APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 2005, p. 5465–5475
`0099-2240/05/$08.00⫹0 doi:10.1128/AEM.71.9.5465–5475.2005
`Copyright © 2005, American Society for Microbiology. All Rights Reserved.
`
`Vol. 71, No. 9
`
`Generation and Phenotypic Characterization of Aspergillus nidulans
`Methylisocitrate Lyase Deletion Mutants: Methylisocitrate
`Inhibits Growth and Conidiation
`Matthias Brock*
`University Hannover, Institute for Microbiology, Herrenha¨user Str. 2, 30419 Hannover, Germany
`
`Received 10 January 2005/Accepted 22 February 2005
`
`Propionate is a very abundant carbon source in soil, and many microorganisms are able to use this as the
`sole carbon source. Nevertheless, propionate not only serves as a carbon source for filamentous fungi but also
`acts as a preservative when added to glucose containing media. To solve this contradiction between carbon
`source and preservative effect, propionate metabolism of Aspergillus nidulans was studied and revealed the
`methylcitrate cycle as the responsible pathway. Methylisocitrate lyase is one of the key enzymes of that cycle.
`It catalyzes the cleavage of methylisocitrate into succinate and pyruvate and completes the ␣-oxidation of
`propionate. Previously, methylisocitrate lyase was shown to be highly specific for the substrate (2R,3S)-2-
`methylisocitrate. Here, the identification of the genomic sequence of the corresponding gene and the generation
`of deletion mutants is reported. Deletion mutants did not grow on propionate as sole carbon and energy source
`and were severely inhibited during growth on alternative carbon sources, when propionate was present. The
`strongest inhibitory effect was observed, when glycerol was the main carbon source, followed by glucose and
`acetate. In addition, asexual conidiation was strongly impaired in the presence of propionate. These effects
`might be caused by competitive inhibition of the NADP-dependent isocitrate dehydrogenase, because the Ki of
`(2R,3S)-2-methylisocitrate, the product of the methylcitrate cycle, on NADP-dependent isocitrate dehydroge-
`nase was determined as 1.55 ␮M. Other isomers had no effect on enzymatic activity. Therefore, methylisocitrate
`was identified as a potential toxic compound for cellular metabolism.
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`Propionate is a common carbon source in soil (9) and can be
`metabolized by a variety of microorganisms. Aerobic metabo-
`lism of propionate generally starts with the activation of pro-
`pionate to the corresponding coenzyme A (CoA) ester propio-
`nyl-CoA as summarized in reference 32. One of the major
`pathways is that of the coenzyme B12-dependent methylmalo-
`nyl-CoA pathway leading to the citric acid cycle intermediate
`succinyl-CoA. The other main pathway is the ␣-oxidation of
`propionate to pyruvate via the methylcitrate cycle. This path-
`way is found in bacteria, as well as in fungi, and involves a
`condensation of propionyl-CoA and oxaloacetate to methylci-
`trate (7, 17, 32). This reaction is catalyzed by a cycle specific
`methylcitrate synthase. The next step is the dehydration of
`methylcitrate to methylaconitate via a specific methylcitrate
`dehydratase. The rehydration of methylaconitate to methyli-
`socitrate was shown at least in Escherichia coli and Salmonella
`enterica serovar Typhimurium to be catalyzed by the citric acid
`cycle aconitase AcnB (8, 16). The last pathway-specific reac-
`tion, catalyzed by a methylisocitrate lyase, is the cleavage of
`methylisocitrate into pyruvate and succinate. Methylisocitrate
`lyases have been well characterized, especially from bacterial
`but also from fungal sources (6, 13, 14, 22, 27, 28). However,
`only one gene sequence coding for a fungal methylisocitrate
`lyase has been published. It was shown that the so-called non-
`functional isocitrate lyase 2 from Saccharomyces cerevisiae pos-
`sesses methylisocitrate lyase activity (22). The protein se-
`
`* Present address: Leibniz Institute for Natural Product Research
`and Infection Biology, Hans Knoell Institute, Beutenbergstr. 11a,
`D-07745 Jena, Germany. Phone (3641) 656815. Fax: (3641) 656825.
