Nitrogen-Fixing Nodules with Ensifer adhaerens Harboring Rhizobium tropici Symbiotic Plasmids

Ensifer adhaerens is a soil bacterium that attaches to other bacteria and may cause lysis of these other bacteria. Based on the sequence of its small-subunit rRNA gene, E. adhaerens is related to Sinorhizobium spp. E. adhaerens ATCC 33499 did not nodulate Phaseolus vulgaris (bean) or Leucaena leucocephala, but with symbiotic plasmids from Rhizobium tropici CFN299 it formed nitrogen-fixing nodules on both hosts. The nodule isolates were identified as E. adhaerens isolates by growth on selective media.     

Study of Bacillus subtilis Endospores in Soil by Use of a Modified Endospore Stain

The Schaeffer-Fulton endospore stain was modified so that it would stain Bacillus subtilis endospores in soil smears. The modified stain differentiated among dormant spores, spores undergoing activation, and spores which had germinated but had not yet shown outgrowth. These differentiations were seen for spores in soil and for pure spore preparations in the laboratory. This stain was used to show reversible B. subtilis spore activation promoted by an Ensifer adhaerens-like indigenous bacterium in soil and by pure cultures of E. adhaerens added to spores in the laboratory. Under the specific conditions in the laboratory, spore germination did not proceed beyond the activation stage, and relatively little change occurred in the numbers of both E. adhaerens and B. subtilis. This was also true in soil, although some germination with destruction of spores and vegetative cells did occur if the soil had been nutritionally enriched by preincubation with incorporated ground alfalfa.     

Paenibacillus taohuashanense sp. nov., a nitrogen-fixing species isolated from rhizosphere soil of the root of Caragana kansuensis Pojark

A nitrogen-fixing bacterium, designated strain gs65^T, was isolated from a rhizosphere soil sample of Caragana kansuensis Pojark. Phylogenetic analysis based on a fragment of the nifH gene and the full-length 16S rRNA gene sequence revealed that strain gs65^T is a member of the genus Paenibacillus. High levels of 16S rRNA gene similarity were found between strain gs65^T and Paenibacillus borealis DSM 13188^T (97.5 %), Paenibacillus odorifer ATCC BAA-93^T (97.3 %), Paenibacillus durus DSM 1735^T (97.0 %) and Paenibacillus sophorae DSM23020^T (96.9 %). Levels of 16S rRNA gene sequence similarity between strain gs65^T and the type strains of other recognized members of the genus Paenibacillus were below 97.0 %. Levels of DNA–DNA relatedness between strain gs65^T and P. borealis DSM 13188^T, P. odorifer ATCC BAA-93^T (97.3 %), P. durus DSM 1735^T and P. sophorae DSM23020^T were 35.9, 38.0, 34.2 and 35.5 % respectively. The DNA G+C content of strain gs65^T was determined to be 51.6 mol%. The major fatty acids were found to be iso-C_14:0, anteiso-C_15:0 and iso-C_16:0. On the basis of its phenotypic characteristics and levels of DNA–DNA hybridization, strain gs65^T is considered to represent a novel species of the genus Paenibacillus, for which the name Paenibacillus taohuashanense sp. nov. is proposed. The type strain is gs65^T (=CGMCC 1.12175^T = DSM 25809^T).     

Analysis of nifH Gene Pool Complexity in Soil and Litter at a Douglas Fir Forest Site in the Oregon Cascade Mountain Range

