Involvement of the ammonium transporter AmtB in nitrogenase regulation and ammonium excretion in Pseudomonas stutzeri A1501

Zhang, Tao; Yan, Yongliang; He, Sheng; Ping, Shuzhen; Alam, Khandakar Mohiul; Han, Yunsheng; Liu, Xiaodong; Lu, Wei; Zhang, Wei; Chen, Ming; Xiang, Wensheng; Wang, Xiangjing; Lin, Min

doi:10.1016/j.resmic.2012.05.002

Cited by 33 publications

(39 citation statements)

References 44 publications

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“…In several diazotroph species, disruption of the NH 4 + transporter, AmtB, results in extracellular NH 4 + accumulation during N 2 fixation (Yakunin and Hallenbeck, 2002;Zhang et al, 2012;Barney et al, 2015). We deleted the genes for both AmtB homologs in R. palustris Nx, resulting in R. palustris NxΔAmtB, and found that NH 4 + accumulated to nearly three times to that of R. palustris Nx in monocultures (Supplementary Figure 2).…”

Section: Resultsmentioning

confidence: 99%

“…Metabolite excretion is a mechanism by which cooperative interactions can be initiated and sustained (Sachs et al, 2004). There is accumulating evidence that some diazotrophs excrete NH 4 + during N 2 fixation (Adam et al, 2016) and that AmtB functions to reacquire NH 4 + and limit its availability to nearby organisms (Supplementary Figure 2) (Yakunin and Hallenbeck, 2002;Zhang et al, 2012;Barney et al, 2015). As proposed in the Black Queen hypothesis (Morris et al, 2012), this NH 4 + leakage makes diazotrophs well-suited to establish nascent mutualisms, both in natural and synthetic communities.…”

Section: Discussionmentioning

confidence: 96%

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Microbial mutualism dynamics governed by dose-dependent toxicity of cross-fed nutrients

et al. 2016

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Microbial interactions, including mutualistic nutrient exchange (cross-feeding), underpin the flow of energy and materials in all ecosystems. Metabolic exchanges are difficult to assess within natural systems. As such, the impact of exchange levels on ecosystem dynamics and function remains unclear. To assess how cross-feeding levels govern mutualism behavior, we developed a bacterial coculture amenable to both modeling and experimental manipulation. In this coculture, which resembles an anaerobic food web, fermentative Escherichia coli and photoheterotrophic Rhodopseudomonas palustris obligately cross-feed carbon (organic acids) and nitrogen (ammonium). This reciprocal exchange enforced immediate stable coexistence and coupled species growth. Genetic engineering of R. palustris to increase ammonium cross-feeding elicited increased reciprocal organic acid production from E. coli, resulting in culture acidification. Consequently, organic acid function shifted from that of a nutrient to an inhibitor, ultimately biasing species ratios and decreasing carbon transformation efficiency by the community; nonetheless, stable coexistence persisted at a new equilibrium. Thus, disrupting the symmetry of nutrient exchange can amplify alternative roles of an exchanged resource and thereby alter community function. These results have implications for our understanding of mutualistic interactions and the use of microbial consortia as biotechnology.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 96%

Microbial mutualism dynamics governed by dose-dependent toxicity of cross-fed nutrients

et al. 2016

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“…There is an active debate, as well as contrasting results, concerning the role that amtB plays in the transport of ammonia or ammonium in various bacteria (38)(39)(40)(41), including reports of increasing extracellular ammonium when amtB is deleted while also demonstrating that cells can obtain ammonium from the extracellular space even in the absence of amtB (42,47). The amtB gene has been implicated in the release of ammonium previously in other bacteria.…”

Section: Discussionmentioning

confidence: 99%

“…Based on this finding and on what is already known about these various genes and the nature of the nitrogen screen, three potential explanations were considered to explain the nitrogen secreting phenotype following amtB disruption. First, deleting amtB might result in increased extracellular ammonium if loss of this gene hindered the ability of the strain to recover ammonia or ammonium that leaks from the cell by natural processes, as has been described in other strains (42). Second, amtB might play a role in the transport of an alternative nitrogen compound, which requires the amtB still present in the A. vinelandii AZBB063 biosensor strain to transport this compound back into the cell.…”

Section: Urea As a Terminal Nitrogen Compoundmentioning

confidence: 97%

Gene Deletions Resulting in Increased Nitrogen Release by Azotobacter vinelandii: Application of a Novel Nitrogen Biosensor

