Proteomic and molecular investigation on the physiological adaptation of Comamonas sp. strain CNB-1 growing on 4-chloronitrobenzene

Comamonas sp. strain CNB-1 can utilize 4-chloronitrobenzene (4CNB) as sole carbon and nitrogen source for growth. Previous studies were focused on 4CNB degradative pathway and have showed that CNB-1 contained a plasmid pCNB1 harboring the genes (cnbABCaCbDEFGH, cnbZ) for the enzymes involving in 4CNB degradation, but only three gene products (CnbCa, CnbCb, and CnbZ) were identified in CNB-1 cells. Comamonas strain CNB-2 that lost pCNB1 was not able to grow on 4CNB. In this study, physiological adaptation to 4CNB by CNB-1 was investigated with proteomic and molecular tools. Comparative proteomes of strains CNB-1 and CNB-2 grown on 4CNB and/or succinate revealed that adaptation to 4CNB by CNB-1 included specific degradative pathway and general physiological responses: (1) Seven gene products (CnbA, CnbCa, CnbCb, CnbD, CnbE, CnbF, and CnbZ) for 4CNB degradation were identified in 4CNB-grown cells, and they were constitutively synthesized in CNB-1. Two genes cnbE and cnbF were cloned and simultaneously expressed in E. coli. The CnbE and CnbF together catalyzed the conversion of 2-oxohex-4-ene-5-chloro-1,6-dioate into 2-oxo-4-hydroxy-5-chloro-valeric acid; (2) Enzymes involving in glycolysis, tricarboxylic acid cycle, and synthesis of glutamate increased their abundances in 4CNB-grown cells.     

Effect of a Sinorhizobium meliloti strain with a modified putA gene on the rhizosphere microbial community of alfalfa

The success of a rhizobial inoculant in the soil depends to a large extent on its capacity to compete against indigenous strains. M403, a Sinorhizobium meliloti strain with enhanced competitiveness for nodule occupancy, was recently constructed by introducing a plasmid containing an extra copy of a modified putA (proline dehydrogenase) gene. This strain and M401, a control strain carrying the same plasmid without the modified gene, were used as soil inoculants for alfalfa in a contained field release experiment at León, Spain. In this study, we determined the effects of these two strains on the indigenous microbial community. 16S rRNA genes were obtained from the rhizosphere of alfalfa inoculated with strain M403 or strain M401 or from noninoculated plants by amplification of DNA from soil with bacterial group-specific primers. These genes were analyzed and compared by restriction fragment length polymorphism and temperature gradient gel electrophoresis. The results allowed us to differentiate between alterations in the microbial community apparently caused by inoculation and by the rhizosphere effect and seasonal fluctuations induced by the alfalfa plants and by the environment. Only moderate inoculation-dependent effects could be detected, while the alfalfa plants appeared to have a much stronger influence on the microbial community.     

Effect of a Sinorhizobium meliloti Strain with a Modified putA Gene on the Rhizosphere Microbial Community of Alfalfa

The success of a rhizobial inoculant in the soil depends to a large extent on its capacity to compete against indigenous strains. M403, a Sinorhizobium meliloti strain with enhanced competitiveness for nodule occupancy, was recently constructed by introducing a plasmid containing an extra copy of a modified putA (proline dehydrogenase) gene. This strain and M401, a control strain carrying the same plasmid without the modified gene, were used as soil inoculants for alfalfa in a contained field release experiment at León, Spain. In this study, we determined the effects of these two strains on the indigenous microbial community. 16S rRNA genes were obtained from the rhizosphere of alfalfa inoculated with strain M403 or strain M401 or from noninoculated plants by amplification of DNA from soil with bacterial group-specific primers. These genes were analyzed and compared by restriction fragment length polymorphism and temperature gradient gel electrophoresis. The results allowed us to differentiate between alterations in the microbial community apparently caused by inoculation and by the rhizosphere effect and seasonal fluctuations induced by the alfalfa plants and by the environment. Only moderate inoculation-dependent effects could be detected, while the alfalfa plants appeared to have a much stronger influence on the microbial community.     

