Prevalence of 1-aminocyclopropane-1-carboxylate deaminase in Rhizobium spp

This is the first report documenting the presence of 1-aminocyclopropane-1-carboxylate (ACC) deaminase in Rhizobium. This enzyme, previously found in free-living bacteria, yeast and fungi, degrades ACC, the immediate precursor of ethylene in higher plants. Thirteen different rhizobial strains were examined by Southern hybridization, Western blots and ACC deaminase enzyme assay. Five of them tested positive for ACC deaminase. Induction of the expression of ACC deaminase was examined in one of the positively tested strains, Rhizobium leguminosarum bv. viciae 128C53K. This rhizobial ACC deaminase had a trace basal level of expression without ACC, but could be induced by a concentration of ACC as low as 1 μM. The more ACC added to this Rhizobium the higher the expression level of the ACC deaminase. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of Rhizobium inoculation methodologies on nodulation and growth of Leucaena leucocephala

The aim of this study was to evaluate the effect of five methods of Rhizobium inoculum application on nodulation and nitrogen fixation in Leucaena leucocephala seedlings cultivated for 6 months in the greenhouse. Plants inoculated with alginate beads were significantly more developed and more nodulated than plants inoculated with the other methodologies used.     

Genes encoding a fructose-1,6-bisphosphate aldolase and a fructose-1,6-bisphosphatase are present within the gene cluster for mimosine degradation in Rhizobium sp. strain TAL1145

Rhizobium sp. strain TAL1145 can catabolize mimosine, a toxic amino acid produced by the tree-legume leucaena. The mid and pyd genes involved in mimosine degradation in TAL1145 are located in two clusters within a 25-kb region in the chromosome, which was cloned in plasmid pUHR263. A 5.5-kb EcoRI fragment, located between the mid and pyd genes in pUHR263, was characterized by sequencing and transposon-insertion mutagenesis and six open reading frames (ORF) were identified. Based on high homologies with other known proteins and conserved signature domains, ORF1 and ORF2 were identified as fba and fbp genes, encoding fructose-1,6-bisphosphate aldolase (FBA) and fructose-1,6-bisphosphatase (FBP), respectively. The fba mutant showed a slightly reduced growth rate compared to TAL1145 while the fbp mutant did not show any growth defects. Both mutants could catabolize mimosine and formed normal nitrogen-fixing nodules on leucaena, suggesting that these genes are not involved in mimosine degradation and symbiosis.     

Isolation and characterization of an unusual 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase gene from Enterobacter cloacae UW4

A genomic library of the 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase-containing plant growth-promoting bacterium Enterobacter cloacae UW4 in pUC19 in Escherichia coli was screened for the ability to utilize ACC as a sole source of nitrogen. One of the clones that was isolated contained a plasmid with an insert of approximately 0.8 kb that conferred ACC deaminase activity. Sequence analysis revealed that this DNA fragment contains an open-reading frame of 696 nucleotides predicted to encode a protein of 232 amino acids, a member of the amidohydrolase protein superfamily, i.e., a deaminase that contains a mononuclear or binuclear metal center as compared to the canonical ACC deaminase which contains pyridoxal phosphate as a co-factor.     

A histidine kinase sensor protein gene is necessary for induction of low pH tolerance in Sinorhizobium sp. strain BL3

The aim of this investigation was to identify and isolate genes involved in acid tolerance from Sinorhizobium sp. strain BL3. It was hypothesized that acid tolerance of strain BL3 could be enhanced by high level expression of certain genes involved in acid tolerance, following insertion of these genes in a multiple copy plasmid. A cosmid clone library of BL3 was introduced into BL3, and the transconjugant colonies were selected at low pH. A single cosmid containing genes for acid tolerance was isolated from 40 different colonies. By transposon–insertion mutagenesis, subcloning and DNA sequencing, a gene involved in acid tolerance, actX, was identified in a 4.4-kb fragment of this cosmid. The actX mutant of BL3 showed increased acid sensitivity and was complemented by the 4.4-kb subcloned fragment. Phaseolus lathyroides seedlings inoculated with recombinant strains containing multiple copies of actX showed increased symbiotic performance at low pH. By constructing an actX::gus fusion, it was shown that actX was induced at low pH. actX encodes a putative histidine kinase sensor protein of a two-component regulatory system. The method of gene identification used in this study for isolation of actX may be applied for the isolation of other genes involved in tolerance to adverse environmental factors.     

