Sequence of the E. coli O104 antigen gene cluster and identification of O104 specific genes

The Escherichia coli O104 polysaccharide is an important antigen, which contains sialic acid and is often associated with EHEC clones. Sialic acid is a component of many animal tissues, and its presence in bacterial polysaccharides may contribute to bacterial pathogenicity. We sequenced the genes responsible for O104 antigen synthesis and have found genes which from their sequences are identified as an O antigen polymerase gene, an O antigen flippase gene, three CMP-sialic acid synthesis genes, and three potential glycosyl transferase genes. The E. coli K9 group IB capsular antigen has the same structure as the O104 O antigen, and we find using gene by gene PCR that the K9 gene cluster is essentially the same as that for O104. It appears that the distinction between presence as group IB capsule or O antigen for this structure does not involve any difference in genes present in the O antigen gene cluster. By PCR testing against representative strains for the 166 E. coli O antigens and some randomly selected Gram-negative bacteria, we identified three O antigen genes which are highly specific to O104/K9. This work provides the basis for a sensitive test for rapid detection of O104 E. coli. This is important both for decisions on patient care as early treatment may reduce the risk of life-threatening complications and for a faster response in control of food borne outbreaks.     

Nitrobacter winogradskyi cytochrome c oxidase genes are organized in a repeated gene cluster

Cytochrome c oxidase (EC 1.9.3.1) is one of the components of the electron transport chain by which Nitrobacter, a facultative lithoautotrophic bacterium, recovers energy from nitrite oxidation. The genes encoding the two catalytic core subunits of the enzyme were isolated from a Nitrobacter winogradskyi gene library. Sequencing of one of the 14 cloned DNA segments revealed that the subunit genes are side by side in an operon-like cluster. Remarkably the cluster appears to be present in at least two copies per genome. It extends over a 5–6 kb length including, besides the catalytic core subunit genes, other cytochrome oxidase related genes, especially a heme O synthase gene. Noteworthy is the new kind of gene order identified within the cluster. Deduced sequences for the cytochrome oxidase subunits and for the heme O synthase look closest to their counterparts in other -subdivision Proteobacteria, particularly the Rhizobiaceae. This confirms the phylogenetic relationships established only upon 16S rRNA data. Furthermore, interesting similarities exist between N. winogradskyi and mitochondrial cytochrome oxidase subunits while the heme O synthase sequence gives some new insights about the other similar published -subdivision proteobacterial sequences.Abbreviations COI cytochrome oxidase subunit I - COII cytochrome oxidase subunit II - COIII cytochrome oxidase subunit III - HOS Heme O synthase - ORF open reading frame - SDS sodium dodecyl sulfate     

嗜热链球菌IMAU20246胞外多糖基因簇及其表达分析北大核心

【目的】嗜热链球菌IMAU20246是一株具有良好发酵特性且高产胞外多糖(exopolysaccharides,EPS)的菌株,但其EPS基因簇及合成途径尚不清晰。因此可通过全基因组测序及生物信息学分析菌株基因组序列,探究EPS合成及调控机制。【方法】本实验对嗜热链球菌IMAU20246进行全基因组测序并进行生物信息学分析,解析EPS生物合成相关基因簇及EPS合成途径,同时采用实时荧光定量PCR技术(quantitative real-time PCR,qRT-PCR)对其不同时间点EPS基因簇的表达进行定量分析。【结果】嗜热链球菌IMAU20246基因组中有一个18.1 kb的EPS生物合成基因簇,编码15个与EPS生物合成相关的基因。嗜热链球菌IMAU20246通过转运葡萄糖、甘露糖、果糖、半乳糖、乳糖、海藻糖、纤维二糖及蔗糖合成UDP-葡萄糖、dTDP-葡萄糖、dTDP-鼠李糖、UDP-半乳糖、UDP-呋喃半乳糖、UDP-N-乙酰葡萄糖胺和UDP-N-乙酰半乳糖胺等7种糖核苷酸。qRT-PCR的结果表明,EPS基因簇中的基因在细胞生长阶段均能表达,特别是糖基转移酶基因epsE、epsF、epsH和epsJ在培养6 h时表达量最高,此时EPS产量达到最高。【结论】本研究从基因组解析了嗜热链球菌IMAU20246 EPS基因簇及其合成途径,为菌株的进一步开发提供了理论依据。     

