Structure and Function of sanV: a gene involved in nikkomycin biosynthesis of Streptomyces ansochromogenes

A 6.3-kb BamHI- BglII DNA fragment was cloned from cos20 by using chromosome walking strategy. It was partially sequenced with the result that there is a possible ORF of 1272 nucleotides. The ORF designated sanV was deposited in GenBank under accession no. AF469955. Database search indicated that the deduced protein of sanV shows 28% identity and 44% similarity over 405 amino acid residues to the large component (E) of glutamate mutases from Clostridium cochlearium. Gene disruption was performed to study the function of sanV. It was found that sanV disruptants exhibited much poorer inhibition to the test strain than the wild-type S. ansochromogenes 7100. Furthermore, HPLC analysis indicated that the sanV disruptants almost did not produce nikkomycins X and Z, whereas they accumulated new nikkomycins O(x) and O(z), which revealed that sanV is an important gene involved in the biosynthesis of the peptidyl moiety of nikkomycins.     

Roles of the three Mycobacterium smegmatis katG genes for peroxide detoxification and isoniazid susceptibility

Three different katG sequences (katGI, katGII and katGIII) were identified in the Mycobacterium smegmatis genome. The contributions of the three katG genes to survival of the bacterium were examined by constructing disruptants of these three genes. The katGIII sequence did not produce a functional catalase‐peroxidase. Analyses of peroxidase activity and mRNA expression revealed that in wild type M. smegmatis, expression dominance between KatGI and KatGII was switched in the exponential and stationary growth phases. Susceptibility of the M. smegmatis gene disruptants to hydrogen peroxide (H₂O₂) was tested in two growth phases. In the exponential phase, the katGI‐null strain was more susceptible to H₂O₂ than the katGII‐null strain, indicating that KatGI plays a more important role in survival than KatGII in this growth phase. In contrast, in the stationary phase, growth of the katGII‐null strain was inhibited at lower concentrations of H₂O₂. These results suggest that M. smegmatis has two types of catalase‐peroxidases, expressions of which are controlled under different gene regulatory systems. Isoniazid (INH) susceptibilities of the katG‐null strains were also examined and it was found that katGI is a major determinant of M. smegmatis susceptibility to INH.

     

The Screening of Hydrogen Peroxide-Producing Lactic Acid Bacteria and Their Application to Inactivating Psychrotrophic Food-Borne Pathogens

Lactic acid bacteria were isolated from various food samples and evaluated for hydrogen peroxide (H₂O₂) production. Cells suspended in 0.5% (wt/vol) glucose plus 0.5% (wt/vol) lactate (pH 7.0) were incubated for 5 h at 37°C under aeration. Among 193 strains, 27 strains accumulated 201-300 ppm H₂O₂, and 4 strains accumulated more than 301 ppm H₂O₂ in the cell suspensions. Among the 9 high-level H₂O₂-producing strains, 8 strains were identified as Lactococcus lactis subsp. lactis. The cell-free filtrate from Lc. lactis subsp. lactis AI 62, which contained approximately 350 ppm H₂O₂, was evaluated for antimicrobial activity against Enterococcus faecalis, Ent. faecium, enterotoxigenic Escherichia coli, Listeria ivanovii, Staphylococcus aureus, Yersinia enterocolitica, and Aeromonas hydrophila. After 1 h incubation at 30°C in the cell-free filtrate, the initial viable cell counts of the target bacteria (5.53–6.00 log cfu/mL) were reduced by 0.12-5.00 log units, except in the case of enterococci. The sensitivity varied with the bacterial species and pH. The enterococci were resistant to the treatment. Our results show that H₂O₂ accumulated by lactic acid bacteria in a cell suspension is very effective in reducing the viable cell count of food-borne pathogens.Received: 7 October 2002 / Accepted: 4 November 2002     

Nutritional control of nikkomycin and juglomycin production by Streptomyces tendae in continuous culture

