Nucleotide sequence of the tcmII-tcmIV region of the tetracenomycin C biosynthetic gene cluster of Streptomyces glaucescens and evidence that the tcmN gene encodes a multifunctional cyclase-dehydratase-O-methyl transferase.

Mutations in the tcmII-tcmIV region of the Streptomyces glaucescens chromosome block the C-3 and C-8 O-methylations of the polyketide antibiotic tetracenomycin C (Tcm C). The nucleotide sequence of this region reveals the presence of two genes, tcmN and tcmO, whose deduced protein products display similarity to the hydroxyindole O-methyl transferase of the bovine pineal gland, an enzyme that catalyzes a phenolic O-methylation analogous to those required for the biosynthesis of Tcm C. The deduced product of the tcmN gene also has an N-terminal domain that shows similarity to the putative ActVII and WhiE ORFVI proteins of Streptomyces coelicolor. The tcmN N-terminal domain can be separated from the remainder of the tcmN gene product, and when coupled on a plasmid with the Tcm C polyketide synthase genes (tcmKLM), this domain enables high-level production of an early, partially cyclized intermediate of Tcm C in a Tcm C- null mutant or in a heterologous host (Streptomyces lividans). By analogy to fatty acid biosynthesis, the tcmKLM polyketide synthase gene products are probably sufficient to produce the linear decaketide precursor of Tcm C; thus, the tcmN N-terminal domain is most likely responsible for one or more of the early cyclizations and, perhaps, the attendant dehydrations that lead to the partially cyclized intermediate. The tcmN gene therefore appears to encode a multifunctional cyclase-dehydratase-3-O-methyl transferase. The tcmO gene encodes the 8-O-methyl transferase.     

Structural and functional analysis of tetracenomycin F2 cyclase from Streptomyces glaucescens. A type II polyketide cyclase

Tetracenomycin F2 cyclase (tcmI gene product), catalyzes an aromatic rearrangement in the biosynthetic pathway for tetracenomycin C in Streptomyces glaucescens. The x-ray structure of this small enzyme has been determined to 1.9-A resolution together with an analysis of site-directed mutants of potential catalytic residues. The protein exhibits a dimeric betaalphabeta ferredoxin-like fold that utilizes strand swapping between subunits in its assembly. The fold is dominated by four strands of antiparallel sheet and a layer of alpha-helices, which creates a cavity that is proposed to be the active site. This type of secondary structural arrangement has been previously observed in polyketide monooxygenases and suggests an evolutionary relationship between enzymes that catalyze adjacent steps in these biosynthetic pathways. Mutational analysis of all of the obvious catalytic bases within the active site suggests that the enzyme functions to steer the chemical outcome of the cyclization rather than providing a specific catalytic group. Together, the structure and functional analysis provide insight into the structural framework necessary to perform the complex rearrangements catalyzed by this class of polyketide cyclases.     

Adenylyl cyclase Rv1264 from Mycobacterium tuberculosis has an autoinhibitory N-terminal domain

Mycobacterium tuberculosis contains 15 class III adenylyl cyclase genes. The gene Rv1264 is predicted to be composed of two distinct protein modules. The C terminus seems to code for a catalytic domain belonging to a subfamily of adenylyl cyclase isozymes mostly found in Gram-positive bacteria. The expressed protein was shown to function as a homodimeric adenylyl cyclase (1 micromol of cAMP x mg(-1) x min(-1)). In analogy to the structure of the mammalian adenylyl cyclase catalyst, six amino acids were targeted by point mutations and found to be essential for catalysis. The N-terminal region represents a novel protein domain, the occurrence of which is restricted to several adenylyl cyclases present in Gram-positive bacteria. The purified full-length enzyme was 300-fold less active than the catalytic domain alone. Thus, the N-terminal domain appeared to be autoinhibitory. The N-terminal domain contains three prominent polar amino acid residues (Asp(107), Arg(132), and Arg(191)) that are invariant in all seven sequences of this domain currently available. Mutation of Asp(107) to Ala relaxed the inhibition and resulted in a 6-fold increase in activity of the Rv1264 holoenzyme, thus supporting the role of this domain as a potential novel regulator of adenylyl cyclase activity.     