`E-mail: Matthias.brock@hki-Jena.de.
`
`quence showed a high degree of similarity to isocitrate lyases
`from other sources but solely exhibited activity toward meth-
`ylisocitrate. Extracts of cells, which carried a deletion of the
`ICL2 gene, showed a drastic reduction of methylisocitrate
`lyase activity compared to the wild type, when grown at the
`carbon sources threonine or ethanol. Unfortunately, no growth
`experiments had been performed to study the effect of the
`mutation on growth in the presence of propionate. Due to the
`high sequence similarity of this methylisocitrate lyase to isocit-
`rate lyases,
`it was not possible to predict whether other
`uncharacterized fungal proteins display isocitrate lyase or
`methylisocitrate lyase activity. Although the specific methyli-
`socitrate lyase from Aspergillus nidulans was purified and char-
`acterized (6), it was difficult at that time to gain access to the
`genome database from A. nidulans. Therefore, it was not pos-
`sible to identify the corresponding gene and to study the phe-
`notype of a deletion mutant.
`In the food and feed industry, propionate is commonly used
`as a preservative against molds. This is surprising because of
`the ability of filamentous fungi to use propionate as the sole
`carbon source. Nevertheless, cometabolism of glucose and pro-
`pionate strongly affects the growth rate in a manner, which is
`strictly dependent on the amount of propionate present (5, 7).
`It has been shown that propionyl-CoA possesses a severe neg-
`ative effect on the pyruvate dehydrogenase complex from var-
`ious organisms (2, 3, 5, 24). Deletion of methylcitrate synthase
`led to an inability of A. nidulans to remove propionyl-CoA,
`causing a strong accumulation of this compound. Therefore,
`the negative effect of propionyl-CoA on growth could at least
`in part be explained by a blockage of the pyruvate dehydroge-
`nase complex. In addition to the growth-inhibitory effect of
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`BROCK
`
`Strain
`
`A26 (mclA⫹)
`RMS011 (mclA⫹)
`SMI45 (mclA⫹)
`SMBA9 (⌬mclA)
`SMBA10 (⌬mclA)
`SRF200 (mclA⫹)
`
`APPL. ENVIRON. MICROBIOL.
`
`TABLE 1. A. nidulans strains used in this studya
`
`Genotype
`
`Source or reference
`
`biA1
`pabaA1 yA2 ⌬argB::trpC⌬B trpC801 veA1
`pabaA1 yA2 wA3 veA1
`pabaA1 yA2 ⌬argB::trpC⌬B ⌬mclA::argB trpC801 veA1
`pabaA1 yA2 ⌬argB::trpC⌬B ⌬mclA::argB trpC801 veA1
`pyrG89 ⌬argB::trpC⌬B pyroA4 veA1
`
`Fungal Genetics Stock Center, Kansas City, KS
`30
`M. Kru¨ger, Marburg, Germany
`This study
`This study
`18
`
`a Methylisocitrate lyase-positive strains A26 and RMS011 were used as controls for excretion of methylisocitrate, growth inhibition, and induction of NADP-
`dependent isocitrate dehydrogenase.
`
`it also disturbed formation of polyketides,
`propionyl-CoA,
`most likely via competition of propionyl-CoA with the natural
`substrates acetyl- and malonyl-CoA for the respective binding
`sites (5, 7, 36, 37).
`In the present study the main interest was the investigation
`of the phenotypic effects caused by the accumulation of meth-
`ylisocitrate. Previously, it was shown that NADP-dependent
`isocitrate dehydrogenase from bovine heart mitochondria and
`rat liver cytosol is strongly inhibited by ␣-threo-methylisocitrate
`with a Ki of ⬍1 ␮M (1, 25). NADP-dependent isocitrate de-
`hydrogenase also exists in A. nidulans, and the cytosolic, per-
`oxisomal, and mitochondrial enzymes are all produced from a
`single gene (31). The exact function of this enzyme has not yet
`been proven. However, a general function may be the shuttle
`of reducing equivalents between NADP in different cellular
`compartments, because the compartmental membranes are
`impermeable for pyrimidine nucleotides and other small mol-
`ecules. There are good indications that the mitochondrial en-
`zyme is involved in the NADPH-dependent synthesis of gluta-
`mate from ␣-ketoglutarate (23). The peroxisomal enzyme
`might provide NADPH for the degradation of unsaturated
`fatty acids and the cytoplasmic enzyme provides NADPH for
`anabolic processes and for the reduction of glutathione and
`thioredoxin (19, 31).
