一株从红藻中分离出的产芳基硫酸酯酶的海单胞菌FW-1的全基因组测序及结果分析

【目的】海单胞菌Marinomonas sp. FW-1是1株经验证可以获得高活性芳基硫酸酯酶的菌株。为深入研究FW-1菌株产芳基硫酸酯酶机制,进一步筛选高活性的芳基硫酸酯酶基因片段,有必要解析FW-1菌株的全基因组序列信息。【方法】本研究采用高通量测序技术对FW-1进行全基因组测序,使用相关软件对测序数据进行基因组装、基因预测与功能注释、COG聚类分析等。结合异源表达的方法对其不同基因片段所产生的芳基硫酸酯酶活性进行分析。【结果】全基因组测序结果表明该基因组大小为3964876 bp,GC含量为44.03%,编码3590个蛋白基因,含有78个tRNA和25个rRNA操纵子。从全基因组测序结果中找到22个可能具有芳基硫酸酯酶活性的基因,对其中4个进一步异源表达后发现FW-1中至少含有的3个具有芳基硫酸酯酶活性的基因,其均含有芳基硫酸酯酶的特异性氨基酸基团C-X-P-X-R基团。【结论】本研究首次报道了1株含有多个芳基硫酸酯酶基因序列的菌株FW-1的全基因组序列,分析了基因组的基本特征,为芳基硫酸酯酶的进一步应用提供了思路。     

Three knotted1-like homeobox genes in Arabidopsis

Five arabidopsis kn1-like homeobox genes were cloned through low-stringency screening of Arabidopsis cDNA libraries with the kn1 homeobox from maize. These five genes were named KNAT1-5 (for kn1-like Arabidopsis thaliana). An analysis of KNAT1 and 2 has been presented previously [19]. Here we present an analysis of the genes KNAT3, 4 and 5. On the basis of sequence and expression patterns, these three genes belong to the class II subfamily of kn1-like homeobox genes [16]. Low-stringency Southern analysis suggests several additional members of the class II genes exist in the Arabidopsis genome. The predicted amino acid sequences of the three genes share extensive homology outside of the homeodomain, including 84% between KNAT3 and KNAT4. Northern analysis shows that although all three genes are expressed in all tissues examined, the level of KNAT3 RNA is highest in young siliques, inflorescences and roots, KNAT4 RNA level is strongest in leaves and young siliques, and KNAT5 RNA level is highest in roots. The specificity of these patterns was confirmed by RNA fingerprint analysis. KNAT3 and 4 are light-regulated as they show reduced expression in etiolated seedlings and also in hy3, cop1 and det1 mutant backgrounds.     

Physiological analysis of Methylobacterium extorquens AM1 grown in continuous and batch cultures

Chemostat cultures of Methylobacterium extorquens AM1 grown on methanol or succinate at a range of dilution rates were compared to batch cultures in terms of enzyme levels, poly-β-hydroxybutyrate content, and intracellular concentrations of adenine and pyridine nucleotides. In both chemostat and batch cultures, enzymes specific to C₁ metabolism were up-regulated during growth on methanol and down-regulated during growth on succinate, polyhydroxybutyrate levels were higher on succinate, intracellular ATP levels and the energy charge were higher during growth on methanol, while the pools of reducing equivalents were higher during growth on succinate. For most of the tested parameters, little alteration occurred in response to growth rate. Overall, we conclude that the chemostat cultivation conditions developed in this study roughly mimic the growth in batch cultures, but provide a better control over the culturing conditions and a better data reproducibility, which are important for integrative functional studies. This study provides baseline data for future work using chemostat cultures, defining key similarities and differences in the physiology compared to existing batch culture data.     

Mapping of pgi and gpd genes involved in C-1 assimilation in the obligate methylotroph Methylobacillus flagellatum

Homologous matings with plasmids R68.45 and pULB113, and also with Hfr type donor were employed for mapping pgi and gpd genes involved in C-1 metabolism in the obligate methylotroph Methylobacillus flagellatum. A preliminary map of the late chromosomal region was constructed on the basis of these experimental results. The C-1 markers were linked to methionine and leucine auxotrophy and nalidixic acid resistance markers. The phenomenon of retrotransfer, or shuttle transfer of chromosomal markers by Inc P1 plasmids, revealed earlier, was demonstrated for M. flagellatum.     

