3种水稻土中7株固氮蓝细菌的分离与特征

【背景】蓝细菌是水生和陆地生态系统中生物固氮的主要贡献者。【目的】增加对稻田土壤固氮蓝细菌的了解,获得用于进一步研究的可培养固氮蓝细菌菌株。【方法】选择3种具有不同固氮能力的水稻土,采用BG11-N培养基分离培养固氮蓝细菌菌株,对新分离菌株进行形态特征观察,通过基因组DNA的nifH基因扩增明确其固氮潜力,进一步采用乙炔还原法和~(15)N_2示踪法定量测定其固氮能力,通过基因组DNA的16SrRNA基因序列比对进行鉴定。【结果】在光照培养条件下,采用BG11-N培养基共分离纯化得到自养菌株7株,细胞呈圆形或椭圆形、单列、无分枝、丝状和念珠状,在固体培养基上形成团垫状菌落。新分离菌株在BG11-N培养基中生长状况良好,以基因组DNA为模板可扩增出nifH基因,乙炔还原法和~(15)N_2示踪法测定结果显示具有较高固氮能力,同时具有铁载体生成能力。结合16S rRNA基因序列比对和形态特征,7株菌被初步鉴定隶属于念珠藻科(Nostocaceae)。【结论】从水稻土中分离到在稻田生物固氮中发挥重要作用的蓝细菌(念珠藻科)菌株,可培养固氮蓝细菌菌株固氮能力较高,兼具铁载体生成能力,可作为进一步深入研究的微生物资源,具有潜在的研究应用价值。     

Co-induction of DNA relaxation and synthesis of DnaK and GroEL proteins inEscherichia coli by expression of LetD (CcdB) protein, an inhibitor of DNA gyrase encoded by the F factor

We examined the influence of overexpression of LetD (CcdB) protein, an inhibitor of DNA gyrase encoded by the F factor ofEscherichia coli, on DNA supercoiling and induction of heat shock proteins. Cells were transformed with a plasmid carrying the structural gene for LetD protein under control of thetac promoter, and LetD protein was induced by adding isopropyl-d-thiogalactopyranoside (IPTG) to the culture medium. Analysis by agarose gel electrophoresis in the presence of chloroquine revealed relaxation of plasmid DNA in cells depending on the concentration of IPTG employed for induction. Protein pulse-labeling experiments with [³⁵S]methionine and cysteine revealed that synthesis of DnaK and GroEL proteins was also induced by IPTG, and concentrations necessary for DNA relaxation and induction of the heat shock proteins were much the same. Expression of mutant LetD protein lacking two amino acid residues at the C-terminus induced neither DNA relaxation nor the synthesis of DnaK and GroEL proteins. Induction of wild-type LetD protein but not mutant LetD protein markedly enhanced synthesis of ³². We interpret these results to mean that DNA relaxation in cells caused by the expression of LetD protein induces heat shock proteins via increased synthesis of ³².     

Progress on the genetics of the N2-fixing actinorhizal symbiont Frankia

The actinomycete Frankia is of fundamental and ecological interests for several reasons including its wide distribution, its ability to fix nitrogen, differentiate into sporangium and vesicle (specialized cell for nitrogen-fixation), and to nodulate plants from about 24 genera. Here, we present a review on the genetics performed so far on Frankia. At the end of July 2001, 293 kbp of Frankia DNA sequences were found in the databases. Thirty five percent of these sequences corresponded to full gene or gene cluster sequences. These genes could be divided according to their role into 6 key activities: gene translation (rrnA and tRNA ^pro gene), proteolysis (pcr genes), assimilation of ammonium (glnA and glnII), protection against superoxide ions (sodF), nitrogen fixation (nif cluster), and plasmid replication. We present a review of these genetic islands; their function, expression, localization and particular properties are discussed. A comparative analysis of Frankia nif genes from various strains and species is presented. An improved nomenclature for some of these genes is suggested to avoid conflicts. Frankia plasmids DNA sequences are also presented. The novel trends in Frankia genetics are described.     

