Cytokinin production by Agrobacterium and Pseudomonas spp.

The production of cytokinins by plant-associated bacteria was examined by radioimmunoassay. Strains producing trans-zeatin were identified in the genera Agrobacterium and Pseudomonas. Agrobacterium tumefaciens strains containing nopaline tumor-inducing plasmids, A. tumefaciens Lippia isolates, and Agrobacterium rhizogenes strains produced trans-zeatin in culture at 0.5 to 44 micrograms/liter. Pseudomonas solanacearum and Pseudomonas syringae pv. savastanoi produced trans-zeatin at levels of up to 1 mg/liter. In vitro cytokinin biosynthetic activity was measured for representative strains and was found to correlate with trans-zeatin production. The genetic locus for trans-zeatin secretion (tzs) was cloned from four strains: A. tumefaciens T37, A. rhizogenes A4, P. solanacearum K60, and P. syringae pv. savastanoi 1006. Southern blot analysis showed substantial homology of the Agrobacterium tzs genes to each other but not to the two Pseudomonas genes.     

发酵生产辅酶Q10高产菌株的选育

以实验室保存的类球红细菌(Rhodobacter sphaeroides)JDW61为出发菌株,考察了紫外、紫外结合氯化锂和亚硝基胍对菌株产生辅酶Q10能力的诱变效应,并结合辅酶Q10的合成途径设计了快速筛选辅酶Q10高产菌株的模型,获得一株辅酶Q10产量提高的突变株CP222,该菌株摇瓶发酵的辅酶Q10产量为276.14mg·L-1,较出发菌株提高了190%,并且遗传性能稳定。     

Cloning and functional expression of the dps gene encoding decaprenyl diphosphate synthase from Agrobacterium tumefaciens

A newly isolated gene from Agrobacterium tumefaciens (A. tumefaciens), which encoded a decaprenyl diphosphate synthase, was cloned in Escherichia coli (E. coli), and its nucleotide sequence was determined. DNA sequence analysis revealed an open reading frame of 1077 bp capable of encoding a 358-amino-acid protein with a calculated isoelectric point of pH 5.16 and a molecular mass of 38 960 Da. The primary structure of the enzyme shared significant homology with prenyl diphosphate synthases from various sources. The deduced amino acid sequence included oligopeptide DDxxD aspartate-rich domains conserved in the majority of prenyl diphosphate synthases. High levels of the active enzyme were expressed in the soluble fraction and were readily purified to homogeneity by Ni-NTA chromatography. E. coli JM109 harboring the dps gene produced ubiquinone-10 in addition to endogenous ubiquinone-8, while E. coli JM109 harboring the dps gene mutated on the DDxxD domain lost the ability to produce ubiquinone-10, which suggests that the A. tumefaciens dps gene is functionally expressed in E. coli and that it encodes a decaprenyl diphosphate synthase.     

Transfer RNA Is the Source of Extracellular Isopentenyladenine in a Ti-Plasmidless Strain of Agrobacterium tumefaciens

Even in the absence of the classical Ti plasmid-encoded cytokinin biosynthetic genes ipt and tzs, Agrobacterium tumefaciens strains still release significant amounts of the cytokinin isopentenyladenine (iP) into the culture medium (R.W. Kaiss-Chapman and R.O. Morris [1977] Biochem Biophys Res Commun 76: 453-459). A potential source of the iP is isopentenylated transfer RNA (tRNA), which, in turn, is synthesized by the activity of tRNA:isopentenyltransferase encoded by the bacterial miaA gene. To determine whether secreted iP had its origin in isopentenylated tRNA, a miaA- deletion/insertion mutant was prepared and reconstructed in Agrobacterium tumefaciens in vivo. The mutant no longer possessed tRNA:isopentenylation activity and no longer released iP into the extracellular medium. Transfer RNA therefore makes a small but significant contribution to the total amount of cytokinin normally secreted by Agrobacterium strains. tRNA-mediated synthesis may also account for cytokinin production by other plant-associated bacteria, such as Rhizobia, that have been reported to secrete similarly low levels of nonhydroxylated cytokinins.     

