RESUMO
Microbial inoculants are attracting growing interest in agriculture, but their efficacy remains unreliable in relation to their poor survival, partly due to the competition with the soil resident community. We hypothesised that recurrent inoculation could gradually alleviate this competition and improve the survival of the inoculant while increasing its impact on the resident bacterial community. We tested the effectiveness of such strategy with four inoculation sequences of Pseudomonas fluorescens strain B177 in soil microcosms with increasing number and frequency of inoculation, compared to a non-inoculated control. Each sequence was carried out at two inoculation densities (106 and 108 cfu.g soil-1). The four-inoculation sequence induced a higher abundance of P. fluorescens, 2 weeks after the last inoculation. No impact of inoculation sequences was observed on the resident community diversity and composition. Differential abundance analysis identified only 28 out of 576 dominants OTUs affected by the high-density inoculum, whatever the inoculation sequence. Recurrent inoculations induced a strong accumulation of nitrate, not explained by the abundance of nitrifying or nitrate-reducing microorganisms. In summary, inoculant density rather than inoculation pattern matters for inoculation effect on the resident bacterial communities, while recurrent inoculation allowed to slightly enhance the survival of the inoculant and strongly increased soil nitrate content.
Assuntos
Inoculantes Agrícolas , Pseudomonas fluorescens , Solo , Nitratos , Agricultura , Microbiologia do SoloRESUMO
The incorporation of plant residues into soil not only represents an opportunity to limit soil organic matter depletion resulting from cultivation but also provides a valuable source of nutrients such as nitrogen. However, the consequences of plant residue addition on soil microbial communities involved in biochemical cycles other than the carbon cycle are poorly understood. In this study, we investigated the responses of one N-cycling microbial community, the nitrate reducers, to wheat, rape, and alfalfa residues for 11 months after incorporation into soil in a field experiment. A 20- to 27-fold increase in potential nitrate reduction activity was observed for residue-amended plots compared to the nonamended plots during the first week. This stimulating effect of residues on the activity of the nitrate-reducing community rapidly decreased but remained significant over 11 months. During this period, our results suggest that the potential nitrate reduction activity was regulated by both carbon availability and temperature. The presence of residues also had a significant effect on the abundance of nitrate reducers estimated by quantitative PCR of the narG and napA genes, encoding the membrane-bound and periplasmic nitrate reductases, respectively. In contrast, the incorporation of the plant residues into soil had little impact on the structure of the narG and napA nitrate-reducing community determined by PCR-restriction fragment length polymorphism (RFLP) fingerprinting. Overall, our results revealed that the addition of plant residues can lead to important long-term changes in the activity and size of a microbial community involved in N cycling but with limited effects of the type of plant residue itself.
Assuntos
Nitrato Redutase/metabolismo , Fixação de Nitrogênio , Plantas/metabolismo , Microbiologia do Solo , Biota , DNA Bacteriano/análise , DNA Bacteriano/genética , Plantas/microbiologia , Reação em Cadeia da Polimerase , Solo/análise , Fatores de TempoRESUMO
The main objective of this study was to determine how the size, structure, and activity of the nitrate reducer community were affected by adoption of a conservative tillage system as an alternative to conventional tillage. The experimental field, established in Madagascar in 1991, consists of plots subjected to conventional tillage or direct-seeding mulch-based cropping systems (DM), both amended with three different fertilization regimes. Comparisons of size, structure, and activity of the nitrate reducer community in samples collected from the top layer in 2005 and 2006 revealed that all characteristics of this functional community were affected by the tillage system, with increased nitrate reduction activity and numbers of nitrate reducers under DM. Nitrate reduction activity was also stimulated by combined organic and mineral fertilization but not by organic fertilization alone. In contrast, both negative and positive effects of combined organic and mineral fertilization on the size of the nitrate reducer community were observed. The size of the nitrate reducer community was a significant predictor of the nitrate reduction rates except in one treatment, which highlighted the inherent complexities in understanding the relationships the between size, diversity, and structure of functional microbial communities along environmental gradients.