Nitrogen-fixing microbial populations in a Douglas fir forest on the western slope of the Oregon Cascade Mountain Range were analyzed. The complexity of the nifH gene pool (nifH is the marker gene which encodes nitrogenase reductase) was assessed by performing nested PCR with bulk DNA extracted from plant litter and soil. The restriction fragment length polymorphisms (RFLPs) of PCR products obtained from litter were reproducibly different than the RFLPs of PCR products obtained from the underlying soil. The characteristic differences were found during the entire sampling period between May and September. RFLP analyses of cloned nifH PCR products also revealed characteristic patterns for each sample type. Among 42 nifH clones obtained from a forest litter library nine different RFLP patterns were found, and among 64 nifH clones obtained from forest soil libraries 13 different patterns were found. Only two of the patterns were found in both the litter and the soil, indicating that there were major differences between the nitrogen-fixing microbial populations. A sequence analysis of clones representing the 20 distinct patterns revealed that 19 of the patterns had a proteobacterial origin. All of the nifH sequences obtained from the Douglas fir forest litter localized in a distinct phylogenetic cluster characterized by the nifH sequences of members of the genera Rhizobium, Sinorhizobium, and Azospirillum. The nifH sequences obtained from soil were found in two additional clusters, one characterized by sequences of members of the genera Bradyrhizobium, Azorhizobium, Herbaspirillum, and Thiobacillus and the other, represented by a single nifH clone, located between the gram-positive bacteria and the cyanobacteria. Our results revealed the distinctness of the nitrogen-fixing microbial populations in litter and soil in a Douglas fir forest; the differences may be related to special requirements for degradation and mineralization processes in the plant litter.     

nifH Sequences and Nitrogen Fixation in Type I and Type II Methanotrophs

Some methane-oxidizing bacteria (methanotrophs) are known to be capable of expressing nitrogenase and utilizing N₂ as a nitrogen source. However, no sequences are available for nif genes in these strains, and the known nitrogen-fixing methanotrophs are confined mainly to a few genera. The purpose of this work was to assess the nitrogen-fixing capabilities of a variety of methanotroph strains. nifH gene fragments from four type I methanotrophs and seven type II methanotrophs were PCR amplified and sequenced. Nitrogenase activity was confirmed in selected type I and type II strains by acetylene reduction. Activities ranged from 0.4 to 3.3 nmol/min/mg of protein. Sequence analysis shows that the nifH sequences from the type I and type II strains cluster with nifH sequences from other gamma proteobacteria and alpha proteobacteria, respectively. The translated nifH sequences from three Methylomonas strains show high identity (95 to 99%) to several published translated environmental nifH sequences PCR amplified from rice roots and a freshwater lake. The translated nifH sequences from the type II strains show high identity (94 to 99%) to published translated nifH sequences from a variety of environments, including rice roots, a freshwater lake, an oligotrophic ocean, and forest soil. These results provide evidence for nitrogen fixation in a broad range of methanotrophs and suggest that nitrogen-fixing methanotrophs may be widespread and important in the nitrogen cycling of many environments.     

Pannonibacter indica sp. nov., a highly arsenate-tolerant bacterium isolated from a hot spring in India

A novel aerobic bacterium, strain HT23^T, able to grow on 500 mM sodium arsenate was isolated from a hot-spring sediment sample collected from Athamallik, Orissa, India. Cells of this isolate were Gram negative. Heterotrophic growth was observed at pH 6.0–11.0 and 20–45 °C. Optimum growth was observed at 37 °C and pH 7.0–10.0. The major polar lipids are diphosphatidyl glycerol, phosphatidyl glycerol, phosphatidyl ethanolamine, phosphatidyl choline and phosphatidyl monomethyl ethanolamine. The major isoprenoid quinone was Q-10. 16S rRNA gene sequence analysis indicated that the bacterium clustered with the genus Pannonibacter and showed 98.9 % similarity with Pannonibacter phragmitetus C6-19^T (DSM 14782^T) and 98 % with the P. phragmitetus group B and P. phragmitetus group E strains. Levels of DNA–DNA relatedness between the strain HT23^T and P. phragmitetus C6-19^T (DSM 14782^T) and other strains of P. phragmitetus group B and group E strains were below 55 %. On the basis of phenotypic and chemotaxonomic characteristics, 16S rRNA gene sequence analysis and DNA–DNA hybridization data, strain HT23^T is considered to represent a novel species of the genus Pannonibacter, for which the name Pannonibacter indica sp. nov. is proposed. The type strain is HT23^T (=JCM 16851^T = DSM 23407^T = LMG 25769^T).     