Characterization and expression in Streptomyces lividans of cefD and cefE genes from Nocardia lactamdurans: the organization of the cephamycin gene cluster differs from that in Streptomyces clavuligerus

Summary The cefD and cefE genes of Nocardia lactamdurans, which encode isopenicillin N epimerase and deacetoxycephalosporin C synthase respectively, have been located 0.63 kb upstream from the lysine-6-amino-transferase (lat) gene. cefD contains an open reading frame (ORF) of 1197 nucleotides (nt) encoding a protein of 398 amino acids with a M_r of 43 622. The deduced amino acid sequence exhibits 62.2% identity to the cefD gene product of Streptomyces clavuligerus. The sequence SXHKXL in isopenicillin N epimerase resembles the consensus sequence for pyridoxal phosphate binding found in several amino acid decarboxylases from Enterobacteria. cefE contains an ORF of 945 nt encoding a protein of 314 amino acids with a M_r of 34532, which is similar to the deacetoxycephalosporin C synthase of S. clavuligerus. Expression of both genes, cefD and cefE, in S. lividans transformants, results in deacetoxycephalosporin C synthase and isopenicillin N epimerase activities that are 10–12 times higher than those in N. lactamdurans. The cefD and cefE genes of N. lactamdurans are closely linked but the overall organization of the cephamycin gene cluster differs in N. lactamdurans and S. clavuligerus.     

Cloning and partial characterization of the putative rifamycin biosynthetic gene cluster from the actinomycete Amycolatopsis mediterranei DSM 46095

The actinomycete Amycolatopsis mediterranei produces the commercially and medically important polyketide antibiotic rifamycin, which is widely used against mycobacterial infections. The rifamycin biosynthetic (rif) gene cluster has been isolated, cloned and characterized from A. mediterranei S699 and A. mediterranei LBGA 3136. However, there are several other strains of A. mediterranei which also produce rifamycins. In order to detect the variability in the rif gene cluster among these strains, several strains were screened by PCR amplification using oligonucleotide primers based on the published DNA sequence of the rif gene cluster and by using dEBS II (second component of deoxy-erythronolide biosynthase gene) as a gene probe. Out of eight strains of A. mediterranei selected for the study, seven of them showed the expected amplification of the DNA fragments whereas the amplified DNA pattern was different in strain A. mediterranei DSM 46095. This strain also showed striking differences in the banding pattern obtained after hybridization of its genomic DNA against the dEBS II probe. Initial cloning and characterization of the 4-kb DNA fragment from the strain DSM 46095, representing a part of the putative rifamycin biosynthetic cluster, revealed nearly 10% and 8% differences in the DNA and amino acid sequence, respectively, as compared to that of A. mediterranei S699 and A. mediterranei LBGA 3136. The entire rif gene cluster was later cloned on two cosmids from A. mediterranei DSM 46095. Based on the partial sequence analysis of the cluster and sequence comparison with the published sequence, it was deduced that among eight strains of A. mediterranei, only A. mediterranei DSM 46095 carries a novel rifamycin biosynthetic gene cluster.     

The cycHJKL gene cluster plays an essential role in the biogenesis of c-type cytochromes in Bradyrhizobium japonicum

We present an extended genetic analysis of the previously identified cycH locus in Bradyrhizobium japonicum. Three new open reading frames found in an operon-like structure immediately adjacent to the 3 end of cycH were termed cycJ, cycK and cycL. A deletion mutant (cycHJKL) and biochemical analysis of its phenotype showed that the genes of the cluster are essential for the biogenesis of cellular c-type cytochromes. Mutations in discrete regions of each of the genes were also constructed and shown to affect anaerobic respiration with nitrate and the ability to elicit an effective symbiosis with soybean, both phenotypes being a consequence of defects in cytochrome c formation. The CycK and CycL proteins share up to 53% identity in amino acid sequence with the Rhodobacter capsulatus Ccll and Cc12 proteins, respectively, which have been shown previously to be essential for cytochrome c biogenesis, where-as cycJ codes for a novel protein of 169 amino acids with an M_r of 17857. Localisation studies revealed that CycJ is located in the periplasmic space; it is probably anchored to the cytoplasmic membrane via an N-terminal hydrophobic domain. Based on several considerations discussed here, we suggest that the proteins encoded by the cycHJKL-cluster may be part of a cytochrome c-haem lyase complex whose active site faces the periplasm.     

