Heterologous expression of the bchM gene product from Rhodobacter capsulatus and demonstration that it encodes S-adenosyl-L-methionine:Mg-protoporphyrin IX methyltransferase.

The bacteriochlorophyll biosynthesis gene, bchM, from Rhodobacter capsulatus was previously believed to code for a polypeptide involved in formation of the cyclopentone ring of protochlorophyllide from Mg-protoporphyrin IX monomethyl ester. In this study, R. capsulatus bchM was expressed in Escherichia coli and the gene product was subsequently demonstrated by enzymatic analysis to catalyze methylation of Mg-protoporphyrin IX to form Mg-protoporphyrin IX monomethyl ester. Activity required the substrates Mg-protoporphyrin IX and S-adenosyl-L-methionine. 14C-labeled product was formed in incubations containing 14C-methyl-labeled S-adenosyl-L-methionine. On the basis of these and previous results, we also conclude that the bchH gene, which was previously reported to code for Mg-protoporphyrin IX methyltransferase, is most likely involved in the Mg chelation step.     

Formation of the isocyclic ring of chlorophyll by isolated Chlamydomonas reinhardtii chloroplasts

Chlamydomonas reinhardtii chloroplasts catalyzed two sequential steps of Chl biosynthesis, S-adenosyl-l-methionine:Mg-protoporphyrin IX methyltransferase and Mg-protoporphyrin IX monomethyl ester oxidative cyclase. A double mutant strain of C. reinhardtii was constructed which has a cell wall deficiency and is unable to form chlorophyll in the dark. Dark-grown cells were disrupted with a BioNeb nebulizer under conditions which lysed the plasma membrane but not the chloroplast envelope. Chloroplasts were purified by Percoll density gradient centrifugation. The purified chloroplasts were used to define components required for the biosynthesis of Mg-2,4-divinylpheoporphyrin a ₅ (divinyl protochlorophyllide) from Mg-protoporphyrin IX. Product formation requires the addition of Mg-protoporphyrin IX, the substrate for S-adenosyl-l-methionine:Mg-protoporphyrin IX methyltransferase which produces Mg-protoporphyrin IX monomethyl ester. The Mg-protoporphyrin IX monomethyl ester that is generated in situ is the substrate for Mg-protoporphyrin IX monomethyl ester oxidative cyclase. The reaction product was identified as Mg-2,4-divinylpheoporphyrin a ₅ (divinyl protochlorophyllide) by excitation and emission spectrofluorometry and HPLC on ion-paired reverse-phase and polyethylene columns. Mg-2,4-divinylpheoporphyrin a ₅ formation by the coupled enzyme system required O₂ and was stimulated by the addition of NADP⁺, an NADPH regenerating system, and S-adenosyl-l-methionine. Product was formed at a relatively steady rate for at least 60 min.Abbreviations MgDVP Mg-2,4-divinylpheoporphyrin a ₅ (divinyl protochlorophyllide) - SAM S-adenosyl-l-methionine     

Magnesium insertion by magnesium chelatase in the biosynthesis of zinc bacteriochlorophyll a in an aerobic acidophilic bacterium Acidiphilium rubrum

To elucidate the mechanism for formation of zinc-containing bacteriochlorophyll a in the photosynthetic bacterium Acidiphilium rubrum, we isolated homologs of magnesium chelatase subunits (bchI, -D, and -H). A. rubrum bchI and -H were encoded by single genes located on the clusters bchP-orf168-bchI-bchD-orf320-crtI and bchF-N-B-H-L as in Rhodobacter capsulatus, respectively. The deduced sequences of A. rubrum bchI, -D, and -H had overall identities of 59. 8, 40.5, and 50.7% to those from Rba. capsulatus, respectively. When these genes were introduced into bchI, bchD, and bchH mutants of Rba. capsulatus for functional complementation, all mutants were complemented with concomitant synthesis of bacteriochlorophyll a. Analyses of bacteriochlorophyll intermediates showed that A. rubrum cells accumulate magnesium protoporphyrin IX monomethyl ester without detectable accumulation of zinc protoporphyrin IX or its monomethyl ester. These results indicate that a single set of magnesium chelatase homologs in A. rubrum catalyzes the insertion of only Mg(2+) into protoporphyrin IX to yield magnesium protoporphyrin IX monomethyl ester. Consequently, it is most likely that zinc-containing bacteriochlorophyll a is formed by a substitution of Zn(2+) for Mg(2+) at a step in the bacteriochlorophyll biosynthesis after formation of magnesium protoporphyrin IX monomethyl ester.     

