William J. Page

Production of poly-?-hydroxybutyrate by Azotobacter vinelandii strain UWD during growth on molasses and other complex carbon sources

SummaryAzotobacter vinelandii strain UWD formed >2 mg/ml poly-β-hydroxybutyrate (pHB) during exponential growth in media containing ammonium acetate and 1% w/v glucose, fructose, sucrose, or maltose, and >1.5 mg/ml with 1% w/v sodium gluconate or glycerol. After acetate exhaustion, pHB formation accompanied carbohydrate utilization and pHB rapidly accounted for 53%–70% of the cell mass. Strain UWD also formed >2 mg/ml pHB when it was grown with 2% w/v corn syrup, cane molasses, beet molasses, or malt extract. Beet molasses had a growth stimulatory effect which promoted higher yields of pHB/ml and a high ratio of pHB/protein. Malt extract also promoted higher yields of pHB/ml. In this case, pHB formation was no longer subject to acetate repression and the cells contained a higher ratio of pHB/protein. This study shows that unrefined carbon sources support pHB formation in strain UWD and that the yields of pHB were comparable to or better than those obtained with refined carbon sources.

Production of poly-β-hydroxybutyrate by Azotobacter vinelandii UWD in media containing sugars and complex nitrogen sources

SummaryVigorously aerated batch cultures of Azotobacter vinelandii UWD formed < 1 g poly-β-hydroxybutyrate (PHB)/l in media containing pure sugars and ≤ 3 g PHB/l in media containing cane molasses, corn syrup or malt extract. However, > 7 g PHB/l was formed when the medium contained 5% beet molasses. Increased yields of PHB were promoted in the media containing pure or unrefined sugars by the addition of complex nitrogen sources. The greatest effect was obtained with 0.05–0.2% fish peptone (FP), proteose peptone no. 3 or yeast extract. Peptones caused a ≤ 1.6-fold increase in residual non-PHB biomass and up to a 25-fold increase in PHB content. Hence the increased PHB formation was not simply due to stimulation of culture growth. The amount of PHB per cell protein formed by UWD in media containing FP was greatest in glucose = corn syrup > malt extract > sucrose = fructose = cane molasses > maltose, as carbon sources. The addition of FP to medium containing beet molasses did not stimulate PHB yield. The peptone effect was most significant in well-aerated cultures, which were fixed nitrogen and consuming glucose at a high rate. An explanation for the peptone effect on PHB yield stimulation is proposed.

Production of poly-b-hydroxybutyrate by Azotobacter vinelandii in a two-stage fermentation process

Poly-b-hydroxybutyrate (PHB) production in Azotobacter vinelandii UWD, a mutant that produces PHB constitutively, was suppressed by high aeration of beet molasses medium. Thus a two-stage process was designed using aeration to promote growth and suppress PHB production in the first phase, while lower aeration of raw sugar medium containing fish peptone was used to promote PHB formation in the second phase. A PHB yield of 36 g/l and productivity of > 1 g polymer l -1 .h was obtained by this approach.

