We identified Thr130 within the replication-dependent histone H2A as a candidate phosphorylation site and found that the abundance of this trypanosomal γH2A increased in vivo in response to DNA damage. Nuclear γH2A foci mark the sites of putative natural replication fork stalling, sites of meganuclease-induced DNA double strand breaks and sites of methyl methanesulphonate-induced DNA damage. Naturally occurring and meganuclease-induced γH2A and RAD51 double-positive repair foci are typically found in S-phase or G2 nuclei. The results link trypanosomal γH2A, with an unusual histone modification motif, to DNA damage sensing and mitotic checkpoint signaling.DNA damage and repair in trypanosomatids impacts virulence, drug resistance and antigenic variation but, currently, little is known about DNA damage responses or cell cycle checkpoints in these divergent protozoa. One of the earliest markers of DNA damage in eukaryotes is γH2A(X), a serine phosphorylated histone H2A (variant). Here, we report the identification and initial characterization of γH2A in  The octameric nucleosomes are comprised of two units of each core histone, H2A, H2B, H3 and H4, and approximately 150 bp of DNA is wrapped on the outer face of each of these nucleosomes. Histone post-translational modification or replacement by histone variants can lead to the specialization of chromatin domains to facilitate processes such as DNA repair, chromosome segregation or transcription control. Thus, chromatin components and chromatin structure have a major impact on the DNA damage response through cell cycle checkpoint control and the recruitment of repair factors 139 phosphorylated version of the histone variant H2AX Drosophila melanogaster, the equivalent of γH2AX is a Ser137 phosphorylated version of H2AZ Saccharomyces cerevisiaeSchizosaccharomyces pombe128/129 phosphorylated version of the major, replication-dependent histone. Cells with H2AX mutations T. brucei γH2A.γH2AX in mammalian cells is a Ser22.1T. brucei Lister 427, MITat1.2 (clone 221a), bloodstream form cells were grown in HMI-11 and transformed as described T. brucei cultures at 0.0003% for 24 h. Phleomycin (Sigma) was applied at 1 μg ml−1 for 18 h. For meganuclease-induction, tetracycline (Sigma) was applied at 1 μg ml−1 for 12 h. RAD51, with Green Fluorescent Protein fused to the C-terminus, was expressed using the pNATRAD51-GFP construct, a derivative of pNATxGFPRAD51-GFP was linearized by digestion with NsiI prior to transfection. Oligonucleotide details are available on request.2.2T. brucei γH2A antibody was used at a 1:200 dilution and secondary goat anti-rabbit IgG HRP (Bio-Rad) was used at a 1:2000 dilution. For the peptide competition assay, primary antibody was pre-incubated with 40 ng ml−1 of the appropriate peptide in wash buffer for 1 h at room temperature prior to incubation with the immunoblot.Anti-γH2A antisera were raised in rabbits according to a 90-day protocol using a KLH-conjugated phospho-peptide, C-KHAKA[pT]PSV (Open BioSystems). Antiserum with an enzyme-linked immunosorbent assay (ELISA) titre of 1:204,800 was affinity purified using the corresponding peptides (Open Biosystems). Western blotting was carried out as described 2.3Immunofluorescence detection and imaging were carried out as described 33.1H2A gene are annotated; these are also on chromosome 7 but at a distal locus relative to H2AZ. All predicted T. brucei H2A protein sequences are identical in the genome reference strain C-terminus. Trypanosomatid histones are probably the most divergent eukaryotic histones known C-terminus of trypanosomal H2A  fused to the C-terminus. Cells with and without meganuclease-induced breaks were then examined for γH2A and RAD51 signals by microscopy. GFP. It was also noted that these signals were almost always at the edge of the DAPI-stained nuclear region  in eukaryotes, ranging from humans to yeast, is a serine-phosphorylated histone H2A variant or the major, replication-dependent histone H2A. The latter is the case in budding yeast and also appears to be the case in several protozoal species. In


Hepatitis E virus (HEV) is a causative agent of hepatitis E. Recently, a novel hepatitis E-like virus was isolated from Norway rats in Germany. However, the antigenicity, pathogenicity and epidemiology of this virus are unclear because of the lack of a cell-culture system in which to grow it. In this study, an N-terminally truncated ORF2 protein was expressed in insect Tn5 cells using a recombinant baculovirus expression system and a large amount of 53 kDa protein was expressed and efficiently released into the supernatant. Electron microscopic analyses of the purified 53 kDa protein revealed that the protein self-assembled into two types of empty HEV-like particles (rat HEVLPs). The smaller rat HEVLPs were estimated to be 24 nm in diameter, which is similar to the size of genotype G1, G3 and G4 HEVLPs. The larger rat HEVLPs were estimated to measure 35 nm in diameter, which is similar to the size of native rat HEV particles. An ELISA to detect antibodies was established using rat HEVLPs as the antigens, which demonstrated that rat HEVLPs were cross-reactive with G1, G3 and G4 HEVs. Detection of IgG and IgM antibodies was performed by examination of 139 serum samples from wild rats trapped in Vietnam, and it was found that 20.9 % (29/139) and 3.6 % (5/139) of the samples were positive for IgG and IgM, respectively. In addition, rat HEV RNA was detected in one rat serum sample that was positive for IgM. These results indicated that rat HEV is widespread and is transmitted among wild rats. Hepevirus in the family Hepeviridae (Hepatitis E virus (HEV) is the causative agent of hepatitis E, a viral disease that manifests as acute hepatitis . The diseviridae . ORF3, wTo date, at least four genotypes of HEV, G1–G4, have been isolated from humans, and G3 and G4 HEVs have been isolated from pigs, wild boar and wild deer  cells were infected at an m.o.i. of 10 with recombinant baculoviruses Ac[ORF2] and Ac[ΔORF2] containing the full-length and N-terminal 100 aa-deleted ORF2 of rat HEV, and the infected cells were incubated at 26.5 °C to express the full-length ORF2 and N-terminally truncated ORF2. The cells were harvested daily up to day 10 post-infection (p.i.), and the proteins generated in the infected cells and supernatant were analysed by Western blotting. In Ac[ORF2]-infected Tn5 cells, a protein band with a molecular mass of 69 kDa (p69) appeared at 2 days p.i., and reached a peak on day 3 p.i. The molecular mass of p69 was in agreement with that calculated for the full-length rat ORF2; however, p69 was not detected in the supernatant (data not shown).In the Ac[ΔORF2]-infected Tn5 cells, a major protein with a molecular mass of 58 kDa (p58) was detected in the cells on day 2 p.i., and expression levels reached a peak at day 4 p.i. . A prote−3 . These results indicated that the p53 protein self-assembled into VLPs and demonstrated that the ORF2 gene encoded the rat HEV capsid protein. No HEVLPs were obtained from either Ac[ΔORF2]-infected or Ac[ORF2]-infected Spodoptera frugiperda-derived (Sf9) cells (data not shown).The culture medium of Ac[ΔORF2]-infected Tn5 cells was harvested at 7 days p.i., and p53 was purified by CsCl-gradient centrifugation as described in Methods. The p53 protein was broadly distributed in fractions 5–20, but mainly in fractions 19 and 20 . However−3 . To iden−3 . The morThe rat HEV capsid protein p53 reacted with anti-G1 HEV antibody, as determined by Western blotting , which sA492) of the IgG of these serum samples were between 0.016 and 0.147, with a mean value±sd of 0.052±0.043. The cut-off value for IgG was set at 0.181, 3 sd above the mean A492 value . Among the 139 rat serum samples, 16 were from Rattus tanezumi and 123 from Rattus norvegicus; the IgG-positive rates were 25.0 % (4/16) and 20.3 % (25/123), respectively. The IgG-positive rates were not significantly different between R. tanezumi and R. norvegicus (P>0.05). These results suggested that rat HEV infection is widespread and that transmission is ongoing among wild rats in Vietnam.A total of 139 serum samples collected from wild rats in Vietnam were examined, and 20.9 % (29/139) of the samples were found to be positive for IgG antibody, whereas 3.6 % (5/139) were positive for IgM antibody . All fivThe IgM-positive serum samples were selected to detect rat HEV RNA using a nested broad-spectrum RT-PCR, and one serum sample was found to be positive for rat HEV. A total of 901 nt corresponding to nt 4108–5008 of the rat HEV genome (GenBank accession no. GU345042) comprising the C terminal ORF1 (814 nt), the junction region (27 nt) and the N terminal ORF2 (60 nt) were compared with the corresponding sequences of other HEVs. A phylogenetic analysis based on these 901 nt indicated that the Vietnamese rat strain formed a cluster with other rat HEVs . This stRat HEV is a new genotype of HEV, and nucleotide sequence identities with HEV G1–4 were 55.1–55.9 %. Because no cell-culture system has yet been developed for rat HEV, it remains necessary to express the capsid protein and generate VLPs in order to analyse the antigenicity and immunogenicity of this pathogen; these recombinant molecules are also extremely useful for seroepidemiological studies of rat HEV infection in wild rats.For the production of VLPs, the full-length rat HEV ORF2 was initially expressed by a recombinant baculovirus; however, the recombinant protein derived from this gene was not released into the culture supernatant and did not form VLPs. In the case of G1, G3 and G4 HEVs, an N terminal 111 aa-deleted ORF2 protein has been found to be released efficiently into the supernatant and to self-assemble into VLPs. Therefore, we employed the same strategy for the current analysis. When the deduced amino acid sequence of the rat HEV ORF2 (GenBank accession no. GU345042) was aligned with that of representatives of HEV G1, G3 and G4 , we found that aa 101 in the rat HEV ORF2 corresponded to aa 112 in the G1, G3 and G4 HEV ORF2. Therefore, we expressed the N-terminal 100 aa-deleted rat HEV ORF2 using a recombinant baculovirus expression system. As expected, the recombinant protein, p53, was released into the supernatant and formed VLPs . Deletio−3  domain, the middle (M) domain and the protruding (P) domain. The outer surface of the particles, which is a target for antibodies, is formed primarily by the M and P domains . The full-length ORF2 was then was cloned into the vector pUC57 (GeneScript) to generate the plasmid pUC57-rat-ORF2. A DNA fragment encoding the N-terminal 100 aa-truncated rat HEV ORF2 was amplified by PCR using pUC57-rat-ORF2 with forward primer rat-E-F2 (5′-AAGGATCCATGGCACAGGCACCGGCGCCTA-3′) and reverse primer rat-E-R1 (5′-ATCTAGATCAGACACTATCGGCGGCTGCTG-3′). The amplified DNA fragment was purified using a PCR purification kit (Qiagen). The full-length and N-terminal 100 aa-truncated ORF2 were digested with BamHI and XbaI and ligated into a baculovirus transfer vector, pVL1393 (Pharmingen), to yield plasmids pVL1393-ORF2 and pVL1393-ΔORF2, respectively.The full-length ORF2 of rat HEV containing a Autographa californica nuclear polyhedrosis virus DNA  and either pVL1393-ORF2 or pVL1393-ΔORF2 by a Lipofectin-mediated method as specified by the manufacturer (Gibco-BRL). The cells were incubated at 26.5 °C in TC-100 medium (Gibco-BRL) supplemented with 8 % FBS and 0.26 % bactotryptose phosphate broth (Difco Laboratories). The recombinant virus was plaque purified three times in Sf9 cells and