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Transformation of the herbicide 2,4-D[14C] was studied in the cultures of Azotobacter chroococcum, Azotobacter vinelandii and Azotobacter agile. These cultures assimilated 2,4-D and metabolized it. The products of transformation included phenol derivatives, water-soluble products, and carbon dioxide. About 15% of the herbicide taken up by the cells was bound to protein.
Cohen, Gary H. (University of Vermont, Burlington), and Donald B. Johnstone. Extracellular polysaccharides of Azotobacter vinelandii. J. Bacteriol. 88:329-338. 1964.-Extracellular polysaccharides synthetized by Azotobacter vinelandii strains 155, 102, and 3A were shown to be carboxylic acid heteropolysaccharides of apparent high molecular weight. Cells were grown in a nitrogen-free, mineral broth medium with 2% sucrose. Extracellular slime was recovered by centrifugation and purified by repeated alcohol precipitation and Sevag deproteinization. Capsular polysaccharide was recovered from washed cells by mild alkaline digestion. Methods of isolation and purification appeared to provide polysaccharide showing no evidence of heterogeneity when examined by chemical and physical methods. Infrared analysis of purified slime from the three strains suggested fundamental structural similarities. Colorimetric, paper chromatographic, and enzymatic analyses on both intact and acid-hydrolyzed slime polysaccharide indicated that the polymers contained in common galacturonic acid, [alpha] d-glucose, and rhamnose at a ratio of approximately 43:2:1, as well as a hexuronic acid lactone, probably mannurono-lactone. However, as shown by chemical and infrared analysis, minor differences did exist; namely, slime from strain 155 and 102 contained o-acetyl groups, whereas slime from strain 3A contained none. A sialic acid-like component (1.5% of dry weight of the polysaccharide, calculated as N-acetyl neuraminic acid), was found only in the slime of strain 155. Capsular polysaccharide composition closely resembled that for slime. It is of interest that the major slime components were identical whether the energy source provided for the cells was sucrose, glucose, fructose, or ethanol.
Quantitative N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) oxidase and superoxide dismutase (SOD) analyses were performed on representative organisms of the family Azotobacteraceae. Azotobacter vinelandii, Azotobacter chroococcum, Azotobacter paspali, and Derxia gummosa exhibited high quantitative TMPD oxidase activities, and their extracts possessed very active and electrophoretically homogeneous (single gel band) Fe-type SODs. Azomonas macrocytogenes extracts had similar single Fe-type SODs, and their cells exhibited no TMPD-dependent cytochrome oxidase activity. Nitrogen-fixing cells of Beijerinckia indica, Beijerinckia derxii, and Beijerinckia mobilis exhibited minimal TMPD oxidation capabilities (rates equivalent to the TMPD autooxidation reaction), and these extracts also possessed very active SODs but only of the Mn metallotype.
Socolofsky, M. D. (University of Texas, Austin) and Orville Wyss. Resistance of the Azotobacter cyst. J. Bacteriol. 84:119-124. 1962-The Azotobacter cysts were found to be more resistant than the vegetative cells to various harmful agents. Studies involving ultraviolet irradiation indicated that cysts required twice as great a dosage, as correspondingly treated vegetative cells, to be 90% inactivated. The acquisition of ultraviolet resistance during the encystment process was gradual and appeared to be related to the formation of exine and intine. A slow loss of ultraviolet resistance during germination was also noted. The cysts exhibited no marked resistance to heat, although they were extremely resistant to gamma radiation, sonic treatment, and desiccation. Evidence was presented indicating that the cyst is not a bacterial endospore. The encystment process may confer a survival advantage upon the organism by coupling the low endogenous respiration rate with the ability to withstand desiccation.