The Complete Genome of Comamonas testosteroni Reveals Its Genetic Adaptations to Changing Environments

Members of the gram-negative, strictly aerobic genus Comamonas occur in various environments. Here we report the complete genome of Comamonas testosteroni strain CNB-2. Strain CNB-2 has a circular chromosome that is 5,373,643 bp long and has a G+C content of 61.4%. A total of 4,803 open reading frames (ORFs) were identified; 3,514 of these ORFs are functionally assigned to energy production, cell growth, signal transduction, or transportation, while 866 ORFs encode hypothetical proteins and 423 ORFs encode purely hypothetical proteins. The CNB-2 genome has many genes for transportation (22%) and signal transduction (6%), which allows the cells to respond and adapt to changing environments. Strain CNB-2 does not assimilate carbohydrates due to the lack of genes encoding proteins involved in glycolysis and pentose phosphate pathways, and it contains many genes encoding proteins involved in degradation of aromatic compounds. We identified 66 Tct and nine TRAP-T systems and a complete tricarboxylic acid cycle, which may allow CNB-2 to take up and metabolize a range of carboxylic acids. This nutritional bias for carboxylic acids and aromatic compounds enables strain CNB-2 to occupy unique niches in environments. Four different sets of terminal oxidases for the respiratory system were identified, and they putatively functioned at different oxygen concentrations. This study conclusively revealed at the genomic level that the genetic versatility of C. testosteroni is vital for competition with other bacteria in its special niches.The members of the genus Comamonas are gram-negative, strict aerobes and frequently occur in diverse habitats, including activated sludge, marshes, marine habitats, and plant and animal tissues (4, 12, 13). They grow on organic acids, amino acids, and peptone, but they rarely attack carbohydrates. Some species, such as Comamonas testosteroni, can also mineralize complex and xenobiotic compounds, such as testosterone (17) and 4-chloronitrobenzene (CNB) (54). Their diversified niches make Comamonas species environmentally important and also suggest that the genus Comamonas represents a group of bacteria that can adapt very well, both ecologically and physiologically, to environments.To understand better how environmental microbes adapt to their environments, many well-known environmental microbes, such as Pseudomonas putida (53) and Rhodococcus sp. strain RAH1 (31), have been sequenced. The genome data for these organisms, as well as other environmental microbes, provide not only an understanding of physiological and environmental functions at the genetic level but also a starting point for systems biology analyses of these microbes. Until now, none of the Comamonas species has been sequenced, although these organisms represent an important group of environmental microbes.C. testosteroni strain CNB-1 was isolated from CNB-contaminated activated sludge and grows with CNB as a sole source of carbon and nitrogen, and it has been used successfully for rhizoremediation of CNB-polluted soil (25). Strain CNB-1 has a circular chromosome and a large plasmid, and the genes involved in the degradation of CNB on plasmid pCNB1 were identified previously (28). In the present study, the genome of strain CNB-2, which was derived from strain CNB-1, was sequenced, and a genome analysis was performed parallel to physiological experiments. The aim of this work was to obtain genetic insight into how C. testosteroni adapts to changing and diverse environments.     

Assimilation of aromatic compounds by Comamonas testosteroni: characterization and spreadability of protocatechuate 4,5-cleavage pathway in bacteria

Comamonas testosteroni strain CNB-1 was isolated from activated sludge and has been investigated for its ability to degrade 4-chloronitrobenzene. Results from this study showed that strain CNB-1 grew on phenol, gentisate, vanillate, 3-hydroxybenzoate (3HB), and 4-hydroxybenzoate (4HB) as carbon and energy sources. Proteomic data and enzyme activity assays suggested that vanillate, 3HB, and 4HB were degraded in strain CNB-1 via protocatechuate (PCA) 4,5-cleavage pathway. The genetics and biochemistry of the PCA 4,5-cleavage pathway were investigated. Results showed that the 4-oxalomesaconate (OMA) hydratase from C. testosteroni takes only enol-OMA as substrate. A previously functionally unknown gene pmdU encodes an OMA tautomerase and catalyzes conversion of OMA_keto into OMA_enol. The 4-carboxy-4-hydroxy-2-oxoadipate (CHA) aldolase is encoded by pmdF and catalyzes the last step of the PCA 4,5-cleavage pathway. We explored the 1,183 microbial genomes at GenBank for potential PCA 4,5-cleavage pathways, and 33 putative pmd clusters were found. Results suggest that PCA 4,5-cleavage pathways are mainly distributed in α- and β-Proteobacteria.     

