The bacterial phleomycin resistance geneble as a dominant selectable marker inChlamydomonas

A chimeric gene composed of the coding sequence of theble gene fromStreptoalloteichus hindustanus fused to the 5 and 3 untranslated regions of theChlamydomonas reinhardtii nuclear geneRBCS2 has been constructed. Introduction of this chimeric gene into the nuclear genome ofC. reinhardtii by co-transformation with theARG7 marker yields Arg⁺ transformants of which approximately 80% possess theble gene. Of these co-transformants, approximately 3% display a phleomycin-resistant (Pm^R) phenotype. Western blot analysis using antibodies against theble gene product confirms the presence of the protein in the Pm^R transformants and genetic analysis demonstrates the co-segregation of theble gene with the phenotype in progeny arising from the mating of a Pm^R transformant to wild-type strains. Direct selection of Pm^R transformants was achieved by allowing an 18-h period for recovery and growth of transformed cells prior to selection. This work represents the first demonstration of stable expression and inheritance of a foreign gene in the nuclear genome ofC. reinhardtii and provides a useful dominant marker for nuclear transformation.     

The potato mitochondrial initiator methionine tRNA gene and its flanking regions: an illustration of the diversity of mitochondrial genome rearrangements among plant species

The initiator methionine transfer RNA (tRNAf ^Met) gene was identified on a 347 bpEco RI-Hind III DNA fragment of the potato mitochondrial (mt) genome. The sequence of this gene shows 1 to 7 nucleotide differences with the other plant mt tRNAsf ^Met or tRNAf ^Met genes studied so far. Whereas the tRNAf ^Met gene is present as a single copy in the potato mt genome, a tRNA pseudogene corresponding to 60% of a complete tRNA (from the 5 end to the variable region) and located at 105 nucleotides upstream of the tRNAf ^Met gene on the opposite strand was shown to be repeated at least three times. Furthermore, the physical environment of the tRNAf ^Met gene in the mt genome is very different among plants, which suggests that the tRNAf ^Met gene region has often been implicated in recombination events of plant mt genomes leading to important rearrangements in gene order.     

Surrogate transformation of perennial ryegrass, Lolium perenne, using genetically modified Acremonium endophyte.

Summary Conditions have been developed for transforming protoplasts of the perennial ryegrass endophyteAcremonium strain 187BB. Unlike most other ryegrass endophytes, this strain does not produce the lolitrem B neurotoxin and is therefore suitable as a host for surrogate introduction of foreign genes into grasses. Transformation frequencies of 700–800 transformants/g DNA were obtained for both linear and circular forms of pAN7-1, a hygromycin (hph) resistant plasmid. Up to 80% of the linear transformants were stable on further culturing but only 25% of the circular transformants retained hygromycin resistance. Integration of pAN7-1 into the genome was confirmed by Southern blotting and probing of genomic digests of transformant DNA. Both single and tandemly repeated copies of the plasmid were found in the genome and both the number and sites of integration varied among the transformants. At least 13 chromosomes were identified in 187BB using contour-clamped homogeneous electric field (CHEF) gel electrophoresis. Probing of Southern blots of these gels confirmed that pAN7-1 had integrated into different chromosomes. The -glucuronidase (GUS) gene,uidA, was also introduced into 187BB by co-transformation of pNOM-2 with pAN7-1. GUS activity was detected by growing the transformants on plates containing 5-bromo-4-chloro-3-indolyl -D-glucuronic acid and by enzyme assays of mycelial extracts. Severalhph- anduidA-containing transformants were reintroduced into ryegrass seedlings and expression of GUS visualized in vivo, demonstrating that 187BB can be used as a surrogate host to introduce foreign genes into perennial ryegrass. Molecular analysis of fungal isolates from the leaf sheath confirmed that the pattern of pAN7-1 and pNOM-2 hybridizing fragments was identical to that observed in the fungus used as inoculum.     

