The extracellular β‐1,3‐endoglucanase EngA is involved in autolysis of Aspergillus nidulans

Aims: To elucidate the roles of the β‐1,3‐endoglucanase EngA in autolysis of the filamentous fungus Aspergillus nidulans and to identify the common regulatory elements of autolytic hydrolases. Methods and Results: A β‐1,3‐endoglucanase was purified from carbon‐starving cultures of A. nidulans. This enzyme is found to be encoded by the engA gene (locus ID: AN0472.3). Functional and gene‐expression studies demonstrated that EngA is involved in the autolytic cell wall degradation resulting from carbon starvation of the fungus. Moreover, regulation of engA is found to be dependent on the FluG/BrlA asexual sporulation signalling pathway in submerged culture. The deletion of either engA or chiB (encoding an endochitinase) caused highly reduced production of hydrolases in general. Conclusions: The β‐1,3‐endoglucanase EngA plays a pivotal role in fungal autolysis, and activities of both EngA and ChiB are necessary to orchestrate the expression of autolytic hydrolases. The production of cell wall–degrading enzymes was coordinately controlled in a highly sophisticated and complex manner. Significance and Impact of the Study: No information was available on the autolytic glucanase(s) of the euascomycete A. nidulans. This study demonstrates that EngA is a key element in fungal autolysis, and normal activities of both EngA and ChiB are crucial for balanced production of hydrolases.     

Characterization and heterologous expression of an age-dependent fungal/bacterial type chitinase of Aspergillus nidulans

Under carbon starvation, Aspergillus nidulans produced a fungal/bacterial type chitinase, ChiB. The chiB gene was cloned and subcloned into pJC40 expression vector containing a 10XHis fusion tag, and the ChiB protein was expressed heterologously in Escherichia coli. Recombinant and native ChiB enzymes shared the same optimal pH ranges and showed similar substrate specificities with endo-acting cleavage patterns.     

Reciprocal oxylipin-mediated cross-talk in the Aspergillus–seed pathosystem

In Aspergilli, mycotoxin production and sporulation are governed, in part, by endogenous oxylipins (oxygenated, polyunsaturated fatty acids and metabolites derived therefrom). In Aspergillus nidulans , oxylipins are synthesized by the dioxygenase enzymes PpoA, PpoB and PpoC. Structurally similar oxylipins are synthesized in seeds via the action of lipoxygenase (LOX) enzymes. Previous reports have shown that exogenous application of seed oxylipins to Aspergillus cultures alters sporulation and mycotoxin production. Herein, we explored whether a plant oxylipin biosynthetic gene ( ZmLOX3 ) could substitute functionally for A. nidulans ppo genes. We engineered ZmLOX3 into wild-type A. nidulans , and into a Δ ppoAC strain that was reduced in production of oxylipins, conidia and the mycotoxin sterigmatocystin. ZmLOX3 expression increased production of conidia and sterigmatocystin in both backgrounds. We additionally explored whether A. nidulans oxylipins affect seed LOX gene expression during Aspergillus colonization. We observed that peanut seed pnlox2–3 expression was decreased when infected by A. nidulans Δ ppo mutants compared with infection by wild type. This result provides genetic evidence that fungal oxylipins are involved in plant LOX gene expression changes, leading to possible alterations in the fungal/host interaction. This report provides the first genetic evidence for reciprocal oxylipin cross-talk in the Aspergillus –seed pathosystem.     