Layne, Joseph S. (University of Mississippi School of Medicine, Jackson), and Emmett J. Johnson. Natural factors involved in the induction of cyst formation in Azotobacter. J. Bacteriol. 87:684-689. 1964.-Recent reports have stimulated an interest in the natural factors responsible for the induction of a resistant form in the genus Azotobacter; therefore, an investigation of these factors was undertaken. A. agilis (vinelandii) was grown in Burk's nitrogen-free medium from which one mineral, or a combination of two minerals, was omitted. These media were made with triple-distilled, deionized water and were sterilized by filtration. After 18 hr of growth, up to 75% of the cells present were resistant forms if a single mineral was omitted, and up to 95% if two minerals were omitted. Formation of resistant forms was also induced by reduction of the sucrose content of the medium. When the sucrose content was reduced from the normal 0.5% to 0.1%, up to 60% resistant forms were obtained. Further reduction of the sucrose content to 0.05% brought about the formation of up to 80% resistant forms. Electron micrographs show that cysts grown in liquid media in response to mineral deficiencies lack the characteristic exine and intine of the cysts grown on the more commonly used solid medium containing butanol as the carbon source.
Layne, Joseph S. (University of Mississippi School of Medicine, Jackson), and Emmett J. Johnson. Resistant properties of Azotobacter cysts induced in response to mineral deficiencies. J. Bacteriol. 88:956-959. 1964.-Cysts produced in response to mineral deficiencies, and apparently lacking the characteristic exine and intine, possess the same degree of resistance to heat, mechanical disruption, desiccation, the action of lysozyme, and the combined action of ethylenediaminetetraacetic acid (EDTA) and lysozyme as do butanol-induced cysts. Slight differences in the behavior of the two varieties of cysts toward EDTA were observed. Since no significant differences seem to exist in resistant properties between cysts induced in response to mineral deficiencies and n-butanol-induced cysts, it would seem that the current concepts attributing the resistant properties of the Azotobacter cyst to the exine and intine require modification.
Cohen, Gary H. (University of Vermont, Burlington), and Donald B. Johnstone. Capsular polysaccharide of Azotobacter agilis. J. Bacteriol. 88:1695-1699. 1964.-Capsular polysaccharide from Azotobacter agilis strain 132 was recovered from washed cells by alkaline digestion. The polysaccharide was purified by centrifugation, repeated alcohol precipitation, Sevag deproteinization, and treatment with ribonuclease and charcoal-cellulose. Methods of isolation and purification appeared to provide a polymer showing no evidence of heterogeneity when examined by chemical and physical methods. Colorimetric, paper chromatographic, and enzymatic analyses on both intact and acid-hydrolyzed polysaccharide indicated that the polymer contained galactose and rhamnose at a molar ratio of approximately 1.0:0.7. A sialic acid-like component was also present in the polysaccharide. The study shows significant differences in the chemical composition of the extra-cellular polysaccharide of A. agilis and that of A. vinelandii. This adds further biochemical evidence for the right of these species to independent status.
Vela, Gerard R. (School of Aerospace Medicine, Brooks Air Force Base, Tex.), and Orville Wyss. Radiation resistance of soil Azotobacter. J. Bacteriol. 89:1280-1285. 1965.-Quantitative recovery of Azotobacter from soils subjected to gamma-radiation from a cobalt-60 source showed the soil populations to be much more highly resistant than isolates from such cultures grown on laboratory media. Even in the encysted state, the laboratory populations were reduced 10,000-fold by exposure to 200 kr, whereas the soil populations were not measurably reduced by that dose.
The ntrA, ntrB and ntrC products are responsible for regulating the transcription of many genes involved in the assimilation of poor nitrogen sources in enteric bacteria. The presence of a similar system in the non-enteric bacterium Azotobacter vinelandii is reported here. Genes analogous to ntrA and ntrC were isolated from an A. vinelandii gene library by complementation of Escherichia coli mutants. The gene encoding glutamine synthetase, glnA, was also isolated and found to be adjacent to ntrC but distant from ntrA, as it is in enteric organisms. The cloned Azotobacter genes also complemented Klebsiella pneumoniae mutants and hybridized to K. pneumoniae ntrA, ntrC and glnA gene probes. The role of ntrA and ntrC in A. vinelandii was established by using Tn5 insertions in the cloned genes to construct mutants by marker exchange. These mutants show that both ntrA and ntrC are required for the utilization of nitrate as a nitrogen source. However, ntrC is not required for nitrogen fixation by A. vinelandii, in contrast with K. pneumoniae where both ntrA and ntrC are essential.