Summary Continuous cultures with Streptomyces tendae revealed some interesting facts. In a continuous culture running for more than 2500 h the production of either nikkomycins or juglomycins could be selected by varying the feed composition. Decreasing the phosphate supply in the feed broth from the initial concentration of 2.5 mm to 1.0 mm enhanced the productivity of nikkomycins and decreased the productivity of juglomycins. When switching back to the initial conditions of the experiment after 2000 h nearly the same production behaviour as at the beginning of the fermentation could be observed. This indicated a stable behaviour of the population with regard to nikkomycin productivity. The long continuous fermentation showed the ability of S. tendae Tü 901/8c to produce nikkomycin at a high level for at least 1500 h. In a second continuous culture it was shown that the productivity of the nikkomycins and juglomycins decreased and increased, respectively, with increasing dilution rate. Comparing batch cultures with continuous fermentations, higher juglomycin productivity was found in the latter. These facts indicate that the strain responds to complex interacting physiological controls, by producing either nikkomycins or juglomycins in a higher amount. Offprint requests to: D. Hege-Treskatis     

Genetic Organization of Genes Encoding Phenol Hydroxylase, Benzoate 1,2-Dioxygenase Alpha Subunit and Its Regulatory Proteins in Acinetobacter calcoaceticus PHEA-2

Acinetobacter calcoaceticus PHEA-2 is a phenol-degrading bacterium isolated from the wastewater from an oil refinery. A 10-kb XhoI fragment consisting of nine complete Open Reading Frames (ORFs) and one partial ORF was screened from a lambda library of PHEA-2 by Southern hybridization. The sequence analyses revealed that ORF2–ORF7, designated mphKLMNOP, are homologous to dmpKLMNOP of Pseudomonas sp. CF600 and mopKLMNOP of Acinetobacter calcoaceticus NCIB8250, sharing 38%–72% and 58.5%–93.5% respectively. The products encoded by dmp and mop genes convert phenol to catechol. The mph-operon and downstream ORFs, ORF9 and ORF10, sharing high identities to benM and benA, which encode ben-operon regulatory protein and benzoate 1,2-dioxygenase alpha subunit respectively, are separated by ORF8, whose function is unknown. The organization of the mph and ben operons is different from that described previously. Received: 8 April 2002 / Accepted: 8 May 2002     

Cloning and sequencing the genes encoding uptake-hydrogenase subunits of Rhodocyclus gelatinosus

Summary Rhodocyclus gelatinosus grew photosynthetically in the light and consumed H₂ at a rate of about 665 nmol/min per mg protein. The uptake-hydrogenase (H₂ase) was found to be membrane bound and insensitive to inhibition by CO. The structural genes of R. gelatinosus uptake-H₂ase were isolated from a 40 kb cosmid gene library of R. gelatinosus DNA by hybridization with the structural genes of uptake-H₂ase of Bradyrhizobium japonicum and Rhodobacter capsulatus. The R. gelatinosus genes were localized on two overlapping DNA restriction fragments subcloned into pUC18. Two open reading frames (ORF1 and ORF2) were observed. ORF1 contained 1080 nucleotides and encoded a 39.4 kDa protein. ORF2 had 1854 nucleotides and encoded a 68.5 kDa protein. Amino acid sequence analysis suggested that ORF1 and ORF2 corresponded to the small (HupS) and large (HupL) subunits, respectively, of R. gelatinosus uptake-H₂ase. ORF1 was approximately 80% homologous with the small, and ORF2 was maximally 68% homologous with the large subunit of typical membrane-bound uptake-H₂ases.     

青花菜谷胱甘肽-S-转移酶基因克隆及其表达分析

为探讨青花菜在模拟酸雨胁迫下谷胱甘肽-S-转移酶的表达变化,克隆了青花菜谷胱甘肽-S-转移酶基因(glutathione-S-transferase,GST)的cDNA序列全长,并进行了生物信息学和表达分析。结果表明:青花菜GST基因cDNA全长为915bp,开放阅读框为642bp,编码213个氨基酸,推测分子式为C1091H1719N289O306S5,分子量为23 940.7,没有跨膜螺旋区域和信号肽。系统进化树分析表明,该青花菜基因GST与芥菜的GST聚类关系最近。实时荧光定量PCR结果显示,在模拟酸雨胁迫下,GST基因的表达量在胁迫初期显著增大,随时间延长开始下降,表明其参与了青花菜抗酸雨的应答反应。     

A peptide-mediated and hydroxyl radical HO*-involved oxidative degradation of cellulose by brown-rot fungi