Expression of poly-3-(R)-hydroxyalkanoate (PHA) polymerase and acyl-CoA-transacylase in plastids of transgenic potato leads to the synthesis of a hydrophobic polymer,presumably medium-chain-length PHAs

Medium-chain-length poly-3-(R)-hydroxyalkanoates (mcl-PHAs) belong to the group of microbial polyesters. The minimum gene-set for the accumulation of mcl-PHAs from de novo fatty acid biosynthesis has been identified in prokaryotes [B. Rehm et al. (1998) J. Biol Chem 273:24044–24051] as consisting of the Pha-C1 polymerase and the ACP-CoA-transacylase. In this paper, the synthesis of mcl-PHAs has been attempted in transgenic potato (Solanum tuberosum L.) using the same set of genes that were introduced into potato by particle bombardment. Polymer contents of transgenic lines were analysed by gas chromatography and by a new simple method employing a size-exclusion filter column. The expression of the Pha-C1 polymerase and the ACP-CoA-transacylase in the plastids of transgenic potato led to the synthesis of a hydrophobic polymer composed of mcl-hydroxy-fatty acids with carbon chain lengths ranging from C-6 to C-12 in leaves of the selected transgenic lines. We strongly suggest that the polymer observed consists of mcl-PHAs and that this report establishes for the first time a possible route for the production of mcl-PHAs from de novo fatty acid biosynthesis in plants.     

Proteomic and molecular investigation on the physiological adaptation of Comamonas sp. strain CNB-1 growing on 4-chloronitrobenzene

Comamonas sp. strain CNB-1 can utilize 4-chloronitrobenzene (4CNB) as sole carbon and nitrogen source for growth. Previous studies were focused on 4CNB degradative pathway and have showed that CNB-1 contained a plasmid pCNB1 harboring the genes (cnbABCaCbDEFGH, cnbZ) for the enzymes involving in 4CNB degradation, but only three gene products (CnbCa, CnbCb, and CnbZ) were identified in CNB-1 cells. Comamonas strain CNB-2 that lost pCNB1 was not able to grow on 4CNB. In this study, physiological adaptation to 4CNB by CNB-1 was investigated with proteomic and molecular tools. Comparative proteomes of strains CNB-1 and CNB-2 grown on 4CNB and/or succinate revealed that adaptation to 4CNB by CNB-1 included specific degradative pathway and general physiological responses: (1) Seven gene products (CnbA, CnbCa, CnbCb, CnbD, CnbE, CnbF, and CnbZ) for 4CNB degradation were identified in 4CNB-grown cells, and they were constitutively synthesized in CNB-1. Two genes cnbE and cnbF were cloned and simultaneously expressed in E. coli. The CnbE and CnbF together catalyzed the conversion of 2-oxohex-4-ene-5-chloro-1,6-dioate into 2-oxo-4-hydroxy-5-chloro-valeric acid; (2) Enzymes involving in glycolysis, tricarboxylic acid cycle, and synthesis of glutamate increased their abundances in 4CNB-grown cells.     

Overexpression of CYP710A1 and CYP710A4 in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis> plants increases the level of stigmasterol at the expense of sitosterol

Sitosterol and stigmasterol are major sterols in vascular plants. An altered stigmasterol:sitosterol ratio has been proposed to influence the properties of cell membranes, particularly in relation to various stresses, but biosynthesis of stigmasterol is poorly understood. Recently, however, Morikawa et al. (Plant Cell 18:1008–1022, 2006) showed in Arabidopsis thaliana that synthesis of stigmasterol and brassicasterol is catalyzed by two separate sterol C-22 desaturases, encoded by the genes CYP710A1 and CYP710A2, respectively. The proteins belong to a small cytochrome P450 subfamily having four members, denoted by CYP710A1-A4, and are related to the yeast sterol C-22 desaturase Erg5p acting in ergosterol synthesis. Here, we report on our parallel investigation of the Arabidopsis CYP710A family. To elucidate the function of CYP710A proteins, transgenic Arabidopsis plants were generated overexpressing CYP710A1 and CYP710A4. Compared to wild-type plants, both types of transformant displayed a normal phenotype, but contained increased levels of free stigmasterol and a concomitant decrease in the level of free sitosterol. CYP710A1 transformants also displayed higher levels of esterified forms of stigmasterol, cholesterol, 24-methylcholesterol and isofucosterol. The results confirm the findings of Morikawa et al. (Plant Cell 18:1008–1022, 2006) regarding the function of CYP710A1 in stigmasterol synthesis, and show that CYP710A4 also has this capacity. Furthermore, our results suggest that an increased stigmasterol level alone is sufficient to stimulate esterification of other major sterols.     