Small Cab-like proteins regulating tetrapyrrole biosynthesis in the cyanobacterium Synechocystis sp. PCC 6803

In the cyanobacterium Synechocystis sp. PCC 6803 five open reading frames (scpA–scpE) have been identified that code for single-helix proteins resembling helices I and III of chlorophyll a/b-binding (Cab) antenna proteins from higher plants. They have been named SCPs (small Cab-like proteins). Deletion of a single scp gene in a wild-type or in a photosystem I-less (PS I-less) strain has little effect. However, the effects of functional deletion of scpB or scpE were remarkable under conditions where chlorophyll availability was limited. When cells of a strain lacking PS I and chlL (coding for a polypeptide needed for light-independent protochlorophyllide reduction) were grown in darkness, the phycobilin and protochlorophyllide levels decreased upon deletion of scpB or scpE and the protoheme level was reduced in the strain lacking scpE. Addition of -aminolevulinic acid (ALA) in darkness drastically increased the level of Mg-protoporphyrin IX and Mg-protoporphyrin IX monomethyl ester in the PS I-less/chlL ^–/scpE ^– strain, whereas PChlide accumulated in the PS I-less/chlL ^–/scpB ^– strain. In the PS I-less/chlL ^– control strain ALA supplementation did not lead to large changes in the levels of tetrapyrrole biosynthesis intermediates. We propose that ScpE and ScpB regulate tetrapyrrole biosynthesis as a function of pigment availability. This regulation occurs primarily at an early step of tetrapyrrole biosynthesis, prior to ALA. In view of the conserved nature of chlorophyll-binding sites in these proteins, it seems likely that regulation by SCPs occurs as a function of chlorophyll availability, with SCPs activating chlorophyll biosynthesis steps when they do not have pigments bound.     

RegA control of bacteriochlorophyll and carotenoid synthesis in Rhodobacter capsulatus

We provide in vivo genetic and in vitro biochemical evidence that RegA directly regulates bacteriochlorophyll and carotenoid biosynthesis in Rhodobacter capsulatus. beta-Galactosidase expression assays with a RegA-disrupted strain containing reporter plasmids for Mg-protoporphyrin IX monomethyl ester oxidative cyclase (bchE), Mg-protoporphyrin IX chelatase (bchD), and phytoene dehydrogenase (crtI) demonstrate RegA is responsible for fourfold anaerobic induction of bchE, threefold induction of bchD, and twofold induction of crtI. Promoter mapping studies, coupled with DNase I protection assays, map the region of RegA binding to three sites in the bchE promoter region. Similar studies at the crtA and crtI promoters indicate that RegA binds to a single region equidistant from these divergent promoters. These results demonstrate that RegA is directly responsible for anaerobic induction of bacteriochlorophyll biosynthesis genes bchE, bchD, bchJ, bchI, bchG, and bchP and carotenoid biosynthesis genes crtI, crtB, and crtA.     

Anaerobic protoporphyrin biosynthesis does not require incorporation of methyl groups from methionine.

It was recently reported (H. Akutsu, J.-S. Park, and S. Sano, J. Am. Chem. Soc. 115:12185-12186, 1993) that in the strict anaerobe Desulfovibrio vulgaris methyl groups from exogenous L-methionine are incorporated specifically into the 1 and 3 positions (Fischer numbering system) on the heme groups of cytochrome c3. It was suggested that under anaerobic conditions, protoporphyrin IX biosynthesis proceeds via a novel pathway that does not involve coproporphyrinogen III as a precursor but instead may use precorrin-2 (1,3-dimethyluroporphyrinogen III), a siroheme and vitamin B12 precursor which is known to be derived from uroporphyrinogen III via methyl transfer from S-adenosyl-L-methionine. We have critically tested this hypothesis by examining the production of protoporphyrin IX-based tetrapyrroles in the presence of exogenous [14C]methyl-L-methionine under anaerobic conditions in a strict anaerobe (Chlorobium vibrioforme) and a facultative anaerobe (Rhodobacter capsulatus). In both organisms, 14C was incorporated into the bacteriochlorophyll precursor, Mg-protoporphyrin IX monomethyl ester. However, most of the label was lost upon base hydrolysis of this compound to yield Mg-protoporphyrin IX. These results indicate that although the administered [14C]methyl-L-methionine was taken up, converted into S-adenosyl-L-methionine, and used for methyl transfer reactions, including methylation of the 6-propionate of Mg-protoporphyrin IX, methyl groups were not transferred to the porphyrin nucleus of Mg-protoporphyrin IX. In other experiments, a cysG strain of Salmonella typhimurium, which cannot synthesize precorrin-2 because the gene encoding the enzyme that catalyzes methylation of uroporphyrinogen III at positions 1 and 3 is disrupted, was capable of heme-dependent anaerobic nitrate respiration and growth on the nonfermentable substrate glycerol, indicating that anaerobic biosynthesis of protoporphyrin IX-based hemes does not require the ability to methylate uroporphyrinogen III. Together, these results indicate that incorporation of L-methionine-deprived methyl groups into porphyrins or their precursors is not generally necessary for the anaerobic biosynthesis of protoporphyrin IX-based tetrapyrroles.     

