Ubiquinone （Coenzyme Q） Biosynthesis in Chlamydophila pneumoniae AR39： Identification of the ubiD Gene

Ubiquinone is an essential electron carrier in prokaryotes.Ubiquinone biosynthesis involves atleast nine reactions in Escherichia coli.3-octaprenyl-4-hydroxybenzoate decarboxylase (UbiD) is an importantenzyme on the pathway and deletion of the ubiD gene in E.coli gives rise to ubiquinone deficiency in vivo.A protein from Chlamydophila pneumoniae AR39 had significant similarity compared with protein UbiDfrom E.coli.Based on this information,the protein-encoding gene was used to swap its counterpart inE.coli,and gene expression in resultant strain DYC was confirmed by RT-PCR.Strain DYC grew usingsuccinate as carbon source and rescued ubiquinone content in vivo,while ubiD deletion strain DYD did not.Results suggest that the chlamydial protein exerts the function of UbiD.     

Characterization of a hypervariable region in the genome of Chlamydophila pneumoniae

Chlamydophila pneumoniae displays surprisingly little genomic variation, as seen by comparisons of the published genomes from three different isolates and sequencing of four different genes from different isolates. We have in the present study, however, demonstrated genomic variation between 10 C. pneumoniae isolates in the 11690-bp region between the two outer membrane protein genes pmp1 and pmp2. This region of the C. pneumoniae CWL-029 isolate contains seven C. pneumoniae-specific open reading frames (hb1-7, encoding hydrophobic beta-sheet-containing proteins). We identified additionally 12 open reading frames in the C. pneumoniae CWL-029 genome encoding hypothetical proteins with similarity to the seven hypothetical Hb-proteins. Compared to other isolates, genomic variation is seen to cause frame-shifting of three of the 19 hb-open reading frames, which are proposed to be three full-length genes and eight frame-shifted pseudogenes. The hypothetical proteins encoded by these proposed genes contain an N-terminally located highly hydrophobic stretch of 50-60 residues. A similar motif is found in all identified Chlamydia inclusion membrane proteins and therefore the Hb-proteins are candidate inclusion proteins.     

Cloning and characterization of a gene from Pasteurella haemolytica A1 involved in lipopolysaccharide biosynthesis

Abstract A Pasteurella haemolytica A1 gene involved in the biosynthesis of a moiety on the core of the lipopolysaccharide molecule has been cloned and characterized. Escherichia coli clones which carry this gene showed an alteration of its lipopolysaccharide migration profile on tricine SDS-PAGE and exhibited resistance to the core-specific phage U3. In addition, lipopolysaccharide extracted from the E. coli clones was recognized by an anti-corespecific antiserum, but not by antiserum specific for the O antigen of P. haemolytica A1 lipopolysaccharide. Nucleotide sequence analysis of the cloned DNA identified an open reading frame ( lpsA ) coding for a protein of 263 amino acids which showed significant homology with a Haemophilus influenzae type b lipooligosaccharide biosynthesis gene. PCR amplification of genomic DNA, using primers based on the P. haemolytica A1 lpsA sequence, yielded products from only the A biotypes of P. haemolytica .     

Erythema nodosum caused by ascariasis and Chlamydophila pneumoniae pulmonary infection – a case report

Erythema nodosum belongs to a group of relatively common hypodermal inflammations. It occurs mainly among women, particularly young women. The etiology of the disease is not clear. Most frequently, changes appear on the surface of the frontal part of the shins. Initially, red nodules change in color to dark brown and then to yellow and green. There is neither dissolution nor cicatrization of the exanthema. Regression is frequent. We present a case of erythema nodosum caused by Ascaris lumbricoides infection as well as by an early Chlamydophila pneumoniae infection, whose etiology has rarely been described in the literature. We were not able to confirm which factor was responsible for the occurrence of the skin changes as treatment of both infections was effective and all skin changes later disappeared completely. Particular attention should be paid to the fact that precise diagnosis of a patient and the search for etiologic factors, even rare ones, are crucial to obtain good results with treatment of erythema nodosum.     

