Ultrastructure and composition of the conidial wall of Cladosporium cladosporioides

Transmission electron microscopy revealed that the conidial wall of Cladosporium cladosporioides was constituted of an electron-lucent inner layer and an electron-dense outer layer. The conidial surface is covered by rodlet fascicles which can be removed by ultrasonication. Ultrastructurally, the 100,000 X g ultracentrifugation pellet of the ultrasonicated extract containing the rodlet layer appeared as an amorphous structure containing probably internal wall material anchoring the rodlet fascicles on the wall. The total conidial wall was essentially composed of beta(1----3)glucans and melanin. Lipid, salt, and galactan represented the main components of the 100,000 X g ultracentrifugation pellet of the ultrasonicated extract. Cladosporium cladosporioides produced melanin via the pentaketide pathway. Tricyclazole inhibited melanin synthesis but did not interfere with allergen production. This suggests that the wall components associated with melanin are not allergenic factors.     

Purification and characterization of a novel chlorpyrifos hydrolase from Cladosporium cladosporioides Hu-01

Chlorpyrifos is of great environmental concern due to its widespread use in the past several decades and its potential toxic effects on human health. Thus, the degradation study of chlorpyrifos has become increasing important in recent years. A fungus capable of using chlorpyrifos as the sole carbon source was isolated from organophosphate-contaminated soil and characterized as Cladosporium cladosporioides Hu-01 (collection number: CCTCC M 20711). A novel chlorpyrifos hydrolase from cell extract was purified 35.6-fold to apparent homogeneity with 38.5% overall recovery by ammoniumsulfate precipitation, gel filtration chromatography and anion-exchange chromatography. It is a monomeric structure with a molecular mass of 38.3 kDa. The pI value was estimated to be 5.2. The optimal pH and temperature of the purified enzyme were 6.5 and 40°C, respectively. No cofactors were required for the chlorpyrifos-hydrolysis activity. The enzyme was strongly inhibited by Hg2?, Fe3?, DTT, β-mercaptoethanol and SDS, whereas slight inhibitory effects (5-10% inhibition) were observed in the presence of Mn2?, Zn2?, Cu2?, Mg2?, and EDTA. The purified enzyme hydrolyzed various organophosphorus insecticides with P-O and P-S bond. Chlorpyrifos was the preferred substrate. The Km and Vmax values of the enzyme for chlorpyrifos were 6.7974 μM and 2.6473 μmol·min?1, respectively. Both NH2-terminal sequencing and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometer (MALDI-TOF-MS) identified an amino acid sequence MEPDGELSALTQGANS, which shared no similarity with any reported organophosphate-hydrolyzing enzymes. These results suggested that the purified enzyme was a novel hydrolase and might conceivably be developed to fulfill the practical requirements to enable its use in situ for detoxification of chlorpyrifos. Finally, this is the first described chlorpyrifos hydrolase from fungus.     

Development of a fungal spore aerosol generator: test with Cladosporium cladosporioides and Penicillium citrinum

As the first step to develop efficient means to control fungal spore bioaerosols, we designed, manufactured, and evaluated a fungal spore aerosol generator. We studied the physical and biological properties of the fungal spore bioaerosols on two common fungal species. The results demonstrated that the fungal spore bioaerosol generator effectively produces fungal spore bioaerosols.     

Production of fructooligosaccharides by mycelium-bound transfructosylation activity present in Cladosporium cladosporioides and Penicilium sizovae

Different filamentous fungi isolated from molasses and jams (kiwi and fig) were screened for fructooligosaccharides (FOS) producing activity. Two strains, identified as Penicilium sizovae (CK1) and Cladosporium cladosporioides (CF₂15), were selected on the basis of the FOS yield and kestose/nystose ratio. In both strains the activity was mostly mycelium-bound. Starting from 600 g/L of sucrose, maximum FOS yield was 184 and 339 g/L for P. sizovae and C. cladosporioides, respectively. Interestingly, the highest FOS concentration with C. cladosporioides was reached at 93% sucrose conversion, which indicated a notable transglycosylation to hydrolysis ratio. The main FOS in the reaction mixtures were identified by HPAEC–PAD chromatography. C. cladosporioides synthesized mainly 1-kestose (158 g/L), nystose (97 g/L), ¹F-fructosylnystose (19 g/L), 6-kestose (12 g/L), neokestose (10 g/L) and a disaccharide (34 g/L) that after its purification and NMR analysis was identified as blastose [Fru-β(2 → 6)-Glc]. P. sizovae was very selective for the formation of ¹F-FOS (in particular 1-kestose) with minor contribution of neoFOS and negligible of levan-type FOS.     