Mitochondrial Genome of <Emphasis Type="Italic">Ciona savignyi</Emphasis> (Urochordata,Ascidiacea, Enterogona): Comparison of Gene Arrangement and tRNA Genes with <Emphasis Type="Italic">Halocynthia roretzi</Emphasis> Mitochondrial Genome

The complete nucleotide sequence of the urochordate Ciona savignyi (Ascidiacea, Enterogona) mitochondrial (mt) genome (14,737 bp) was determined. The Ciona mt genome does not encode a gene for ATP synthetase subunit 8 but encodes an additional tRNA^Gly gene (anticodon UCU), as is the case in another urochordate, Halocynthia roretzi (Ascidiacea, Pleurogona), mt genome. In addition, the Ciona mt genome encodes two tRNA^Met genes; anticodon CAT and anticodon TAT. The tRNA^Cys gene is thought to lack base pairs at the D-stem. Thus, the Ciona mt genome encodes 12 protein, 2 rRNA, and 24 tRNA genes. The gene arrangement of the Ciona mt genome differs greatly from those of any other metazoan mt genomes reported to date. Only three gene boundaries are shared between the Halocynthia and the Ciona mt genomes. Molecular phylogenetic analyses based on amino acid sequences of mt protein genes failed to demonstrate the monophyly of the chordates.     

Genomic characterization of thermophilic Geobacillus species isolated from the deepest sea mud of the Mariana Trench

The thermophilic strains HTA426 and HTA462 isolated from the Mariana Trench were identified as Geobacillus kaustophilus and G. stearothermophilus, respectively, based on physiologic and phylogenetic analyses using 16S rDNA sequences and DNA–DNA relatedness. The genome size of HTA426 and HTA462 was estimated at 3.23–3.49 Mb and 3.7–4.49 Mb, respectively. The nucleotide sequences of three independent -phage inserts of G. stearothermophilus HTA462 have been determined. The organization of protein coding sequences (CDSs) in the two -phage inserts was found to differ from that in the contigs corresponding to each insert assembled by the shotgun clones of the G. kaustophilus HTA426 genome, although the CDS organization in another insert is identical to that in the HTA426 genome.     

Morphological and molecular characterization of fertile tetraploid somatic hybrids produced by protoplast electrofusion and PEG-induced fusion between Lycopersicon esculentum Mill. and Lycopersicon peruvianum Mill.

Summary Mesophyl protoplasts of two genotypes of cultivated tomato (Lycopersicon esculentum Mill.) and one of its wild relative species (Lycopersicon peruvianum Mill.) were fused by using electrofusion and polyethyleneglycol-induced fusion. Forty-three fertile tetraploid somatic hybrid plants, each deriving from separate calli, were recovered from both fusion procedures. Electrofusion appeared more efficient than chemical fusion for the production of somatic hybrids. These plants appeared morphologically similar, whatever the fusion procedure and tomato genotype. They had intermediate leaf, inflorescence, and flower morphology. After self-pollination, the hybrids set fruit of intermediate size and color. The hybrid nature of these plants was confirmed by isoelectric focusing of the Rubisco small subunits used as nuclear markers. L. esculentum and L. peruvianum were distinguished by means of two chloroplast markers: CF1-ATPase subunit as analyzed by isoelectro-focusing and ct DNA restriction patterns. All hybrids displayed both ct markers of only one parent with no biased transmission. Mitochondrial (mt) DNAs were prepared from flower buds by using miniaturized CsCl gradients. Preliminary analysis indicated that mt genomes from the hybrids all differed from those of both parents. mt DNA Sall restriction enzyme analysis revealed that all but two hybrids contained one novel fragment of 13.5 kb. Gene mapping experiments showed that the mt apocytochrome b and ATPase subunit 9 homologies in the somatic hybrid mt DNA resembled L. esculentum and L. peruvianum, respectively; the mt nad5 probe distinguished at least four distinct patterns in the hybrids. These results indicated that mt DNA rearrangements involving intergenomic recombinations occurred through protoplast fusion. A greater mt DNA polymorphism was induced with chemical fusion than with electrofusion.     