肺炎嗜衣原体主要外膜蛋白的克隆表达与抗原性研究

肺炎嗜衣原体主要外膜蛋白是其特征抗原之一。实验中通过PCR方法从肺炎嗜衣原体基因组中扩增主要外膜蛋白基因,插入pET32a(+)表达载体,转化BL21(DE3)感受态细胞,得到表达56kD融合蛋白的工程菌株。该菌株的表达量可达53%,提纯后的主要外膜蛋白纯度可达90%以上,在Western Blotting试验和胶体金免疫层析试验中显示了良好的抗原性。     

Identification of an additional protein involved in mannan biosynthesis

Galactomannans comprise a β‐1,4‐mannan backbone substituted with α‐1,6‐galactosyl residues. Genes encoding the enzymes that are primarily responsible for backbone synthesis and side‐chain addition of galactomannans were previously identified and characterized. To identify additional genes involved in galactomannan biosynthesis, we previously performed deep EST profiling of fenugreek (Trigonella foenum‐graecum L.) seed endosperm, which accumulates large quantities of galactomannans as a reserve carbohydrate during seed development. One of the candidate genes encodes a protein that is likely to be a glycosyltransferase. Because this protein is involved in mannan biosynthesis, we named it ‘mannan synthesis‐related’ (MSR). Here, we report the characterization of a fenugreek MSR gene (TfMSR) and its two Arabidopsis homologs, AtMSR1 and AtMSR2. TfMSR was highly and specifically expressed in the endosperm. TfMSR, AtMSR1 and AtMSR2 proteins were all determined to be localized to the Golgi by fluorescence confocal microscopy. The level of mannosyl residues in stem glucomannans decreased by approximately 40% for Arabidopsis msr1 single T‐DNA insertion mutants and by more than 50% for msr1 msr2 double mutants, but remained unchanged for msr2 single mutants. In addition, in vitro mannan synthase activity from the stems of msr1 single and msr1 msr2 double mutants also decreased. Expression of AtMSR1 or AtMSR2 in the msr1 msr2 double mutant completely or partially restored mannosyl levels. From these results, we conclude that the MSR protein is important for mannan biosynthesis, and offer some ideas about its role.     

Engineering of ubiquinone biosynthesis using the yeast coq2 gene confers oxidative stress tolerance in transgenic tobacco

Ubiquinone (UQ), an electron carrier in the respiratory chain ranging from bacteria to humans, shows antioxidative activity in vitro, but its physiological role in vivo is not yet clarified in plants. UQ biosynthesis was modified by overexpressing the yeast gene coq2, which encodes p-hydroxybenzoate:polyprenyltransferase, to increase the accumulation of UQ-6 in yeast and UQ-10 in tobacco. The yeast and tobacco transgenic lines showed about a three- and six-fold increase in UQ, respectively. COQ2 polypeptide, the localization of which was forcibly altered to the endoplasmic reticulum, had the same or a greater effect as mitochondria-localized COQ2 on the increase in UQ in both the yeast and tobacco transformants, indicating that the UQ intermediate is transported from the endoplasmic reticulum to the mitochondria. Plants with a high UQ level are more resistant to oxidative stresses caused by methyl viologen or high salinity. This is attributable to the greater radical scavenging ability of the transgenic lines when compared with the wild type.     

Suppression of a key gene involved in chlorophyll biosynthesis by means of virus-inducing gene silencing

The ChlH gene coding the H subunit of magnesium chelatase, an enzyme involved in chlorophyll biosynthesis, was silenced in Nicotiana benthamiana plants by infection with tobacco mosaic virus vectors (pTMV-30b) containing 67, 214 or 549 nt long ChlH inserts. Silencing of the nuclear ChlH gene induced a chimeric phenotype with green and yellow/white leaves associated with alterations of chloroplast shape and ultrastructure. The symptoms became first evident around veins of young leaves, and only later in the mesophyll tissues. The efficiency of gene silencing was not dependent on the insert orientation, but was strongly correlated with the size of the ChlH insert, providing a flexible method to modulate the level of gene suppression. Silencing efficiency seemed to be strongly dependent on endogenous ChlH mRNA level of the target tissue. Silencing of the ChlH gene with the longest fragment of 549 nt also lowered the accumulation of ChlD and chlorophyll synthetase mRNAs, i.e. other genes involved in chlorophyll biosynthesis.     

Cloning and characterization of a potato StAN11 gene involved in anthocyanin biosynthesis regulation

