microbial communities influenced by changes in atmospheric conditions? (iii) Can we identify known and novel ice-nucleating microbes in the atmosphere by testing for correlations between taxa abundances and the concentrations of biological ice nuclei? To address these questions, we combined epifluorescence microscopy, tagged pyrosequencing, Sanger sequencing, and an ice nucleation assay with atmospheric measurements to characterize the microbial communities at a high-elevation research site.  相似文献   

4.
Rice (Oryza sativa L.) is, on a global scale, one of the most important food crops. Although endophytic fungi and bacteria associated with rice have been investigated, little is known about the endophytic fungi of wild rice (Oryza granulate) in China. Here we studied the root endophytic mycobiota residing in roots of O. granulate by the use of an integrated approach consisting of microscopy, cultivation, ecological indices, and direct PCR. Microscopy confirmed the ubiquitousness of dark septate endophytes (DSEs) and sclerotium-like structures in root tissues. Isolations from 204 root segments from 15 wild rice plants yielded 58 isolates, for which 31 internal transcribed spacer (ITS)-based genotypes were recorded. The best BLAST match indicated that 34.5% of all taxa encountered may represent hitherto undescribed species. Most of the fungi were isolated with a very low frequency. Calculation of ecological indices and estimation of taxon accumulation curves indicated a high diversity of fungal species. A culture-independent approach was also performed to analyze the endophytic fungal community. Three individual clone libraries were constructed. Using a threshold of 90% similarity, 35 potentially different sequences (phylotypes) were found among 186 positive clones. Phylogenetic analysis showed that frequently detected clones were classified as Basidiomycota, and 60.2% of total analyzed clones were affiliated with unknown taxa. Exophiala, Cladophialophora, Harpophora, Periconia macrospinosa, and the Ceratobasidium/Rhizoctonia complex may act as potential DSE groups. A comparison of the fungal communities characterized by the two approaches demonstrated distinctive fungal groups, and only a few taxa overlapped. Our findings indicate a complex and rich endophytic fungal consortium in wild rice roots, thus offering a potential bioresource for establishing a novel model of plant-fungal mutualistic interactions.The majority of terrestrial plant roots are intimately associated with mycorrhizal fungi, and many aspects of the ecological roles played by these mycorrhizal fungi are well understood. In recent years, however, endophytic fungi have been gaining increasing interest. There is accumulating evidence that plant roots usually harbor mycorrhizal as well as endophytic fungi (29, 30, 34, 39, 52, 63). Dark septate endophytes (DSEs), which are characterized by dark pigmented hyphae and sclerotium-like structures, are believed to represent primary nonmycorrhizal root-inhabiting fungi (23). In some cases, DSEs are even more frequent than mycorrhizal fungi (68).Endophytic fungi have frequently been reported to be associated with crop plants, including wheat (Triticum aestivum), wild barley (Hordeum brevisubulatum and Hordeum bogdanii), soya bean (Glycine max), and maize (Zea mays) (6, 9, 11, 13, 21, 26, 27, 33, 36, 67). Some of the endophytic fungi in these crops conferred resistance of the plant to insect or fungal pathogens (55).Domesticated from the wild grass Oryza rufipogon 10,000 to 14,000 years ago, rice is today the main staple for more than 3 billion people (i.e., half of the world''s population). Its consumption exceeds 100 kg per capita annually in many Asian countries, and it is the principal food for most of the world''s poorest people, particularly in Asia. The association of arbuscular mycorrhizal fungi and endophytic bacteria with rice plants has been well documented (15, 32, 35, 44, 53, 56, 60). Less, however, is known about its fungal endophytes. Fungal endophytes have been detected in cultivated rice (Oryza sativa L.) (12, 14, 37, 61, 70), and antagonistic or plant growth-stimulating properties have been claimed for some of these isolates. For example, endophytic Fusarium spp. from cultivated rice roots proved to be effective in biocontrol of a root-knot nematode (28). The occurrence of mycorrhizal and endophytic fungi in a variety of rice cultivars has also recently been reported (63).Nondomesticated, wild plant species may live in symbiosis with a unique and rich mycoflora that may have been lost during breeding of the cultivars used in agriculture (20, 59). The purpose of this research was to characterize the endophytic fungal community of the roots of rare (nearly extinct) wild rice (Oryza granulate) from a nature reserve in Yunnan, China. Our results showed that arbuscular mycorrhizal fungi were apparently absent from wild rice roots. This finding was confirmed by standard root staining techniques and molecular detection using the arbuscular mycorrhizal (AM)-specific primer pairs (69). The characterization of root endophytes in wild rice as reported in this study will improve our knowledge concerning the ecology and evolution of mutualistic plant-fungus interactions.  相似文献   

5.
