Morphogenesis of Cysts in Azotobacter vinelandii

Cultures of Azotobacter vinelandii were induced to encystment with β-hydroxybutyrate. The morphological events in the transition from cell to cyst were observed by electron microscopy of thin sections. Upon induction of encystment, cells became rounded and nonmotile. The exine coat developed by the continuous excretion of membranous components into the capsule surrounding the cell.     

Cysts of Azotobacter vinelandii with Double Coats

Cyst germination was studied in cultures of Azotobacter vinelandii ATCC 12837. The existence of cysts enclosed by double coats is described.     

Giant Cysts and Cysts with Multiple Central Bodies in Azotobacter vinelandii

Cyst germination in Azotobacter vinelandii ATCC 12837 was studied by using phase contrast and electron microscopy. Germination in this organism was accompanied by the formation of large cyst forms of two different types: giant cysts and cysts containing multiple central bodies. Previously, these two types have been reported only when yeast extract was added to the encystment medium. In this study, we observed giant cysts and cysts with multiple central bodies in nitrogen-free liquid medium. The germination of "normal" cysts is often preceded by enlargement to the giant form and division of the central body to produce cysts with multiple central bodies. Structures similar in appearance to ribosomal aggregates were observed only in cysts undergoing pregermination transformations.     

Regulation of Nitrogen Fixation in Azotobacter vinelandii OP and in an Apparently Partially Constitutive Mutant

Methylamine and 2-methylalanine appeared to act as co-repressors of nitrogenase in Azotobacter vinelandii OP. They inhibited the growth of this organism on molecular nitrogen but not on nitrate, ammonia, or Casamino Acids; they prevented the formation of nitrogenase by cells transferred from repression to induction conditions; and they did not inhibit the activity of nitrogenase in vitro. A mutant of strain OP, selected on the basis of its relative resistance to methylalanine, appeared partially constitutive because nitrogenase in this strain was less sensitive to repressors than was the enzyme in the wild-type strain.     

Surface Structure of Azotobacter vinelandii Cysts as Revealed by Freeze-Cleaving

Micrographs of freeze-cleaved Azotobacter vinelandii cysts reveal that the surface is composed of several overlapping layers. This observation is consistent with the previously proposed structure of the outer cyst coat.     

Nitrogen fixation by Azotobacter vinelandii in tungsten-containing medium

Nitrogenase was isolated and purified from wild-type and a tungsten-resistant mutant (LM2) of Azotobacter vinelandii strain OP derepressed on medium containing 1-10 mM W. While the enzyme from the wild-type strain contained the polypeptides of the conventional enzyme, metal analysis of component 1 demonstrated the existence of one atom each of molybdenum and tungsten. Furthermore, the ESR spectrum of this protein contained three signals, two of which originated from S = 3/2 spin states. One of these signals is nearly identical to that of the conventional MoFe-protein while the other is hypothesized to originate from a W-containing cofactor. In spite of the presence of W, the substrate reduction pattern of this enzyme is the same as that of the conventional enzyme.     

Sequence and molecular analysis of the nifL gene of Azotobacter vinelandii

In both Klebsiella pneumoniae and Azotobacter vinelandii the nifL gene, which encodes a negative regulator of nitrogen fixation, lies immediately upstream of nifA. We have sequenced the A. vinelandii nifL gene and found that it is more homologous in its C-terminal domain to the histidine protein kinases (HPKs) than Is K. pneumoniae NifL. In particular A. vinelandii NifL contains a conserved histidine at a position shown to be phosphorylated in other systems. Both NifL proteins are homologous in their N-termini to a part of the Halobacterium halobium bat gene product; Bat is involved in regulation of bacterio-opsin, the expression of which is oxygen sensitive. The same region showed homology to the haembinding N-terminai domain of the Rhizobium meliloti fixL gene product, an oxygen-sensing protein. Like K. pneumoniae NifL, A. vinelandii NifL is shown here to prevent expression of nif genes in the presence of NH⁺₄ or oxygen. The sequences found homologous in the C-terminal regions of NifL, FixL and Bat might therefore be involved in oxygen binding or sensing. An in-frame deletion mutation in the nifL coding region resulted in loss of repression by NH⁺₄ and the mutant excreted high amounts of ammonia during nitrogen fixation, thus confirming a phenotype reported earlier for an insertion mutation. In addition, nifLA are cotranscribed in A. vinelandii as in K. pneumoniae, but expression from the A. vinelandii promoter requires neither RpoN nor NtrC.     