Localization of tRNA genes on the Petunia hybrida 3704 mitochondrial genome

22 tRNA genes corresponding to 17 tRNA species were localized on the master circle of Petunia hybrida mitochondrial (mt) DNA. Genes for trnN, trnM, trnS-GGA, trnW and trnH are of the chloroplast-like type and presumably originate from promiscuous chloroplast (cp) DNA sequences inserted into the petunia mitochondrial genome. A comparison of the mt tRNAs or tRNA genes population present in two monocotyledonous plants (wheat and maize) and two dicotyledonous plants (petunia and potato) show slight differences in the genetic origin of individual tRNAs. The organization of the petunia mt tRNA genes as well as the number of tRNA gene copies, compared to other plant species, is discussed.     

Alteration of mitochondrial genomes containing atpA genes in the sexual progeny of cybrids between Raphanus sativus cms line and Brassica napus cv. Westar

Summary We have investigated the fate of the mitochondrial genomes of cybrids derived from donor-recipient protoplast fusion between X-irradiated Raphanus sativus (cms line) and iodoacetamide-treated Brassica napus cv. Westar. Two out of ten fusion products were male-sterile with the diploid chromosome number of B. napus. The mitochondrial (mt) genomes of the cybrids and their progeny were further analyzed by DNA-DNA hybridizaion using the pea mitochondrial ATPase subunit gene (atpA) as a probe. One cybrid, 18-3, had a 3.0 kb fragment characteristic of B. napus and a 2.0 kb non-parental fragment when the BamHI-digested DNA was hybridized with the probe. In the first-backcrossed progeny of this cybrid, the hybridization pattern was not stably inherited. A 4.0 kb radish fragment, not detectable in the cybrid, appeared in one of the BC₁ generation siblings, and the 2.0 kb non-parental fragment was lost in another. The hybridization patterns in BC₁ progeny siblings of cybrid 12-9 were also varied. The alteration of mtDNA in the cybrid progeny continued to the BC₂ generation. There was no clear evidence of a heteroplasmic state or of sub-stoichiometric molecules in the mt genome of cybrid 18-3. A possible cause of the observed alteration in the mt genome is discussed.     

In vitro reconstitution of rice anthranilate synthase: distinct functional properties of the α subunits OASA1 and OASA2

Anthranilate synthase (AS) is a key enzyme in the biosynthesis of various indole compounds including tryptophan. AS consists of two subunits, and , and converts chorismate to anthranilate. Two or more AS -subunit genes have been identified and characterized in several land plants. Although subunits of AS induced by elicitation have been suggested to play significant roles in secondary metabolism, the biochemical and precise functional properties of individual AS isozymes have remained unclear. We have previously identified and characterized two AS -subunit genes (OASA1 and OASA2) in rice (Oryza sativa). To provide further insight into the enzymatic functions of AS isozymes in rice, we have now isolated rice cDNAs encoding the AS subunits OASB1 and OASB2 and reconstituted AS isozymes in vitro with the wheat germ cell-free system for protein expression. Both OASB subunits conferred glutamine-dependent AS activity on either OASA1 or OASA2, indicating the absence of a marked functional difference between the two subunits in terms of amidotransferase activity. Furthermore, both OASA subunits required assembly with a subunit to achieve maximal enzymatic activity even with NH ₄ ⁺ as the amino donor. The V _max and K _i for tryptophan of the OASA1-OASB1 isozyme with glutamine as the amino donor, however, were 2.4 and 7.5 times, respectively, those of OASA2-OASB1, suggesting that AS isozymes containing OASA1 possess a higher activity and are less sensitive to feedback inhibition than those containing OASA2. Our biochemical characterization of reconstituted AS isozymes has thus revealed distinct functional properties of these isozymes in rice.     

