Optimal conditions for induction of competence in nitrogen-fixing <i>Azotobacter vinelandii</i>

Page, William

doi:10.1139/m82-059

Cited by 41 publications

(44 citation statements)

References 19 publications

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“…16 This study 12,17 This study 14,17 This study 14 This study This study 16 This study 26 This study This (23). When A. vinelandii strains were transformed with pMJH3 containing an insertion of TnS-B21, transformants were selected initially on modified Burk medium containing tetracycline.…”

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confidence: 99%

The gene encoding dinitrogenase reductase 2 is required for expression of the second alternative nitrogenase from Azotobacter vinelandii

1991

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Under diazotrophic conditions in the absence of molybdenum (Mo) and vanadium (V), Azotobacter vinelandii reduces N2 to NH4+ by using nitrogenase 3 (encoded by anfHDGK). However, dinitrogenase reductase 2 (encoded by vnfH) is also expressed under these conditions even though this protein is a component of the Vcontaining alternative nitrogenase. Mutant strains that lack dinitrogenase reductase 2 (VnfH-) grow slower than the wild-type strain in N-free, Mo-, and V-deficient medium. In this medium, these strains synthesize dinitrogenase reductase 1 (a component of the Mo-containing nitrogenase encoded by nifH), even though this component is not normally synthesized in the absence of Mo. Strains that lack both dinitrogenase reductases 1 and 2 (NifH-VnfH-) are unable to grow diazotrophically in Mo-and V-deficient medium. In this medium, NifH-VnfH-strains containing an anfH--lacZ transcriptional fusion exhibited less than 3% of the ,B-galactosidase activity observed in the wild type with the same fusion. P-Galactosidase activity expressed by VnfHmutants containing the anJH-lacZ fusion ranged between 57 and 78% of that expressed by the wild type containing the samne fusion. Thus, expression of dinitrogenase reductase 2 seems to be required for transcription of the anfHDGK operon, although, in Vnft-mutants, dinitrogenase reductase 1 appears to serve this function. Active dinitrogenase reductase 1 or 2 is probably required for this function since a nijM deletion mutant containing the anffl-lacZ fusion was unable to synthesize ,-galactosidase above background levels. An anfA deletion strain containing the anpfH-lacZ fusion exhibited ,B-galactosidase activity at 16% of that of the wild type containing the same fusion. However, in the presence of NH4', the ,B-galactosidase activity expressed by this strain more than doubled. This indicates that AnfA is required not only for normal levels of anfHDGK transcription but also for NH4'-and, to a lesser extent, Mo-mediated repression of this transcription.The regulation of the expression of the three nitrogenases in Azotobacter vinelandii is responsive to the presence or absence of ammonium (NH4'), molybdenum (Mo), and vanadium (V) in the culture medium. The synthesis of all three nitrogenases is repressed by NH4'. Nitrogenase 1 is found in cells grown in the presence of Mo and nitrogenase 2 is expressed in the presence of V but in the absence of Mo, whereas nitrogenase 3 is synthesized only in the absence of both Mo and V (4, 9, 13, and references therein). Our knowledge of the molecular basis for nitrogen and metal regulation in A. vinelandii is still rudimentary. The regulatory genes nifA, vnfA, and anfA have been identified, and some of their functions have been described on the basis of the phenotypes of NifA-, VnfA-, and AnfA-mutants (1, 14). The nifA gene product is required for transcription of the structural genes for nitrogenase 1 (1). NifA binds to an upstream activator sequence (6) or indirectly represses the synthesis of nitrogenase 1 in cells grown in Mo-deficient me...