Variation of Microbial Rhizosphere Communities in Response to Crop Species, Soil Origin, and Inoculation with Sinorhizobium meliloti L33

Abstract A greenhouse study with soil–plant microcosms was conducted in order to compare the effect of crop species, soil origin, and a bacterial inoculant on the establishment of microbial communities colonizing plant roots. Two crop species, alfalfa (Medicago sativa) and rye (Secale cereale), were grown separately in two soils collected from agricultural fields at different locations and with differing histories of leguminous crop rotation. A subset of microcosms was inoculated at 10⁶ cfu g^-1 soil with the luciferase marker gene-tagged Sinorhizobium meliloti strain L33, a symbiotic partner of M. sativa. Microbial consortia were collected from the rhizospheres of alfalfa after 10 weeks of incubation and from rye after 11 weeks. S. meliloti L33 populations were one to two orders of magnitude higher in the rhizospheres of alfalfa than of rye. In soil with previous alfalfa cultivation, 80% of the alfalfa nodules were colonized by indigenous bacteria, while in the other soil alfalfa was colonized almost exclusively (>90%) with S. meliloti L33. Three community-level targeting approaches were used to characterize the variation of the extracted microbial rhizosphere consortia: (1) Community level physiological profiles (CLPP), (2) fatty acid methyl ester analysis (FAME), and (3) diversity of PCR amplified 16S rRNA target sequences from directly extracted ribosomes, determined by temperature gradient gel electrophoresis (TGGE). All approaches identified the crop species as the major determinant of microbial community characteristics. Consistently, the influence of soil was of minor importance, while a modification of the alfalfa-associated microbial community structure after inoculation with S. meliloti L33 was only consistently observed by using TGGE. Received: 20 October 1999; Accepted: 15 January 2000; Online Publication: 18 July 2000     

Influence of arbuscular mycorrhiza and Rhizobium on phytoremediation by alfalfa of an agricultural soil contaminated with weathered PCBs: a field study

A field experiment was conducted to study the effects of inoculation with the arbuscular mycorrhizal fungus Glomus caledonium and/or Rhizobium meliloti on phytoremediation of an agricultural soil contaminated with weathered PCBs by alfalfa grown for 180 days. Planting alfalfa (P), alfalfa inoculated with G. caledonium (P + AM), alfalfa inoculated with R. meliloti (P + R), and alfalfa co-inoculated with R. meliloti and G. caledonium (P+AM+R) decreased significantly initial soil PCB concentrations by 8.1, 12.0, 33.8, and 43.5%, respectively. Inoculation with R. meliloti and/or G. caledonium (P+AM+R) increased the yield of alfalfa, and the accumulation of PCBs in the shoots. Soil microbial counts and the carbon utilization ability of the soil microbial community increased when alfalfa was inoculated with R. meliloti and/or G. caledonium. Results of this field study suggest that synergistic interactions between AMF and Rhizobium may have great potential to enhance phytoremediation by alfalfa of an agricultural soil contaminated with weathered PCBs.     

Influence of intercropping and intercropping plus rhizobial inoculation on microbial activity and community composition in rhizosphere of alfalfa (Medicago sativa L.) and Siberian wild rye (Elymus sibiricus L.)