designated Ac[ORF2] and Ac[ΔORF2], respectively. To achieve large-scale expression, an insect cell line from Trichoplusia ni, Tn5 (Invitrogen), was used (Sf9 cells (RIKEN Cell Bank) were co-transfected with linearized wild-type was used . Tn5 celwas used . VLPs ofp-toluidine were used for detection of antibody binding (Bio-Rad Laboratories).The proteins in the cell lysates and culture medium were separated by SDS-PAGE with a 5–20 % acrylamide gradient gel and stained with Coomassie blue. For Western blot analysis, proteins in the gel were electrophoretically transferred onto a nitrocellulose membrane. The membrane was then soaked with 5 % skimmed milk in 50 mM Tris/HCl (pH 7.4), 150 mM NaCl, and incubated with a rabbit anti-G1 HEVLP polyclonal antibody as described previously  gradient and centrifuged at 32 000 r.p.m. for 2 h in a Beckman SW55Ti rotor. For CsCl-gradient centrifugation, 4.5 ml of each sample was mixed with 2.1 g CsCl and centrifuged at 35 000 r.p.m. for 24 h at 10 °C in the same rotor. The gradient was fractionated into 250 µl aliquots, and each fraction was weighed to estimate the buoyant density and isopycnic point. Each fraction was diluted with EX-CELL 405 medium and centrifuged for 2 h at 50 000 r.p.m. in a Beckman TLA55 rotor to sediment the HEVLPs.Recombinant baculovirus-infected Tn5 cells were harvested on day 7 p.i. The intact cells, cell debris and progeny baculoviruses were removed by centrifugation at 10 000 Purified HEVLPs were placed on a carbon-coated grid for 45 s, rinsed with distilled water, stained with a 2 % uranyl acetate solution and examined under a JEOL TEM-1400 electron microscope operating at 80 kV.The proteins separated by SDS-PAGE were visualized by staining with GelCode Blue Staining Reagent (Pierce) and purified by sucrose-gradient centrifugation. N-terminal amino acid microsequencing was carried out using 100 pmol protein by Edman automated degradation on an Applied Biosystems Model 477 Protein Sequencer.Rabbits were immunized with rat, G1, G3 and G4 HEVLPs. Immunization was performed by one percutaneous injection of purified HEVLPs with a dose of 500 µg per rabbit. Rats were immunized with the recombinant rat HEVLPs by intramuscular injection at a dose of 200 µg per rat, and booster injections were carried out at 4 and 6 weeks after the first injection with half doses of rat HEVLPs. All of the injections, including booster injections, were carried out without adjuvant. Immunized animals were bled 3 weeks after the last injection.R. norvegicus and 16 were identified as R. tanezumi. All of the serum samples were stored at −80 °C until use.A total of 130 serum samples from laboratory rats  were collected at the Division for Experimental Animal Research of the National Institute of Infectious Diseases of Japan. A total of 139 serum samples from wild rats were collected in Vietnam . With regard to the rat species sampled, 123 were identified as −1, 100 µl per well) and incubated overnight at 4 °C. Unbound HEVLPs were removed and the plates washed twice with 10 mM PBS containing 0.05 % Tween 20 (PBS-T) and then blocked with 200 µl 5 % skimmed milk (Difco Laboratories) dissolved in PBS-T for 1 h at 37 °C. After the plates had been washed four times with PBS-T, diluted rat serum samples (100 µl per well) were added in duplicate. The plates were incubated at 37 °C for 1 h and then washed three times as described above. The wells were incubated with 100 µl HRP-conjugated goat anti-rat IgG (H+L)  or HRP-conjugated goat anti-rat IgM  diluted in PBS-T containing 1 % skimmed milk. The plates were incubated at 37 °C for 1 h and washed four times with PBS-T. One hundred microlitres of substrate orthophenylenediamine  and 5 µl H2O2 (30 % in 12.5 ml substrate buffer) were added to each well. The plates were incubated in a dark room at room temperature for 30 min, and then 50 µl 2 M H2SO4 was added to each well. Absorbance was measured at 492 nm. The cut-off values for IgG and IgM were determined as described previously  were coated with the purified rat HEVLPs (1 µg ml− reverse transcriptase (Invitrogen), 1 µl oligo(dT) primer, 1 µl RNaseOUT (Invitrogen), 2 µl 0.1 M DTT, 4 µl 5× RT buffer (Invitrogen), 1 µl 10 mM dNTPs, 5 µl RNA and 5 µ1 distilled water.Total RNA was extracted using a QIAamp viral RNA mini kit (Qiagen) and resuspended in 20 µl DNase-, RNase- and proteinase-free water. Reverse transcription was performed at 42 °C for 50 min, followed by 70 °C for 15 min in a 20 µl reaction mixture containing 1 µl Superscript II RNase HA nested broad-spectrum RT-PCR analysis was performed to amplify a portion of ORF1, based on a method described previously with slight modifications . The reverse primers were designed according to the German rat HEV strain (GenBank accession no. GU354042). The first PCR analysis was carried out in a 50 µl volume reaction mixture with an external forward primer, rat-HEV-F10 , and an external reverse primer, rat-HEV-R7 . Each cycle consisted of 95 °C for 30 s, 55 °C for 60 s and 72 °C for 4 min, followed by 72 °C for 7 min. Two microlitres of the first PCR product was used for the nested PCR with an internal forward primer, HEV-F11 , and an internal reverse primer, rat-HEV-R9 . Each cycle consisted of 95 °C for 30 s, 55 °C for 45 s and 72 °C for 60 s, followed by 72 °C for 7 min.Nucleotide sequencing of the PCR products was carried out using primers HEV-csn, HEV-casn, HEV-F11 and HEV-R9 on an ABI 3130 Genetic Analyzer automated sequencer (Applied Biosystems) and a BigDye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems) according to the manufacturer’s instructions.R. norvegicus and R. tanezumi were performed with an unpaired Student’s t-test.Comparisons of the rate of positivity between different areas and between


KCNE1, a small transmembrane protein, can coassemble with hERG. However, it is not known how KCNE1 variants influence the channel's response to premature stimuli or if they influence the sensitivity of hERG to pharmacological inhibition. Accordingly, whole-cell patch-clamp measurements of hERG current (IhERG) were made at 37°C from hERG channels coexpressed with either wild-type (WT) KCNE1 or with one of three KCNE1 variants . Under both conventional voltage clamp and ventricular action potential (AP) clamp, the amplitude of IhERG was smaller for A8V, D76N, and D85N KCNE1 + hERG than for WT KCNE1 + hERG. Using paired AP commands, with the second AP waveform applied at varying time intervals following the first to mimic premature ventricular excitation, the response of IhERG carried by each KCNE1 variant was reduced compared to that with WT KCNE1 + hERG. The IhERG blocking potency of the antiarrhythmic drug quinidine was similar between WT KCNE1 and the three KCNE1 variants. However, the IhERG inhibitory potency of the antibiotic clarithromycin and of the prokinetic drug cisapride was altered by KCNE1 variants. These results demonstrate that naturally occurring KCNE1 variants can reduce the response of hERG channels to premature excitation and also alter the sensitivity of hERG channels to inhibition by some drugs linked to acquired LQTS. IKr) contributes significantly to ventricular action potential (AP) repolarization and to setting the duration of the QT interval of the ECG  contributes to net membrane conductance for some time following complete AP repolarization, with the consequence that the channel can act to offset potentially arrhythmogenic premature depolarizations early in diastole  drugs and also for a wide range of noncardiac drugs linked to the acquired (drug-induced) form of the LQTS. Consequently, drugs in development are routinely tested for their ability to inhibit IKr/hERG (Witchel and Hancox The rapid delayed rectifier potassium current (IhERG) at physiological temperature that is similar to IKr  KCNE1   is a 129 amino acid protein that is best known as the i et al.  providedIKr/hERG in vivo and in vitro, at present there is no information as to whether or not KCNE1 variants can influence the protective role of IKr/hERG against arrhythmogenic premature stimulation. Neither it is known whether KCNE1 variants exert a direct influence on the sensitivity of hERG to pharmacological blockade. This study was undertaken to address these issues, by comparing IhERG elicited when hERG was expressed with three naturally occurring KCNE1 variants  with that with WT KCNE1, using a combination of conventional voltage and AP voltage clamp measurements at physiological temperature.Despite evidence that KCNE1 may influence The common S38 KCNE1 polymorphism (in pCR3.1) was kindly donated by Dr. F. Toyoda . The more usual G38 KCNE1 WT variant was generated from this using QuickChange® site-directed mutagenesis . This construct was then used as WT-KCNE1 and was also used as the template for construction of the A8V, D75N, and D85N KCNE1 mutations. For each variant, a pair of complementary oligonucleotide primers  and 100 μg/mL geneticin , and kept in a 5% CO2/95% O2 incubator at 37°C. Cells were passaged using enzyme free cell dissociation solution  and plated in 40-mm petri dishes containing DMEM medium supplemented with 10% fetal bovine serum and 50 μg/mL gentamycin. Cells at >80% confluence were transiently transfected with 0.5 μg of KCNE1 plasmids and 1 μg of green fluorescent protein  using Lipofectamine 2000  according to the manufacturer's instructions. Cells were incubated at 37°C for at least 12 h prior to electrophysiological recording.Human embryonic kidney (HEK 293) cells stably expressing hERG channels  were maintained in Dulbecco's minimum essential medium  supplemented with 10% fetal bovine serum , 50 2, 1 MgCl2, 10 Glucose, and 5 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (titrated to pH of 7.45 with NaOH). The pipette solution contained (in mmol/L): 130 KCl, 1 MgCl2, 5 EGTA, 5 MgATP, and 10 HEPES (titrated to pH of 7.2 with KOH). Patch pipette resistance ranged from 2 to 4 MΩ. Series resistance values lay between 3 and 7 MΩ and were compensated by 60–80%. Data digitization rates were 10–25 kHz with an appropriate bandwidth of 2–10 kHz set on the amplifier. The ventricular AP waveforms for single and paired AP clamp experiments were identical to those used in previous investigations from our group , cisapride (monohydrate), and clarithromycin were obtained from Sigma-Aldrich  and were dissolved in deionized water (quinidine) and dimethyl sulfoxide  to give an initial stock solution of 100 mmol/L, 10 mmol/L, and 50 mmol/L, respectively. Stock solutions were diluted appropriately and aliquots of drug stock solutions were added to Tyrode's solution to produce the final concentrations mentioned in the Results section. The final DMSO concentration in test solutions (for cisapride and clarithromycin) was 0.1% in experiments on WT KCNE1 and on the different KCNE1 variants. DMSO at concentrations up to 1% has been demonstrated previously not to affect significantly the potency of hERG blocking drugs , Excel 2007, Origin 7, and Prism v3  software. Data are presented as the mean ± standard error of the mean (SEM). For concentration–response relations, SEM values presented in the Results represent SEM values for the fit to the mean plotted data. Statistical comparisons were made using one-way or two-way analyses of variance (ANOVA) followed by Bonferroni's post hoc comparisons tests as appropriate. IhERG activation was fitted with a Boltzmann equation of the form:Mathematical fits to the data were as follows: The voltage dependence of I is the peak IhERG tail current amplitude at the test voltage