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At least 595 records · Page 33Linked to original sources
PubMed2000

Dynamic accumulation and degradation of poly(3-hydroxyalkanoate)s in living cells of Azotobacter vinelandii UWD characterized by (13)C NMR.

The synthesis and degradation of poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-hydroxyvalerate) (P(HB-co-HV)) by Azotobacter vinelandii UWD were investigated using natural abundance solution (13)C nuclear magnetic resonance (NMR) in vivo in shake flask culture and in fermenter culture. The synthesis and the degradation of poly(3-hydroxyalkanoate)s (PHA) monomers hydroxybutyrate (HB) and hydroxyvalerate (HV) had different rates. The amount of HB and HV increased dramatically in the initial degradation stage. The results suggest that the intracellular PHA of strain UWD was the subject of dynamic metabolic processing. (13)C NMR in vivo analysis provided a rapid, easy, accurate, non-destructive method to obtain valuable information on the metabolism of PHA.

Azotobacter vinelandii↗
PubMed2001

Biosynthesis of poly-beta-hydroxybutyrate (PHB) is controlled by CydR (Fnr) in the obligate aerobe Azotobacter vinelandii.

CydR is an Fnr-like protein in the obligatory aerobic nitrogen-fixing bacterium Azotobacter vinelandii. The cydR mutant overproduces the cytochrome bd terminal oxidase. Using two-dimensional polyacrylamide gel electrophoresis, we showed that beta-ketothiolase and acetoacetyl-CoA reductase were also overexpressed in the cydR mutant. Fumarase C and a coenzyme A transferase, possibly succinyl-SCoA transferase, were decreased in this mutant. Enzyme assays confirmed the elevated beta-ketothiolase and acetoacetyl-CoA reductase activities in this mutant. The cydR mutant accumulated poly-beta-hydroxybutyrate throughout the exponential growth phase, unlike the wild-type strain that only accumulated poly-beta-hydroxybutyrate during stationary phase. The results demonstrate that CydR controls poly-beta-hydroxybutyrate synthesis in A. vinelandii.

Azotobacter vinelandii↗
PubMed2002

Characterization of Azotobacter vinelandii aggregation in submerged culture by digital image analysis.

A simple and accurate method for determining the distribution of sizes of single cells and aggregates of Azotobacter vinelandii by image analysis has been developed. A staining procedure using methylene blue helps to enhance the contrast between aggregates and background without altering aggregate size distribution. Sample dilution affected the distribution of the population and therefore should be avoided. Mixing and aeration conditions during culture play an important role in the aggregation of A. vinelandii. Cells grown under mild mixing conditions (unbaffled flasks) presented a thick slime layer and formed aggregates of up to 35 microm of average equivalent diameter. In contrast, under strong agitation conditions (baffled flasks) practically no aggregates were formed throughout cultivation. The method described can be used for the characterization of aggregation of other microbial cultures.

Anaerobiosis↗
PubMed2007

Microscopical investigation of poly(3-hydroxybutyrate) granule formation in Azotobacter vinelandii.

Poly(3-hydroxybutyrate) (PHB) granule formation in Azotobacter vinelandii was investigated by laser scanning fluorescence microscopy after staining the cells with Nilered and Baclight. Cells that had been starved for a carbon source for > or =3 days were almost free of PHB granules. Formation of visible PHB granules started within 1-2 h after transfer of the cells to a medium permissive for PHB accumulation. Fluorescent PHB granules at the early stages of formation were exclusively found in the cell periphery of the 2-3 mum ovoid-shaped cells. After 3 h of PHB accumulation or later, PHB granules were also found to be detached from the cell periphery. Our results indicate that PHB granule formation apparently begins at the inner site of the cytoplasmic membrane. This finding is different from previous assumptions that PHB granule formation occurs randomly in the cytoplasm of PHB-accumulating bacteria.

Azotobacter vinelandii↗
PubMed1992

Crystallographic structure of the nitrogenase iron protein from Azotobacter vinelandii.