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The gene encoding dinitrogenase reductase 2 is required for expression of the second alternative nitrogenase from Azotobacter vinelandii

1991

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“…vinalandii strains UW, UW1 or UWlO for transformation by pKT210 required a period of growth under oxygen-and iron-limited conditions (Page, 1982(Page, , 1985Page & von Tigerstrom, 1978), since no Cmr or Smr transformants of strain UW 10 grown under conditions of iron-limitation and high aeration, or in iron-containing Burk medium, were detected.…”

Section: J L D O R a N A N D O T H E R Smentioning

confidence: 99%

“…The basis for this decline is not understood; however it may be related to the effects of prolonged O?-starvation (Page, 1982) rather than a loss of competence-related function.…”

Section: Plasmid Transformation Of Azotobacter 2067mentioning

confidence: 99%

“…The initial studies of plasmid DNA transformation of Azotobacter vinelandii, conducted by David et al (1981), revealed that cells made artificially competent by a modification of the CaC1,-dependent treatment of Cohen et al (1972) were transformed by plasmid DNA at frequencies one to three orders of magnitude lower than those observed using chromosomal markers to transform A. vinelandii which had been naturally grown to competence (Page, 1985;Page & von Tigerstrom, 1979). The inherent limitations of plasmid transfer from Escherichia coli to A. vinelandii via conjugation (David et al, 1981 ;Kennedy & Robson, 1983) may mean that transformation of naturally competent A. vinelandii is the method of choice for the introduction of plasmids into this organism.…”

Section: Introductionmentioning

confidence: 99%

“…vinelandii OP (capsule-) derivative strains UW, UWl and UWlO are genetically transformed at high frequencies with single chromosomal markers ( 10-3-10-2 transformants per viable cell) when competent cells are prepared naturally, by growth in iron-deficient medium under conditions of oxygen limitation (Page, 1982(Page, , 1985Page & von Tigerstrom, 1979); this frequency corresponds to 1 04-I O5 transformants per pg DNA. Considering the high background of homologous 'non-marker' DNA present in preparations of chromosomal DNA, and the ability of this DNA to compete with and inhibit transformation by 'marker' DNA (Doran & Page, 1983), it might be expected that nearly all cells in the population attain a competent state.…”

Section: Introductionmentioning

confidence: 99%

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Plasmid Transformation of Azotobacter vinelandii OP

Doran

Bingle²,

Roy³

et al. 1987

Microbiology

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Azotobacter vinelandii OP which had been naturally induced to competence by growth in ironand molybdenum-limited medium was transformed with the broad-host-range cloning vector pKT210. However, the transformation frequency at nearly saturating levels of DNA was 1000-fold lower for pKT210 than for a single chromosomal DNA marker (nif+). Plasmid-and chromosomal-DN A-mediated transformation events were competitive, magnesium-dependent, 42 "C-sensitive processes specific to double-stranded DNA, suggesting a common mechanism of DNA binding and uptake. The low frequency of plasmid transformation was not related to restriction of transforming DNA or to the growth period allowed for phenotypic expression. Covalently-closed-circular and open-circular forms of pKT2 10 transformed cells equally well whereas EcoRI-or HindIII-linearized pKT210 transformed cells with two to three times greater efficiency. Genetic transformation was enhanced 10-to 50-fold when pKT210 contained an insert fragment of A . vinelandii nif DNA, indicating that A. vinelandii possessed a homologyfacilitated transformation system. However, all transformants failed to maintain the plasmidencoded antibiotic resistance determinants, and extrachromosomal plasmid DNA was not recovered from these cells. Flush-ended pKT210 was not active in transformation; however, competent cells were transformed to Nif+ by HincII-digested plasmid DNA containing the cloned A. vinelandii nif-I0 marker.

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Biochemical Genetics of Nitrogen Fixation

Brill¹

1983

Structure and Function of Plant Genomes

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Optimal conditions for induction of competence in nitrogen-fixing Azotobacter vinelandii

Cited by 41 publications

References 19 publications

The gene encoding dinitrogenase reductase 2 is required for expression of the second alternative nitrogenase from Azotobacter vinelandii

The gene encoding dinitrogenase reductase 2 is required for expression of the second alternative nitrogenase from Azotobacter vinelandii

Plasmid Transformation of Azotobacter vinelandii OP

Biochemical Genetics of Nitrogen Fixation

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