Cloning of the gene for phosphoenolpyruvate carboxylase from Azotobacter chroococcum, an enzyme important in aerobic nitrogen fixation

Summary Azotobacter chroococcum Fos 189 is a Tn1-induced mutant which, unlike the parent strain MCD1, does not fix nitrogen in air when provided with glucose or pyruvate as sole carbon sources. Fos 189 showed 5% of parental activity for phosphoenolpyruvate carboxylase though PEP synthetase activity was normal. The A. chroococcum phosphoenolpyruvate carboxylase (ppc) gene was isolated after complementation of an appropriate Escherichia coli mutant using a broad host range gene bank prepared from A. chroococcum genomic DNA. The gene was localised by transposon mutagenesis and subcloning on a minimum DNA fragment of 6.6 kb. Broad host range plasmids containing the A. chroococcum ppc gene complemented the mutation in Fos 189 thereby restoring aerotolerant nitrogen fixation.     

Cloning and expression of the SalI restriction-modification genes of Streptomyces albus G

Summary The Streptomyces albus G genes (salR and salM) for the class II restriction enzyme SalI (SalGI) and its cognate modification enzyme were cloned in Streptomyces lividans 66. Selection was initially for the salR gene. From a library of S. albus G DNA in the high copy number plasmid pIJ486 several clones of S. lividans were obtained that were resistant to phage C31 unmodified at the many SalI sites in its DNA, but were sensitive to modified phages last propagated on a restriction-deficient, modification-proficient mutant of S. albus G. SalI activity was detected in cell-free extracts of the clones, though only at levels comparable with that in S. albus G. Five different recombinant plasmids were isolated, with inserts of 5.6, 5.7, 8.9, 10 and 18.9 kb that contained a common region of 4.5 kb. These plasmids could not be digested by SalI, although the vector has four recognition sites for this enzyme, indicating that the salM gene was also cloned and expressed. Subcloning experiments in S. lividans indicated the approximate location of salR and salM, and in Escherichia coli led to detectable expression of salM but not of salR. A variety of previously isolated S. albus G mutants affected in aspects of SalI-specific restriction and modification were complemented by the cloned DNA; they included a mutant temperature-sensitive for growth apparently because of a mutation in salM. Southern blotting showed that DNA homologous to the cloned sal genes was present in Xanthomonas and Rhodococcus strains, but not detectably in Herpetosiphon strains, all of which produce SalI isoschizomers.     

Ammonium sensing in nitrogen fixing bacteria: Functions of theglnB andglnD gene products

A plentiful supply of fixed nitrogen as ammonium (or other compounds such as nitrate or amino acids) inhibits nitrogen fixation in free-living bacteria by preventing nitrogenase synthesis and/or activity. Ammonium and nitrate have variable effects on the ability ofRhizobiaceae (Rhizobium, Bradyrhizobium andAzorhizobium) species to nodulate legume hosts and on nitrogen fixation capacity in bacteroid cells contained in nodules or in plant-free bacterial cultures. In addition to effects on nitrogen fixation, excess ammonium can inhibit activity or expression of other pathways for utilization of nitrogenous compounds such as nitrate (through nitrate and nitrite reductase), or glutamine synthetase (GS) for assimilation of ammonium. This paper describes the roles of two key genesglnB andglnD, whose gene products sense levels of fixed nitrogen and initiate a cascade of reactions in response to nitrogen status. While work onEscherichia coli and other enteric bacteria provides the model system,glnB and, to a lesser extent,glnD have been studied in several nitrogen fixing bacteria. Such reports will be reviewed here. Recent results on the identity and function of theglnB andglnD gene products inAzotobacter vinelandii (a free-living soil diazotroph) and inRhizobium leguminosarum biovarviciae, hereinafter designatedR.l. viciae will be presented. New data suggests thatAzotobacter vinelandii probably contains aglnB-like gene and this organism may have twoglnD-like genes (one of which was recently identified and namednfrX). In addition, evidence for uridylylation of theglnB gene product (the PII protein) ofR. l. viciae in response to fixed nitrogen deficiency is presented. Also, aglnB mutant ofR. l. viciae has been isolated; its characteristics with respect to expression of nitrogen regulated genes is described.     

Cloning and expression of sucrose phosphorylase gene from Bifidobacterium longum in E. coli and characterization of the recombinant enzyme

A DNA fragment, which complemented the growth of E. coli both on M9 medium containing raffinose and on LB medium containing ampicillin, IPTG and 5-bromo-4-chloro-3-indoxyl--d-galactoside, was isolated from the genomic library of Bifidobacterium longum SJ32, which had been digested with EcoRI. In the cloned DNA fragment, a gene encoding a sucrose phosphorylase (splP) and a partially cloned putative sucrose regulator gene (splR) were identified using the deletion analysis and sequence analysis. A 56 kDa protein was synthesized in E. coli and partially purified by DEAE-ion exchange chromatography. The partially purified enzyme did not react with melibiose, melezitoze and raffinose but did with sucrose. It had transglucosylation activity in addition to hydrolytic activity.     

