Biological nitrogen fixation and phosphate solubilization by bacteria isolated from tropical soils

Introduction

In addition to fixing atmospheric nitrogen, some bacterial isolates can also solubilize insoluble phosphates, further contributing to plant growth.

Aims

The objectives of this study were the following: isolate, select, and identify nodulating bacteria in the cowpea that are efficient not only in biological nitrogen fixation (BNF) but also in the solubilization of insoluble inorganic phosphates; identify and quantify the organic acids produced; and establish the relationship between those acids and the solubilizing capacity.

Methods

The bacteria were captured from two soils containing high concentrations of insoluble phosphorus from the cities of Lavras and Patos de Minas, using the cowpea [Vigna unguiculata (L.) Walp.] as bait. We obtained 78 strains, which were characterized according to their cultural attributes in culture medium 79 with the strains UFLA 03-84, INPA 03-11B, and BR3267 (approved by the Ministry of Livestock and Supply Agriculture—MAPA, as inoculants for the cowpea) and Burkholderia cepacia (LMG1222^T), which was used as a positive control for phosphate solubilization. Strains that were selected for their efficiency in both processes were identified by 16S rDNA sequence analysis. We evaluated the symbiotic efficiency (BNF) in a greenhouse and the solubilization efficiency of CaHPO₄, Al(H₂PO₄)₃, and FePO₄.2H₂O in solid and liquid GELP media. Strains that excelled at the solubilization of these phosphate sources were also evaluated for the production of the following organic acids: oxalic, citric, gluconic, lactic, succinic, and propionic.

Results

The presence of Acinetobacter, Bacillus, Firmicutes, Microbacterium, Paenibacillus, and Rhizobium was detected by 16S rDNA sequencing and analysis. Bacterial strains obtained from cowpea nodules varied greatly in the efficiency of their BNF and phosphate solubilization processes, especially in the strains UFLA 03-09, UFLA 03-10, UFLA 03-12, and UFLA 03-13, which were more efficient in both processes. More strains were able to solubilize insoluble inorganic calcium and iron phosphates in liquid medium than in solid medium. The production of organic acids was related to the solubilization of CaHPO₄ and FePO₄.2H₂O for some strains, and the type and concentration of the acid influenced this process.

Conclusions

These are the first results obtained with bacterial isolates from tropical soils in which the production of organic acids was detected and quantified to examine the solubilization of insoluble inorganic phosphates.     

Seedling growth and soil nutrient availability in exotic and native tree species: implications for afforestation in southern China

Background and aims

The relationship between tree species and soil nutrient availability is critical for evaluating plantation succession and promoting forest restoration. This study was conducted to evaluate the impact of exotic and native tress species on soil nutrient availability.

Methods

Four exotic species (Eucalyptus urophylla, E. tereticornis, Acaia auriculaeformis, A. mangium) and four native species (Castanopsis fissa, Schima superba, C. hystrix, Michelia macclurei) were planted and grown for one-year. Soil solution (DOC, DON, NH₄?N, NO₃?N) was sampled and analyzed during the study. After the experiment, soil properties were determined, and plant tissues were analyzed.

Results

DOC levels were greater in soils with trees planted than controls without trees. Compared to native species, exotic species had much faster growth rates and greatly reduced DON and NO₃?N concentrations. Exotic species always had less P concentrations in leaves and stems than native species. Furthermore, N-fixing A. auriculaeformis led to greater soil available P compared to other species.

Conclusions

Based on these findings, we provide some recommendations for afforestation practice. This study highlights that a better understanding of the pros and cons of exotic species would be beneficial to advance afforestation in China and the world.     

Aluminum tolerance of wheat does not induce changes in dominant bacterial community composition or abundance in an acidic soil

Aims

Aluminum-tolerant wheat plants often produce more root exudates such as malate and phosphate than aluminum-sensitive ones under aluminum (Al) stress, which provides environmental differences for microorganism growth in their rhizosphere soils. This study investigated whether soil bacterial community composition and abundance can be affected by wheat plants with different Al tolerance.