Induced expression of the Aspergillus nidulans QUTE gene introduced by transformation into Neurospora crassa

Summary Theqa-2 gene ofNeurospora crassa encodes catabolic dehydroquinase which catabolizes dehydroquinic acid to dehydroshikimic acid. TheQUTE gene ofAspergillus nidulans corresponds to theqa-2 gene ofN. crassa. The plasmid pEH1 containing theQUTE gene fromA. nidulans was used to transform aqa-2 ^– strain ofN. crassa. In Southern blot analyses, DNAs isolated from these transformants hybridized specifically to theQUTE gene probe. Northern blot analyses indicated thatQUTE mRNA was produced in the transformants. The functional integrity of theQUTE gene inN. crassa was indicated by transformants which had regained the ability to grow on quinic acid as sole carbon source. Enzyme assays indicated that the specific activities of catabolic dehydroquinase induced by quinic acid in the transformants ranged from 4% to 32% of that induced in wild-typeN. crassa. The evidence that theQUTE structural gene ofA. nidulans is inducible when introduced into theN. crassa genome implies that theN. crassa qa activator protein can recognize, at least to a limited extent, DNA binding sequences 5 to theQUTE gene.     

Nutritional Requirements of <Emphasis Type="Italic">Allisonella histaminiformans,</Emphasis> a Ruminal Bacterium that Decarboxylates Histidine and Produces Histamine

A xylose ABC (ATP-binding cassette) transport operon, xylFGH, was cloned from Thermoanaerobacter ethanolicus, a thermophilic ethanol-producing eubacterium. The cistrons code for a periplasmic D-xylose-binding protein (XylF, partial sequence of 250 amino acids), ATP-binding protein (XylG, 505 amino acids), and integral membrane protein (XylH, 388 amino acids). These results, together with previous work, indicate that duplicate copies of both xylF and xylH are present in the T. ethanolicus chromosome, suggesting ancient gene duplication or lateral gene transfer events. XylG resembles other eubacterial monosaccharide ABC-ATPases in that its two nucleotide-binding domains (NBDs) are highly homologous, yet significantly different with respect to putative catalytic residues. Unlike most other integral membrane ABC transport proteins, XylH apparently contains 11 or 12 transmembrane segments (TMS) and is similar to a small group of ABC permeases that defy the 2 × 6 helix paradigm. This is the first report of a monosaccharide ABC transport operon in a thermophilic anaerobic eubacterium.     

A novel class of <Emphasis Type="Italic">Helitron</Emphasis>- related transposable elements in maize contain portions of multiple pseudogenes

We recently described a maize mutant caused by an insertion of a Helitron type transposable element (Lal, S.K., Giroux, M.J., Brendel, V., Vallejos, E. and Hannah, L.C., 2003, Plant Cell, 15: 381–391). Here we describe another Helitron insertion in the barren stalk1 gene of maize. The termini of a 6525 bp insertion in the proximal promoter region of the mutant reference allele of maize barren stalk1 gene (ba1-ref) shares striking similarity to the Helitron insertion we reported in the Shrunken-2 gene. This insertion is embedded with pseudogenes that differ from the pseudogenes discovered in the mutant Shrunken-2 insertion. Using the common terminal ends of the mutant insertions as a query, we discovered other Helitron insertions in maize BAC clones. Based on the comparison of the insertion site and PCR amplified genomic sequences, these elements inserted between AT dinucleotides. These putative non-autonomous Helitroninsertions completely lacked sequences similar to RPA (replication protein A) and DNA Helicases reported in other species. A blastn analysis indicated that both the 5 and 3 termini of Helitrons are repeated in the maize genome. These data provide strong evidence that Helitron type transposable elements are active and may have played an essential role in the evolution and expansion of the maize genome.     