Highly active antiretroviral therapy (HAART) can reduce human immunodeficiency virus type 1 (HIV-1) viremia to clinically undetectable levels. Despite this dramatic reduction, some virus is present in the blood. In addition, a long-lived latent reservoir for HIV-1 exists in resting memory CD4+ T cells. This reservoir is believed to be a source of the residual viremia and is the focus of eradication efforts. Here, we use two measures of population structure—analysis of molecular variance and the Slatkin-Maddison test—to demonstrate that the residual viremia is genetically distinct from proviruses in resting CD4+ T cells but that proviruses in resting and activated CD4+ T cells belong to a single population. Residual viremia is genetically distinct from proviruses in activated CD4+ T cells, monocytes, and unfractionated peripheral blood mononuclear cells. The finding that some of the residual viremia in patients on HAART stems from an unidentified cellular source other than CD4+ T cells has implications for eradication efforts.Successful treatment of human immunodeficiency virus type 1 (HIV-1) infection with highly active antiretroviral therapy (HAART) reduces free virus in the blood to levels undetectable by the most sensitive clinical assays (18, 36). However, HIV-1 persists as a latent provirus in resting, memory CD4+ T lymphocytes (6, 9, 12, 16, 48) and perhaps in other cell types (45, 52). The latent reservoir in resting CD4+ T cells represents a barrier to eradication because of its long half-life (15, 37, 40-42) and because specifically targeting and purging this reservoir is inherently difficult (8, 25, 27).In addition to the latent reservoir in resting CD4+ T cells, patients on HAART also have a low amount of free virus in the plasma, typically at levels below the limit of detection of current clinical assays (13, 19, 35, 37). Because free virus has a short half-life (20, 47), residual viremia is indicative of active virus production. The continued presence of free virus in the plasma of patients on HAART indicates either ongoing replication (10, 13, 17, 19), release of virus after reactivation of latently infected CD4+ T cells (22, 24, 31, 50), release from other cellular reservoirs (7, 45, 52), or some combination of these mechanisms. Finding the cellular source of residual viremia is important because it will identify the cells that are still capable of producing virus in patients on HAART, cells that must be targeted in any eradication effort.Detailed analysis of this residual viremia has been hindered by technical challenges involved in working with very low concentrations of virus (13, 19, 35). Recently, new insights into the nature of residual viremia have been obtained through intensive patient sampling and enhanced ultrasensitive sequencing methods (1). In a subset of patients, most of the residual viremia consisted of a small number of viral clones (1, 46) produced by a cell type severely underrepresented in the peripheral circulation (1). These unique viral clones, termed predominant plasma clones (PPCs), persist unchanged for extended periods of time (1). The persistence of PPCs indicates that in some patients there may be another major cellular source of residual viremia (1). However, PPCs were observed in a small group of patients who started HAART with very low CD4 counts, and it has been unclear whether the PPC phenomenon extends beyond this group of patients. More importantly, it has been unclear whether the residual viremia generally consists of distinct virus populations produced by different cell types.Since the HIV-1 infection in most patients is initially established by a single viral clone (23, 51), with subsequent diversification (29), the presence of genetically distinct populations of virus in a single individual can reflect entry of viruses into compartments where replication occurs with limited subsequent intercompartmental mixing (32). Sophisticated genetic tests can detect such population structure in a sample of viral sequences (4, 39, 49). Using two complementary tests of population structure (14, 43), we analyzed viral sequences from multiple sources within individual patients in order to determine whether a source other than circulating resting CD4+ T cells contributes to residual viremia and viral persistence. Our results have important clinical implications for understanding HIV-1 persistence and treatment failure and for improving eradication strategies, which are currently focusing only on the latent CD4+ T-cell reservoir.  相似文献   

6.
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.  相似文献   

7.
Cytotoxicity and proliferation capacity are key functions of antiviral CD8 T cells. In the present study, we investigated a series of markers to define these functions in virus-specific CD8 T cells. We provide evidence that there is a lack of coexpression of perforin and CD127 in human CD8 T cells. CD127 expression on virus-specific CD8 T cells correlated positively with proliferation capacity and negatively with perforin expression and cytotoxicity. Influenza virus-, cytomegalovirus-, and Epstein-Barr virus/human immunodeficiency virus type 1-specific CD8 T cells were predominantly composed of CD127+ perforin/CD127 perforin+, and CD127/perforin CD8 T cells, respectively. CD127/perforin and CD127/perforin+ cells expressed significantly more PD-1 and CD57, respectively. Consistently, intracellular cytokine (gamma interferon, tumor necrosis factor alpha, and interleukin-2 [IL-2]) responses combined to perforin detection confirmed that virus-specific CD8 T cells were mostly composed of either perforin+/IL-2 or perforin/IL-2+ cells. In addition, perforin expression and IL-2 secretion were negatively correlated in virus-specific CD8 T cells (P < 0.01). As previously shown for perforin, changes in antigen exposure modulated also CD127 expression. Based on the above results, proliferating (CD127+/IL-2-secreting) and cytotoxic (perforin+) CD8 T cells were contained within phenotypically distinct T-cell populations at different stages of activation or differentiation and showed different levels of exhaustion and senescence. Furthermore, the composition of proliferating and cytotoxic CD8 T cells for a given antiviral CD8 T-cell population appeared to be influenced by antigen exposure. These results