The hydrogenase gene cluster ofRhizobium leguminosarum bv.viciae contains an additional gene (hypX), which encodes a protein with sequence similarity to the N10-formyltetrahydrofolate-dependent enzyme family and is required for nickel-dependent hydrogenase processing and activity

Plasmid pAL618 contains the genetic determinants for H₂ uptake (hup) fromRhizobium leguminosarum bv.viciae, including a cluster of 17 genes namedhupSLCDEFGHIJK-hypABFCDE. A 1.7-kb segment of insert DNA located downstream ofhypE has now been sequenced, thus completing the sequence of the 20 441-bp insert DNA in plasmid pAL618. An open reading frame (designatedhypX) encoding a protein with a calculated M_r of 62 300 that exhibits extensive sequence similarity with HoxX fromAlcaligenes eutrophus (52% identity) andBradyrhizobium japonicum (57% identity) was identified 10 bp downstream ofhypE. Nodule bacteroids produced byhypX mutants in pea (Pisum sativum L.) plants grown at optimal nickel concentrations (100 µM) for hydrogenase expression, exhibited less than 5% of the wild-type levels of hydrogenase activity. These bacteroids contained wild-type levels of mRNA from hydrogenase structural genes (hupSL) but accumulated large amounts of the immature form of HupL protein. The Hup-deficient mutants were complemented for normal hydrogenase activity and nickel-dependent maturation of HupL by ahypX gene provided in trans. From expression analysis ofhypX-lacZ fusion genes, it appears thathypX gene is transcribed from the FnrN-dependenthyp promoter, thus placinghypX in thehyp operon (hypBFCDEX). Comparisons of the HypX/HoxX sequences with those in databases provided unexpected insights into their function in hydrogenase synthesis. Similarities were restricted to two distinct regions in the HypX/HoxX sequences. Region I, corresponding to a sequence conserved in N₁₀-formyltetrahydrofolate-dependent enzymes involved in transferring one-carbon units (C₁), was located in the N-terminal half of the protein, whereas region II, corresponding to a sequence conserved in enzymes of the enoyl-CoA hydratase/isomerase-family, was located in the C-terminal half. These similarities strongly suggest that HypX/HoxX have dual functions: binding of the C₁ donor N₁₀-formyl-tetrahydrofolate and transfer of the C₁ to an unknown substrate, and catalysis of a reaction involving polarization of the C=O bond of an X-CO-SCoA substrate. These results also suggest the involvement of a small organic molecule, possibly synthesized with the participation of an X-CO-SCoA precursor and of formyl groups, in the synthesis of the metal-containing active centre of hydrogenase.     

pMH2, a small plasmid bearing the nif gene cluster of Enterobacter agglomerans 333 as an excisable cassette

A small plasmid containing the entire nif gene cluster of Enterobacter agglomerans 333 as an excisable cassette has been constructed, using pACYC177 as a vector. Two cosmid clones taken from a gene library of E. agglomerans plasmid pEA3 were used as a source of nif genes. A SmaI fragment of peaMS2-2, containing the H,D,K,Y,E,N,X,U,S,V,W,Z,M,L,A and B genes and an ApaI fragment of peaMS2-16 containing nifA,B,Q,F and J were selected to construct pMH2. The resulting plasmid of 33 kb carries the complete nif gene cluster as a nif cassette on a single XbaI fragment. The nif construct pMH2 in Escherichia coli strains has significant nitrogenase activity compared to wild-type E. agglomerans 333. The nif gene cluster construct was found to be very stable.     

Cloning and characterisation of the cytochrome c gene of Aspergillus nidulans

The cytochrome c gene (cycA) of the filamentous fungus Aspergillus nidulans has been isolated and sequenced. The gene is present in a single copy per haploid genome and encodes a polypeptide of 112 amino acid residues. The nucleotide sequence of the A. nidulans cycA gene shows 87% identity to the DNA sequence of the Neurospora crassa cytochrome c gene, and approximately 72% identity to the sequence of the Saccharomyces cerevisiae iso-1-cytochrome c gene (CYC1). The S. cerevisiae CYC1 gene was used as a heterologous probe to isolate the homologous gene in A. nidulans. The A. nidulans cytochrome c sequence contains two small introns. One of these is highly conserved in terms of position, but the other has not been reported in any of the cytochrome c genes so far sequenced. Expression of the cycA gene is not affected by glucose repression, but has been shown to be induced approximatly tenfold in the presence of oxygen and three- to fourfold under heatshock conditions.     

