Cloning and sequence analysis of the putative rifamycin polyketide synthase gene cluster from Amycolatopsis mediterranei

The 54-kbp Type I polyketide synthase gene cluster, most probably involved in rifamycin biosynthesis by Amycolatopsis mediterranei, was cloned in E. coli and completely sequenced. The DNA encodes five closely packed, very large open reading frames reading in one direction. As expected from the chemical structure of rifamycins, ten polyketide synthase modules and a CoA ligase domain were identified in the five open reading frames which contain one to three polyketide synthase modules each. The order of the functional domains on the DNA probably reflects the order in which they are used because each of the modules contains the predicted acetate or propionate transferase, dehydratase, and β-ketoacyl-ACP reductase functions, required for the respective step in rifamycin biosynthesis.     

Nonactin biosynthesis: the potential nonactin biosynthesis gene cluster contains type II polyketide synthase-like genes

Nonactin is the parent compound of a group of highly atypical polyketide metabolites produced by Streptomyces griseus subsp. griseus ETH A7796. In this paper we describe the isolation, sequencing, and analysis of 15? omitted?559 bp of chromosomal DNA, containing the potential nonactin biosynthesis gene cluster, from S. griseus subsp. griseus ETH A7796. Fourteen open reading frames were observed in the DNA sequence. Significantly, type II polyketide synthase (PKS) homologues were discovered in an apparent operon structure, which also contained the nonactate synthase gene (nonS), clustered with the tetranactin resistance gene. The deduced products of two of the genes (nonK and nonJ) are quite unusual ketoacyl synthase (KAS) alpha and KASbeta homologues. We speculate that nonactic acid, the polyketide precursor of nonactin, is synthesized by a type II PKS system.     

The gene encoding a Prevotella loescheii lectin-like adhesin contains an interrupted sequence which causes a frameshift.

We cloned and sequenced the Prevotella loescheii gene plaA, which encodes a lectin-like adhesin that mediates the coaggregation of P. loescheii 1295 with Streptococcus oralis 34. A probe derived from the N-terminal amino acid sequence of the purified adhesin was used to identify the plaA gene from a P. loescheii genomic library constructed in lambda GEM-11. Sequence analysis of plaA indicates that the initial translation product contains a 22-amino-acid leader. The reading frame of the plaA gene is interrupted after amino acid 28 of the mature protein by a TAA termination codon. Amplification of the P. loescheii genomic DNA in the region surrounding this codon by the polymerase chain reaction followed by DNA sequencing of the cloned DNA fragment established that this stop codon was not an experimental artifact. A frameshift beginning 29 bp downstream of the ochre terminator was required to access the only large open reading frame in the gene. Amino acid sequences of six purified peptides derived by limited proteolysis of adhesin with endoproteinase Lys-C matched the downstream amino acid sequence derived by translation of the large open reading frame. The gene coding sequence of 2.4 kb contains sufficient information for the synthesis of an 89-kDa protein. A putative rho-independent terminator (delta G = -25.5 kcal/mol [ca. -107 kJ/mol]) was detected 38 bp downstream from the plaA stop codon.     

A gene cluster inChlorobium vibrioforme encoding the first enzymes of chlorophyll biosynthesis

A cloned 5.8-kb genomic fragment of the green sulfur bacteriumChlorobium vibrioforme encodes the genes for three enzymes catalyzing early steps in the biosynthetic pathway of tetrapyrroles, common to chlorophyll and heme. ThehemA, hemC andhemD genes encode the enzymes glutamyl tRNA dehydrogenase, porphobilinogen deaminase and uroporphyrinogen III synthase, respectively. The cloned genes were expressed in transformedEscherichia coli orSalmonella typhimurium and conferred autotrophy on the respective auxotrophs. Activities of the enzymes encoded by the cloned genes were demonstrated in vitro, with cell extracts obtained from the transformed enterobacteria. The proximity of these genes indicates that they form a cluster inChlorobium vibrioforme, while in most other organisms they appear to be scattered. The presence of this cluster may imply coordinate regulation of the genes involved and they may constitute an operon.     

The duck beta-globin gene cluster contains a single enhancer element

An erythroid-specific enhancer was previously identified in the 3'-flanking region of the beta adult gene in chicken and duck, by transfection into AEV transformed chicken erythroblasts. Here we show that the duck enhancer is equally active in erythroid human K562 cells, presenting an embryonic/fetal program of globin gene expression. Furthermore, no other enhancer was found within the 20 kb of DNA including four beta-like globin genes as well as a 1.5 kb upstream and a 3 kb downstream sequence.     