Alfalfa–Siberian wild rye intercropping is the predominant cropping system used to produce forage in China. In this study, the effects of intercropping and intercropping-rhizobial inoculation on soil enzyme activities, microbial biomass and bacterial community composition in the rhizosphere were examined. In both treatments, the yield of alfalfa, microbial biomass and activities of soil urease, invertase and alkaline phosphatase in the alfalfa rhizosphere were markedly increased, whereas there was a slight increase in the yield of Siberian wild rye, few impacts on soil microbial biomass, and decreased enzyme activities (except for urease) in the Siberian wild rye rhizosphere. Terminal restriction fragment length polymorphism (T-RFLP) of 16S rRNA genes indicated that Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, Firmicutes, Actinobacteria and Bacteroidetes were the major bacterial groups in the rhizosphere of both plants. However, intercropping and rhizobial inoculation induced some shifts in the relative abundance of them. Nitrosomonas and Nitrosospira groups were detected in all treatments by the T-RFLP patterns of ammonia monooxygenase subunit A ( amoA ) gene, but the relative abundance of Nitrosomonas increased and that of Nitrosospira decreased in the intercropping-rhizobial inoculation treatment. Both treatments tended to increase the diversity of amoA . Conclusively, the two treatments clearly affected soil microbial composition and soil enzyme activities, which might be reflected in changes in yield.     

Mapping the Centimeter-Scale Spatial Variability of PAHs and Microbial Populations in the Rhizosphere of Two Plants

Rhizoremediation uses root development and exudation to favor microbial activity. Thus it can enhance polycyclic aromatic hydrocarbon (PAH) biodegradation in contaminated soils. Spatial heterogeneity of rhizosphere processes, mainly linked to the root development stage and to the plant species, could explain the contrasted rhizoremediation efficiency levels reported in the literature. Aim of the present study was to test if spatial variability in the whole plant rhizosphere, explored at the centimetre-scale, would influence the abundance of microorganisms (bacteria and fungi), and the abundance and activity of PAH-degrading bacteria, leading to spatial variability in PAH concentrations. Two contrasted rhizospheres were compared after 37 days of alfalfa or ryegrass growth in independent rhizotron devices. Almost all spiked PAHs were degraded, and the density of the PAH-degrading bacterial populations increased in both rhizospheres during the incubation period. Mapping of multiparametric data through geostatistical estimation (kriging) revealed that although root biomass was spatially structured, PAH distribution was not. However a greater variability of the PAH content was observed in the rhizosphere of alfalfa. Yet, in the ryegrass-planted rhizotron, the Gram-positive PAH-degraders followed a reverse depth gradient to root biomass, but were positively correlated to the soil pH and carbohydrate concentrations. The two rhizospheres structured the microbial community differently: a fungus-to-bacterium depth gradient similar to the root biomass gradient only formed in the alfalfa rhizotron.     

滴灌对苜蓿根际土壤细菌多样性和群落结构的影响

【背景】细菌作为土壤微生物中的重要类群,能够有效促进土壤物质循环和能量流动,细菌多样性以及群落结构能够反映土壤的质量状况。【目的】了解滴灌条件下苜蓿根际土壤细菌群落结构及多样性变化,探讨土壤环境因子对细菌群落结构的影响。【方法】基于细菌16Sr RNAV3-V4区高通量测序技术,分析比较滴灌与自然降雨两种模式下生长的苜蓿根际与非根际土壤中细菌多样性和群落分布规律,然后采用冗余分析(Redundancy analysis,RDA)探讨土壤环境因子与细菌多样性的关系。【结果】苜蓿根际土壤中细菌多样性丰富,滴灌根际土壤中细菌多样性显著高于自然降雨根际土壤;土壤样品中共检测到细菌46门53纲116目220科469属,主要的优势菌门为变形菌门(Proteobacteria,25.27%-34.42%),其中α-变形菌纲(Alphaproteobacteria,11.41%-18.97%)为优势亚群,鞘氨醇单胞菌属(Sphingomonas,1.00%-4.54%)为优势属。相较于自然降雨,滴灌条件下苜蓿根际土壤细菌的6个门和16个属的群落结构发生显著变化;此外,RDA分析表明,不同环境因子对微生物群落的影响不同,滴灌根际土壤中9个细菌属的丰度与全磷、全钾、有效磷、碱解氮、有机质、土壤中性磷酸酶以及土壤脲酶的含量显著正相关。【结论】滴灌作为新型节水技术,在促进植物生长、提高产量、节约成本的基础上增加了植物根际土壤中细菌多样性和丰度,该结果为新型灌溉体制的改革以及土壤微生物资源的开发利用提供科学数据。     