The bialaphos biosynthetic genes ofStreptomyces hygroscopicus: Molecular cloning and characterization of the gene cluster

Summary We have isolated and studied the organization ofStreptomyces hygroscopicus genes responsible for the biosynthesis of the antibiotic herbicide bialaphos. Bialaphos production genes were cloned from genomic DNA using a plasmid vector (pIJ702). Three plasmids were isolated which restored productivity toS. hygroscopicus mutants blocked at different steps of the biosynthetic pathway. Subcloning experiments using other nonproducing mutants showed that four additional bialaphos production genes were also contained on these plasmids. A gene conferring resistance to bialaphos, which was independently cloned using the plasmid vector pIJ61, and an antibiotic-sensitive host (S. lividans), was also linked to the production genes. Cosmids were isolated which defined the location of these genes in a 16 kb cluster.     

Genetic and physical mapping of an hydrogenase gene cluster from Rhodobacter capsulatus

Summary An hydrogenase-deficient (Hup^–) mutant of Rhodobacter capsulatus was obtained by adventitious insertion of IS21 DNA into an hydrogenase structural gene (hup) of the wild-type strain 1310. The resulting Hup^– mutant, strain JP91, selected by its inability to grow autotrophically (Aut^– phenotype) together with other Hup^– mutant strains obtained by classical ethyl methane sulphonate mutagenesis were used in R plasmid-mediated conjugation experiments to map the hup/aut loci on the chromosome of R. capsulatus. The hup genes tested in this study were found to cluster on the chromosome in the proximity of the his-1 marker. A cluster of hup genes comprising the structural genes was isolated from a gene bank constructed in the cosmid vector pHC79 with 40 kb insert DNA. The clustered hup genes, characterized by hybridization studies and complementation analyses of the R. capsulatus Hup^– mutants, span 15–20 kb of DNA.     

The nifH, nifM and nifN genes of Azotobacter vinelandii: Characterisation by Tn5 mutagenesis and isolation from pLAFR1 gene banks

Summary Tn5 was introduced into Azotobacter vinelandii on a suicide vector, pGS9. Three Nif^- mutants were found to carry Tn5 in nifH (MV6), in nifN (MV22), and in or near nifM (MV21), from the results of hybridisation experiments. For MV21 and MV22 this was also shown by complementation with the nif genes of Klebsiella pneumoniae on pRD1. MV6 failed to synthesis the nifH, D and K gene products. MV6 and MV22 fixed nitrogen in the absence of supplied molybdenum while mutant MV21 did not, suggesting that the nifM gene product may be required for the alternative nitrogenase system synthesised in azotobacteria under conditions of molybdenum deprivation. Reconstitution experiments with mutant extracts showed that MV22 (nifN ^-) lacked the FeMo cofactor and that MV21 (NifM^-) synthesised inactive Fe protein. These biochemical phenotypes are identical to those of the K. pneumoniae nifN and nifM mutants, respectively, demonstrating that these genes have the same function in both K. pneumoniae and A. vinelandii. Complementation of the A. vinelandii mutants with pLAFR1 gene banks of A. vinelandii or a. chroococcum yielded three cosmids of interest. pLV10 complemented UW91, a nifH mutant, and corrected the defect in MV6 after recombination with the mutant genome. It also carried nifD (but not nifK) and about 18 kb of DNA upstream from nifH. pLV1 from the A. vinelandii gene bank complemented both MV21 and MV22 as did pLC11, isolated from the A. chroococcum gene bank. Both pLV1 and pLC11 carried part of the nif cluster downstream of nifHDK which also includes nifEN and nifMVS on about 22 kb of DNA.     