advance our understanding of the relationship between cytotoxicity, proliferation capacity, the levels of senescence and exhaustion, and antigen exposure of antiviral memory CD8 T cells.Cytotoxic CD8 T cells are a fundamental component of the immune response against viral infections and mediate an important role in immunosurveillance (7, 10, 55), and the induction of vigorous CD8 T-cell responses after vaccination is thought to be a key component of protective immunity (37, 41, 49, 50, 58, 60, 69). Cytotoxic CD8 T cells exert their antiviral and antitumor activity primarily through the secretion of cytotoxic granules containing perforin (pore-forming protein) and several granule-associated proteases, including granzymes (Grms) (5, 15, 20, 44). Several studies have recently advanced the characterization of the mechanism of granule-dependent cytotoxic activity and performed a comprehensive investigation of the content of cytotoxic granules in human virus-specific CD8 T cells (2, 19, 29, 44, 53).Heterogeneous profiles of cytotoxic granules have been identified in different virus-specific memory CD8 T cells and associated with distinct differentiation stages of memory CD8 T cells (2, 19, 29, 44). Furthermore, we have observed a hierarchy among the cytotoxic granules in setting the efficiency of cytotoxic activity and demonstrated that perforin (and to a lesser extent GrmB) but not GrmA or GrmK were associated with cytotoxic activity (29). Recently, a novel mechanism of perforin-dependent granule-independent CTL cytotoxicity has also been demonstrated (45).Major advances in the characterization of antigen (Ag)-specific CD4 and CD8 T cells have been made recently and have aimed at identifying functional profiles that may correlate with protective CD8 T-cell responses (1, 3, 4, 12, 13, 24, 28, 36-38, 40, 41, 49, 50, 56-58, 60, 64, 68). In particular, the functional characterization of antigen-specific T cells was mainly performed on the basis of (i) the pattern of cytokines secreted (i.e., gamma interferon [IFN-γ], tumor necrosis factor alpha [TNF-α], interleukin-2 [IL-2], or macrophage inflammatory protein 1β [MIP-1β]), (ii) the proliferation capacity, and (iii) the cytotoxic capacity (13, 28, 59). Of note, degranulation activity (i.e., CD107a mobilization following specific stimulation) has been used as a surrogate marker of cytotoxic activity (11, 13).The term “polyfunctional” has been used to define T-cell immune responses that, in addition to typical effector functions such as secretion of IFN-γ, TNF-α, or MIP-1β and cytotoxic activity (measured by the degranulation capacity), comprise distinct T-cell populations able to secrete IL-2 and retain proliferation capacity (13, 28, 49, 50). Some evidence indicates that a hallmark of protective immune responses is the presence of polyfunctional T-cell responses (59). Furthermore, the ability to secrete IL-2 was shown to be linked to proliferation capacity, and both factors have been associated with protective antiviral immunity (13, 28, 49, 50). Although a lack of correlation between degranulation activity and GrmB expression was reported in mice (65), the relationship between degranulation activity and perforin expression has never been comprehensively investigated in mice and in humans.The private α chain of the IL-7 receptor (IL-7Rα, also called CD127) has been suggested to selectively identify CD8 T cells that will become long-lived memory cells (6, 34, 36). Moreover, it was shown in mice (34, 36) and humans (14, 48, 63) that the CD127high memory-precursor CD8 T cells produced IL-2 in contrast to CD127low effector CD8 T cells. Of interest, CD127 expression has also been shown to correlate with Ag-specific proliferation capacity in mice (34, 36). A similar correlation was observed in humans, although only for polyclonal stimulations (48). With the exception of studies performed in HIV-1 infection, where an association between CD127 expression and HIV-1 viremia has been shown (21, 22, 42, 48, 54), very limited information is available on the CD127 expression in human virus-specific CD8 T cells other that HIV-1.Although cytotoxic activity and proliferation capacity are key components of the antiviral cellular immune response, the relationship between these functions has been only investigated in nonprogressive HIV-1 infection (46), where these two functions were shown to be related. However, it still remains to be determined whether these functions are mediated by the same or by different T-cell populations.In the present study, we performed a comprehensive characterization of virus-specific CD8 T-cell responses against HIV-1, cytomegalovirus (CMV), Epstein Barr virus (EBV), and influenza virus (Flu) in order to (i) analyze the degree of concordance between degranulation activity and perforin/Grm expression; (ii) identify the relevance of CD127 in identifying virus-specific CD8 T cells endowed with proliferation capacity; (iii) delineate the relationship between proliferation capacity, cytotoxic activity, activation/differentiation stage, and level of exhaustion of CD8 T cells; and (iv) determine the influence of antigen exposure in shaping the functional composition of virus-specific CD8 T cells.Our data indicate that cytotoxic (as defined by perforin expression) and proliferating (as defined by CD127 expression or IL-2 secretion) virus-specific CD8 T cells are contained within distinct CD8 T-cell populations. Furthermore, the proportion of proliferating and cytotoxic T cells within a given virus-specific CD8 T-cell population appears to be influenced by antigen exposure. These results advance our understanding of the relationship between cytotoxicity, proliferative capacity, differentiation stage, and Ag exposure of memory CD8 T cells.  相似文献   

