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PubMed2006

Identification and characterization of an Azotobacter vinelandii type I secretion system responsible for export of the AlgE-type mannuronan C-5-epimerases.

Alginate is a linear copolymer of beta-d-mannuronic acid and its C-5-epimer, alpha-l-guluronic acid. During biosynthesis, the polymer is first made as mannuronan, and various fractions of the monomers are then epimerized to guluronic acid by mannuronan C-5-epimerases. The Azotobacter vinelandii genome encodes a family of seven extracellular such epimerases (AlgE1 to AlgE7) which display motifs characteristic for proteins secreted via a type I pathway. Putative ATPase-binding cassette regions from the genome draft sequence of the A. vinelandii OP strain and experimentally verified type I transporters from other species were compared. This analysis led to the identification of one putative A. vinelandii type I system (eexDEF). The corresponding genes were individually disrupted in A. vinelandii strain E, and Western blot analysis using polyclonal antibodies against all AlgE epimerases showed that these proteins were present in wild-type culture supernatants but absent from the eex mutant supernatants. Consistent with this, the wild-type strain and the eex mutants produced alginate with about 20% guluronic acid and almost pure mannuronan (< or =2% guluronic acid), respectively. The A. vinelandii wild type is able to enter a particular desiccation-tolerant resting stage designated cyst. At this stage, the cells are surrounded by a rigid coat in which alginate is a major constituent. Such a coat was formed by wild-type cells in a particular growth medium but was missing in the eex mutants. These mutants were also found to be unable to survive desiccation. The reason for this is probably that continuous stretches of guluronic acid residues are needed for alginate gel formation to take place.

Azotobacter vinelandii↗
PubMed2006

Peptidyl-prolyl cis/trans isomerase-independent functional NifH mutant of Azotobacter vinelandii.

Peptidyl-prolyl cis/trans isomerases (PPIases) play a pivotal role in catalyzing the correct folding of many prokaryotic and eukaryotic proteins that are implicated in a variety of biological functions, ranging from cell cycle regulation to bacterial infection. The nif accessory protein NifM, which is essential for the biogenesis of a functional NifH component of nitrogenase, is a PPIase. To understand the nature of the molecular signature that defines the NifM dependence of NifH, we screened a library of nifH mutants in the nitrogen-fixing bacterium Azotobacter vinelandii for mutants that acquired NifM independence. Here, we report that NifH can acquire NifM independence when the conserved Pro258 located in the C-terminal region of NifH, which wraps around the other subunit in the NifH dimer, is replaced by serine.

Amino Acid Sequence↗
PubMed2006

Controlled expression and functional analysis of iron-sulfur cluster biosynthetic components within Azotobacter vinelandii.

A system for the controlled expression of genes in Azotobacter vinelandii by using genomic fusions to the sucrose catabolic regulon was developed. This system was used for the functional analysis of the A. vinelandii isc genes, whose products are involved in the maturation of [Fe-S] proteins. For this analysis, the scrX gene, contained within the sucrose catabolic regulon, was replaced by the contiguous A. vinelandii iscS, iscU, iscA, hscB, hscA, fdx, and iscX genes, resulting in duplicate genomic copies of these genes: one whose expression is directed by the normal isc regulatory elements (Pisc) and the other whose expression is directed by the scrX promoter (PscrX). Functional analysis of [Fe-S] protein maturation components was achieved by placing a mutation within a particular Pisc-controlled gene with subsequent repression of the corresponding PscrX-controlled component by growth on glucose as the carbon source. This experimental strategy was used to show that IscS, IscU, HscBA, and Fdx are essential in A. vinelandii and that their depletion results in a deficiency in the maturation of aconitase, an enzyme that requires a [4Fe-4S] cluster for its catalytic activity. Depletion of IscA results in a null growth phenotype only when cells are cultured under conditions of elevated oxygen, marking the first null phenotype associated with the loss of a bacterial IscA-type protein. Furthermore, the null growth phenotype of cells depleted of HscBA could be partially reversed by culturing cells under conditions of low oxygen. Conserved amino acid residues within IscS, IscU, and IscA that are essential for their respective functions and/or whose replacement results in a partial or complete dominant-negative growth phenotype were also identified using this system.

Aconitate Hydratase↗
PubMed1969

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 polybeta-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.

Azotobacter↗
PubMed1969

Chemical composition of Azotobacter vinelandii cysts.