Introduction and maintenance of prokaryotic DNA inUstilago violacea

Summary A strain of the basidiomycete,Ustilago violacea, was transformed with a prokaryotic plasmid, pMP4-1, which confers resistance to neomycin.U. violacea transformants were selected at a frequency of 5 per g pMP4-1 DNA. Such transformants were at least 8-fold more resistant to neomycin than was the untransformed recipientU. violacea. Enzyme activity associated with the neomycin resistance gene was also found in the transformants. Southern DNA-DNA hybridization detected pMP4-1-derived sequences in both nuclear and mitochondrially-associated DNAs from transformants. The patterns of hybridization suggested integration of pMP4-1 sequences into the respective genomes. DNA from the nuclear fraction ofU. violacea transformants failed to produceE. coli transformants resistant to neomycin or to carbenicillin. In contrast, DNA from the mitochondrially-associated fraction inU. violacea transformants producedE. coli transformants resistant to neomycin. TheE. coli transformants contained a pMP4-1-derivative, pWP8, which was subsequently shown by Southern blot analysis to harborU. violacea mitochondrial DNA. Thus, a prokaryotic plasmid can be used to transform the eukaryoteU. violacea and acquire endogenous sequences from this organism.     

Overproduction of recombinant laccase using a homologous expression system in <Emphasis Type="Italic">Coriolus versicolor</Emphasis>

One of the major extracellular enzymes of the white-rot fungus Coriolus versicolor is laccase, which is involved in the degradation of lignin. We constructed a homologous system for the expression of a gene for laccase III (cvl3) in C. versicolor, using a chimeric laccase gene driven by the promoter of a gene for glyceraldehyde-3-phosphate dehydrogenase (gpd) from this fungus. We transformed C. versicolor successfully by introducing both a gene for hygromycin B phosphotransferase (hph) and the chimeric laccase gene. In three independent experiments, we recovered 47 hygromycin-resistant transformants at a transformation frequency of 13 transformants g^–1 of plasmid DNA. We confirmed the introduction of the chimeric laccase gene into the mycelia of transformants by a polymerase chain reaction in nine randomly selected transformants. Overproduction of extracellular laccase by the transformants was revealed by a colorimetric assay for laccase activity. We examined the transformant (T2) that had the highest laccase activity and found that its activity was significantly higher than that of the wild type, particularly in the presence of copper (II). Our transformation system should contribute to the efficient production of the extracellular proteins of C. versicolor for the accelerated degradation of lignin and aromatic pollutants.     

Cloning and expression of Candida albicans ADE2 and proteinase genes on a replicative plasmid in C. albicans and in Saccharomyces cerevisiae.

Summary A plasmid vector (denoted pRC2312) was constructed, which replicates autonomously in Escherichia coli, Saccharomyces cerevisiae and Candida albicans. It contains LEU2, URA3 and an autonomously replicating sequence (ARS) from C. albicans for selection and replication in yeasts, and bla (ampicillin resistance) and ori for selection and replication in E. coli. S. cerevisiae AH22 (Leu^–) was transformed by pRC2312 to Leu^– at a frequency of 1.41 × 10⁵ colonies per g DNA. Transformation of C. albicans SGY-243 (Ura-) to Ura⁺ with pRC2312 resulted in smaller transformant colonies at a frequency of 5.42 × 10³ per g DNA where the plasmid replicated autonomously in transformed cells, and larger transformant colonies at a frequency of 32 per g DNA, in which plasmid integrated into the genome. Plasmid copy number in yeasts was determined by a DNA hybridization method and was estimated to be 15±3 per haploid genome in S. cerevisiae and 2–3 per genome in C. albicans replicative transformants. Multiple tandem integration occurred in integrative transformants and copy number of the integrated sequence was estimated to be 7–12 per diploid genome. The C. albicans ADE2 gene was ligated into plasmid pRC2312 and the construct transformed Ade^– strains of both C. albicans and S. cerevisiae to Ade⁺. The vector pRC2312 was also used to clone a fragment of C. albicans genomic DNA containing an aspartic proteinase gene. C. albicans transformants harboring this plasmid showed a two-fold increase in aspartic proteinase activity. However S. cerevisiae transformants showed no such increase in proteinase activity, suggesting the gene was not expressed in S. cerevisiae.     