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Sulfate-reducing bacteria (SRB) play a major role in the coupled biogeochemical cycling of sulfur and chalcophilic metal(loid)s. By implication, they can exert a strong influence on the speciation and mobility of multiple metal(loid) contaminants. In this study, we combined DsrAB gene sequencing and sulfur isotopic profiling to identify the phylogeny and distribution of SRB and to assess their metabolic activity in salt marsh sediments exposed to acid mine drainage (AMD) for over 100 years. Recovered dsrAB sequences from three sites sampled along an AMD flow path indicated the dominance of a single Desulfovibrio species. Other major sequence clades were related most closely to Desulfosarcina, Desulfococcus, Desulfobulbus, and Desulfosporosinus species. The presence of metal sulfides with low δ34S values relative to δ34S values of pore water sulfate showed that sediment SRB populations were actively reducing sulfate under ambient conditions (pH of ∼2), although possibly within less acidic microenvironments. Interestingly, δ34S values for pore water sulfate were lower than those for sulfate delivered during tidal inundation of marsh sediments. 16S rRNA gene sequence data from sediments and sulfur isotope data confirmed that sulfur-oxidizing bacteria drove the reoxidation of biogenic sulfide coupled to oxygen or nitrate reduction over a timescale of hours. Collectively, these findings imply a highly dynamic microbially mediated cycling of sulfate and sulfide, and thus the speciation and mobility of chalcophilic contaminant metal(loid)s, in AMD-impacted marsh sediments.Salt marshes exhibit high primary production rates (1, 101) and form biogeochemical “transition zones” for nutrient production, transport, and cycling between terrestrial and coastal marine environments (41, 66, 100). These zones also serve to reduce the flux of potentially toxic metals in contaminated groundwater to estuaries (12, 99, 106). Both functions depend strongly on microbial activity, especially that of sulfate-reducing bacteria (SRB) (42, 62, 67). SRB recycle much of the sedimentary organic carbon pool in marsh sediments (42-44) and indirectly inhibit production of the greenhouse gas methane (37, 71). They can restrict the mobility of dissolved contaminant metals by inducing precipitation of poorly soluble metal sulfides, and studies have examined their use in constructed wetlands to bioremediate acid mine drainage (AMD) and other metalliferous waste streams (11, 35, 40, 46, 50, 76, 90, 94, 104). However, the high acidity and metal concentrations inherent to AMD can inhibit SRB growth (15, 88, 98), and preferential growth of iron- and sulfur-oxidizing bacteria over SRB has been observed in some treatment wetlands (39).For natural salt marshes, 16S ribosomal nucleic acid- and phospholipid fatty acid (PLFA)-based analyses have shown that SRB commonly comprise a significant fraction of the microbial community (13, 24, 31, 34, 51, 58). Studies of salt marsh dissimilatory sulfite reductase genes (dsrAB), a highly conserved functional phylogenetic marker of prokaryotic sulfate reducers (49, 57, 102, 103, 107), have revealed both novel and deeply branching clades (3). Studies of mining-impacted sites at pH 2.0 to 7.8 (5, 7, 39, 70, 72, 77, 84), of soils and geothermal settings at a pH of ∼4 (55, 68), of metal-contaminated estuaries at pH 6.8 to 7.2 (65), and of hypersaline lakes at pH 7.5 (56) further outline the distribution and tolerance of specific groups and species of SRB under geochemically stringent conditions. Other findings point toward the existence of deltaproteobacteria in environments at a pH of ∼1 (10), although it is unknown if these include SRB. SRB diversity in salt marshes under long-term contamination by AMD has not been well investigated. Such studies may provide useful information for bioremediation projects in estuarine environments, as well as general insights into relationships between SRB physiology and the geochemistry of AMD.We studied the diversity of SRB, based on phylogenetic analysis of recovered DsrAB gene sequences (∼1.9 kb), in natural salt marsh sediments of the San Francisco Bay impacted by AMD for over 100 years. Sulfur isotope ratio and concentration measurements of pore water sulfate and metal sulfide minerals provided information about the spatial and temporal extent of active bacterial sulfate reduction (BSR) in sediment cores taken from specific sites along an AMD flow path. Collectively, the results revealed a tidal marsh system characterized by rapidly cycling bacterial sulfate reduction and sulfide reoxidation associated with oscillating tidal inundation and groundwater infiltration.  相似文献   

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The Escherichia coli HtrA protein is a periplasmic protease/chaperone that is upregulated under stress conditions. The protease and chaperone activities of HtrA eliminate or refold damaged and unfolded proteins in the bacterial periplasm that are generated upon stress conditions. In the absence of substrates, HtrA oligomerizes into a hexameric cage, but binding of misfolded proteins transforms the hexamers into bigger 12-mer and 24-mer cages that encapsulate the substrates for degradation or refolding. HtrA also undergoes partial degradation as a consequence of self-cleavage of the mature protein, producing short-HtrA protein (s-HtrA). The aim of this study was to examine the physiological role of this self-cleavage process. We found that the only requirement for self-cleavage of HtrA into s-HtrA in vitro was the hydrolysis of protein substrates. In fact, peptides resulting from the hydrolysis of the protein substrates were sufficient to induce autocleavage. However, the continuous presence of full-length substrate delayed the process. In addition, we observed that the hexameric cage structure is required for autocleavage and that s-HtrA accumulates only late in the degradation reaction. These results suggest that self-cleavage occurs when HtrA reassembles back into the resting hexameric structure and peptides resulting from substrate hydrolysis are allosterically stimulating the HtrA proteolytic activity. Our data support a model in which the physiological role of the self-cleavage process is to eliminate the excess of HtrA once the stress conditions cease.The cell envelope of gram-negative bacteria mediates the communication of the cell with the environment, and it is responsible for many vital functions, including nutrient uptake and interaction with other bacteria and host cells. These activities are performed by a large collection of proteins that make the periplasm a cellular compartment with an even higher protein concentration than the cytoplasm (2). Bacteria are frequently exposed to multiple stresses such as heat shock, osmotic stress, and pH changes and are regularly