Methods

Two wheat varieties, Atlas 66 (Al-tolerant) and Scout 66 (Al-sensitive), were grown for 60 days in acidic soils amended with or without CaCO₃. Plant growth, soil pH, exchangeable Al content, bacterial community composition and abundance were investigated.

Results

Atlas 66 showed better growth and lower rhizosphere soil pH than Scout 66 irrespective of CaCO₃ amendment or not, while there was no significant difference in the exchangeable Al content of rhizosphere soil between the two wheat lines. The dominant bacterial community composition and abundance in rhizosphere soils did not differ between Atlas 66 and Scout 66, although the bacterial abundance in rhizosphere soil of both wheat lines was significantly higher than that in bulk soil. Sphingobacteriales, Clostridiales, Burkholderiales and Acidobacteriales were the dominant bacteria phylotypes.

Conclusions

The difference in wheat Al tolerance does not induce the changes in the dominant bacterial community composition or abundance in the rhizosphere soils.     

Elevated CO2 improves root growth and cadmium accumulation in the hyperaccumulator Sedum alfredii

Aims

This study examined the effect of elevated CO₂ on plant growth, root morphology and Cd accumulation in S. alfredii, and assessed the possibility of using elevated CO₂ as fertilizer to enhance phytoremediation efficiency of Cd-contaminated soil by S. alfredii.

Methods

Both soil pot culture and hydroponic experiments were carried out to characterize plant biomass, root morphological parameters, and cadmium uptake in S. alfredii grown under ambient (350 μL L^?1) or elevated (800 μL L^?1) CO₂.

Results

Elevated CO₂ prompted the growth of S. alfredii, shoot and root biomass were increased by 24.6–36.7% and 35.0–52.1%, respectively, as compared with plants grown in ambient CO₂. After 10 days growth in medium containing 50 μM Cd under elevated CO₂, the development of lateral roots and root hairs were stimulated, additionally, root length, surface area, root volume and tip number were increased significantly, especially for the finest diameter roots. The total Cd uptake per pot was significantly greater under elevated CO₂ than under ambient CO₂. After 60 d growth, Cd phytoextraction efficiency was increased significantly in the elevated CO₂ treatment.

Conclusions

Results suggested that the use of elevated CO₂ may be a useful way to improve phytoremediation efficiency of Cd-contaminated soil by S. alfredii.     

Isolation of ACC deaminase-producing habitat-adapted symbiotic bacteria associated with halophyte Limonium sinense (Girard) Kuntze and evaluating their plant growth-promoting activity under salt stress

Background and aims

Many plant growth-promoting endophytes (PGPE) possessing 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity can reduce the level of stress ethylene and assist their host plants cope with various biotic and abiotic stresses. However, information about the endophytic bacteria colonizing in the coastal halophytes is still very scarce. This study aims at isolating efficient ACC deaminase-producing plant growth-promoting (PGP) bacterial strains from the inner tissues of a traditional Chinese folk medicine Limonium sinense (Girard) Kuntze, a halophyte which has high economic and medicinal values grown in the coastal saline soils. Their PGP activity and effects on host seed germination and seedling growth under salinity stress were also evaluated.

Methods

A total of 126 isolates were obtained from the surface sterilized roots, stems and leaves of L. sinense (Girard) Kuntze. They were initially selected for their ability to produce ACC deaminase as well as other PGP properties such as production of indole-3-acetic acid (IAA), N₂-fixation, and phosphate-solubilizing activities and subsequently identified by the 16S rRNA gene sequencing. For selected strains, seed germination, seedling growth, and flavonoids production in axenically growth L. sinense (Girard) Kuntze seedlings at different NaCl concentrations (0–500 mM) were quantified.

Results

Thirteen isolates possessing ACC deaminase activity were obtained. The 16S rRNA gene sequencing analysis showed them to belong to eight genera: Bacillus, Pseudomonas, Klebsiella, Serratia, Arthrobacter, Streptomyces, Isoptericola, and Microbacterium. Inoculation with four of the selected ACC deaminase-producing strains not only stimulated the growth of the host plant but also influenced the flavonoids accumulation. All four strains could colonize and can be re-isolated from the host plant interior tissues.