The push-pull mechanism of bacteriophage Ø29 DNA injection

Regulators of G-protein signalling play a crucial role in controlling the degree of heterotrimeric G-protein signalling. In addition to the previously studied flbA, we have identified three genes (rgsA, rgsB and rgsC) encoding putative RGS proteins in the genome of Aspergillus nidulans. Characterization of the rgsA gene revealed that RgsA downregulates pigment production and conidial germination, but stimulates asexual sporulation (conidiation). Deletion of rgsA (DeltargsA) resulted in reduced colony size with increased aerial hyphae, elevated accumulation of brown pigments as well as enhanced tolerance of conidia and vegetative hyphae against oxidative and thermal stress. Moreover, DeltargsA resulted in conidial germination in the absence of a carbon source. Deletion of both flbA and rgsA resulted in an additive phenotype, suggesting that the G-protein pathways controlled by FlbA and RgsA are different. Morphological and metabolic alterations caused by DeltargsA were suppressed by deletion of ganB encoding a Galpha subunit, indicating that the primary role of RgsA is to control negatively GanB-mediated signalling. Overexpression of rgsA caused inappropriate conidiation in liquid submerged culture, supporting the idea that GanB signalling represses conidiation. Our findings define a second and specific RGS-Galpha pair in A. nidulans, which may govern upstream regulation of fungal cellular responses to environmental changes.     

Production and characterization of pure Clostridium spore suspensions

Aims:  A general protocol was derived for optimizing the production of pure, high concentration Clostridium endospore suspensions.
Methods and Results:  Two sporulation methods were developed that yielded high concentrations of notably pure Clostridium sporogenes , C. hungatei and C. GSA-1 (Greenland ice core isolate) spore suspensions (10 ml of 10⁹ spores ml⁻¹ with >99% purity each). Each method was derived by evaluating combinations of three sporulation conditions, including freeze drying of inocula, heat shock treatment of cultures, and subsequent incubation at suboptimal temperatures that yielded the highest percentage of sporulation. Pure spore suspensions were characterized in terms of dipicolinic acid content, culturability, decimal reduction time ( D ) value for heat inactivation (100°C) and hydrophobicity.
Conclusions:  While some Clostridium species produce a high percentage of spores with heat shock treatment and suboptimal temperature incubation, other species require the additional step of freeze drying the inocula to achieve a high percentage of sporulation.
Significance and Impact of the Study:  Pure Clostridium spore suspensions are required for investigating species of medical and environmental importance. Defining the conditions for optimal spore production also provides insight into the underlying mechanisms of Clostridium sporulation.     

Differential Roles of the ChiB Chitinase in Autolysis and Cell Death of Aspergillus nidulans