Measurement of serum levels of natalizumab, an immunoglobulin G4 therapeutic monoclonal antibody

Chlorophyllide a is a metabolite late in the biosynthesis of chlorophylls and bacteriochlorophylls. Isolation procedures for chlorophyllide a from Rhodobacter capsulatus CB1200 and barley (Hordeum vulgare L.) are described and compared. R. capsulatus CB1200 is a double mutant in the bacteriochlorophyllide a biosynthetic pathway, and chlorophyllide a is excreted by the cells when grown in Tween 80-containing liquid medium. It was purified by liquid or solid phase extraction, yielding 7 mg of chlorophyllide a from 1 L of culture. In a second approach, intrinsic chlorophyllase activity was used to dephytylate chlorophyll in an acetonic preparation of leaves of wild-type or chlorophyll b-deficient barley. Purification was achieved by liquid phase extraction, yielding 14 μg of chlorophyllide a per gram of barley leaves. Chlorophyllide a was identified by thin layer chromatography, absorption spectroscopy, and mass spectrometry.     

Inactivation of Mg chelatase during transition from anaerobic to aerobic growth in Rhodobacter capsulatus

The facultative photosynthetic bacterium Rhodobacter capsulatus can adapt from an anaerobic photosynthetic mode of growth to aerobic heterotrophic metabolism. As this adaptation occurs, the cells must rapidly halt bacteriochlorophyll synthesis to prevent phototoxic tetrapyrroles from accumulating, while still allowing heme synthesis to continue. A likely control point is Mg chelatase, the enzyme that diverts protoporphyrin IX from heme biosynthesis toward the bacteriochlorophyll biosynthetic pathway by inserting Mg(2+) to form Mg-protoporphyrin IX. Mg chelatase is composed of three subunits that are encoded by the bchI, bchD, and bchH genes in R. capsulatus. We report that BchH is the rate-limiting component of Mg chelatase activity in cell extracts. BchH binds protoporphyrin IX, and BchH that has been expressed and purified from Escherichia coli is red in color due to the bound protoporphyrin IX. Recombinant BchH is rapidly inactivated by light in the presence of O(2), and the inactivation results in the formation of a covalent adduct between the protein and the bound protoporphyrin IX. When photosynthetically growing R. capsulatus cells are transferred to aerobic conditions, Mg chelatase is rapidly inactivated, and BchH is the component that is most rapidly inactivated in vivo when cells are exposed to aerobic conditions. The light- and O(2)-stimulated inactivation of BchH could account for the rapid inactivation of Mg chelatase in vivo and provide a mechanism for inhibiting the synthesis of bacteriochlorophyll during adaptation of photosynthetically grown cells to aerobic conditions while still allowing heme synthesis to occur for aerobic respiration.     

Recent advances in chlorophyll biosynthesis

The importance of chlorophyll (Chl) to the process of photosynthesis is obvious, and there is clear evidence that the regulation of Chl biosynthesis has a significant role in the regulation of assembly of the photosynthetic apparatus. The understanding of Chl biosynthesis has rapidly advanced in recent years. The identification of genetic loci associated with each of the biochemical steps has been accompanied by a greater appreciation of the role of Chl biosynthesis intermediates in intracellular signaling. The purpose of this review is to provide a source of information for all the steps in Chl and bacteriochlorophyll a biosynthesis, with an emphasis on steps that are believed to be key regulation points.     