Conclusions

These results demonstrate that ACC deaminase-producing habitat-adapted symbiotic bacteria isolated from halophyte could enhance plant growth under saline stress conditions and the PGPE strains could be appropriate as bioinoculants to enhance soil fertility and protect the plants against salt stress.     

Canalization effect in the coagulation cascade and the interindividual variability of oral anticoagulant response. a simulation Study

Background

The aim of the study was to find a simple intravenous glucose tolerance test (IVGTT) that can be used to estimate insulin sensitivity.

Methods

In 20 healthy volunteers aged between 18 and 51 years (mean, 28) comparisons were made between kinetic parameters derived from a 12-sample, 75-min IVGTT and the M_bw (glucose uptake) obtained during a hyperinsulinemic euglycemic glucose clamp. Plasma glucose was used to calculate the volume of distribution (V _d) and the clearance (CL) of the injected glucose bolus. The plasma insulin response was quantified by the area under the curve (AUC_ins). Uptake of glucose during the clamp was corrected for body weight (M_bw).

Results

There was a 7-fold variation in M_bw. Algorithms based on the slope of the glucose-elimination curve (CL/V _d) in combination with AUC_ins obtained during the IVGTT showed statistically significant correlations with M_bw, the linearity being r² = 0.63-0.83. The best algorithms were associated with a 25-75^th prediction error ranging from -10% to +10%. Sampling could be shortened to 30-40 min without loss of linearity or precision.

Conclusion

Simple measures of glucose and insulin kinetics during an IVGTT can predict between 2/3 and 4/5 of the insulin sensitivity.     

Genomic analysis of human lung fibroblasts exposed to vanadium pentoxide to identify candidate genes for occupational bronchitis

Background

Exposure to vanadium pentoxide (V₂O₅) is a cause of occupational bronchitis. We evaluated gene expression profiles in cultured human lung fibroblasts exposed to V₂O₅ in vitro in order to identify candidate genes that could play a role in inflammation, fibrosis, and repair during the pathogenesis of V₂O₅-induced bronchitis.

Methods

Normal human lung fibroblasts were exposed to V₂O₅ in a time course experiment. Gene expression was measured at various time points over a 24 hr period using the Affymetrix Human Genome U133A 2.0 Array. Selected genes that were significantly changed in the microarray experiment were validated by RT-PCR.

Results

V₂O₅ altered more than 1,400 genes, of which ~300 were induced while >1,100 genes were suppressed. Gene ontology categories (GO) categories unique to induced genes included inflammatory response and immune response, while GO catogories unique to suppressed genes included ubiquitin cycle and cell cycle. A dozen genes were validated by RT-PCR, including growth factors (HBEGF, VEGF, CTGF), chemokines (IL8, CXCL9, CXCL10), oxidative stress response genes (SOD2, PIPOX, OXR1), and DNA-binding proteins (GAS1, STAT1).

Conclusion

Our study identified a variety of genes that could play pivotal roles in inflammation, fibrosis and repair during V₂O₅-induced bronchitis. The induction of genes that mediate inflammation and immune responses, as well as suppression of genes involved in growth arrest appear to be important to the lung fibrotic reaction to V₂O₅.     

5-(4-alkyl-1,2,3-triazol-1-yl)methyl derivatives of 2′-deoxyuridine as inhibitors of viral and bacterial growth

A series of 5-(4-alkyl-1,2,3-triazol-1-yl)methyl derivatives of 2′-deoxyuridine have been synthesized by the interaction of 3′,5′-diacetyl-5-azidomethyl-2′-deoxyuridine with the corresponding 1-alkynes in a biphasic methylene chloride—water system catalyzed by Cu(I) followed by the deblocking with a water-alcohol ammonia solution. A low cytotoxicity of the compounds in Vero, Jurkat, and A549 cell cultures has been shown. The 2′-deoxyuridine derivatives exhibited an antiherpetic activity in vitro toward two laboratory strains of human herpes simplex virus type 1 (HSV-1): acyclovir-sensitive (HSV-1/L₂) and acyclovir-resistant (HSV-1/L₂R_ACV). They also inhibited the growth of some bacteria (Mycobacterium smegmatis, Staphylococcus aureus, Micrococcus luteus, and Leuconostoc mesenteroides) and yeasts Saccharomyces cerevisiae in vitro.     