The nitrogenase enzyme system catalyzes the ATP (adenosine triphosphate)-dependent reduction of dinitrogen to ammonia during the process of nitrogen fixation. Nitrogenase consists of two proteins: the iron (Fe)-protein, which couples hydrolysis of ATP to electron transfer, and the molybdenum-iron (MoFe)-protein, which contains the dinitrogen binding site. In order to address the role of ATP in nitrogen fixation, the crystal structure of the nitrogenase Fe-protein from Azotobacter vinelandii has been determined at 2.9 angstrom (A) resolution. Fe-protein is a dimer of two identical subunits that coordinate a single 4Fe:4S cluster. Each subunit folds as a single alpha/beta type domain, which together symmetrically ligate the surface exposed 4Fe:4S cluster through two cysteines from each subunit. A single bound ADP (adenosine diphosphate) molecule is located in the interface region between the two subunits. Because the phosphate groups of this nucleotide are approximately 20 A from the 4Fe:4S cluster, it is unlikely that ATP hydrolysis and electron transfer are directly coupled. Instead, it appears that interactions between the nucleotide and cluster sites must be indirectly coupled by allosteric changes occurring at the subunit interface. The coupling between protein conformation and nucleotide hydrolysis in Fe-protein exhibits general similarities to the H-Ras p21 and recA proteins that have been recently characterized structurally. The Fe-protein structure may be relevant to the functioning of other biochemical energy-transducing systems containing two nucleotide-binding sites, including membrane transport proteins.

Adenosine Diphosphate↗
PubMed1990

Possible mechanism of mannose inhibition of sucrose-supported growth in N2-fixing Azotobacter vinelandii.

When mannose was added to a sucrose-supported culture of Azotobacter vinelandii under N2-fixing conditions, cell growth was inhibited. The degree of inhibition was proportional to the amount of mannose and to the aeration rate (T.-Y. Wong, Appl. Environ. Microbiol. 54:473-475, 1988). In this report, we demonstrate that once inside the cell, mannose was phosphorylated to mannose 6-phosphate. It was then isomerized to fructose 6-phosphate and to glucose 6-phosphate. Mannose inhibited sucrose uptake noncompetitively. The decrease in sucrose uptake after mannose addition coincided with a lower rate of respiration and a decrease in nitrogenase activity. The decrease in sucrose uptake and in the ATP pool may decrease the electron flow and reduce protection of the nitrogenase from O2. Cells became very sensitive to O2, and therefore, cell growth was inhibited under high aeration conditions.

Azotobacter↗
PubMed1990

Melibiose is hydrolyzed exocellularly by an inducible exo-alpha-galactosidase in Azotobacter vinelandii.

Azotobacter vinelandii hydrolyzed melibiose exocellularly, leading to an accumulation of free glucose and galactose in the medium. This enzyme could also be induced by galactose, raffinose, and stachyose. The alpha-galactosidase activity could be detected quantitatively by using p-nitrophenyl-alpha-galactopyranoside as a substrate for intact cells. Chloramphenicol totally inhibited the induction of this enzyme. However, benzyl alcohol inhibited the secretion of this enzyme but did not inhibit the biosynthesis of the enzyme.

Azotobacter↗
PubMed1992

Excretion of ammonium by a nifL mutant of Azotobacter vinelandii fixing nitrogen.

A mutation in the gene upstream of nifA in Azotobacter vinelandii was introduced into the chromosome to replace the corresponding wild-type region. The resulting mutant, MV376, produced nitrogenase constitutively in the presence of 15 mM ammonium. When introduced into a nifH-lacZ fusion strain, the mutation permitted beta-galactosidase production in the presence of ammonium. The gene upstream of nifA is therefore designated nifL because of its similarity to the Klebsiella pneumoniae nifL gene in proximity to nifA, in mutant phenotype, and in amino acid sequence of the gene product. The A. vinelandii nifL mutant MV376 excreted significant quantities of ammonium (approximately 10 mM) during diazotrophic growth. In contrast, ammonium excretion during diazotrophy was much lower in a K. pneumoniae nifL deletion mutant (maximum, 0.15 mM) but significantly higher than in NifL+ K. pneumoniae. The expression of the A. vinelandii nifA gene, unlike that of K. pneumoniae, was not repressed by ammonium.

Ammonia↗
PubMed1992

Formation of poly(hydroxybutyrate-co-hydroxyvalerate) by Azotobacter vinelandii UWD.