Conversion of an RFLP marker for the barley stem rust resistance geneRpg1 to a specific PCR-amplifiable polymorphism

TheRpg1 gene in barley has provided satisfactory levels of stem rust resistance for the last 50 years. The appearance of a new race of stem rust that is virulent toRpg1 has resulted in efforts to incorporate new stem rust resistance genes into barley. Marker-assisted selection may provide the only means of combining this useful gene with resistance genes for which no virulent races have been identified. Several RFLP markers have been identified as linked to theRpg1 locus. One of these, ABG704 was converted into a post-amplification restriction polymorphism. To generate a specific PCR-amplifiable polymorphism the sequence of the ABG704 locus from four barley cultivars was determined. Primers were developed that can detect a single-base difference between resistant and susceptible cultivars. The successful conversion of an RFLP marker to an allele-specific PCR-based marker not only demonstrates that this type of conversion is possible for cereals, but also results in an immediately useful marker for application to plant breeding programmes.     

桑树磷脂酶MaPLA1-2D亚型4个基因的克隆与胁迫应答分析

磷脂酶(phospholipase)是一类在植物生长发育和胁迫应答中起重要调控作用的磷脂水解酶,也是一类重要的信号转导酶。而磷脂酶A1(PLA1)在植物应答生物胁迫和非生物胁迫中的功能研究鲜见报道。研究从桑树(Morus alba L.)中克隆了磷脂酶PLA1的1个亚型MaPLA1-2D基因,对其进行了序列分析、组织表达、胁迫诱导表达和蛋白亚细胞定位分析。结果表明,桑树PLA1-2D亚型基因包括4个成员,命名为MaPLA1-2D.1~MaPLA1-2D.4。4个基因在桑树根和叶中高水平表达,蛋白亚细胞定位在叶绿体。序列和进化分析表明MaPLA1-2D基因4个成员与拟南芥AtDAD1基因的保守结构域序列具有较高相似度且进化关系紧密。MaPLA1-2D基因4个成员的启动子含有多种胁迫应答顺式元件和激素响应元件;胁迫诱导表达模式分析表明MaPLA1-2D基因表达受干旱和脱落酸处理显著诱导。以上结果说明,MaPLA1-2D基因与拟南芥DAD同源,可能在桑树非生物胁迫应答中发挥重要功能。     

Characterization of CONSTITUTIVE PHOTOMORPHOGENESIS AND DWARFISM Homologs in Rice (Oryza sativa L.)

To enhance our understanding of brassinosteroid (BR) biosynthesis in rice, we attempted to identify putative rice homologs of Arabidopsis CYP90A1/ CPD and related mutants. Two candidate genes, designated CYP90A3/OsCPD1 and CYP90A4/OsCPD2, are located on chromosomes 11 (2.0 cM) and 12 (1.9 cM), respectively. Based on sequence similarity with the Arabidopsis CYP90A1/CPD gene, we predict that the CYP90A3/OsCPD1 and CYP90A4/OsCPD2 gene products function as C-23α hydroxylases in the BR biosynthesis pathway. Both are broadly expressed in wild-type rice, and their expression is regulated by a feedback mechanism. A retrotransposon insertion mutant of CYP90A3/OsCPD1, oscpd1-1, did not produce any BR-deficient phenotype or feedback upregulation of genes for BR biosynthesis enzymes. These results indicate that if, as predicted, the CYP90A3/OsCPD1 and CYP90A4/OsCPD2 genes do function in the BR biosynthesis pathway, they may each have enough capacity to catalyze BR biosynthesis on their own. As a consequence, the oscpd1-1 mutant may not be deficient in endogenous BRs. Interestingly, BR biosynthesis enzymes except C-6 oxidase are encoded by plural genes in rice but by single genes in Arabidopsis (again, except C-6 oxidase). On the basis of these findings, we discuss the differences in BR biosynthesis between rice and Arabidopsis.     