Vm; Imax is the maximal tail current observed; V0.5 is the voltage at which IhERG was half-maximally activated; and k is the slope factor for the fitted relationship.where IhERG inactivation was fitted with a modified Boltzmann equation:The voltage dependence of V0.5 is the half-maximal inactivation voltage and other terms have same meaning as for equation where IhERG recovery from inactivation was fitted with a one-phase exponential association:The time course of y is the current amplitude at time x; ymax is the maximal IhERG transient observed; and τ presents the time constant of IhERG recovery from inactivation.where The fractional block of hERG tail current by drugs was determined using an equation of the form:IhERG(Control) is the amplitude of Itail in control and IhERG(Drug) is the Itail amplitude in the presence of the drug.where IhERG fractional block was determined by fitting data with a Hill equation of the form:The relationship between drug concentration and y is fractional block at a given concentration  of Drug ([Drug]), IC50 is the Drug concentration producing half-maximal inhibition of IhERG, and h is the Hill coefficient for the fit to the plotted data. No more than two drug concentrations were studied in a single experiment and consequently fits with equation where IhERG amplitude under AP clamp  studied, the response was smaller than that for WT KCNE1 or hERG alone. Mean data for peak IhERG density during repolarization are shown in Figure IhERG magnitude by each KCNE1 mutation compared to WT KCNE1 or hERG alone.The effect of WT KCNE1 and KCNE1 variants on IhERG currents  for IhERG with WT KCNE1 and the three variants studied, elicited by conventional voltage clamp (protocol shown in lower traces). Similar to the data with AP clamp, current magnitude was suppressed by the A8V, D76N, and D85N KCNE1 variants. Figure I–V) relations for end pulse current  for WT KCNE1-hERG, −22.64 ± 1.13 mV  for A8V KCNE1-hERG, −25.94 ± 1.63 mV  for D76N KCNE1-hERG, and −22.33 ± 1.99 mV  for D85N KCNE1-hERG. There was no significant difference for the V0.5 and k values among these four groups. However, these values exhibited a modest negative shift compared to that for WT IhERG alone (V0.5 of −16.06 ± 0.97 mV and k of 6.23 ± 0.23). Our data for hERG expressed alone compared to that with hERG + WT KCNE1 coexpression in respect of current amplitude and activation V0.5 shift are consistent with prior findings under conventional voltage clamp  data were then plotted as shown in Figure V0.5 of −60.75 ± 2.19 mV  for WT KCNE1-hERG, −60.06 ± 1.21 mV  for A8V KCNE1-hERG (V0.5P > 0.05 vs. WT), −69.53 ± 1.48 mV  for D76N KCNE1-hERG (V0.5P < 0.01 vs. WT and vs. A8V), and −64.93 ±1.24 mV  for D85N KCNE1-hERG (V0.5P > 0.05 vs. WT). Thus, the inactivation curve was negatively shifted for D76N KCNE1 compared with WT KCNE1 and with A8V KCNE1. The time course of development of inactivation was compared between WT KCNE1 and the three other variants by monoexponential fitting of the decline of the IhERG elicited by the third step following a repolarization voltage of −120 mV. The time constant of IhERG inactivation at −120 mV was 1.15 ± 0.12 msec for WT KCNE1-hERG, 1.29 ± 0.09 msec for A8V KCNE1-hERG, 1.64 ± 0.16 msec for D76N KCNE1-hERG, and 1.19 ± 0.10 for D85N KCNE1-hERG. Though inactivation of D76N KCNE1-hERG appeared to develop slightly slower than that for the other variants, there was no significant difference in the time constant among the four variants (P > 0.05).Figure IhERG from inactivation was evaluated with the protocol shown in the inset of Figure IhERG transient records for WT KCNE1 and A8V KCNE1 are shown in Figure IhERG recovery following repolarizing steps of different durations was measured and normalized to the maximal transient current observed during the protocol and was plotted against the corresponding duration of the repolarization step  for WT KCNE1-hERG, 1.68 ± 0.09 msec (n = 7 cells) for A8V KCNE1-hERG, 2.23 ± 0.16 msec (n = 8 cells) for D76N KCNE1-hERG, and 2.03 ± 0.08 mV (n = 7 cells) for D85N KCNE1-hERG. The recovery from inactivation of IhERG with D76N KCNE1 was slower than that with A8V KCNE1 (P < 0.05), but not than that with WT KCNE1.The time course of recovery of IhERG to premature stimuli is shown in Figure IhERG transients prior to a sustained current component during the AP. The magnitude of the response first increased as the second stimulus was applied closer to 90% repolarization of the initial AP (APD90), with a time-dependent profile similar to that reported previously  for WT KCNE1-hERG, 0.47 ± 0.10 μmol/L (h = 0.69 ± 0.09) for A8V KCNE1-hERG, 0.44 ± 0.11 μmol/L (h = 0.73 ± 0.12) for D76N KCNE1-hERG, and 0.46 ± 0.10 μmol/L (h = 0.69 ± 0.09) for D85N KCNE1-hERG.The IhERG blocking effect of the macrolide antibiotic clarithromycin has been shown to differ between WT and mutant KCNE2  and of 80.26 ± 9.20 μmol/L (h = 1.15 ± 0.12) for A8V KCNE1. Thus, the A8V mutation decreased sensitivity of IhERG to clarithromycin in comparison with that of WT KCNE1. Figure IhERG inhibitory effect of clarithromycin .The IhERG records with WT and A8V KCNE1 in the absence and presence of 100 nmol/L cisapride. The drug's action was significantly greater for A8V than WT-KCNE1 IhERG. Figure IhERG blocking potency of cisapride was enhanced for the mutant KCNE1 variants compared to WT KCNE1. The IC50 values derived from the concentration–response relations for cisapride's action were 59.27 ± 14.86 nmol/L (h = 0.78 ± 0.14) for WT KCNE1-hERG, 28.13 ± 8.17 nmol/L (h = 0.81 ± 0.17) for A8V KCNE1-hERG, 29.08 ± 10.07 nmol/L (h = 1.01± 0.28) for D76N KCNE1-hERG, and 25.53 ±6.87 nmol/L (h = 1.12 ± 0.25) for D85N KCNE1-hERG.Cisapride is a gastric prokinetic drug that has been withdrawn from use due to acquired LQTS and associated torsades de pointes (TdP) arrhythmia Henney . It is aIhERG amplitude compared to that with WT KCNE1, with only modest accompanying changes in IhERG kinetics; (2) that the three KCNE1 variants studied here suppressed the IhERG response to premature stimuli compared to the response with WT KCNE1; and (3) the sensitivity of IhERG to pharmacological inhibition by cisapride and clarithromycin, but not quinidine, differed between variants and WT KCNE1 expression conditions.The principal findings of this study are as follows: (1) that two KCNE1 variants associated with LQTS (A8V and D76N) and the D85N KCNE1 polymorphism decreased IhERG by WT KCNE1, McDonald et al. , while leftward shifted IhERG activation compared to hERG expressed alone was observed for both WT KCNE1 and for the three variant KCNE1 forms studied. Thus, alterations to macroscopic IhERG kinetics are unlikely to be primarily responsible for the differences in IhERG density shown in Figure In the first report of the functional modulation of d et al.  found a RG Figs. –4 showedIhERG density. To our knowledge, there is also a lack of information at this time regarding effects of KCNE1 variants on single-channel conductance of hERG. Further work is now warranted to determine whether either of these factors contribute to effects of KCNE1 variants on IhERG magnitude, or whether membrane hERG protein and single-channel conductance are unaltered, and instead KCNE1 variants alter the relative proportions of active and dormant hERG channels in the membrane compared to WT KCNE1  during which IhERG/IKr would provide reduced protection from unwanted premature excitation.hERG channels are well established to remain available early in diastole, due to rapid recovery from inactivation and comparatively slow deactivation, and can generate outward macroscopic nts Fig. . As IhERTo our knowledge, this is the first study to demonstrate a direct effect of KCNE1 variants on the sensitivity of hERG channels to pharmacological blockade. Questions arising from our observations are how KCNE1 exerts its modulatory effect on hERG drug sensitivity and how the responses vary between the drugs tested and between different KCNE1 variants?IhERG block through binding within the channel's inner cavity  with both reduced current amplitude , D85N KCNE1 may influence acquired LQTS susceptibility to at least some drugs through a direct effect on hERG/IKr inhibition. Accordingly, future investigations should determine the effects of this polymorphism on IhERG sensitivity to a wide range of drugs associated with acquired LQTS.It is widely recognized that only a small proportion of people who receive drugs associated with acquired LQTS develop significantly prolonged QTe” Roden . Our finta Figs. –8 sugges


Previously, we showed that MGO-mediated modification of αA-crystallin increased its chaperone function. We identified MGO-modified arginine residues in αA-crystallin and found that replacing such arginine residues with alanine residues mimicked the effects of MGO on the chaperone function. Arginine 12 (R12) is a conserved amino acid residue in Hsp27 as well as αA- and αB-crystallin. When treated with MGO at or near physiological concentrations (2–10 µM), R12 was modified to hydroimidazolone in all three small heat shock proteins. In this study, we determined the effect of arginine substitution with alanine at position 12 (R12A to mimic MGO modification) on the structure and chaperone function of these proteins. Among the three proteins, the R12A mutation improved the chaperone function of only αA-crystallin. This enhancement in the chaperone function was accompanied by subtle changes in the tertiary structure, which increased the thermodynamic stability of αA-crystallin. This mutation induced the exposure of additional client protein binding sites on αA-crystallin. Altogether, our data suggest that MGO-modification of the conserved R12 in αA-crystallin to hydroimidazolone may play an important role in reducing protein aggregation in the lens during aging and cataract formation.Methylglyoxal (MGO) is an α-dicarbonyl compound present ubiquitously in the human body. MGO reacts with arginine residues in proteins and forms adducts such as hydroimidazolone and argpyrimidine  Small heat shock proteins are a family of stress proteins. α-Crystallin and Hsp27 are the major small heat shock proteins in humans. These proteins are beneficial in preventing cellular damage for various diseases α-Crystallin is a major protein of vertebrate eye lenses, although its presence in other organs such as the brain, heart, kidney, spleen and thymus has also been recognized A large number of advanced glycation end products (AGEs) can be found in the aged human lens The lens contains relatively high levels of methylglyoxal (MGO). The reported levels are 1–2 µM In general, it is believed that AGE formation is a cause for lens protein aging and cataract formation. However, we and others have observed that MGO-AGE formation in αA-crystallin makes it a better chaperone Unlike α-crystallin, Hsp27 is ubiquitously expressed throughout the human body. We have shown that Hsp27 is particularly vulnerable to MGO modification in kidney mesangial cells Altogether, it is clear now that MGO modification of the small heat shock proteins results in an improvement in their key functions. Whether the improvement in the chaperone function of small heat shock proteins occurs via modification of a conserved arginine residue and whether physiological levels of MGO could improve the chaperone function through a hydroimidazolone modification is not known. In this study, we modified human Hsp27 and αA- and αB-crystallin with 2–10 µM MGO and identified hydroimidazolone AGEs using mass spectrometry. Interestingly, the