Effects of the Inoculant Strain Sphingomonas paucimobilis 20006FA on Soil Bacterial Community and Biodegradation in Phenanthrene-contaminated Soil

The effects of the inoculant strain Sphingomonas paucimobilis 20006FA (isolated from a phenanthrene-contaminated soil) on the dynamics and structure of microbial communities and phenanthrene elimination rate were studied in soil microcosms artificially contaminated with phenanthrene. The inoculant managed to be established from the first inoculation as it was evidenced by denaturing gradient gel electrophoresis analysis, increasing the number of cultivable heterotrophic and PAH-degrading cells and enhancing phenanthrene degradation. These effects were observed only during the inoculation period. Nevertheless, the soil biological activity (dehydrogenase activity and CO₂ production) showed a late increase. Whereas gradual and successive changes in bacterial community structures were caused by phenanthrene contamination, the inoculation provoked immediate, significant, and stable changes on soil bacterial community. In spite of the long-term establishment of the inoculated strain, at the end of the experiment, the bioaugmentation did not produce significant changes in the residual soil phenanthrene concentration and did not improve the residual effects on the microbial soil community.     

Effect of Dissemination of 2,4-Dichlorophenoxyacetic Acid (2,4-D) Degradation Plasmids on 2,4-D Degradation and on Bacterial Community Structure in Two Different Soil Horizons

Transfer of the 2,4-dichlorophenoxyacetic acid (2,4-D) degradation plasmids pEMT1 and pJP4 from an introduced donor strain, Pseudomonas putida UWC3, to the indigenous bacteria of two different horizons (A horizon, depth of 0 to 30 cm; B horizon, depth of 30 to 60 cm) of a 2,4-D-contaminated soil was investigated as a means of bioaugmentation. When the soil was amended with nutrients, plasmid transfer and enhanced degradation of 2,4-D were observed. These findings were most striking in the B horizon, where the indigenous bacteria were unable to degrade any of the 2,4-D (100 mg/kg of soil) during at least 22 days but where inoculation with either of the two plasmid donors resulted in complete 2,4-D degradation within 14 days. In contrast, in soils not amended with nutrients, inoculation of donors in the A horizon and subsequent formation of transconjugants (10⁵ CFU/g of soil) could not increase the 2,4-D degradation rate compared to that of the noninoculated soil. However, donor inoculation in the nonamended B-horizon soil resulted in complete degradation of 2,4-D within 19 days, while no degradation at all was observed in noninoculated soil during 89 days. With plasmid pEMT1, this enhanced degradation seemed to be due only to transconjugants (10⁵ CFU/g of soil), since the donor was already undetectable when degradation started. Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes showed that inoculation of the donors was followed by a shift in the microbial community structure of the nonamended B-horizon soils. The new 16S rRNA gene fragments in the DGGE profile corresponded with the 16S rRNA genes of 2,4-D-degrading transconjugant colonies isolated on agar plates. This result indicates that the observed change in the community was due to proliferation of transconjugants formed in soil. Overall, this work clearly demonstrates that bioaugmentation can constitute an effective strategy for cleanup of soils which are poor in nutrients and microbial activity, such as those of the B horizon.     

Association of microbial community composition and activity with lead,chromium, and hydrocarbon contamination