Characterization of the fixABC region of Azorhizobium caulinodans ORS571 and identification of a new nitrogen fixation gene

Summary The fast growing strain, Azorhizobium caulinodans ORS571, isolated from stem nodules of the tropical legume Sesbania rostrata, can grow in the free-living state at the expense of molecular nitrogen. Five point mutants impaired in nitrogen fixation in the free-living state have been complemented by a plasmid containing the cloned fix-ABC region of strain ORS571. Genetic analysis of the mutants showed that one was impaired in fixC, one in fixA and the three others in a new gene, located upstream from fixA and designated nifO. Site-directed Tn5 mutagenesis was performed to obtain Tn5 insertions in fixB and fixC. The four genes are required for nitrogen fixation both in the free-living state and under symbiotic conditions. The nucleotide sequence of nifO was established. The gene is transcribed independently of fixA and does not correspond to fixX, recently identified in Rhizobium meliloti and R. leguminosarum. Biochemical analysis of the five point mutants showed that they synthesized normal amounts of nitrogenase components. It is unlikely that fixA, fixC and nifO are involved in electron transport to nitrogenase. FixC could be required for the formation of a functional nitrogenase component 2.     

Variation in moss-associated nitrogen fixation in boreal forest stands

Traditionally it has been thought that most boreal forest communities lack a significant input of biologically fixed nitrogen. Recent discoveries of nitrogen fixation by cyanobacteria associated with mosses have resulted in a re-evaluation of this view. While it is recognized that rates of nitrogen fixation in mosses can be highly variable, there is little understanding as to why this occurs. I monitored nitrogen fixation, using acetylene reduction, in wet lowland and dry upland boreal forest communities, in central Canada, over a growing season. At the peak of nitrogen fixation in mid summer, Sphagnum capillifolium had an 11 times higher rate of fixation than Pleurozium schreberi. Variation in canopy openness and precipitation had no effect on rates of fixation over the growing season. In P. schreberi fixation rates did not vary between sites. Temperature had a positive effect on fixation rates in both S. capillifolium and P. schreberi, but the effect was 4 times more pronounced in S. capillifolium. Seasonal rates of nitrogen fixation were estimated at 193 mg N m⁻² for S. capillifolium and 23 mg N m⁻² for P. schreberi. With moderate increases in climate warming, predicted increases in nitrogen fixation in S. capillifolium are sufficient to raise its decomposition rate. Increased temperatures may therefore act synergistically to change boreal systems from a sink to a source of carbon.     

Visualization of site-specific recombination catalyzed by a recombinase from Zygosaccharomyces rouxii in Arabidopsis thaliana

Excision of a DNA segment can occur in Arabidopsis thaliana by reciprocal recombination between two specific recombination sites (RSs) when the recombinase gene (R) from Zygosaccharomyces rouxii is expressed in the plant. To monitor recombination events, we generated several lines of transgenic Arabidopsis plants that carried a cryptic -glucuronidase (GUS) reporter gene which was designed in such a way that expression of the reporter gene could be induced by R gene-mediated recombination. We also made several transgenic lines with an R gene linked to the 35S promoter of cauliflower mosaic virus. Each transgenic line carrying the cryptic reporter gene was crossed with each line carrying the R gene. Activity of GUS in F₁ and F₂ progeny was examined histochemically and recombination between two RSs was analyzed by Southern blotting and the polymerase chain reaction. In seedlings and plantlets of F₁ progeny and most of the F₂ progeny, a variety of patterns of activity of GUS, including sectorial chimerism in leaves, was observed. A small percentage of F₂ individuals exhibited GUS activity in the entire plant. This pattern of expression was ascribed to germinal recombination in the F₁ generation on the basis of an analysis of DNA structure by Southern blotting. These results indicate that R gene-mediated recombination can be induced in both somatic and germ cells of A. thaliana by cross-pollination of parental transgenic lines.     