Nitrite and Nitrous Oxide Reductase Regulation by Nitrogen Oxides in Rhodobacter sphaeroides f. sp. denitrificans IL106

We have cloned the nap locus encoding the periplasmic nitrate reductase in Rhodobacter sphaeroides f. sp. denitrificans IL106. A mutant with this enzyme deleted is unable to grow under denitrifying conditions. Biochemical analysis of this mutant shows that in contrast to the wild-type strain, the level of synthesis of the nitrite and N(2)O reductases is not increased by the addition of nitrate. Growth under denitrifying conditions and induction of N oxide reductase synthesis are both restored by the presence of a plasmid containing the genes encoding the nitrate reductase. This demonstrates that R. sphaeroides f. sp. denitrificans IL106 does not possess an efficient membrane-bound nitrate reductase and that nitrate is not the direct inducer for the nitrite and N(2)O reductases in this species. In contrast, we show that nitrite induces the synthesis of the nitrate reductase.     

Cloning and characterization of the dxs gene, encoding 1-deoxy-d-xylulose 5-phosphate synthase from Agrobacterium tumefaciens, and its overexpression in Agrobacterium tumefaciens

A newly isolated gene dxs11 from Agrobacterium tumefaciens (KCCM 10413), an organism with potential for the industrial production of ubiquinone-10 (UbiQ(10)), encoding a 1-deoxy-d-xylulose 5-phosphate synthase (Dxs), was cloned in Escherichia coli and its nucleotide sequence was determined. DNA sequence analysis revealed an open reading frame of 1920bp, capable of encoding a polypeptide of 640 amino acids residues with a calculated isoelectric point of pH 5.63 and a molecular mass of 68,054Da. The homodimeric enzyme was overexpressed in E. coli and purified as an active soluble form. The enzyme required thiamine diphosphate and a divalent metal ion, either Mg(2+) or Mn(2+), for enzymatic activity. The enzyme had an optimal pH and temperature of 8.0 and 37 degrees C, respectively, with a k(cat) of 26.8s(-1) and a k(cat)/K(m) of 0.67 and 1.17s(-1)M(-1) for pyruvate and d-glyceraldehyde 3-phosphate, respectively. A. tumefaciens Dxs showed a comparable catalytic efficiency to other Dxs proteins. The dxs11 gene was transformed into A. tumefaciens KCCM 10413, and the resulting recombinant, A. tumefaciens pGX11, showed higher UbiQ(10) production (502.4mg/l) and content (8.3mg/gDCW) than A. tumefaciens KCCM 10413, by 21.9 and 23.9%, respectively. This work describes Dxs from A. tumefaciens, an organism with the potential for industrial UbiQ(10) production, and the first metabolic engineering study with the non-mevalonate pathway enzyme in A. tumefaciens.     

Structural and Functional Analyses of Photosynthetic Regulatory Genes regA and regB from Rhodovulum sulfidophilum, Roseobacter denitrificans, and Rhodobacter capsulatus

Genes coding for putative RegA, RegB, and SenC homologues were identified and characterized in the purple nonsulfur photosynthetic bacteria Rhodovulum sulfidophilum and Roseobacter denitrificans, species that demonstrate weak or no oxygen repression of photosystem synthesis. This additional sequence information was then used to perform a comparative analysis with previously sequenced RegA, RegB, and SenC homologues obtained from Rhodobacter capsulatus and Rhodobacter sphaeroides. These are photosynthetic bacteria that exhibit a high level of oxygen repression of photosystem synthesis controlled by the RegA-RegB two-component regulatory system. The response regulator, RegA, exhibits a remarkable 78.7 to 84.2% overall sequence identity, with total conservation within a putative helix-turn-helix DNA-binding motif. The RegB sensor kinase homologues also exhibit a high level of sequence conservation (55.9 to 61.5%) although these additional species give significantly different responses to oxygen. A Rhodovulum sulfidophilum mutant lacking regA or regB was constructed. These mutants produced smaller amounts of photopigments under aerobic and anaerobic conditions, indicating that the RegA-RegB regulon controls photosynthetic gene expression in this bacterium as it does as in Rhodobacter species. Rhodobacter capsulatus regA- or regB-deficient mutants recovered the synthesis of a photosynthetic apparatus that still retained regulation by oxygen tension when complemented with reg genes from Rhodovulum sulfidophilum and Roseobacter denitrificans. These results suggest that differential expression of photosynthetic genes in response to aerobic and anaerobic growth conditions is not the result of altered redox sensing by the sensor kinase protein, RegB.     