Cysts of Azotobacter vinelandii ATCC 12837 were germinated by exposure to 3.0 mm ethylenediaminetetraacetic acid (EDTA)-tris(hydroxymethyl)aminomethane buffer at pH 7.8, and their outer coats (exines) were purified by differential and isopycnic centrifugation. Electron micrographs of exine showed it to consist of multilayers of a three-membered sheet structure whose thickness was 7.0 to 7.5 nm. The inner, less electron-dense layer (intine) was also prepared from cysts by EDTA treatment, centrifugation, concentration, and dialysis. The exine consisted of 32% carbohydrate, 28% protein, 30% lipid, and 3.2% ash, with the ash comprised of 1.62% calcium, 0.02% magnesium, and 0.34% phosphorus. The amino acid composition of exine was similar to that of gram-negative bacterial cell walls. The intine consisted of 44% carbohydrate, 9.1% protein, 37% lipid, and 4.1% ash, with the ash comprised of 2.45% calcium, 0.02% magnesium, and 0.38% phosphorus. The carbohydrates of both exine and intine contained glucose, mannose, xylose, and rhamnose. Glucosamine and galactosamine were found only in the exines. The fatty acids consisted of normal, iso, and anteiso saturated fatty acids with 10 to 18 carbon atoms and mono-unsaturated C(11), C(16), and C(18) fatty acids. The exines contained mostly bound lipid, but intines contained primarily free lipid.

Amino Acids↗
PubMed1970

Outer layers of the Azotobacter vinelandii cyst.

Ruthenium red stained a capsule external to the exine of the Azotobacter cyst. The central body is therefore surrounded by three layers, the intine, the exine, and the capsule, all containing acid mucopolysaccharide. Vesicles that appear to originate from the contracting cell membrane of the central body may account for the lipid content of the intine. The exine is composed of laminated sheets that tend to fragment into hexagonal pieces.

Azotobacter↗
PubMed1970

Pyridine nucleotide transhydrogenase from Azotobacter vinelandii.

A method is described for the partial purification of pyridine nucleotide transhydrogenase from Azotobacter vinelandii (ATCC 9104) cells. The most highly purified preparation catalyzes the reduction of 300 mumoles of nicotinamide adenine dinucleotide (NAD(+)) per min per mg of protein under the assay conditions employed. The enzyme catalyzes the reduction of NAD(+), deamino-NAD(+), and thio-NAD(+) with reduced nicotinamide adenine dinucleotide phosphate (NADPH) as hydrogen donor, and the reduction of nicotinamide adenine dinucleotide phosphate (NADP(+)) and thio-NAD(+) with reduced NAD (NADH) as hydrogen donor. The reduction of acetylpyridine AD(+), pyridinealdehyde AD(+), acetylpyridine deamino AD(+), and pyridinealdehydedeamino AD(+) with NADPH as hydrogen donor was not catalyzed. The enzyme catalyzes the transfer of hydrogen more readily from NADPH than from NADH with different hydrogen acceptors. The transfer of hydrogen from NADH to NADP(+) and thio-NAD(+) was markedly stimulated by 2'-adenosine monophosphate (2'-AMP) and inhibited by adenosine diphosphate (ADP), adenosine triphosphate (ATP), and phosphate ions. The transfer of hydrogen from NADPH to NAD(+) was only slightly affected by phosphate ions and 2'-AMP, except at very high concentrations of the latter reagent. In addition, the transfer of hydrogen from NADPH to thio-NAD(+) was only slightly influenced by 2'-AMP, ADP, ATP, and other nucleotides. The kinetics of the transhydrogenase reactions which utilized thio-NAD(+) as hydrogen acceptor and NADH or NADPH as hydrogen donor were studied in some detail. The results suggest that there are distinct binding sites for NADH and NAD(+) and perhaps a third regulator site for NADP(+) or 2'-AMP. The heats of activation for the transhydrogenase reactions were determined. The properties of this enzyme are compared with those of other partially purified transhydrogenases with respect to the regulatory functions of 2'-AMP and other nucleotides on the direction of flow of hydrogen between NAD(+) and NADP(+).

Adenine Nucleotides↗
PubMed1970

Morphogenesis of cysts in Azotobacter vinelandii.

Cultures of Azotobacter vinelandii were induced to encystment with beta-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.

Azotobacter↗
PubMed1970

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.

Acetates↗
PubMed1971

Physiological studies of encystment in Azotobacter vinelandii.

Azotobacter vinelandii, in late exponential growth phase, encysts when the glucose in the medium is replaced with beta-hydroxybutyrate. A final cell division then occurs without apparent deoxyribonucleic acid (DNA) synthesis, resulting in a reduction from two to one nucleoids per cell and a final DNA content of 3.2 x 10(-14) g per cell. This is also the DNA content per cyst. A beta-hydroxybutyrate dehydrogenase is derepressed by the addition of the inducer and is identical to the enzyme in acetate-grown cells in its pH optimum, Michaelis constant for substrate, temperature-activity response, and mobility during electrophoresis in acrylamide gel.