Assuntos
Agricultura/métodos , Biodiversidade , Nitratos/metabolismo , Microbiologia do Solo , Madagáscar , Nitritos/metabolismo , OxirreduçãoRESUMO
To determine to which extent root-derived carbon contributes to the effects of plants on nitrate reducers and denitrifiers, four solutions containing different proportions of sugar, organic acids and amino acids mimicking maize root exudates were added daily to soil microcosms at a concentration of 150 microg C g(-1) of soil. Water-amended soils were used as controls. After 1 month, the size and structure of the nitrate reducer and denitrifier communities were analysed using the narG and napA, and the nirK, nirS and nosZ genes as molecular markers respectively. Addition of artificial root exudates (ARE) did not strongly affect the structure or the density of nitrate reducer and denitrifier communities whereas potential nitrate reductase and denitrification activities were stimulated by the addition of root exudates. An effect of ARE composition was also observed on N(2)O production with an N(2)O:(N(2)O + N(2)) ratio of 0.3 in microcosms amended with ARE containing 80% of sugar and of 1 in microcosms amended with ARE containing 40% of sugar. Our study indicated that ARE stimulated nitrate reduction or denitrification activity with increases in the range of those observed with the whole plant. Furthermore, we demonstrated that the composition of the ARE affected the nature of the end-product of denitrification and could thus have a putative impact on greenhouse gas emissions.
Assuntos
Bactérias/classificação , Bactérias/isolamento & purificação , Biodiversidade , Nitratos/metabolismo , Nitritos/metabolismo , Raízes de Plantas/microbiologia , Microbiologia do Solo , Bactérias/metabolismo , Proteínas de Bactérias/genética , Carbono/metabolismo , DNA Bacteriano/química , DNA Bacteriano/genética , Dados de Sequência Molecular , Oxirredução , Oxirredutases/genética , Análise de Sequência de DNA , Zea maysRESUMO
We investigated the effects of internal primer-template mismatches on the efficiency of PCR amplification using the 16S rRNA gene as the model template DNA. We observed that the presence of a single mismatch in the second half of the primer extension sequence can result in an underestimation of up to 1,000-fold of the gene copy number, depending on the primer and position of the mismatch.
Assuntos
Pareamento Incorreto de Bases/genética , Primers do DNA/genética , Reação em Cadeia da Polimerase/métodos , Dosagem de Genes/genética , Pseudomonas aeruginosa/genética , RNA Ribossômico 16S/genéticaRESUMO
In this study, we report the cloning and characterization of denitrifying gene clusters of Pseudomonas fluorescens C7R12 containing the narXLDKGHJI, nirPOQSM, norCB and nosRZDFYL genes. While consensus sequences for Fnr-like protein binding sites were identified in the promoter regions of the nar, nir, nor and nos genes, consensus sequences corresponding to the NarL binding sites were identified only upstream the nar genes. Monitoring by mRNA analysis the expression of the narG, nirS, norB and nosZ structural genes suggests a sequential induction of the denitrification system in P. fluorescens.
Assuntos
Genes Bacterianos , Família Multigênica , Pseudomonas fluorescens/genética , Sequência de Bases , Clonagem Molecular , DNA Bacteriano/genética , Dados de Sequência Molecular , Nitrogênio/metabolismo , Regiões Promotoras Genéticas , Pseudomonas fluorescens/metabolismo , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência do Ácido NucleicoRESUMO
The dissimilative membrane-bound nitrate reductase from Pseudomonas fluorescens strain AK15 was purified and the alpha subunit of the enzyme partially sequenced. On the basis of this partial amino acid sequence and of conserved stretches of amino acids between Escherichia coli and Bacillus subtilis, degenerate primers were design to amplify the narG gene and part of the narH gene in a PCR approach. The deduced amino acid sequence of narG shows 72% and 52% and narH 78% and 62% identity to the homologous subunit of E. coli and B. subtilis, respectively.
Assuntos
Genes Bacterianos/genética , Nitrato Redutases/genética , Nitrato Redutases/isolamento & purificação , Pseudomonas fluorescens/enzimologia , Sequência de Aminoácidos , Clonagem Molecular , Dados de Sequência Molecular , Nitrato Redutase , Pseudomonas fluorescens/genética , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
Little information is available regarding the landscape-scale distribution of microbial communities and its environmental determinants. However, a landscape perspective is needed to understand the relative importance of local and regional factors and land management for the microbial communities and the ecosystem services they provide. In the most comprehensive analysis of spatial patterns of microbial communities to date, we investigated the distribution of functional microbial communities involved in N-cycling and of the total bacterial and crenarchaeal communities over 107 sites in Burgundy, a 31,500 km(2) region of France, using a 16 × 16 km(2) sampling grid. At each sampling site, the abundance of total bacteria, crenarchaea, nitrate reducers, denitrifiers- and ammonia oxidizers were estimated by quantitative PCR and 42 soil physico-chemical properties were measured. The relative contributions of land use, spatial distance, climatic conditions, time, and soil physico-chemical properties to the spatial distribution of the different communities were analyzed by canonical variation partitioning. Our results indicate that 43-85% of the spatial variation in community abundances could be explained by the measured environmental parameters, with soil chemical properties (mostly pH) being the main driver. We found spatial autocorrelation up to 739 km and used geostatistical modelling to generate predictive maps of the distribution of microbial communities at the landscape scale. The present study highlights the potential of a spatially explicit approach for microbial ecology to identify the overarching factors driving the spatial heterogeneity of microbial communities even at the landscape scale.