Organisation and functions of the actV A region of the actinorhodin biosynthetic gene cluster of Streptomyces coelicolor

Summary Sequence analysis of the actVA region of the actinorhodin biosynthetic gene cluster of Streptomyces coelicolor revealed a succession of six open reading frames (ORFs), all running in the same direction and extending over 5.32 kb. The protein product of actVA-ORF1 strongly resembles that of another gene, elsewhere in the act cluster (actII-ORF2), which codes for a trans-membrane protein previously implicated in actinorhodin export from the mycelium. This suggests that the two gene products may co-operate in actinorhodin export, perhaps being sufficient for self-protection of the organism against suicide. At least four of the other five ORFs are implicated in the control of the C-6 and C-8 ring-hydroxylation reactions, lacking in actVA mutants, that occur at middle to late stages in the actinorhodin biosynthetic pathway. This conclusion was reached by genetic mapping of actVA mutants to actVA-ORF3 and-ORF5 (and perhaps -ORF4), and by the finding of strong resemblances between the protein products of actVA-ORF2 and -ORF6 and the products of genes of the oxytetracycline or tetracenomycin gene clusters that have been implicated in ring-hydroxylation reactions in the biosynthesis of these other aromatic polyketide antibiotics.     

Organization and expression of mouse Hox3 cluster genes

Summary The three yolk protein genes (yp) of Drosophila melanogaster are transcribed in a sex- and tissue-limited fashion. We have searched for cis-regulatory sequences in regions flanking yp1 and yp2 to identify the elements that confer female-specific expression in the fat body. One such 127 by element has previously been identified in this region. We show here the existence of two additional regions which confer female fat body-specific expression on an Adh reporter gene and on the native yp2 gene, respectively. This suggests some redundancy in the regulation of expression of the yp genes. Computer searches for putative binding sites for the DSX protein, which regulates sex-specific expression of the yp genes, revealed several such sites in our constructs. However, the significance of these is unclear since many such sites also occur in genes which one would not expect to be regulated in a sex-specific manner (e.g. Adh, Actin 5C). We suggest that DSX acts in concert with other proteins to mediate sex- and tissue-specific expression of the yp genes.     

Nucleotide sequence of the recF gene cluster from Staphylococcus aureus and complementation analysis in Bacillus subtilis recF mutants

We have determined the nucleotide sequence of a 3.5 kb segment in the recF region of the Staphylococcus aureus chromosome. The gene order at this locus, dnaA-dnaN-recF-gyrB is similar to that found in the replication origin region of many other bacteria. S. aureus RecF protein (predicted molecular mass 42.3 kDa), has 57% amino acid sequence identity with the Bacillus subtilis RecF protein (42.2 kDa), but only 26% with the Escherichia coli RecF protein (40.5 kDa). We have shown that the S. aureus recF gene partially complements the defect of a B. subtilis recF mutant, but does not complement an E. coli recF strain. The amino acid sequence alignment of seven available RecF proteins (five of them from bacteria of gram-negative origin) allowed us to identify eight highly conserved regions ( to ) and to predict five new conserved regions within the gram-positive group (a to f). We suggest that the basic mechanism of homologous recombination is conserved among free-living bacteria.     

A gene cluster involved in nogalamycin biosynthesis fromStreptomyces nogalater: sequence analysis and complementation of early-block mutations in the anthracycline pathway

We have analyzed an anthracycline biosynthesis gene cluster fromStreptomyces nogalater. Based on sequence analysis, a contiguous region of 11 kb is deduced to include genes for the early steps in anthracycline biosynthesis, a regulatory gene (snoA) promoting the expression of the biosynthetic genes, and at least one gene whose product might have a role in modification of the glycoside moiety. The three ORFs encoding a minimal polyketide synthase (PKS) are separated from the regulatory gene (snoA) by a comparatively AT-rich region (GC content 60%). Subfragments of the DNA region were transferred toStreptomyces galilaeus mutants blocked in aclacinomycin biosynthesis, and to a regulatory mutant ofS. nogalater. TheS. galilaeus mutants carrying theS. nogalater minimal PKS genes produced auramycinone glycosides, demonstrating replacement of the starter unit for polyketide biosynthesis. The product ofsnoA seems to be needed for expression of at least the genes for the minimal PKS.     