Role of cytosine deaminase and β-alanine-pyruvate transaminase in pyrimidine base catabolism by Burkholderia cepacia

A determination of the possible role of the salvage enzyme cytosine deaminase or -alanine-pyruvate transaminase in the catabolism of the pyrimidine bases uracil and thymine by the opportunistic pathogen Burkholderia cepacia ATCC 25416 was undertaken. It was of interest to learn whether these enzymes were influenced by cell growth on pyrimidine bases and their respective catabolic products to the same degree as the pyrimidine reductive catabolic enzymes were. It was found that cytosine deaminase activity was influenced very little by cell growth on the pyrimidines tested. Using glucose as the carbon source, only B. cepacia growth on 5-methylcytosine as a nitrogen source increased deaminase activity by about three-fold relative to (NH₄)₂SO₄-grown cells. In contrast, the activity of –alanine-pyruvate transaminase was observed to be at least double in glucose-grown ATCC 25416 cells when pyrimidine bases and catabolic products served as nitrogen sources instead of (NH₄)₂SO₄. Transaminase activity in the B. cepacia glucose-grown cells was maximal after the strain was grown on either uracil or 5-methylcytosine as a nitrogen source compared to (NH₄)₂SO₄-grown cells. A possible role for -alanine-pyruvate transaminase in pyrimidine base catabolism by B. cepacia would seem to be suggested from the similarity in how its enzyme activity responded to cell growth on pyrimidine bases and catabolic products when compared to the response of the three reductive catabolic enzymes.     

Nitrogen fixation by the hydrogen-oxidizing bacterium Alcaligenes latus

The aerobic hydrogen-oxidizing bacterium Alcaligenes latus represented by three strains was found to be able to grow with dinitrogen as the sole nitrogen source: The doubling time of total (Kjeldahl) nitrogen during growth on glucose at 30°C under an atmosphere containing 2% (v/v) oxygen in dinitrogen amounted to 39 h, while that in the presence of ammonium was 3 h. Nitrogen fixation did apparently not occur under air. During diazotrophic growth the cells accumulated up to 75% (w/dry weight) poly--hydroxybutyric acid. The efficiency of nitrogen fixation varied between 10 and 15 mg N per g glucose utilized. The specific nitrogenase activity measured in the acetylene reduction assay amounted to 5–17 nmol C₂H₄ formed per min and mg protein.     

Sequence and functional analysis of the left-hand part of the T-region from the nopaline-type Ti plasmid,pTiC58

The Agrobacterium tumefaciens nopaline strain C58 transfers a large, 29 kb T-DNA into plant cells during infection. Part of this DNA (the `common DNA') is also found on the T-DNA of octopine strains, the remaining DNA is nopaline strain-specific. Up to now, only parts of the C58 T-DNA and related T37 T-DNA have been sequenced. We have sequenced the remainder of the nopaline-specific T-DNA (containing genes a to d) and acs to iaaM. Gene c codes for a new unknown T-DNA protein. Gene a is homologous to the agrocinopine synthase gene. Genes b, c, d and e are part of a larger family: they are related to the T-DNA genes 5, rolB, lso and 3. Genes 5, rolB and lso induce or modify plant growth and have been called T-DNA oncogenes. Our studies show that gene 3 (located on the TR-DNA of octopine strains) is also oncogenic. Although the b–e T-DNA fragment from C58 and its individual genes lack growth-inducing activity, an a-acs deletion mutant was distinctly less virulent on Kalanchoe daigremontiana and showed reduced shoot formation on Kalanchoe tubiflora. Shoot formation could be restored by genes c and c in co-infection experiments. Contrary to an earlier report, a C58 e gene deletion mutant was fully virulent on all plants tested.     

Construction of a gene bank and identification of the ribulose bisphosphate carboxylase/oxygenase genes from the nutritionally versatile cyanobacterium Chlorogloeopsis fritschii

A gene bank of the nutritionally versatile, nitrogen-fixing cyanobacterium Chlorogloeopsis fritschii was constructed in Charon 4A. 2,800 recombinants containing 10–20 kbp C. fritschii DNA fragments were screened by Southern hybridization using probes containing the genes for the large (LSU) and small (SSU) subunits of ribulose bisphosphate carboxylase/oxygenase (RuBisCO) from Anacystis nidulans. A single recombinant plaque (CDG1) containing a 10.9 kbp EcoR1 fragment from C. fritschii hybridized to both the LSU and SSU probes, indicating a possible linkage of these RuBisCO genes in C. fritschii. RuBisCO activity and protein were detected in CDG1 lysates of Escherichia coli. Hybridization was also obtained between C. fritschii DNA and the LSU probe from Chlamydomonas reinhardtii, although no homology was detected using the LSU probe from maize or the SSU probe from pea.Abbreviations RuBisCO d-ribulose 1,5-bisphosphate carboxylase/oxygenase - RuBP d-ribulose 1,5-bisphosphate - LSU large subunit of RuBisCO - SSU small subunit of RuBisCO - SDS sodium dodecyl sulphate - DOC deoxycholate     