Microbial community composition and activity were characterized in soil contaminated with lead (Pb), chromium (Cr), and hydrocarbons. Contaminant levels were very heterogeneous and ranged from 50 to 16,700 mg of total petroleum hydrocarbons (TPH) kg of soil(-1), 3 to 3,300 mg of total Cr kg of soil(-1), and 1 to 17,100 mg of Pb kg of soil(-1). Microbial community compositions were estimated from the patterns of phospholipid fatty acids (PLFA); these were considerably different among the 14 soil samples. Statistical analyses suggested that the variation in PLFA was more correlated with soil hydrocarbons than with the levels of Cr and Pb. The metal sensitivity of the microbial community was determined by extracting bacteria from soil and measuring [(3)H]leucine incorporation as a function of metal concentration. Six soil samples collected in the spring of 1999 had IC(50) values (the heavy metal concentrations giving 50% reduction of microbial activity) of approximately 2.5 mM for CrO(4)2- and 0.01 mM for Pb2+. Much higher levels of Pb were required to inhibit [14C]glucose mineralization directly in soils. In microcosm experiments with these samples, microbial biomass and the ratio of microbial biomass to soil organic C were not correlated with the concentrations of hydrocarbons and heavy metals. However, microbial C respiration in samples with a higher level of hydrocarbons differed from the other soils no matter whether complex organic C (alfalfa) was added or not. The ratios of microbial C respiration to microbial biomass differed significantly among the soil samples (P < 0.05) and were relatively high in soils contaminated with hydrocarbons or heavy metals. Our results suggest that the soil microbial community was predominantly affected by hydrocarbons.     

丛毛单胞菌CNB-1合成PHA及相关基因的研究

分析了丛毛单胞菌(Comamonas sp.)CNB-1菌株在不同条件下合成聚羟基烷酸(polyhydroxyalkanoic acids,PHAs)的组分和含量,同时克隆了与PHA合成相关的基因。结果表明,该菌可以多种短链有机酸及醇类为碳源合成PHA多聚物或共聚物,以戊酸和1,4-丁二醇为底物时,可达菌体干重的57%;同时发现小分子醇类的存在能显著促进PHA的合成,推测与醇类氧化过程中提供了更多的还原力有关。为了克隆相关基因,利用已知phaC的保守区简并引物筛选基因组文库,将得到的阳性克隆质粒测序,发现phaC、phaA、phaB组成一个基因簇phaC-A-B。将phaC、phaA、phaB连接到pET载体在E.coli中共表达,重组E.coli菌株能合成PHA;将这3个基因单独连接到pET载体,在E.coli中表达后检测到相应酶活,分别约为原始菌株的4.1、71和2882倍。     

Bacterial community structure of a pesticide-contaminated site and assessment of changes induced in community structure during bioremediation

The introduction of culture-independent molecular screening techniques, especially based on 16S rRNA gene sequences, has allowed microbiologists to examine a facet of microbial diversity not necessarily reflected by the results of culturing studies. The bacterial community structure was studied for a pesticide-contaminated site that was subsequently remediated using an efficient degradative strain Arthrobacter protophormiae RKJ100. The efficiency of the bioremediation process was assessed by monitoring the depletion of the pollutant, and the effect of addition of an exogenous strain on the existing soil community structure was determined using molecular techniques. The 16S rRNA gene pool amplified from the soil metagenome was cloned and restriction fragment length polymorphism studies revealed 46 different phylotypes on the basis of similar banding patterns. Sequencing of representative clones of each phylotype showed that the community structure of the pesticide-contaminated soil was mainly constituted by Proteobacteria and Actinomycetes. Terminal restriction fragment length polymorphism analysis showed only nonsignificant changes in community structure during the process of bioremediation. Immobilized cells of strain RKJ100 enhanced pollutant degradation but seemed to have no detectable effects on the existing bacterial community structure.     

桑树根际土壤微生物对间作和施氮的响应

利用Biolog-Ecoplate^TM结合主成分与冗余分析法,研究与苜蓿间作和施氮对桑树根际土壤微生物功能多样性及其与环境因子的影响.结果表明: 与单作无氮相比,单作施氮和间作(无氮和施氮)明显降低桑树根际土壤pH值,显著提高有机质含量、有效氮、含水量和过氧化物酶、脲酶的活性.同时,单作施氮和间作(无氮和施氮)提高了桑树根际土壤微生物的AWCD值、多样性指数和利用碳源比例,且间作施氮增加其提高的程度.主成分分析表明,施氮和间作改变了根际土壤微生物利用碳源强度和种类,其中单作施氮和与间作无氮处理的碳源利用情况相似,但后者对衣康酸和D-葡糖胺酸的利用率(>4%)显著高于前者.冗余分析表明,单作无氮桑树根际土壤微生物多样性与土壤多酚氧化酶活性呈正相关,与土壤含水量呈负相关.单作施氮与间作无氮处理的桑树根际微生物多样性与土壤pH值和含水量呈显著正相关,与土壤氮素营养呈负相关.间作施氮处理的桑树根际微生物多样性与土壤氮素营养呈正相关,而与土壤pH值呈显著负相关.     