A special low-molecular-weight peptide named Gt factor, was isolated and purified from the extracellular culture of brown-rot fungi Gloeophyllum trabeum via gel filtration chromatography and HPLC. It has been shown to reduce Fe³⁺ to Fe²⁺. Electron paramagnetic resonance (EPR) spectroscopy revealed Gt factor was able to drive H₂O₂ generation via a superoxide anion O₂ ^.- intermediate and mediate the formation of hydroxyl radical HO^. in the presence of O₂. All the results indicated that Gt factor could oxidize the cellulose, disrupt the inter- and intrahydrogen bonds in cellulose chains by a HO^. -involved mechanism. This resulted in depolymerization of the cellulose, which made it accessible for further enzymatic hydrolysis.     

Genetic analysis of glyphosate tolerance in Halomonas variabilis strain HTG7

Summary A highly glyphosate-tolerant bacterium strain HTG₇ was isolated from glyphosate-polluted soil in north China, and identified as Halomonas variabilis. It was a Gram-negative motile rod giving convex colony. The strain HTG₇ could tolerate up to 900 mM glyphosate in minimal medium. The 16S rDNA sequence was amplified by PCR using universal primers. The region essential for glyphosate tolerance was localized to a 3.5-kb fragment from a cosmid library of HTG₇. The DNA fragment consisted of one complete open reading frame (ORF) and one partial ORF. The partial ORF was homologous to prephenate dehydrogenase of Pseudomonas aeruginosa PA01. The complete ORF contained the tyrA and aroA genes. Only the 1.35-kb aroA encoding EPSP synthase conferred glyphosate tolerance, and complemented with E. coli aroA mutant ER2799. E. coli JM109 harboring aroA grew well in Mops supplemented with 80 mM glyphosate.     

Within-stem oxygen concentration and sap flow in four temperate tree species: does long-lived xylem parenchyma experience hypoxia?

Oxygen levels as low as 1–5% (gaseous mole fraction) occur in secondary xylem, but it is not known if there is a consistent pattern of decline in O₂ from the cambium toward the pith, or whether parenchyma cells experience hypoxic conditions deep within the stem. We developed a system for repeated in situ measurement of O₂ at different depths within stems of Acer rubrum, Fraxinus americana, Tsuga canadensis, and Quercus rubra. In summer during active transpiration, O₂ declined from the cambium toward the heartwood boundary in F. americana, T. canadensis and Q. rubra, but remained constant in A. rubrum. Average sapwood O₂ was about 10%, with the lowest values observed in the innermost sapwood around 3–5%. Before spring leaf flush, O₂ content in the outer sapwood was reduced in Q. rubra and T. canadensis relative to summer, and was occasionally lower than in the inner sapwood. Sapwood respiration in T. canadensis was constant above 5% O₂, but reduced by about 65% at 1% O₂. In F. americana, sapwood respiration was constant above 10% O₂ but reduced by 25% at 5% O_2, and by 75% at 1% O₂, the most extreme inhibition observed. However, when prolonged (72 h) exposure to 1%, 5% and 10% O₂ was followed by re-equilibration to 10% O₂, no inhibition was found. Given the minor (and reversible) effect of low O₂ on parenchyma metabolism at levels common in the inner sapwood, it is unlikely that O₂ content severely limits parenchyma respiration or leads to parenchyma cell death during sapwood senescence. Within-stem O₂ levels may instead be most relevant to metabolism in the cambial zone and phloem, for which sapwood could serve as a significant source of O₂.     

Molecular characterization of co-transcribed genes from Streptomyces tendae Tü901 involved in the biosynthesis of the peptidyl moiety of the peptidyl nucleoside antibiotic nikkomycin

