Effect of Oxygen Concentration and Growth Rate on Glucose Metabolism, Poly- -hydroxybutyrate Biosynthesis and Respiration of Azotobacter beijerinckii

Carter, Ian S.; Dawes, E. A.

doi:10.1099/00221287-110-2-393

Cited by 28 publications

(13 citation statements)

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“…These limitations include oxygen deprivation, nitrogen deprivation, sulfate limitation, and magnesium limitation (3,4,10,15,(18)(19)(20)(21)24). Under limiting environmental conditions, PHB may constitute as much as 80% of the dry cell weight.…”

Section: Poly-p-hydroxybutyrate (Phb) a Homopolymer Of D-(-)-mentioning

confidence: 99%

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Cloning and expression in Escherichia coli of the Alcaligenes eutrophus H16 poly-beta-hydroxybutyrate biosynthetic pathway

1988

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The poly-I-hydroxybutyrate (PHB) biosynthetic pathway from Alcaligenes eutrophus H16 has been cloned and expressed in Escherichia coli. Initially, an A. eutrophus H16 genomic library was constructed by using cosmid pVK102, and cosmid clones that encoded the PHB biosynthetic pathway were sought by assaying for the first enzyme of the pathway, I-ketothiolase. Six enzyme-positive clones were identified. Three of these clones manifested acetoacetyl coenzyme A reductase activity, the second enzyme of the biosynthetic pathway, and accumulated PHB. PHB was produced in the cosmid clones at approximately 50% of the level found in A. eutrophus. One cosmid clone was subjected to subcloning experiments, and the PHB biosynthetic pathway was isolated on a 5.2-kilobase KpnI-EcoRI fragment. This fragment, when cloned into small multicopy vectors, can direct the synthesis of PHB in E. coli to levels approaching 80% of the bacterial cell dry weight.Poly-p-hydroxybutyrate (PHB), a homopolymer of D-(-)-3-Hydroxybutyrate, is a storage material produced by a variety of bacteria in response to environmental stress. The presence of PHB in bacteria was first recognized by Lemoigne in 1926 (11), and it has since been identified in more than 20 bacterial genera, including Azotobacter, Bacillus, Beijerinckia, Alcaligenes, Pseudomonas, Rhizobium, and Rhodospirillum (4).The PHB pathway and its regulation have been studied extensively in Alcaligenes eutrophus H16 and Azotobacter beijerinckii (3,4,9,10,15,(18)(19)(20)(21)24). The pathway consists of a biosynthetic portion and a degradative portion and is made up of five enzymes. One of these enzymes, 0-ketothiolase, is both the entry point and the exit point of the cycle (4). In the biosynthetic part of the pathway, ,-ketothiolase catalyzes the reversible condensation of two acetyl coenzyme A (CoA) molecules to acetoacetyl-CoA. Acetoacetyl-CoA is subsequently reduced to D-(-)-3-hydroxybutyryl-CoA by acetoacetyl-CoA reductase, and PHB is then produced by the polymerization of 0-hydroxybutyrylCoA via the action of PHB synthetase.Studies in A. eutrophus H16 and in Azotobacter beijerinckii have shown the PHB pathway to be regulated in response to several types of environmental limitation. These limitations include oxygen deprivation, nitrogen deprivation, sulfate limitation, and magnesium limitation (3,4,10,15,(18)(19)(20)(21)24). Under limiting environmental conditions, PHB may constitute as much as 80% of the dry cell weight. When limiting conditions are relaxed, PHB quantities decrease to preinduction levels (4). Induction studies in which 1-ketothiolase and acetoacetyl-CoA reductase were studied have revealed that both enzymatic activities increase markedly in response to PHB-stimulating limitations (4,10,15,19).These experiments indicate that the PHB pathway may exhibit a mode of transcriptional control that is similar to that of other metabolic pathways that are induced by environmental stress. Examples of such pathways include the heat shock regulon, the pho regulon, and the carbon starvati...

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Section: Poly-p-hydroxybutyrate (Phb) a Homopolymer Of D-(-)-mentioning

confidence: 99%

“…The PHB pathway and its regulation have been studied extensively in Alcaligenes eutrophus H16 and Azotobacter beijerinckii (3,4,9,10,15,(18)(19)(20)(21)24). The pathway consists of a biosynthetic portion and a degradative portion and is made up of five enzymes.…”

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confidence: 99%

Cloning and expression in Escherichia coli of the Alcaligenes eutrophus H16 poly-beta-hydroxybutyrate biosynthetic pathway

1988

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“…Growth was monitored by the absorbance at 680 nm, using 1 cm cuvettes in a Pye Unicam SP500 spectrophotonieter. Poly-a-hydroxybutyrate was estimated by the method of Carter & Dawes (1979), using a 5 ml culture sample. For estimation of polyphosphate, a complete 1 1 culture was centrifuged, polyphosphate was extracted and hydrolysed as described by Herbert et af.…”