Ensifer adhaerens Predatory Activity Against Other Bacteria in Soil, as Monitored by Indirect Phage Analysis

An indirect phage analysis procedure was used to detect and follow the activity of the bacterial predator Ensifer adhaerens in situ in natural soil. The soil was percolated with an aqueous suspension of washed bacterial host cells so that the E. adhaerens cells naturally present in the soil would multiply in response to the host cells. The natural phage development which ensued against these multiplying E. adhaerens cells in the soil was then monitored by noting plaques which developed when the percolation fluid was plated with laboratory strains of E. adhaerens on laboratory media. The activities of the other members of the predation system that includes E. adhaerens (Streptomyces sp. strain 34 and a myxobacter) could not be monitored directly by phage analysis because phage were not found for them. Indirect monitoring was possible, however, because they were susceptible to attack by E. adhaerens. In general, the results were in agreement with previous observations by other methods of the predation sequence. E. adhaerens attacked Micrococcus luteus, Streptomyces sp. strain 34, and the myxobacter but did not attack several other possible species of hosts. It also did not respond to percolation of the soil with various nutrient solutions. E. adhaerens phage activity was not present in half of the soils percolated with M. luteus cells. This seemed to reflect too great a phage-host specificity for the technique as regards these soils, because E. adhaerens-like bacteria other than the strains used for plaquing were present in at least some of these soils. Although E. adhaerens did not attack Escherichia coli or Pseudomonas aeruginosa in soil, there was an overproduction of E. adhaerens phage if these bacteria were percolated simultaneously with M. luteus cells. The possibility is discussed that this represents an activation by M. luteus (or by a heat-extractable factor from it) of other bacterial predators that attack E. coli or P. aeruginosa and that these predators subsequently are themselves attacked by E. adhaerens.     

Abundance of Microbes Involved in Nitrogen Transformation in the Rhizosphere of Leucanthemopsis alpina (L.) Heywood Grown in Soils from Different Sites of the Damma Glacier Forefield

Glacier forefields are an ideal playground to investigate the role of development stages of soils on the formation of plant–microbe interactions as within the last decades, many alpine glaciers retreated, whereby releasing and exposing parent material for soil development. Especially the status of macronutrients like nitrogen differs between soils of different development stages in these environments and may influence plant growth significantly. Thus, in this study, we reconstructed major parts of the nitrogen cycle in the rhizosphere soil/root system of Leucanthemopsis alpina (L.) Heywood as well as the corresponding bulk soil by quantifying functional genes of nitrogen fixation (nifH), nitrogen mineralisation (chiA, aprA), nitrification (amoA AOB, amoA AOA) and denitrification (nirS, nirK and nosZ) in a 10-year and a 120-year ice-free soil of the Damma glacier forefield. We linked the results to the ammonium and nitrate concentrations of the soils as well as to the nitrogen and carbon status of the plants. The experiment was performed in a greenhouse simulating the climatic conditions of the glacier forefield. Samples were taken after 7 and 13 weeks of plant growth. Highest nifH gene abundance in connection with lowest nitrogen content of L. alpina was observed in the 10-year soil after 7 weeks of plant growth, demonstrating the important role of associative nitrogen fixation for plant development in this soil. In contrast, in the 120-year soil copy numbers of genes involved in denitrification, mainly nosZ were increased after 13 weeks of plant growth, indicating an overall increased microbial activity status as well as higher concentrations of nitrate in this soil.     