旱地小麦根际细菌中产生1-氨基环丙烷-1-羧酸(ACC)脱氨酶菌株的分离和鉴定

采用富集定向筛选法,从旱地小麦的根际土壤中分离到2株产生1-氨基环丙烷-1-羧酸(ACC)脱氨酶的菌株AS和CS。经测定菌株AS和CS的ACC脱氨酶的比活力分别为0.018 6 U/mg和0.016 7 U/mg蛋白。根据培养特征观察和生理生化指标测定结果,结合16S rDNA碱基序列测定和系统发育同源性分析,确定菌株AS和菌株CS分别属于霍氏肠杆菌(Enterobacter hormaechei)和变形斑沙雷氏菌(Serratia proteamaculans)。     

Characterization of 1-aminocyclopropane-1-carboxylate deaminase producing methylobacteria from phyllosphere of rice and their role in ethylene regulation

The presence of 1-aminocyclopropane-1-carboxylate deaminase (ACCD) activity among the phyllosphere methylobacteria of rice was detected and its role in regulating plant ethylene level was assessed. Eighteen methylobacterial isolates from four different cultivars of rice were isolated and screened for ACCD. The 16S rRNA homology of ACCD positive methylobacterial isolate closely related to the species Methylobacterium radiotolerans. The accD gene sequence homology of the isolate was 98% similar to Rhizobium leguminosarum. Foliar spray of ACCD positive methylobacterial isolates enhanced the root and shoot length of rice and tomato seedlings under gnotobiotic condition and lower the ethylene level (60–80%) in the plant species.     

Regulation of ethylene levels in canola (Brassica campestris) by 1-aminocyclopropane-1-carboxylate deaminase-containing Methylobacterium fujisawaense

We report the presence of ACC deaminase in Methylobacterium fujisawaense and its lowering of ethylene levels and promotion of root elongation in canola seedlings under gnotobiotic conditions. To test a part of the previous model proposed for ACC deaminase producing bacteria with Methylobacterium, ACC levels and various enzyme activities were monitored in canola. Lower amounts of ACC were present in the tissues of seeds treated with M. fujisawaense strains than in control seeds treated with MgSO₄. Though the increased activities of ACC synthase in the tissue extracts of the treated seedlings might be due to bacterial indole-3-acetic acid, the amount of ACC was reduced due to bacterial ACC deaminase activity. The activities of ACC oxidase, the enzyme catalyzing conversion of ACC to ethylene remained lower in M. fujisawaense treated seedlings. This consequently lowered the ethylene in plants and prevented ethylene inhibition of root elongation. Our results collectively suggest that Methylobacterium commonly found in soils, as well as on the surfaces of leaves, seeds, and in the rhizosphere of a wide variety of plants could be better exploited to promote plant growth.     

Characterization of 1-aminocyclopropane-1-carboxylate (ACC) deaminase containing <Emphasis Type="Italic">Methylobacterium oryzae</Emphasis> and interactions with auxins and ACC regulation of ethylene in canola (<Emphasis Type="Italic">Brassica campestris</Emphasis>)

The possible interaction of the plant hormones auxin and ethylene and the role of 1-aminocyclopropane-1-carboxylate (ACC) deaminase containing bacteria on ethylene production in canola (Brassica campestris) in the presence of inhibitory concentrations of growth regulators were investigated. The effects of auxin (indole-3-acetic acid and 2,4-dichlorophenoxy acetic acid), auxin transport inhibitor 2-(p-chlorophenoxy)-2-methylpropionic acid, ethylene precursor 1-aminocyclopropane-1-carboxylate and ethylene synthesis inhibitor l-α-(2-aminoethoxyvinyl)glycine hydrochloride on root elongation were concentration dependent. Exogenous addition of growth regulators influences the enzyme activities of ethylene production and we have presented here evidences that support the hypothesis that inhibitory effects of auxin on root elongation are independent of ethylene. Additionally, we have proved that inoculation of ACC deaminase containing Methylobacterium oryzae sequester ACC exuded from roots and hydrolyze them lowering the concentration of ACC in root exudates. However, the inhibitory actions of exogenous additions of auxins could not be ameliorated by bacterial inoculation that reduces ethylene concentration in canola seedlings.     

The Rhizobium meliloti early nodulation genes (nodABC) are nitrogen-regulated: Isolation of a mutant strain with efficient nodulation capacity on alfalfa in the presence of ammonium

Summary The presence of combined nitrogen in the soil suppresses the formation of nitrogen-fixing root nodules by Rhizobium. We demonstrate that bacterial genes determining early nodulation functions (nodABC) as well as the regulatory gene nodD3 are under nitrogen (NH ₄ ⁺ ) control. Our results suggest that the gene product of nodD3 has a role in mediating the ammonia regulation of early nod genes. The general nitrogen regulatory (ntr) system as well as a chromosomal locus mutated in Rhizobium meliloti were also found to be involved in the regulation of nod gene expression. A R. meliloti mutant with altered sensitivity to ammonia regulation was isolated, capable of more efficient nodulation of alfalfa than the wild-type strain in the presence of 2 mM ammonium sulfate.     

