Characterization of Iron Uptake from Ferrioxamine B by Chlorella vulgaris

Iron uptake from two Fe³⁺-hydroxamate siderophores, ferrioxamine B and Fe³⁺-rhodotorulate, by iron-stressed Chlorella vulgaris (ATCC strain 11468) was evaluated with some comparison to iron uptake from synthetic and organic acid ferric chelates. Iron-stress induced iron uptake from ferrioxamine B. Dissipation of the electrochemical gradient, via uncouplers, inhibited iron uptake. Respiratory inhibitors gave variable results, an indication that a direct link to respiration was not apparent. Vanadate inhibition of iron uptake indicated that an ATPase or phosphate intermediate could be involved in the uptake mechanism. Divalent cations manifested variable effects dependent on the cation and chelator used. These data confirm that C. vulgaris has an inducible iron-uptake system for Fe³⁺-hydroxamic acid siderophores which may involve a different mechanism than that observed for other chelates.     

Evaluation of Reductive Release as a Mechanism for Iron Uptake from Ferrioxamine B by Chlorella vulgaris

The involvement of ferric reduction in the iron uptake mechanism of iron-stressed Chlorella vulgaris from ferrioxamine B was investigated. Some comparative data for ferric-citrate was also obtained. EPR and a spectrophotometric assay were used to measure Fe³⁺ reduction. These two methods differed in the absolute quantity but not in effectors of ferric reduction. The mechanism governing ferric reduction was investigated by use of respiratory inhibitors, uncouplers, alternative electron acceptors, and ATPase inhibitors. Reduction appears to play a role in iron uptake from both Fe³⁺-deferrioxamine B and Fe³⁺-citrate; however, the involvement of photoreduction in Fe³⁺-citrate uptake implies multiple reductive mechanisms could be involved.     

Inhibition of lipases from Chromobacterium viscosum and Rhizopus oryzae by tetrahydrolipstatin

Tetrahydrolipstatin is known as an inhibitor for pancreatic lipase but not for microbial lipases. In this paper we demonstrate that in the presence of water-insoluble substrates like tributyrin or olive oil, tetrahydrolipstatin inhibits the lipases of Chromobacterium viscosum and Rhizopus oryzae, although with different potency. In contrast to porcine pancreatic lipase, which forms an irreversible and covalent enzyme-inhibitor complex with tetrahydrolipstatin, the inhibition of the microbial lipases is reversible as the inhibitor can be removed from the enzyme-inhibitor complex by solvent extraction. Moreover, after inhibition of Chromobacterium viscosum lipase tetrahydrolipstatin remains chemically unchanged.     

Expression and characterization of fumarase (FUMR) from Rhizopus oryzae

Fumarase catalyzes the reversible hydration of fumarate to l-malate in Rhizopus oryzae. A recombinant pET22b-fumR harboring a fumarase gene (fumR) from R. oryzae was constructed for high level expression in E. coli BL21 (DE3). The FUMR activity was optimal at 30°C and pH 7.2. The enzyme was stable below 45°C and at pH 3.0-9.0. No effects of Zn(2+), Fe(2+), or EDTA were observed on enzyme activity. A slight inhibition of FUMR activity was seen with Mg(2+), while Ca(2+) had a small stimulatory effect. The K(m) for l-malic acid and fumaric acid were 0.46 mM and 3.07 mM, respectively. The activity of FUMR catalyzing hydration of fumarate to l-malate was completely inhibited by 2mM fumaric acid. The unique enzymatic properties suggested that overexpression of FUMR could enhance fumaric acid accumulation in R. oryzae.     

