Members of the genus Arthrobacter grow anaerobically using nitrate ammonification and fermentative processes: anaerobic adaptation of aerobic bacteria abundant in soil

Members of the genus Arthrobacter are usually regarded as obligate aerobic bacteria. The anaerobic growth and energy metabolism of two Arthrobacter species were investigated. Arthrobacter globiformis utilized both nitrate ammonification and lactate, acetate and ethanol producing fermentation processes for anaerobic growth. Only nitrate supported anaerobic growth of Arthrobacter nicotianae. Anaerobically induced respiratory nitrate reductase activity was detected in both strains. Neither of the tested strains used the alternative electron acceptors fumarate, dimethylsulfoxide or trimethylamine-N-oxide.     

美洲大蠊成虫肠道可培养细菌多样性研究

【目的】了解美洲大蠊成虫肠道可培养细菌的多样性。【方法】运用纯培养法、数值分类和16S rRNA基因序列的系统发育分析对样品中可培养细菌多样性进行研究。【结果】从NA培养基中分离得到54株细菌,根据形态观察和部分生理生化特性,选取32个代表性菌株进行16S rRNA基因序列的系统发育多样性分析。结果表明,数值分类中的代表菌株在82%相似水平上可分为12个表观群;这些分离菌株代表20个物种,属于4个大的系统发育类群(Proteobacteria,Bacteroidetes,Firmicutes,Actinobacteria)的10个科、15个属。多数菌株属于Proteobacteria门(15株,占46.9%)和Bacteroidetes门(10株,占31.3%)。【结论】美洲大蠊成虫肠道内存在较为丰富的细菌多样性。     

Diversity of antagonistic bacteria isolated from medicinal plant Peganum harmala L.

The antimicrobial activity of plant extract of Peganum harmala, a medicinal plant has been studied already. However, knowledge about bacterial diversity associated with different parts of host plant antagonistic to different human pathogenic bacteria is limited. In this study, bacteria were isolated from root, leaf and fruit of plant. Among 188 bacterial isolates isolated from different parts of the plant only 24 were found to be active against different pathogenic bacteria i.e. Escherichia coli, Methicillin-resistant Staphylococcus aureus (MRSA), Enterococcus faecium, Enterococcus faecalis and Pseudomonas aeruginosa. These active bacterial isolates were identified on the basis of 16S rRNA gene analysis. Total population of bacteria isolated from plant was high in root, following leaf and fruit. Antagonistic bacteria were also more abundant in root as compared to leaf and fruit. Two isolates (EA5 and EA18) exhibited antagonistic activity against most of the targeted pathogenic bacteria mentioned above. Some isolates showed strong inhibition for one targeted pathogenic bacterium while weak or no inhibition for others. Most of the antagonistic isolates were active against MRSA, following E. faecium, P. aeruginosa, E. coli and E. faecalis. Taken together, our results show that medicinal plants are good source of antagonistic bacteria having inhibitory effect against clinical bacterial pathogens.     

Expression analysis of a high-affinity nitrate transporter isolated from Arabidopsis thaliana by differential display

We used the differential display technique on total RNAs from roots of Arabidopsis thaliana (L.) Heynh. plants which had or had not been induced for 2 h by nitrate. One isolated cDNA clone, designated Nrt2:1At, was found to code for a putative high-affinity nitrate transporter. Two genomic sequences homologous to Nrt2:1At were found to be localized on the same fragment of chromosome 1 in the Arabidopsis genome. Expression analyses of both low- and high-affinity nitrate transporter genes, respectively Nrt1:1At (previously named Chl1) and Nrt2:1At, were carried out on plants grown under different nitrogen regimes. In this paper, we show that both genes are induced by very low levels of nitrate (50 μM KNO₃). However, stronger induction was observed with Nrt2:1At than with Nrt1:1At. Moreover, these two genes, although both over-expressed in a nitrate-reductase-deficient mutant, were differently regulated when N-sufficient wild-type or mutant plants were transferred to an N-free medium. Indeed, the steady-state amounts of Nrt1:1At mRNA declined whereas the amount of Nrt2:1At mRNA increased, probably reflecting the de-repression of the high-affinity transport system during N-starvation. Received: 4 May 1998 / Accepted: 26 August 1998     

