Determination of the most frequent N<sub>2</sub>-fixing bacteria in a rice rhizosphere

Bally, René; Thomas-Bauzon, Danièle; Heulin, T.; Balandreau, J.; Richard, C; Ley, J. De

doi:10.1139/m83-143

Cited by 146 publications

(48 citation statements)

References 26 publications

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“…Rhizobium etli CFN42 was grown in tryptone-yeast extract medium supplemented with 6 mM CaCl 2 (9). For cloning purposes, Escherichia coli strains containing recombinant plasmids were grown in 2YT (yeast extract [5 ]) broth or agar at 37°C with chloramphenicol at 100 g ml Ϫ1 (53).…”

Section: Methodsmentioning

confidence: 99%

Bacteriophage Prevalence in the Genus Azospirillum and Analysis of the First Genome Sequence of an Azospirillum brasilense Integrative Phage

Boyer

Haurat

Samain

et al. 2008

Appl Environ Microbiol

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The prevalence of bacteriophages was investigated in 24 strains of four species of plant growth-promoting rhizobacteria belonging to the genus Azospirillum. Upon induction by mitomycin C, the release of phage particles was observed in 11 strains from three species. Transmission electron microscopy revealed two distinct sizes of particles, depending on the identity of the Azospirillum species, typical of the Siphoviridae family. Pulsed-field gel electrophoresis and hybridization experiments carried out on phage-encapsidated DNAs revealed that all phages isolated from A. lipoferum and A. doebereinerae strains had a size of about 10 kb whereas all phages isolated from A. brasilense strains displayed genome sizes ranging from 62 to 65 kb. Strong DNA hybridizing signals were shown for most phages hosted by the same species whereas no homology was found between phages harbored by different species. Moreover, the complete sequence of the A. brasilense Cd bacteriophage (⌽Ab-Cd) genome was determined as a double-stranded DNA circular molecule of 62,337 pb that encodes 95 predicted proteins. Only 14 of the predicted proteins could be assigned functions, some of which were involved in DNA processing, phage morphogenesis, and bacterial lysis. In addition, the ⌽Ab-Cd complete genome was mapped as a prophage on a 570-kb replicon of strain A. brasilense Cd, and a region of 27.3 kb of ⌽Ab-Cd was found to be duplicated on the 130-kb pRhico plasmid previously sequenced from A. brasilense Sp7, the parental strain of A. brasilense Cd.

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Section: Methodsmentioning

confidence: 99%

Bacteriophage Prevalence in the Genus Azospirillum and Analysis of the First Genome Sequence of an Azospirillum brasilense Integrative Phage

Boyer

Haurat

Samain

et al. 2008

Appl Environ Microbiol

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“…A. lipoferum 4T retains the ability to efficiently colonize rice roots (2). Like 4V I , A. lipoferum 4T was found to be genetically very close to A. lipoferum 4B (3,8,28), suggesting that A. lipoferum 4T could in fact be a 4V I variant of strain 4B generated within the soil ecosystem.…”

Section: ϫ3mentioning

confidence: 95%

“…The 4V I variant exhibits pleiotropic modifications; it gained assimilation of certain sugars but lost the ability to assimilate other sugars (3), to reduce triphenyl tetrazolium chloride, to bind some dyes, to swim (4), and to reduce nitrous oxide (our unpublished results). A. lipoferum 4T, a nonswimming strain displaying all of the features of the 4V I variant, and strain A. lipoferum 4B have been isolated simultaneously from rice rhizosphere at the same frequency (8). A. lipoferum 4T retains the ability to efficiently colonize rice roots (2).…”

Section: ϫ3mentioning

confidence: 99%

Phase Variation and Genomic Architecture Changes inAzospirillum

et al. 2006

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The plant growth-promoting rhizobacterium Azospirillum lipoferum 4B generates in vitro at high frequency a stable nonswimming phase variant designated 4V I , which is distinguishable from the wild type by the differential absorption of dyes. The frequency of variants generated by a recA mutant of A. lipoferum 4B was increased up to 10-fold. The pleiotropic modifications characteristic of the phase variant are well documented, but the molecular processes involved are unknown. Here, the objective was to assess whether genomic rearrangements take place during phase variation of strain 4B. The random amplified polymorphic DNA (RAPD) profiles of strains 4B and 4V I differed. RAPD fragments observed only with the wild type were cloned, and three cosmids carrying the corresponding fragments were isolated. The three cosmids hybridized with a 750-kb plasmid and pulse-field gel electrophoresis analysis revealed that this replicon was missing in the 4V I genome. The same rearrangements took place during phase variation of 4BrecA. Large-scale genomic rearrangements during phase variation were demonstrated for two additional strains. In Azospirillum brasilense WN1, generation of stable variants was correlated with the disappearance of a replicon of 260 kb. For Azospirillum irakense KBC1, the variant was not stable and coincided with the formation of a new replicon, whereas the revertant recovered the parental genomic architecture. This study shows large-scale genomic rearrangements in Azospirillum strains and correlates them with phase variation.