A simple procedure for the isolation of streptomycin resistant plants in Solanaceae

A system has been developed for rapid selection of streptomycin resistant mutants, as adventitious shoots arising from explants of several Solanaceous species. Efficient mutagenesis was achieved by incubating shoot culture-derived leaf strips with 1 or 5 mM nitroso-methylurea, for 90 or 120 min. In Nicotiana tabacum and Lycopersicon peruvianum these treatments resulted in white or variegated adventitious shoots from up to 3.5% of explants placed on medium promoting shoot regeneration. Chlorophyll deficiencies were only observed very rarely in Solanum nigrum. Streptomycin resistant shoots were obtained from leaf explants placed on medium containing 500 mg l^-1 streptomycin sulphate, under which conditions explants are bleached and adventitious shoot development suppressed. Green adventitious s shoots appeared at a frequency dependent both on the mutagenic treatment and on the species. The best response was with S. nigrum where >70% of the explants produced streptomycin resistant shoots, most of which retained their resistance on subsequent testing. Maternal inheritance of streptomycin resistance has been confirmed for several N. tabacum and S. nigrum mutants, and there is also evidence for paternal transmission in the latter species. The procedure has been successfully extended to other species, including N. sylvestris and N. plumbaginifolia, and also to obtain spectinomycin resistant mutants.Communicated by R. Hagemann     

Comparative analysis of the structure and chromosomal assignment ofCD1: an evidence for different evolutionary histories between classic CD1 and CD1D class genes

The non-MHC-encoded CD1 family has recently emerged as a novel antigen-presenting system that is distinct from MHC class I and class II molecules. In the present study, we determined the genomic structure of that rat CD1, and compared with those of other previously reported CD1 genes. Rat CD1 was extremely similar to mouse CD1 genes, especially to CD1D1. It is of interest that a tyrosine-based motif for endosomal localization, identified in the human CD1b cytoplasmic tail, was conserved in all CD1 molecules except for CD1a, that was encoded by a single short exon. Comparison of the overall exon-intron organization of CD1 genes revealed that the length of the introns was also characteristic to each of the two classes of CD1 genes; classic (CD1A, CD1B, CD1C and CD1E), and CD1D, which have been categorized by comparison of coding regions. These findings support a hypothesis that the two classes have different evolutionary histories. In contrast to the absence of the classic CD1 genes in rats and mice, the entire region of nonpolymorphic CD1D gene has been conserved through mammalian evolution. Furthermore, we determined chromosomal localization of rat CD1 gene using the fluorescence in situ hybridization method with several probes derived from genomic rat CD1 clones. Similar to human and mouse CD1, rat CD1 mapped outside the MHC loci despite the structural and functional resemblance to MHC. Conserved syntheny of chromosomal segments of RNO2 and MMU3 is implied.     

Harvesting of Chlorella vulgaris using a bioflocculant from Paenibacillus sp. AM49

Microbial flocculants for harvesting mass cultured Chlorella vulgaris were screened and that from Paenibacillussp. AM49 was identified as the best. The flocculation efficiency of this bioflocculant increased with the pH within a range of pH 5–11 and was 83%, which was higher than the 72% and 78% produced by aluminum sulfate and polyacrylamide, respectively. The highest flocculation efficiency was with 6.8 mm CaCl₂ as co-flocculant. The bioflocculant from Paenibacillussp. AM49 can be used effectively to harvest C. vulgaris from large-scale cultures.     

Spectrophotometric estimation of 4-androstene-3,17-dione and 1,4-androstadiene-3,17-dione during C-1(2)-dehydrogenation by Mycobacterium fortuitum NRRL B-8153

A spectrophotometric method for simultaneously estimating 4-androstene-3,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD) in a binary mixture has been developed using sulphuric acid chromogens. The method has been used to estimate both AD and ADD during C-1(2)-dehydrogenation by Mycobacterium fortuitum NRRL B-8153.The authors are with the School of Life Sciences, Devi Ahilya Vishwavidyalaya. Vigyan Bhawan, Khandwa Road, Indore-452 001, India.     