Molecular phylogeny of Bradyrhizobium bacteria isolated from root nodules of tribe Genisteae plants growing in southeast Poland

The phylogeny of 16 isolates from root nodules of Genista germanica, Genista tinctoria, Cytisus ratisbonensis, and Cytisus scoparius growing in southeast Poland was estimated by comparative sequence analysis of core (16S rDNA, atpD, glnII, recA) and symbiosis-related (nodC, nodZ, nifH) genes. All the sequences analyzed placed the studied rhizobia in the genus Bradyrhizobium. Phylogenetic analysis of individual and concatenated housekeeping genes showed that the Genisteae microsymbionts form a homogeneous group with Bradyrhizobium japonicum strains. The phylogeny of nodulation and nitrogen fixation genes indicated a close relationship of the examined rhizobia with B. japonicum, Bradyrhizobium canariense, Bradyrhizobium cytisi, Bradyrhizobium rifense and Bradyrhizobium lupini strains infecting other plants of the tribe Genisteae. For the first time, the taxonomic position of G. germanica and C. ratisbonensis rhizobia, inferred from multigenic analysis, is described. The results of the phylogenetic analysis based on the protein-coding gene sequences presented in this study also indicate potential pitfalls concerning the choice of marker and reference strains, which may lead to conflicting conclusions in species delineation.     

Expression of the nitrate transporter nrt2 gene from the symbiotic basidiomycete Hebeloma cylindrosporum is affected by host plant and carbon sources

Although the function of the extramatrical mycelium of ectomycorrhizal fungi is considered essential for the acquisition of nitrogen by forest trees, gene regulation in this fungal compartment is poorly characterized. In this study, the expression of the nitrate transporter gene nrt2 from the ectomycorrhizal basidiomycete Hebeloma cylindrosporum was shown to be regulated by plant host and carbon sources. In the presence of a low fructose concentration, nrt2 expression could not be detected in the free-living mycelium but was high in the extramatrical symbiotic mycelium associated to the host plant Pinus pinaster. In the absence of nitrogen or in the presence of nitrate, high sugar concentrations in the medium were able to enhance nrt2 expression. Nevertheless, in the presence of high fructose concentration, high ammonium concentration still completely repressed nrt2 expression indicating that the nitrogen repression overrides sugar stimulation. This is the first report revealing an effect of host plant and of carbon sources on the expression of a fungal nitrate transporter-encoding gene.     

The reactivation of nitrate reductase from spinach (Spinacea oleracea L.) inactivated by NADH and cyanide: effects of peroxidase and associated systems

Nitrate reductase of spinach (Spinacea oleracea L.) leaves which had been inactivated in vitro by treatment with NADH and cyanide, was reactivated by incubation with oxidant systems and measured as FMNH₂-dependent activity. Ferricyanide, a purely chemical oxidant, produced rapid maximal reactivation (100%) which was 90% complete in less than 3 min. Reactivation occurred slowly and less completely (30–75% in 30 or 60 min) when the enzyme was incubated with pure horseradish peroxidase alone, depending on using one or 20 units and time. Addition of glucose and glucose oxidase to generate hydrogen peroxide increased reactivation slightly (10–15%) with 20 units of peroxidase but more (30–50%) with one unit and to 75–90% of ferricyanide values. Adding catalase decreased reactivation by more than half either with or without glucose oxidase. Glucose and glucose oxidase alone did not cause reactivation. Addition of superoxide dismutase increased reactivation from 50–75% of ferricyanide values with one unit of peroxidase alone but had no effect on greater reactivation obtained in the presence of glucose oxidase. The addition of p-cresol and manganese together increased reactivation with one unit of peroxidase and in the presence of glucose oxidase by about double, but omission of manganese had no effect. However, as shown previously, although trivalent manganese was formed, the residual presence of manganous ions inhibited reactivation. Nevertheless, peroxidase systems either alone or with additionally generated hydrogen peroxide can induce substantial reactivation of nitrate reductase in physiologically relevant conditions.Abbreviations EDTA ethylenediaminetetraacetic acid - FMN flavine mononucleotide     