Autolysis is a natural event that occurs in most filamentous fungi. Such self-degradation of fungal cells becomes a predominant phenomenon in the absence of the regulator of G protein signaling FlbA in Aspergillus nidulans. Among a number of potential hydrolytic enzymes in the A. nidulans genome, the secreted endochitinase ChiB was shown to play a major role in autolysis. In this report, we investigate the roles of ChiB in fungal autolysis and cell death processes through genetic, biochemical, and cellular analyses using a set of critical mutants. Determination of mycelial mass revealed that, while the flbA deletion (ΔflbA) mutant autolyzed completely after a 3-day incubation, the ΔflbA ΔchiB double mutant escaped from hyphal disintegration. These results indicate that ChiB is necessary for the ΔflbA-induced autolysis. However, importantly, both ΔflbA and ΔflbA ΔchiB strains displayed dramatically reduced cell viability compared to the wild type. These imply that ChiB is dispensable for cell death and that autolysis and cell death are separate processes. Liquid chromatography-tandem mass spectrometry analyses of the proteins that accumulate at high levels in the ΔflbA and ΔflbA ΔchiB mutants identify chitinase (ChiB), dipeptidyl peptidase V (DppV), O-glycosyl compound hydrolase, β-N-acetylhexosaminidase (NagA), and myo-inositol-1-phosphate synthase (InoB). Functional characterization of these four genes reveals that the deletion of nagA results in reduced cell death. A working model bridging G protein signaling and players in autolysis/cell death is proposed.Autolysis can be described as enzymatic self-degradation of the cells. It involves the activation of several key enzyme classes, resulting in the catabolism of macromolecules within the cell (11, 12, 23). Autolysis is observed in most filamentous fungi at the late stages of cultures and is affected by aging, programmed cell death, development, nutrient limitation, and numerous other factors (39). Despite its fundamental importance in growth, differentiation, secondary metabolism, and heterologous protein production, autolysis is a poorly understood feature of fungal biology (26, 39). It is anticipated that the elucidation of the molecular mechanisms governing fungal autolysis would have great impacts on both fundamental and applied aspects of filamentous fungi.The Aspergillus nidulans ΔflbA mutant exhibits autolysis as a predominant phenotype (18). FlbA is a regulator of G protein signaling (RGS) that negatively controls vegetative growth signaling, primarily mediated by a heterotrimeric G protein composed of FadA (Gα) and SfaD::GpgA (Gβγ) (31, 33, 34, 42, 43). Loss of flbA function causes prolonged activation of G protein signaling, which results in uncontrolled proliferation of hyphal mass, the blockage of sporulation, and hyphal disintegration (autolysis). Because nearly the entire mycelium disappears during autolysis of ΔflbA mutant strains, some component of this phenomenon is hypothesized to involve enzymatic degradation as observed during autolysis of aging fungal cultures (11, 12, 23).In A. nidulans, the last step of autolysis is thought to be the degradation of the cell wall of empty hyphae, which is associated with increased proteinase and chitinase activities (10). There are 15 potential chitinase open reading frames (ORFs) in the genome of A. nidulans. Among these, the class V endochitinase ChiB was shown to play an important role in autolysis. The deletion of chiB considerably reduced the intracellular and extracellular chitinase activities, and the levels of ChiB significantly increased when the fungal cells were starved for carbon sources, an induced condition for hyphal autolysis of A. nidulans (41).In the present study we addressed two primary questions: (i) does ChiB function in the accelerated autolysis and/or cell death caused by loss of FlbA and (ii) are there other hydrolytic enzymes involved in autolysis and/or cell death in A. nidulans? In order to investigate a hypothetical connection between FlbA-controlled autolysis and the ChiB activity, we carried out genetic, biochemical, and cellular studies using the ΔchiB, ΔflbA, ΔflbA ΔchiB (double deletion), and chiB overexpression mutants. We found that, while ChiB plays a crucial role in ΔflbA-induced autolysis, ChiB is dispensable for cell death, corroborating the idea that cell death and autolysis are independent processes in A. nidulans (8). We further present the identification and partial functional characterization of four gene encoding the proteins accumulate at high level in ΔflbA and/or ΔflbA ΔchiB strains and propose a working model linking G protein signaling and autolysis.     

Reciprocal oxylipin-mediated cross-talk in the Aspergillus-seed pathosystem.

In Aspergilli, mycotoxin production and sporulation are governed, in part, by endogenous oxylipins (oxygenated, polyunsaturated fatty acids and metabolites derived therefrom). In Aspergillus nidulans, oxylipins are synthesized by the dioxygenase enzymes PpoA, PpoB and PpoC. Structurally similar oxylipins are synthesized in seeds via the action of lipoxygenase (LOX) enzymes. Previous reports have shown that exogenous application of seed oxylipins to Aspergillus cultures alters sporulation and mycotoxin production. Herein, we explored whether a plant oxylipin biosynthetic gene (ZmLOX3) could substitute functionally for A. nidulans ppo genes. We engineered ZmLOX3 into wild-type A. nidulans, and into a DeltappoAC strain that was reduced in production of oxylipins, conidia and the mycotoxin sterigmatocystin. ZmLOX3 expression increased production of conidia and sterigmatocystin in both backgrounds. We additionally explored whether A. nidulans oxylipins affect seed LOX gene expression during Aspergillus colonization. We observed that peanut seed pnlox2-3 expression was decreased when infected by A. nidulansDeltappo mutants compared with infection by wild type. This result provides genetic evidence that fungal oxylipins are involved in plant LOX gene expression changes, leading to possible alterations in the fungal/host interaction. This report provides the first genetic evidence for reciprocal oxylipin cross-talk in the Aspergillus-seed pathosystem.     