Azotobacter vinelandii UWD formed polyhydroxyalkanoate (PHA) copolymers containing beta-hydroxybutyrate and beta-hydroxyvalerate (HV) when grown in a medium containing glucose as the primary C source and valerate (pentanoate) as a precursor. Copolymer was not formed when propionate was added to the glucose medium but was formed when heptanoate, nonanoate, or trans-2-pentenoate was present. Optimal levels of HV were formed when valerate was added at the time of maximum PHA synthesis, although HV incorporation was not dependent on glucose catabolism. HV content in the polymer was increased from 17 to 24 mol% by adding 10 to 40 mM valerate to glucose medium, but HV insertion into the polymer occurred at a fixed rate. Similarly, the addition of valerate to a fed-batch culture of strain UWD in beet molasses in a fermentor produced 19 to 22 g of polymer per liter, containing 8.5 to 23 mol% HV after 38 to 40 h. The synthesis of HV in these cultures also occurred at a fixed rate (2.3 to 2.8 mol% h-1), while the maximum PHA production rate was 1.1 g liter-1 h-1. During synthesis of copolymer in batch or fed-batch culture, the yield from conversion of glucose into PHA (YP/S) remained at maximum theoretical efficiency (greater than or equal to 0.33 g of PHA per g of glucose consumed). Up to 45 mol% C source, but the PHA produced amounted to less than 1 g/liter. The combination of 30 mM valerate as a sole C source and 0.5 mM 4-pentenoate increased the HV content in the polymer to 52 mol%.(ABSTRACT TRUNCATED AT 250 WORDS)

Azotobacter vinelandii↗
PubMed2000

Role of molybdate and other transition metals in the accumulation of protochelin by Azotobacter vinelandii.

Both molybdate and iron are metals that are required by the obligately aerobic organism Azotobacter vinelandii to survive in the nutrient-limited conditions of its natural soil environment. Previous studies have shown that a high concentration of molybdate (1 mM) affects the formation of A. vinelandii siderophores such that the tricatecholate protochelin is formed to the exclusion of the other catecholate siderophores, azotochelin and aminochelin. It has been shown previously that molybdate combines readily with catecholates and interferes with siderophore function. In this study, we found that the manner in which each catecholate siderophore interacted with molybdate was consistent with the structure and binding potential of the siderophore. The affinity that each siderophore had for molybdate was high enough that stable molybdo-siderophore complexes were formed but low enough that the complexes were readily destabilized by Fe(3+). Thus, competition between Fe(3+) and molybdate did not appear to be the primary cause of protochelin accumulation; in addition, we determined that protochelin accumulated in the presence of vanadate, tungstate, Zn(2+), and Mn(2+). We found that all five of these metal ions partially inhibited uptake of (55)Fe-protochelin and (55)Fe-azotochelin complexes. Also, each of these metal ions partially inhibited the activity of ferric reductase, an enzyme important in the deferration of ferric siderophores. Our results suggest that protochelin accumulates in the presence of molybdate because protochelin uptake and conversion into its component parts, azotochelin and aminochelin, are inhibited by interference with ferric reductase.

Azotobacter vinelandii↗
PubMed2000

Effect of oxygen on formation and structure of Azotobacter vinelandii alginate and its role in protecting nitrogenase.

The activity of nitrogenase in the nitrogen-fixing bacterium Azotobacter vinelandii grown diazotrophically under aerobic conditions is generally considered to be protected against O(2) by a high respiration rate. In this work, we have shown that a high rate of respiration is not the prevailing mechanism for nitrogenase protection in A. vinelandii grown in phosphate-limited nitrogen-free chemostat culture. Instead, the formation of alginate appeared to play a decisive role in protecting the nitrogenase that is required for cell growth in this culture. Depending on the O(2) tension and cell growth rate, the formation rate and composition of alginate released into the culture broth varied significantly. Furthermore, transmission electron microscopic analysis of cell morphology and the cell surface revealed the existence of an alginate capsule on the surface of A. vinelandii. The composition, thickness, and compactness of this alginate capsule also varied significantly. In general, increasing O(2) tension led to the formation of alginate with a higher molecular weight and a greater L-guluronic acid content. The alginate capsule was accordingly thicker and more compact. In addition, the formation of the alginate capsule was found to be strongly affected by the shear rate in a bioreactor. Based on these experimental results, it is suggested that the production of alginate, especially the formation of an alginate capsule on the cell surface, forms an effective barrier for O(2) transfer into the cell. It is obviously the quality, not the quantity, of alginate that is decisive for the protection of nitrogenase.

Alginates↗
PubMed2001

Poly(3-hydroxybutyrate) synthesis genes in Azotobacter sp. strain FA8.

Genes responsible for the synthesis of poly(3-hydroxybutyrate) (PHB) in Azotobacter sp. FA8 were cloned and analyzed. A PHB polymerase gene (phbC) was found downstream from genes coding for beta-ketothiolase (phbA) and acetoacetyl-coenzyme A reductase (phbB). A PHB synthase mutant was obtained by gene inactivation and used for genetic studies. The phbC gene from this strain was introduced into Ralstonia eutropha PHB-4 (phbC-negative mutant), and the recombinant accumulated PHB when either glucose or octanoate was used as a source of carbon, indicating that this PHB synthase cannot incorporate medium-chain-length hydroxyalkanoates into PHB.

