Phylogeny of 16S rRNA and nifH genes and regulation of nitrogenase activity by oxygen and ammonium in the genus Paenibacillus

Xie, Jianbo; Bai, L. Q.; Wang, L. Y.; Chen, S. F.

doi:10.1134/s0026261712060173

Cited by 14 publications

(18 citation statements)

References 22 publications

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“…WLY78 is a gram-positive, facultative anaerobic, endospore-forming bacterium isolated from the rhizosphere of bamboo [14]. This bacterium has potential use in agriculture, since it is able to fix nitrogen and also produces antimicrobial substances.…”

Section: Resultsmentioning

confidence: 99%

“…WLY78 was isolated from the rhizosphere of bamboo in Beijing, China by enrichment in nitrogen-free medium after heating at 100°C for 10 min [14]. Strain WLY78 is similar to P. polymyxa based on 16S rDNA phylogeny and whole genome sequencing.…”

Section: Methodsmentioning

confidence: 99%

“…Nitrogen-deficient medium contained 2 mM glutamate as nitrogen source in nitrogen-free medium. Nitrogen-excess medium contained 100 mM NH 4 Cl in nitrogen-free medium [14].…”

Section: Methodsmentioning

confidence: 99%

“…Then, 1 ml of the culture was transferred to a 25-ml test tube and the test tube was sealed with robber stopper. The headspace in the tube was then evacuated and replaced with argon gas [14]. After incubating the cultures for 6–8 h at 30°C with shaking at 250 rpm, C 2 H 2 (10% of the headspace volume) was injected into the test tubes.…”

Section: Methodsmentioning

confidence: 99%

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A Minimal Nitrogen Fixation Gene Cluster from Paenibacillus sp. WLY78 Enables Expression of Active Nitrogenase in Escherichia coli

et al. 2013

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Most biological nitrogen fixation is catalyzed by molybdenum-dependent nitrogenase, an enzyme complex comprising two component proteins that contains three different metalloclusters. Diazotrophs contain a common core of nitrogen fixation nif genes that encode the structural subunits of the enzyme and components required to synthesize the metalloclusters. However, the complement of nif genes required to enable diazotrophic growth varies significantly amongst nitrogen fixing bacteria and archaea. In this study, we identified a minimal nif gene cluster consisting of nine nif genes in the genome of Paenibacillus sp. WLY78, a gram-positive, facultative anaerobe isolated from the rhizosphere of bamboo. We demonstrate that the nif genes in this organism are organized as an operon comprising nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA and nifV and that the nif cluster is under the control of a σ70 (σA)-dependent promoter located upstream of nifB. To investigate genetic requirements for diazotrophy, we transferred the Paenibacillus nif cluster to Escherichia coli. The minimal nif gene cluster enables synthesis of catalytically active nitrogenase in this host, when expressed either from the native nifB promoter or from the T7 promoter. Deletion analysis indicates that in addition to the core nif genes, hesA plays an important role in nitrogen fixation and is responsive to the availability of molybdenum. Whereas nif transcription in Paenibacillus is regulated in response to nitrogen availability and by the external oxygen concentration, transcription from the nifB promoter is constitutive in E. coli, indicating that negative regulation of nif transcription is bypassed in the heterologous host. This study demonstrates the potential for engineering nitrogen fixation in a non-nitrogen fixing organism with a minimum set of nine nif genes.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…Nitrogen-deficient medium contained 2 mM glutamate as nitrogen source in nitrogen-free medium. Nitrogen-excess medium contained 100 mM NH 4 Cl in nitrogen-free medium [14].…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

A Minimal Nitrogen Fixation Gene Cluster from Paenibacillus sp. WLY78 Enables Expression of Active Nitrogenase in Escherichia coli

et al. 2013

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“…Then, 3-5 ml of suspension was transferred to a 26 ml test tube which was sealed with rubber stopper. The headspace in the tube was then vacuumed and filled with argon gas (42). After C 2 H 2 (10 % of the headspace volume) was injected into the test tubes, the cultures were incubated at 30°C and with shaking at 250 rpm.…”

Section: Methodsmentioning

confidence: 99%

The role of Fnr paralogs for controlling anaerobic metabolism in diazotrophPaenibacillus polymyxaWLY78