不同动物肠道优势需氧菌对黄豆苷原转化菌株转化能力的影响

摘要: 【目的】探讨不同动物肠道优势需氧菌对黄豆苷原转化菌株转化能力的影响。【方法】有氧条件下,采用稀释涂布法分别从ICR 小鼠、芦花鸡、长白猪和獭兔等4 种健康动物肠道中分离优势需氧菌,将不同动物的优势需氧菌分别与不同类型黄豆苷原转化菌株进行厌氧混合培养,高效液相色谱检测培养液中黄豆苷原的转化情况。【结果】16S rRNA 基因序列分析,结合形态学及相关理化特性分析表明,分离的22 株优势需氧菌分属埃希氏菌属(10 株) 、变形菌属(5 株) 、肠球菌属(4 株) 、芽胞杆菌属(2 株) 和假单胞菌属(     

Iron assimilation in plants: reduction of a ferriphytosiderophore by NADH:nitrate reductase from squash

NADH:nitrate reductase (EC 1.6.6.1) from squash (Cucurbita maxima Duch., cv. Buttercup) can catalyze the reduction of a ferriphytosiderophore from barley (Hordeum vulgare L. cv. Europa). Maximal activity occurs at pH 6, with an apparentK _m andV _max of 76 M and 21 nmol·min^-1·(mg protein)^-1, respectively. The ferriphytosiderophore strongly inhibits nitrate reduction catalyzed by nitrate reductase at the optimal pH for nitrate reduction, i.e. 7.5. On the contrary, nitrate is a poor inhibitor of ferriphytosiderophore reduction catalyzed by nitrate reductase at the optimal pH for this reaction, pH 6.0. Thus, squash has the potential to assimilate the iron from a ferriphytosiderophore synthesized by another plant.     

Inhibition of the growth of Paenibacillus larvae, the causal agent of American foulbrood of honeybees, by selected strains of aerobic spore-forming bacteria isolated from apiarian sources

The bacterium Paenibacillus larvae, the causative agent of American foulbrood disease of honeybee larvae, occurs throughout the world and is found in many beekeeping areas of Argentina. The potential as biocontrol agents of antagonic aerobic spore-forming bacteria isolated from honey samples and other apiarian sources were evaluated. Each isolate was screened against one strain of Paenibacillus larvae (ATCC 9545) by using a perpendicular streak technique. Ten randomly selected bacterial strains from the group that showed the best antagonistic effect to P. larvae ATCC 9545 were selected for further study. These were identified as Bacillus subtilis (m351), B. pumilus (m350), B. licheniformis (m347), B. cereus (mv33), B. cereus (m387), B. cereus (m6c), B. megaterium (m404), Brevibacillus laterosporus (BLAT169), B. laterosporus (BLAT170), and B. laterosporus (BLAT171). The antagonistic strains were tested against 17 P. larvae strains from different geographical origins by means of a spot test in wells. The analysis of variance and posterior comparison of means by Tukey method (P < 0.01) showed that the best antagonists were B. megaterium (m404), B. licheniformis (m347), B. cereus (m6c), B. cereus (mv33), and B. cereus (m387).     

Glutamate dehydrogenase from the aerobic hyperthermophilic archaeon Aeropyrum pernix K1: enzymatic characterization, identification of the encoding gene, and phylogenetic implications

NADP-dependent glutamate dehydrogenase (l-glutamate: NADP oxidoreductase, deaminating, EC 1.4.1.4) from the aerobic hyperthermophilic archaeon Aeropyrum pernix K1 (JCM 9820) was purified to homogeneity for characterization. The enzyme retained its full activity on heating at 95°C for 30 min, and the maximum activity in l-glutamate deamination was obtained around 100°C. The enzyme showed a strict specificity for l-glutamate and NADP on oxidative deamination and for 2-oxoglutarate and NADPH on reductive amination. The K _m values for NADP, l-glutamate, NADPH, 2-oxoglutarate, and ammonia were 0.039, 3.3, 0.022, 1.7, and 83 mM, respectively. On the basis of the N-terminal amino acid sequence, the encoding gene was identified in the A. pernix K1 genome, cloned, and expressed in Escherichia coli. Analysis of the nucleotide sequence revealed an open reading frame of 1257 bp starting with a minor TTG codon and encoding a protein of 418 amino acids with a molecular weight of 46 170. Phylogenetic analysis revealed that the glutamate dehydrogenase from A. pernix K1 clustered with those from aerobic Sulfolobus solfataricus, Sulfolobus shibatae, and anaerobic Pyrobaculum islandicum in Crenarchaeota, and it separated from another cluster of the enzyme from Thermococcales in Euryarchaeota. The branching pattern of the enzymes from A. pernix K1, S. solfataricus, S. shibatae, and Pb. islandicum in the phylogenetic tree coincided with that of 16S rDNAs obtained from the same organisms. Received: April 24, 2000 / Accepted: August 10, 2000     