only conserved arginine residue that was modified to hydroimidazolone by MGO was R12 in all three proteins. To determine if the hydroimidazolone modification of this arginine residue is responsible for the improvement of the chaperone function, we replaced R12 with alanine (to mimic the hydroimidazolone modification) and explored the effect of this mutation on the structure and chaperone function of Hsp27 and αA- and αB-crystallin.in vivoMGO is derived mostly from triose phosphate intermediates of glycolysis by non-enzymatic mechanisms We first determined the “first hit” arginine residues for modification to hydroimidazolone. To accomplish this, we modified the proteins with 2, 5 and 10 µM of MGO. With 2 µM MGO, we found that 6, 6 and 8 arginine residues were modified to hydroimidazolone in αA- and αB-crystallin and Hsp27, respectively . With 10R12A, αA-crystallinR12A (αAR12A) and αB-crystallinR12A (αBR12A) mutant proteins in E. coli BL21(DE3). We then purified the proteins by sequential chromatographic methods (gel filtration and ion-exchange chromatography), as previously described The modification of arginine residues to hydroimidazolone converts the positive charge on arginine to a neutral charge. Previously, we reported that the substitution of MGO-modifiable arginine residues with neutral alanine residues enhanced the chaperone function of αA-crystallin, similarly to MGO-modification R12A mutant showed a 61%, 15% and 10% increase in the chaperone function compared to the Wt protein with CS, γ-crystallin and LDH, respectively, as client proteins , it showed a slight reduction in the chaperone function against the other two client proteins tested  were nearly identical to that of their Wt protein counterparts. Hsp27R12A had a decreased (∼65%) surface hydrophobicity compared to its Wt counterpart (To understand the molecular basis behind the enhancement in the chaperone function of αAnterpart . These rd) using the Scatchard equation:n is the number of binding site and Kd is the dissociation constant. The stoichiometry of n and Kd obtained from the Scatchard plot (n) per subunit of αAR12A increased from 3.61 in Wt to 4.68, and the association constant increased from 0.118 µM−1 (Kd = 8.44 µM) in Wt to 0.124 µM−1 (Kd = 8.07 µM) in αAR12A. The n and Kd values decreased ∼25% and ∼13%, respectively, in αBR12A. In contrast, Hsp27R12A showed no changes in either of these parameters when compared to its Wt counterpart  for 1 hr at 60°C with various concentrations of γ-crystallin (2–18 µM). The unbound (S) and bound γ-crystallin were determined by filtration, as described in ard plot  is 3.61 ard plot . We notenterpart . From thR12A, αBR12A and Hsp27R12A increased ∼27%, 8% and 10%, respectively, compared to the corresponding Wt proteins. Moreover, the λmax of the tryptophan fluorescence spectra of the wild-type proteins did not alter due to the mutation. The changes in fluorescence intensity may reflect changes in the microenvironment of W9  and W16 (in Hsp27), which are located close to the mutation sites. The near-UV CD spectra of these three proteins (both wild type and mutant) agreed with our intrinsic fluorescence data (data not shown). However, these changes in tryptophan fluorescence (perturbation in tertiary structure) did not correlate with the changes in the chaperone function. While some studies showed a direct relationship between an increase in tryptophan fluorescence with improved chaperone function, others did not find such a relationship We used tryptophan (W) fluorescence along with near- and far-UV CD techniques to determine if there were any changes in the tertiary and secondary structures of the Wt proteins compared to the mutant proteins. The intrinsic fluorescence spectra indicated some differences between Wt and mutant proteins . The fluQuantitative analysis of the far-UV CD data using the CONTINLL program showed that Hsp27 and αA- and αB-crystallin are major β-sheet proteins . The dati.e., the oligomeric assembly) of these three small heat shock proteins. From the data in h) was slightly increased only with αAR12A  from the sigmoidal analysis of the denaturation profiles revealed that the C1/2 value increased from 2.34 M of urea for Wt αA-crystallin to 2.72 M of urea for the R12A mutant  at a urea concentration of zero. The fitted parameters are listed in 0 is comparable to that we and others have previously reported 0value for αAR12A increased to 25.54 kJ/mol, indicating an enhancement in thermodynamic stability by ∼4.5 kJ/mol. However, the R12A substitution had no effect on the structural stability of the other two small heat shock proteins  in all three small heat shock proteins, this modification is likely beneficial only for αA-crystallin. Because αA-crystallin is predominantly found in the eye lens, MGO-induced enhancement in the chaperone function of this protein may be important in maintaining the transparency of the lens.Citrate synthase (CS), lactate dehydrogenase (LDH), dithiothreitol (DTT), lysozyme and bovine insulin were obtained from Sigma-Aldrich Chemical Co., LLC . CS was dialyzed in 40 mM HEPES buffer, pH 7.4, for 24 hr before use. 2-(p-toluidino) naphthalene-6-sulfonate (TNS) was obtained from Molecular Probes . Bovine γ-crystallin were purified from lenses, as previously described Small heat shock proteins  were incubated with either 2, 5 or 10 µM MGO  for 3 days at 37°C. The incubated samples were subjected to SDS-PAGE (12% gel) under reducing conditions.Gel pieces containing αA-crystallin, αB-crystallin and Hsp27 cut from the SDS-PAGE were first destained with 50% acetonitrile in 100 mM ammonium bicarbonate, and then 100% acetonitrile. Then, the proteins were reduced by 20 mM DTT at room temperature for 60 min, followed by the alkylation of the proteins using 50 mM iodoacetamide for 30 min in the dark. The reaction reagents were removed, and the gel pieces were washed with 100 mM ammonium bicarbonate and dehydrated in acetonitrile. Sequencing grade modified trypsin  in 50 mM ammonium bicarbonate was added to the dried gel pieces and incubated at 37°C overnight. Proteolytic peptides extracted from the gel with 50% acetonitrile in 5% formic acid were then dried and dissolved in 0.1% formic acid. Liquid chromatography-tandem mass spectrometric analysis of the resulting peptides was performed on a LTQ Orbitrap Velos  equipped with nanoACQUITY UPLC  system. The spectra were acquired by data dependent methods consisting of a full scan and MS/MS on the ten most abundant precursor ions at the normalized collision energy of 35%. The data that were obtained were submitted to Mascot Daemon  for identification of the hydroimidazolone modification on arginine residues. The modification sites were then verified by manual interpretation of the MS/MS spectra.The previously described constructs for Wt αA- and αB-crystallin were used as templates 5-GGCCATATGGACGTGACCATCCAGCACαA FP: 3-CCCAAGCTTGGACGAGGGAGCCGAGGTGαARP: 5-GGCCATATGGACATCGCCATCCACCACαB FP: 3-CCCAAGCTTTTTCTTGGGGGCTGCGGTGACαB RP: 5- GGCCATATGACCGAGCGCCGCGTCCCCTTCTCGHsp27 FP: 3- CCCAAGCTTTTACTTGGCGGCAGTCTCATCGGATTTHsp27 RP: The amplified PCR product was cloned into the pET23a vector using NdeI and HindIII restriction sites. The R12A mutants were generated by site-directed mutagenesis using the respective Wt cDNA as the template. The following primers were used.R12AFP:5-TGGTTCAAGGCCACCCTGGGGαAR12ARP:3-CCCCAGGGTGGCCTTGAACAAαAR12AFP:5-TGGATCCGCGCCCCCTTCTTTαBR12ARP:3-AAAGAAGGGGGCGCGGATCCAαBR12A FP:5-TCGCTCCTGCGGGGCCCCAGCHsp27R12A RP:3-GCTGGGGCCCCGCAGGAGCGAHsp27E.coli DH5alpha cells. Plasmids from the resulting colonies were sequenced to confirm the presence of the mutation. The recombinant proteins were overexpressed in E.coli BL21(DE3) by induction with 250 µM IPTG when the OD600 nm of the culture in LB broth reached ∼0.6. The bacterial pellet obtained after centrifugation at 10,000 g was suspended in 50 mM Tris, pH 8.0 containing 50 mM NaCl, 2 mM EDTA and 10 µl/ml of a protease inhibitor cocktail (Sigma). Lysozyme was added at 0.3 mg/ml to the cell suspension and incubated for 10 min at 37°C, followed by sonication on ice at 40 duty cycles at 30% amplitude. Benzonase nuclease (1.0 µl) was then added to the resulting cell lysate and incubated at 37°C in a shaker for 20 min, which was followed by the addition of sodium deoxycholate at 1.0 mg/ml and a subsequent incubation for 10 min at 37°C. DTT was then added to the lysate at a 5 mM concentration and incubated for 10 min at 37°C. The cell lysate was then centrifuged at 20,000 g for 30 min at 4°C. DNA in the lysate was precipitated by adding 0.2% polyethyleneimine followed by centrifugation at 20,000 g for 15 min. Ammonium sulfate was added to the lysate to reach 70% saturation, and the suspension was then left at 4°C overnight and then centrifuged at 20,000 g for 5 min. The resulting pellet was suspended in 50 mM sodium phosphate buffer (pH 7.4), which contained 150 mM NaCl and 5 mM DTT, and was then centrifuged at 20,000 g for 5 min. The supernatant was filtered with a 0.45 µm filter and applied onto a Superdex-200 prep grade  gel filtration column that was pre-equilibrated with 50 mM sodium phosphate buffer pH 7.4. Fractions of 2.0 ml were collected and their OD280 nm was recorded. The peak fractions were pooled and dialyzed overnight at 4°Cin 20 mM Tris, pH 8.0 that contained 0.1 mM EDTA. The dialyzed sample was applied onto a Q-Sepharose  anion exchange column equilibrated with 20 mM Tris, pH 8.0 with 0.1 mM EDTA. The bound protein was eluted with a 0–1 M NaCl gradient. The protein peak fractions were pooled and dialyzed in PBS containing 0.1 mM EDTA and stored in aliquots at −80°C.The resulting PCR product was digested with DpnI and then transformed into w) and the hydrodynamic radius (Rh) of Wt and mutant proteins were determined using ASTRA (5.3.4) software developed by Wyatt Technology Corp.The molar mass and the hydrodynamic radius of wild-type and R12A mutants of α-crystallin and Hsp27 were estimated by multi-angle light scattering measurements as previously described The far-UV CD spectra were measured at 25°C using a Jasco 815 spectropolarimeter . The spectra were collected from 250 to 200 nm using a cylindrical quartz cell of 2 mm path length. Proteins (0.2 mg/ml) were dissolved in 10 mM phosphate buffer (pH 7.2). The resultant spectra after five scans were analyzed for secondary structure by the curve-fitting program CONTINLL The near-UV CD spectra were measured at 25°C using the same spectropolarimeter as stated above. The spectra were measured with a 0.5 mg/ml protein solution in 50 mM phosphate buffer (pH 7.2). The reported spectra were the average of 5 scans.The intrinsic tryptophan fluorescence spectra of proteins (0.1 mg/ml) in 50 mM phosphate buffer (pH 7.2) at 25°C were recorded using a Fluoromax-4P spectrofluorometer . The excitation wavelength was set to 295 nm, and the emission spectra were recorded between 310 and 400 nm. Data were collected at a 0.5 nm wavelength resolution.The surface hydrophobicity of the different protein solutions (0.1 mg/ml of the wild-type and R12A mutants) was measured using a hydrophobic probe, TNS , as previously described The chaperone assays were carried out as previously described d) were determined by a similar procedure The chaperone-substrate binding study was performed by a membrane filtration method that we recently described The structural stability of Wt and mutant proteins was determined by equilibrium chemical denaturation experiment. Wt and mutant proteins  were incubated separately with various urea concentrations (0–7 M) for 18 hrs at 25°C. Tryptophan fluorescence spectra of all samples were taken in the 300–400 nm region using 295 nm as the excitation wavelength. The equilibrium unfolding profile was fitted according to a three state model