`By creating a mutant with a deletion of the gene coding for
`methylisocitrate lyase, it was possible to study the effect of
`intracellularly generated methylisocitrate on growth and devel-
`opment with a focus on NADP-dependent isocitrate dehydro-
`genase.
`
`MATERIALS AND METHODS
`
`Identification and deletion of the methylisocitrate lyase coding region. The
`gene coding for methylisocitrate lyase was identified from N-terminal sequencing
`of the purified protein and a subsequent BLAST search against the A. nidulans
`genome as described in Results.
`In all transformation steps for generation of the deletion construct, E. coli
`MRF’ XL1-Blue (MBI Fermentas, St. Leon-Rot, Germany) was used. The cod-
`ing region including 1,154 bp upstream of the ATG start codon and 1,415 bp
`downstream of the TGA stop codon was amplified by DyNAzyme EXT DNA
`polymerase (BioCat, Heidelberg, Germany). The oligonucleotides used were
`MICLup2Pst (5⬘-CTA CGC TGC AGG CAC TCA TGA AG-3⬘; the PstI restric-
`tion site is in boldface) and AnMICLnest_down (5⬘-GGC AAT TCA CCG TCA
`AGG AC-3⬘). As template DNA genomic DNA from A. nidulans RMS011 was
`used. The resulting PCR product was cloned into the PCR2.1 vector (Invitrogen,
`Karlsruhe, Germany). Positive clones were analyzed by PstI restriction, releasing
`a fragment of 4,452 bp, which contained the mclA gene, the whole upstream
`region, and 1,287 bp of the downstream region (which possessed an internal PstI
`restriction site). The fragment was subcloned into a previously PstI-restricted
`pUC19 vector (Invitrogen). In order to exclude the coding region from the
`construct and for introduction of a NotI restriction site, an inverse PCR on the
`vector was performed with a proofreading polymerase (Accuzyme polymerase;
`
`Bioline, Luckenwalde, Germany). Oligonucleotides were NotMICLup (5⬘-CCG
`CGT GGA GTA CTG GAA GG-3⬘; reads toward the upstream region) and
`NotMICLdown (5⬘-CCG CTT TGA TGT GAG CGT TCG-3⬘; reads toward the
`downstream region), which both contained a half NotI restriction site (indicated
`in boldface). The resulting PCR product was eluted from an agarose gel and
`phosphorylated with polynucleotide kinase as described by the manufacturer
`(New England Biolabs, Frankfurt, Germany). The phosphorylated product was
`self-ligated with T4 DNA ligase (NEB) and yielded a vector with a pUC19
`backbone, 1,131-bp upstream region, and 1,269-bp downstream region, both
`regions separated by a newly generated NotI restriction site. In this NotI site the
`argB gene from vector pAlcArg (7) was subcloned, leading to the final deletion
`construct. The deletion part was removed from the pUC-backbone by PstI
`restriction and used for transformation of A. nidulans RMS011 (Table 1).
`Transformation of A. nidulans was performed by standard methods (34).
`Genomic DNA was isolated by standard procedures and subjected to XbaI
`restriction. A digoxigenin-labeled probe was amplified by PCR with digoxigenin-
`11-dUTP in the nucleotide mix and oligonucleotides NotMICLdown (see above)
`and MICLdown (5⬘-CTG CAG GCC GGC CAA GG-3⬘). This probe was specific
`for the downstream region. A Southern blot was performed on the restricted
`DNA, and bands were detected after hybridization with alkaline phosphatase-
`linked anti-digoxigenin Fab fragments (Roche Diagnostics, Mannheim, Ger-
`many) by use of CDPstar as described in the manufacturer’s protocol (Roche
`Diagnostics).