challenged by the host immune system. Thus, the maintenance of periplasmic proteins in a fully functional state is a challenging task undertaken by the protein quality control system (5). It is generally accepted that under stress conditions misfolded proteins, protein fragments, and mislocalized membrane proteins appear, activating a stress response through three different signal transduction pathways (σE, Cpx, and Bae) (21, 22). Activation of this stress response in the periplasm triggers the upregulation of molecular chaperones, peptidases, proteases, and other enzymes with a role in eliminating or refolding damaged periplasmic proteins.The Escherichia coli HtrA protein (also called DegP or protease Do) is a periplasmic protein (4) that is upregulated under stress conditions such as heat shock (14, 15). HtrA functions as a chaperone and a protease in a temperature-dependent fashion (24). Recent studies have also shown that HtrA substrates targeted for degradation or refolding are recognized differently, suggesting that the mechanisms through which HtrA recognizes the substrate may play a role in the protease-chaperone switch (8).HtrA contains an N-terminal protease domain, followed by the PDZ1 and PDZ2 domains. In the absence of substrates, HtrA oligomerizes into a hexameric cage (12) that represents the resting state of the protein (10, 13). Upon binding to protein substrates, HtrA transforms into bigger cages formed by 12 or 24 monomers that encapsulate substrates for degradation or refolding (9, 13).HtrA is a 474-residue protein whose first 26 amino acids are removed at the N terminus most likely by a signal peptidase rendering the mature 48-kDa protein (14, 15). This form of the protein will hereon be referred to as full-length HtrA. However, it has been described (11, 23) that mature HtrA undergoes partial degradation both in vivo and in vitro as a consequence of self-cleavage occurring after Cys69 and Gln82 of the mature protein. These forms of the protein have been named short-HtrA (s-HtrA) (23).A similar phenomenon of autocleavage has been observed in other members of the HtrA family such as the human homologs HtrA1 (7) and HtrA2 proteins (6). The autocleavage process is not specific for proteases of the HtrA family. Several prokaryotic proteins involved in regulation of gene expression, such as the SOS response proteins LexA (16-18) and UmuD (3), are inactivated through a self-cleaving mechanism. Conversely, many mammalian proteases are produced as longer inactive precursors and depend on an intramolecular cleavage event to become active. This is the case for some gastric proteases such as pepsin and chymosin or the lysosome cathepsins D and E (1).Although autocleavage as a mechanism of activation or inactivation of certain proteases is well documented, the physiological role and the events triggering the self-cleavage of HtrA are poorly understood. In this study, we observed that the hexameric cage structure is required to observe autocleavage of HtrA. In addition, we analyzed the conditions that led to self-cleavage of HtrA and we found that the only requirement to observe accumulation of the s-HtrA form in vitro was the hydrolysis of protein substrates. In fact, peptides resulting from the degradation of protein substrates were sufficient to induce autocleavage. Therefore, considering the current functional model for HtrA (9, 13), our data suggest that the physiological role of the HtrA autocleavage is to eliminate the excess of HtrA protein expressed under stress conditions when the enzymatic activities of the protein are no longer needed.  相似文献   

13.
The purpose of the present study was to investigate the inhibition of Vibrio by Roseobacter in a combined liquid-surface system. Exposure of Vibrio anguillarum to surface-attached roseobacters (107 CFU/cm2) resulted in significant reduction or complete killing of the pathogen inoculated at 102 to 104 CFU/ml. The effect was likely associated with the production of tropodithietic acid (TDA), as a TDA-negative mutant did not affect survival or growth of V. anguillarum.Antagonistic interactions among marine bacteria are well documented, and secretion of antagonistic compounds is common among bacteria that colonize particles or surfaces (8, 13, 16, 21, 31). These marine bacteria may be interesting as sources for new antimicrobial drugs or as probiotic bacteria for aquaculture.Aquaculture is a rapidly growing sector, but outbreaks of bacterial diseases are a limiting factor and pose a threat, especially to young fish and invertebrates that cannot be vaccinated. Because regular or prophylactic administration of antibiotics must be avoided, probiotic bacteria are considered an alternative (9, 18, 34, 38, 39, 40). Several microorganisms have been able to reduce bacterial diseases in challenge trials with fish or fish larvae (14, 24, 25, 27, 33, 37, 39, 40). One example is Phaeobacter strain 27-4 (17), which inhibits Vibrio anguillarum and reduces mortality in turbot larvae (27). The antagonism of Phaeobacter 27-4 and the closely related Phaeobacter inhibens is due mainly to the sulfur-containing tropolone derivative tropodithietic acid (TDA) (2, 5), which is also produced by other Phaeobacter strains and Ruegeria mobilis (28). Phaeobacter and Ruegeria strains or their DNA has been commonly found in marine larva-rearing sites (6, 17, 28).Phaeobacter and Ruegeria (Alphaproteobacteria, Roseobacter clade) are efficient surface colonizers (7, 11, 31, 36). They are abundant in coastal and eutrophic zones and are often associated with algae (3, 7, 41). Surface-attached Phaeobacter bacteria may play an important role in determining the species composition of an emerging biofilm, as even low densities of attached Phaeobacter strain SK2.10 bacteria can prevent other marine organisms from colonizing solid surfaces (30, 32).In continuation of the previous research on roseobacters as aquaculture probiotics, the purpose of this study was to determine the antagonistic potential of Phaeobacter and Ruegeria against Vibrio anguillarum in liquid systems that mimic a larva-rearing environment. Since production of TDA in liquid marine broth appears to be highest when roseobacters form an air-liquid biofilm (5), we addressed whether they could be applied as biofilms on solid surfaces.  相似文献   

14.