Assuntos
Fenômenos Fisiológicos Bacterianos , Crenarchaeota/fisiologia , Ecossistema , Nitrogênio/metabolismo , Microbiologia do Solo , Bactérias/classificação , Bactérias/genética , Crenarchaeota/genética , Crenarchaeota/isolamento & purificação , França , RNA Ribossômico 16S/genética , Solo/químicaRESUMO
Although the impact of bioremediation of PCB-contaminated sites on the indigenous microbial community is a key question for soil restoration, it remains poorly understood. Therefore, a small-scale bioremediation assay made of (a) a biostimulation treatment with carvone, soya lecithin and xylose and (b) two bioaugmentation treatments, one with a TSZ7 mixed culture and another with a Rhodococcus sp. Z6 pure strain was set up. Changes in the structure of the global soil microbial community and in the abundances of different taxonomic phyla were monitored using ribosomal intergenic spacer analysis (RISA) and real-time PCR. After an 18-month treatment, the structure of the bacterial community in the bioremediated soils was significantly different from that of the native soil. The shift observed in the bacterial community structure using RISA analysis was in accordance with the monitored changes in the abundances of 11 targeted phyla and classes. Actinobacteria, Bacteriodetes and α- and γ-Proteobacteria were more abundant under all three bioremediation treatments, with Actinobacteria representing the dominant phylum. Altogether, our results indicate that bioremediation of PCB-contaminated soil induces significant changes in the structure and abundance of the total microbial community, which must be addressed to implement bioremediation practices in order to restore soil functions.
Assuntos
Biodegradação Ambiental , Recuperação e Remediação Ambiental/métodos , Bifenilos Policlorados/metabolismo , Rhodococcus/metabolismo , Microbiologia do Solo , Poluentes do Solo/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Extracting DNA directly from micro-organisms living in soil is a crucial step for the molecular analysis of soil microbial communities. However, the use of a plethora of different soil DNA extraction protocols, each with its own bias, makes accurate data comparison difficult. To overcome this problem, a method for soil DNA extraction was proposed to the International Organization for Standardization (ISO) in 2006. This method was evaluated by 13 independent European laboratories actively participating in national and international ring tests. The reproducibility of the standardized method for molecular analyses was evaluated by comparing the amount of DNA extracted, as well as the abundance and genetic structure of the total bacterial community in the DNA extracted from 12 different soils by the 13 laboratories. High quality DNA was successfully extracted from all 12 soils, despite different physical and chemical characteristics and a range of origins from arable soils, through forests to industrial sites. Quantification of the 16S rRNA gene abundances by real time PCR and analysis of the total bacterial community structure by automated ribosomal intergenic spacer analysis (A-RISA) showed acceptable to good levels of reproducibility. Based on the results of both ring-tests, the method was unanimously approved by the ISO as an international standard method and the normative protocol will now be disseminated within the scientific community. Standardization of a soil DNA extraction method will improve data comparison, facilitating our understanding of soil microbial diversity and soil quality monitoring.
Assuntos
DNA/isolamento & purificação , Técnicas Microbiológicas/métodos , Técnicas Microbiológicas/normas , Microbiologia do Solo , Técnicas de Tipagem Bacteriana/métodos , Impressões Digitais de DNA/métodos , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , DNA Ribossômico/genética , DNA Ribossômico/isolamento & purificação , RNA Ribossômico 16S/genética , Reprodutibilidade dos TestesRESUMO
The saline-sodic soil of the former Lake Texcoco, a large area exposed to desertification, is a unique environment, but little is known about its microbial ecology. The objective of this study was to examine bacterial community structure, activity, and function when biosolids were added to microcosms. The application rates were such that 0, 66, 132, or 265 mg total Nk g(-1) were added with the biosolids (total C and N content 158 and 11.5 g kg(-1) dry biosolids, respectively). Approximately 60% of the biosolids were mineralized within 90 days. Microbial respiration and to a lesser extent ammonification and nitrification, increased after biosolids application. The rRNA intergenic spacer analysis (RISA) patterns for the biosolids and unamended soil bacterial communities were different, indicating that the microorganisms in the biosolids were distinct from the native population. It appears that the survival of the allochthonous microorganisms was short, presumably due to the adverse soil conditions.