Genome analyses of three strains of Rhodobacter sphaeroides: evidence of rapid evolution of chromosome II

Three strains of Rhodobacter sphaeroides of diverse origin have been under investigation in our laboratory for their genome complexities, including the presence of multiple chromosomes and the distribution of essential genes within their genomes. The genome of R. sphaeroides 2.4.1 has been completely sequenced and fully annotated, and now two additional strains (ATCC 17019 and ATCC 17025) of R. sphaeroides have been sequenced. Thus, genome comparisons have become a useful approach in determining the evolutionary relationships among different strains of R. sphaeroides. In this study, the concatenated chromosomal sequences from the three strains of R. sphaeroides were aligned, using Mauve, to examine the extent of shared DNA regions and the degree of relatedness among their chromosome-specific DNA sequences. In addition, the exact intra- and interchromosomal DNA duplications were analyzed using Mummer. Genome analyses employing these two independent approaches revealed that strain ATCC 17025 diverged considerably from the other two strains, 2.4.1 and ATCC 17029, and that the two latter strains are more closely related to one another. Results further demonstrated that chromosome II (CII)-specific DNA sequences of R. sphaeroides have rapidly evolved, while CI-specific DNA sequences have remained highly conserved. Aside from the size variation of CII of R. sphaeroides, variation in sequence lengths of the CII-shared DNA regions and their high sequence divergence among strains of R. sphaeroides suggest the involvement of CII in the evolution of strain-specific genomic rearrangements, perhaps requiring strains to adapt in specialized niches.     

Structure of the photosynthetic reaction centre from Rhodobacter sphaeroides reconstituted with anthraquinone as primary quinone Q(A)

In the photosynthetic reaction centre (RC) from the purple bacterium Rhodobacter sphaeroides, the primary quinone, a ubiquinone-10 (Q(A)), has been substituted by anthraquinone. Three-dimensional crystals have been grown from the modified RC and its structure has been determined by X-ray crystallography to 2.4 A resolution. The bindings of the head-group from ubiquinone-10 and of the anthraquinone ring are very similar. In particular, both rings are parallel to each other and the hydrogen bonds connecting the native ubiquinone-10 molecule to AlaM260 and HisM219 are conserved in the anthraquinone containing RC. The space of the phytyl tail missing in the anthraquinone exchanged RC is occupied by the alkyl chain of a detergent molecule. Other structural changes of the Q(A)-binding site are within the limit of resolution. Our structural data bring strong credit to the very large amount of spectroscopic data previously achieved in anthraquinone-replaced RCs and which have participated in the determination of the energetics of the quinone system in bacterial RCs.     

氩离子激光照射对类球红细菌的诱变效应及对辅酶Q10产生量的影响

以低产量辅酶Q10类球红细菌为亲本,以氩离子激光为诱变源,对其幅照诱变,结果发现:亲本株发生了明显的诱变效应,出现了不同的色素突变表型。诱变后的色素突变株不仅遗传性状稳定,且辅酶Q10产量比亲本株有明显提高。对其中的黄色突变株发酵液进行辅酶Q10提取及测定,结果显示:其辅酶Q10产量比亲本株提高102.10%,经发酵条件初步优化,其最高产量可达26.39 mg/L发酵液。     

Identification of two new denitrifying strains of Rhodobacter sphaeroides

Abstract Highly specific polyclonal and antibodies against either nitrate, nitrite or nitrous oxide reductases from a photosynthetic denitrifying bacterium Rhodobacter sphaeroides f. sp. denitrificans were used to show the presence of immunologically reactive proteins in strains that Pellerin and Gest had shown to grow in the dark with nitrate as a terminal acceptor [9]. Two strains of this bacterium, namely 81-3 and 2.4.3 synthesized the three denitrifying enzymes and were capable of denitrification. Strains 81-1 and 2.4.1 (neotype) both expressed nitrate reductase activities but nitrite reductase was not detected since these strains did not reduce nitrite. They also did not grow in the dark with nitrate as a terminal acceptor. Each of strains 81-1, 81-3, 2.4.1 and 2.4.3 contain four plasmids. R. sphaeroides f. sp. denitrificans , however, contains only one large 108 kb plasmid, which is distinctly different in size from those detected in the other strains. This indicates that the 108 kb plasmid is not necessarily specific for denitrification.     