Nucleotide sequence and deduced functions of a set of cotranscribed genes of Streptomyces coelicolor A3(2) including the polyketide synthase for the antibiotic actinorhodin.

A 5.3-kb region of the Streptomyces coelicolor actinorhodin gene cluster, including the genes for polyketide biosynthesis, was sequenced. Six identified open reading frames (ORF1-6) were related to genetically characterized mutations of classes actI, VII, IV, and VB by complementation analysis. ORF1-6 run divergently from the adjacent actIII gene, which encodes the polyketide synthase (PKS) ketoreductase, and appear to form an operon. The deduced gene products of ORF1-3 are similar to fatty acid synthases (FAS) of different organisms and PKS genes from other polyketide producers. The predicted ORF5 gene product is similar to type II beta-lactamases of Bacillus cereus and Bacteroides fragilis. The ORF6 product does not resemble other known proteins. Combining the genetical, biochemical, and similarity data, the potential activities of the products of the six genes can be postulated as: 1) condensing enzyme/acyl transferase (ORF1 + ORF2); 2) acyl carrier protein (ORF3); 3) putative cyclase/dehydrase (ORF4); 4) dehydrase (ORF5); and 5) "dimerase" (ORF6). The data show that the actinorhodin PKS consists of discrete monofunctional components, like that of the Escherichia coli (Type II) FAS, rather than the multifunctional polypeptides for the macrolide PKSs and vertebrate FASs (Type I).     

Nucleotide sequences and heterologous expression of tcmG and tcmP, biosynthetic genes for tetracenomycin C in Streptomyces glaucescens.

The nucleotide sequence of the tcmIII, tcmIc, and tcmVII region of the tetracenomycin (TCM) C gene cluster of Streptomyces glaucescens ETH 22794 (GLA.0) revealed the presence of two genes, tcmP and tcmG. The deduced product of tcmG resembles flavoprotein hydroxylases found in several other bacteria, whereas the predicted amino acid sequence of tcmP is not significantly similar to those of any known proteins in the available data bases. Southern blot hybridization revealed an approximately 180-bp deletion in a tcmIII (tcmG) mutant and a 1,800-bp insertion in a tcmVII (tcmP) mutant. Heterologous expression of tcmG and tcmP in Streptomyces lividans and tcmP in Escherichia coli established that tcmP encodes an O-methyltransferase, catalyzing the methylation of the C-9 carboxy group of TCM E to yield TCM A2, and that tcmG is responsible for the hydroxylation of TCM A2 at positions C-4, C-4a, and C-12a to give TCM C. These are the final two steps of TCM C biosynthesis.     

Genomic structure and expression of the soluble guanylyl cyclase alpha2 subunit gene in the medaka fish Oryzias latipes

A cDNA clone encoding the soluble guanylyl cyclase alpha2 subunit was isolated from medaka fish (Oryzias latipes) and designated as OlGCS-alpha2. The OlGCS-alpha2 cDNA was 3,192 bp in length and the open reading frame (ORF) encodes a protein of 805 amino acids. The deduced amino acid sequence has high similarity to that of the mammalian alpha2 subunit gene except for the N-terminal regulatory domain. The C-terminal 5 amino acids, "RETSL", which have been reported to interact with the post synaptic density protein (PSD)-95 were conserved. An RNase protection assay with adult fish organs showed that OlGCS-alpha2 was expressed mainly in the brain and testis. The complete nucleotide sequence (about 41 kbp) of the OlGCS-alpha2 genomic DNA clone isolated from a medaka fish BAC library indicated that the OlGCS-alpha2 gene consisted of 9 exons and 8 introns. The 5'-flanking region and larger introns, such as introns 1, 4, and 7, contained the several fragments conserved in the nucleotide sequences of Rex6 (non-long terminal repeat retrotransposon), MHC class I genomic region, and OlGC1, the medaka fish homolog of the mammalian guanylyl cyclase B gene. Linkage analysis on the medaka fish chromosome demonstrated that the OlGCS-alpha2 gene was mapped to LG13; this mapping position was different from those for the OlGCS-alpha1 and OlGCS-beta1 genes (LG1).     