`Isolation of RNA, reverse transcription, and sequencing of cDNA. Strain A26
`(Table 1) was grown for 40 h on minimal medium containing 10 mM glucose and
`100 mM propionate. The mycelium was harvested and frozen in liquid nitrogen,
`and ca. 0.1 g was ground to a fine powder. For RNA extraction, the RNeasy Plant
`Minikit (QIAGEN, Hilden, Germany) was used. An aliquot of the RNA was
`used as a template for first-strand cDNA synthesis with the sequence specific
`oligonucleotide cDNAmicl_down (5⬘-CAT ACA TAC ATT CGA ACG CTC
`AC-3⬘) and SuperScript II reverse transcriptase as described in the manufactur-
`er’s protocol (Invitrogen). Second-strand synthesis and amplification of the
`cDNA was performed by use of DyNAzyme EXT DNA polymerase and the
`sequence
`specific oligonucleotides
`cDNAmicl_down and cDNAmicl_up
`(5⬘-CAG TAC TCC ACG CCA GAC-3⬘). The resulting PCR product was cloned
`into the pDrive cloning vector (QIAGEN) and sequenced from both strands by
`SeqLab (Go¨ttingen, Germany).
`Phenotypic characterization of methylisocitrate lyase mutants and sexual
`crossing. Growth experiments were performed as described earlier (5). Mutant
`strains were inoculated in replicate cultures, and mycelium was harvested after
`growth for the indicated time (result section). Carbon sources used for growth
`were glucose, sodium acetate, sodium propionate, and glycerol in the concen-
`trations and combinations described for each experiment. For determination of
`growth inhibition, mycelium was harvested and dried for at least 12 h at 80°C and
`weighed. Biomass obtained from glucose, acetate, or glycerol as the sole carbon
`source was set as 100%, respectively.
`The ability of the mutant strains to form conidia was determined from solid
`plates containing 2% agar and the indicated carbon sources. Spore suspensions
`were point inoculated, and plates were incubated for 3 days at 37°C (the growth
`time for plates containing propionate as sole carbon source was prolonged to 7
`days).
`Crossing of the yellow strain SMBA9 and the green strain SRF200, which
`carries an intact methylisocitrate lyase locus, was performed by standard proce-
`dures. In brief, both strains were inoculated on plates, which allowed both strains
`to grow. Agar blocks were removed from areas, where mycelium of both strains
`was found and transferred to an agar plate that lacked p-aminobenzoic acid and
`uracil. Only mycelium, which contained nuclei from both strains was able to grow
`on these plates. The plates were sealed and incubated for 10 days at 37°C, which
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`VOL. 71, 2005
`
`A. NIDULANS METHYLISOCITRATE LYASE DELETION MUTANTS
`
`5467
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`induced the formation of cleistothecia. Mature cleistothecia were isolated and
`plated on nonselective media. A successful crossing event was monitored by
`green and yellow colonies in a 1:1 deviation. Single colonies were analyzed for
`their phenotypes on selective media. Pictures of the colonies were taken with a
`digital camera (Nikon Coolpix 995).
`Purification of isocitrate lyase. Isocitrate lyase from A. nidulans was purified
`from strain SMI45, which was grown for 40 h in minimal medium containing 100
`mM acetate and 100 mM propionate as carbon sources. This composition of the
`medium induced both isocitrate lyase and methylisocitrate lyase activity (5).
`During the purification procedure fractions were checked for both activities.
`Approximately 4 g of drypressed mycelium was ground to a fine powder under
`liquid nitrogen and resuspended in 50 ml of buffer A (50 mM Tris-HCl, 2 mM
`MgCl2, 2 mM dithiothreitol [pH 8.0]). Cell debris was removed by centrifugation
`at 25,000 ⫻ g at 4°C for 20 min. The supernatant was subjected to fractionated
`(NH4)2SO4 precipitation from 0 to 40% and from 40 to 75% saturation. The
`pellet from the second precipitation was resolved in 5 ml of buffer A and loaded
`on a Phenyl-Sepharose column (bed size, 20 ml) previously equilibrated with
`buffer A containing 1 M (NH4)2SO4. Proteins were eluted with a decreasing
`(NH4)2SO4 gradient from 1 to 0 M. Isocitrate lyase containing fractions were
`pooled, concentrated, and desalted by use of centrifugal filter devices (30-kDa
`cutoff; Millipore, Schwalbach, Germany) and buffer A. The concentrated sample
`was loaded onto a ResourceQ column (bed size, 1 ml; Amersham Biosciences
`Europe, Freiburg, Germany) and eluted against a sodium chloride gradient from
`0 to 0.2 M in buffer A. Enzyme-containing fractions were collected, desalted, and
`again subjected to chromatography on a ResourceQ column.