Mg-chelatase of tobacco: identification of a Chl D cDNA sequence encoding a third subunit, analysis of the interaction of the three subunits with the yeast two-hybrid system, and reconstitution of the enzyme activity by co-expression of recombinant CHL D, CHL H and CHL I

Mg-protoporphyrin IX chelatase catalyzes insertion of the magnesium ion into protoporphyrin IX, the last common intermediate precursor in chlorophyll and heme biosynthesis, to form Mg-protoporphyrin IX. In Rhodobacter sphaeroides, and Synechocystis, the three open reading frames bchD/chlD, bchH/chlH and bchl/chll encode proteins which are required for in vitro Mg-chelatase activity. In higher plants also, three proteins are necessary for the Mg chelation, and genes homologous to bchH and bchl have been isolated previously. In this study, a novel tobacco cDNA sequence homologous to bchD is isolated and initially characterized. Together with the tobacco clones encoding the other two subunits, full-length cDNAs are now available for the first time for all three subunits of one plant species. The CHL D polypeptide deduced from the open reading frame encodes a protein of 758 aa (82.9 kDa) with an amino terminal extension that resembles a plastid transit peptide. Sequence comparison of tobacco CHL D revealed similarities to the D subunit of Rhodobacter and Synechocystis of 44% and 75%. The amino terminal half of CHL D shows significant similarity (46%) to the entire CHL I peptide sequence, indicating a gene duplication from an ancestral gene. The carboxy terminal half seemed to be unique. Both parts of CHL D are linked with a glutamine/asparagine/proline-rich region flanked by a highly acid-rich segment. Protein-protein interaction among the three subunits CHL D, H and I was studied using the yeast two-hybrid system. Physical interaction was demonstrated between CHL D and CHL I indicating that CHL D is part of the Mg-chelatase. Heterodimer formation of CHL H with CHL I or CHL D could not be demonstrated by transactivation of the lacZ reporter gene. Homodimerization of the CHL D subunit was indicated in the more sensitive assay on X-Gal-containing agar plates. In vitro Mg²⁺ insertion into protoporphyrin IX was demonstrated in protein extracts of yeast strains expressing the three subunits of tobacco Mg-chelatase. The reconstitution of the recombinant enzyme activity required additional ATP.     

An analysis of precursors accumulated by several chlorophyll biosynthetic mutants of maize

Summary Several mutants of maize defective in chlorophyll synthesis are analysed. By feeding shoots of dark-grown seedlings -aminolevulinic acid, the regulatory step in chlorophyll biosynthesis is bypassed and chlorophyll precursors accumulate. In normal plants this results in a buildup of protoporphyrin IX and protochlorophyllide, while mutants accumulate precursors, depending on the site of the mutant-induced lesion. Mutants at three loci, l ^*-Blandy4, 113, and oy, are defective in conversion of protoporphyrin IX to Mg-protoporphyrin. Mutants at the oro and oro2 loci are defective in conversion of Mg-protoporphyrin monomethyl ester to protochlorophyllide. A dominant modifier gene, Orom, which allows oro seedlings to bypass their lesion is also described.Journal Paper No J-9076 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa Project No. 2035     

The reduction of vinyl side-chains of Mg-protoporphyrin IX monomethyl ester in vitro

Portions of crude homogenates of etiolated wheat seedlings incubated with Mg-protoporphyrin IX and $\text{S-}\text{adenosyl}\text{-}\text{L}\text{-}\text{methionine}$ and then added to other portions of the same crude homogenates that were pretreated with [1-³H]ethanol and yeast alcohol dehydrogenase provided, after a short reaction period, ³H-labeled Mg-protoporphyrin IX monomethyl ester. The ³H-labeled Mg-protoporphyrin IX monomethyl ester thus obtained was shown to contain the ³H in one reduced (to ethyl) vinyl side-chain. Subsequently, ³H-labeled Mg-monoethyl-(monodivinyl)-protoporphyrin IX monomethyl ester was obtained when Mg-protoporphyrin IX monomethyl ester and [³H]NADH were added to dialyzed crude homogenates of etiolated wheat seedlings. Insignificant amounts of ³H were incorporated into poprhyrin substrates when Mg-2,4-divinylpheoporphyrin a₅ or [³H]NADPH were substituted in reaction mixtures for Mg-protoporphyrin IX monomethyl ester or [³H]NADPH, respectively. The results of these and further experiments suggest that an NADPH-dependent enzyme in the crude homogenates of etiolated wheat seedlings was capable of catalyzing the reduction to ethyl of one vinyl side-chain of Mg-protoporphyrin IX monomethyl ester. These findings suggest that the 4-vinyl side-chain reductive reaction likely occurs after the biosynthesis IX monomethyl ester, but before isocyclic ring formation in the pathway to chlorophyll a.     