Anthocyanins are a class of products of plant secondary metabolism and are responsible for tubers color in potato.The biosynthesis of anthocyanins is a complex Researchbiological process,in which multiple genes are involved including structural genes and regulatory genes.In this study,StAN11,a WD40-repeat gene,was cloned from potato cultivar Chieftain(Solanum tuberosum L.).StAN11(HQ599506)contained no intron and its open reading frame(ORF)was 1,029 bp long,encoding a putative protein of 342 amino acids.In order to verify its role in anthocyanin biosynthesis,StAN11 was inserted behind the CaMV-35S promoter of pCMBIA1304 and the recombination vector was introduced into the potato cultivar Désirée plants by Agrobacterium-mediated transformation.The color of transgenic tuber skin was significantly deepened,compared to the wild-type control,which was highly consistent with the accumulation of anthocyanin and expression of StAN11 in transgenic lines tuber skin.Further analysis on the expression of Flavonone-3-hydroxylase(F3H),Dihydroflavonol reductase(DFR),Anthocyanidin synthase(ANS),and Flavonoid 3-O-glucosyl transferase(3GT)in transgenic plants revealed that only DFR was upregulated.This result suggested that StAN11 regulated anthocyanin biosynthesis in potato by controlling DFR expression and accumulation of anthocyanin could be increased through overexpression of StAN11 in the tubers with the genetic background of anthocyanin biosynthesis.     

Identification and characterization of a rhamnosyltransferase involved in rutin biosynthesis in Fagopyrum esculentum (common buckwheat)

Rutin, a 3-rutinosyl quercetin, is a representative flavonoid distributed in many plant species, and is highlighted for its therapeutic potential. In this study, we purified uridine diphosphate-rhamnose: quercetin 3-O-glucoside 6″-O-rhamnosyltransferase and isolated the corresponding cDNA (FeF3G6″RhaT) from seedlings of common buckwheat (Fagopyrum esculentum). The recombinant FeF3G6″RhaT enzyme expressed in Escherichia coli exhibited 6″-O-rhamnosylation activity against flavonol 3-O-glucoside and flavonol 3-O-galactoside as substrates, but showed only faint activity against flavonoid 7-O-glucosides. Tobacco cells expressing FeF3G6″RhaT converted the administered quercetin into rutin, suggesting that FeF3G6″RhaT can function as a rhamnosyltransferase in planta. Quantitative PCR analysis on several organs of common buckwheat revealed that accumulation of FeF3G6″RhaT began during the early developmental stages of rutin-accumulating organs, such as flowers, leaves, and cotyledons. These results suggest that FeF3G6″RhaT is involved in rutin biosynthesis in common buckwheat.     

Cloning, sequencing and function ofsanA, a gene involved in nikkomycin biosynthesis ofStreptomyces ansochromogenes

Several genetically stable mutants blocked in nikkomycin biosynthesis were obtained after the slightly germinated spores of Streptomyces ansochromogenes, a nikkomycin producer, were treated with ultra violet radiation. One of the mutants is the same in morpholotical differentiation as the wild type strain and is designated as NBB19. A DMA library was constructed using plasmid plJ702 as cloning vector, NBB19 as cloning recipient. A 6 kb DNA fragment which can genetically complement NBB19 was cloned when screening the library for antifungal activity. Sequence analysis showed that the 3 kb Bgl II-Sal I fragment contains one complete ORF (ORF1) and one partial ORF (ORF2). ORF1 is designated as sanA. sanA is 1 365 bp, encoding a protein consisting of 454 amino acid residues. Database searching indicated that sanA is homologous to the hypothetical methyltransferase in Pyrococcus horikoshli with 25% identities and 41% positives. Disruptant of sanA lost the ability to synthesize nikkomycin. It indicated that sa     

Characterization of a methyltransferase involved in herboxidiene biosynthesis

The herboxidiene biosynthetic gene cluster contains a regulatory gene and six biosynthetic genes that encode three polyketide synthases (HerB, HerC and HerD) and three tailoring enzymes (HerE, HerF and HerG). Through single crossover recombination, an integrative plasmid was inserted into the genome of Streptomyces chromofuscus ATCC 49982 between herE and herF, resulting in low-level expression of herF and the downstream herG. The mutant strain produced two new compounds, 18-deoxy-25-demethyl-herboxidiene and 25-demethyl-herboxidiene. HerF was expressed in Escherichia coli and biochemically characterized as the dedicated methyltransferase in herboxidiene biosynthesis. It prefers 25-demethyl-herboxidiene to 18-deoxy-25-demethyl-herboxidiene, suggesting that C-25 methylation is the last tailoring step.     