Effect of Aeration on Growth and Aflatoxin Production by Aspergillus flavus in Submerged Culture

Aspergillus flavus ATCC 15517 produced up to 212 mg per liter of total aflatoxin in submerged culture in aerated (3,000, 6,000, 9,000, and 12,000 ml/min) and agitated medium in 14-liter fermentors with 10 liters of medium consisting of 2% yeast extract and 10% sucrose. Aflatoxin production increased with time. A maximum of 212 mg/liter was produced at 9,000 ml/min aeration, whereas the yield decreased substantially at the lower aeration rates. Two other strains of A. flavus synthesized aflatoxin in smaller quantities.     

Surface motility and associated surfactant production in Agrobacterium vitis

Aims:  Agrobacterium vitis is the causal agent of crown gall of grapevine. Surface motility (swarming), an important mechanism for bacterial colonization of new environments and a previously unknown behaviour of Ag. vitis , was demonstrated.
Methods:  Surface motility assays were performed on half-strength potato dextrose agar (Difco) containing 0·75% agar. To test for surfactant production, a drop-collapse test was used. Quorum-sensing (QS) negative and complemented mutants were tested for swarming activity.
Results:  Ninety-one Agrobacterium strains representing – Agrobacterium tumefaciens (17 strains), Agrobacterium rhizogenes (14 strains) and Ag. vitis (60 strains) were tested for swarming and production of surfactant. All Ag. vitis strains expressed a surface-related motility. In contrast, none of 17 strains of Ag. tumefaciens or 14 strains of Ag. rhizogenes exhibited this behaviour. Surface motility in Ag. vitis was associated with surfactant secretion; both of which are regulated by a QS system previously associated with induction of a hypersensitive response on tobacco and necrosis on grape. An aviR (belongs to luxR family) mutant was surface motility negative and did not produce surfactant. An avsI mutant (autoinducer synthase) was also surface motility negative and was complemented with an Ag. tumefaciens clone expressing avsI .
Conclusions:  Agrobacterium vitis is able to produce a characteristic swarming phenotype that is regulated by a complex QS system.
Significance and Impact of the Study:  Swarming activity is unique to Ag. vitis among Agrobacterium sp. and may be associated with the ability of the pathogen to colonize grapevines.     

Re-design of Rhodobacter sphaeroides dimethyl sulfoxide reductase. Enhancement of adenosine N1-oxide reductase activity

The periplasmic DMSO reductase from Rhodobacter sphaeroides f. sp. denitrificans has been expressed in Escherichia coli BL21(DE3) cells in its mature form and with the R. sphaeroides or E. coli N-terminal signal sequence. Whereas the R. sphaeroides signal sequence prevents formation of active enzyme, addition of a 6x His-tag at the N terminus of the mature peptide maximizes production of active enzyme and allows for affinity purification. The recombinant protein contains 1.7-1.9 guanines and greater than 0.7 molybdenum atoms per molecule and has a DMSO reductase activity of 3.4-3.7 units/nmol molybdenum, compared with 3.7 units/nmol molybdenum for enzyme purified from R. sphaeroides. The recombinant enzyme differs from the native enzyme in its color and spectrum but is indistinguishable from the native protein after redox cycling with reduced methyl viologen and Me2SO. Substitution of Cys for the molybdenum-ligating Ser-147 produced a protein with DMSO reductase activity of 1.4-1.5 units/nmol molybdenum. The mutant protein differs from wild type in its color and absorption spectrum in both the oxidized and reduced states. This substitution leads to losses of 61-99% of activity toward five substrates, but the adenosine N1-oxide reductase activity increases by over 400%.     