Analysis of denitrification genes and comparison of nosZ, cnorB and 16S rDNA from culturable denitrifying bacteria in potato cropping systems

Bacterial denitrification in agricultural soils is a major source of nitrous oxide, a potent greenhouse gas. This study examined the culturable bacterial population of denitrifiers in arable field soils in potato (Solanum tuberosum L.) production and denitrification genes (nir, nor and nos) and 16S rDNA in those isolates. Enrichments for culturable denitrifiers yielded 31 diverse isolates that were then analysed for denitrification genes. The nitrous oxide reductase (nosZ) gene was found in all isolates. The majority of isolates ( approximately 90%) contained the cnorB nitric oxide reductase gene, with the remainder containing the qnorB gene. Nitrite reductase genes (nirS and nirK) were amplifiable from most of the isolates, and were segregated between species similar to previously isolated denitrifiers. Isolated strains were preliminarily identified using fatty acid methyl ester analysis and further identified using 16S rDNA sequencing. The majority of isolates (21) were classified as Pseudomonas sp., with smaller groups of isolates being most similar to Bosea spp. (4), Achromobacter spp. (4) and two isolates closely related to Sinorhizobium/Ensifer spp. Phylogenetic trees were compared among nosZ, cnorB and 16S rDNA genes for a subset of Pseudomonas strains. The trees were mostly congruent, but some Pseudomonas sp. isolates grouped differently depending on the gene analysed, indicating potential horizontal gene transfer of denitrification genes. Although Bosea spp. are known denitrifiers, to the best of our knowledge this is the first report of isolation and sequencing of denitrification genes from this bacterial genus.     

Metabolic activators of spinach leaf nitrate reductase: Effects on enzymatic activity and dephosphorylation by endogenous phosphatases

Nitrate reductase (NR; EC 1.6.6.1) in spinach (Spinacia oleracea L.) leaves was inactivated in the dark and reactivated by light in vivo. When extracted from dark leaves, NR activity was lower and more strongly inhibited by Mg²⁺ relative to the enzyme extracted from leaves harvested in the light. When dark extracts were desalted at pH 6.5 and preincubated at 25° C prior to assay, enzyme activity (assayed either in the presence or absence of Mg²⁺) remained essentially constant, i.e. there was no spontaneous reactivation in vitro. However, addition of certain metabolites resulted in a time- and concentration-dependent activation of NR in vitro. Effective activators included inorganic phosphate (Pi), 5-AMP, and certain of its derivatives such as FAD and pyridine nucleotides (both oxidized and reduced forms). All of the activators increased NR activity as assayed in the absence of Mg²⁺, whereas some activators (e.g. Pi, 5-AMP and FAD) also reduced Mg²⁺ inhibition. The reduction of Mg²⁺ inhibition was also time-dependent and was almost completely prevented by a combination of okadaic acid plus KF, suggesting the involvement of dephosphorylation catalyzed by endogenous phosphatase(s). In contrast, the activation of NR (assayed minus Mg²⁺) was relatively insensitive to phosphatase inhibitors, indicating a different mechanism was involved. Compounds that were not effective activators of NR included sulfate, ribose-5-phosphate, adenosine 5-monosulfate, coenzyme A, ADP and ATP. We postulate that NR can exist in at least two states that differ in enzymatic activity. The activators appear to interact with the NR molecule at a site distinct from the NADH active site, and induce a slow conformational change (hysteresis) that increases NR activity (assayed in the absence of Mg²⁺). Possibly as a result of the conformational change caused by certain activators, the regulatory phospho-seryl groups are more readily dephosphorylated by endogenous phosphatases, thereby reducing sensitivity to Mg²⁺ inhibition. Preliminary results suggest that light/dark transitions in vivo may alter the distribution of NR molecules between the low- and high-activity forms.Abbreviations AP₅A P¹, P⁵-di(adenosine-5)pentaphosphate - DTT dithiothreitol - Mops 3-(N-morpholino)propanesulfonic acid - NR NADH:nitrate reductase - NRA nitrate reductase activity Cooperative investigations of the U.S. Department of Agriculture, Agricultural Research Service, and the North Carolina Agricultural Research Service, Raleigh, NC 27695-7643. This work was also supported in part by grants from the U.S. Department of Energy (Grant DE-AIO5-91 ER 20031) and USDA-NRI (Grant 93-373-5-9231). The authors thank Dr. W.M. Kaiser (Lehrstuhl Botanik I der Universität, Würzburg, Germany) for discussions and Dr. C. Lillo (Rogaland University Center, Stavanger, Norway) for sharing results prior to publication.     