`Purity of the active fractions was determined by sodium dodecyl sulfate-
`polyacrylamide gel electrophoresis (20). Further identification was made by
`peptide mass analysis. The purified protein band was excised from the gel and
`sent to the Ludwig-Maximilians-Universita¨t Mu¨nchen. The protein was digested
`with trypsin and peptides were subjected to matrix-assisted laser desorption
`ionization–time of flight analysis. The peptide masses were than compared to
`nonredundant protein databases.
`Enzyme assays and determination of methylisocitrate from the growth me-
`dium. The activities of isocitrate lyase and methylisocitrate lyase were deter-
`mined as described before (5). The Km value of isocitrate lyase for the substrate
`D-isocitrate was identified by varying the substrate concentration of D,L-isocitrate
`in the assay from 1 to 0.1 mM, giving an effective substrate concentration of
`D-isocitrate in the range of 0.5 and 0.05 mM. The Km value for (2R,3S)-2-
`methylisocitrate was determined with stereoisomeric pure substrate (10) in a
`range of 1.5 and 0.25 mM.
`Methylisocitrate lyase activity in mutant strains was defined with 0.5 mM
`methylisocitrate in the assay, which would have been sufficient for maximum
`activity of methylisocitrate lyase (Km ⫽ 31 ␮M) (6).
`The concentration of methylisocitrate in the growth medium was tested by
`enzymatic methods after harvest of the mycelium. Samples of 50 to 100 ␮l were
`applied to a test containing NADH and lactate dehydrogenase. The assay was
`started by the addition of purified methylisocitrate lyase from E. coli, and the
`decrease in absorbance at 340 nm was monitored. Concentrations of methyli-
`socitrate from the complete culture broth were calculated and referred to the
`mycelial dry weight.
`NADP-dependent isocitrate dehydrogenase was assayed by monitoring the
`formation of NADPH in a slightly modified procedure as described in reference
`1. The final assay with a volume of 1 ml contained: 50 mM Na-HEPES (pH 7.5),
`2 mM D,L-isocitrate, 1 mM MnCl2 (or 2 mM MgCl2), 1 mM NADP, and crude
`extract. For determination of the Km value, all components were kept constant
`except the concentration of isocitrate, which was used in an effective concentra-
`tion (D-isocitrate) between 1.0 and 0.025 mM. The inhibition constant of meth-
`ylisocitrate was determined by preincubating the respective assay mixture with
`25, 50, or 100 ␮M (2R,3S)-2-methylisocitrate and starting the reaction with an
`effective concentration of D-isocitrate in the range of 1.5 and 0.375 mM. The Ki
`was calculated from the factor of the increase of the Km with respect to the
`inhibitor concentration used.
`Protein concentrations were determined by use of Bio-Rad protein assay
`concentrate (Bio-Rad Laboratories, Munich, Germany) as described in the man-
`ufacturer’s protocol with bovine serum albumin as a standard.
`
`RESULTS
`
`Identification of the gene coding for methylisocitrate lyase.
`Methylisocitrate lyase was purified from A. nidulans wild type
`as described in an earlier publication (6). After blotting of the
`purified enzyme on a polyvinylidene difluoride membrane
`
`(Millipore), the purified enzyme was subjected to N-terminal
`sequencing (kindly carried out by D. Linder, Justus-Liebig-
`University, Giessen, Germany) and revealed the putative pep-
`tide sequence SPSSLPPVQPP.