Acetyl-CoA C-Acyltransferase↗
PubMed2003

mRNA extraction and reverse transcription-PCR protocol for detection of nifH gene expression by Azotobacter vinelandii in soil.

The study of free-living nitrogen-fixing organisms in bulk soil is hampered by the great diversity of soil microbial communities and the difficulty of relating nitrogen fixation activities to individual members of the diazotroph populations. We developed a molecular method that allows analysis of nifH mRNA expression in soil in parallel with determinations of nitrogen-fixing activity and bacterial growth. In this study, Azotobacter vinelandii growing in sterile soil and liquid culture served as a model system for nifH expression, in which sucrose served as the carbon source and provided nitrogen-limited conditions, while amendments of NH(4)NO(3) were used to suppress nitrogen fixation. Soil RNA extraction was performed with a new optimized direct extraction protocol that yielded nondegraded total RNA. The RNA extracts were of high purity, free of DNA contamination, and allowed highly sensitive and specific detection of nifH mRNA by a reverse transcription-PCR. The level of nifH gene expression was estimated by PCR amplification of reverse-transcribed nifH mRNA fragments with A. vinelandii-specific nifH primers. This new approach revealed that nifH gene expression was positively correlated with bulk nitrogen fixation activity in soil (r(2) = 0.72) and in liquid culture (r(2) = 0.84) and therefore is a powerful tool for studying specific regulation of gene expression directly in the soil environment.

Azotobacter vinelandii↗
PubMed2005

Identification of a positive transcription regulatory element within the coding region of the nifLA operon in Azotobacter vinelandii.

Nitrogen fixation in Azotobacter vinelandii is regulated by the nifLA operon. NifA activates the transcription of nif genes, while NifL antagonizes the transcriptional activator NifA in response to fixed nitrogen and molecular oxygen levels. However, transcriptional regulation of the nifLA operon of A. vinelandii itself is not fully understood. Using the S1 nuclease assay, we mapped the transcription start site of the nifLA operon, showing it to be similar to the sigma54-dependent promoters. We also identified a positive cis-acting regulatory element (+134 to +790) of the nifLA operon within the coding region of the nifL gene of A. vinelandii. Deletion of this element results in complete loss of promoter activity. Several protein factors bind to this region, and the specific binding sites have been mapped by DNase I foot printing. Two of these sites, namely dR1 (+134 to +204) and dR2 (+745 to +765), are involved in regulating the nifLA promoter activity. The absence of NtrC-like binding sites in the upstream region of the nifLA operon in A. vinelandii makes the identification of these downstream elements a highly significant finding. The interaction of the promoter with the proteins binding to the dR2 region spanning +745 to +765 appears to be dependent on the face of the helix as introduction of 4 bases just before this region completely disrupts promoter activity. Thus, the positive regulatory element present within the BglII-BglII fragment may play, in part; an important role in nifLA regulation in A. vinelandii.

Azotobacter vinelandii↗
PubMed1972

Improved staining of extracellular polymer for electron microscopy: examination of Azotobacter, Zoogloea, Leuconostoc, and Bacillus.

Phase contrast, ultraviolet microscopy, and freeze-etching were used to determine the amount of exocellular polymer surrounding unfixed cells of four genera of bacteria: Azotobacter vinelandii, Zoogloea ramigera, Leuconostoc mesenteroides, and an acid-tolerant, floc-forming Bacillus species. Thin-sectional electron microscopy was employed to measure the effectiveness of a modified ruthenium red staining method. The results obtained with this modification of ruthenium red staining technique were compared to results obtained when previously proposed ruthenium red methods of fixation were employed. The results of these relations were then compared to the amounts of exocellular material as determined with phase-contrast microscopy, ultraviolet microscopy, and freeze-etching. The data obtained indicate that improved fixation of exocellular polymer is achieved when cells are pretreated with ruthenium red as described herein. In addition, the modified methods also reveal cytological detail not apparent when other methods of ruthenium fixation are employed.

Azotobacter↗
PubMed1974

Survival of Azotobacter in dry soil.

Detection of viable Azotobacter in soils stored in the laboratory for more than 10 years suggests that these bacteria can remain dormant in nature for prolonged periods of time. Studies of dried cultures show that cysts of A. vinelandii 12837 remain viable for at least 10 years, whereas vegetative cells do not survive for even 1 year under the same conditions.

Azotobacter↗