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“…In the rice root zone, N 2 fixation is associated with the activity of N 2 -fixing heterotrophic bacteria (4,20). It has been reported that a large percentage of the total aerobic heterotrophic population in the root is diazotrophic (1,10,17). However, studies on the rhizospheric N 2 -fixing microflora have until now suffered from the use of selective media for counts and isolations, because it is widely believed that only a small percentage of natural prokaryotes may actually be culturable (18,19).…”

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confidence: 99%

Remarkable N2-fixing bacterial diversity detected in rice roots by molecular evolutionary analysis of nifH gene sequences

et al. 1995

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To demonstrate the extent of phylogenetic diversity of diazotrophic bacteria associated with rice roots, we characterized phylogenetically 23 nifH gene sequences obtained by PCR amplification of mixed organism DNA extracted directly from rice roots without culturing the organisms. The analyses document the presence of eight novel NifH types, which appear to be a variety of significant components of the diazotrophic community, dominated mainly by proteobacteria.The method developed by Pace et al. to determine species diversity and composition, using rRNA (or ribosomal DNA) isolated directly from nature has opened a window into the world of unculturable bacteria, although this method has the disadvantage that organisms whose genes have been isolated by the method cannot be studied for any other trait (11,16). This report extends the method by applying it to a functionally important gene, nifH, to determine the importance and diversity of nitrogen-fixing bacteria associated with rice roots. The nitrogenase iron protein gene nifH is one of the oldest existing and functioning genes in the history of gene evolution, and the outline of the NifH tree is reported to be largely consistent with the 16S rRNA phylogeny (21,22). These features prompted us to study the genetic diversity of N 2 -fixing bacteria by the molecular evolutionary analysis of nifH sequences amplified directly from rice root DNA, because the rice root DNA contains not only plant DNA but also microbial DNA in the roots. In the rice root zone, N 2 fixation is associated with the activity of N 2 -fixing heterotrophic bacteria (4,20). It has been reported that a large percentage of the total aerobic heterotrophic population in the root is diazotrophic (1, 10, 17). However, studies on the rhizospheric N 2 -fixing microflora have until now suffered from the use of selective media for counts and isolations, because it is widely believed that only a small percentage of natural prokaryotes may actually be culturable (18,19). Here we report a study of N 2 -fixing bacterial diversity by analysis of nifH gene sequences without a cultivation technique.DNA extraction. Rice, Oryza sativa L. cv. nihonnbare, was raised under flooded conditions in the Kyushu University Farm. Rice plants taken at the heading stage in September were dug out from a wetland rice field, and the roots were washed to remove the attached soil. The washed roots were cut into segments, frozen with liquid nitrogen, and ground to a fine powder in a mortar and pestle. The fine powder was suspended in extraction buffer (100 mM Tris, 100 mM EDTA, 250 mM NaCl, 100 g of proteinase K per ml) supplemented with Sarkosyl (1% final concentration) and lysed by incubation at 55ЊC for 1 h. Treatment of the lysate with RNase A was followed by chloroform extraction and isopropanol precipitation. Crude DNA was purified by phenol extraction, chloroform extraction, and isopropanol precipitation.PCR amplification of nifH genes. The primers for PCR amplification were chosen by careful inspection of the 37 published ni...

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Determination of the most frequent N₂-fixing bacteria in a rice rhizosphere

Cited by 146 publications

References 26 publications

Bacteriophage Prevalence in the Genus Azospirillum and Analysis of the First Genome Sequence of an Azospirillum brasilense Integrative Phage

Bacteriophage Prevalence in the Genus Azospirillum and Analysis of the First Genome Sequence of an Azospirillum brasilense Integrative Phage

Phase Variation and Genomic Architecture Changes inAzospirillum

Remarkable N2-fixing bacterial diversity detected in rice roots by molecular evolutionary analysis of nifH gene sequences

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Determination of the most frequent N2-fixing bacteria in a rice rhizosphere

Cited by 146 publications

References 26 publications

Bacteriophage Prevalence in the Genus Azospirillum and Analysis of the First Genome Sequence of an Azospirillum brasilense Integrative Phage

Bacteriophage Prevalence in the Genus Azospirillum and Analysis of the First Genome Sequence of an Azospirillum brasilense Integrative Phage

Phase Variation and Genomic Architecture Changes inAzospirillum

Remarkable N2-fixing bacterial diversity detected in rice roots by molecular evolutionary analysis of nifH gene sequences

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Determination of the most frequent N₂-fixing bacteria in a rice rhizosphere