Prion strain interference can influence the emergence of a dominant strain from a mixture; however, the mechanisms underlying prion strain interference are poorly understood. In our model of strain interference, inoculation of the sciatic nerve with the drowsy (DY) strain of the transmissible mink encephalopathy (TME) agent prior to superinfection with the hyper (HY) strain of TME can completely block HY TME from causing disease. We show here that the deposition of PrPSc, in the absence of neuronal loss or spongiform change, in the central nervous system corresponds with the ability of DY TME to block HY TME infection. This suggests that DY TME agent-induced damage is not responsible for strain interference but rather prions compete for a cellular resource. We show that protein misfolding cyclic amplification (PMCA) of DY and HY TME maintains the strain-specific properties of PrPSc and replicates infectious agent and that DY TME can interfere, or completely block, the emergence of HY TME. DY PrPSc does not convert all of the available PrPC to PrPSc in PMCA, suggesting the mechanism of prion strain interference is due to the sequestering of PrPC and/or other cellular components required for prion conversion. The emergence of HY TME in PMCA was controlled by the initial ratio of the TME agents. A higher ratio of DY to HY TME agent is required for complete blockage of HY TME in PMCA compared to several previous in vivo studies, suggesting that HY TME persists in animals coinfected with the two strains. This was confirmed by PMCA detection of HY PrPSc in animals where DY TME had completely blocked HY TME from causing disease.Prions are infectious agents of animals, including humans, which are comprised of PrPSc, a misfolded isoform of the noninfectious host encoded protein PrPC (17, 24, 50, 63). Prion diseases of humans are unique neurodegenerative disorders in that they can have either a sporadic, familial, or infectious etiology. Prions cause disease in economically important domestic and wild animal species such as bovine spongiform encephalopathy in cattle and chronic wasting disease in wild and captive cervids (20, 62). Prion diseases can be zoonotic as illustrated by the transmission of bovine spongiform encephalopathy to humans that resulted in the emergence of variant Creutzfeldt-Jacob disease (14, 19, 22, 23, 46, 61, 68). Prion diseases are inevitably fatal and there are currently no effective treatments (21).Prion strains are defined by a characteristic set of features that breed true upon experimental passage (33, 34). Strain-specific differences have been identified in incubation period, clinical signs, agent distribution, overdominance, host range, neuropathology, and biochemical properties of PrPSc (5, 10, 11, 13, 28, 34, 42, 44). Strain-specific conformations of PrPSc are hypothesized to encode prion strain diversity; however, it is not understood how these differences result in the distinct strain properties (11, 19, 40, 47, 59, 66).Prion strain interference may be involved in the emergence of a dominant strain from a mixture as could occur during prion adaptation to a new host species or during prion evolution (4, 36, 43, 48, 56). In the natural prion diseases, there are examples where an individual host may be infected with more than one prion strain (15, 25, 55, 57, 58). Experimentally, coinfection or superinfection of prion strains can result in interference where a blocking, long incubation period strain extends the incubation period or completely blocks a superinfecting, short incubation period strain from causing disease (26, 27). Prion interference has been described in experimental studies of mice and hamsters infected with a wide variety of prion strains and routes of inoculation, suggesting it may be a common property of prion disease (3, 27, 52, 53, 60).It has been proposed that prion strains compete for a shared “replication site”; however, mechanistic details are not known, and it is unclear whether the blocking strain destroys or occupies the replication sites required for the superinfecting strain (28). The transport to and relative onset of replication of interfering strains in a common population of neurons is an important factor that can determine which strain will emerge (8). In the present study, we sought to determine whether the blocking strain disables transport and spread of the superinfecting strain or whether prion interference is due to competition for a cellular resource.  相似文献   

15.