`The exact interpretation of the sequence data was difficult
`because of the high amount of proline, which weakens the
`signal and leads to a high background. Nevertheless, a BLAST
`search against fungal proteins (http://www.ncbi.nlm.nih.gov
`/BLAST/) revealed a single hit on the hypothetical protein
`AN8755.2 (accession no. EAA60548) from the annotated A.
`nidulans genome (http://www.broad.mit.edu/annotation/fungi
`/aspergillus/index.html). Sequencing of the cDNA obtained
`with specific oligonucleotides confirmed the predicted se-
`quence with an exception at the C-terminal end (accession no.
`AJ890109; protein ID no. CAI65406). At this site the sequence
`of the hypothetical protein is extended by 10 amino acids,
`which is mainly due to a wrong intron prediction at the C-
`terminal end. The protein consists of 604 amino acids with a
`molecular mass of 66.9 kDa, which is in good agreement with
`66 ⫾ 3 kDa as determined from sodium dodecyl sulfate-poly-
`acrylamide gel electrophoresis (6). However, a mitochondrial
`localization of the native enzyme was predicted because that
`seems to be the compartment in which the methylcitrate cycle
`takes place (7, 22). Therefore, the protein was scanned for a
`putative mitochondrial targeting sequence with the program
`MITOPROT (http://ihg.gsf.de/ihg/mitoprot.html). With a
`probability of 0.9992 (maximum ⫽ 1.0) the protein is trans-
`ported to mitochondria with a cleavage site of the leader pep-
`tide at position 41 (MLRSIPRRVPRRLPIFTTTATAGGPSR
`LAQRAFTCGYLRM/SPSSL. . .), which is in perfect agreement
`with the result from N-terminal sequencing. The native protein
`therefore has a molecular subunit size of 62.4 kDa, which is
`also consistent with that determined from gel electrophoresis.
`The corresponding gene is located at the right arm of chro-
`mosome III and spans a region of 2,081 nucleotides (including
`five introns) on contig 1.161 (positions 60367 to 62448).
`Prediction of a conserved sequence motif for fungal meth-
`ylisocitrate lyases. In previous publications, the crystal struc-
`ture of isocitrate lyases from Mycobacterium tuberculosis (26)
`and from A. nidulans (4) had been determined. In addition, the
`structure of bacterial methylisocitrate lyases from E. coli and
`Salmonella enterica serovar Typhimurium had also been deter-
`mined (14, 28). Interestingly, the sequence identity of the E.
`coli methylisocitrate lyase (PrpB) to the respective isocitrate
`lyase (AceA) was only 27%, but the structural identity was
`extremely high. Furthermore, a high sequence identity was
`found within the amino acids of the active site.
`Isocitrate lyases contain conserved tryptophan, phenylala-
`nine, and threonine residues, which are responsible for correct
`orientation of the glyoxylate moiety in the active site. In con-
`trast, in bacterial methylisocitrate lyases the amino acids phe-
`nylalanine, leucine, and proline replaced these residues. Mod-
`eling of pyruvate into the active site revealed that these
`exchanges lead to a hydrophobic binding pocket, which gives
`more space for the additional methyl group of pyruvate (14).
`An alignment of the methylisocitrate lyase from S. cerevisiae
`(ICL2) revealed that the first tryptophan residue from isocit-
`rate lyases was still conserved but that phenylalanine and thre-
`onine were replaced by leucine and serine. Both changes may
`also provide more space for an additional methyl group. To
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`FIG. 1. Partial alignment of fungal methylisocitrate lyases and isocitrate lyases. Putative active site residues are shown in boldface. Residues,
`which may specify the acceptance of the methyl group from methylisocitrate are in boldface and shaded. AnMICL, methylisocitrate lyase from A.
`nidulans (accession no. CAI65406); NcMICL, hypothetical methylisocitrate lyase from N. crassa (accession no. XP_331680); MgMICL, hypothet-
`ical methylisocitrate lyase from M. grisea (accession no. EAA47373); ScMICL, methylisocitrate lyase from S. cerevisiae (accession no. NP_015331);
`YlMICL, hypothetical methylisocitrate lyase from Y. lipolytica (Accession: XP_506117); IclAn, isocitrate lyase from A. nidulans (accession no.