High-level synthesis of active adenylate cyclase toxin of Bordetella pertussis in a reconstructed Escherichia coli system

The Bordetella pertussis adenylate cyclase(Cya) toxin-encoding locus (cya) is composed of five genes. The cyaA gene encodes a virulence factor (CyaA), exhibiting adenylate cyclase, hemolytic and invasive activities. The cyaB, D and E gene products are necessary for CyaA transport, and the cyaC gene product is required to activate CyaA. We reconstructed, in Escherichia coli, the cya locus of B. pertussis by cloning the different genes on appropriate vectors under the control of strong promoters and E. coli-specific translation initiation signals. We show that in the absence of additional gene products, CyaA is synthesized at high levels, is endowed with adenylate cyclase activity, but is devoid of invasive and hemolytic activities. CyaC is sufficient to confer upon the adenylate cyclase holotoxin full invasive and partial hemolytic activities. Coexpression of the cyaB, D and E genes neither stimulates nor potentiates the activation brought about by CyaC. This reconstructed system should help to elucidate both the mechanism and the structural requirements of holotoxin activation.     

The carboxyl region contains the catalytic domain of the membrane form of guanylate cyclase

A polypeptide containing the catalytic domain of an atrial natriuretic peptide receptor guanylate cyclase has been produced using a bacterial expression system. A carboxyl fragment of the membrane form of guanylate cyclase from rat brain, which contains a region homologous to soluble guanylate and adenylate cyclases, was expressed in Escherichia coli with a double plasmid system that encodes T7 RNA polymerase (Tabor, S., and Richardson, C.C. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 1074-1078). Application of this expression system permitted exclusive radiolabeling of the cloned gene product, thereby providing a means to evaluate the level of expression and stability of encoded proteins. Fusion proteins were formed with the T7 bacteriophage gene 10 product and the 293 carboxyl-terminal residues of guanylate cyclase and two deletional mutants encoding 105 and 69 residues. Extracts prepared from bacteria expressing the carboxyl region, but not those expressing further deletions in this region, had substantial guanylate cyclase activity. There was no associated adenylate cyclase activity, suggesting that the catalytic domain retained its enzymatic specificity. These results provide direct evidence that the carboxyl portion of the membrane form of guanylate cyclase contains a catalytic domain. Homologous regions of the soluble form of guanylate cyclase and adenylate cyclase are likely to have enzymatic properties.     

Mutants of the carotene cyclase domain of al-2 from Neurospora crassa

Phytoene synthase and carotene cyclase, two key enzymes in carotenoid biosynthesis, are encoded by two separate genes in bacteria and plants, but by a single bifunctional gene in fungi. The cyclase function has been demonstrated for the products of the genes crtYB from the basidiomycete Xanthophyllomyces dendrorhous, and for carRA and carRP from the zygomycetes Phycomyces blakesleeanus and Mucor circinelloides, respectively. These three genes are highly similar to al-2 from Neurospora crassa. Taking advantage of the high proportion of the final product of the carotenoid pathway that accumulates Neurospora when mycelium is illuminated at low temperature, we have isolated two mutants with a pale reddish pigmentation. Both mutants are complemented by the wild-type al-2 gene, and carry mutations in the al-2 domain to which cyclase activity has been attributed in other fungi. The mutants lack neurosporaxanthin and accumulate an unidentified reddish carotenoid, as shown by column chromatography and HPLC. The chemical and spectrophotometrical properties of this carotenoid are consistent with the absence of carotenoid cyclization, and indicate that the product of al-2 is bifunctional. The existence of a single gene responsible for phytoene synthase and carotene cyclase thus seems to be a widespread trait among filamentous fungi, as shown by the examples now known in a basidiomycete, two zygomycetes and one ascomycete.     

Regulatory function of the Saccharomyces cerevisiae RAS C-terminus.

