Anaerobic growth of Escherichia coli K12 with fumarate as terminal electron acceptor. Genetic studies with menaquinone and fluoroacetate-resistant mutants.

Fifteen independent menaquinone biosynthesis mutants (men) of Escherichia coli K12, selected for their inability to use fumarate as terminal electron acceptor, were investigated. Two nutritionally distinct groups were detected. The major group (13 mutants) responded to 1,4-dihydroxy-2-naphthoate (DHN), 2-succinylbenzoate (SB) and its dilactone, whereas the minor group (2 mutants) only responded to DHN. DHN was at least five times more effective than SB but it inhibited growth at concentrations greater than 10 microM. For anaerobic growth on glucose minimal medium the auxotrophs responded to much lower concentrations of DHN and SB and these intermediates could be replaced by uracil. Anaerobic growth tests showed that glycerol, formate and H2 are good substrates for E. coli when fumarate is the ultimate electron acceptor but growth with lactate or with fumarate alone is poor. All 15 men mutations were located between glpT and purF at approximately 49 min in the E. coli linkage map. Cotransduction frequencies with relevant markers were: nalA (21%), glpT (35%) and purF (15%). The presence of at least three genetically distinct classes (menC and menD, SB-requirers; menB, DHN-requirers) was indicated using abortive transduction as a complementation test and three-factor genetic analysis. The relative orientation nalA...menC-(D,B)...purF was indicated. Fluoroacetate-resistant mutants were isolated and four different classes were identified: ack, lacking acetate kinase; pta, lacking phosphotransacetylase; facA, lacking both of these activities; and facB, which retained both of these enzyme activities. Some of the pta mutants and all of the facA mutants failed to grow on media containing fumarate as terminal electron acceptor or anaerobically on glucose minimal medium. All four types had genetic lesions clustered between the men and purF sites. Average cotransduction frequencies with relevant markers were: nalA (4%), men (27 to 35%) and purF (71 to 80%).     

Identification of the dehydrin gene family from grapevine species and analysis of their responsiveness to various forms of abiotic and biotic stress

ABSTRACT: BACKGROUND: Dehydrins (DHNs) protect plant cells from desiccation damage during environmental stress, and also participate in host resistance to various pathogens. In this study, we aimed to identify and characterize the DHN gene families from Vitis vinifera and wild V. yeshanensis, which is tolerant to both drought and cold, and moderately resistant to powdery mildew. RESULTS: Four DHN genes were identified in both V. vinifera and V. yeshanensis, which shared a high sequence identity between the two species but little homology between the genes themselves. These genes were designated DHN1, DHN2, DHN3 and DHN4. All four of the DHN proteins were highly hydrophilic and were predicted to be intrinsically disordered, but they differed in their isoelectric points, kinase selectivities and number of functional motifs. Also, the expression profiles of each gene differed appreciably from one another. Grapevine DHN1 was not expressed in vegetative tissues under normal growth conditions, but was induced by drought, cold, heat, embryogenesis, as well as the application of abscisic acid (ABA), salicylic acid (SA), and methyl jasmonate (MeJA). It was expressed earlier in V. yeshanensis under drought conditions than in V. vinifera, and also exhibited a second round of up-regulation in V. yeshanensis following inoculation with Erysiphe necator, which was not apparent in V. vinifera. Like DHN1, DHN2 was induced by cold, heat, embryogenesis and ABA; however, it exhibited no responsiveness to drought, E. necator infection, SA or MeJA, and was also expressed constitutively in vegetative tissues under normal growth conditions. Conversely, DHN3 was only expressed during seed development at extremely low levels, and DHN4 was expressed specifically during late embryogenesis. Neither DHN3 nor DHN4 exhibited responsiveness to any of the treatments carried out in this study. Interestingly, the presence of particular cis-elements within the promoter regions of each gene was positively correlated with their expression profiles. CONCLUSIONS: The grapevine DHN family comprises four divergent members. While it is likely that their functions overlap to some extent, it seems that DHN1 provides the main stress-responsive function. In addition, our results suggest a close relationship between expression patterns, physicochemical properties, and cis-regulatory elements in the promoter regions of the DHN genes.     