`EAA62727); IclNc, isocitrate lyase from N. crassa (accession no. CAA44573); IclMg, isocitrate lyase from M. grisea (accession no. AAN28719);
`IclSc, isocitrate lyase from S. cerevisiae (accession no. NP_010987); IclYl, isocitrate lyase from Y. lipolytica (accession no. XP_501923).
`
`check whether this is a general motif of fungal methylisocitrate
`lyases, a BLAST search against fungal databases was per-
`formed using the sequences from S. cerevisiae and A. nidulans
`as a template. The hypothetical methylisocitrate lyases from
`Neurospora crassa, Magnaporthe grisea, and Yarrowia lipolytica
`were identified by similarity and by their putative mitochon-
`drial targeting sequence. An alignment of the residues, which
`are proposed to be involved in substrate binding of these meth-
`ylisocitrate lyases against their isocitrate lyase counterparts, is
`shown in Fig. 1.
`From this sequence alignment we predict a sequence motif
`[GF(V/T)(L/M)QL(I/V)SLAG(L/V)H; amino acids providing
`the space for the methyl group are indicated in boldface] to be
`specific for fungal methylisocitrate lyases.
`Deletion of the methylisocitrate lyase coding region and
`identification of mutant strains. The coding region of the
`methylisocitrate lyase gene (mclA) with flanking regions was
`amplified from genomic DNA of strain RMS011 (Table 1).
`Thereby, a PstI restriction site ca. 1.15 kb upstream of the start
`ATG was introduced and, additionally, the endogenous PstI
`restriction site ca. 1.29 kb downstream of the stop codon was
`included. This enabled the subcloning into pUC19 vector. The
`coding region of the mclA gene was removed by PCR, gener-
`ating a NotI restriction site in which the argB gene from A.
`nidulans (coding for carbamoyl transferase and leading to ar-
`ginine prototrophy of transformants) was cloned. In this con-
`struct, a 1.13-kb upstream and a 1.27-kb downstream fragment
`of the mclA flanking regions surrounded the argB gene. For
`transformation of A. nidulans strain, RMS011 the vector back-
`bone of the deletion construct was removed by PstI restriction,
`and only the deletion part was used further. Transformation of
`protoplasts was performed as described elsewhere (34).
`Genomic DNA from transformants was isolated, XbaI re-
`stricted, and subjected to Southern analysis using a digoxige-
`nin-labeled probe against the downstream fragment. The wild
`type was supposed to yield a 1.8-kb fragment, whereas this
`fragment was expected to shift to 9.6 kb in case of a homolo-
`gous integration of the deletion construct into the mclA locus.
`Transformants SMBA9 and SMBA10 exactly showed the ex-
`
`pected pattern, whereas others seem to posses either tandem
`and/or ectopic integrations (Fig. 2).
`Phenotypic characterization of methylisocitrate lyase dele-
`tion mutants. In order to study the phenotypes caused by a
`deletion of the mclA gene, growth experiments were per-
`formed on solid and liquid media. Liquid media were used to
`investigate a potential growth inhibitory effect by determina-
`tion of the dried biomass, whereas solid agar plates were used
`to study the effect of propionate on asexual development. The
`effects of different carbon sources on development are illus-
`trated in Fig. 3A. With propionate as the sole carbon and
`energy source the deletion strains did not produce any visible
`biomass or conidia, not even after incubation for more than 5
`days. Therefore, we conclude that the methylisocitrate lyase is
`essential for the utilization of propionate. In contrast, no phe-
`notype was visible, when glucose or acetate was used as sole
`carbon sources. This finding implies that methylisocitrate lyase
`is specifically involved in the methylcitrate cycle and not re-
`quired for the citric acid or glyoxylate cycle. Addition of 20
`mM propionate to glucose medium totally abolished the for-
`mation of conidia. However, a microscopic investigation of the
`colonies showed that development of conidiophores, including
`metulae and phialides was not disturbed (data not shown).
`Nevertheless, an extra addition of 50 mM acetate led to a slight
`restoration of conidiation. This is in agreement with the com-
`petition of acetate and propionate for the activation to the
`corresponding CoA ester as shown previously (5). Further-
`more, when acetate was the main carbon source, propionate
`only showed a strong reduction of conidiation when used in the
`same or even in a higher concentration.