Aspergillus niger is known to be subjected to tight regulation, and the acid production profiles are fine-tuned to respond to the ambient pH. Based on transcriptome data, putative trans-acting pH responding transcription factors were listed and through knock out studies, mutants exhibiting an oxalate overproducing phenotype were identified. The yield of oxalate was increased up to 158% compared to the wild type and the corresponding transcription factor was therefore entitled Oxalic Acid repression Factor, OafA. Detailed physiological characterization of one of the ΔoafA mutants, compared to the wild type, showed that both strains produced substantial amounts of gluconic acid, but the mutant strain was more efficient in re-uptake of gluconic acid and converting it to oxalic acid, particularly at high pH (pH 5.0). Transcriptional profiles showed that 241 genes were differentially expressed due to the deletion of oafA and this supported the argument of OafA being a trans-acting transcription factor. Furthermore, expression of two phosphoketolases was down-regulated in the ΔoafA mutant, one of which has not previously been described in fungi. It was argued that the observed oxalate overproducing phenotype was a consequence of the efficient re-uptake of gluconic acid and thereby a higher flux through glycolysis. This results in a lower flux through the pentose phosphate pathway, demonstrated by the down-regulation of the phosphoketolases. Finally, the physiological data, in terms of the specific oxygen consumption, indicated a connection between the oxidative phosphorylation and oxalate production and this was further substantiated through transcription analysis.Acid formation in  Aspergillus niger is an industrially important organism, used as cell factory of a wide range of commercial enzymes as well as productions of million tons of citric acid A. niger in the biotech industry, strain improvement is a key component in process optimization. Traditionally, it has been approached by genetic engineering of a single or few metabolic genes; however, this strategy struggles to overcome the superjacent regulation thus the outcome has frequently shown to be of limited success. Another strategy entails of direct manipulation of transcription factors (TFs), since these proteins have the potential of controlling several fluxes in an organism. Modulation of TFs as a strategy for metabolic engineering has been demonstrated by Schuurmans et al. 2008, where the authors deleted one TF and overexpressed another to improve ethanol production in Saccharomyces cerevisiaeS. cerevisiaeS. cerevisiae have been public available since 1996 Neurospora crassa being the earliest to be publically available E. coli has 48 A. niger has approximately 1000 A. niger. Deleting prtT significantly lowers the protease activity without a noteworthy effect on the physiology of the fungus A. niger being highly dependent on pH of the culture medium Transcription factors can be grouped as cis- or trans-acting. Cis-acting TFs are characterized by their local response, inducing an entire cluster including the transcription factor itself as in the case of many secondary metabolite clusters A. niger was cultivated at three different pH set points ΔoafA, and the reference strain, were subjected to detailed physiological characterization.Based on transcriptome data from a previous study where A. niger ATCC 1015 was used as wild type strain (obtained from the IBT collection as IBT 28639). The ΔoafA strains were generated from the ATCC 1015 strain. All strains were maintained as frozen spore suspensions at −80°C in 20% glycerol.3, 0.52 g/L KCl, 0.52 g/L MgSO4 · 7H2O, 1 mL/L of 1% thiamine solution, 1 mL/L trace element solution. Trace element solution: 22 g/L ZnSO4 · 7 H2O, 11 g/L H3BO3, 5 g/L MnCl2 · 4 H2O, 5 g/L FeSO4 · 7 H2O, 1.7 g/L CoCl2 · 6 H2O, 1.6 g/L CuSO4 · 5 H2O, 1.5 g/L Na2MoO4 · 2 H2O, 50 g/L Na4EDTA.Transformation medium: 182.2 g/L sorbitol, 10 g/L glucose monohydrate, 6 g/L NaNO3, 1 g/L K2HPO4, 0.5 g/L MgSO4 · 7 H2O, 0.5 g/L KCl, 0.01 g/L FeSO4 · 7H2O, 15 g/L Agar, 1 mL/L trace element solution. 0.4 g/L CuSO4 · 5 H2O, 0.04 g/L Na2B2O7 · 10 H2O, 0.8 g/L FeSO4 · 7 H2O, 0.8 g/L MnSO4 · H2O, 0.8 g/L Na2MoO4 · 2 H2O, 8.0 g/L ZnSO4 · 7 H2O. pH adjusted to 6.2 prior to autoclavation.Czapek yeast extract (CYA) media: 30 g/L Sucrose, 5 g/L Yeast extract, 3 g/L NaNO3, 0.52 g/L KCl, 0.52 g/L MgSO4 · 7H2O, 1 mL/L of 1% thiamine solution, 1 mL/L trace element solution. Trace element solution: 22 g/L ZnSO4 · 7 H2O, 11 g/L H3BO3, 5 g/L MnCl2 · 4 H2O, 5 g/L FeSO4 · 7 H2O, 1.7 g/L CoCl2 · 6 H2O, 1.6 g/L CuSO4 · 5 H2O, 1.5 g/L Na2MoO4 · 2 H2O, 50 g/L Na4EDTA. As buffer 19.52 g/L 2-(N-morpholino)ethanesulfonic acid (MES) was used. pH was adjusted to 6.0 prior to autoclavation.Minimal screenings medium: 10 g/L glucose monohydrate, 6 g/L NaNO3, 1 g/L K2HPO4 · 3 H2O, 0.5 g/L MgSO4 · 7 H2O, 0.05 g/L FeSO4 · 7 H2O, 0.2 g/L KCl, 1 mL/L Trace metal solution. Trace element solution: 22 g/L ZnSO4 · 7 H2O, 11 g/L H3BO3, 5 g/L MnCl2 · 4 H2O, 5 g/L FeSO4 · 7 H2O, 1.7 g/L CoCl2 · 6 H2O, 1.6 g/L CuSO4 · 5 H2O, 1.5 g/L Na2MoO4 · 2 H2O, 50 g/L Na4EDTA. As buffer 19.52 g/L 2-(N-morpholino)ethanesulfonic acid (MES) was used. pH was adjusted to 6.0 prior to autoclavation.Complex screening medium (Watman medium): 30 g/L Sucrose, 5 g/L Corn steep liquor, 2 g/L Yeast extract, 3 g/L Peptone, 2 g/L Glucose, 2 g/L NaNO4)2SO4, 1.5 g/L KH2PO4, 1.0 g/L MgSO4 · 7 H2O, 1.0 g/L NaCl, 0.1 g/L CaCl2, 0.1 mL Antifoam 204 (sigma), 1 mL/L trace element solution. Trace element solution: 0.4 g/L CuSO4 · 5 H2O, 0.04 g/L Na2B2O7 · 10 H2O, 0.8 g/L FeSO4 · 7 H2O, 0.8 g/L MnSO4 · H2O, 0.8 g/L Na2MoO4 · 2 H2O, 8.0 g/L ZnSO4 · 7 H2O.Batch cultivation medium: 20 g/L glucose, 7.3 g/L . Blotting was done according to standard methods 1.5 µg genomic DNA was isolated and digested with appropriate restriction enzymes (SmaI and NdeI). Sequence information for restriction digest of the target loci was obtained from the 3 conidia/mL and incubated without agitation at 30°C for 5 days. At the end of the experiment, extracellular samples for pH and HPLC-measurements were collected.Fresh conidia were added to 8 mL of minimal- or complex screenings medium in a 50 mL sterile falcon tube (BD Biosciences) to a final concentration of 1·10Batch cultivations were performed in 2 L Sartorius fermenters with a working volume of 1.5 L, equipped with two Rushton six-blade disc turbines. The bioreactor was sparged with air, and the concentrations of oxygen and carbon dioxide in the exhaust gas were measured in a gas analyzer ). The temperature was maintained at 30°C during the cultivation and pH was controlled by automatic addition of 2 M NaOH and 1 M HCl, respectively. Initial conditions in the bioreactor were pH: 3.0; stirring rate: 100 rpm; and aeration: 0.2 volumes of air per volume of fluid per minute (vvm). After germination, the stirring rate was gradually increased to 800 rpm and the air flow to 1 vvm. The pH was adjusted to 2.5 or 6.0 with addition of 2 M NaOH or 1 M HCl over 2 hours.−1). The first steady state, pH 2.5, was obtained after five residence times (50 hours), whereas the second steady state was achieved after three additional residence times (30 hours).The chemostat cultivations were initiated as batch cultivations and in late exponential phase, supply of additional substrate through the feed was initiated. The feed medium was similar to the batch medium with the exception of the glucose concentration being 8 g/L. Pumps were controlled to a rate of 150 g/h . Initially, the filters were pre-dried in a microwave oven at 150 W for 20 min, and then weighed. A known weight of cell culture was filtered, and the residue was washed with distilled water. Finally, the filter was dried in the microwave at 150 W for 20 min and the dry weight was determined.−1.For quantification of the extracellular metabolites, a culture sample was taken and immediately filtered through a 0.45 µm-pore-size nitrocellulose filter (Osmonics). The filtrate was frozen and kept at −20°C until analysis. Glucose, oxalate, citrate, gluconate, glycerol and acetate concentrations were detected and quantified by refractive index and UV using an Aminex HPX-87H cationic-exchange column  eluted at 35°C, with 5 mM H2SO4 at a flow rate of 0.6 mL min+ through a coupled reaction with glucose-6-phosphate dehydrogenase and formation of NADH was determined spectrophotometrically by measuring the increase in absorbance at 340 nm.The applied HPLC method is unable to separate glucose and gluconate. Therefore, in samples where both glucose and gluconate were present, gluconate was quantified based on the UV-spectrum from the HPLC-analysis, whereas glucose was determined using a glucose enzymatic assay . This assay was based on NADFor gene expression analysis, mycelium was harvested at steady state by filtration through sterile Mira-Cloth . Liquid was quickly removed from the mycelium by squeezing, and it was then subsequently frozen in liquid nitrogen. Samples were stored at −80°C until RNA extraction.Total RNA was extracted from 40 to 50 mg of frozen mycelium as described in 150 ng of total RNA in 1.5 µl was labeled according to the One Color Labeling for Expression Analysis, Quick Amp Low Input (QALI) version 6.5 May 2010 from Agilent Technologies. Yield and specific activity was determined on a NanoDrop ND-1000 and verified on Qubit 2.0. 1.65 µg labeled cRNA was fragmented at 60°C on a heating block and the cRNA was prepared for hybridization according to the QALI protocol.100 µl sample was loaded on a 4×44 Agilent Gasket Slide situated in an Agilent Technologies hybridization chamber. The 4×44 Array was placed on top of the Gasket Slide. The Array was hybridized at 65°C for 17 hours in an Agilent Technologies Hybridization oven. The array was washed following the QALI protocol and scanned in a G2505C Agilent Technologies Micro Array Scanner.The raw array signal was processed by first removing the background noise using the normexp method, and signal between arrays made comparable using the quantile normalization method, as implemented in the Limma package t tests between sets of biological replicates from each pH level. Empiric Bayesian statistics were used to moderate the standard errors within each gene, and Benjamini-Hochberg's method P<0.05 was set to assess statistical significance.Statistical analysis was applied to determine genes subjected to differential transcriptional regulation. The limma package A. niger ATCC 1015 v3.0 gene models. The significance level was selected to P<0.05 and calculated using hypergeometric testing with Benjamini & Hochberg correction.Significantly regulated subsets of genes were examined for GO-term enrichment by using Cytoscape with BiNGO plugin . GO-term assignments were based on automatic annotation of the A. niger is known for its acid formation and recent studies have shown that a up to 6228 genes are influenced by ambient pH and the physiological response results in an acid production profile that ensures optimal acidification of the surrounding medium A. niger. Further analysis of the data from A. niger ATCC 1015. The corresponding transcription factor was entitled OafA .The genes encoding the predicted TFs were individually