Identification and Characterization of an<i>Azotobacter vinelandii</i>Type I Secretion System Responsible for Export of the AlgE-Type Mannuronan C-5-Epimerases

Gimmestad, Martin; Steigedal, Magnus; Ertesvåg, Helga; Moreno, Soledad; Christensen, Bjørn E.; Espı́n, Guadalupe; Valla, Svein

doi:10.1128/jb.00236-06

Cited by 31 publications

(40 citation statements)

References 63 publications

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“…This broad screening potential could have further benefits in identifying additional potential "nitrogen shuttle" compounds that might have further utility for providing nitrogen in coculture. A. vinelandii also produces additional nitrogen shuttle compounds such as siderophores that are suitable for supporting the growth of algae in coculture (11), as well as additional extracellular proteins (49). In these studies, the levels of nitrogen released to the culture medium by the amtB disruption were very low (i.e., a low micromolar range) and might be missed by less sensitive assays.…”

Section: Discussionmentioning

confidence: 84%

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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Section: Discussionmentioning

confidence: 84%

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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“…The N-terminal module shares 29% sequence identity with the characterized G-specific alginate lyase AlyA from Klebsiella pneumoniae (5) and 28% identity with an MG-specific alginate lyase from Pseudomonas aeruginosa (45). The RTX module is characteristic for a group of proteins (including the A. vinelandii AlgE epimerases) that is secreted by type I secretion systems (12,14,18) and contains several copies of a nine-amino-acid Ca 2ϩ -binding motif (RTX motif) (6). AlyA3 contains nine putative copies of this repeat (underlined in Fig.…”

Section: H-nmrmentioning

confidence: 99%

“…Gene replacement vectors for alyA1, alyA2, and alyA3 were constructed, as described in Table 1, and designated pMG134, pMG136, and pMG138, respectively. They were linearized by MluI and EcoRI (pMG134), BamHI and EcoRI (pMG136), or EcoRI only (pMG138) and transferred to A. vinelandii by transformation, as described earlier (18).…”

Section: Growth Of Bacteriamentioning

confidence: 99%

Characterization of Three New Azotobacter vinelandii Alginate Lyases, One of Which Is Involved in Cyst Germination

et al. 2009

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Alginates are polysaccharides composed of 1-4-linked ␤-D-mannuronic acid and ␣-L-guluronic acid. The polymer can be degraded by alginate lyases, which cleave the polysaccharide using a ␤-elimination reaction. Two such lyases have previously been identified in the soil bacterium Azotobacter vinelandii, as follows: the periplasmic AlgL and the secreted bifunctional mannuronan C-5 epimerase and alginate lyase AlgE7. In this work, we describe the properties of three new lyases from this bacterium, AlyA1, AlyA2, and AlyA3, all of which belong to the PL7 family of polysaccharide lyases. One of the enzymes, AlyA3, also contains a C-terminal module similar to those of proteins secreted by a type I secretion system, and its activity is stimulated by Ca 2؉ . All three enzymes preferably cleave the bond between guluronic acid and mannuronic acid, resulting in a guluronic acid residue at the new reducing end, but AlyA3 also degrades the other three possible bonds in alginate. Strains containing interrupted versions of alyA1, alyA3, and algE7 were constructed, and their phenotypes were analyzed. Genetically pure alyA2 mutants were not obtained, suggesting that this gene product may be important for the bacterium during vegetative growth. After centrifugation, cultures from the algE7 mutants form a large pellet containing alginate, indicating that AlgE7 is involved in the release of alginate from the cells. Upon encountering adverse growth conditions, A. vinelandii will form a resting stage called cyst. Alginate is a necessary part of the protective cyst coat, and we show here that strains lacking alyA3 germinate poorly compared to wild-type cells.

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“…During cultivation of A. chroococcum at higher oxygen concentration, an alginate capsule, with a relatively high guluronic acid content is formed on the cell surface. A similar effect was also observed during cultivation of Azotobacter vinelandii (Segura et al, 2003a;Gimmestad et al, 2006). This alginate capsule can be released into the medium and it makes an effective barrier that restricts oxygen transfer into the cell and consequently, more intensive aeration has to be used to ensure sufficient oxygen supply during cultivation (Sabra et al, 2001).…”

Section: Introductionmentioning

confidence: 66%

Cultivation of the bacterium Azotobacter chroococcum for preparation of biofertilizers

Oros¹,

Pavlečić²,

antek³

et al. 2011

Afr. J. Biotechnol.

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The biomass of the bacterium Azotobacter chroococcum can be used as a biofertilizer due to its ability to fix nitrogen from the atmosphere. In order to optimize the production of bacterial biomass for this purpose, a cultivation of A. chroococcum was done by using different media and cultivation techniques (batch, fed batch and repeated batch). Chemically defined and complex media with 20 g/l of sugar were selected as the most appropriate media for batch cultivation in stirred tank bioreactor. In order to obtain higher fed batch and repeated batch techniques were examined. During these cultivations, increase of bioprocess efficiency parameters (yield coefficient and productivity) were observed compared with the batch cultivation. On the basis of the obtained results, repeated batch technique appeared to be the most suitable for the bacterial biomass production at industrial scale.

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Identification and Characterization of anAzotobacter vinelandiiType I Secretion System Responsible for Export of the AlgE-Type Mannuronan C-5-Epimerases

Cited by 31 publications

References 63 publications

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

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

Characterization of Three New Azotobacter vinelandii Alginate Lyases, One of Which Is Involved in Cyst Germination

Cultivation of the bacterium Azotobacter chroococcum for preparation of biofertilizers

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