Biogenesis of mitochondria 19 the effects of unsaturated fatty acid depletion on the lipid composition and energy metabolism of a fatty acid desaturase mutant of

1. The lipid composition of a mutant ofSaccharomyces cerevisiae which cannot synthesize unsaturated fatty acid (UFA) can be extensively manipulated by growing the organism in the presence of added fatty acids.
2. Growth of the mutant is supported by a wide range of unsaturated fatty acids including oleic, palmitoleic, petroselenic, 11-eicosaenoic, ricinoleic, arachidonic, clupanodonic, linoleic and linolenic acids; 9- and 10-hydroxystearic acids support growth less effectively, but erucic, nervonic, elaidic and saturated fatty acids (C_8∶0?C_20∶0)^* are ineffective. All the fatty acids which support growth are incorporated into cell lipids, apparently without further metabolism.
3. The effects of altered lipid composition on the energy metabolism of yeast cells were investigated. Cells containing less than approximately 20% of their fatty acids as UFA cannot grow on non-fermentable substrates, and their growth on glucose is restricted to that which can be supported by fermentation alone.
4. UFA-depleted cells contain mitochondria which are apparently normal in morphology, furthermore they have normal levels of cytochromesa+a ₃,b,c ₁ andc and respire at normal rates. This suggests that the lesion in energy metabolism produced by UFA-depletion may be the loss of the ability of the mitochondria to couple respiration to phosphorylation.
5. UFA-depleted cells incorporate added UFA into their cell lipids and subsequently regain the ability to grow on non-fermentable substrates, showing that the lesion in energy metabolism is fully reversible.

     

Soil microbial residue dynamics after 3-year elevated O3 exposure are plant species-specific

Background and aims

Rising ozone (O₃) concentrations poses a great threat to crop growth and ecosystem carbon storage, but the underlying mechanism remains unclear. Identifying the impact of elevated O₃ on soil microbial residues may advance our knowledge of microbial-mediated soil organic matter (SOM) turnover. In this paper, we aimed to investigate the effects of elevated O₃ on the accumulation of amino sugars in the soil of the two wheat cultivars (Tritcium aestivum L.) with different ozone-tolerances.

Methods

Using the O₃-Free Air Concentration Enrichment technique, we investigated the response of amino sugars to elevated O₃ in a soil planted with two wheat cultivars of different ozone-tolerance [ozone-sensitive Yannong 19 (Y19) and ozone-tolerant Yangmai 16 (Y16)]. This study was conducted during the wheat growing season (jointing stage and ripening stage) of 2010 after exposure to elevated O₃ for 3 years. Soil amino sugars were measured by gas chromatography technique.

Results

After exposure to elevated O₃, the contents of total amino sugars decreased at the wheat jointing stage, and increased at the wheat ripening stage in the Y16 cultivar. In contrast, no significant effect of elevated O₃ was found in the Y19 cultivar. The Glucosamine/Galactosamine and fungal carbon/bacterial carbon ratios decreased under elevated O₃. The findings indicated that elevated O₃ altered the microbial process of SOM turnover and bacteria contributed more to SOM cycling than fungi under elevated O₃ conditions.

Conclusions

The effect of elevated O₃ on the SOM turnover was wheat cultivar-specific. Thus, belowground processes of SOM turnover should be considered when selecting a tolerant wheat cultivar under elevated O₃ regimes from a view of long-term ecosystem stability.     

Net root growth and nutrient acquisition in response to predicted climate change in two contrasting heathland species

Background and aims

Accurate predictions of nutrient acquisition by plant roots and mycorrhizas are critical in modelling plant responses to climate change.