deleted through a bipartite gene knockout approach ΔoafA strain was subjected to detailed physiological characterization to explore the cellular performance of the oxalate overproducing strain from a quantitative perspective.The constructed ΔoafA strain was compared in pH-controlled batch cultivations, in duplicates. A. niger has been reported to produce a range of different acids in high amounts at pH 6.0 ΔoafA strain. Representative profiles of the biomass concentration, sugar concentration, carbon dioxide formation and acid formation during these batch cultivations are shown in The physiology of the wild type strain and the ΔoafA strain was that the majority of glucose in the exponential growth phase was converted into gluconate and biomass, where gluconate formation accounted for approximately 90% of the carbon conversion. After complete utilization of glucose, gluconate was metabolized resulting in mainly oxalate formation, but citrate was also detected in both set of cultures, −1 and 0.25±0.02 h−1 for the WT and the ΔoafA strain respectively, so deleting oafA showed no effect on the growth rate. However, a notable difference was observed in the formation of oxalate as the yield of oxalate on glucose was increased by 87% from 0.13 Cmol Cmol−1 to 0.25 Cmol Cmol−1 and the volumetric oxalate formation rate was doubled from 0.11±0.02 gOx (L h)−1 in the WT to 0.22±0.00 gOx (L h)−1 in the ΔoafA strain.Characteristic for both the wild type and the An interesting observation from the results of the batch cultivations was that acid production, except for gluconate formation, was mainly measured in stationary phase after glucose completion. From ΔoafA and the wild type during growth at both high and low pH, carbon limited chemostat cultivations were carried out. An important feature in the presented characterization was the application of chemostat cultures, as it can be difficult to obtain and maintain steady states working with filamentous fungi, because of the complex morphology of the cells To compare the acid production profiles of 2 at comparable yields. However, low citrate production was observed in the wild type, a product not detected in ΔoafA. During the first steady state at pH 2.5, achieved at 73 h (5 retention times), the mutant had an unchanged product profile with identical quantities in comparison with the wild type. The second steady at state occurred at 126 hours, 3 retention times after pH was changed to pH 5.0. As it was evident in the high pH batch cultivation, ΔoafA had a highly increased oxalate formation (158%) compared to WT, whereas both citrate and gluconate formation were reduced. A summary of growth and yield coefficients can be found in ΔoafA mutant and the wild type strain is increased oxalate formation in the ΔoafA mutant at the expense of gluconate, particularly in the second steady state at pH 5.0. The validity of the observed oxalate overproducing phenotype in the ΔoafA mutant is substantiated by the carbon balances, which closes for each distinctive phase.From ΔoafA thus exhibits an oxalate overproducer phenotype at high pH in both the reported batch and chemostat culture, as well as in the initial screening. To investigate the transcriptional aspects of this phenotype and to obtain further insight to OafA's function as a transcription factor, biomass samples were taken from all biological triplicates at steady state conditions  and subjected to transcriptome analysis.The A. niger genes analyzed are presented in the Data from the biological triplicates of the two strains at pH 2.5 and 5.0 were statistically analyzed, and genes that were significantly regulated  in pair-wise comparisons between two strains were identified across pH. The raw data of the expression levels of the entire oafA mutant at the first steady state (pH 2.5), a notably low number of significantly regulated genes was identified. Seven genes with a significant change in expression level, where four have been annotated, were identified. They included: down-regulation of a putative lysophospholipase, a polyketide syntease and intriguingly oxaloacetate acetyl hydrolase (oahA), the enzyme converting oxaloacetate to oxalate.Comparing the wild type with the −4, cluster freq 19/64, genome freq. 818/6268) associated with transport processes and within this subset three of these putative transporters were annotated as hexose transporters.At pH 5.0, 241 genes were significant changed in expression levels, 121 genes being up-regulated and 120 being down-regulated. A Gene Ontology overrepresentation analysis on the subset of up-regulated genes revealed an enrichment of 19 genes . Additionally, two genes encoding for epigenic regulation, a RNA-dependent RNA polymerase (Gene ID: 176277) and a histone acetyltransferase (Gene ID: 53882) were also found to be up-regulated.ΔoafA strain was shown to be a significant increase in oxalate formation. It was therefore surprising that the gene encoding oahA  was not up-regulated. Yet a slight up-regulation of a cytosolic malate dehydrogenase (MDH), the enzyme responsible for converting malate to oxaloacetate, was measured.The phenotypic trait of the −5, cluster freq 28/52, total freq 1300/6268). Two of these genes, a NADH-dehydrogenase and monooxygenase were associated with the oxidative phosphorylation. A catalase and a chloroperoxidase were additionally found down-regulated, a further indication of reduced oxidative phosphorylation.An ontology overrepresentation analysis on the subset of down-regulated genes showed an enrichment of 28 genes associated with oxidoreductase activity  and an Acyl-CoA syntase (129581), both enzymes responsible for converting acetate-phosphate into acetyl-CoA, was observed as well.oafA.Deletion of the putative trans-acting transcription factor entitled oxalic acid repression factor, OafA, resulted in an oxalate overproducing strain. The physiology of the constructed strain was characterized in detail, in both batch and chemostat cultivations, and compared to wild type physiology. In addition, transcriptome analysis at steady state conditions at both high and low pH highlighted the transcriptional response caused by the deletion of ΔoafA mutant showed an altered organic acid production profile at high pH compared to the wild type strain. All of the acid yields were affected, but in particular the oxalate yield increased  in the ΔoafA mutant. Two types of phosphoketolases have been described in the literature. Type 1 (EC 4.1.2.9) catalyzes, in the presence of inorganic phosphate, an irreversible cleavage of D-xylulose-5-phosphate into acetyl phosphate and glyceraldehyde-3-phosphate. Type 2 (EC 4.1.2.22) also termed fructose-6-phosphate phosphoketolase catalyzes as well in presence of phosphate, an irreversible cleavage of D-fructose 6-phosphate into acetyl phosphate and D-erythrose 4-phosphate. The activity of type 1 phosphoketolases is well characterized in a wide range of bacterial species and recently indications of this activity were found in Penicillium chrysogenumDeletion of An interesting finding in relation to Gene ID 197387 was that the open reading frame (ORF) of this gene was located next to the ORF of an acetate kinase. The close linkage of the ORFs of phosphoketolase and acetate kinase is found in many bacteria ΔoafA mutant. The metabolic response caused by the deletion of oafA is presented in ΔoafA mutant, none of the genes encoding glycolytic enzymes were up-regulated. As described earlier, an up-regulation of sugar permeases were argued to mediate an increased reuptake of gluconate in the ΔoafA mutant compared to the wild type strain. Furthermore, since the cultures were carried out as glucose limited chemostats, the glucose uptake rate was fixed but the reuptake of gluconate by the mutant strain could result in a higher amount of carbon metabolized through the glycolysis without a significant up-regulation of the glycolytic genes, subsequently leading to the observed overproduction of oxalate.The product in common for the two phosphoketolase enzymes is acetyl-phosphate that can be converted to acetyl-CoA through two enzymatic steps catalyzed by acetate-kinase and acetyl-CoA synthase, respectively. Together with the two genes encoding the phosphoketolases, the genes encoding acetate-kinase and acetyl-CoA synthase were also down-regulated in the oafA mutant compared to the wild type strain. The primary outcome of the PP-pathway is generation of reducing equivalents in the form of NADPH, used mainly in reductive biosynthesis reactions e.g. amino acid biosynthesis. A reduced flux through the PP-pathway would therefore result in a lower biomass yield and this further substantiates the argument of a lower PP-pathway in the oafA mutant, as the biomass yield, YSX; in the ΔoafA strain was reduced compared to YSX of the wild type. Finally, in the reverse study where oahA was deleted in A. niger eliminating oxalic acid production, an increase in the PP-pathway flux of 10% was detected oafA mutant and the wild type strain, it is argued that the increased oxalate formation in the oafA mutant is a consequence of an effective re-uptake of gluconate and thereby a higher flux through glycolysis resulting in a lower flux through the PP-pathway, demonstrated by down-regulation of the two phosphoketolases.In continuation of the proposed higher flux through glycolysis, the measured down-regulation of the two phosphoketolase genes indicate a lower flux through the pentose phosphate pathway in the ΔoafA strain, down-regulation of the oxidative phosphorylation was observed both physiologically by a 35% decrease in oxygen consumption per cmol glucose (soY) and transcriptionally as down-regulation of a NADH dehydrogenase and monooxygenase, both with ubiquinone binding. The decreased activity of the oxidative phosphorylation is further supported by the down-regulation of genes encoding a catalase and a chloroperoxidase. These enzymes are highly conserved in organisms exposed to oxygen and aid removal of reactive oxygen species that is an inevitable byproduct of the cytochrome c oxidase complex. To our knowledge a connection between oxidative phosphorylation and oxalate production has not been described, however the mutant response presented here provides indications of such a connection.The oxidative phosphorylation is responsible for the major ATP generation during aerobic metabolism by coupling reoxidation of NADH to ATP synthesis. In the ΔoafA mutant strain, with focus on the mutant being an oxalate overproducing strain.From the results described above it is concluded that OafA is a trans-acting transcription factor since deletion of the responsible gene resulted in 241 genes being differently expressed compared to the wild type strain. In addition, the function of OafA as a transcription factor was further underlined by the altered acid production profile in the ΔoafA strain. Furthermore, the oxidative phosphorylation was down-regulated in the oafA mutant and this indicates an interesting correlation between the oxidative phosphorylation and organic acid formation.Through transcription analysis, reduced phosphoketolase activity together with increased reuptake of gluconate were identified as being the main players in the metabolic response, resulting in the observed oxalate overproducing phenotype of the Table S1A. niger genes. In addition, complete lists of the significantly regulated genes at the two different pH's are also presented.The raw data for the expression level of all analyzed (XLSX)Click here for additional data file.