Six genes (nikA, nikB, nikD, nikE, nikF, and nikG) from Streptomyces tendae Tü901 were identified by sequencing the region surrounding the nikC gene, which encodes L-lysine 2-aminotransferase, previously shown to catalyze the initial reaction in the biosynthesis of hydroxypyridylhomothreonine, the peptidyl moiety of the peptidyl nucleoside antibiotic nikkomycin. These genes, together with the nikC gene, span a DNA region of 7.87 kb and are transcribed as a polycistronic mRNA in a growth-phase–dependent manner. The sequences of the deduced proteins NikA and NikB exhibit significant similarity to those of acetaldehyde dehydrogenases and 4-hydroxy-2-oxovalerate aldolases, respectively, which are involved in meta-cleavage degradation of aromatic hydrocarbons. The predicted NikD gene product shows sequence similarity to monomeric sarcosine oxidases, and the deduced NikE protein belongs to the superfamily of adenylate-forming enzymes. The nikF gene and the nikG gene encode a cytochrome P450 monooxygenase and a ferredoxin, respectively. Disruption of any of the genes nikA, nikB, nikD, nikE and nikF by insertion of a kanamycin resistance cassette abolished formation of the biologically active nikkomycins I, J, X, and Z. The nikA, nikB, nikD, and nikE mutants accumulated the nucleoside moieties nikkomycins C_x and C_z. In the nikD and nikE mutants nikkomycin production (nikkomycins I, J, X, Z) could be restored by feeding with picolinic acid and hydroxypyridylhomothreonine, respectively. The nikF mutant exclusively produced novel derivatives, nikkomycins L_x and L_z, which contain pyridylhomothreonine as the peptidyl moiety. Our results indicate that the nikA, nikB, nikD, nikE, nikF, and nikG genes, in addition to nikC, function in the biosynthetic pathway leading to hydroxypyridylhomothreonine; the putative activities of each of their products are discussed. Received: 1 February 1999 / Accepted: 29 April 1999     

枸杞WRKY_3基因克隆及组织表达分析

以枸杞为材料,采用PCR及RACE方法,克隆了枸杞WRKY转录因子基因cDNA序列,命名为Lb WRKY3,GenBank登录号为KX196192。在生物信息学分析的基础上,进行亚细胞定位、基因表达分析。结果显示:(1)Lb WRKY3开放阅读框ORF长度为1 068bp,编码356个氨基酸。(2)生物信息学分析显示,Lb WRKY3编码蛋白具有一个WRKY结构域,二级结构中不规则卷曲结构所占比例最大(58.67%),延伸链结构次之(18.88%),α螺旋比例为15.82%,β转角最少,仅为6.63%;Lb WRKY3蛋白与案头菊WRKY蛋白、黄花蒿WRKY蛋白相似性较高。(3)亚细胞定位显示,Lb WRKY3蛋白定位于细胞核。(4)实时定量PCR分析表明,Lb WRKY3在根中表达量最高,在花中表达量最低;在枸杞果实发育过程中Lb WRKY3均有表达,表达量随果实成熟逐渐升高,并于35d达到峰值;Lb WRKY3基因在果实中的表达具有组织特异性表达特性(果肉果皮种子)。研究表明,Lb WRKY3基因参与了枸杞果实生长发育调控。     

Cloning and Nucleotide Sequence of a New Insertion Sequence, IS231N, from a Non-Serotypable Strain of Bacillus thuringiensis

A new IS231 variant, IS231N, has been isolated from an autoagglutinable, non-serotypable strain of B. thuringiensis. IS231N is 1654 bp in length and is delimited by two incomplete 20-bp inverted repeats (IR_L and IR_R) with two mismatches. No direct repeats (DRs) were found at the right and left borders of IS231N. Surprisingly, IS231N contains three open reading frames (ORFs) that could code for polypeptides of 329 (ORF1), 118 (ORF2), and 17 (ORF3) amino acids, respectively. IS231N lacks the 5th conserved amino acid domain, called C2, owing to the addition of an adenine residue at nucleotide 1319. IS231N shows the highest nucleotide identity (99%) with IS231M, another insertion sequence previously isolated from the same bacterial strain. IS231N, however, shares only 83% amino acid identity with IS231M because of nucleotide substitutions and additions. The ORF1 of IS231N has five fewer amino acids than ORF1 of IS231M. Furthermore, the ORF2-3 putative fusion product in IS231N contains eight fewer amino acids than ORF2 in IS231M. The dendrogram showing the evolutionary relationship between members of the IS231 family and IS231N indicates that IS231N is phylogenetically more closely related to IS231M (83%), followed by IS231F(74%), and is more distant from IS231V and W(46%). Received: 4 January 2001/Accepted: 6 February 2001     