Assuntos
Bactérias/crescimento & desenvolvimento , Água Doce/microbiologia , Nitrogênio/metabolismo , Salinidade , Esgotos/microbiologia , Microbiologia do Solo , Amônia/metabolismo , Carbono/metabolismo , Dióxido de Carbono/metabolismo , DNA Espaçador Ribossômico/genética , México , VolatilizaçãoRESUMO
Dissimilatory nitrate reduction is catalyzed by a membrane-bound and a periplasmic nitrate reductase. We set up a real-time PCR assay to quantify these two enzymes, using the narG and napA genes, encoding the catalytic subunits of the two types of nitrate reductases, as molecular markers. The narG and napA gene copy numbers in DNA extracted from 18 different environments showed high variations, with most numbers ranging from 2 x 10(2) to 6.8 x 10(4) copies per ng of DNA. This study provides evidence that, in soil samples, the number of proteobacteria carrying the napA gene is often as high as that of proteobacteria carrying the narG gene. The high correlation observed between narG and napA gene copy numbers in soils suggests that the ecological roles of the corresponding enzymes might be linked.
Assuntos
Nitrato Redutase/metabolismo , Proteobactérias/enzimologia , Microbiologia do Solo , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , DNA Bacteriano/genética , Meio Ambiente , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Nitrato Redutase/biossíntese , Nitrato Redutase/genética , Proteínas Periplásmicas/metabolismo , Raízes de Plantas/microbiologia , Reação em Cadeia da Polimerase , Proteobactérias/classificação , Proteobactérias/genéticaRESUMO
During the last decade, application of molecular methods using cultivation-independent approaches has provided new insights into the composition and structure of denitrifying communities in various environments. However, little is known about their abundance, and quantification is still performed using cultivation-based approaches, which are not only biased by the inability to cultivate of many micro-organisms but also fastidious and time-consuming. Two types of cultivation-independent approaches have recently been developed to quantify denitrifiers. The first type, which is based on the hybridization technique, comprises the use of Southern hybridization and DNA arrays. The second type, based on PCR, comprises the use of MPN (most probable number)-PCR, competitive PCR or real-time PCR. In this review, these different approaches will be presented with examples of their application in environmental studies.
Assuntos
Meio Ambiente , Genes Bacterianos , Nitratos/metabolismo , Nitritos/metabolismo , Microbiologia do Solo , Hibridização de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase/métodosRESUMO
Changes in community structure and activity of the dissimilatory nitrate-reducing community were investigated across a glacier foreland in the Central Alps to gain insight into the successional pattern of this functional group and the driving environmental factors. Bulk soil and rhizosphere soil of Poa alpina was sampled in five replicates in August during the flowering stage and in September after the first snowfalls along a gradient from 25 to 129 years after deglaciation and at a reference site outside the glacier foreland (>2000 years deglaciated). In a laboratory-based assay, nitrate reductase activity was determined colorimetrically after 24 h of anaerobic incubation. In selected rhizosphere soil samples, the community structure of nitrate-reducing microorganisms was analysed by restriction fragment length polymorphism (RFLP) analysis using degenerate primers for the narG gene encoding the active site of the membrane-bound nitrate reductase. Clone libraries of the early (25 years) and late (129 years) succession were constructed and representative clones sequenced. The activity of the nitrate-reducing community increased significantly with age mainly due to higher carbon and nitrate availability in the late succession. The community structure, however, only showed a small shift over the 100 years of soil formation with pH explaining a major part (19%) of the observed variance. Clone library analysis of the early and late succession pointed to a trend of declining diversity with progressing age. Presumably, the pressure of competition on the nitrate reducers was relatively low in the early successional stage due to minor densities of microorganisms compared with the late stage; hence, a higher diversity could persist in this sparse environment. These results suggest that the nitrate reductase activity is regulated by environmental factors other than those shaping the genetic structure of the nitrate-reducing community.