Cloning and sequencing of the genes encoding the large and the small subunits of the H2 uptake hydrogenase (hup) of Rhodobacter capsulatus

Summary The structural genes (hup) of the H₂ uptake hydrogenase of Rhodobacter capsulatus were isolated from a cosmid gene library of R. capsulatus DNA by hybridization with the structural genes of the H₂ uptake hydrogenase of Bradyrhizobium japonicum. The R. capsulatus genes were localized on a 3.5 kb HindIII fragment. The fragment, cloned onto plasmid pAC76, restored hydrogenase activity and autotrophic growth of the R. capsulatus mutant JP91, deficient in hydrogenase activity (Hup^-). The nucleotide sequence, determined by the dideoxy chain termination method, revealed the presence of two open reading frames. The gene encoding the large subunit of hydrogenase (hupL) was identified from the size of its protein product (68108 dalton) and by alignment with the NH₂ amino acid protein sequence determined by Edman degradation. Upstream and separated from the large subunit by only three nucleotides was a gene encoding a 34 256 dalton polypeptide. Its amino acid sequence showed 80% identity with the small subunit of the hydrogenase of B. japonicum. The gene was identified as the structural gene of the small subunit of R. capsulatus hydrogenase (hupS). The R. capsulatus hydrogenase also showed homology, but to a lesser extent, with the hydrogenase of Desulfovibrio baculatus and D. gigas. In the R. capsulatus hydrogenase the Cys residues, (13 in the small subunit and 12 in the large subunit) were not arranged in the typical configuration found in [4Fe–4S] ferredoxins.     

Expression of the cbbLcbbS and cbbM genes and distinct organization of the cbb Calvin cycle structural genes of Rhodobacter capsulatus

Rhodobacter capsulatus fixes CO₂ via the Calvin reductive pentose phosphate pathway and, like some other nonsulfur purple bacteria, is known to synthesize two distinct structural forms of ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO). Cosmid clones that hybridized to form I (cbbLcbbS) and form II (cbbM) RubisCO gene probes were isolated from a genomic library of R. capsulatus strain SB1003. Southern blotting and hybridization analysis with gene-specific probes derived from Rhodobacter sphaeroides revealed that R. capsulatus cbbM is clustered with genes encoding other enzymes of the Calvin cycle, including fructose 1,6/sedoheptulose 1,7-bisphosphatase (cbbF), phosphoribulokinase (cbbP), transketolase (cbbT), glyceraldehyde-3-phosphate dehydrogenase (cbbG), and fructose 1,6-bisphosphate aldolase (cbbA), as well as a gene (cbbR) encoding a divergently transcribed LysR-type regulatory protein. Surprisingly, a cosmid clone containing the R. capsulatus form I RubisCO genes (cbbL and cbbS) failed to hybridize to the other cbb structural gene probes, unlike the situation with the closely related organism R. sphaeroides. The form I and form II RubisCO genes were cloned into pUC-derived vectors and were expressed in Escherichia coli to yield active recombinant enzyme in each case. Complementation of a RubisCO-deletion strain of R. sphaeroides to photosynthetic growth by R. capsulatus cbbLcbbS or cbbM was achieved using the broad host-range vector, pRK415, and R. sphaeroides expression vector pRPS-1. Received: 6 June 1995 / Accepted: 29 September 1995     

拟南芥黄化突变体chlm-4的基因定位与分析

叶绿素是光合作用必需的重要色素,其合成需要Mg-原卟啉Ⅸ甲基转移酶等一系列酶的催化。我们通过筛选拟南芥EMS（乙基磺酸甲酯,ethyl-methane sulphonate）突变体库,分离到一个黄化突变体。该突变体叶绿素含量显著减少,叶绿体内垛叠的基粒缺失。遗传学分析表明,该突变体的黄化表型是由单基因控制的隐性性状。利用图位克隆的方法最终将基因定位在第IV条染色体分子标记F13M23和T30C3之间114kb的区间内,其中包含编码Mg-原卟啉Ⅸ甲基转移酶的CHLM基因。通过测序及等位分析表明该突变体是chlm的等位突变体,命名为chlm-4。在chlm-4中,CHLM蛋白的Gly59突变成Glu59,说明Gly59对于Mg-原卟啉Ⅸ甲基转移酶功能的行使是必需的。     

Introduction of a new branchpoint in tetrapyrrole biosynthesis in Escherichia coli by co-expression of genes encoding the chlorophyll-specific enzymes magnesium chelatase and magnesium protoporphyrin methyltransferase.