Overexpression of a cyanobacterial phospho<Emphasis Type="Italic">enol</Emphasis>pyruvate carboxylase with diminished sensitivity to feedback inhibition in<Emphasis Type="Italic"> Arabidopsis</Emphasis> changes amino acid metabolism

Phosphoenolpyruvate carboxylase (EC 4.1.1.31) from Synechococcus vulcanus (SvPEPC) is a unique enzyme, being almost insensitive to feedback inhibition at neutral pH. In order to assess its usefulness in metabolic engineering of plants, SvPEPC was expressed in Arabidopsis thaliana (L.) Heynh. under the control of the cauliflower mosaic virus 35S promoter. About one-third of the transformants of the T1 generation showed severe visible phenotypes such as leaf bleaching and were infertile when grown on soil. However, no such phenotype was observed with Arabidopsis transformed with Zea mays L. PEPC (ZmPEPC) for C4 photosynthesis, which is normally sensitive to a feedback inhibitor, l-malate. For the SvPEPC transformants of the T2 generation, which had been derived from fertile T1 transformants, three kinds of phenotype were observed when plants were grown on an agar medium containing sucrose: Type-I plants showed poor growth and a block in true leaf development; Type-II plants produced a few true leaves, which were partially bleached; Type-III plants were apparently normal. In Type-I plants, total PEPC activity was increased about 2-fold over the control plant but there was no such increase in Type-III plants. The phenotypes of Type-I plants were rescued when the sucrose-containing agar medium was supplemented with aromatic amino acids. Measurement of the free amino acid content in whole seedlings of Type-I transformants revealed that the levels of the aromatic amino acids Phe and Tyr were lowered significantly as compared with the control plants. In contrast, the levels of several amino acids of the aspartic and glutamic families, such as Asn, Gln and Arg, were markedly enhanced (4- to 8-fold per plant fresh weight). However, when the medium was supplemented with aromatic amino acids, the levels of Asn, Gln, and Arg decreased to levels slightly higher than those of control plants, accompanied by growth recovery. Taken together, it can be envisaged that SvPEPC is capable of efficiently exerting its activity in the plant cell environment so as to cause imbalance between aromatic and non-aromatic amino acid syntheses. The growth inhibition of Type-I plants was presumed to be primarily due to a decreased availability of phosphoenolpyruvate, one of the precursors for the shikimate pathway for the synthesis of aromatic amino acids and phenylpropanoids. The possible usefulness of SvPEPC as one of the key components for installing the C₄-like pathway is proposed.Abbreviations CaMV Cauliflower mosaic virus - GUS -Glucuronidase - Kan Kanamycin - 2-ME 2-Mercaptoethanol - MS/G medium 1/2 Murashige–Skoog and 1/2 Gamborg mixed medium - PEP Phosphoenolpyruvate - PEPC Phosphoenolpyruvate carboxylase - Sv Synechococcus vulcanus - ZmPEPC Maize PEPC involved in C₄ photosynthesis     

Transposition of the maize autonomous element Activator in transgenic Nicotiana plumbaginifolia plants

The maize autonomous transposable element Ac was introduced into haploid Nicotiana plumbaginifolia via Agrobacterium tumefaciens transformation of leaf disks. All the regenerated transformants (R0) were diploid and either homozygous or heterozygous for the hygromycin resistance gene used to select primary transformants. The Ac excision frequency was determined using the phenotypic assay of restoration of neomycin phosphotransferase activity and expression of kanamycin resistance among progeny seedlings. Some of the R0 plants segregated kanamycin-resistant seedlings in selfed progeny at a high frequency (34 to 100%) and contained one or more transposed Ac elements. In the primary transformants Ac transposition probably occurred during plant regeneration or early development. Other R0 transformants segregated kanamycin-resistant plants at a low frequency ( 4%). Two transformants of this latter class, containing a unique unexcised Ac element, were chosen for further study in the expectation that their kanamycin resistant progeny would result from independent germinal transposition events. Southern blot analysis of 32 kanamycin-resistant plants (R1 or R2), selected after respectively one or two selfings of these primary transformants, showed that 27 had a transposed Ac at a new location and 5 did not have any Ac element. Transposed Ac copy number varied from one to six and almost all transposition events were independent. Southern analysis of the R2 and R3 progeny of these kanamycin-resistant plants showed that Ac continued to transpose during four generations, and its activity increased with its copy number. The frequency of Ac transposition, from different loci, remained low ( 7%) from R0 to R3 generations when only one Ac copy was present. The strategy of choosing R0 plants that undergo a low frequency of germinal excision will provide a means to avoid screening non-independent transpositions and increase the efficiency of transposon tagging.     