植物病原真菌1,8-间苯二酚黑色素研究进展

黑色素是一种广泛分布于生物体中的酚类聚合物疏水色素,分为1,8-间苯二酚(1,8-dihydroxynaphthalene,DHN)黑色素和3,4-二羟基苯丙氨酸(3,4-dihydroxyphenylalanine,L-DOPA)黑色素两种,其中DHN黑色素多存在于子囊菌门的植物病原真菌中。基因组和转录组技术的发展及功能基因组研究的深入,使DHN黑色素合成途径上关键基因在不同病原真菌中被鉴定,而且黑色素与真菌抗逆、发育和致病的关系受到越来越多的关注。本文阐述了DHN黑色素合成途径及其在真菌抗辐射与抗极端温度中的作用,以及黑色素对真菌侵染和细胞发育的影响,旨在加深人们对黑色素介导真菌与环境和寄主协同进化的认识,这对黑色素的基础研究和开发利用具有重要意义。     

Characterization of the Neurospora crassa DHN melanin biosynthetic pathway in developing ascospores and peridium cells

Neurospora crassa contains all four enzymes for the synthesis of DHN (dihydroxynaphthalene), the substrate for melanin formation. We show that the DHN melanin pathway functions during N. crassa female development to generate melanized peridium and ascospore cell walls. N. crassa contains one polyketide synthase (PER-1), two polyketide hydrolases (PKH-1 and PKH-2), two THN (tetrahydroxynaphthalene) reductases (PKR-1 and PKR-2), and one scytalone dehydratase (SCY-1). We show that the PER-1, PKH-1, PKR-1 and SCY-1 are required for ascospoer melanization. We also identified the laccase that functions in the conversion of DHN into melanin via a free radical oxidative polymerization reaction, and have named the gene lacm-1 (laccase for melanin formation-1). In maturing perithecia, we show that LACM-1 is localized to the peridium cell wall space while the DHN pathway enzymes are localized to intracellular vesicles. We present a model for melanin formation in which melanin is formed within the cell wall space and the cell wall structure is similar to “reinforced concrete” with the cell wall glucan, chitin, and glycoproteins encased within the melanin polymer. This arrangement provides for a very strong and resilient cell wall and protects the glucan/chitin/glycoprotein matrix from digestion from enzymes and damage from free radicals.     

Evidence for 1,8-dihydroxynaphthalene melanin in three halophilic black yeasts grown under saline and non-saline conditions

The ascomycetous black yeasts Hortaea werneckii, Phaeotheca triangularis, and Trimmatostroma salinum are halophilic fungi that inhabit hypersaline water of solar salterns. They are characterized by slow, meristematic growth and very thick, darkly pigmented cell walls. The dark pigment, generally thought to be melanin, is consistently present in their cell walls when they grow under saline and non-saline conditions. We used the inhibitor tricyclazole to test the fungi in this study for the presence of 1,8-dihydroxynaphthalene (DHN)-melanin biosynthesis, since fungal melanins reportedly are derived either from DHN, tyrosine via 3,4-dihydroxyphenylalanine, gamma-glutaminyl-3,4-dihydroxybenzene, or catechol. Tricyclazole-treated cultures of the fungi were reddish-brown in color and contained typical intermediates of the DHN-melanin pathway, as demonstrated by high-performance liquid chromatography. This investigation showed that the three fungi synthesized DHN-melanin under saline and non-saline growth conditions.     