Oxygen and carbon dioxide exchanges in crassulacean-acid-metabolism-plants

The 24 h O₂ uptake and release together with the CO₂ balance have been measured in two CAM plants, one a non-succulent Sempervivum grandifolium, the other a succulent Prenia sladeniana. The O₂ uptake was estimated by the use of ¹⁸O₂. It was found that the mean hourly O₂ uptake in the light was 7 times that in the dark for Sempervivum and 5 times that for Prenia, after correction for the lightdark temperature difference. It was estimated that oxygen uptake in the light was 2.4 times greater than oxygen release (=net photosynthesis) in Sempervivum and 1.4 times greater in Prenia. In both plants there was a positive carbon balance over the 24 h period under the experimental conditions. It was estimated that malate formed during the night could, if completely oxidized to CO₂ and water, account for 74% of the light phase O₂ uptake in Sempervivum. In Prenia the O₂ uptake was more than sufficient to account for a full oxidation of malate.Abbreviations CAM Crassulacean acid metabolism - PAR photosynthetically active radiation - PEP phosphoenolpyruvate - RrBP ribulose-1,5-bisphosphate - TCA tricarboxylic acid cycle     

Nitrogenase activity and root nodule metabolism in response to O2 and short-term N2 deprivation in dark-treated Frankia-Alnus incana plants

Inhibition of nitrogenase (EC 1.18.6.1) activity by O₂ has been suggested to be an early response to disturbance in carbon supply to root nodules in the Frankia‐Alnus incana symbiosis. Intact nodulated root systems of plants kept in prolonged darkness of 22 h were used to test responses to O₂ and short‐term N₂ deprivation (1 h in Ar:O₂). By using a Frankia lacking uptake hydrogenase it was possible to follow nitrogenase activity over time as H₂ evolution in a gas exchange system. Respiration was simultaneously recorded as CO₂ evolution. Dark‐treated plants had lower initial nitrogenase activity in N₂:O₂ (68% of controls), which declined further during a 1‐h period in the assay system in N₂:O₂ at 21 and 17% O₂, but not at 13% O₂. When dark‐treated plants were deprived of N₂ at 21 and 17% O₂ nitrogenase activity declined rapidly to 61 and 74%, respectively, after 20 min, compared with control plants continuously kept in their normal light regime. In contrast, there was no decline in dark‐treated plants at 13% O₂, and only a smaller and temporary decline in control plants at 21% O₂. When dark‐treated plants were kept at 21% O₂ during 45 min prior to N₂ deprivation at 17% O₂ the decline was abolished. This supports the idea that the decline in nitrogenase activity observed in N₂:O₂ at 21% O₂ and during N₂ deprivation was caused by O₂, which affected a sensitive nodule fraction. Nodule contents of the amino acids Gln and Cit decreased during N₂ deprivation, suggesting decreased assimilation of NH₄⁺. Contents of ATP and ADP in nodules were not affected by short‐term N₂ deprivation. ATP/ADP ratios were about 5 indicating a highly aerobic metabolism in the root nodule. We conclude that nitrogenase activity of Alnus plants exposed to prolonged darkness becomes more sensitive to inactivation by O₂. It seemed that dark‐treated plants could not adjust their nodule metabolism at higher perceived pO₂ and during cessation of NH₄⁺ production.     

毛竹PeSCL3基因表达特征及其启动子活性研究

为探讨毛竹(Phyllostachys edulis)SCL3基因的表达特征及其启动子活性,采用同源克隆的方法从毛竹中分离到SCL3同源基因Pe SCL3的编码区(ORF)和上游启动子序列(Pe SCL3p)。序列分析表明,Pe SCL3的ORF为1335 bp,推测编码含444氨基酸的蛋白,该蛋白与水稻(Oryza sativa)的SCL3同源性高达93.9%。Pe SCL3p长度为1358 bp,含有脱落酸(ABA)应答元件ABRE、赤霉素(GA3)应答元件GARE-motif和P-box、干旱诱导MYB结合位点等多种作用元件。实时定量PCR分析结果表明,Pe SCL3在毛竹叶中的表达丰度最高,其次是茎和根,而鞘中的最低;Pe SCL3的表达受GA3的抑制,受ABA、Na Cl和干旱的诱导。转Pe SCL3p∷GUS拟南芥(Arabidposis thaliana)的GUS染色结果表明,根尖、顶端生长点和子叶叶柄均被染成蓝色,尤其根尖的染色最深。这表明Pe SCL3对毛竹的生长发育,尤其是根系,可能起着重要的调控作用。     