Assuntos
Bactérias/classificação , Nitrato Redutase/metabolismo , Raízes de Plantas/microbiologia , Poa/microbiologia , Microbiologia do Solo , Bactérias/genética , Bactérias/metabolismo , Análise Discriminante , Ecossistema , Variação Genética/genética , Alemanha , Camada de Gelo , Dados de Sequência Molecular , Método de Monte Carlo , Nitrato Redutase/genética , Filogenia , Polimorfismo de Fragmento de Restrição , Fatores de TempoRESUMO
Nitrous oxide (N2O) is an important greenhouse gas in the troposphere controlling ozone concentration in the stratosphere through nitric oxide production. In order to quantify bacteria capable of N2O reduction, we developed a SYBR green quantitative real-time PCR assay targeting the nosZ gene encoding the catalytic subunit of the nitrous oxide reductase. Two independent sets of nosZ primers flanking the nosZ fragment previously used in diversity studies were designed and tested (K. Kloos, A. Mergel, C. Rösch, and H. Bothe, Aust. J. Plant Physiol. 28:991-998, 2001). The utility of these real-time PCR assays was demonstrated by quantifying the nosZ gene present in six different soils. Detection limits were between 10(1) and 10(2) target molecules per reaction for all assays. Sequence analysis of 128 cloned quantitative PCR products confirmed the specificity of the designed primers. The abundance of nosZ genes ranged from 10(5) to 10(7) target copies g(-1) of dry soil, whereas genes for 16S rRNA were found at 10(8) to 10(9) target copies g(-1) of dry soil. The abundance of narG and nirK genes was within the upper and lower limits of the 16S rRNA and nosZ gene copy numbers. The two sets of nosZ primers gave similar gene copy numbers for all tested soils. The maximum abundance of nosZ and nirK relative to 16S rRNA was 5 to 6%, confirming the low proportion of denitrifiers to total bacteria in soils.
Assuntos
Bacillus/enzimologia , Oxirredutases/genética , Reação em Cadeia da Polimerase/métodos , Proteobactérias/enzimologia , Microbiologia do Solo , Bacillus/genética , Benzotiazóis , Primers do DNA , Diaminas , Genes de RNAr/genética , Dados de Sequência Molecular , Nitrato Redutase/genética , Nitrito Redutases/genética , Compostos Orgânicos , Filogenia , Proteobactérias/genética , Quinolinas , RNA Ribossômico 16S/genética , Sensibilidade e Especificidade , Análise de Sequência de DNA , Solo/análiseRESUMO
The ability to respire nitrate when oxygen is limited has been described in taxonomically diverse microorganisms including members of the alpha-, beta-, gamma- and epsilon-proteobacteria, high and low GC Gram-positive bacteria and even Archaea. Respiratory nitrate reduction is the first step of the denitrification pathway, which is important since it is the main biological process responsible for the return of fixed nitrogen to the atmosphere, thus completing the nitrogen cycle. During the last decade, considerable knowledge has been accumulated on the biochemistry and genetics of the nitrate reductases. In this paper, we summarize the recent progress in molecular approaches for studying the ecology of the nitrate-reducing community in the environment.
Assuntos
Bactérias/metabolismo , Óxido Nítrico/metabolismo , Bactérias/classificação , Bactérias/genética , Genes Bacterianos , Oxirredução , Filogenia , Reação em Cadeia da PolimeraseRESUMO
A total of 1246 Pseudomonas strains were isolated from the rhizosphere of two perennial grasses (Lolium perenne and Molinia coerulea) with different nitrogen requirements. The plants were grown in their native soil under ambient and elevated atmospheric CO2 content (pCO2) at the Swiss FACE (Free Air CO2 Enrichment) facility. Root-, rhizosphere-, and non-rhizospheric soil-associated strains were characterized in terms of their ability to reduce nitrate during an in vitro assay and with respect to the genes encoding the membrane-bound (named NAR) and periplasmic (NAP) nitrate reductases so far described in the genus Pseudomonas. The diversity of corresponding genes was assessed by PCR-RFLP on narG and napA genes, which encode the catalytic subunit of nitrate reductases. The frequency of nitrate-dissimilating strains decreased with root proximity for both plants and was enhanced under elevated pCO2 in the rhizosphere of L. perenne. NAR (54% of strains) as well as NAP (49%) forms were present in nitrate-reducing strains, 15.5% of the 439 strains tested harbouring both genes. The relative proportions of narG and napA detected in Pseudomonas strains were different according to root proximity and for both pCO2 treatments: the NAR form was more abundant close to the root surface and for plants grown under elevated pCO2. Putative denitrifiers harbored mainly the membrane-bound (NAR) form of nitrate reductase. Finally, both narG and napA sequences displayed a high level of diversity. Anyway, this diversity was correlated neither with the root proximity nor with the pCO2 treatment.