Use of site-specific recombination to regenerate selectable markers

Summary A method which allows the repeated use of a single selectable marker in DNA transformations was demonstrated. This marker regeneration method employed portions of the Saccharomyces cerevisiae 2 m circle plasmid: the inverted repeat sequences (FRTs), and the FLP gene whose product, a site-specific recombinase, catalyzes recombination events between FRTs. When FRTs were oriented as direct repeats and integrated into the genome of the yeast Pichia pastoris, FLP-mediated recombination resulted in the efficient and precise deletion of DNA located between the repeats. In the example described, the S. cerevisiae ARG4 gene, placed between a set of FRTs and integrated into Pichia in a prior transformation, was deleted by FLP, thereby regenerating an arginine-requiring phenotype in the P. pastoris strain.     

The gene for the α-subunit of ATPase: a site of homologous recombination in plant mitochondrial DNA also functions in somatic hybrid cells

Summary Segments of mitochondrial DNA (mtDNA) carrying the gene for the -subunit of F₁-ATPase (atpA) were detected by Southern hybridization with atpA from pea as probe. In the case of Nicotiana langsdorffii, we identified four fragments that are derived from combinations of two different 5 and two different 3 flanking regions of atpA. All four types share the coding region, suggesting that they result from homologous recombination in the coding region of atpA. By contrast, N. glauca generated only one analogous fragment, which indicated the existence of only a single type of atpA in N. glauca. In the case of somatic hybrids obtained by fusion between protoplasts from N. langsdorffii and N. glauca, analysis with EcoRI or HindIII detected three new fragments in addition to the parental fragments. These new fragments can be explained by homologous recombination within the coding region of atpA. Our results show that the coding region of atpA is involved not only in intragenomic homologous recombination but can also be involved in homologous recombination between two parental mitochondrial genomes of somatic hybrids.     

Molecular studies in Pelargonium (Geraniaceae). A taxonomic appraisal of section Ciconium and the origin of the ‘Zonal’ and ‘Ivy-leaved’ cultivars

An RFLP analysis of chloroplast and nuclear DNA was followed in this taxonomic appraisal of section Ciconium. The section appears to be a monophyletic one, which divides into two groups, or sub-sections. The Zonal Geranium, P. × hortorum is derived from section Ciconium, and the species most likely to be its ancestors are shown to be P. inquinans and P. zonale. Maternal inheritance of chloroplasts was demonstrated by comparing chloroplast DNA from interspecific hybrids with their parents. These data also suggest that P. inquinans was probably the maternal parent of P. × hortorum in the original crosses. The Ivy-leaved cultivars appear to be derived from P. peltatum, with some contribution from P. zonale and/or P. × hortorum.     

New Escherichia coli gyrA and gyrB mutations which have a graded effect on DNA supercoiling

Summary We isolated new gyrA and gyrB mutations in Escherichia coli which have a graded effect on DNA supercoiling. The mutants, selected respectively for resistance to nalidixic acid and coumermycin, were sorted by means of a rapid in vivo assay of DNA gyrase activity (Aleixandre and Blanco 1987). Cells carrying a gyrB (Cou^r) mutation usually showed a decrease in DNA supercoiling, which would indicate a reduction in gyrase activity. In contrast, most of the gyrA (Nal^r) mutations had no significant effect on DNA supercoiling. Moreover, they conferred a high level of resistance to nalidixic acid and other quinolones, thus being similar to the gyrA(Nal^r) mutants currently used. We also detected rare gyrA mutants showing a reduction in DNA gyrase activity. These mutants were, in addition, resistant to only low concentrations of quinolones, which allowed us to use the phenotype of partial quinolone resistance as an indicator to score gyrA mutations affecting DNA supercoiling. When gyrB mutations were introduced into the gyrA mutants, these became more sensitive to quinolones and a decrease in supercoiling was observed. Moreover, the topA10 mutation sensitized gyrA(Nal^r) cells to quinolones. We conclude therefore that the GyrA-dependent quinolone resistance is diminished as a consequence of the reduction either in topoisomerase I or gyrase activities.