Methods

We conducted a field experiment with the aim to investigate root nutrient uptake in a future climate and studied root production by ingrowth cores, mycorrhizal colonization, and fine root N and P uptake by root assay of Deschampsia flexuosa and Calluna vulgaris.

Results

Net root growth increased under elevated CO₂, warming and drought, with additive effects among the factors. Arbuscular mycorrhizal colonization increased in response to elevated CO₂, while ericoid mycorrhizal colonization was unchanged. The uptake of N and P was not increased proportionally with root growth after 5 years of treatment.

Conclusions

While aboveground biomass was unchanged, the root growth was increased under elevated CO₂. The results suggest that plant production may be limited by N (but not P) when exposed to elevated CO₂. The species-specific response to the treatments suggests different sensitivity to global change factors, which could result in changed plant competitive interactions and belowground nutrient pool sizes in response to future climate change.     

Increased photosynthetic capacity in response to nitrate is correlated with enhanced cytokinin levels in rice cultivar with high responsiveness to nitrogen nutrients

Background and aims

Ammonium (NH₄ ⁺) is the preferred nitrogen nutrient over nitrate (NO₃ ^–) in Oryza sativa L. (rice), but photosynthetic capacity is enhanced by partial NO₃ ^– nutrition (PNN). The role of cytokinin in the effects of PNN on photosynthetic capacity is unknown.

Methods

We investigated effects of PNN on six cytokinin fractions in roots, xylem sap, and leaves and on the expression of eight cytokinin synthesis genes in the roots of Nanguang and Elio rice cultivars. The effect of exogenous cytokinin (6-BA) on leaf growth and photosynthetic activity was examined.

Results

Cell expansion and CO₂ assimilation in the first fully expanded leaf were enhanced by PNN in Nanguang but not in Elio. The concentrations of cytokinins in roots, xylem sap, and leaves of Nanguang increased approximately 25–34 % with PNN compared with sole NH₄ ⁺, but no difference was observed in Elio. Exogenous 6-BA counteracted the effects of sole NH₄ ⁺ on leaf growth and photosynthetic activity in both cultivars. OsIPT3 was the key NO₃ ^–-responsive cytokinin synthesis gene in cv. Nanguang.

Conclusions

High NO₃ ^– responsiveness is associated with increased cytokinin synthesis and transport from the root to the leaf and is strongly related to a higher photosynthetic capacity in cv. Nanguang.     

In vitro antimicrobial activity of light-activated phthalocyanines

Background

Photodynamic antimicrobial therapy (PACT) is proposed as a topical, non-invasive approach suitable for treatment of locally occurring infection. Research of photosensitizers, (PS) as well as their development, is aimed at finding effective antimicrobial substances which would have a broad-spectrum potency. The aim of this paper is to evaluate the antimicrobial effect of phthalocyanine (Pc) derivatives.

Methods

Fifteen different Pc compounds were investigated. Their photokilling activity was tested on Staphylococcus aureus, Escherichia coli and Candida albicans. After treating of microbial cells with Pc at the concentrations: 1 mg/l, 2 mg/l, 4 mg/l, 8 mg/l for 30 minutes, the cultures were irradiated with low-power laser light at a wavelength of 670 nm (20 J/cm², 40 J/cm²). The effectiveness of photoinactivation was evaluated based on the decrease in number (log₁₀) of viable bacteria.

Results

Eight Pc compounds tested showed antibacterial effects against S. aureus, but only four were effective against E. coli and two against C. albicans. The most effective photosensitizers were amphiphilic sulphonated zinc Pc compounds [(3-diethylammonium)-propylsulphonamide citrate (Pc3) and cationic tetramethylenepyridinium chloride of hydroxyaluminum Pc (Pc7)].

Conclusions

The most efficient phthalocyanines (Pc3, Pc7) cause a significant decrease in viable counts of all tested microbes.     

Cefotaxime prevents microbial contamination and improves microspore embryogenesis in wheat and triticale

Key message

Cefotaxime (100 mg/l) mitigate occasional gram negative bacterial contamination in wheat and triticale microspore culture and most importantly it increases cell growth and green plant production.