Section: E T H O D Smentioning

confidence: 99%

Adenine Nucleotide Pools During Starvation of Beneckea natriegens

1980

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The marine bacterium Beneckea natriegens was grown in batch culture on a glucose/ NH,+/salts medium; growth terminated due to either carbon or nitrogen depletion from the medium. Nitrogen-limited cultures converted part of the excess glucose into glycogen whereas the carbon-limited cultures formed little glycogen. Glycogen-rich cultures survived longer than glycogen-poor cultures during starvation. Little protein was utilized during starvation and RNA was degraded as the primary endogenous source of energy. Glycogen was consumed only when the RNA content had decreased to about a third of the growth value.The adenine nucleotide content of nitrogen-limited cultures increased at the start of the stationary phase but the energy charge remained at the growth value of 0.9 to 0.95. The maximum size of the adenine nucleotide pool depended on the concentration of glucose remaining in the medium at the start of the stationary phase but a limiting value of about 60 pmol ATP (g protein)-l was attained, compared with 12 to 14 pmol ATP (g protein)-l in exponentially growing cultures. During extended starvation of both glycogen-rich and glycogen-poor cultures, there was a large decrease in adenine nucleotide content, but the energy charge remained above 0-6 even when viability was very low. I N T R O D U C T I O NThere are many factors that can cause loss of viability of bacteria (Dawes, 1976). During long-term starvation, endogenous metabolites such as non-essential RNA, protein and reserve polymers (glycogen, poly-P-hydroxybutyrate, triacylglycerols and polyphosphate) act as energy sources permitting essential maintenance processes to occur, thereby preserving viability (Dawes & Senior, 1973). Under these circumstances loss of viability may be related to the inability to regenerate ATP due to depletion of endogenous metabolites and reserve polymers. This would be seen as a decrease in the adenine nucleotide content or as a decrease in the ATP : ADP and ATP : AMP ratios, which may be measured as a decrease in energy charge (Atkinson, 1968(Atkinson, , 1978Chapman & Atkinson, 1977;Knowles, 1977Knowles, ,1979.There have been few studies on the adenylate pools of starving bacteria. Chapman et uZ. (1 97 1) showed that, following nitrogen depletion from a glucose-containing growth medium, there was a gradual decrease in the intracellular energy charge of Escherichia cofi strain B from a growth value of about 0-8 to between 0.6 and 0-5 after 60 to 80 h, without loss of viability. The intracellular adenine nucleotide content also decreased during this period. After 60 to 80 h, a rapid decrease in energy charge occurred, coincident with a loss of viability. Montague & Dawes (1974) showed a similar relationship of decrease in energy charge and loss of viability during starvation of Peptococcus pre'votii, which does not form reserve polymers but utilizes RNA as the sole endogenous source of energy. 7 Present address: The College of the Resurrection, Mirfield, West Yorkshire WF14 OBN.

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“…KEYWORDS hydrogen, in vivo nitrogenase activity, molybdenum, vanadium, iron A zotobacter species are archetypes of aerobic diazotrophic heterotrophy, studied for over 100 years for their ability to fix nitrogen in the presence of oxygen (1)(2)(3)(4), and their physiology has been discussed in detail by our group previously (5,6). A. vinelandii encodes three versions of the nitrogenase complex, each employing a different metalcontaining cofactor in its active site.…”

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Azotobacter vinelandii Nitrogenase Activity, Hydrogen Production, and Response to Oxygen Exposure

Natzke

Noar

Bruno-Bárcena

2018

Appl Environ Microbiol

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selectively utilizes three types of nitrogenase (molybdenum, vanadium, and iron only) to fix N, with their expression regulated by the presence or absence of different metal cofactors in its environment. Each alternative nitrogenase isoenzyme is predicted to have different electron flux requirements based on measurements, with the molybdenum nitrogenase requiring the lowest flux and the iron-only nitrogenase requiring the highest. Here, prior characterized strains, derepressed in nitrogenase synthesis and also deficient in uptake hydrogenase, were further modified to generate new mutants lacking the ability to produce poly-β-hydroxybutyrate (PHB). PHB is a storage polymer generated under oxygen-limiting conditions and can represent up to 70% of the cells' dry weight. The absence of such granules facilitated the study of relationships between catalytic biomass and product molar yields across different adaptive respiration conditions. The released hydrogen gas observed during growth, due to the inability of the mutants to recapture hydrogen, allowed for direct monitoring of nitrogenase activity for each isoenzyme. The data presented here show that increasing oxygen exposure limits equally the activities of all nitrogenase isoenzymes, while under comparative conditions, the Mo nitrogenase enzyme evolves more hydrogen per unit of biomass than the alternative isoenzymes. has been a focus of intense research for over 100 years. It has been investigated for a variety of functions, including agricultural fertilization and hydrogen production. All of these endeavors are centered around 's ability to fix nitrogen aerobically using three nitrogenase isoenzymes. The majority of research up to this point has targeted measurements of the molybdenum nitrogenase, and robust data contrasting how oxygen impacts the activity of each nitrogenase isoenzyme are lacking. This article aims to provide nitrogenase activity data using a real-time evaluation of hydrogen gas released by derepressed nitrogenase mutants lacking an uptake hydrogenase and PHB accumulation.

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Effect of Oxygen Concentration and Growth Rate on Glucose Metabolism, Poly- -hydroxybutyrate Biosynthesis and Respiration of Azotobacter beijerinckii

Cited by 28 publications

References 13 publications

Cloning and expression in Escherichia coli of the Alcaligenes eutrophus H16 poly-beta-hydroxybutyrate biosynthetic pathway

Cloning and expression in Escherichia coli of the Alcaligenes eutrophus H16 poly-beta-hydroxybutyrate biosynthetic pathway

Adenine Nucleotide Pools During Starvation of Beneckea natriegens

Azotobacter vinelandii Nitrogenase Activity, Hydrogen Production, and Response to Oxygen Exposure

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