Activating mutations (valine 19 or leucine 68) were introduced into the Saccharomyces cerevisiae RAS1 and RAS2 genes. In addition, a deletion was introduced into the wild-type gene and into an activated RAS2 gene, removing the segment of the coding region for the unique C-terminal domain that lies between the N-terminal 174 residues and the penultimate 8-residue membrane attachment site. At low levels of expression, a dominant activated phenotype, characterized by low glycogen levels and poor sporulation efficiency, was observed for both full-length RAS1 and RAS2 variants having impaired GTP hydrolytic activity. Lethal CDC25 mutations were bypassed by the expression of mutant RAS1 or RAS2 proteins with activating amino acid substitutions, by expression of RAS2 proteins lacking the C-terminal domain, or by normal and oncogenic mammalian Harvey ras proteins. Biochemical measurements of adenylate cyclase in membrane preparations showed that the expression of RAS2 proteins lacking the C-terminal domain can restore adenylate cyclase activity to cdc25 membranes.     

Disruption of an aromatase/cyclase from the oxytetracycline gene cluster of Streptomyces rimosus results in production of novel polyketides with shorter chain lengths.

Oxytetracycline is a polyketide antibiotic made by Streptomyces rimosus. From DNA sequencing, the gene product of otcD1 is deduced to function as a bifunctional cyclase/aromatase involved in ring closure of the polyketide backbone. Although otcD1 is contiguous with the ketoreductase gene, they are located an unusually large distance from the genes encoding the "minimal polyketide synthase" of the oxytetracycline gene cluster. A recombinant, disrupted in the genomic copy of otcD1, made four novel polyketides, all of shorter chain length (by up to 10 carbons) than oxytetracycline. All four novel structures contained the unusual carboxamido group, typical of oxytetracycline. This implies that the carboxamido group is present at the start of biosynthesis of oxytetracycline, a topic that has been debated in the literature. Loss of the cyclase protein has a profound influence on the length of polyketide chain assembled, implying that OtcD1 plays a greater role in the overall integrity of the quaternary structure of the polyketide complex than hitherto imagined.     

Identification of the missing trans-acting enoyl reductase required for phthiocerol dimycocerosate and phenolglycolipid biosynthesis in Mycobacterium tuberculosis

Phthiocerol dimycocerosates (DIM) and phenolglycolipids (PGL) are functionally important surface-exposed lipids of Mycobacterium tuberculosis. Their biosynthesis involves the products of several genes clustered in a 70-kb region of the M. tuberculosis chromosome. Among these products is PpsD, one of the modular type I polyketide synthases responsible for the synthesis of the lipid core common to DIM and PGL. Bioinformatic analyses have suggested that this protein lacks a functional enoyl reductase activity domain required for the synthesis of these lipids. We have identified a gene, Rv2953, that putatively encodes an enoyl reductase. Mutation in Rv2953 prevents conventional DIM formation and leads to the accumulation of a novel DIM-like product. This product is unsaturated between C-4 and C-5 of phthiocerol. Consistently, complementation of the mutant with a functional pks15/1 gene from Mycobacterium bovis BCG resulted in the accumulation of an unsaturated PGL-like substance. When an intact Rv2953 gene was reintroduced into the mutant strain, the phenotype reverted to the wild type. These findings indicate that Rv2953 encodes a trans-acting enoyl reductase that acts with PpsD in phthiocerol and phenolphthiocerol biosynthesis.     

Different chromosomal localization of two adenylyl cyclase genes expressed in human brain

Summary Recently, we characterized a cDNA clone that encodes a human brain adenylyl cyclase (HBAC1). In the present study, we identified a second population of mRNA suspected to encode a new brain adenylyl cyclase (HBAC2). The amino acid sequence of HBAC2 displays significant homology with HBAC1 in the highly conserved adenylyl cyclase domain (250 aminio acids), found in the 3 cytoplasmic domain of all mammalian adenylyl cyclases. However, outside this domain, the homology is extremely low, suggesting that the corresponding mRNA originates from a different gene. We report here the first chromosomal localization of the adenylyl cyclase genes determined by in situ hybridization of human metaphase chromosomal spreads using human brain cDNA probes specific for each mRNA. The probe corresponding to HBAC1 exhibited a strong specific signal on chromosome 8q24, with a major peak in the band q24.2. In contrast, the HBAC2 probe hybridized to chromosome 5p15, with a major peak in the band p15.3. The two cDNAs hybridized at the two loci without any cross reactivity. Thus, in human brain, a heterogeneous population of adenylyl cyclase mRNAs is expressed, and the corresponding genes might be under the control of independent regulatory mechanisms.Abbreviations C catalytic part of adenylyl cyclase - BBAC bovine brain - HBAC human brain - ROAC rat olfactory - RLAC rat liver - RTAC rat testis adenylyl cyclase - G guanine nucleotide GTP binding protein (s, stimulatory; i, inhibitory)     