Efficacy of different fungicides,botanical extracts and bio-control agents against Cladosporium cladosporioides,the causal agent of Cladosporium rot in grapes

The current research aims to find out different effective and suitable fungicides, botanical extracts and bio-control agents against Cladosporium cladosporioides, the causal agent of Cladosporium rot in grapes. For this purpose, different fungicides including, Antracol, Alliette, Melody duo, Cabriotop and Topsin-M at the rates of 100, 200 and 300 ppm, as well as botanical extracts including, Garlic, Ginger, Onion, Eucalyptus and Neem at the rates of 5, 10 and 15% were tested using food poisoning method. Bio-control agents such as Pestalotiopsis sp., Neurospora sp., Fusarium sp., Arthrinium sp. and Hypocrea lixii were also evaluated against the pathogen. Pathogenicity test was also performed to see the disease severity in grapes. Minimum linear colony growth of C. cladosporioides was observed as 39, 30.5 and 21 mm for Melody duo, respectively followed by Antracol (38.5, 30.5 and 23), Alliette (39, 31.5 and 23.5), Topsin-m (42, 33 and 27.5) and cabriotop (43.5, 36.5 and 26), as compared with the control, which was 90 mm. Whereas Minimum linear colony growth of C. cladosporioides was observed as 50, 32.5 and 19 for Neem, respectively followed by Ginger (56.5, 36.5 and 22.5), Garlic (61, 39 and 24), euclayptus (62, 41.5 and 25) and Onion (64, 43 and 25), maximum linear colony growth (90) was observed under control. Whereas, the minimum linear colony growth of C. cladosporioides was observed for Neurospora sp. (3.7), followed by Arthrinium sp. (7.5), Pestalotiopsis sp. (9), Hypocrea Lixii (9) and Fusarium sp. (9.5), maximum linear colony growth (90) was recorded in control. This study could be helpful for researchers and farming community in future for better management of this disease.     

Edible dates (Phoenix dactylifera), a potentialsource of Cladosporium cladosporioides andSporobolomyces roseus: Implications for public health

The non-steroidal anti-inflammatory drug niflumic acid was found to inhibit growth of the yeast form of Candida albicans. Niflumic acid inhibited respiratory oxygen uptake and it is hypothesised that this was achieved by cytosolic acidification and block of glycolysis. Inhibitory concentrations are compatible with current practice of topical application. This revised version was published online in June 2006 with corrections to the Cover Date.     

Biodegradation of Chlorpyrifos and Its Hydrolysis Product 3,5,6-Trichloro-2-Pyridinol by a New Fungal Strain Cladosporium cladosporioides Hu-01

Intensive use of chlorpyrifos has resulted in its ubiquitous presence as a contaminant in surface streams and soils. It is thus critically essential to develop bioremediation methods to degrade and eliminate this pollutant from environments. We present here that a new fungal strain Hu-01 with high chlorpyrifos-degradation activity was isolated and identified as Cladosporium cladosporioides based on the morphology and 5.8S rDNA gene analysis. Strain Hu-01 utilized 50 mg·L⁻¹ of chlorpyrifos as the sole carbon of source, and tolerated high concentration of chlorpyrifos up to 500 mg·L⁻¹. The optimum degradation conditions were determined to be 26.8°C and pH 6.5 based on the response surface methodology (RSM). Under these conditions, strain Hu-01 completely metabolized the supplemented chlorpyrifos (50 mg·L⁻¹) within 5 d. During the biodegradation process, transient accumulation of 3,5,6-trichloro-2-pyridinol (TCP) was observed. However, this intermediate product did not accumulate in the medium and disappeared quickly. No persistent accumulative metabolite was detected by gas chromatopraphy-mass spectrometry (GC-MS) analysis at the end of experiment. Furthermore, degradation kinetics of chlorpyrifos and TCP followed the first-order model. Compared to the non-inoculated controls, the half-lives (t _1/2) of chlorpyrifos and TCP significantly reduced by 688.0 and 986.9 h with the inoculum, respectively. The isolate harbors the metabolic pathway for the complete detoxification of chlorpyrifos and its hydrolysis product TCP, thus suggesting the fungus may be a promising candidate for bioremediation of chlorpyrifos-contaminated water, soil or crop.     