Small heat shock proteins (sHsps) maintain cellular homeostasis by preventing stress and disease-induced protein aggregation. While it is known that hydrophobicity impacts the ability of sHsps to bind aggregation-prone denaturing proteins, the complex quaternary structure of globular sHsps has made understanding the significance of specific changes in hydrophobicity difficult. Here we used recombinant protein of the lenticular sHsp α A-crystallin from six teleost fishes environmentally adapted to temperatures ranging from -2°C to 40°C to identify correlations between physiological temperature, protein stability and chaperone-like activity. Using sequence and structural modeling analysis we identified specific amino acid differences between the warm adapted zebrafish and cold adapted Antarctic toothfish that could contribute to these correlations and validated the functional consequences of three specific hydrophobicity-altering amino acid substitutions in αA-crystallin. Site directed mutagenesis of three residues in the zebrafish  confirmed that each impacts either protein stability or chaperone-like activity or both, with the V62T substitution having the greatest impact. Our results indicate a role for changing hydrophobicity in the thermal adaptation of α A-crystallin and suggest ways to produce sHsp variants with altered chaperone-like activity. These data also demonstrate that a comparative approach can provide new information about sHsp function and evolution. Small heat shock proteins (sHsps) play a central role in protein homeostasis by preventing the stress and age-related aggregation of denaturing proteins through their chaperone-like activity Biophysical approaches have been used to investigate how α-crystallins and other sHsps interact with compromised target proteins to prevent aggregation Danio rerio) contains two αB-crystallin proteins with divergent structures and function To identify specific changes in hydrophobicity affecting sHsp function we compared αA-crystallins from ectothermic teleost fishes adapted to environmental temperatures spanning −2°C to 40°C. While the zebrafish  relative to the rest could be due to excessive thermal denaturation. An almost identical pattern was seen when lactalbumin was used as a target protein but shifted 5°C lower, suggesting that the correlation between chaperone-like activity and the physiological temperature of each species is due to the αA-crystallins themselves and not a byproduct of each target protein  had lower thermal stability than those from the three warmest species  using the bovine αA-crystallin monomer as a structural template. Modeled structures were refined, equilibrated using molecular dynamics, and protein stabilization energies were estimated as described in Methods. We found a significant correlation (R2 = 0.8074) between each species' mean environmental temperature and the stabilization energy of its αA-crystallin, with greater relative stability in proteins from warmer species , and sequence identity between the fishes and human sequence ranged from 68.4% to 73.3%. Variation between the six fish species occurred at 56 of 177 amino acid positions in the alignment, presenting a large number of potentially adaptive substitutions. Only 5 of these 56 variable positions have been studied in the literature , (rat: PDB id: 2WJ5) and (cow: PDB id:3L1E). The mammalian structures are very similar to a recent crystal structure for a partial zebrafish αA-crystallin (PDB id: 3N3E) . StabiliDissostichus). Our hypothesis was that these substitutions would both decrease the upper level of thermal stability while increasing chaperone-like activity compared to wildtype zebrafish αA-crystallin. The V62T substitution fit this hypothesis, significantly enhancing chaperone-like activity at 25° and 30°C . RT-PCR amplification with primers  specific to a conserved region of the previously cloned zebrafish αA-crystallin Danio, Dissostichus and Notothenia were produced previously et al. −1 with 20 mM Tris, pH 8.5 with stepwise concentrations of 0.1 M, 0.2 M and 0.3 M NaCl. Fractions from the 0.3 M NaCl buffer containing αA-crystallin were concentrated to 1 ml in Amicon centrifugal filters  and further purified on a 90 cm×2.5 cm size exclusion column containing Sephacryl S-200 High Resolution bedding material  at a flow rate of 0.4 ml/min at 8°C. Fractions containing purified αA-crystallin were concentrated to approximately 5 mg/ml using Amicon centrifugal filters  and concentration was determined by spectrophotometry (1 OD280 = 1 mg ml−1). The purity of each purified protein was assessed by SDS-PAGE. Animal use in this study was approved by Ashland University's Institutional Animal Care and Use Committee (permit # 6).αA-crystallins from −1) and insulin  as the target protein. Insulin assays were performed with a mass ratio of 3∶1 target to crystallin in a buffer containing 50 mM sodium phosphate, 0.1 M NaCl, pH 7.0. The insulin was dissolved in 0.1 N NaOH, and 0.5 M NaPO4 (pH 6.8) was added to raise pH to 7.7. Lactalbumin assays used either a 10∶1 or 20∶1 ratio of target to crystallin in a buffer containing 50 mM sodium phosphate, 0.1 M NaCl, pH 6.75. Lactalbumin was dissolved directly in this buffer prior to use. Aggregation was induced with 20 mM DTT and monitored by measuring light scattering at 360 nm on a Shimadzu UV 1601 with a CPS 240 A temperature controller. All reactions were in a total of 500 µl using a 5 mm path length cuvette. Percent protection was calculated from the assay data by measuring the proportion of target protein aggregation prevented by each crystallin. Two way ANOVA was used to calculate statistical differences between αA-crystallins .Chaperone-like activity of each αA-crystallin was assayed be measuring their ability to prevent the chemically induced aggregation of insulin and/or lactalbumin. These target proteins were used because they aggregate across a broad range of temperatures (20 to 40°C) when chemically denatured with DTT. Other commonly used target proteins, such as lysozyme, do not aggregate well at temperatures below 30°C. Neither target protein used aggregated well below 20°C or above 40°C, so this was the range used in the assays. Three replicates were performed with all αA-crystallins at each temperature for both a-lactalbumin . Structure of the bovine αA-crystallin (PDB ID: 3L1E) was used as the structural template to model protein structures of six fish αA-crystallins. Structure visualization was performed using the module incorporated in the UCSF Chimera, build 1.4.1 Multiple sequence alignments were produced with ClustalW Structures of wild type proteins and the zebrafish missense variants were refined using the minimization procedure performed with the Impact module of the Maestro program package . Hydrogen atoms were added to the structures. Protein structures were regularized by an energy minimization procedure using the OPLS_2005 potentials, the 12 Å non-bonded cut-offs, the distance-dependent dielectric constant and 100 steepest descent steps of minimization followed by 200 steps of conjugated gradient in the presence of explicit 7453 SPC water molecules in the 50 Å×70 Å×70 Å on the final step. All bonds were constrained by the linear constraint solver algorithm. Temperature was kept constant to 298.15 K. Finally, the quality of modeled structures tested with the program Procheck The evaluation of protein stabilization free energy for the six fish α-crystallin domains was accessed with the FoldX force field, based on an empirical energy function derived from experimental work on proteins in silico translated amino acid sequences. Corresponding nucleotide sequences were then aligned by codon constraint to each other using the amino acid alignment via CodonAlign 2.0 For phylogenetic reconstruction, αA-crystallins from six ectothermic fish species and endothermic human . Because we were aware that hydrophobicity and chaperone properties are likely linked Molecular adaptation analysis was performed using TreeSAAP 3.2 Fluorescence emission spectra were recorded with a Cary Eclipse fluorescence spectrophotometer . Protein samples of 0.1 mg/mL in 10 mM phosphate buffer, pH 7.0, were used. Excitation was fixed at 295 nm and emission scanned from 310 to 400 nm. Binding of the hydrophobic probe bis-ANS   was assessed by recording fluorescence spectra with excitation wavelength fixed at 390 nm and emission spectra recorded between 420 and 600 nm as follows: 10 µL of 20 mM methanolic solution of Bis-ANS was added to a 1 mL protein (0.1 mg/mL) solution in 10 mM phosphate buffer, pH 7.4, and incubated for 2 h.To investigate secondary and tertiary structural changes of recombinant zebrafish αA-wild-type and our three variants their near- and far-UV CD spectra were recorded using a Jasco 500 A spectropolarimeter. Near-UV CD spectra were recorded with protein concentrations of 2.8, 2.0, 2.8, and 2.1 mg/mL, respectively, in 10 mM potassium phosphate buffer, pH 7.4 at room temperature. The reported CD spectra are the average of seven smoothed scans with an optical path of 1 cm. Far-UV CD spectra were recorded with protein concentrations of 0.09, 0.05, 0.04, 0.04 mg/mL, respectively, in 10 mM potassium phosphate buffer, pH 7.0 at 25°C. The reported spectra are the average of 12 smoothed scans with an optical path of 0.2 cm.Table S1αA-crystallin modifications previously reported in the literature. Modified αA-crystallins and their corresponding chaperone-like activity are shown. Each of these modifications were produced in mammalian αA-crystallins. The three variants produced in this study are novel in that their functional effects were predicted by comparing amino acid sequences from six different species.(DOC)Click here for additional data file.


Absidia cylindrospora culture gave S-(+)-1,2,3,4-tetrahydro-1-naftol of 92 % ee, whereas longer biotransformation time resulted in decrease of ee value. 3-Day transformation of β-tetralone by the same strain gave predominantly S-(−)-1,2,3,4-tetrahydro-2-naftol, whereas after 9 days of the reaction, the R-enantiomer with 85 % ee was isolated. Transformation of β-tetralone by Chaetomium sp. KCh 6651 gave pure (S)-(−)-1,2,3,4-tetrahydro-2-naftol in high yield at the concentration of 1 g/l. In this process, a non-selective carbonyl reduction was observed, followed by a selective oxidation of the R-alcohol.α-Tetralone and β-tetralone were subjected to biotransformation by 14 fungal strains. Enantiomeric purity of the products depended on the reaction time. 3-Day transformation of α-tetralone in  Enantiomerically pure alcohols are useful chiral building blocks for asymmetric synthesis of bioactive compounds of complicated structures , 22, 24.1) and β-tetralone (3) as substrates for the tests because products of their reduction are used as chiral synthons in pharmaceutical industry  in 10 % H2SO4. Products were separated by column chromatography using silica gel  and hexane/acetone mixture  as the developing system. Composition of biotransformation mixtures was established by GC. GC analysis was performed using a Hewlett-Packard 5890A (Series II) GC instrument fitted with a flame ionization detector. The chiral, capillary column  was used to determinate the composition and enantiomeric excesses of product mixtures. Authentic reference samples of the racemic alcohol were prepared by reducing the ketones with sodium borohydride in methanol. The NMR spectra were recorded on a DRX 500 MHz Bruker spectrometer and measured in CDCl3. Optical rotations were measured on an Autopol IV automatic polarimeter (Rudolph). The absolute configuration of the products was determined by comparison of their optical rotational values with literature data.The course of biotransformation was controlled by means of TLC. Analytical TLC was carried out on silica gel G 60 F3 (3 × 10 ml). The extracts were dried over MgSO4, concentrated in vacuo, and analyzed by GC. All the experiments were repeated three times.Erlenmeyer flasks (300 ml), each containing 100 ml of the medium consisting of 3 g glucose and 1 g aminobac dissolved in water, were inoculated with a suspension of microorganisms and then incubated for 3–7 days at 25 °C on a rotary shaker (190 rpm). After full growth of the culture (about 12 g of cell dry weight/l), 20, 40, 60, 80, and 100 mg of a substrate dissolved in 1 ml of acetone was added. After 1, 3, 6, and 9 days of incubation under the above conditions, portions of 10 ml of the transformation mixture were taken out and extracted with CHClC. betulinum culture (the strain selected for detailed studies). After 6 days of incubation, the mixtures were extracted with CHCl3 (3 × 300 ml), dried (MgSO4), and concentrated in vacuo. The transformation products were separated by column chromatography and analyzed .The same transformations were performed on the preparative scale in 2,000-ml flasks, each containing 500 ml of the cultivation medium. The cultures were incubated under the same conditions and then 200 mg of substrate dissolved in 2 ml of acetone was added to the grown HOH signals in the 1H NMR spectra.It was proved that the obtained products were the secondary alcohols. The locations and orientations of the introduced hydroxyl groups were determined on the basis of