Insertional mutagenesis of Bordetella pertussis adenylate cyclase.

We developed an improved method of linker insertion mutagenesis for introducing 2 or 16 codons into the Bordetella pertussis cyaA gene which encodes a calmodulin-dependent adenylate cyclase. A recombinant kanamycin resistance cassette, containing oligonucleotide linkers, was cloned in plasmids which carried a truncated cyaA gene, fused at its 3' end to the 5' end of the Escherichia coli lacZ gene, specifying the alpha-peptide. This construction permitted a double selection for in-frame insertions by using screening for kanamycin resistance and for lactose-positive phenotype, resulting from alpha-complementation. We showed that most of the two-amino acid insertions within the N-terminal moiety of the catalytic domain of adenylate cyclase abolished enzymatic activity and/or altered the stability of the protein. All two-amino acid insertions within the C-terminal part of adenylate cyclase resulted in fully stable and active enzymes. These results confirm the modular structure of the catalytic domain of adenylate cyclase, previously proposed on the basis of proteolytic studies. Two-amino acid insertions between residues 247-248 and 335-336 were shown to affect the calmodulin responsiveness of adenylate cyclase, suggesting that the corresponding region in the enzyme is involved in the binding of calmodulin or in the process of calmodulin activation. In addition, we have identified within the primary structure of adenylate cyclase several permissive sites which tolerate 16-amino acid insertions without interfering with the catalytic activity or calmodulin binding. By inserting foreign antigenic determinants into these permissive sites the resulting recombinant adenylate cyclase toxin could be used to deliver specific epitopes into antigen-presenting cells.     

F1C fimbriae of a uropathogenic Escherichia coli strain: genetic and functional organization of the foc gene cluster and identification of minor subunits.

The genetic organization of the foc gene cluster has been studied; six genes involved in the biogenesis of F1C fimbriae were identified. focA encodes the major fimbrial subunit, focC encodes a product that is indispensable for fimbria formation, focG and focH encode minor fimbrial subunits, and focI encodes a protein which shows similarities to the subunit protein FocA. Apart from the FocA major subunits, purified F1C fimbriae contain at least two minor subunits, FocG and FocH. Minor proteins of similar size were observed in purified S fimbriae. Remarkably, some mutations in the foc gene cluster result in an altered fimbrial morphology, i.e., rigid stubs or long, curly fimbriae.     

The domino gene of Drosophila encodes novel members of the SWI2/SNF2 family of DNA-dependent ATPases, which contribute to the silencing of homeotic genes

The Drosophila domino gene has been isolated in a screen for mutations that cause hematopoietic disorders. Generation and analysis of loss-of-function domino alleles show that the phenotypes are typical for proliferation gene mutations. Clonal analysis demonstrates that domino is necessary for cell viability and proliferation, as well as for oogenesis. domino encodes two protein isoforms of 3202 and 2498 amino acids, which contain a common N-terminal region but divergent C termini. The common region includes a 500 amino acid DNA-dependent ATPase domain of the SWI2/SNF2 family of proteins, which function via interaction with chromatin. We show that, although domino alleles do not exhibit homeotic phenotypes by themselves, domino mutations enhance Polycomb group mutations and counteract Trithorax group effects. The Domino proteins are present in large complexes in embryo extracts, and one isoform binds to a number of discrete sites on larval polytene chromosomes. Altogether, the data lead us to propose that domino acts as a repressor by interfering with chromatin structure. This activity is likely to be performed as a subunit of a chromatin-remodeling complex.