Changes in Nitrogen-Fixing and Ammonia-Oxidizing Bacterial Communities in Soil of a Mixed Conifer Forest after Wildfire

This study was undertaken to examine the effects of forest fire on two important groups of N-cycling bacteria in soil, the nitrogen-fixing and ammonia-oxidizing bacteria. Sequence and terminal restriction fragment length polymorphism (T-RFLP) analysis of nifH and amoA PCR amplicons was performed on DNA samples from unburned, moderately burned, and severely burned soils of a mixed conifer forest. PCR results indicated that the soil biomass and proportion of nitrogen-fixing and ammonia-oxidizing species was less in soil from the fire-impacted sites than from the unburned sites. The number of dominant nifH sequence types was greater in fire-impacted soils, and nifH sequences that were most closely related to those from the spore-forming taxa Clostridium and Paenibacillus were more abundant in the burned soils. In T-RFLP patterns of the ammonia-oxidizing community, terminal restriction fragments (TRFs) representing amoA cluster 1, 2, or 4 Nitrosospira spp. were dominant (80 to 90%) in unburned soils, while TRFs representing amoA cluster 3A Nitrosospira spp. dominated (65 to 95%) in fire-impacted soils. The dominance of amoA cluster 3A Nitrosospira spp. sequence types was positively correlated with soil pH (5.6 to 7.5) and NH₃-N levels (0.002 to 0.976 ppm), both of which were higher in burned soils. The decreased microbial biomass and shift in nitrogen-fixing and ammonia-oxidizing communities were still evident in fire-impacted soils collected 14 months after the fire.     

Paenibacillus beijingensis sp. nov., a novel nitrogen-fixing species isolated from jujube garden soil

A novel Gram-positive, rod-shaped, motile, spore-forming, nitrogen-fixing bacterium, designated strain 7188^T, was isolated from jujube rhizosphere soil in Beijing, China. The strain grew at 4–40 °C and pH 6–12, with an optimum of 30 °C and pH 7.0, respectively. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that strain 7188^T is a member of the genus Paenibacillus. Levels of 16S rRNA gene sequence similarities between strain 7188^T and the type strains of all recognized members of the genus Paenibacillus were below 96 %. The major cellular fatty acids were anteiso-C_15:0, anteiso-C_17:0 and C_16:0. The predominant menaquinone was MK-7. The DNA G+C content of strain 7188^T was 60.3 mol%. The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and unknown aminophospholipids. The diamino acid in the cell wall peptidoglycan is meso-diaminopimelic acid. On the basis of these results, strain 7188^T is considered to represent a novel species of the genus Paenibacillus, for which the name Paenibacillus beijingensis sp. nov. is proposed. The type strain is 7188^T (=ACCC 03082^T = DSM 24997^T).     

Paenibacillus beijingensis sp. nov., a nitrogen-fixing species isolated from wheat rhizosphere soil

A novel nitrogen-fixing bacterium, BJ-18^T, was isolated from wheat rhizosphere soil. Strain BJ-18^T was observed to be Gram-positive, facultatively anaerobic, motile and rod-shaped (0.4–0.9 μm × 2.0–2.9 μm). Phylogenetic analysis based on a partial nifH gene sequence and an assay for nitrogenase activity showed its nitrogen-fixing capacity. Phylogenetic analysis based on full 16S rRNA gene sequences suggested that strain BJ-18^T is a member of the genus Paenibacillus. High similarity of 16S rRNA gene sequence was found between BJ-18^T and Paenibacillus peoriae DSM 8320^T (99.05 %), Paenibacillus jamilae DSM 13815^T (98.86 %), Paenibacillus brasiliensis DSM 13188^T (98.55 %), Paenibacillus polymyxa DSM 36^T (98.74 %), Paenibacillus terrae DSM 15891^T (97.99 %) and Paenibacillus kribbensis JCM 11465^T (97.92 %), whereas the similarity was below 96.0 % between BJ-18^T and the other Paenibacillus species. DNA–DNA relatedness between strain BJ-18^T and P. peoriae DSM 8320^T, P. jamilae DSM 13815^T, P. brasiliensis DSM 13188^T, P. polymyxa DSM 36^T, P. kribbensis JCM 11465^T and P. terrae DSM 15891^T was determined to be 43.6 ± 2.7, 34.2 ± 5.3, 47.9 ± 6.6, 36.8 ± 3.5, 27.4 ± 4.3 and 23.6 ± 4.1 % respectively. The DNA G+C content of BJ-18^T was determined to be 45.8 mol %. The major fatty acid was identified as anteiso-C_15:0 (67.1 %). The polar lipids present in strain BJ-18^T were identified as diphosphatidylglycerol, phosphatidyl methylethanolamine, phosphatidylethanolamine and phosphatidylglycerol. The phenotypic and genotypic characteristics, and DNA–DNA relatedness data, suggest that BJ-18^T represents a novel species of the genus Paenibacillus, for which the name Paenibacillus beijingensis sp. nov. (Type strain BJ-18^T=DSM25425^T=CGMCC 1.12045^T) is proposed.     