Shi

Hao

et al. 2020

Preprint

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Fnr is a transcriptional regulator that controls the expression of a variety of genes in response to oxygen limitation in bacteria. Genome sequencing revealed four genes (fnr1, fnr3, fnr5 and fnr7) coding for Fnr proteins in Paenibacillus polymyxa WLY78. Fnr1 and Fnr3 showed more similarity to each other than to Fnr5 and Fnr7. Also, Fnr1 and Fnr3 exhibited high similarity with Bacillus cereus Fnr and Bacillus subtilis Fnr in sequence and structures. Deletion analysis showed that the four fnr genes, especially fnr1 and fnr3, have significant impacts on the growth and nitrogenase activity. Single deletion of fnr1 or fnr3 led to 50% reduction in nitrogenase activity and double deletion of fnr1 and fnr3 resulted to 90% reduction in activity. Both of the aerobically purified His-tagged Fnr1 and His-tagged Fnr3 in Escherichia coli could bind to the specific DNA promoter. Genome-wide transcription analysis showed that Fnr1 and Fnr3 indirectly activated expression of nif (nitrogen fixation) genes and Fe transport genes under anaerobic condition. Fnr1 and Fnr3 inhibited expression of the genes involved in aerobic respiratory chain and activated expression of genes responsible for anaerobic electron acceptor genes.IMPORTANCEPaenibacillus is a genus of Gram-positive, facultative anaerobic and endospore-forming bacteria. The members of nitrogen-fixing Paenibacillus have great potential use as a bacterial fertilizer in agriculture. However, the functions of fnr gene(s) in nitrogen fixation and other metabolisms in Paenibacillus spp. are not known. Here, we revealed that copy numbers vary largely among different Paenibacillus species and strains. Deletion and complementation analysis demonstrated that fnr1 and fnr3 have significant impacts on the growth and nitrogenase activity. Both of the aerobically purified His-tagged Fnr1 and His-tagged Fnr3 purified in Escherichia coli could bind to the specific DNA promoter as Bacillus cereus Fnr did. Fnr1 and Fnr3 indirectly activated nif expression under anaerobic condition. Fnr1 and Fnr3 directly or indirectly activated or inhibited expression of many important genes involved in respiration, energy metabolism, Fe uptake and potentially specific electron transport for nitrogenase under anaerobic condition. This study not only reveals the roles of fnr genes in nitrogen fixation and anaerobic metabolism, but also provides insight into the evolution and regulatory mechanisms of fnr in Paenibacillus.

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Paenibacillus helianthi sp. nov., a nitrogen fixing species isolated from the rhizosphere of Helianthus annuus L.

Ambrosini

Sant’Anna

Heinzmann

et al. 2018

Antonie van Leeuwenhoek

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Three facultatively anaerobic endospore-forming bacteria were isolated from the rhizosphere of sunflowers grown in fields of Rio Grande do Sul State, Brazil. The designated type strain P26E was previously identified as a sunflower growth promoting bacterium and is able to fix nitrogen and to excrete ammonia. According to analyses of 16S rRNA gene sequences, P26E presented similarity values above 98.8% in relation to Paenibacillus azotifigens NF2-4-5, Paenibacillus graminis RSA19, Paenibacillus jilunlii Be17, Paenibacillus salinicaeni LAM0A28, and Paenibacillus sonchi X19-5. Phylogenetic reconstructions based on 16S rRNA gene and core proteome data showed that the strains P26E, P3E and P32E form a distinct clade, which did not include any type strain of the currently described Paenibacillus species. Also, genomic comparisons using average nucleotide identity (ANI), Orthologous ANI and in silico DNA-DNA hybridization revealed similarity ranges below the recommended thresholds when the three isolates from sunflower were compared to their close relatives. The DNA G + C content of strain P26E was determined to be 49.4 mol%. The major cellular fatty acids are anteiso-C and iso-C, representing about 58 and 14% of the total fatty acids in P26E, respectively. Based on different taxonomic genomic metrics, phylogeny, and phenotypic data, we propose that strain P26E (= DSM 102269 = BR10509) represents a novel species within the genus Paenibacillus, for which the name Paenibacillus helianthi sp. nov. is proposed.

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Phylogeny of 16S rRNA and nifH genes and regulation of nitrogenase activity by oxygen and ammonium in the genus Paenibacillus

Cited by 14 publications

References 22 publications

A Minimal Nitrogen Fixation Gene Cluster from Paenibacillus sp. WLY78 Enables Expression of Active Nitrogenase in Escherichia coli

A Minimal Nitrogen Fixation Gene Cluster from Paenibacillus sp. WLY78 Enables Expression of Active Nitrogenase in Escherichia coli

The role of Fnr paralogs for controlling anaerobic metabolism in diazotrophPaenibacillus polymyxaWLY78

Paenibacillus helianthi sp. nov., a nitrogen fixing species isolated from the rhizosphere of Helianthus annuus L.

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