AgTHR4, a new selection marker for transformation of the filamentous fungusAshbya gossypii, maps in a four-gene cluster that is conserved betweenA. gossypii andSaccharomyces cerevisiae

Single-read sequence analysis of the termini of eight randomly picked clones ofAshbya gossypii genomic DNA revealed seven sequences with homology toSaccharomyces cerevisiae genes (15% to 69% on the amino acid level). One of these sequences appeared to code for the carboxy-terminus of threonine synthase, the product of theS. cerevisiae THR4 gene (52.4% identity over 82 amino acids). We cloned and sequenced the complete putativeAgTHR4 gene ofA. gossypii. It comprises 512 codons, two less than theS. cerevisiae THR4 gene. Overall identity at the amino acid sequence level is 67.4%. A continuous stretch of 32 amino acids displaying complete identity between these two fungal threonine synthases presumably contains the pyridoxal phosphate attachment site. Disruption of theA. gossypii gene led to threonine auxotrophy, which could be complemented by transformation with replicating plasmids carrying theAgTHR4 gene and variousS. cerevisiae ARS elements. Using these plasmids only very weak complementation of aS. cerevisiae thr4 mutation was observed. Investigation of sequences adjacent to theAgTHR4 gene identified three additional ORFs. Surprisingly, the order and orientation of these four ORFs is conserved inA. gossypii andS. cerevisiae.     

Oenothera mitochondrial orf454, a gene involved in cytochrome c biogenesis corresponds to orf169 and orf322 of Marchantia

We have characterized a mitochondrial gene in Oenothera, designated orf454, capable of encoding a component of the cytochrome c biogenesis system. This open reading frame is interrupted by an intron of 941 nucleotides showing high similarity to a group II intron residing in the rpl2 gene. RNA editing, which is observed at 18 cytidine positions within the orf454 reading frame, improves the similarity to protein-coding sequences in bacteria and higher plants and removes the last 16 amino acids. orf454 also shows high sequence similarity to two overlapping reading frames (orf169 and orf322) of Marchantia mitochondria. These ORFs belong to an operon-like cluster of genes in the liverwort that is not conserved in Oenothera mitochondria. However, in bacteria these reading frames are organized like the Marchantia gene cluster. It has been shown by genetical analysis in Rhodobacter capsulatus that these genes are essential for cytochrome c biogenesis. Genes of bacterial operons — ccl1 in Rhodobacter and yejR and nrfE in Escherichia coli — show high sequence similarity to the mitochondrial reading frames orf577 and orf454 of Oenothera. orf454, which we describe here, is homologous to the C-terminal region of these bacterial genes, while the previously described orf577 is homologous to the N-terminal region.     

Cloning and analysis of geldanamycin partial biosynthetic gene cluster of streptomyces hygroscopicus 17997

A geldanamycin (GDM)-producing strain, Streptomyces hygroscopicus 17997, was isolated from the soil of Yunnan, China, by the researchers of the Institute of Medicinal Biotechnology, CAMS & PUMC. GDM is an ansamycin antibiotic, which has the ability to bind with Hsp90 (heat shock protein 90) and alter its function. Hsp90 plays a key role in regulating the physiology of cells exposed to environmental stress and in maintaining the malignant phenotype of tumor cells. As an inhibitor of Hsp90, GDM possesses potent antitumoral and antiviral bioactivity, but the hypatotoxicity and poor solubility in water limit its clinical use. To accomplish the structural modification of GDM by genetic means, an attempt to obtain the biosynthetic gene cluster of GDM from S. hygroscopicus 17997 was made. In this study, a pair of primers was designed according to a conserved sequence of one of the possible post-PKS (polyketides synthase) modification genes, the carbamoyltransferase (CT) gene (gdmN) in GDM biosynthesis. The 732-bp PCR product was obtained from the S. hygroscopicus 17997 genomic DNA. Through the colony-PCR Binary Search Method, using the CT gene primers, six positive cosmid clones, CT1-6, were identified from the S. hygroscopicus 17997 cosmid genomic library. The CT-4 positive cosmid was then sub-cloned and sequenced. Approximately 28.356 kb of foreign gene sequence from CT-4 cosmid and by further PCR extension reaction was obtained. According to BLAST analysis, this sequence contains 13 possible ORFs, and they are believed to be involved in GDM production. The obtained possible GDM biosynthetic gene cluster in S. hygroscopicus 17997 will facilitate the further functional analysis of the genes and the modification of the structure of GDM through combinatorial biosynthesis.