Abstract

Isolated microspore culture is a promising option to rapidly fix the product of meiotic recombination of F₁ hybrids, in the process of varietal development. Clean culture and high embryogenesis rate are essential to commercial triticale and wheat microspore cultures. So, this study investigated (1) contaminants from isolated microspores cultures, (2) two antibiotics to control bacterial growth, and (3) the contribution of antibiotics to increased microspore-derived embryo-like structures (ELS), green and albino plants. Five species of bacteria were identified in contaminated cultures (Erwinia aphidicola, Pantoea agglomerans, Pseudomonas sp., Staphylococcus epidermis and Staphylococcus warneri) using fatty acid analysis and 16S ribosomal RNA sequences analysis, and yeast. Antibacterial susceptibility test using Cefotaxime and Vancomycin resulted in strong inhibition of 24 bacterial isolates, using Cefotaxime at 100 mg/l, but not Pseudomonas sp. Other antibiotic treatments inhibited bacterial growth at least partially. Microspore induction medium supplemented with the same antibiotics treatments resulted in successful microspore embryogenesis and green plant production. Antibiotic treatments were first tested in triticale and then validated in wheat cultivars AC Carberry and AC Andrew. Induction medium supplemented with Cefotaxime at 50 and 100 mg/l substantially increased the formation of ELS and green plants in triticale and wheat, respectively. Incidentally, it also affected the occurrence of albinism in all genotypes. Our results demonstrated dual purpose of Cefotaxime for isolated microspore culture, most importantly it increases cell growth and success of microspore cultures in triticale and wheat genotypes, but would also prevent accidental loss of cultures with most common bacterial contaminants.     

Soil nitrogen balance resulting from N fixation and rhizodeposition by the symbiotic association Anthyllis vulneraria/Mesorhizobium metallidurans grown in highly polluted Zn,Pb and Cd mine tailings

Background and aims

The association of the legume Anthyllis vulneraria and the grass Festuca arvernensis, was found to be very efficient for the phytostabilisation of highly multi-metal contaminated mine tailings. Our objective was to quantify the contribution of Anthyllis inoculated with its symbiotic bacteria Mesorhizobium metallidurans to the soil N pool and to test whether a starter nitrogen fertilization may improve symbiotic nitrogen fixation and the growth of Festuca.

Methods

Plants of Festuca and of Anthyllis inoculated with M. metallidurans were grown separately during eight months in pots filled with mine contaminated soil. Estimation of the N fluxes was realized using ¹⁵?N isotopic methods.

Results

Starter N fertilization (28 kg N ha^?1) improved symbiotic N₂ fixation and the growth of both species. Belowground N balance (N rhizodeposition – soil N uptake) of the non-fertilized Anthyllis at maturity was negative (?30.6 kg N ha^?1). However, the amount of N derived from fixation, including above- and belowground parts, was 78.6 kg N ha^?1, demonstrating the ability of this symbiotic association to improve soil N content after senescence.

Conclusions

i) soil N enrichment by the N₂-fixing symbiotic association occurs after plant senescence, when decaying leaves and shoots are incorporated into the soil; ii) application of a starter fertilization is an efficient solution to improve phytostabilisation of highly contaminated sites.     

Adaptive capabilities of microorganisms of salt lakes of the Altai Region under conditions of early Mars

Adaptive capacity of bacteria and archaea from salt lakes of the Altai Region are discussed. It is established that halophilic archaea (genus Halorubrum) and halotolerant bacteria (genus Halomonas) grow in a wide range of pH and mineralization (in the presence of Cl^?, SO ₄ ^2? , ClO ₄ ^? , Mg²⁺) and survive at low temperatures with a minor decrease in viability.     