Barney

Eberhart

Ohlert

et al. 2015

Appl Environ Microbiol

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Azotobacter vinelandii is a widely studied model diazotrophic (nitrogen-fixing) bacterium and also an obligate aerobe, differentiating it from many other diazotrophs that require environments low in oxygen for the function of the nitrogenase. As a freeliving bacterium, A. vinelandii has evolved enzymes and transporters to minimize the loss of fixed nitrogen to the surrounding environment. In this study, we pursued efforts to target specific enzymes and further developed screens to identify individual colonies of A. vinelandii producing elevated levels of extracellular nitrogen. Targeted deletions were done to convert urea into a terminal product by disrupting the urease genes that influence the ability of A. vinelandii to recycle the urea nitrogen within the cell. Construction of a nitrogen biosensor strain was done to rapidly screen several thousand colonies disrupted by transposon insertional mutagenesis to identify strains with increased extracellular nitrogen production. Several disruptions were identified in the ammonium transporter gene amtB that resulted in the production of sufficient levels of extracellular nitrogen to support the growth of the biosensor strain. Further studies substituting the biosensor strain with the green alga Chlorella sorokiniana confirmed that levels of nitrogen produced were sufficient to support the growth of this organism when the medium was supplemented with sufficient sucrose to support the growth of the A. vinelandii in coculture. The nature and quantities of nitrogen released by urease and amtB disruptions were further compared to strains reported in previous efforts that altered the nifLA regulatory system to produce elevated levels of ammonium. These results reveal alternative approaches that can be used in various combinations to yield new strains that might have further application in biofertilizer schemes. N utrient requirements are directly linked to biomass production, and any potential increased improvement in the scale of biomass yield will necessitate a proportional increase in the demand for essential nutrients. For all photosynthetic organisms (photoautotrophs such as land plants, algae, and cyanobacteria) with requisite light energy and water, nitrogen is the most limiting and expensive nutrient input for aquaculture and agricultural production alike (1). A majority of our current nitrogen fertilizer production is tied to the burning of fossil fuels to generate ammonia from molecular nitrogen (N 2 gas) through the Haber-Bosch process, which accounts for 3 to 5% of world natural gas consumption, or about 1 to 2% of total worldwide energy expenditures (1-3). In developed countries, industrial nitrogen production is accompanied by a huge economic and energetic cost overall (2), while this key nutrient limits agricultural productivity in developing countries, where energy and infrastructure costs prohibit the utilization of the Haber-Bosch process to produce ammonia from atmospheric nitrogen on a large scale.The development of biological approaches to improve biof...

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“…This observation was further supported by the findings that the presence of low concentrations of nitrate [22] or ammonium [23] did not abolish nitrogen fixation ability of P. stutzeri A1501 while there is evidence that constitutive expression of nifA regulatory gene may result in enhanced nitrogen fixation even under high-ammonia conditions [24]. Recently, it has also been shown that constitutive expression of nifA enhanced ammonium excretions by an amtB1- amtB2 double mutant [25]. Furthermore, integration of a large fragment of P. stutzeri A1501 nitrogen fixation island (52 genes corresponding to PST_1302-PST1306 and PST_1313-PST_1359 regions) into a random genome site of P. fluorescence Pf-5 converted this bacterium to nitrogen fixer and ammonia producer [26].…”

Section: Introductionmentioning

confidence: 74%

The Nitrogen-Fixation Island Insertion Site Is Conserved in Diazotrophic Pseudomonas stutzeri and Pseudomonas sp. Isolated from Distal and Close Geographical Regions

et al. 2014

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The presence of nitrogen fixers within the genus Pseudomonas has been established and so far most isolated strains are phylogenetically affiliated to Pseudomonas stutzeri. A gene ortholog neighborhood analysis of the nitrogen fixation island (NFI) in four diazotrophic P. stutzeri strains and Pseudomonas azotifigens revealed that all are flanked by genes coding for cobalamin synthase (cobS) and glutathione peroxidise (gshP). The putative NFIs lack all the features characterizing a mobilizable genomic island. Nevertheless, bioinformatic analysis P. stutzeri DSM 4166 NFI demonstrated the presence of short inverted and/or direct repeats within both flanking regions. The other P. stutzeri strains carry only one set of repeats. The genetic diversity of eleven diazotrophic Pseudomonas isolates was also investigated. Multilocus sequence typing grouped nine isolates along with P. stutzeri and two isolates are grouped in a separate clade. A Rep-PCR fingerprinting analysis grouped the eleven isolates into four distinct genotypes. We also provided evidence that the putative NFI in our diazotrophic Pseudomonas isolates is flanked by cobS and gshP genes. Furthermore, we demonstrated that the putative NFI of Pseudomonas sp. Gr65 is flanked by inverted repeats identical to those found in P. stutzeri DSM 4166 and while the other P. stutzeri isolates harbor the repeats located in the intergenic region between cobS and glutaredoxin genes as in the case of P. stutzeri A1501. Taken together these data suggest that all putative NFIs of diazotrophic Pseudomonas isolates are anchored in an intergenic region between cobS and gshP genes and their flanking regions are designated by distinct repeats patterns. Moreover, the presence of almost identical NFIs in diazotrophic Pseudomonas strains isolated from distal geographical locations around the world suggested that this horizontal gene transfer event may have taken place early in the evolution.

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Involvement of the ammonium transporter AmtB in nitrogenase regulation and ammonium excretion in Pseudomonas stutzeri A1501

Cited by 33 publications

References 44 publications

Microbial mutualism dynamics governed by dose-dependent toxicity of cross-fed nutrients

Microbial mutualism dynamics governed by dose-dependent toxicity of cross-fed nutrients

Gene Deletions Resulting in Increased Nitrogen Release by Azotobacter vinelandii: Application of a Novel Nitrogen Biosensor

The Nitrogen-Fixation Island Insertion Site Is Conserved in Diazotrophic Pseudomonas stutzeri and Pseudomonas sp. Isolated from Distal and Close Geographical Regions

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