The Polyketide Synthase Gene pks4 of Trichoderma reesei Provides Pigmentation and Stress Resistance

Species of the fungal genus Trichoderma (Hypocreales, Ascomycota) are well-known for their production of various secondary metabolites. Nonribosomal peptides and polyketides represent a major portion of these products. In a recent phylogenomic investigation of Trichoderma polyketide synthase (PKS)-encoding genes, the pks4 from T. reesei was shown to be an orthologue of pigment-forming PKSs involved in synthesis of aurofusarin and bikaverin in Fusarium spp. In this study, we show that deletion of this gene in T. reesei results in loss of green conidial pigmentation and in pigmentation alteration of teleomorph structures. It also has an impact on conidial cell wall stability and the antagonistic abilities of T. reesei against other fungi, including formation of inhibitory metabolites. In addition, deletion of pks4 significantly influences the expression of other PKS-encoding genes of T. reesei. To our knowledge, this is the first indication that a low-molecular-weight pigment-forming PKS is involved in defense, mechanical stability, and stress resistance in fungi.     

一个推测的拟南芥漆酶基因在巴斯德毕赤酵母中的表达

At5g01040基因是一个推测的拟南芥漆酶基因。根据其编码序列设计引物,利用RT—PCR方法扩增出1755bp的片段。测序结果表明,该片段存在3个突变位点,其中一个突变位点改变了所编码的氨基酸。将该片段克隆到表达载体pPICZaB上,电击转化毕赤酵母。经筛选获得80个候选重组菌株,其中18个菌株的培养液中存在漆酶活性,说明所克隆的基因在毕赤酵母中实现了分泌表达,证实了At5g01040编码的蛋白具有漆酶活性。     

Isolation and Characterization of a Putative Class E Gene from Taihangia rupestris

Studies In model plants showed that SEPALLATA （SEP） genes are required for the Identification of floral organs and the determination of floral meristems In Arabidopsis. In this paper a SEP homolog, TrSEP3, was Isolated from a China-specific species, Taihangla rupestrisi Yü et LI. Phylogenetlc analysis showed that the gene belongs to the SEP3-clade of SEP （previous AGL2） subfamily. In situ hybridization was used to reveal the potential functional specification, and the results showed that TrSEP3 expression was first observed in floral meristems and then confined to the floral primordla of the three inner whorls. In the matured flower, TrSEP3 was strongly expressed In the tips of pistils and weak In stamens and petals. The evolution force analysis shows that TrSEP3 might undergo a relaxed negative selection. These results suggested that TrSEP3 may not only function In determining the identity of floral merlstems and the primordia of three inner whorls, but also function In matured reproductive organs.     

一个受褐飞虱取食诱导的水稻GST类似蛋白基因的克隆

BpHi006AcDNA长度为1943bp,具有一个795bp组成的完整的读码框,其表达受褐飞虱取食的诱导。BpHi006A蛋白含有谷胱苷肽S转移酶(glutathione Stransferase)的N末端结构域和C末端结构域,为谷胱苷肽S转移酶超家族的成员。BpHi006A蛋白与拟南芥四氯氢醌还原性脱卤素酶相关蛋白有61％的一致性,序列分析表明这两种蛋白质构成一类新的植物GST。     

Inactivation of a Novel Gene Produces a Phenotypic Variant Cell and Affects the Symbiotic Behavior of Xenorhabdus nematophilus

Xenorhabdus nematophilus is an insect pathogen that lives in a symbiotic association with a specific entomopathogenic nematode. During prolonged culturing, variant cells arise that are deficient in numerous properties. To understand the genetic mechanism underlying variant cell formation, a transposon mutagenesis approach was taken. Three phenotypically similar variant strains of X. nematophilus, each of which contained a single transposon insertion, were isolated. The insertions occurred at different locations in the chromosome. The variant strain, ANV2, was further characterized. It was deficient in several properties, including the ability to produce antibiotics and the stationary-phase-induced outer membrane protein, OpnB. Unlike wild-type cells, ANV2 produced lecithinase. The emergence of ANV2 from the nematode host was delayed relative to the emergence of the parental strain. The transposon in ANV2 had inserted in a gene designated var1, which encodes a novel protein composed of 121 amino acid residues. Complementation analysis confirmed that the pleiotropic phenotype of the ANV2 strain was produced by inactivation of var1. Other variant strains were not complemented by var1. These results indicate that inactivation of a single gene was sufficient to promote variant cell formation in X. nematophilus and that disruption of genetic loci other than var1 can result in the same pleiotropic phenotype.     