Abundance and diversity of nitrogen-fixing bacteria in rhizosphere and bulk paddy soil under different duration of organic management

Denaturing gradient gel electrophoresis (DGGE) and quantitative real-time PCR (qPCR) approaches were used to assess respectively the molecular diversity and quantity of the nifH gene sequences in rhizosphere and bulk paddy soil under conventional management and different duration of organic management (2, 3, 5, 9 years). The phylogenetic distribution of clones based on nifH gene sequence showed that taxonomic groups were consisted of Alphaproteobacteria (27.6%), Betaproteobacteria (24.1%) and Gammaproteobacteria (48.3%). Members of the order Rhizobiales and Pseudomonadales were prevalent among the dominant diazotrophs. When the quantity of the nifH gene sequences was determined by qPCR, 2.27 × 10⁵ to 1.14 × 10⁶ copies/g of soil were detected. Except for 2 years organically managed soil, nifH gene copy numbers in organic soil, both rhizosphere and bulk, were significantly higher than in CM soil. Moreover, nifH gene copy numbers in the organic rhizosphere soil (3, 5, 9 years) were significantly higher than in bulk soil. The abundance and diversity of nitrogen-fixing bacteria tended to increase with duration of organic management but the highest number of nifH gene copies was observed in the rhizosphere and bulk soil of 5 years organic management. In addition, analysis of variance and canonical correspondence analysis (CCA) showed that C/N, C and N were important factors influencing the abundance and community structure of nitrogen-fixing bacterial.     

Alteromonas australica sp. nov., isolated from the Tasman Sea

A non-pigmented, motile, Gram-negative bacterium designated H 17^T was isolated from a seawater sample collected in Port Phillip Bay (the Tasman Sea, Pacific Ocean). The new organism displayed optimal growth between 4 and 37 °C, was found to be neutrophilic and slightly halophilic, tolerating salt water environments up to 10 % NaCl. Strain H 17^T was found to be able to degrade starch and Tween 80 but unable to degrade gelatin or agar. Phosphatidylglycerol (27.7 %) and phosphatidylethanolamine (72.3 %) were found to be the only associated phospholipids. The major fatty acids identified are typical for the genus Alteromonas and include C_16:0, C_16:1ω7, C_17:1ω8 and C_18:1ω7. The G+C content of the DNA was found to be 43.4 mol%. A phylogenetic study, based on the 16S rRNA gene sequence analysis and Multilocus Phylogenetic Analysis, clearly indicated that strain H 17^T belongs to the genus Alteromonas. The DNA?DNA relatedness between strain H 17^T and the validly named Alteromonas species was between 30.7 and 46.4 mol%. Based on these results, a new species, Alteromonas australica, is proposed. The type strain is H 17^T (= KMM 6016^T = CIP 109921^T).     

Utilizing pyrene-degrading endophytic bacteria to reduce the risk of plant pyrene contamination

Aims

Endophytic bacteria are ubiquitous in plants, but little information is available on the influence of endophytic bacteria on the uptake and metabolism of PAH by plants. Thus, we seek to investigate whether the colonization of a target plant by a PAH-degrading endophytic bacterium would improve the PAH metabolism of the plant and reduce the risk of plant PAH contamination.

Methods

A pyrene-degrading endophyte was isolated from PAH-contaminated plants using enrichment culture. After root inoculation with the isolated bacterium, greenhouse container experiments were conducted. Pyrene residues in soil and plant samples were analyzed by HPLC.

