Plasmid Transformation of Azotobacter vinelandii OP

Doran, James L.; Bingle, Wade H.; Roy, Kanak; Hiratsuka, Koji; Page, William

doi:10.1099/00221287-133-8-2059

Cited by 15 publications

(8 citation statements)

References 42 publications

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“…These data suggest that formation of the array was not promoted by the C1-counter ion. This was confirmed by the observation that CaSO, also mediated S-layer reassembly on calcium-limited cells (Doran, 1983). After incubation with divalent cations, cells were washed with BBP04 containing the appropriate cation in order to expose the Slayer (Bingle et af., 1984).…”

Section: Methodsmentioning

confidence: 68%

“…To preclude unforeseen artifacts resulting from this washing procedure, the order of the cation addition and washing steps was reversed; identical results were obtained. The 42 "C heating step in this washing procedure (Bingle et al, 1984) was not necessary in order to visualize by freeze-etch electron microscopy, the regular surface array that was formed upon the addition of Ca2+ to calcium-limited cells (Doran, 1983). This indicated that formation and visualization of the regular array was a Ca2+-dependent, not a heat-dependent, event.…”

Section: Methodsmentioning

confidence: 95%

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Role of Calcium in Assembly of the Azotobacter vinelandii Surface Array

1987

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Azotobacter vinelandii grown in a defined medium lacking calcium did not exhibit the tetragonally-arranged surface layer present on calcium-sufficient cells, although each cell type possessed equivalent amounts of surface-localized S-protein. The addition of Ca2+ or Sr2+ to calcium-limited cells suspended in buffer resulted in formation of the S-layer, whereas a similar addition of Mg2+ or Be2+ did not. Incubation of cells with 35SO$-during Ca2+-mediated in vim reassembly of the S-layer confirmed that the array was formed from previously synthesized, surface-localized S-protein. Rate-zonal sedimentation of S-protein extracted from calciumlimited cells demonstrated tetrameric S-protein subunits characteristic of the native array. Sprotein on the surface of calcium-limited cells was not more susceptible to iodination or proteolytic degradation than that on calcium-sufficient cells. These data suggested minimal alteration of the surface layer beyond disorganization of the S-protein subunits. Calcium limitation caused only a minor perturbation of the outer membrane and did not prevent the outer membrane from serving as a template for the in vitro reassembly of externally supplied S-protein subunits. Notably, Mg2+ or Ca2+ mediated in vitro reassembly of the S-layer and produced a layer that was more loosely attached than the native array. These data support the hypothesis that calcium is specifically required for the in vivo assembly of S-protein subunits into a tetragonal surface array.

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Section: Methodsmentioning

confidence: 68%

Section: Methodsmentioning

confidence: 95%

Role of Calcium in Assembly of the Azotobacter vinelandii Surface Array

1987

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“…Whether or not DNA in Acinetobacter is translocated in a single-or double-stranded form has yet to be determined, and this issue is still rather controversial for other Gram-negative bacteria (Doran et al, 1987;Goodgal, 1982). One way to obtain more information about this process is to investigate the effects of plasmid topology on transformation.…”

Section: Efect Of Dna Topologymentioning

confidence: 99%

Physiological characterization of natural transformation in Acinetobacter calcoaceticus

Palmen

Vosman

Buijsman

et al. 1993

Journal of General Microbiology

163

153

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~Acinetobacter calcoaceticus BD413 develops competence for natural transformation immediately after the start of the exponential growth-phase and remains competent up to a few hours into the stationary phase, after which competence gradually declines. The transformation frequencies obtained strongly depend on the kind of transforming DNA and the incubation time with DNA. Up to 25% of the cells in a culture can be transformed. DNA uptake in Acinetobacter does not display sequence specificity, is Mg2+-, Mn2+-or Ca2+dependent and is uncoupler sensitive. The transforming DNA enters the cells in single-stranded form. These properties constitute a unique combination, not previously observed in other bacteria, and make A. calcoaceticus ideally suited for detailed studies of the bioenergetics of DNA translocation.

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“…vinelandii genome by random insertion [9]. Therefore, other synthetic biology researchers working with magnetosomes will now be able to use our simple insertion kit to conveniently express various proteins on magnetosomes.…”

Section: Discussionmentioning

confidence: 99%

Characterization ofAzotobacter vinelandiiand Kits for Its Synthetic Biology Applications

Jfc

Lcu

et al. 2017

Preprint

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Azotobacter vinelandii, a Gram-negative aerobic bacterium with an intracellular anaerobic environment that maintains the oxygen-sensitive enzymatic cascades for nitrogen fixation, could be used to express oxygen-sensitive proteins. However, little is known about the properties of A. vinelandii for synthetic biology applications. We therefore first characterized and optimized the conditions for growing and screening BioBrick constructs in A. vinelandii in the presence of 2 antibiotics, ampicillin and chloramphenicol, and then developed two sets of BioBricks for regulated protein expression. The first kit used T7 RNA polymerase, whose expression is under the control of a nitrogen-repressible nifH promoter. The commonly used T7-dependent system in Escherichia coli can then be used in A. vinelandii. Because its intracellular anaerobic environment is favorable for processes such as magnetosome biogenesis, we attempted to migrate the biogenesis machineries from the magnetotactic bacterium Magnetospirillum gryphiswaldense to A. vinelandii. During this undertaking, another insertion kit construct was developed to allow protein conjugation onto magnetosomes. The kit consists of mamC, a gene encoding a transmembrane protein on magnetosomes, and multiple restriction sites downstream of mamC for fusing a gene of interest. This insertion kit allows the attachment of any desired protein onto the magnetosome membrane by fusing with the mamC protein. We demonstrated the function of this kit by fusing mamC to a GFP nanobody. This kit will facilitate the conjugation of any target protein onto magnetosomes for downstream applications in the future.Financial DisclosureWe received sponsorship from the 2012–15 Teaching Development Grants Triennium, Faculty of Engineering and Biochemistry Program, School of Life Sciences, The Chinese University of Hong Kong. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.Competing InterestsThe authors declare that no competing interests exist.Ethics StatementN/A.Data AvailabilityAll data are fully available without restriction.

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Plasmid Transformation of Azotobacter vinelandii OP

Cited by 15 publications

References 42 publications

Role of Calcium in Assembly of the Azotobacter vinelandii Surface Array

Role of Calcium in Assembly of the Azotobacter vinelandii Surface Array

Physiological characterization of natural transformation in Acinetobacter calcoaceticus

Characterization ofAzotobacter vinelandiiand Kits for Its Synthetic Biology Applications

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