Assessing the impact of denitrifier-produced nitric oxide on other bacteria

A series of experiments was undertaken to learn more about the impact on other bacteria of nitric oxide (NO) produced during denitrification. The denitrifier Rhodobacter sphaeroides 2.4.3 was chosen as a denitrifier for these experiments. To learn more about NO production by this bacterium, NO levels during denitrification were measured by using differential mass spectrometry. This revealed that NO levels produced during nitrate respiration by this bacterium were in the low muM range. This concentration of NO is higher than that previously measured in denitrifiers, including Achromobacter cycloclastes and Paracoccus denitrificans. Therefore, both 2.4.3 and A. cycloclastes were used in this work to compare the effects of various NO levels on nondenitrifying bacteria. By use of bacterial overlays, it was found that the NO generated by A. cycloclastes and 2.4.3 cells during denitrification inhibited the growth of both Bacillus subtilis and R. sphaeroides 2.4.1 but that R. sphaeroides 2.4.3 caused larger zones of inhibition in the overlays than A. cycloclastes. Both R. sphaeroides 2.4.3 and A. cycloclastes induced the expression of the NO stress response gene hmp in B. subtilis. Taken together, these results indicate that there is variability in the NO concentrations produced by denitrifiers, but, irrespective of the NO levels produced, microbes in the surrounding environment were responsive to the NO produced during denitrification.     

Histidine Production by Auxotrophic Histidine Analog-resistant Mutants of Corynebacterinm glutamicum

Corynebacterium glutamicum mutants carrying both auxotrophy and histidine analog-resistance were derived by a mutagenic treatment, and their histidine productivity was compared with that of a triazolealanine (TRA)-resistant histidine producer, C. glutamicum KY-10260. As a result, a leucine auxotrophic TRA-resistant mutant, Rα-88 was selected out of 164 auxotrophic derivatives of KY-10260. It produced histidine at a distinctly higher concentration than the parent strain under every condition tested. The concentration reached 11 mg/ml or 5.8% (w/w) of the initial sugar. Addition of an excessive amount of leucine to the medium inhibited the histidine production together with the by-production of valine by this mutant. Thiazolealanine-resistant mutants derived from a tyrosine auxotroph, a phenylalanine auxotroph and a tryptophan auxotroph gave the same or lower production in comparison with KY-10260.     

根癌农杆菌介导的日本曲霉转化体系的建立

【目的】通过根癌农杆菌介导的方法构建日本曲霉转化子库,从而筛选出高产甘没氧化酶的日本曲霉突变菌株。【方法】本文通过三亲杂交的方法将双元载体pBI-hphII转移至根癌农杆菌EHA105中并作为侵染菌株,以日本曲霉As5999为受体菌株,建立了农杆菌介导的日本曲霉转化体系,构建了突变体库,并对影响转化效率的根癌农杆菌浓度,乙酰丁香酮(As)加入与否,共培养时间,共培养温度等因素进行了分析。【结果】对转化子的PCR检测和Southern杂交分析表明,T-DNA已整合进日本曲霉基因组中,随机挑选的9个转化子连续转接10代后均能稳定遗传。【结论】该转化体系的建立为筛选出高产甘油氧化酶的日本曲霉突变菌株奠定了基础。     

Cytochrome aa3 of Rhodobacter sphaeroides as a model for mitochondrial cytochrome c oxidase. The coxII/coxIII operon codes for structural and assembly proteins homologous to those in yeast.

The coxII/coxIII operon of Rhodobacter sphaeroides cytochrome c oxidase has been sequenced and characterized by insertional inactivation/complementation analysis. The organization of the genes in this locus (coxII.orf1.orf3.coxIII) is the same as that of the equivalent operon of Paracoccus denitrificans (ctaC.ctaB.ctaG.ctaE), but unlike that of other bacteria whose cytochrome oxidase genes have been characterized so far. The predicted amino acid sequence homology with eukaryotic oxidases is also higher for Rb. sphaeroides (and P. denitrificans) than for other bacterial versions of the enzyme. The inactivation of coxII results in loss of the characteristic cytochrome oxidase spectrum from membranes of the mutant strain. Full recovery requires introduction into the bacterium of the complete operon containing coxII.orf1.orf3.coxIII; partial complementation yielding a spectrally altered enzyme is achieved with a plasmid containing coxII or coxII.orf1.orf3. These results indicate that the peptides ORF1, ORF3, and COXIII are all required for assembly of native cytochrome c oxidase, suggesting an oxidase-specific assembly or chaperonin function for the ORFs in Rb. sphaeroides similar to that observed for the homologous gene products in yeast, COX10 and COX11.