Comparative Secretome Analysis of Magnaporthe oryzae Identified Proteins Involved in Virulence and Cell Wall Integrity

Plant fungal pathogens secrete numerous proteins into the apoplast at the plant–fungus contact sites to facilitate colonization. However, only a few secretory proteins were functionally characterized in Magnaporthe oryzae, the fungal pathogen causing rice blast disease worldwide. Asparagine-linked glycosylation 3 (Alg3) is an α-1,3-mannosyltransferase functioning in the N-glycan synthesis of N-glycosylated secretory proteins. Fungal pathogenicity and cell wall integrity are impaired in Δalg3 mutants, but the secreted proteins affected in Δalg3 mutants are largely unknown. In this study, we compared the secretomes of the wild-type strain and the Δalg3 mutant and identified 51 proteins that require Alg3 for proper secretion. These proteins were predicted to be involved in metabolic processes, interspecies interactions, cell wall organization, and response to chemicals. Nine proteins were selected for further validation. We found that these proteins were localized at the apoplastic region surrounding the fungal infection hyphae. Moreover, the N-glycosylation of these proteins was significantly changed in the Δalg3 mutant, leading to the decreased protein secretion and abnormal protein localization. Furthermore, we tested the biological functions of two genes, INV1 (encoding invertase 1, a secreted invertase) and AMCase (encoding acid mammalian chinitase, a secreted chitinase). The fungal virulence was significantly reduced, and the cell wall integrity was altered in the Δinv1 and Δamcase mutant strains. Moreover, the N-glycosylation was essential for the function and secretion of AMCase. Taken together, our study provides new insight into the role of N-glycosylated secretory proteins in fungal virulence and cell wall integrity.     

Purification and characterization of protease from a newly isolated Rhizopus oryzae

A newly isolated Rhizopus oryzae was found to exhibit some unusual phenomenon of secreting alkaline protease which was purified and characterized. The molecular weight was determined to be 28,600 dalton in gel electrophoresis. The enzyme is stable in the pH range from 3 to 11 and most active at pH 8. The temperature optimum of this thermostable biocatalyst is at 60 °C. The enzyme is sensitive to metal chelators, most of the metal ions (excepting a few monovalent cations) and inhibitor like PMSF. This indicates that the protease of isolated Rhizopus oryzae falls under alkaline serine group.     

Distinct Determinants in HIV-1 Vif and Human APOBEC3 Proteins Are Required for the Suppression of Diverse Host Anti-Viral Proteins

Background

APOBEC3G (A3G) and related cytidine deaminases of the APOBEC3 family of proteins are potent inhibitors of many retroviruses, including HIV-1. Formation of infectious HIV-1 requires the suppression of multiple cytidine deaminases by Vif. HIV-1 Vif suppresses various APOBEC3 proteins through the common mechanism of recruiting the Cullin5-ElonginB-ElonginC E3 ubiquitin ligase to induce target protein polyubiquitination and proteasome-mediated degradation. The domains in Vif and various APOBEC3 proteins required for APOBEC3 recognition and degradation have not been fully characterized.

Methods and Findings

In the present study, we have demonstrated that the regions of APOBEC3F (A3F) that are required for its HIV-1-mediated binding and degradation are distinct from those reported for A3G. We found that the C-terminal cytidine deaminase domain (C-CDD) of A3F alone is sufficient for its interaction with HIV-1 Vif and its Vif-mediated degradation. We also observed that the domains of HIV-1 Vif that are uniquely required for its functional interaction with full-length A3F are also required for the degradation of the C-CDD of A3F; in contrast, those Vif domains that are uniquely required for functional interaction with A3G are not required for the degradation of the C-CDD of A3F. Interestingly, the HIV-1 Vif domains required for the degradation of A3F are also required for the degradation of A3C and A3DE. On the other hand, the Vif domains uniquely required for the degradation of A3G are dispensable for the degradation of cytidine deaminases A3C and A3DE.

Conclusions

Our data suggest that distinct regions of A3F and A3G are targeted by HIV-1 Vif molecules. However, HIV-1 Vif suppresses A3F, A3C, and A3DE through similar recognition determinants, which are conserved among Vif molecules from diverse HIV-1 strains. Mapping these determinants may be useful for the design of novel anti-HIV inhibitors.     