The araC regulatory gene mRNA contains a leader sequence

Summary An estimation of the size of the araC gene in Escherichia coli B/r was made by sub-cloning restriction fragments of the araC-containing hybrid plasmid pTB1 into the plasmid pBR322. Plasmids which contained a functional araC gene were identified by genetic complementation tests. DNA sequence analysis of the promoter-proximal region of the araC gene revealed that araC mRNA contains a 150 nucleotide leader.     

Tagging of a nitrogen pathway-specific regulator gene in Tolypocladium inflatum by the transposon Restless

Restless is an endogenous hAT transposon found in the cyclosporin-producing fungus Tolypocladium inflatum. This element is present in about 15 copies in a particular strain (ATCC34921) which was used for successful gene tagging. We have isolated a T. inflatum mutant with a defect in nitrogen metabolism. This mutant carries a copy of the Restless element in a gene encoding a C6 zinc-finger protein. The deduced amino acid sequence of the gene product shows a significant similarity to the NIT4 protein of Neurospora crassa, which is a regulator of nitrogen metabolism. The wild-type T. inflatum gene was shown to complement a nit-4 mutant of N. crassa. From these data, we conclude that the T. inflatum gene also encodes a regulator of nitrogen metabolism, which was named tnir1 (Tolypocladium nitrogen regulator 1). To the best of our knowledge, this is the first fungal gene to be identified by transposon-directed gene tagging. A general method for gene tagging using an endogenous fungal transposon is presented. Received: 20 October 1999 / Accepted: 15 December 1999     

DNA from human lymphocytes contains a sequence, homologous to immunoglobulin gene kappa

DNA released by human lymphocytes and hyman blood plasma DNA were examined by the electrophoresis, electron microscopy and Southern blot hybridization. The data obtained suggested that DNA released by lymphocytes contains covalently closed circular molecules. By the technique of the Southern blot analysis it was shown that DNA released by lymphocytes and human blood plasma DNA contain discretely sized molecules homologous to the C kappa fragment of the human Ig gene.     

The engL gene cluster of Clostridium cellulovorans contains a gene for cellulosomal manA

A five-gene cluster around the gene in Clostridium cellulovorans that encodes endoglucanase EngL, which is involved in plant cell wall degradation, has been cloned and sequenced. As a result, a mannanase gene, manA, has been found downstream of engL. The manA gene consists of an open reading frame with 1,275 nucleotides encoding a protein with 425 amino acids and a molecular weight of 47, 156. ManA has a signal peptide followed by a duplicated sequence (DS, or dockerin) at its N terminus and a catalytic domain which belongs to family 5 of the glycosyl hydrolases and shows high sequence similarity with fungal mannanases, such as Agaricus bisporus Cel4 (17.3% identity), Aspergillus aculeatus Man1 (23.7% identity), and Trichoderma reesei Man1 (22.7% identity). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and N-terminal amino acid sequence analyses of the purified recombinant ManA (rManA) indicated that the N-terminal region of the rManA contained a DS and was truncated in Escherichia coli cells. Furthermore, Western blot analysis indicated that ManA is one of the cellulosomal subunits. ManA production is repressed by cellobiose.     

Elucidation of a carotenoid biosynthesis gene cluster encoding a novel enzyme, 2,2'-beta-hydroxylase, from Brevundimonas sp. strain SD212 and combinatorial biosynthesis of new or rare xanthophylls

A carotenoid biosynthesis gene cluster mediating the production of 2-hydroxyastaxanthin was isolated from the marine bacterium Brevundimonas sp. strain SD212 by using a common crtI sequence as the probe DNA. A sequence analysis revealed this cluster to contain 12 open reading frames (ORFs), including the 7 known genes, crtW, crtY, crtI, crtB, crtE, idi, and crtZ. The individual ORFs were functionally analyzed by complementation studies using Escherichia coli that accumulated various carotenoid precursors due to the presence of other bacterial crt genes. In addition to functionally identifying the known crt genes, we found that one (ORF11, named crtG) coded for a novel enzyme, carotenoid 2,2'-beta-hydroxylase, which showed intriguingly partial homology with animal sterol-C5-desaturase. When this crtG gene was introduced into E. coli accumulating zeaxanthin and canthaxanthin, the resulting transformants produced their 2-hydroxylated and 2,2'-dihydroxylated products which were structurally novel or rare xanthophylls, as determined by their nuclear magnetic resonance and high-performance liquid chromatography/photodiode array detector/atmospheric pressure chemical ionization mass spectrometry spectral data. The new carotenoid produced was suggested to have a strong inhibitory effect on lipid peroxidation.