`A strong negative effect on biomass formation caused by the
`addition of propionate was observed when strains were grown
`in liquid media. It has been shown that propionate possesses
`some inhibitory effect on growth of an A. nidulans wild-type
`strains when glucose was the main carbon source (7). A 40%
`reduction of biomass was observed when 50 mM propionate
`was added, and a 60% reduction was observed in the presence
`of 100 mM propionate. On the other hand, the addition of
`various amounts of propionate had no effect on biomass for-
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`FIG. 2. Southern analysis of transformants. (A) Southern blot of different transformants in comparison to the original mclA⫹ strain RMS011.
`Strains A9 and A10 show the expected shift from 1.8 to 9.6 kb. (B) Diagram of the wild type and a transformant with a homologous integration
`of the deletion construct.
`
`mation when acetate was the main carbon source. Compared
`to that the methylisocitrate lyase mutants were severely af-
`fected in growth when glucose-propionate or acetate-propi-
`onate served as carbon sources. The strongest inhibitory effect
`was noticed when glycerol was the main carbon source (Table
`2). This insinuates a severe inhibition of mitochondrial metab-
`olism because glycerol is, like glucose, metabolized via the
`citric acid cycle without the gain of energy from cytoplasmic
`glycolysis.
`Confirmation of mutant phenotype by sexual crossing. In
`order to confirm that the observed phenotypes were due to a
`deletion of the genomic locus coding for methylisocitrate lyase
`and were not caused by other secondary effects, the mutant
`strain SMBA9 was crossed with strain SRF200. The transfor-
`
`mant strain SMBA9 was prototroph for arginine because the
`argB gene was used as a selection marker during transforma-
`tion. Nevertheless, the original copy of argB in this strain was
`still deleted. Strain SRF200 also carried a deleted argB locus.
`Due to that constellation, all arginine prototrophic progenies
`of the sexual crossing were supposed to carry a deleted meth-
`ylisocitrate lyase gene and should show a sporulation defect in
`the presence of propionate. Because of the different conidial
`colors of the two strains (yellow for SMBA9 and green for
`SRF200), ascospores deriving from a crossing event of the two
`partners were easily identified by the deviation of green and
`yellow spore color of the offsprings from a single cleistoth-
`ecium. For crossing, the carbon source solely consisted of glu-
`cose. Under this condition, no phenotype in cleistothecia for-
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`FIG. 3. Phenotypes of A. nidulans strains grown on different carbon sources. (A) The mclA⫹ strain RMS011 and the mclA deletion strain
`SMBA10 were point inoculated onto media containing different carbon sources (G, glucose; P, propionate; A, acetate; Glyc, glycerol; numbers
`denote millimolar concentrations of carbon sources). The mutant strain is severely affected in growth and conidiation upon the addition of
`propionate. Propionate alone does not serve as a carbon source for the mutant strain. For further explanations refer to the text. (B) Progenies of
`a sexual cross of the deletion strain SMBA9 and the green mclA⫹ strain SRF200. Arginine auxotrophic and prototrophic strains were selected and
`point inoculated onto glucose medium with or without arginine and with an addition of propionate. All strains show the expected phenotypes (see
`also panel A, G50 and G50 P20).
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`3 the purification factor for methylisocitrate lyase never ex-
`ceeded a value of 2, whereas that of isocitrate lyase was 57-
`fold. The purified enzyme was subjected to peptide mass anal-
`ysis by matrix-assisted laser desorption ionization–time of
`flight (kindly performed by the Zentrallabor fu¨r Proteinanaly-
`tik, Ludwig-Maximilians-Universita¨t Mu¨nchen, Munich, Ger-
`many). Peptide masses showed a sequence coverage of 57%,
`resembling an unambiguous identification of the protein as
`isocitrate lyase. The Km values for the substrates isocitrate and
`methylisocitrate were determined as 0.35 and 3.33 mM, respec-
`tively. The virtual Vmax with the substrate methylisocitrate was
`1.34 U