米根霉富马酸酶三维结构的同源建模与分析

利用Modeller7v7软件对米根霉（Rhizopus oryzoe）富马酸酶（fumarase）进行了三级结构的同源建模并对结果的空间和能量上的合理性进行了验证,进一步对酶的结构域和催化活性位点进行了研究。结果表明：富马酸酶由三个结构域组成,中心区域为一个由五个几乎平行的α螺旋组成的独特的束型结构,其催化活性位点是由三个亚基上的氨基酸相互靠近共同组成的。为以后有针对性的进行富马酸酶的定点突变提高富马酸产量提供分子水平上的理论指导。     

Molecular cloning of endo-beta-D-1,4-glucanase genes,rce1, rce2, and rce3, from Rhizopus oryzae

Three endoglucanase genes, designated the rce1, rce2, and rce3 genes, were isolated from Rhizopus oryzae as the first cellulase genes from the subdivision ZYGOMYCOTA: All the amino acid sequences deduced from the rce1, rce2, and rce3 genes consisted of three distinct domains: cellulose binding domains, linker domains, and catalytic domains belonging to glycosyl hydrolase family 45. The rce3 gene had two tandem repeated sequences of cellulose binding domains, while rce1 and rce2 had only one. rce1, rce2, and rce3 had various lengths of linker sequences.     

The Pre-S1 and Antigenic Loop Infectivity Determinants of the Hepatitis B Virus Envelope Proteins Are Functionally Independent