Effect of physiological conditions on the autolysis ofStaphylococcus aureus strains

The effect of physiological conditions on autolysis and autolytic activity in various strains ofStaphylococcus aureus was determined. The rate of whole cell autolysis ofS. aureus was growth phase dependent and a maximum rate was observed in early stationary phase cultures. However, the autolysins extracted by the freeze-thaw method (cell-wall bound autolytic activity) did not show any significant increase in activity. The addition of NaCl to the growth medium enhanced the rate of autolysis with the highest rate being displayed by cultures grown in 1.5 M NaCl. However, lower autolytic activity was found in the freeze-thaw extracts of cultures grown at higher concentrations of NaCl. The rate of autolysis of cultures grown at 30°C was higher than cultures grown at 37 or 43°C. Thus, the rate of autolysis seems to be independent of the bacterial growth rate. Cultures grown in slightly acidic conditions showed a faster rate of autolysis compared to cultures grown under alkaline conditions. SDS-polyacrylamide gel containing 0.2% crude cell-wall ofS. aureus did not show any obvious correlation with the appearance of any particular lytic band in the zymogram to autolytic activity or rate of autolysis of cultures grown under various environmental conditions. A nonhemolytic phenotype, mutations in the accessory gene regulator, and lysogeny (phages ø11, ø12, ø13) had no obvious effect either on the rate of autolysis or on the pattern of lytic bands in the zymograms.     

FluG-BrlA途径参与构巢曲霉无性发育机制的研究进展

构巢曲霉是丝状真菌的模式生物,已对其无性发育机制进行了比较充分的研究。本文以FluG-BrlA途径参与构巢曲霉无性发育机制的研究为切入点,综述了构巢曲霉无性发育中心调控路径中各主要成员如brlA、abaA、wetA,中心调控路径修饰基因如stuA、medA及中心调控路径激活因子fluG、flbA-E的研究进展,绘制出构巢曲霉无性发育相关基因遗传位置模式图。研究将为其它丝状真菌无性发育机制的研究提供参考。     

Activation of caspase-like activity and poly (ADP-ribose) polymerase degradation during sporulation in Aspergillus nidulans

Mycelium vacuolization, protein degradation, and as the final stage autolysis, often accompanies developmental changes in fungi and similarities between autolysis and apoptosis have previously been suggested. Caspases are the key executors of apoptosis and in this study caspase-like activities were detected in protein extracts from Aspergillus nidulans during sporulation. This was shown by hydrolysis of the fluorescent DEVD- and IETD-AFC peptide substrates specific for caspase 3- and 8-like activities, respectively. These activities were repressed by the caspase 3 and 8 specific irreversible peptide inhibitors DEVD-fmk and IETD-fmk, but were not affected by the unspecific inhibitor E-64. Isoelectric focusing of protein extracts followed by activity staining revealed the presence of two bands with caspase-like activity. One of the proteins degraded both caspase 3 and caspase 8 specific substrates whereas the other only degraded the caspase 8 substrate. Searches in an A. nidulans genome database revealed two genes encoding metacaspase proteins with predicted sizes of 45 kDa that could be responsible for the measured caspase-like activities. The searches also found a single gene encoding a poly (ADP-ribose) polymerase (PARP) protein with a predicted size of 81 kDa. PARP is one of the known target proteins inactivated by caspase degradation in animal cells. Western blotting of fungal extracts using a bovine PARP antibody confirmed the presence of a fungal PARP-like protein of about 81 kDa. By Western blotting it was shown that this PARP-like protein band was present only at early time points until the start of conidia formation and the accompanying increase in caspase-like activity. Thereafter, a degradation product of about 60 kDa appeared indicating that the degradation of the fungal PARP-like protein was specific. The PARP antibody also recognized an 85 kDa protein band that was not degraded, and which conceivably represents a modified form of the 81 kDa PARP. Fungal extracts high in caspase-like activity could degrade both the fungal 81 kDa PARP and bovine PARP. In the presence of the caspase 3 inhibitor DEVD-fmk this degradation was delayed. Thus, as in animal apoptotic cells, caspase activities are involved in fungal mycelium self-activated proteolysis.     