After exhaustion of the C-source, terminating the accumulation of the extracellular polysaccharide, the excretion of the epimerase is increased, accordingly the moiety of guluronosyls of the alginate is augmented. Epimerization reactions with an epimerase preparation with commercial alginates from algae lead to higher conversion rates than with alginate from Azotobacter vinelandii. It is reasoned that the acetyl groups of bacterial alginate perform a steric hinderance. Because of the small amount of guluronosyls in Azotobacter alginate, these alginates do not form gels with Ca++.
A spontaneous pleiotropic Nif- mutation in Rhodospirillum rubrum has been partially characterized biochemically and by complementation analysis with recombinant plasmids carrying Azotobacter vinelandii DNA in the vicinity of ORF12 [Jacobson et al. (1989) J. Bacteriol 171: 1017-1027]. In addition to being unable to grow on N2 as a nitrogen source the phenotypic characterization of this and other metronidazole enriched spontaneous mutants showed (a) no nitrogenase activity, (b) the absence of NifHDK polypeptides, (c) a slower growth rate on NH4+, (d) approximately 50% higher glutamine synthetase (GS) activity than the wild-type, which was repressible, (e) an inability to switch-off GS activity in response to an NH4+ up-shift, and (f) an inability to modify (32P-label) the GS polypeptide. The apparent relationship between the absence of nifHDK expression and the absence of GS adenylylation cannot be explained in terms of the current model for nif gene regulation. However, R. rubrum transconjugants receiving A. vinelandii DNA which originated immediately upstream from nifH, restored all aspects of the wild-type phenotype. These data suggest a here-to-fore unrecognized relationship between nif expression and GS switch-off (adenylylation) activity, and the existence of a previously unidentified regulatory locus in Azotobacter that complements this mutation.
Azotobacter vinelandii genes contained within the major nif-cluster and designated orf6, nifU, nifS, nifV, orf7, orf8, nifW, nifZ, nifM, and orf9 are organized into at least two overlapping transcriptional units. Nitrogenase derepressed crude extracts of Azotobacter vinelandii mutant strains having individual deletions located within nifU, nifS, nifV, nifW, nifZ, or nifM were examined for nitrogenase component protein activities. The results of these experiments indicated that, in A. vinelandii, the nifU, nifS and nifM gene products are required for the full activation or the catalytic stability of the nitrogenase Fe protein. Deletion of the nifV gene resulted in lower MoFe protein activity, probably resulting from the accumulation of an altered FeMo-cofactor. The nifW and nifZ gene products were required for the full activation or catalytic stability of the MoFe protein. Deletion of nifZ alone or nifM alone did not appear to affect FeMo-cofactor biosynthesis. However, deletion of both nifZ and nifM eleminated either FeMo-cofactor biosynthesis or the insertion of FeMo-cofactor into the apo-MoFe protein. Other genes contained within the nifUSVWZM gene cluster (orf6, orf7, orf8, and orf9) were not required for Mo-dependent diazotrophic growth.
Several regulatory gene mutants of Azotobacter vinelandii were tested for ability to synthesize functional nitrogenase-1 (Nif phenotype), nitrogenase-2 (Vnf), or nitrogenase-3 (Anf). While nifA mutants were Nif-, Vnf+, and Anf+/-, and ntrC mutants were Nif+, Vnf+, and Anf+, nifA ntrC double mutants were Nif-, Vnf-, and Anf-. A vnfA mutant was Nif+, Vnf+/-, and Anf+/-, and an anfA strain was Nif+, Vnf+, and Anf-. lacZ fusions in the nifH, vnfH, vnfD, anfH, and nifM genes of Azotobacter vinelandii were constructed and introduced into wild-type and regulatory mutants of A. vinelandii. Expression of these operons correlated with the growth phenotype of the regulatory mutants. Apparently either NifA or NtrC can activate expression of nifM. Also, expression of the anf operon required the NifA transcriptional activator, although there are no NifA binding sites at appropriate locations upstream of anfH (or anfA). The results confirm previous reports that VnfA and AnfA are required for expression of vnf and anf genes, respectively, and that VnfA is involved in repression of the nifHDK operon in the absence of molybdenum and of the anfHDGK operon in the presence of vanadium.