Control of polyhydroxyalkanoate synthesis in Azotobacter vinelandii strain UWD

polyhydroxyalkanoate (PHA) in mutant Azotobacter vinelandii UWD, the kinetic properties of 3-ketothiolase, acetoacetyl-CoA reductase, and /3hydroxybutyrate dehydrogenase were examined. The regulatiorn of the condensation of acetyl-CoA mediated by 3-ketothiolase was narmal, in that it was negatively regulated by free CoA, but inhibition was overeome by higher concentrations of acetyl-CoA. Acetoacetyl-CoA from this reaction was reduced to 3-hydroxybutyryl-CoA by an NADPH-specif ic acetoacetyl-CoA reductase. This enzyme also reduced 3-ketovaleryl-CoA derived from the /?-oxidation of C, C, or C, n-alkanoates, but at only 16% of the rate found with the C,-substrate. The acetoacetyl-CoA reductase was determined to be an allosteric enzyme that bound NADPH and acetoacetyl-CoA at multiple binding sites irl a general hybrid Ping-Pong random mechanism. The enzyme was negatively regulaqed by acetoacetyl-CoA, but this was overcome at high concentrations of NADPH, The activity of pyridine nucleotide transhydrogenase was determined to be important for the conversion of NADH in these mutant cells to NADPH and for decreasing the availability of NADP+, which was a negative regulator of the acetoacetyl-CoA reductase. The combination of high acetoacetyl-CoA, the UWD mutation, transhydrogenase activity, and high NADPH appeared to be the conditions promoting PHA formation by strain UWD during active growth on glucose. Degradation of PHA in strain UWD did not appear to be regulated at the level of /3-hydroxybutyrate dehydrogenase. This enzyme was unaffected by NADH, was inhibited only 13% by pyruvate and its activity was enhanced by NADPH. The thiolysis of acetoacetyl-CoA also was unusual, in that 3ketothiolase was not inhibited by acetoacetyl-CoA, but free CoA was a competitive inhibitor in a bireactant Ping-Pong mechanism. This inhibition was overcome by higher concentrations of the normal first substrate, acetoacetylCoA. Thus a single thiolase was used for the condensation of acetyl-CoA and the thiolysis of acetoacetyl-CoA, derived from PHA depolymerization or from the /?-oxidation of n-alkanoates.

Dual regulation of catecholate siderophore biosynthesis in Azotobacter vinelandii by iron and oxidative stress.

Azotobacter vinelandii forms both catecholate and azotobactin siderophores during iron-limited growth. Azotobactin is repressed by about 3 microM iron, but catecholate siderophore synthesis continues up to a maximum of 10 microM iron. This suggests that catecholate siderophore synthesis is regulated by other factors in addition to the ferric uptake repressor (Fur). In this study the first gene required for catecholate siderophore biosynthesis, which encodes an isochorismate synthase (csbC), was isolated. The region upstream of csbC contained a typical sigma(70) promoter, with an iron-box overlapping the -35 sequence and a Sox-box (Box 1) overlapping the -10 sequence. Another Sox-box was found further upstream of the -35 sequence (Box 2). Also upstream, an unidentified gene (orfA) was detected which would be transcribed from a divergent promoter, also controlled by an iron-box. The activity of csbC and a csbC::luxAB fusion was negatively regulated by iron availability and upregulated by increased aeration and by superoxide stress. The iron-box in the csbC promoter was 74% identical to the Fur-binding consensus sequence and bound the Fur protein of Escherichia coli with relatively high affinity. Both Box 1 and Box 2 were in good agreement with the consensus sequence for binding the SoxS protein of E. coli and Box 1 was in very good agreement with the Sox-box found in the fpr promoter of A. vinelandii, which is also regulated by superoxide stress. Both Sox-boxes bound a protein found in A. vinelandii cell extracts, with Box 1 exhibiting the higher binding affinity. The Sox protein identified in this assay appeared to be constitutive, rather than inducible by superoxide stress. This indicates that the Sox response in A. vinelandii is different from that in E. coli. These data support the hypothesis that catecholate siderophore biosynthesis is under dual control, repressed by a Fur-iron complex and activated by another DNA-binding protein in response to superoxide stress. The interaction between these regulators is likely to account for the delay in ferric repression of catecholate siderophore production, since these siderophores have an additional role to play in the protection of iron-limited cells against oxidative damage.

The catecholate siderophores of Azotobacter vinelandii : their affinity for iron and role in oxygen stress management