The impact of transgenic white spruce [Picea glauca (Moench) Voss] containing the endochitinase gene (ech42) on soil fungal biomass and on the ectendomycorrhizal fungi Wilcoxina spp. was tested using a greenhouse trial. The measured level of endochitinase in roots of transgenic white spruce was up to 10 times higher than that in roots of nontransformed white spruce. The level of endochitinase in root exudates of three of four ech42-transformed lines was significantly greater than that in controls. Analysis soil ergosterol showed that the amount of fungal biomass in soil samples from control white spruce was slightly larger than that in soil samples from ech42-transformed white spruce. Nevertheless, the difference was not statistically significant. The rates of mycorrhizal colonization of transformed lines and controls were similar. Sequencing the internal transcribed spacer rRNA region revealed that the root tips were colonized by the ectendomycorrhizal fungi Wilcoxina spp. and the dark septate endophyte Phialocephala fortinii. Colonization of root tips by Wilcoxina spp. was monitored by real-time PCR to quantify the fungus present during the development of ectendomycorrhizal symbiosis in ech42-transformed and control lines. The numbers of Wilcoxina molecules in the transformed lines and the controls were not significantly different (P > 0.05, as determined by analysis of covariance), indicating that in spite of higher levels of endochitinase expression, mycorrhization was not inhibited. Our results indicate that the higher levels of chitinolytic activity in root exudates and root tissues from ech42-transformed lines did not alter the soil fungal biomass or the development of ectendomycorrhizal symbiosis involving Wilcoxina spp.White spruce [Picea glauca (Moench) Voss] is a tree species with an extensive distribution in boreal and subboreal forests and with significant ecological roles (37, 38). It is also an important commercial species for production of pulpwood and construction-grade lumber. However, in nurseries and plantations, white spruce is sensitive to multiple fungal diseases (23, 29, 42, 62, 76). Climate change scenarios suggest that diseases could result in increased mortality in conifer forests (22, 48). Genetic engineering offers a potential means to mitigate biotic and abiotic stresses.During the last 2 decades, chitinase genes isolated from plants, fungi, or bacteria have been studied and used to transform crops or trees in order to increase their resistance to plant-pathogenic fungi. One potential goal is improving white spruce tolerance to fungal infection through insertion of a chitinase gene. Chitin is a biopolymer of β-(1-4)-linked molecules of N-acetylglucosamine (NAG), a derivative of glucose, and is the primary constituent of the fungal cell wall and arthropod exoskeleton (3, 51). Chitinases are plant defense pathogenesis-related proteins (6, 11) that break down the chitin chain either by cleavage of internal glycoside bonds (endochitinases), by hydrolysis of the nonreducing end of the chitin chain (exochitinases), or by hydrolysis of NAG oligomers and trimers into NAG monomers (chitobiases). Endo- and exochitinase genes have been well characterized using sugar beet (Beta vulgaris) (44) and the filamentous fungal genus Trichoderma (14, 24, 69). Chitinolytic genes have been inserted into the genomes of cultivated plants and trees in an attempt to boost plant chitinase activity. Among the different genes involved in the production of chitinolytic enzymes, the ech42 endochitinase gene from Trichoderma harzianum has been inserted into plant genomes to enhance their resistance against phytopathogenic fungi. In McIntosh apple cultivars transformed with the ech42 gene there was limited attack by the apple scab fungus Venturia inaequalis (5). Transgenic black spruce (Picea mariana) expressing the ech42 gene was more resistant to the root rot pathogen Cylindrocladium floridanum (45).However, field deployment of crops and trees genetically transformed to improve nonspecific resistance against phytopathogenic fungi has raised concerns about the impact on nontarget fungi, including potentially beneficial symbionts. This is particularly worrisome when nonspecific constitutive promoters control expression of the resistance gene and the gene is expressed in all tissues from roots to leaves. As a consequence, the natural colonization of such transformed plants by endophytic or mycorrhizal fungi can be altered.Mycorrhizal fungi play a key role in plant nutrition (55) by mobilizing and transferring nutrients to the host through an intimate and highly organized association with plant roots (52, 63). Furthermore, their involvement in soil nutrient recycling (56) makes mycorrhizal symbiosis a major ecological process that is important for the health of soil and forest ecosystems. Crops, fruits, and forest trees exhibit mycorrhizal colonization by arbuscular mycorrhizae, ectomycorrhizae, and ectendomycorrhizae (EEM). While numerous studies have addressed the impact of transgenic plants on arbuscular mycorrhizae (10, 26, 64, 68, 72, 73) and ectomycorrhizae (32, 43, 50, 60), no previous study focused on EEM.Ectendomycorrhizal fungi can be distinguished from ectomycorrhizae by the presence of a thin or fragmented mantle and intracellular penetration into root cortical cells. All EEM fungi identified so far belong to the Ascomycetes, and these fungi are represented by several genera of Helotiales and Pezizales (77). EEM fungi are prevalent in conifer and deciduous tree nurseries (27, 39, 40, 70) and are also very common on seedling root tips at disturbed sites (15, 16, 19). The prevalence of EEM fungi on seedling roots, from which the genus Wilcoxina is frequently recovered (16, 67), suggests that they can play a significant role in establishment and growth of seedlings (77) and provide protection against root diseases (31, 61). Consequently, the potentially negative effects of chitinase-transformed trees on ectendomycorrhizal fungi could be detrimental to plant health.The present study addressed the potential impact of ech42-transformed white spruce on soil fungal biomass and ectendomycorrhizal symbiosis. It was hypothesized that (i) the soil fungal biomass in a transgenic white spruce rhizosphere is less than the soil fungal biomass in a control tree rhizosphere and (ii) the development of Wilcoxina spp. on root tips of transgenic white spruce is less important than the development of Wilcoxina spp. on root tips of control trees. To test these hypotheses, 5-year-old white spruce trees transformed with the 35S promoter-ech42 construct were analyzed by performing a greenhouse trial. The amount of soil fungal biomass was estimated using measurements of ergosterol in soil. A real-time PCR method was developed to detect changes in the quantity of ectendomycorrhizal hyphae involved in colonization of transgenic white spruce root tips.  相似文献   

16.