The genes encoding the three Mg chelatase subunits, ChlH, ChlI and ChlD, from the cyanobacterium Synechocystis PCC6803 were all cloned in the same pET9a-based Escherichia coli expression plasmid, forming an artificial chlH-I-D operon under the control of the strong T7 promoter. When a soluble extract from IPTG-induced E. coli cells containing the pET9a-ChlHID plasmid was assayed for Mg chelatase activity in vitro, a high activity was obtained, suggesting that all three subunits are present in a soluble and active form. The chlM gene of Synechocystis PCC6803 was also cloned in a pET-based E. coli expression vector. Soluble extract from an E. coli strain expressing chlM converted Mg-protoporphyrin IX to Mg-protoporphyrin monomethyl ester, demonstrating that chlM encodes the Mg-protoporphyrin methyltransferase of Synechocystis. Co-expression of the chlM gene together with the chlH-I-D construct yielded soluble protein extracts which converted protoporphyrin IX to Mg-protoporphyrin IX monomethyl ester without detectable accumulation of the Mg-protoporphyrin IX intermediate. Thus, active Mg chelatase and Mg-protoporphyrin IX methyltransferase can be coupled in E. coli extracts. Purified ChlI, -D and -H subunits in combination with purified ChlM protein were subsequently used to demonstrate in vitro that a molar ratio of ChlM to ChlH of 1 to 1 results in conversion of protoporphyrin IX to Mg-protoporphyrin monomethyl ester without significant accumulation of Mg-protoporphyrin.     

Involvement of tetrapyrroles in inter-organellar signaling in plants and algae

For the assembly of a functional chloroplast, the coordinated expression of genes distributed between nucleus and chloroplasts is a prerequisite. While the nucleus plays an undisputed dominant role in controling biogenesis and functioning of chloroplasts, plastidic signals appear to control the expression of a subset of nuclear genes; the majority of which encodes chloroplast constituents. Tetrapyrrole biosynthesis intermediates are attractive candidates for one type of plastidic signal ever since an involvement of Mg–porphyrins in signaling from chloroplast to nucleus was first demonstrated in Chlamydomonas reinhardtii. Since then, Mg-protoporphyrin IX has been shown to exert a regulatory function on nuclear genes in higher plants as well. Here we review evidence for the role played by tetrapyrroles in inter-organellar communication. We also report on a screening for nuclear genes that may be subject to regulation by tetrapyrroles. This revealed that (i) >HEMA, the gene encoding the first enzyme specific for porphyrin biosynthesis is induced by Mg-protoporphyrin IX, (ii) several nuclear HSP70 genes are regulated by tetrapyrroles. Members of the gene family induced by the feeding of Mg–rotoporphyrin IX encode chaperones located in either the chloroplast or the cytosol. These results point to an important role of Mg–tetrapyrroles as plastidic signal in controling the initial step of porphyrin biosynthesis, and the synthesis of chaperones involved in protein folding in cytosol/stroma, protein transport into organelles, and the stress response.     

Deletion of the <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> lysine biosynthesis gene <Emphasis Type="Italic">lysF</Emphasis> encoding homoaconitase leads to attenuated virulence in a low-dose mouse infection model of invasive aspergillosis

Aspergillus fumigatus is an important pathogen of the immunocompromised host, causing pneumonia and invasive disseminated disease with high mortality. In order to determine the importance of lysine biosynthesis for growth and pathogenicity, the A. fumigatus lysF gene, encoding a homologue of the A. nidulans homoaconitase LysF, was cloned and characterized. Cosmid cosGTM encoding lysF complemented a lysF mutant of Aspergillus nidulans. A. fumigatus lysF was deleted, resulting in a lysine-auxotroph. This phenotype was complemented to the wild-type by supplementation of the medium with both L-lysine and -aminoadipic acid, or transformation using cosmid cosGTM. To study the virulence of the lysF deletion mutant of A. fumigatus, a low-dose intranasal mouse infection model of invasive aspergillosis was optimized for immunosuppressed BALB/c mice, allowing the application of an infection dose as low as 5×10³ conidia per mouse. In this murine model, the lysF mutant was avirulent, suggesting that lysine biosynthesis, or at least a functional homoaconitase, is important for survival of A. fumigatus in vivo and a potential target for antifungal drugs.