中间锦鸡儿DHN1基因克隆及表达分析

脱水素(DHNs, dehydrins)是LEA II亚家族蛋白,在种子发育后期大量积累,并受不同逆境胁迫处理诱导表达。在中间锦鸡儿干旱胁迫抑制性削减杂交文库中筛选到了一段 DHN1 序列,利用RACE技术克隆获得基因全长序列。序列比对分析表明, CiDHN1 具有开放阅读框891bp,起始密码子为ATG,终止密码子为TAG,编码297个氨基酸,含有5个Y片段和一个K片段,与CiDHN1相似性最高的为山茱萸科主教红端木(Cornus sericea),相似性为39%。系统进化分析显示CiDHN1单独聚为一支,推测该蛋白为新的功能未知蛋白。亚细胞定位结果表明CiDHN1定位在细胞质、质膜和细胞核。荧光定量PCR结果显示 CiDHN1 的表达受冷、脱水和NaCl等非生物胁迫的诱导。 CiDHN1 过量表达拟南芥后,其中表达量最强的株系对200 mmol/L的NaCl处理较为敏感。CiDHN1在中间锦鸡儿抵抗逆境胁迫中的功能有待于进一步研究。     

Photoperiod and temperature differentially regulate the expression of two dehydrin genes during overwintering of birch (Betula pubescens Ehrh.)

The overwintering of trees in northern areas depends on processes regulated by photoperiod and temperature. To identify the physiological and genetic factors involved in this environmental control, three latitudinal ecotypes of pubescent birch (Betula pubescens Ehrh.) growing in a common garden experiment were used. Each ecotype responded to the shortening of the photoperiod according to its specific critical daylength, resulting in the induction of freezing tolerance and dehydration of buds first in the northern ecotype, followed by the central and southern ecotypes, respectively. By contrast, there was no clear difference in the timing of dormancy release, bud rehydration, and deacclimation in the spring, suggesting that these traits were controlled mainly by temperature. To elucidate the role of dehydrins (DHN) in the overwintering process, two DHN genomic clones were isolated from pubescent birch and expression of the corresponding genes, both in field and under controlled conditions, was characterized. BpuDhn1 was found to encode an Y(n)K(n)-type of basic DHN, while BpuDhn2 encoded an acidic, SK(n)-type of DHN. In field-grown trees the level of BpuDhn1 increased in buds during the autumn, while the level of BpuDhn2 was highest during the coldest winter months. Under controlled conditions BpuDhn1 increased in response to the combined effect of short daylength and low, non-freezing temperatures whereas the expression of BpuDhn2 was mainly controlled by low temperature while photoperiod had less effect on its expression. These results suggest that DHNs participate in the sensitive environmental regulation of the overwintering process in birch.     

RNA-mediated gene silencing in the phytopathogenic fungus Bipolaris oryzae

The Ascomycetous fungus Bipolaris oryzae is the causal agent of brown leaf spot disease in rice and is a model for studying photomorphogenetic responses by near-UV radiation. Targeted gene disruption (knockout) for functional analysis of photomorphogenesis-related genes in B. oryzae can be achieved by homologous recombination with low efficiency. Here, the applicability of RNA silencing (knockdown) as a tool for targeting endogenous genes in B. oryzae is reported. A polyketide synthase gene (PKS1), involved in fungal DHN melanin biosynthesis pathways, was targeted by gene silencing as a marker. The silencing vector encoding hairpin RNA of the PKS1 fragment was constructed in a two-step PCR-based cloning, and introduced into the B. oryzae genomic DNA. Silencing of the PKS1 gene resulted in albino phenotypes and reduction of PKS1 mRNA expression. These results demonstrate the applicability of targeted gene silencing as a useful reverse-genetics approach in B. oryzae.     

Differential expression of wheat genes during cold acclimation

Overwintering crops such as winter wheat display significant increase in freezing tolerance during a period of cold acclimation (CA). To gain better understanding of molecular mechanisms of CA, it is important to unravel functions and regulations of CA-associated genes. Differential screening of a cDNA library constructed from cold acclimated crown tissue of winter wheat identified three novel CA-associated cDNA clones. Nucleotide sequence analysis showed that the clones encode a high mobility globular protein (HMGB1), a glycine-rich RNA-binding protein (TaGRP2), and a LEA D-11 dehydrin (DHN14). Accumulation of the three mRNAs during 14 days of CA was differentially regulated. In response to drought, and ABA, DHN14 mRNA rapidly accumulated while HMGB1 and TaGRP2 mRNA levels remained unchanged. The possible functions of each of these genes in cold acclimation are discussed.