Isolation of a Putative Carboxysomal Carbonic Anhydrase Gene from the Cyanobacterium Synechococcus PCC7942

The Type II mutants of the cyanobacterium Synechococcus PCC7942 (G.D. Price, M.R. Badger [1989] Plant Physiol 91: 514-525) are able to accumulate a large pool of inorganic carbon inside the cell, but are unable to utilize it for CO₂ fixation, resulting in a high CO₂-requiring phenotype. We have isolated a 3.5-kb BamHI clone (pT2) that complements the Type II mutants, and complementation analysis with DNA subclones indicated that the complementing region was located in the 0.75-kb XhoI-Bg/II fragment. This same region hybridized to the chloroplastic carbonic anhydrase (CA) gene from spinach on Southern blots and to a mRNA of approximate 1 kb on northern blots. Restriction mapping and sequence analysis revealed that pT2 is the same as a genomic clone (pBM3.8) that complements another high CO₂-requiring (temperature sensitive) mutant, C3P-O (E. Suzuki, H. Fukuzawa, S. Miyachi [1991] Mol Gen Genet 226: 401-408). Recently, a 272-amino acid open reading frame showing 22% homology with pea and spinach chloroplast CA genes was identified in clone pBM3.8 (H. Fukuzawa, E. Suzuki, Y. Komukal, S. Miyachi [1992] Proc Natl Acad Sci USA 89: 4437-4441). CA activity was detected in Escherichia coli cells transformed with subclones of pT2 (pT2-A and pT2-A1) containing the HindIII-Bg/II fragment, and the expressed CA has properties similar to those of the CA activity associated with carboxysomes purified from Synechococcus PCC7942 (G.D. Price, J.R. Coleman, M.R. Badger [1992] Plant Physiol 100: 784-793). Therefore, it is reasonable to conclude that the HindIII-Bg/II fragment codes for the carboxysomal CA gene product. The result is discussed in the context of the role that carboxysomal CA plays in the operation of the CO₂-concentrating mechanism in cyanobacteria.     

Micronuclear and Macronuclear Sequences of a Euplotes crassus Gene Encoding a Putative Nuclear Protein Kinase

The sequences of a 1.8-kbp macronuclear DNA molecule (V3), and the majority of its micronuclear counterpart, are reported. The macronuclear V3 DNA molecule contains an open reading frame that is interrupted by a single intron, while the micronuclear copy is interrupted by four internal eliminated sequences, one of which is located within the intron. The predicted protein product of the macronuclear V3 gene is a 471-amino acid polypeptide that is very similar to a group of protein-serine/threonine kinases from both plant and animal species, some of whose members appear to be involved in cell cycle or growth control.     