In iron-limited medium, Azotobacter vinelandii strain UW produces three catecholate siderophores: the tricatecholate protochelin, the dicatecholate azotochelin and the monocatecholate aminochelin. Each siderophore was found to bind Fe3+ preferentially to Fe2+, in a ligand:Fe ratio of 1:1, 3:2 and 3:1, respectively. Protochelin had the highest affinity for Fe3+, with a calculated proton-independent solubility coefficient of 10439, comparable to ferrioxamine B. Iron-limited wild-type strain UW grown under N2-fixing or nitrogen-sufficient conditions hyper-produced catecholate siderophores in response to oxidative stress caused by high aeration. In addition, superoxide dismutase activity was greatly diminished in iron-limited cells, whereas catalase activity was maintained. The ferredoxin I (Fdl)-negative A. vinelandii strain LM100 also hyper-produced catecholates, especially protochelin, under oxidative stress conditions, but had decreased activities of both superoxide dismutase and catalase, and was about 10 times more sensitive to paraquat than strain UW. Protochelin and azotochelin held Fe3+ firmly enough to prevent its reduction by.O- 2 and did not promote the generation of hydroxyl radical by the Fenton reaction. Ferric-aminochelin was unable to resist reduction by O- 2 and was a Fenton catalyst. These data suggest that under iron-limited conditions, A. vinelandii suffers oxidative stress caused by.O- 2. The catecholate siderophores azotochelin, and especially protochelin, are hyper-produced to offer chemical protection from oxidative damage catalysed by.O- 2 and Fe3+. The results are also consistent with Fdl being required for oxidative stress management in A. vinelandii.

Aminochelin, a Catecholamine Siderophore Produced by Azotobacter vinelandii

SUMMARY: A catecholamine siderophore, named aminochelin, produced by iron-limited Azotobacter vinelandii was purified and tentatively identified as 2,3-dihydroxybenzoylputrescine. This compound was first observed as an ethyl-acetate-insoluble catechol that accounted for 30 to 50% of the total catechol in iron-limited culture supernatant fluids. The purified compound was unstable at neutral to alkaline pH, bound Fe3+, Fe2+ and molybdate, and promoted 55Fe-uptake into iron-limited A. vinelandii. Aminochelin was induced and repressed coordinately with the other catechol siderophore azotochelin. The catechol siderophores were, however, less sensitive to repression by soluble iron than the yellow-green fluorescent peptide siderophore azotobactin.

Production of the triacetecholate siderophore protochelin by Azotobacter vinelandii

Azotobacter vinelandii grown in iron-limited medium containing 1 μm molybdate released the catecholate siderophores azotochelin and aminochelin [bis(2,3-dihydroxybenzoyl-lysine) and 2,3-dihydroxybenzoyl-putrescine, respectively] into the culture fluid. However these catecholates were not observed when the medium contained 1 mm molybdate, but were replaced by another catecholate compound. The appearance of this new compound was not an artifact of extraction of the catecholates from the culture fluid in the presence of high molybdate. Full and partial acid hydrolysis and fast atom bombardment mass spectroscopy showed that the new compound was the tricatecholate protochelin, a product of the condensation of azotochelin and aminochelin. The production of protochelin was iron-repressible and protochelin very rapidly decolorized the Chrome Azurol-S assay. Protochelin promoted the growth of the siderophore-deficient A. vinelandii strain P100 under iron-restricted conditions and promoted 55Fe uptake into iron-limited cells, confirming that protochelin can be used as a siderophore by A. vinelandii.

The effect of substrate on the molecular weight ofpoly-β-hydroxybutyrate produced byAzotobacter vinelandii UWD

SummaryAzotobacter vinelandii UWD produced very high molecular weight (MW) (approx. 4 million Daltons) poly-β-hydroxybutyrate (PHB) when grown in 5% w/v beet molasses medium. The polymer MW decreased as the beet molasses concentration was increased. Similar results were obtained in equivalent concentrations of sucrose (as raw sugar), but the polymer MW was not greater than 1.6 million. This difference was not caused by more severe oxygen-limitation in the beet molasses medium. It appeared that the nonsugar components of beet molasses promoted the formation of higher MW polymer. Fish peptone, a known PHB-yield-promoter in this organism, did not promote the formation of very high MW polymer.

Role of Molybdate and Other Transition Metals in the Accumulation of Protochelin by Azotobacter vinelandii

ABSTRACT 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. vinelandiisiderophores 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 Fe3+. Thus, competition between Fe3+ 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, Zn2+, and Mn2+. We found that all five of these metal ions partially inhibited uptake of55Fe-protochelin and 55Fe-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.