Results

A pyrene-degrading endophytic bacterium, Staphylococcus sp. BJ06, was isolated from Alopecurus aequalis and could degrade 56.0 % of pyrene (50 mg?·?L^?1) within 15 days. BJ06 grew and degraded pyrene efficiently under environmental conditions. The bacterium significantly promoted ryegrass growth and pyrene removal from contaminated soil in container experiments. The pyrene concentrations in ryegrass roots and shoots in endophyte-inoculated planted soil were reduced by 31.01 % and 44.22 %, respectively, compared with endophyte-free planted soil.

Conclusions

We have provided new perspectives on the regulation and control of plant uptake of organic contaminants with endophytic bacteria. The results of this study will be valuable to risk assessments of plant PAH contamination.     

Establishment of Elymus natans improves soil quality of a heavily degraded alpine meadow in Qinghai-Tibetan Plateau, China

Elymus natans is a dominant native species widely planted to restore the heavily degraded alpine meadows in Qinghai-Tibetan plateau. The objective of this study was to determine how E. natans establishment affected the quality and fertility of a heavily degraded soil. Soil samples (at depths of 0–10, 10–20 and 20–30 cm) were collected from the 3- and 7-year-old E. natans re-vegetated grasslands, and in the heavily degraded alpine meadow (control). The establishment of E. natans promoted plant cover and aboveground biomass. Compared to the non-reseeded meadow, the concentration of total organic C increased by 13% in the soil under 3-year-old reseeded E. natans grassland at 0–10 cm, and by 7–33% in the soil under 7-year-old reseeded E. natans grassland at 0–10, 10–20 and 20–30 cm depths. Rapid increases in total and available N were also observed in two E. natans re-vegetated grasslands, especially in the 0–10 cm soil layer. Across three sampling depths, total P concentration was increased by 17–35% and 18–54% in 3- and 7-year-old reseeded soil respectively, compared to the soil of control. After 3 years of E. natans growth, microbial biomass C increased by 13–58% at 0–10 and 10–20 cm layers; while it increased by 43–87% in 7-year-old reseeded treatment at 0–10, 10–20 and 20–30 cm depths relative to control. A similar increasing trend was observed for microbial biomass N and P generally. Significant increase in neutral phosphatase, urease, catalase and dehydrogenase was also found in 3- and 7-year-old re-vegetated grasslands compared with heavily degraded meadow. Our results suggest a significant positive impact of E. natans establishment on soil quality. Thus, E. natans establishment could be an effective and applicable measure in restoring heavily degraded alpine meadow in the region of Qinghai-Tibetan Plateau.     

Azospirillum himalayense sp. nov., a nifH bacterium isolated from Himalayan valley soil,India

Gram-negative, free-living bacterial strain ptl-3^T was isolated from Himalayan valley soil, India. Polyphasic taxonomy was performed including morphological characterization, fatty acid analysis, biochemical tests, 16S rRNA and nifH gene sequence analyses. 16S rRNA gene sequence analysis showed that the strain ptl-3^T belonged to the genus Azospirillum and was closely related to A. brasilense (98.7 % similarity) and A. rugosum (97 % similarity). 16S rRNA gene sequence similarity (96–95 %) was shown with other members of the genus Azospirillum. Major fatty acid 18:1ω7c was also similar to the genus Azospirillum. DNA–DNA relatedness value between strain ptl-3^T and A. brasilense was found to be 47 %. Various biochemical tests showed that the strain ptl-3^T differed from its closely related species A. brasilense. On the basis of phenotypic, chemotaxonomic and molecular genetics evidence, a bacterium with the type strain ptl-3^T is proposed as a novel species of the genus Azospirillum. The name of bacterial strain ptl-3^T has been proposed as Azospirillum himalayense sp. nov. The type strain of ptl-3^T (CCUG 58760^T, KCTC 23189^T) has been submitted to two culture collection centres. The accession numbers for 16S rRNA and nifH gene are GQ 284588 and GQ 249665. respectively.     