Synthesis of Pyrrolo[2′,3′:4,5]Furo[3,2-c]Pyridine-2-Carboxylic Acids

1H-Pyrrolo[2′,3′:4,5]furo[3,2-c]pyridine-2-carboxylic acid (6a) and its 1-methyl (6b) and 1-benzyl (6c) derivatives were synthesized. 3-(5-Methoxycarbonyl-4H-furo[3,2-b]-pyrrole-2-yl)propenoic acid (1) was converted to the corresponding azide 2, which in turn was cyclized to give 3 by heating in diphenylether. The pyridone 3 obtained was aromatized with phosphorus oxychloride, then reduced with zinc in acetic acid to give methyl 1H-pyrrolo[2′,3′:4,5]furo[3,2-c]pyridine-2-carboxylate (5), which by hydrolysis gave the corresponding carboxylic acid 6a.     

D-amino acids in the cell pool of bacteria

About 30 different bacterial species were tested for the possible presence of freed-amino acids in their cell pool. Gram-positive bacteria particularly the species of the genusBacillus have a fairly large pool of freely extractabled-amino acids. Varied quantities of freed-amino acids were detected inBacillus subtilis B₃,Bacillus subtilis Marburg,Bacillus licheniformis, Bacillus brevis, Bacillus stearothermophilus, Lactobacillus fermenti, Lactobacillus delbrueckii, Staphylococcus aureus andClostridium acetobutylicum. The individual components ofd-amino acids were identified in 5Bacillus species referred to above,d-alanine is the major component; the otherd-amino acids identified are aspartic acid, glutamic acid, histidine, leucines, proline, serine and tyrosine. Thed-amino acid pool size inBacillus subtilis B₃ varies with different culture conditions. The pool size is maximum when growth temperature is 30°C and it fluctuates with change in pH of the medium. The maximum quantity ofd-amino acids could be recovered when the culture was at mid log phase. O₂ supply to the medium has little effect ond-amino acid pool size. The starvation of cells leads to depletion of thed-amino acid pool which is exhausted almost completely within 4 hours by incubation in nutrient-free medium.     

Soil N mineralization, nitrification and dynamic changes in abundance of ammonia-oxidizing bacteria and archaea along a 2000 year chronosequence of rice cultivation

Background and Aims

Soil mineralization, nitrification, and dynamic changes in abundance of ammonia-oxidizing bacteria (AOB) and archaea (AOA) were studied to validate our hypothesis that soil mineralization and nitrification decreased along the chronosequence of rice cultivation.

Methods

Paddy soils with a 300, 700 and 2000-year cultivation history (P300, P700 and P2000) were selected to study net mineralization and nitrification processes. Dynamic abundance of AOB and AOA was estimated by quantifying their respective amoA gene copies.

Results

The net mineralization rate was higher for P300 than P700 and P2000. Potential nitrification (N _p ) and average nitrification rates (V _a ) were similar for P300 and P700 soils, but the simulated potential nitrification rate (V _p ) and nitrification rate (k₁) was 72 % and 88 % higher for P300 than P700, respectively. V _a was about 70 % lower than for P2000 than P300 and P700. AOB amoA gene copies were higher for P300 than P700 and P2000, whereas AOA abundance did not show significant differences. AOB abundance showed a positive response to NH₄ supply but AOA did not.

Conclusions

Both N mineralization and nitrification were depressed with increased cultivation time. Archaea responded to mineralization positively rather than nitrification, which suggested that readily mineralized organic matter may play an important role in AOA.     

Sequence Analysis of Leuconostoc mesenteroides Bacteriophage Φ1-A4 Isolated from an Industrial Vegetable Fermentation