The hepatitis B virus (HBV) envelope proteins bear two determinants of viral entry: a receptor-binding site (RBS) in the pre-S1 domain of the large envelope protein and a conformation-dependent determinant, of unknown function, in the antigenic loop (AGL) of the small, middle, and large envelope proteins. Using an in vitro infection assay consisting of susceptible HepaRG cells and the hepatitis delta virus (HDV) as a surrogate of HBV, we first investigated whether subelements of the pre-S1 determinant (amino acids 2 to 75), i.e., the N-terminal myristoyl anchor, subdomain 2-48 (RBS), and subdomain 49-75, were functionally separable. In transcomplementation experiments, coexpression of two distinct infectivity-deficient pre-S1 mutants at the surface of HDV virions failed to restore infectivity, indicating that the myristoyl anchor, the 2-48 RBS, and the 49-75 sequence, likely cooperate in cis at viral entry. Furthermore, we showed that as much as 52% of total pre-S1 in the HDV envelope could bear infectivity-deficient lesions without affecting entry, indicating that a small number of pre-S1 polypeptides—estimated at three to four per virion—is sufficient for infectivity. We next investigated the AGL activity in the small or large envelope protein background (S- and L-AGL, respectively) and found that lesions in S-AGL were more deleterious to infectivity than in L-AGL, a difference that reflects the relative stoichiometry of the small and large envelope proteins in the viral envelope. Finally, we showed that C147S, an AGL infectivity-deficient substitution, exerted a dominant-negative effect on infectivity, likely reflecting an involvement of C147 in intermolecular disulfide bonds.Hepatitis B virus (HBV) remains a major public health concern worldwide, affecting more than 350 millions of chronically infected individuals. Since the discovery of HBV, substantial information has been gathered on the viral replication cycle, but our understanding of the viral entry mechanism remains limited, and the identity of the receptor(s) for HBV is still unknown (15). HBV displays a very narrow host range, which is likely determined at viral entry by a highly specific interaction between the HBV envelope proteins and receptors at the surface of human hepatocytes. The envelope proteins designated large (L-HBsAg), middle (M-HBsAg), and small (S-HBsAg) are membrane-spanning glycoproteins that differ from each other by the size of their N-terminal ectodomain (21). L-HBsAg contains a N-terminal pre-S1, central pre-S2, and C-terminal S domains. M-HBsAg is shorter than L-HBsAg in lacking pre-S1, whereas S-HBsAg consists of the S domain only (Fig. ​(Fig.1).1). Envelope protein synthesis occurs at the endoplasmic reticulum (ER) membrane. Empty subviral particles (SVPs) assemble from aggregates at a pre-Golgi membrane and exit the cell through the secretory pathway (36). Assembly of mature HBV virions requires, in addition to S-HBsAg, the presence of L-HBsAg as a matrix protein for nucleocapsid envelopment (6). Recent findings indicate that HBV virions and SVPs follow distinct pathways for budding: the late endosomal multivesicular bodies (MVBs) for HBV virions, and the MVB-independent secretory pathway for SVPs (26, 28, 46). The HBV envelope proteins can also package the hepatitis delta virus (HDV) ribonucleoprotein (RNP), in case of HBV/HDV coinfection (5, 45), leading to the formation of HDV virions. Whether HDV uses the SVP secretion pathway rather than an MVB-dependent route is uncertain.Open in a separate windowFIG. 1.Schematic representation of HBV envelope proteins. The topology of the L-, M-, and S-HBsAg proteins at the viral membrane is represented. The pre-S2 domain of L- and M-HBsAg, and the determinants of viral entry, pre-S1 and AGL, are indicated. The M-HBsAg protein, represented in gray, is dispensable for infectivity. The myristic acid (Myr) linked to the L-HBsAg N terminus is indicated (closed box). Subdomains 2-48 and 49-75 of the pre-S1 infectivity determinant are indicated. Open boxes represent transmembrane regions in the S domain.L-HBsAg, but not M-HBsAg, is crucial to infectivity of both HBV and HDV particles (13, 31, 41, 42). L-HBsAg contains a major infectivity determinant located between amino acid residues 2 and 75 of its N-terminal pre-S1 domain (4, 30), including a myristoyl anchor linked to glycine-2 (1, 8, 18), a putative receptor binding site (RBS) between positions 2 and 48, and a domain of unknown function between amino acids 49 and 75. To date, the most compelling evidence that pre-S1 mediates receptor binding comes from studies demonstrating that myristoylated synthetic peptides specific for the N-terminal 2-to-48 pre-S1 domain can bind to hepatocyte plasma membranes and block infection in vitro (3, 16, 17) and in vivo (37). Beside pre-S1, a second determinant was recently identified in the antigenic loop (AGL) borne by the three HBV envelope proteins (Fig. ​(Fig.1).1). The AGL participation in viral entry was first established in the HDV model (23) and more recently directly in the HBV model (39). Interestingly, serine substitutions for the AGL cysteine residues, which prove detrimental to the conserved immunodominant “a” determinant, could also block viral entry. Note that the “a” determinant consists in conformational epitopes, which elicit highly neutralizing antibodies (22). Infectivity and the “a” determinant were also lost when virions were treated with membrane-impermeable inhibitors of thiol/disulfide isomerization (2). These findings clearly established a correlation between the AGL cysteine disulfide bonds network, the conformation of the “a” determinant, and infectivity. Hence, the strict conservation of the “a” determinant among all HBV genotypes is related to the AGL function at viral entry. The AGL determinant may operate in association with, or independently of pre-S1, in binding to receptors at the early step of entry and/or in the mechanism of envelope disassembly postentry.In the present study, we investigated the pre-S1 determinant by performing transcomplementation experiments between mutants of 3 pre-S1 subelements: the myristoyl anchor, subdomain 2-48, and subdomain 49-75. We analyzed the activity of the AGL determinant in the S- or L-HBsAg background (S- and L-AGL, respectively), and we examined the effect of introducing increasing amounts of infectivity-deficient pre-S1, or AGL, in the virion''s envelope on infectivity.     

Strain improvement for tannase production from co-culture of Aspergillus foetidus and Rhizopus oryzae

Spores from the co-culture of Aspergillus foetidus and Rhizopus oryzae were subjected to UV, heat and NTG (3-nitro,5-methylguanidine) mutagenesis. A few colonies were screened from the selected media for tannase study. Amongst all, the best mutant isolated from the heat treatment (60 degrees C for 60 min) was SCPR 337. The maximum yield of gallic acid and tannase in case of mutant strain was 95.2% and 53.6 U/ml with an incubation period of 30 h as compared to wild strain where the incubation period was 48 h with an enzyme activity of 44.2 U/ml and gallic acid yield of 94%, respectively. The mutant was sensitive to tetracycline and was also an over-producer of protease and amylase.