<Emphasis Type="Italic"> Fugu rubripes</Emphasis> possesses genes for the entire set of the ITAM-bearing transmembrane signal subunits

The transmembrane signaling subunits (TSSs) bearing the immunoreceptor tyrosine-based activation motif (ITAM) play a crucial role in triggering the effector functions of mammalian leukocytes. The involvement in key immune reactions and obvious extension through duplication events make TSSs valuable markers of the evolution of the immune system. We surveyed the genomic sequences of the teleostean fish Fugu rubripes for the presence of genes encoding these accessory molecules. Automatic gene prediction was not efficient because of the poor ability of the programs used to recognize the short exons encoding the intracellular regions of TSSs. However, the unique compactness of the Fugu genome and the conservation of the exon/intron arrangements of the TSS genes facilitated their recognition by visual inspection of the candidate genomic sequences. Evidence for the presence of the CD3, CD3/, CD79a, CD79b, TCR, FcR, DAP12 and DAP10 genes in the Fugu genome was obtained. Furthermore, conserved synteny for the short regions including the TSS genes was revealed by comparison of the Fugu and human genomes. The data demonstrate that the set of TSSs arose before the teleost–tetrapod split and provide a starting point for experimental investigation of the molecular evolution of the leukocyte-activating receptor complexes from fish species to mammals.Abbreviations TSS Transmembrane signal subunit - ITAM Immunoreceptor tyrosine-based activation motif     

Unusual ADP-forming acetyl-coenzyme A synthetases from the mesophilic halophilic euryarchaeon <Emphasis Type="Italic">Haloarcula marismortui</Emphasis> and from the hyperthermophilic crenarchaeon <Emphasis Type="Italic">Pyrobaculum aerophilum</Emphasis>

ADP-forming acetyl-CoA synthetase (ACD), the novel enzyme of acetate formation and energy conservation in archaea ( ), has been studied only in few hyperthermophilic euryarchaea. Here, we report the characterization of two ACDs with unique molecular and catalytic features, from the mesophilic euryarchaeon Haloarcula marismortui and from the hyperthermophilic crenarchaeon Pyrobaculum aerophilum. ACD from H. marismortui was purified and characterized as a salt-dependent, mesophilic ACD of homodimeric structure (166 kDa). The encoding gene was identified in the partially sequenced genome of H. marismortui and functionally expressed in Escherichia coli. The recombinant enzyme was reactivated from inclusion bodies following solubilization and refolding in the presence of salts. The ACD catalyzed the reversible ADP- and P_i-dependent conversion of acetyl-CoA to acetate. In addition to acetate, propionate, butyrate, and branched-chain acids (isobutyrate, isovalerate) were accepted as substrates, rather than the aromatic acids, phenylacetate and indol-3-acetate. In the genome of P. aerophilum, the ORFs PAE3250 and PAE 3249, which code for and subunits of an ACD, overlap each other by 1 bp, indicating a novel gene organization among identified ACDs. The two ORFs were separately expressed in E. coli and the recombinant subunits (50 kDa) and (28 kDa) were in-vitro reconstituted to an active heterooligomeric protein of high thermostability. The first crenarchaeal ACD showed the broadest substrate spectrum of all known ACDs, catalyzing the conversion of acetyl-CoA, isobutyryl-CoA, and phenylacetyl-CoA at high rates. In contrast, the conversion of phenylacetyl-CoA in euryarchaeota is catalyzed by specific ACD isoenzymes.Dedicated to Prof. Dr. Dr. h.c. mult. Hans Günter Schlegel on the occasion of his 80th birthday.