Assuntos
Variação Genética , Nitrato Redutases/genética , Raízes de Plantas/microbiologia , Poaceae/microbiologia , Pseudomonas/genética , Microbiologia do Solo , Dióxido de Carbono/metabolismo , Contagem de Colônia Microbiana , Primers do DNA , Proteínas de Membrana/metabolismo , Proteínas Periplásmicas/metabolismo , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Pseudomonas/enzimologia , SuíçaRESUMO
Involvement of nitrate reductase and pyoverdine in the competitiveness of the biocontrol strain Pseudomonas fluorescens C7R12 was determined, under gnotobiotic conditions, in two soil compartments (bulk and rhizosphere soil), with the soil being kept at two different values of matric potential (-1 and -10 kPa). Three mutants affected in the synthesis of either the nitrate reductase (Nar(-)), the pyoverdine (Pvd(-)), or both (Nar(-) Pvd(-)) were used. The Nar(-) and Nar(-) Pvd(-) mutants were obtained by site-directed mutagenesis of the wild-type strain and of the Pvd(-) mutant, respectively. The selective advantage given by nitrate reductase and pyoverdine to the wild-type strain was assessed by measuring the dynamic of each mutant-to-total-inoculant (wild-type strain plus mutant) ratio. All three mutants showed a lower competitiveness than the wild-type strain, indicating that both nitrate reductase and pyoverdine are involved in the fitness of P. fluorescens C7R12. The double mutant presented the lowest competitiveness. Overall, the competitive advantages given to C7R12 by nitrate reductase and pyoverdine were similar. However, the selective advantage given by nitrate reductase was more strongly expressed under conditions of lower aeration (-1 kPa). In contrast, the selective advantage given by nitrate reductase and pyoverdine did not differ in bulk and rhizosphere soil, indicating that these bacterial traits are not specifically involved in the rhizosphere competence but rather in the saprophytic ability of C7R12 in soil environments.
Assuntos
Nitrato Redutases/metabolismo , Oligopeptídeos , Pigmentos Biológicos/metabolismo , Pseudomonas fluorescens/crescimento & desenvolvimento , Microbiologia do Solo , Controle de Doenças Transmissíveis , Solanum lycopersicum/microbiologia , Mutação , Nitrato Redutase , Nitrato Redutases/genética , Pigmentos Biológicos/genética , Raízes de Plantas/microbiologia , Pseudomonas fluorescens/genética , Seleção GenéticaRESUMO
We examined the influence of soil aeration state and plant root presence on the comparative survival of wild-type bacteria and isogenic Tn5 (Nir(sup-)) mutants lacking the ability to synthesize nitrite reductase. Two denitrifying Pseudomonas strains with different nitrite reductase types were used. Enumeration of bacteria in sterile and nonsterile soils was based on differential antibiotic resistance. The validity of the bacterial models studied (i.e., equal growth of wild-type and mutant bacteria under aerobic conditions and significantly better growth of wild-type bacteria under denitrifying conditions) was verified in pure-culture studies. In sterile soil, both strains survived better under aerobic than under anaerobic conditions. The lower efficiency of denitrification than O(inf2) respiration in supporting bacterial growth explained this result, and the physical heterogeneity of soil did not strongly modify the results obtained in pure-culture studies. In nonsterile soil, one of the Pseudomonas strains survived better under anaerobic conditions while the other competed equally with the indigenous soil microflora under aerobic and anaerobic conditions. However, when the Nir(sup-)-to-total inoculant ratios (wild type plus Nir(sup-) mutant) were analyzed, it appeared that the presence of nitrite reductase conferred on both Pseudomonas strains a competitive advantage for anaerobic environment or rhizosphere colonization. This is the first attempt to demonstrate with isogenic nondenitrifying mutants that denitrification can contribute to the persistence and distribution of bacteria in fluctuating soil environments.