Fatty acids, unusual glycophospholipids and DNA analyses of thermophilic bacteria isolated from hot springs

The composition of fatty acids in 12 strains of the genera Thermus, Meiothermus, Geobacillus and Alicyclobacillus was analyzed by gas chromatography–mass spectrometry. Major FAs found in the profiles included i-15:0, i-17:0, ai-15:0, i-16:0, 16:0, ai-17:0, together with some minor components. Branched FAs were predominant, forming more than 80% of all FAs measured. Fast atom bombardment-mass spectrometry was used for analysis of unusual glycophospholipids, i.e., acylglycosylcardiolipins from genera Geobacillus and Alicyclobacillus and 1-(hydroxy(2-(O-acylglycosyl-oxy)hexadecyloxy)phosphoryloxy) hexadecan-2-yl esters of C15–C17 acids from genera Thermus and Meiothermus. Cloning and preliminary sequence analysis of 16S rDNA showed that these isolates belong to the genera Thermus, Meiothermus, Geobacillus and Alicyclobacillus.     

Morphology, molecular characteristics and prevalence of a Cystosporogenes species (Microsporidia) isolated from Agrilus anxius (Coleoptera: Buprestidae)

A microsporidium was isolated from the bronze birch borer, Agrilus anxius Gory (Coleoptera: Buprestidae), collected near Sudbury and Sault Ste Marie, Canada. Light and electron microscopic investigations showed that gross pathology and ultrastructure of the investigated Cystosporogenes species was similar to those characterized and described for other Cystosporogenes species. Small subunit rRNA gene sequence data and comparative phylogenetic analysis confirmed that the microsporidian species from A. anxius is most closely related to the genus Cystosporogenes clade of microsporidia. Infection average in the Sudbury and Sault Ste Marie beetle populations was >80% and relatively stable in 2006-2007 but declined in 2008. Field prevalence of the A. anxius isolate, mechanisms that may potentially be involved in its horizontal (autoinfection) and vertical (transovarial) transmission, and disease dynamics are discussed. The congeneric relationship between Agrilus planipennis and A. anxius makes it imperative to study the virulence of this Cystosporogenes species in A. planipennis.     

Morphological and molecular studies of a microsporidium (Nosema sp.) isolated from the thee spot grass yellow butterfly, Eurema blanda arsakia (Lepidoptera: Pieridae)

A microsporidium possessing molecular and morphological characteristics of the genus Nosema was isolated from larvae of the thee-spot grass yellow butterfly, Eurema blanda arsakia. The complete rRNA gene sequences of the E. blanda isolate contained 4,428 base pairs (GenBank Accession No. EU338534). The organization of the rRNA genes is LSU rRNA-ITS-SSU rRNA-IGS-5S, which corresponds with that of Nosema species closely related to Nosema bombycis. Phylogenetic analysis based on rRNA gene sequences show that this isolate is closely related to Nosema bombycis, Nosema plutellae, Nosema spodopterae, and Nosema antheraeae. The ultrastructure of all developmental stages of this microsporidium confirmed its placement in the genus Nosema. The isolate was successfully propagated in cell lines IPLB-LD652Y (Lymantria dispar) and NTU-LY (Lymantria xylina) and, in the in vitro system, it was frequently found to develop in the nuclei of the host cells, a circumstance that seldom occurs in other Nosema species. An extra-cellular vegetative stage of this microsporidium was also observed in the culture medium after 14 days of infection. The ECMDFs might be released from disrupted host cells.