Acrylates↗
PubMed1971

Regulation at the phosphoenolpyruvate branchpoint in Azotobacter vinelandii: phosphoenolpyruvate carboxylase.

Phosphoenolpyruvate carboxylase (EC 4.1.1.31) from Azotobacter vinelandii, like the corresponding enzyme from other organisms, is activated by acetyl coenzyme A and inhibited by l-aspartate. Both modifiers affect primarily the affinity of the enzyme for phosphoenolpyruvate. This is the first enzyme with a strictly anaplerotic (intermediate-replacing) function to be tested for response to the adenylate energy charge; it is entirely insensitive to variation in charge. The results suggest that carboxylation of phosphoenolpyruvate in this organism is controlled by negative feedback from aspartate and by the stimulatory effect of acetyl coenzyme A. The adenylate energy charge may be expected to affect the rate of this reaction indirectly through its effects on the concentrations of acetyl coenzyme A and l-aspartate.

Aspartic Acid↗
PubMed1971

Regulation at the phosphoenolpyruvate branchpoint in Azotobacter vinelandii: pyruvate kinase.

Pyruvate kinase (EC 2.7.1.40) from Azotobacter vinelandii responds sharply to the adenylate energy charge, with a decrease in activity at high values of charge, as expected for an enzyme of an adenosine triphosphate-regenerating sequence. Glycolytic intermediates, especially glucose 6-phosphate, fructose 6-phosphate, and fructose-1,6-diphosphate, strongly stimulate the reaction and overcome the inhibition caused by high values of energy charge. Thus, the properties of this enzyme depend on interaction between energy charge and the concentrations of hexose phosphates. The properties of pyruvate kinase, together with those of phosphoenolpyruvate carboxylase, aspartokinase, and citrate synthase, seem adapted to provide appropriate partitioning of phosphoenolpyruvate between competing pathways in response to metabolic need.

Adenine Nucleotides↗
PubMed1971

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.

Alcohols↗
PubMed1972

Freeze-etching of Azotobacter vinelandii: examination of wall, exine, and vesicles.

The structure of the cell wall, the arrangement of the cyst exine, and the origin and distribution of intine vesicles in Azotobacter vinelandii ATCC 12837 were examined by freeze-etching and conventional electron microscopic techniques. In the vegetative organism the cell wall appears to have a woven texture which disappears during cyst formation. The exine is composed of two different types of material: the outer layer is a fibrous, amorphous layer, and the numerous inner layers form the basic hexagonal structures which unite to form the cyst coat. The presence of intine vesicles in the encysting organism was confirmed in frozen-etched cells. The appearance of frozen-etched cells and cysts and the distribution of capsular material indicate that extracellular polysaccharide is an important factor in cyst formation.

Azotobacter↗
PubMed1972

Effect of peptone on Azotobacter morphology.

Pleomorphism in cultures of Azotobacter vinelandii was induced by addition of Difco peptone to the growth medium. Under the conditions of the study, 5% peptone gave rise to transient forms described as "fungoid cells" which were osmotically fragile. After some 48 hr of culture, they became osmotically stable and resumed a more typical morphology. It was shown that the pleomorphism-inducing principle in peptone was glycine.

Amino Acids↗
PubMed1973

Germination of Azotobacter vinelandii cysts: sequence of macromolecular synthesis and nitrogen fixation.

Azotobacter vinelandii cysts undergo conversion to vegetative cells in Burk's nitrogen-free medium utilizing glucose, sucrose, or acetate. In 1% glucose, this overall process was complete in 8 hr and consisted of a germination and an outgrowth phase. Respiration, ribonucleic acid, and protein synthesis began soon after the addition of the germinant, and these processes proceeded at rates characteristic of the germination. The rates of respiration and synthesis increased sharply between 4 and 5 hr, the beginning of the outgrowth, at which time deoxyribonucleic acid synthesis and nitrogen fixation began. Respiration, macromolecular synthesis and nitrogen fixation continued at high rates until the emergence of vegetative cells from the cyst coats.

Acetates↗
PubMed1973

Internal membrane control in Azotobacter vinelandii.

Azotobacter vinelandii was grown on N(2), NH(4) (+), or NO(3) (-), and an internal membrane network was observed by electron microscopy of thin sections of cells. Cells obtained in early exponential growth contained less internal membrane than did cells from cultures in late exponential growth. It seems likely that O(2) has a role in regulating the amount of internal membrane structure.

Ammonium Chloride↗