Analysis of the <Emphasis Type="Italic">MAT1-2-1</Emphasis> gene of <Emphasis Type="Italic">Colletotrichum lindemuthianum</Emphasis>

A single MAT1-2-1 gene was identified from a mating pair of the filamentous ascomycete Colletotrichum lindemuthianum. The MAT1-2-1 genes from both mating partners carried an open reading frame (ORF) of 870 bp encoding a putative protein of 290 amino acids that includes the highly conserved high mobility group (HMG) domain typical of the fungal MAT1-2-1 genes. Three introns were confirmed within the C. lindemuthianum ORF, two of which were found to be conserved relative to a previously reported MAT1-2-1 gene from C. gloeosporioides. The amino acid sequence of the HMG domain from C. lindemuthianum MAT1-2-1 was also compared with those from other ascomycetes. These results suggest that although the MAT1-2-1 genes are highly conserved among ascomycetes, the mechanism which defines mating partners in the genus Colletotrichum is distinct to the idiomorph system described for other members of this phylum.     

AM1* parameters for bromine and iodine

Our extension of the AM1 semiempirical molecular orbital technique, AM1*, has been parameterized for the elements Br and I. The basis sets for both halogens contain a set of d-orbitals as polarization functions. AM1* performs as well as other MNDO-like methods that use d-orbitals in the basis, and better than those that rely on an sp-basis. Thus, AM1* parameters are now available for H, C, N, O and F (which use the original AM1 parameters), Al, Si, P, S, Cl, Ti, Cu, Zn, Br, Zr, Mo and I. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

AM1* parameters for vanadium and chromium

Our extension of the AM1 semiempirical molecular orbital technique, AM1*, has been parameterized for the elements V and Cr. The basis sets for both metals contain one set each of s-, p- and d-orbitals. AM1* parameters are now available for H, C, N, O and F (which use the original AM1 parameters), Al, Si, P, S, Cl, Ti, V, Cr, Cu, Zn, Br, Zr, Mo and I. The performance and typical errors of AM1* are discussed for V and Cr and compared with available NDDO Hamiltonians. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

AM1* parameters for copper and zinc

Our extension of the AM1 semiempirical molecular orbital technique, AM1*, has been parameterized for the elements Cu and Zn. The basis sets for both metals contain a set of d-orbitals. The zinc parameterization uses a filled d-shell to give 12 valence electrons. Thus, AM1* parameters are now available for H, C, N, O and F (which use the original AM1 parameters), Al, Si, P, S, Cl, Ti, Cu, Zn, Zr and Mo. The performance and typical errors of AM1* are discussed for the newly parameterized elements. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Detection of streptomycin resistance in Mycobacterium tuberculosis clinical isolates from China as determined by denaturing HPLC analysis and DNA sequencing

China is regarded by the World Health Organization as a major hot-spot region for Mycobacterium tuberculosis infection. Streptomycin has been deployed in China for over 50 years and is still widely used for tuberculosis treatment. We have developed a denaturing HPLC (DHPLC) method for detecting various gene mutations conferring drug resistance in M. tuberculosis. The present study focused on rpsL and rrs mutation analysis. Two hundred and fifteen M. tuberculosis clinical isolates (115 proved to be streptomycin-resistant and 100 susceptible by a routine proportional method) from China were tested to determine the streptomycin minimal inhibitory concentration (MIC), and subjected to DHPLC and concurrent DNA sequencing to determine rpsL and rrs mutations. The results showed that 85.2% (98/115) of streptomycin-resistant isolates harbored rpsL or rrs mutation, while rpsL mutation (76.5%, 88/115) dominated. MIC of 98 mutated isolates revealed no close correlation between mutation types and levels of streptomycin resistance. No mutation was found in any of the susceptible isolates. The DHPLC results were completely consistent with those of sequencing. The DHPLC method devised in this study can be regarded as a useful and powerful tool for detection of streptomycin resistance. This is the first report to describe DHPLC analysis of mutations in the rpsL and rrs genes of M. tuberculosis in a large number of clinical isolates.