the chemical shifts and the shapes of Cα]D20 = +19.4°  (86 % ee),  δ (ppm): 1.76–2.01 ; 2.73–2.82 ; 4.75 ; 7.03–7.37 ; 7.45 ; GC: Rt (S) 23.0 min, Rt (R) 23.5 min.Colorless oil [α]D23 = −59.2°  (100 % ee),  [1H NMR (CDCl3) δ (ppm): 1.82 ; 2.05 ; 2.76 ; 2.86 ; 2.96 ; 3.09 ; 4.16 ; 7.10 ; GC: Rt (S) 26.9 min, Rt (R) 27.3 min.Pale yellow oil [% ee (S) ); 1H NMR1) and β-tetralone (3), the biotransformations in Fusarium culmorum [Didymosphaeria igniaria KCh 6670 [In our previous studies on enantiospecific reduction of α-tetralone (culmorum  and DidyKCh 6670  cultures1) and β-tetralone (3), only a few  nor β-tetralone (3) were reduced in the following cultures: D. betulina KCh 6538, A. tiliae KCh 6610, Chalara sp. KCh 6664, N. galligena KCh 6640, C. radicola KCh 6671, S. pastorianus KCh 906, H. uvarum KCh 912, and E. fibulger KCh 913. The strain A. coerulea KCh 93 transformed exclusively α-tetralone.Neither α-tetralone (3) was transformed much quicker than α-tetralone (1) by the majority of the tested fungal strains. Moreover, much higher concentrations of the substrate 3 were tolerated compared to α-tetralone (1), and alcohol 4 was obtained with higher optical purity than product 2. These results are different from the data reported in the literature, concerning reduction with the help of chiral catalysts, where 1 was more effectively reduced than 3 [Comparing the results presented in Tables d than 3 , 8.S)-alcohols according to the Prelog rule.Transformations of both substrates resulted in reduction of their carbonyl groups, leading mainly to the respective -(+)-1,2,3,4-tetrahydro-1-naftol ((S)-2) in 75 % yield and the enantiomeric excess above 86 %. This result was achieved after 6 days of the biotransformation. In the culture of A. cylindrospora KCh 336, the same product (S)-2 was obtained after 3 days with much higher optical purity—above 92 % at 72 % substrate conversion.In the culture of A. coerulea KCh 93, the yield of reduction of 1 increased with time, but the enantiomeric purity of formed alcohol (S)-2 decreased. After 1 day of transformation, the product was obtained with the ee as high as 100 %, but in low yield (19 %). After 9 days of transformation, (S)-2 was obtained in 76 % yield, but in low ee (50 %).In the culture of 3) by the strain of A. cylindrospora KCh 336 -4) was isolated with 85 % of enantiomeric purity (the (R)-4 alcohol represents the anti-Prelog product).Influence of the reaction time on enantiomeric purity of the product was also observed in the case of transformation of β-tetralone (36 Table . After 33) by Chaetomium sp. KCh 6651. In this process, at substrate concentration of 1 g/l, after 6 days we obtained enantiomerically pure (S)-(−)-1,2,3,4-tetrahydro-2-naftol ((S)-4) at 98 % ketone conversion. Tracing the progress of the reaction with time -4) gradually decreased. These facts indicate that (R)-4 was oxidized to the ketone 3, but (S)-4 could not be oxidized under the reaction conditions.Another interesting result was observed in the transformation of β-tetralone . Thus, one possible mechanism of stereoinversion is that oxidoreduction between (R)-4 and 3 is reversible and the reduction of 3 is irreversible  confirmed the conclusion that at first non-selective carbonyl group reduction occurs, followed by selective oxidation of the e Scheme .Scheme 1The corresponding time profiles for the transformation of α-tetralone in the culture of this strain do not feature such prominent variations. In this case, after the third day of transformation, the substrate conversion of 13 % was observed. However, after even longer elapsed time, there was decrease in the percentage of the alcohol in the biotransformation mixture, which indicates increase in the oxidation process rate relative to the reduction.Chaetomium sp. KCh 6651 strain culture leads to the conclusion that the substrate 3 undergoes reduction more efficiently. The alcohol 4 undergoes oxidation with slightly higher enantiospecificity with respect to the alcohol 2.Comparison of the conversion rates of both substrates in the 3 in the culture of C. betulinum KCh 6534 gave optically pure (S)-(−)-1,2,3,4-tetrahydro-2-naftol ((S)-4) in 100 % yield after 6-day biotransformation process. In the case of this strain, as in the culture of Chaetomium sp. KCh 6651 described above, lower enantiomeric excesses of the formed alcohol (S)-4 were initially (after 1 and 3 days) observed. The substrate 1 underwent reduction process in the C. betulinum KCh 6534 strain culture slower with respect to the compound 3. In this case, however, so much pronounced stereoinversion was not observed—the enantiomeric excess increased from 83 % after the first transformation day to 88 % in the ninth day.Transformation of 1) and β-tetralone (3) with particular emphasis on time variation of reaction mixture composition—a feature closely related to enantiospecificity of oxidation of alcohols formed previously in either enantioselective or non-enantioselective reduction .In summary, the manuscript presents the biotransformation of α-tetralone (There are numerous experiments described in the literature related to enantioselective oxidation of one enantiomer using racemic mixture of phenylethanols as the substrate , 11, 19.A. cylindrospora, the ketone is reduced to an S-alcohol with high enantiomeric excess, and simultaneously the obtained S-alcohol is at once oxidized back to the ketone with even higher enatioselectivity. Such mechanism of transformation leads to the observed decrease of enantiomeric excess of the initially formed S-alcohol after sufficiently long time of biotransformation process.In our experiments, we noted the sustained selectivity of dehydrogenases both in reduction and oxidation processes. For example, during transformation of β-tetralone in the culture of


Stagonospora nodorum is required for a variety of phenotypes including pathogenicity, melanisation and asexual differentiation. The roles though of the Gγ and Gβ subunits though were unclear. The objective of this study was to identify and understand the role of these subunits and assess their requirement for pathogenicity and development.It has been well established that the Gα subunit of the heterotrimeric G-protein in the wheat pathogen Gga1 and Gba1 respectively, were identified in the Stagonospora nodorum genome by comparative analysis with known fungal orthologues. A reverse genetics technique was used to study the role of these and revealed that the mutant strains displayed altered in vitro growth including a differential response to a variety of exogenous carbon sources. Pathogenicity assays showed that Stagonospora nodorum strains lacking Gba1 were essentially non-pathogenic whilst Gga1-impaired strains displayed significantly slower growth in planta. Subsequent sporulation assays showed that like the previously described Gα subunit mutants, both Gba1 and Gga1 were required for asexual sporulation with neither mutant strain being able to differentiate either pycnidia nor pycnidiospores under normal growth conditions. Continued incubation at 4°C was found to complement the mutation in each of the G-protein subunits with nearly wild-type levels of pycnidia recovered.G-protein Gγ and Gβ subunits, named This study provides further evidence on the significance of cAMP-dependent signal transduction for many aspects of fungal development and pathogenicity. The observation that cold temperatures can complement the G-protein sporulation defect now provides an ideal tool by which asexual differentiation can now be dissected. Stagonospora (Teleomorph: Phaeosphaeria) nodorum is a necrotrophic fungal pathogen and the causal agent of stagonospora nodorum blotch (SNB) of wheat  containing 1.5% agar. Plates were incubated at 22_C in 12 h cycles of darkness and near-UV light (Phillips TL 40 W/05). Liquid cultures were started with the addition of 107spores to 100 ml CzV8CS and were grown at 22°C shaking at 130 rpm in the dark. For experiments that required defined growth conditions, S. nodorum SN15 was used to inoculate minimal medium (MM), which consisted of 30 g/l sucrose, 2 g/l NaNO3-, 1.0 g/l K2HPO4, 0.5 g/l KCl, 0.5 g/l MgSO4.7H2O, 0.01 g/l ZnSO4.7H2O, 0.01 g/l FeSO4.7H2O and 0.0025 g/l CuSO4.5H2O. Agarose (15 g/l) was added when plates were required.S. nodorum strains to grow on different carbon sources was assayed by inoculation of MM agar plates containing different carbon sources by supplementing a stock of MM without the carbon source with 25 mM of the appropriate carbon source. S. nodorum strains were inoculated onto the above media from minimal medium (25 mM glucose) by excising a region of the agar containing approximately 4 mm2 of the agar surface of the (non-sporulating) growing edge of the mycelia onto the plates. Cultures were grown for 10 days in the dark at 22°C and colony diameters recorded 3, 5 and 10 days from inoculation, and observations of phenotype made. Four replicates were prepared per strain per carbon source assay. All statistical analyses were undertaken using the JMP7 package (SAS Institute). Statistical significance was determined using the Tukey–Kramer analysis.The capacity for the _ C in a 12 hr day/12 hr night cycle. The pathogenicity of the mutants was assayed on detached leaves from 2-week-old wheat seedlings, using a method modified from that described by Benedikz et al. [3) and incubated in a 12 h light/12 h dark cycle at 22°C to enable disease development.Plant material and infection conditions Pots (10 cm diam.) containing Perlite (P500) and grade 2 Vermiculite  were seeded with five seeds of the wheat variety Amery and grown at 20z et al. ,21. The S. nodorum mycelia using a Retsch® MM301 lyser  at 30  and the QIAGEN BioSprint 15 using the BioSprint 15 DNA Plant Kit protocol . DNA concentrations were determined using a NanoDropTM ND-1000 .Genomic DNA (gDNA) was extracted and isolated from S. nodorum Gga1 was synthesized using the 5′ and 3′ UTRs flanking the putative S. nodorum Gga1 (SNOG_00288), and the phleomycin cassette from the plasmid vector previously constructed and described by Solomon et al.[Gga1 gene was performed using a split-marker approach [Gga1 were also amplified, using the PCR primer sets Gga1KO5′F and Gga1KO5′R, and Gga1KO3′F and Gga1KO3′R. Fusion of the resulting PCR products; PHL with Gga1KO3′, and LEO with Gga1KO5′ was achieved by combining equimolar amounts (between 15 and 45 fmol) of each as template in a fusion PCR consisting of 5 μM each of PHLprimer and Gga1KO3′r, or LEOprimer and Gga1KO5′f, 1 U TaKaRa Ex TaqTM DNA polymerase and 1 × TaKaRa PCR Buffer , and 10 mM dNTPs in a final reaction volume of 20 μl. Thermal cycling consisted of 94°C for 2 minutes, 35 rounds of  and a final hold at 72°C for 10 minutes.A construct for the disruption of on et al.. The disapproach . To creaGba1 using the primers GbetaKOF1 and F2 and cloning this into the KpnI and XhoI sites in pBSK-phleo [Gba1 was amplified with GbetaKOR1 and R2 and cloned into Pst1 and BamHI sites subsequent to the 5′ flank cloning. The subsequent construct, pBphleo-GβKO, was transformed into S. nodorum SN15 as described below.The disruption construct was developed by amplifying an 800 bp 5′ flanking region to SK-phleo . SimilarS. nodorum mycelia as described by Solomon et al.[Gba1 or Gga1 in S. nodorum SN15. The screening primers are listed in Table Protoplasts were prepared from on et al.. Transfoon et al.. Fungal S. nodorum.The number of targeted gene insertions following fungal transformation was determined by quantitative real-time PCR (RT-qPCR) as described by . BrieflyTaq Polymerase, QuantiTect SYBR Green PCR Buffer, dNTPs, SYBR Green I dye; Qiagen, Australia), in a reaction volume of 15 μl. Thermal cycling consisted of 95°C for 15 minutes, followed by 40 cycles of .CT Values were determined from reactions consisting of four gDNA amounts  for each template, performed in triplicate. The primer pairs PhleoqPCRf and PhleoqPCRr or ActinqPCRf and ActinqPCRr were each used at 1.2 μM with 1× QuantiTect SYBR Green PCR Master Mix  Gba1 and (B) GgaA with fungal orthologues. Figure S2. (A) Agarose gel electrophoresis of PCR products arising from the amplification of the (A) GgaA locus of the created S. nodorum mutants. Targeted Insertion of the phleomycin cassette in place of the S. nodorum GgaA gene results in a 4196 bp amplicon  , replacing the 1789 bp amplicon of the wild type (WT) SN15. MW, Molecular weight marker; WT, S. nodorum SN15 gDNA; NTC, no template PCR control; the remaining lanes labeled by mutant culture number. Lanes 1, 2, 11, 20, 32, 34, no observed amplification or (B) Gba1 locus of strains transformed with the Gba1 homologous disruption construct. A band of 6.1 kb represents the wildtype locus and 7.6 kb the locus having undergone homologous recombination with the disruption construct. Lane 1, 1 kb ladder; Lane 2, S. nodorum SN15 (wildtype); Lanes 3–8, a representative selection of transformants. Strains represented in lanes 4, 6 and 7 have all undergone homologous recombination and represent Gba1 mutants. Figure S3. Light microscopy of the asexual spores of S. nodorum, harvested from the wild-type SN15 and mutant strains gna1-35, gba1-6 and ggaA-25.Click here for file Table S1. Sequences of primers used in this study.Click here for file