Leaf litter decomposition and microbial activity in nutrient-enriched and unaltered reaches of a headwater stream

SUMMARY 1. Decomposition of red maple ( Acer rubrum ) and rhododendron ( Rhododendron maximum ) leaves and activity of associated microorganisms were compared in two reaches of a headwater stream in Coweeta Hydrologic Laboratory, NC, U.S.A. The downstream reach was enriched with ammonium, nitrate, and phosphate whereas the upstream reach was not altered.
2. Decomposition rate, microbial respiration, fungal and bacterial biomass, and the sporulation rate of aquatic hyphomycetes associated with decomposing leaf material were significantly higher for both leaf types in the nutrient-enriched reach. Species richness and community structure of aquatic hyphomycetes also exhibited considerable changes with an increase in the number of fungal codominants in the nutrient-enriched reach.
3. Fungal biomass was one to two orders of magnitude greater than bacterial biomass in both reaches. Changes in microbial respiration rate corresponded to those in fungal biomass and sporulation, suggesting a primary role of fungi in leaf decomposition.
4. Nutrient enrichment increased microbial activity, the proportion of leaf carbon channelled through the microbial compartment and the decomposition rate of leaf litter.     

苏云金芽孢杆菌chiA,chiB全基因的克隆、表达及其序列分析

以苏云金芽孢杆菌科默尔亚种15A3菌株基因组DNA为模版,用touchdown PCR方法扩增几丁质酶ChiA和ChiB的全基因序列(GenBank登录号:EF103273和DQ512474)。将PCR产物连接pUCm-T克隆载体,获得重组质粒pUCm-chiA和pUCm-chiB,分别转化E.coliXL-Blue。克隆的几丁质酶基因可以利用本身的启动子异源表达各自的蛋白,不需要几丁质作为诱导物。表达的几丁质酶能够分泌到胞外。证明15A3菌株可组成型表达2种几丁质酶。经核苷酸及氨基酸序列分析证明,chiA基因全长1426bp,含有343bp的上游非编码区和1083bp的ORF,编码360个氨基酸。推测成熟蛋白分子量为36kD,只有一个几丁质酶催化域。chiB基因全长2279bp,含有248bp的上游非编码区和2031bp的ORF,编码676个氨基酸。推测成熟蛋白分子量约为70.6kD,具有三个功能域。核苷酸序列分析显示chiA和chiB的启动子所处的位置及转录起始碱基都不相同,-35区相同,而-10区有两个碱基不同,SD序列也不完全一致。     

The autolysis of industrial filamentous fungi

Fungal autolysis is the natural process of self-digestion of aged hyphal cultures, occurring as a result of hydrolase activity, causing vacuolation and disruption of organelle and cell wall structure. Previously, authors have considered individual aspects of fungal lysis, in terms of either an enzyme, a process or an organism. This review considers both the physiology and morphology of fungal autolysis, with an emphasis on correlations between enzymological profiles and the morphological changes occurring during culture degeneration. The involvement of the main groups of autolytic hydrolases is examined (i.e., proteases, glucanases, and chitinases), in addition to the effects of autolysis on the morphology and products of industrial bioprocesses. We call for a concerted approach to the study of autolysis, as this will be fundamental for research to progress in this field. Increased understanding will allow for greater control of the prevention, or induction of fungal autolysis. Such advances will be applicable in the development of antifungal medicines and enable increased productivity and yields in industrial bioprocesses. Using paradigms in existing model systems, including mammalian cell death and aging in yeast, areas for future study are suggested in order to advance the study of fungal cell death.     

Chitinolytic activity in the autolysis of Aspergillus nidulans

Abstract Chitinolytic activity in filtrates of Aspergillus nidulans cultures was studied at the start of the autolysis (maximum dry weight of mycelium) and during autolysis in 24 different media. During the growth the chitinolytic activity was induced only by the presence of ascorbic acid or colloidal chitin in the medium. During autolysis an increasing chitinolytic activity was observed with the incubation time in all the conditions, and synthesis of a β - N -acetylgucosaminidase and endochitinase was detected. The possible induction of these enzymes during A. nidulans autolysis is established.