Nitrogen fixation system of tungsten-resistant mutants of Azotobacter vinelandii.

Mutants of Azotobacter vinelandii ATCC 12837 were isolated which could fix N2 in the presence of high tungsten concentrations. The most studied of these mutants (WD2) grew well in N-free modified Burk broth containing 10 mM W, whereas the wild type would not grow in this medium. WD2 would also grow in Burk N-free broth at about the same rate as the wild type. WD2 in broth containing W exhibited 22% of the whole cell acetylene reduction activity of the wild type in broth containing Mo and showed a lowered affinity for acetylene. Two-dimensional gel electrophoresis experiments showed that N2-fixing cells of WD2 from broth containing W or Mo did not produce significant amounts of component I of native nitrogenase protein. Electron spin resonance spectra of whole cells and cell-free extracts of WD2 from broth containing W lacked any trace of the g = 3.6 resonance associated with FeMoCo.     

Correlation of Ultrastructure in Azotobacter vinelandii with Nitrogen Source for Growth

Azotobacter synthesizes an extensive internal membranous nework when grown with air (N₂), i.e., under conditions when these bacteria fix nitrogen. Very slight quantities of internal membrane, concentrated mainly about the cell periphery, are formed when Azotobacter grows with fixed nitrogen, i.e., ammonia and amino acids. Compared to cells growing with ammonia, cells utilizing atmospheric nitrogen as the sole nitrogen source are smaller in size and volume, grow one-third slower, and lack detectable poly-β-hydroxybutyrate.     

Phospholipids of Azotobacter vinelandii

Analyses of resting cells of Azotobacter vinelandii revealed that numerous phospholipids were present that did not concentrate in the membranous R(3) fraction which carried out electron transport function.     

Ultrastructure of Azotobacter vinelandii

Vegetative cells and cysts of Azotobacter vinelandii 12837 were prepared for electron microscopy by several methods assumed to preserve structural details destroyed by techniques previously reported in the literature. Examination of large numbers of cells and cysts by these methods revealed four structural details not reported previously: intine fibrils, intine vesicles, intine membrane, and microtubules. The intine fibrils form a network in the gel-like homogeneous matrix of the CC2 layer. Intine vesicles which seem to originate in the cell wall complex of the central body are seen in the intine and exine of cysts. Analogous structures are found on vegetative cells. The intine is divided into two chemically distinct areas by the two-layered intine membrane. Microtubules, previously reported only in vegetative cells, were found in cysts.     

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

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 free-living 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.     

Chemotactic Behavior of Azotobacter vinelandii

Chemotaxis was exhibited by Azotobacter vinelandii motile cells. Exposure of cells to sudden increases in attractant concentration suppressed the frequency of tumbling and resulted in smooth swimming. Cells responded chemotactically to a chemical gradient produced during metabolism. Motility occurred over a temperature range of 25 to 37°C with an optimum pH range of between pH 7.0 and 8.0. The average speed of motile cells was determined to be 74 μm/s or 37 body lengths per s. The speed of cells appeared to increase as a function of attractant concentration. Chemotactic systems for fructose, glucose, xylitol, and mannitol were inducible. A. vinelandii exhibited chemotaxis for a number of compounds, including hexoses, hexitols, pentitols, pentoses, disaccharides, and amino sugars. We conclude from these studies that A. vinelandii exhibits a temporal chemotactic sensing system.     

Plasmids of Azotobacter vinelandii.

Four laboratory strains and two isolates of Azotobacter vinelandii were found to contain plasmids. Twenty-five laboratory strains which could fix nitrogen did not have free, covalently closed circular plasmid DNA. The plasmids varied in size from 9 to 52 megadaltons, and each strain yielded only one plasmid. No discernible differences in ability to fix nitrogen were found between plasmid-bearing and cured cultures.     

Endogenous Encystment of Azotobacter vinelandii

When young cells of Azotobacter vinelandii are impinged on membrane filters, washed free of carbon substrate, and placed on a mineral salts basal medium, the culture will proceed to encyst although at a slower rate than if n-butanol were supplied as a substrate. The endogenous cysts are depleted in polyβ-hydroxybutyrate and have a narrower intine but show an increased resistance to desiccation and are susceptible to lysis by chelating agents. Membrane-supported cells reveal details of the encystment process such as the formation of a zone within the capsule prior to exine formation and the early deposition of exine structures.