Effect of dissemination of 2,4-dichlorophenoxyacetic acid (2,4-D) degradation plasmids on 2,4-D degradation and on bacterial community structure in two different soil horizons

Transfer of the 2,4-dichlorophenoxyacetic acid (2,4-D) degradation plasmids pEMT1 and pJP4 from an introduced donor strain, Pseudomonas putida UWC3, to the indigenous bacteria of two different horizons (A horizon, depth of 0 to 30 cm; B horizon, depth of 30 to 60 cm) of a 2,4-D-contaminated soil was investigated as a means of bioaugmentation. When the soil was amended with nutrients, plasmid transfer and enhanced degradation of 2,4-D were observed. These findings were most striking in the B horizon, where the indigenous bacteria were unable to degrade any of the 2,4-D (100 mg/kg of soil) during at least 22 days but where inoculation with either of the two plasmid donors resulted in complete 2,4-D degradation within 14 days. In contrast, in soils not amended with nutrients, inoculation of donors in the A horizon and subsequent formation of transconjugants (10(5) CFU/g of soil) could not increase the 2,4-D degradation rate compared to that of the noninoculated soil. However, donor inoculation in the nonamended B-horizon soil resulted in complete degradation of 2,4-D within 19 days, while no degradation at all was observed in noninoculated soil during 89 days. With plasmid pEMT1, this enhanced degradation seemed to be due only to transconjugants (10(5) CFU/g of soil), since the donor was already undetectable when degradation started. Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes showed that inoculation of the donors was followed by a shift in the microbial community structure of the nonamended B-horizon soils. The new 16S rRNA gene fragments in the DGGE profile corresponded with the 16S rRNA genes of 2,4-D-degrading transconjugant colonies isolated on agar plates. This result indicates that the observed change in the community was due to proliferation of transconjugants formed in soil. Overall, this work clearly demonstrates that bioaugmentation can constitute an effective strategy for cleanup of soils which are poor in nutrients and microbial activity, such as those of the B horizon.     

Secondary successional trajectories of structural and catabolic bacterial communities in oil‐polluted soil planted with hybrid poplar

Poplars have widely been used for rhizoremediation of a broad range of organic contaminants for the past two decades. Still, there is a knowledge gap regarding the rhizosphere‐associated bacterial communities of poplars and their dynamics during the remediation process. It is envisaged that a detailed understanding of rhizosphere‐associated microbial populations will greatly contribute to a better design and implementation of rhizoremediation. To investigate the long‐term succession of structural and catabolic bacterial communities in oil‐polluted soil planted with hybrid poplar, we carried out a 2‐year field study. Hybrid aspen (Populus tremula × Populus tremuloides) seedlings were planted in polluted soil excavated from an accidental oil‐spill site. Vegetated and un‐vegetated soil samples were collected for microbial community analyses at seven different time points during the course of 2 years and sampling time points were chosen to cover the seasonal variation in the boreal climate zone. Bacterial community structure was accessed by means of 16S rRNA gene amplicon pyrosequencing, whereas catabolic diversity was monitored by pyrosequencing of alkane hydroxylase and extradiol dioxygenase genes. We observed a clear succession of bacterial communities on both structural and functional levels from early to late‐phase communities. Sphingomonas type extradiol dioxygenases and alkane hydroxylase homologs of Rhodococcus clearly dominated the early‐phase communities. The high‐dominance/low‐diversity functional gene communities underwent a transition to low‐dominance/high‐diversity communities in the late phase. These results pointed towards increased catabolic capacities and a change from specialist to generalist strategy of bacterial communities during the course of secondary succession.