The use of a double isotope (15N and 34S) labelling technique to assess the suitability of various reference crops for estimating nitrogen fixation in Gliricidia sepium and Leucaena leucocephala

In this study, an approach involving a double isotope (¹⁵N and ³⁴S) labelling technique was used to examine which of five reference crops (Eucalyptus camaldulensis, Cassia siamea, Cassia spectabilis, Lolium perenne and Eucalyptus grandis) would be suitable for measuring N fixed by Gliricidia sepium and Leucaena leucocephala. The rationale is that the ratio of fertilizer-derived S to soil-derived S in a suitable reference crop is similar to that measured in the nitrogen fixing tree (NFT) since the N ratios in the two crop types cannot be measured directly. E. camaldulensis and E. grandis were found to be suitable reference crops because they absorbed fertilizer and soil S in the same ratio as G. sepium and L. leucocephala.     

The effects of the herbicide quinclorac on shoot growth in tomato is alleviated by inhibitors of ethylene biosynthesis and by the presence of an antisense construct to the 1-aminocyclopropane-1-carboxylic acid (ACC) synthase gene in transgenic plants

Reduction of shoot growth, leaf epinasty and chlorosis in young tomato plants (Lycopersicon esculentum Mill. cv. Hellfrucht/Frühstamm) treated hydroponically with 10^-7 M of the herbicide quinclorac were partially compensated when the plants were simultaneously sprayed with salicyclic acid or the oxime ether derivative PACME. Since salicyclic acid and PACME are known inhibitors of ethylene biosynthesis, it is suggested that this pathway is implicated in quinclorac action. Further support for this hypothesis was obtained in experiments with transgenic tomato plants containing an antisense gene to 1-aminocyclopropane-1-carboxylic acid (ACC) synthase in ethylene biosynthesis. When quinclorac was applied via the root antisense plants showed reduced phenotypical alterations compared to those of wild-type plants.     

山西省草地早熟禾白粉菌分离株BGP(TG)的鉴定及致病力分析

【背景】草地早熟禾(Poa pratensis)是一种被广泛使用的兼具观赏性和设施性的冷季型草坪草,白粉病的发生严重降低了草地早熟禾的质量和使用年限。【目的】对山西农业大学温室草地早熟禾白粉病病原物进行分离、纯化、鉴定和致病力分析,为草地早熟禾白粉病的防治提供理论依据。【方法】采用形态特征观察、核糖体内源转录间隔区(ribosomal DNA internally transcribed spacer, rDNA-ITS)序列分析进行分离株BGP(TG)的鉴定;通过分生孢子密集抖落接种法测定BGP(TG)对23份草地早熟禾材料的致病力。【结果】草地早熟禾白粉菌分离株BGP(TG)分生孢子串生、无色、椭圆形,长和宽分别为(23.31−34.67) µm和(11.33−15.77) µm,成熟吸器为指状;rDNA-ITS序列分子发育分析结果表明分离株BGP(TG)与禾布氏白粉菌早熟禾专化型(Blumeria graminis f. sp. poae)聚于同一分支;BGP(TG)对23份草地早熟禾的致病力测定结果显示,白粉病发病率为49.47%−77.72%,病情指数为14.41−53.12;仅‘太行’草地早熟禾对BGP(TG)表现为中抗,‘应县’ ‘黑杰克’和‘浑源’草地早熟禾表现为中感,‘探险家’和‘奖品’草地早熟禾表现为极感,其余17个品种表现为高感,占比达73.91%。【结论】山西省草地早熟禾白粉菌分离株BGP(TG)的致病力较强,进一步挖掘‘太行’草地早熟禾的抗白粉病基因,对草地早熟禾抗白粉病育种工作具有重要意义。     

Thermotolerant poly(3-hydroxybutyrate)-degrading bacteria from hot compost and characterization of the PHB depolymerase of Schlegelella sp. KB1a