Crystal structure of MshB from Mycobacterium tuberculosis, a deacetylase involved in mycothiol biosynthesis

All living species require protection against the damaging effects of the reactive oxygen species that are a natural by-product of aerobic life. In most organisms, glutathione is a critical component of these defences, maintaining a reducing environment inside cells. Some bacteria, however, including pathogenic mycobacteria, use an alternative low molecular mass thiol compound called mycothiol (MSH) for this purpose. Enzymes that synthesize MSH are attractive candidates for the design of novel anti-TB drugs because of the importance of MSH for mycobacterial life and the absence of such enzymes in humans. We have determined the three-dimensional structure of MshB (Rv1170), a metal-dependent deacetylase from Mycobacterium tuberculosis that catalyses the second step in MSH biosynthesis. The structure, determined at 1.9A resolution by X-ray crystallography (R=19.0%, R(free)=21.4%), reveals an alpha/beta fold in which helices pack against a seven-stranded mostly parallel beta-sheet. Large loops emanating from the C termini of the beta-strands enclose a deep cavity, which is the location of the putative active site. At the bottom of this cavity is a metal-binding site associated with a sequence motif AHPDDE that is invariant in all homologues. An adventitiously bound beta-octylglucoside molecule, used in crystallization, enables us to model the binding of the true substrate and propose a metal-dependent mechanistic model for deacetylation. Sequence comparisons indicate that MshB is representative of a wider family of enzymes that act on substituted N-acetylglucosamine residues, including a deacetylase involved in the biosynthesis of glycosylphosphatidylinositol (GPI) anchors in eukaryotes.     

Identification of Escherichia coli ubiB, a gene required for the first monooxygenase step in ubiquinone biosynthesis

It was recently discovered that the aarF gene in Providencia stuartii is required for coenzyme Q (CoQ) biosynthesis. Here we report that yigR, the Escherichia coli homologue of aarF, is ubiB, a gene required for the first monooxygenase step in CoQ biosynthesis. Both the P. stuartii aarF and E. coli ubiB (yigR) disruption mutant strains lack CoQ and accumulate octaprenylphenol. Octaprenylphenol is the CoQ biosynthetic intermediate found to accumulate in the E. coli strain AN59, which contains the ubiB409 mutant allele. Analysis of the mutation in the E. coli strain AN59 reveals no mutations within the ubiB gene, but instead shows the presence of an IS1 element at position +516 of the ubiE gene. The ubiE gene encodes a C-methyltransferase required for the synthesis of both CoQ and menaquinone, and it is the 5' gene in an operon containing ubiE, yigP, and ubiB. The data indicate that octaprenylphenol accumulates in AN59 as a result of a polar effect of the ubiE::IS1 mutation on the downstream ubiB gene. AN59 is complemented by a DNA segment containing the contiguous ubiE, yigP, and ubiB genes. Although transformation of AN59 with a DNA segment containing the ubiB coding region fails to restore CoQ biosynthesis, transformation with the ubiE coding region results in a low-frequency but significant rescue attributed to homologous recombination. In addition, the fre gene, previously considered to correspond to ubiB, was found not to be involved in CoQ biosynthesis. The ubiB gene is a member of a predicted protein kinase family of which the Saccharomyces cerevisiae ABC1 gene is the prototypic member. The possible protein kinase function of UbiB and Abc1 and the role these polypeptides may play in CoQ biosynthesis are discussed.     

Identification and characterization two isoforms of NADH:ubiquinone oxidoreductase from the hyperthermophilic eubacterium Aquifex aeolicus

The NADH:ubiquinone oxidoreductase (complex I) is the first enzyme of the respiratory chain and the entry point for most electrons. Generally, the bacterial complex I consists of 14 core subunits, homologues of which are also found in complex I of mitochondria. In complex I preparations from the hyperthermophilic bacterium Aquifex aeolicus we have identified 20 partially homologous subunits by combining MALDI-TOF and LILBID mass spectrometry methods. The subunits could be assigned to two different complex I isoforms, named NQOR1 and NQOR2. NQOR1 consists of subunits NuoA₂, NuoB, NuoD₂, NuoE, NuoF, NuoG, NuoI₁, NuoH₁, NuoJ₁, NuoK₁, NuoL₁, NuoM₁ and NuoN₁, with an entire mass of 504.17?kDa. NQOR2 comprises subunits NuoA₁, NuoB, NuoD₁, NuoE, NuoF, NuoG, NuoH₂, NuoI₂, NuoJ₁, NuoK₁, NuoL₂, NuoM₂ and NuoN₂, with a total mass of 523.99?kDa. Three Fe-S clusters could be identified by EPR spectroscopy in a preparation containing predominantly NQOR1. These were tentatively assigned to a binuclear center N1, and two tetranuclear centers, N2 and N4. The redox midpoint potentials of N1 and N2 are ?273?mV and ?184?mV, respectively. Specific activity assays indicated that NQOR1 from cells grown under low concentrations of oxygen was the more active form. Increasing the concentration of oxygen in the bacterial cultures induced formation of NQOR2 showing the lower specific activity.