Azotobacter vinelandii strain ATCC 12837 produces peptide siderophores of the general class known as pyoverdines. In the past, it was assumed that a single well-defined pyoverdine was produced by each parent microorganism. However, there are a number of reports of incompletely characterized pyoverdines that demonstrate heterogeneity in pyoverdine preparations obtained from a single organism, but the nature of this phenomena has not been explained. This study shows that A. vinelandii does indeed produce more than one pyoverdine and that these compounds differ in their peptide components. The metabolism of these siderophores suggests that only one of them is a true siderophore while the others are metabolic byproducts. It was demonstrated that this phenomenon is likely due to intrinsic limitations of the synthetase complex involved in the biosynthesis of these compounds. Characterization of two of the major pyoverdines produced demonstrated that they are novel compounds, although they belonged to the Azotobacter-type family of pyoverdines.
During the growth of Azotobacter vinelandii in batch culture in Burk's 2% glucose medium supplemented with 50 mg EDTA per litre, water-insoluble capsular polysaccaride material accumulated in cultures prior to the appearance of water-soluble polysaccharide in the culture medium. On isolation, hydrolysis and chromatography, both these polysaccharides were observed to be composed of carbohydrate monomers having the same chromatographic mobilities as glucose, rhamnos, guluronic acid and mannuronic acid. The activity of GDP-D-mannose dehydrogenase recorded in crude cell-free extracts from Azotobacter vinelandii, when these polysaccharides were produced, may indicate a close similarity between the biosynthetic pathway of alginate synthesis in marine Phaeophyceae and this soil microorganism.
Survival and establishment of inoculant strains of Azotobacter chroococcum NV 11 and its mutant NV 43 were assessed in sterilized soil and rhizosphere soil of wheat plants at 7 and 15 d interval, respectively, after sowing, i.e., up to 45 d under pot-house conditions. There was an apparent decrease in population of both strains in bulk soil but a steady increase was observed in root zones of the wheat plant. 10(3) to 10(4) introduced bacteria per g root were found sticking to roots. Further studies indicated that inoculant strains of Azotobacter could survive, proliferate and establish well in root zones.
Two phosphate-solubilizing analogue-resistant mutants of Azotobacter chroococcum showed a positive interactive effect on sunflower (Helianthus annuus). Plant height, leaf area, seed mass, seed yield, protein and oil content increased significantly by inoculation with A. chroococcum mutants. Both levels of fertilizers applied (80N + 30P2O5 and 80N + 60P2O5 kg/hm2 showed increased seed yield. Likewise, inoculation resulted in a significantly higher yield. Application of P-solubilizing A. chroococcum strains with the lower fertilizer level showed no significant difference on plant growth parameters when compared with the higher fertilizer level, indicating that inoculation of P-solubilizing Azotobacter strains can reduce the necessary levels of fertilization from 60P2O5 to 30P2O5 kg/hm2.
The putative replication origin of Azotobacter vinelandii was cloned as an autonomously replicating fragment after ligation to an antibiotic resistance cartridge. The resulting plasmids could be isolated and labelled by Southern hybridisation with the antibiotic resistance cartridge as probe and also visualised by electron microscopy. These plasmids integrated into the chromosome after a few generations, even in the recA mutant of A. vinelandii. The integrated copy of the plasmid was re-isolated from the chromosome and the DNA and its subfragments were cloned in the plasmid vector pBR322. A 200-bp DNA fragment was sufficient to allow the replication of pBR322 in an Escherichia coli polA strain. Electron microscopic analysis of this plasmid showed that replication initiated mostly within the A. vinelandii DNA fragment. The nucleotide sequence of the putative replication origin and its flanking regions was determined. In the sequence of the 200-bp fragment many of the distinctive features found in other replication origins are lacking. A greater variation from the consensus DnaA binding sequence was observed in A. vinelandii. Direct sequencing of the relevant genomic fragment was also carried after amplifying it from A. vinelandii chromosomal DNA by PCR. This confirmed that no rearrangements had taken place while the cloned fragment was resident in E. coli. It was shown by hybridisation that the 200-bp chromosomal origin fragment of A. vinelandii was present in three other field strains of Azotobacter spp.