Eighteen gram-negative thermotolerant poly(3-hydroxybutyrate) (PHB)-degrading bacterial isolates (T _max60°C) were obtained from compost. Isolates produced clearing zones on opaque PHB agar, indicating the presence of extracellular PHB depolymerases. Comparison of physiological characteristics and determination of 16S rRNA gene sequences of four selected isolates revealed a close relatedness of three isolates (SA8, SA1, and KA1) to each other and to Schlegelella thermodepolymerans and Caenibacterium thermophilum. The fourth strain, isolate KB1a, showed reduced similarities to the above-mentioned isolates and species and might represent a new species of Schlegelella. Evidence is provided that S. thermodepolymerans and C. thermophilum are only one species. The PHB depolymerase gene, phaZ, of isolate KB1a was cloned and functionally expressed in Escherichia coli. Purified PHB depolymerase was most active around pH 10 and 76°C. The DNA-deduced amino acid sequence of the mature protein (49.4 kDa) shared significant homologies to other extracellular PHB depolymerases with a domain substructure: catalytic domain type 2—linker domain fibronectin type 3—substrate-binding domain type 1. A catalytic triad consisting of S₂₀, D₁₀₄, and H₁₃₈ and a pentapeptide sequence (GLS₂₀AG) characteristic for PHB depolymerases (PHB depolymerase box, GLSXG) and for other serine hydrolases (lipase box, GXSXG) were identified.This contribution is dedicated to Hans G. Schlegel in honor of his 80th birthday.Fabian Romen and Simone Reinhardt share first authorship.     

Biosynthesis of medium chain length poly(3-hydroxyalkanoates) (mcl PHAs) from cosmetic co-products by Pseudomonas raguenesii sp. nov., isolated from Tetiaroa, French Polynesia

A new bacterium, designated as strain TE9 was isolated from a microbial mat in French Polynesia and was studied for its ability to synthesize medium chain length poly-β-hydroxyalkanoates (mcl PHAs) during cultivation on cosmetics co-products. The composition of PHAs was analysed by coupled gas chromatography mass spectroscopy (GC/MS), nuclear magnetic resonance (NMR) and Fourier Transform InfraRed (FTIR) spectroscopy. PHAs were composed of C6–C14 3-hydroxyacids monomers, with a predominance of 3-hydroxyoctanoate (3HO), 3-hydroxydecanoate (3HD) and 3-hydroxydodecanoate (3HDD). Differential scanning calorimetry (DSC) experiments allowed the characterization of elastomeric materials with a melting point T_m near 50 °C, enthalpy of fusion ΔH_m from 27 to 32 J/g, and glass transition temperature T_g of −43 °C. Molecular weights ranged from 175,000 to 358,000 g/mol. On the basis of the phenotypical features and genotypic investigations, strain TE9 was assigned to the Pseudomonas genus and the name of Pseudomonas raguenesii sp. nov. is proposed.     

Cloning and expressing DBT (dibenzothiophene) monooxygenase gene (dszC) from Rhodococcus sp. DS-3 in Escherichia coli

Dibenzothiophene (DBT) monooxygenase (DszC) catalysis, the first and also the key step in the microbial DBT desulfurization, is the conversion of DBT to DBT sulfone (DBTO₂). In this study, dszC of a DBT-desulfurizing bacterium Rhodococcus sp. DS-3 was cloned by PCR. The sequence cloned was 99% homologous to Rhodococcus erythropolis IGTS8 that was reported in the Genebank. The gene dszC could be overexpressed effectively after being inserted into plasmid pET28a and transformed into E. coli BL21 strain. The expression amount of DszC was about 20% of total supernatant at low temperature. The soluble DszC in the supernatant was purified by Ni²⁺ chelating His-Tag resin column and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to electronics purity. Only one band was detected by Western-blotting, which is for the antibody released in mouse against purified DszC in the expression product of BL21 (DE3, paC5) and Rhodococcus sp. DS-3. The activity of purified DszC was 0.36 U. DszC can utilize the organic compound such as DBT and methyl-DBT, but not DBT derivates such as DBF, which has no sulfur or inorganic sulfur. __________ Translated from Acta Scientiarum Naturalium Universitatis Nankaiensis, 2005, 38(6): 1–6 [译自: 南开大学学报 (自然科学版), 2005, 38(6): 1–6]     

迟缓爱德华氏菌Eta1-L-Gapdh融合蛋白在蓝藻中的表达

在蓝藻中表达迟缓爱德华氏菌Eta1-L-Gapdh融合蛋白。提取迟缓爱德华氏菌基因组DNA为模板,用PCR技术分别扩增两个已知具有较强免疫原性的基因eta1和gapdh,再采用重叠延伸PCR将这两个基因融合,获得目的融合基因eta1-L-gapdh。将目的基因连接到表达载体pRL489的两个Bam H I酶切位点之间构建表达载体,用质粒提取、PCR、酶切、测序等手段对表达载体进行验证。验证正确的表达载体通过三亲接合转化野生鱼腥藻PCC7120,用新霉素抗性筛选出转基因藻落,通过质粒提取和PCR验证转基因藻。用RT-PCR和Western-blot分别从转录水平和翻译水平对转基因藻中融合基因的表达进行了检测。结果表明,含目的基因的表达载体构建成功,目的基因在蓝藻中转录并表达蛋白,该蛋白在蓝藻中的表达量为2.46%。