Oxygen effects on methane production and oxidation in humid tropical forest soils

We investigated the effects of oxygen (O₂) concentration on methane (CH₄) production and oxidation in two humid tropical forests that differ in long‐term, time‐averaged soil O₂ concentrations. We identified sources and sinks of CH₄ through the analysis of soil gas concentrations, surface emissions, and carbon isotope measurements. Isotope mass balance models were used to calculate the fraction of CH₄ oxidized in situ. Complementary laboratory experiments were conducted to determine the effects of O₂ concentration on gross and net rates of methanogenesis. Field and laboratory experiments indicated that high levels of CH₄ production occurred in soils that contained between 9±1.1% and 19±0.2% O₂. For example, we observed CH₄ concentrations in excess of 3% in soils with 9±1.1% O₂. CH₄ emissions from the lower O₂ sites were high (22–101 nmol CH₄ m^?2 s^?1), and were equal in magnitude to CH₄ emissions from natural wetlands. During peak periods of CH₄ efflux, carbon dioxide (CO₂) emissions became enriched in ¹³C because of high methanogenic activity. Gross CH₄ production was probably greater than flux measurements indicated, as isotope mass balance calculations suggested that 48–78% of the CH₄ produced was oxidized prior to atmospheric egress. O₂ availability influenced CH₄ oxidation more strongly than methanogenesis. Gross CH₄ production was relatively insensitive to O₂ concentrations in laboratory experiments. In contrast, methanotrophic bacteria oxidized a greater fraction of total CH₄ production with increasing O₂ concentration, shifting the δ¹³C composition of CH₄ to values that were more positive. Isotopic measurements suggested that CO₂ was an important source of carbon for methanogenesis in humid forests. The δ¹³C value of methanogenesis was between ?84‰ and ?98‰, which is well within the range of CH₄ produced from CO₂ reduction, and considerably more depleted in ¹³C than CH₄ formed from acetate.     

小麦TaMAPK2基因的克隆及表达分析

该研究从小麦中克隆了1个MAPK基因TaMAPK2。序列分析表明,TaMAPK2基因的ORF为1 110bp,编码369个氨基酸。序列比对分析表明,该基因所编码的蛋白与粗山羊草、水稻、谷子等MAPK蛋白具有较高的一致性,分别为99%、94%、94%。进化树分析表明,TaMAPK2与水稻OsMAPK2的亲缘关系最近。实时荧光定量PCR分析表明,该基因的表达显著受渗透胁迫、低温胁迫、高盐胁迫、乙烯和双氧水诱导,受ABA抑制。研究表明,TaMAPK2可能参与非生物逆境胁迫及相关信号分子应答。     

Involvement of Lysosomal Cysteine Proteases in Hydrogen Peroxide-induced Apoptosis in HL-60 Cells

Hydrogen peroxide is a well-known mediator of apoptosis. As a mechanism for H₂O₂-induced apoptosis, both a mitochondrial Cyt.c-dependent pathway and a lysosome-mediated pathway have been suggested. However, the relative roles of and the relation between these two pathways in H₂O₂-induced apoptosis remain to be discovered. In this study, to find the relative roles of the lysosomal and mitochondrial pathways, the effects of E-64-d, a cell-permeable inhibitor of lysosomal cysteine proteases, on apoptosis caused by H₂O₂ in HL-60 cells were investigated. It was found that the concentration of H₂O₂ strongly affected the inhibitory effect of E-64-d on the apoptosis in HL-60 cells: dose-dependent inhibition (up to 40%) of both DNA fragmentation and caspase-3 activation was observed when a high concentration of H₂O₂ (50 μM) was used to induce apoptosis, but no inhibitory effect was detected when a low concentration (10 μM) was used. Consistent with these observations, apparent lysosomal destabilization was observed only with 50 μM H₂O₂. The release of mitochondrial Cyt.c, in contrast, was observed at both 10 μM and 50 μM. These results indicated that the mitochondrial Cyt.c-mediated pathway predominates in the H₂O₂-induced apoptosis in HL-60 cells and the lysosomal mediated pathway is partially involved when high concentrations of H₂O₂ are used to induce apoptosis.