Analysis of the diversity of diazotrophic bacteria in peat soil by cloning of the <Emphasis Type="Italic">nifH</Emphasis> gene

The diversity of nitrogen-fixing microorganisms in the soil of an oligotrophic Sphagnum peat bog was studied by molecular cloning of fragments of the nifH gene encoding one of the main components of the nitrogenase complex. The fragments were amplified from the DNA isolated from the peat samples collected at the same site in January (library I) and November (library II), 2005. Analysis of the nifH sequence libraries revealed high diversity of diazotrophic bacteria in peat soil: the first library consisted of 237 clones and 55 unique sequence types, the second one included 171 clones and 52 sequence types. Comparison of the two clone libraries showed that the composition and population structure of the nitrogen-fixing community depended greatly on the sampling time; they shared only 11 phylotypes. The sequences of representatives of the class Alphaproteobacteria prevailed in both libraries (27% and 57% of clones in libraries I and II, respectively). Representatives of the classes Deltaproteobacteria and Chlorobea were minor components of library I (6% and 7% of clones, respectively), whereas they prevailed in library II (18% and 24% of clones, respectively). Members of the class Chloroflexi were present only in library I, while members of the classes Bacilli, Clostridia, and Methanomicrobia were present only in library II. Our studies demonstrated that, for complete evaluation of the diversity of natural nitrogen-fixing communities, nifH libraries should consist of at least 200–300 clones.     

Acrylamide biodegradation ability and plant growth-promoting properties of Variovorax boronicumulans CGMCC 4969

Species of the genus Variovorax are often isolated from nitrile or amide-containing organic compound-contaminated soil. However, there have been few biological characterizations of Variovorax and their contaminant-degrading enzymes. Previously, we reported a new soil isolate, Variovorax boronicumulans CGMCC 4969, and its nitrile hydratase that transforms the neonicotinoid insecticide thiacloprid into an amide metabolite. In this study, we showed that CGMCC 4969 is able to degrade acrylamide, a neurotoxicant and carcinogen in animals, during cell growth in a mineral salt medium as well as in its resting state. Resting cells rapidly hydrolyzed 600 mg/L acrylamide to acrylic acid with a half-life of 2.5 min. In in vitro tests, CGMCC 4969 showed plant growth-promoting properties; it produced a siderophore, ammonia, hydrogen cyanide, and the phytohormone salicylic acid. Interestingly, in soil inoculated with this strain, 200 mg/L acrylamide was completely degraded in 4 days. Gene cloning and overexpression in the Escherichia coli strain Rosetta (DE3) pLysS resulted in the production of an aliphatic amidase of 345 amino acids that hydrolyzed acrylamide into acrylic acid. The amidase contained a conserved catalytic triad, Glu59, Lys 134, and Cys166, and an “MRHGDISSS” amino acid sequence at the N-terminal region. Variovorax boronicumulans CGMCC 4969, which is able to use acrylamide for cell growth and rapidly degrade acrylamide in soil, shows promising plant growth-promoting properties. As such, it has the potential to be developed into an effective Bioaugmentation strategy to promote growth of field crops in acrylamide-contaminated soil.     

The effects of pesticides on bacterial nitrogen fixers in peanut-growing area

In the peanut production, the applications of herbicides and fungicides are a common practice. In this work, studies done under field conditions demonstrated that pesticides affected negatively the number and nitrogenase activity of diazotrophic populations of soil. Agrochemical effects were not transient, since these parameters were not recovered to pre-treatment levels even 1 year after pesticides application. Results obtained from greenhouse experiments revealed that the addition of herbicide or fungicides diminished the free-living diazotrophs number reaching levels found in soil amended with the pesticides and that the number of symbiotic diazotrophs was not affected by the insecticide assayed. The soil nitrogenase activity was not affected by fungicides and glyphosate. The effect of pesticides on the nitrogen-fixing bacteria diversity was evaluated both in field and greenhouse experiments. Analysis of clone libraries generated from the amplification of soil nifH gene showed a diminution in the genetic diversity of this bacterial community.