Vegetable fermentations rely on the proper succession of a variety of lactic acid bacteria (LAB). Leuconostoc mesenteroides initiates fermentation. As fermentation proceeds, L. mesenteroides dies off and other LAB complete the fermentation. Phages infecting L. mesenteroides may significantly influence the die-off of L. mesenteroides. However, no L. mesenteroides phages have been previously genetically characterized. Knowledge of more phage genome sequences may provide new insights into phage genomics, phage evolution, and phage-host interactions. We have determined the complete genome sequence of L. mesenteroides phage Φ1-A4, isolated from an industrial sauerkraut fermentation. The phage possesses a linear, double-stranded DNA genome consisting of 29,508 bp with a G+C content of 36%. Fifty open reading frames (ORFs) were predicted. Putative functions were assigned to 26 ORFs (52%), including 5 ORFs of structural proteins. The phage genome was modularly organized, containing DNA replication, DNA-packaging, head and tail morphogenesis, cell lysis, and DNA regulation/modification modules. In silico analyses showed that Φ1-A4 is a unique lytic phage with a large-scale genome inversion (∼30% of the genome). The genome inversion encompassed the lysis module, part of the structural protein module, and a cos site. The endolysin gene was flanked by two holin genes. The tail morphogenesis module was interspersed with cell lysis genes and other genes with unknown functions. The predicted amino acid sequences of the phage proteins showed little similarity to other phages, but functional analyses showed that Φ1-A4 clusters with several Lactococcus phages. To our knowledge, Φ1-A4 is the first genetically characterized L. mesenteroides phage.Bacteriophages are the most abundant biological entities (estimated to be on the order of ≥10³¹) on the planet (9, 18). Phages are ubiquitous in nature and can influence the microbial ecology and genetics of bacteria. Because of their small (usually <60 kb) genomes, phages can provide an excellent model system for studying many biological processes, including DNA replication and genetic evolution. Despite this, many phages remain uncharacterized. Very little is known about phage diversity and phage-host interactions owing to the small number of sequenced phages. Furthermore, the existing phage sequence database is highly biased toward a limited spectrum of phage hosts, namely, Enterobacteriaceae, Bacillus, Staphylococcus, Pseudomonas, Vibrio cholerae, Lactococcus, Streptococcus thermophilus, and S. pyogenes. The majority of host species for sequenced phages are either pathogenic or dairy-related bacteria. Most of the newly sequenced phage genes have no assigned functions or matches in the GenBank database (7).Vegetable fermentations rely on a variety of lactic acid bacteria (LAB). The proper succession of LAB directly determines the quality and safety of the final fermentation products. Leuconostoc mesenteroides initiates most vegetable fermentations. It converts the sugars in vegetables (primarily glucose and fructose) to lactic acid, acetic acid, ethanol, CO₂, and other flavor compounds (22, 58, 59, 60, 61). Acid production lowers the pH of fermenting vegetables and inhibits the growth of many microorganisms, including pathogens. CO₂ production promotes the establishment of an anaerobic environment which favors the growth of other LAB. The metabolites produced by L. mesenteroides largely determine the flavor characteristics of the final products. As fermentation proceeds, L. mesenteroides rapidly dies off. Other LAB, including Lactobacillus plantarum, take over and complete the fermentation.It has been a widely held view that the disappearance of L. mesenteroides and the subsequent bacterial succession in sauerkraut fermentations are due to the inhibitory effect of acids that accumulate during fermentation (54, 61). Little is known about other factors that may play a role in bacterial succession. Recent studies have shown that phages are present in the vegetable fermentations (4, 47, 48, 74, 75). Because of the rapid lytic cycle of these phages, they may significantly impact starter cultures and bacterial succession in vegetable fermentations (56). Phages active against L. mesenteroides have been isolated and characterized (48); however, genome sequences have not been reported.L. mesenteroides phage 1-A4 (designated Φ1-A4) is of particular interest. Φ1-A4 is a lytic phage that was repeatedly isolated during the initial stages of a commercial sauerkraut fermentation. As a result, Φ1-A4 may significantly influence the survival of L. mesenteroides and flavor development during sauerkraut fermentation. It was found that Φ1-A4 infects at least three different strains of L. mesenteroides (48), and therefore it may also promote genetic exchange and genetic diversity in microbial communities (34).The objectives of this study were to determine and analyze the complete genome sequence of Φ1-A4, to experimentally identify the structural protein genes, and to compare the genome organization with that of related phages. To our knowledge, this study represents the first complete genomic and molecular characterization of Leuconostoc phage. The results from this study may provide new insights into our understanding of phage genetics. This study may aid the development of phage control technologies in vegetable and other fermentations that are susceptible to phage attack.