Molecular and Genetic Analyses of the Putative Proteus O Antigen Gene Locus

Proteus species are well-characterized opportunistic pathogens primarily associated with urinary tract infections (UTI) of humans. The Proteus O antigen is one of the most variable constituents of the cell surface, and O antigen heterogeneity is used for serological classification of Proteus isolates. Even though most Proteus O antigen structures have been identified, the O antigen locus has not been well characterized. In this study, we identified the putative Proteus O antigen locus and demonstrated this region''s high degree of heterogeneity by comparing sequences of 40 Proteus isolates using PCR-restriction fragment length polymorphism (RFLP). This analysis identified five putative Proteus O antigen gene clusters, and the probable functions of these O antigen-related genes were proposed, based on their similarity to genes in the available databases. Finally, Proteus-specific genes from these five serogroups were identified by screening 79 strains belonging to the 68 Proteus O antigen serogroups. To our knowledge, this is the first molecular characterization of the putative Proteus O antigen locus, and we describe a novel molecular classification method for the identification of different Proteus serogroups.Proteus species are usually found in soil, water, and sewage and are well-known opportunistic pathogens that most commonly cause urinary tract infections (UTIs) in persons with anatomical and physiological defects of their urinary tracts (15, 28). This genus includes the five named species P. mirabilis, P. vulgaris, P. myxofaciens, P. penneri, and P. hauseri and the three unnamed Proteus genomospecies 4, 5, and 6 (20, 21). Among these, P. mirabilis, P. vulgaris, and P. penneri are the most common human pathogens (28). Among Proteus species, P. mirabilis is most frequently associated with UTIs and is a common cause of catheter-associated UTIs (12).Potential virulence factors and bacterial behaviors associated with the infection processes and disease, including swarming, growth rates, fimbria expression, flagella, and the production of hemolysins, ureases, proteases, and amino acid deaminases, in addition to the expression of lipopolysaccharide (LPS) antigens and capsular polysaccharides (CPSs), have been described in many studies (11, 18, 28). Both LPSs and CPSs have been considered to play an important role in the progression of UTIs, in addition to affecting both kidney and bladder stone formation (7, 25, 35). Furthermore, the LPS O antigen confers protection against serum-mediated bactericidal activity (13, 27), and bacterial LPS released from bacteria is a biologically active endotoxin that causes a broad spectrum of pathophysiological conditions, including septic shock (26). Recently, two additional virulence factors with cytotoxic and agglutination properties, the high-affinity phosphate transporter (Pst) and the autotransporter (Pta), have been described (1, 11).The O antigen located on the cell surface of Gram-negative bacteria consists of oligosaccharide repeats (O unit) that normally contain 2 to 8 sugar residues. The O antigen is one of the most variable constituents on the cell surface, due to variations in the types of sugars present and their arrangements and respective linkages, and is subject to intense selection by the host immune system and bacteriophages. The serological classification scheme established by Kauffman and Perch defines 49 different P. mirabilis and P. vulgaris O serogroups (10), and an additional 11 serogroups were later proposed (23). In the case of P. penneri, an additional 15 O antigen serogroups were described (16, 42; Z. Sidorczyk, K. Zych, K. Kolodziejska, D. Drzewiecka, and A. Zablotni, presented at the Second German-Polish-Russian Meeting on Bacterial Carbohydrates, Moscow, Russia, 10 to 12 September 2002). To date, the O antigen structures of 78 Proteus species have been described (unpublished data), and uronic acid, which can sometimes be substituted for amino acids, was identified as a component of the Proteus O antigen. Although acidic O-specific polysaccharides have been identified in most Proteus O antigens, a study of the genetic locus associated with Proteus O antigens has never been carried out.The genome sequence of P. mirabilis was published for the first time in 2008 (22). In this study, we characterized the putative O antigen locus by analyzing genomic sequences and confirming the locus heterogeneity by carrying out PCR-restriction fragment length polymorphism (RFLP) on 40 strains. Four putative O antigen gene clusters were sequenced and analyzed, and specific primers were identified for Proteus species by screening 79 Proteus strains, confirming that the identified loci were specific to particular serogroups.     

水稻中一个人类肿瘤抑制基因同源物的克隆与表达分析

用同源筛选方法,从水稻 (Oryza sativa L.) 基因组文库中分离到一个与人类肿瘤抑制基因QM具有同源性的基因,命名为OSQM1.该基因包括4个外显子和3个内含子,编码219个氨基酸,其中有46个碱性氨基酸,其等电点高达11.02.同源性搜寻发现此基因存在于真核生物中而且保守性较强,表明它可能具有重要的作用.Northern分析结果表明,它在不同的水稻器官中都有表达,但在花和愈伤组织中的表达水平明显低于其他营养器官.它在根和叶中的表达水平受环境因素的影响.     

水稻中一个人类肿瘤抑制基因同源物的克隆与表达分析(英)

用同源筛选方法 ,从水稻 (OryzasativaL .)基因组文库中分离到一个与人类肿瘤抑制基因QM具有同源性的基因 ,命名为OSQM1。该基因包括 4个外显子和 3个内含子 ,编码 2 19个氨基酸 ,其中有 4 6个碱性氨基酸 ,其等电点高达 11.0 2。同源性搜寻发现此基因存在于真核生物中而且保守性较强 ,表明它可能具有重要的作用。North ern分析结果表明 ,它在不同的水稻器官中都有表达 ,但在花和愈伤组织中的表达水平明显低于其他营养器官。它在根和叶中的表达水平受环境因素的影响。