A family 5 glycoside hydrolase from Clostridium phytofermentans was cloned and engineered through a cellulase cell surface display system in Escherichia coli. The presence of cell surface anchoring, a cellulose binding module, or a His tag greatly influenced the activities of wild-type and mutant enzymes on soluble and solid cellulosic substrates, suggesting the high complexity of cellulase engineering. The best mutant had 92%, 36%, and 46% longer half-lives at 60°C on carboxymethyl cellulose, regenerated amorphous cellulose, and Avicel, respectively.The production of biofuels from nonfood cellulosic biomass would benefit the economy, the environment, and national energy security (17, 32). The largest technological and economical obstacle is the release of soluble fermentable sugars at prices competitive with those from sugarcane or corn kernels (17, 31). One of the approaches is discovering new cellulases from cellulolytic microorganisms, followed by cellulase engineering for enhanced performance on pretreated solid substrates. However, cellulase engineering remains challenging because enzymatic cellulose hydrolysis is complicated, involving heterogeneous substrates (33, 37), different action mode cellulase components (18), synergy and/or competition among cellulase components (36, 37), and declining substrate reactivity over the course of conversion (11, 26). Directed enzyme evolution, independent of knowledge of the protein structure and the enzyme-substrate interactions (6, 34), has been conducted to generate endoglucanase mutants, such as enhanced activities on soluble substrates (14, 16, 22), prolonged thermostability (20), changed optimum pH (24, 28), or improved expression levels (21). Here, we cloned and characterized a family 5 glycoside hydrolase (Cel5A) from a cellulolytic bacterium, Clostridium phytofermentans ISDg (ATCC 700394) (29, 30), and engineered it for enhanced thermostability.  相似文献   

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Nitrate-reducing enrichments, amended with n-hexadecane, were established with petroleum-contaminated sediment from Onondaga Lake. Cultures were serially diluted to yield a sediment-free consortium. Clone libraries and denaturing gradient gel electrophoresis analysis of 16S rRNA gene community PCR products indicated the presence of uncultured alpha- and betaproteobacteria similar to those detected in contaminated, denitrifying environments. Cultures were incubated with H34-hexadecane, fully deuterated hexadecane (d34-hexadecane), or H34-hexadecane and NaH13CO3. Gas chromatography-mass spectrometry analysis of silylated metabolites resulted in the identification of [H29]pentadecanoic acid, [H25]tridecanoic acid, [1-13C]pentadecanoic acid, [3-13C]heptadecanoic acid, [3-13C]10-methylheptadecanoic acid, and d27-pentadecanoic, d25-, and d24-tridecanoic acids. The identification of these metabolites suggests a carbon addition at the C-3 position of hexadecane, with subsequent β-oxidation and transformation reactions (chain elongation and C-10 methylation) that predominantly produce fatty acids with odd numbers of carbons. Mineralization of [1-14C]hexadecane was demonstrated based on the recovery of 14CO2 in active cultures.Linear alkanes account for a large component of crude and refined petroleum products and, therefore, are of environmental significance with respect to their fate and transport (38). The aerobic activation of alkanes is well documented and involves monooxygenase and dioxygenase enzymes in which not only is oxygen required as an electron acceptor but it also serves as a reactant in hydroxylation (2, 16, 17, 32, 34). Alkanes are also degraded under anoxic conditions via novel degradation strategies (34). To date, there are two known pathways of anaerobic n-alkane degradation: (i) alkane addition to fumarate, commonly referred to as fumarate addition, and (ii) a putative pathway, proposed by So et al. (25), involving carboxylation of the alkane. Fumarate addition proceeds via terminal or subterminal addition (C-2 position) of the alkane to the double bond of fumarate, resulting in the formation of an alkylsuccinate. The alkylsuccinate is further degraded via carbon skeleton rearrangement and β-oxidation (4, 6, 8, 12, 13, 21, 37). Alkane addition to fumarate has been documented for a denitrifying isolate (21, 37), sulfate-reducing consortia (4, 8, 12, 13), and five sulfate-reducing isolates (4, 6-8, 12). In addition to being demonstrated in these studies, fumarate addition in a sulfate-reducing enrichment growing on the alicyclic alkane 2-ethylcyclopentane has also been demonstrated (23). In contrast to fumarate addition, which has been shown for both sulfate-reducers and denitrifiers, the putative carboxylation of n-alkanes has been proposed only for the sulfate-reducing isolate strain Hxd3 (25) and for a sulfate-reducing consortium (4). Experiments using NaH13CO3 demonstrated that bicarbonate serves as the source of inorganic carbon for the putative carboxylation reaction (25). Subterminal carboxylation of the alkane at the C-3 position is followed by elimination of the two terminal carbons, to yield a fatty acid that is one carbon shorter than the parent alkane (4, 25). The fatty acids are subject to β-oxidation, chain elongation, and/or C-10 methylation (25).In this study, we characterized an alkane-degrading, nitrate-reducing consortium and surveyed the metabolites of the consortium incubated with either unlabeled or labeled hexadecane in order to elucidate the pathway of n-alkane degradation. We present evidence of a pathway analogous to the proposed carboxylation pathway under nitrate-reducing conditions.  相似文献   

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