Characterization of the gene coding for GDP-mannose dehydrogenase (algD) from Azotobacter vinelandii

Campos, Marı́a Eugenia; Martínez-Salazar, Jaime M.; Lloret, Lourdes; Moreno, Soledad; Núñez, Cinthia; Espı́n, Guadalupe; Soberón-Chávez, Gloria

doi:10.1128/jb.178.7.1793-1799.1996

Cited by 83 publications

(111 citation statements)

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“…After completion of this process, the cells are at a resting stage designated cyst, and in these cysts the cells are surrounded by a rigid coat consisting of layers (intine and exine) in which alginate is a major component. As expected, mutants unable to produce alginate also fail to form cysts (6).…”

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confidence: 87%

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Identification and Characterization of anAzotobacter vinelandiiType I Secretion System Responsible for Export of the AlgE-Type Mannuronan C-5-Epimerases

et al. 2006

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Alginate is a linear copolymer of ␤-D-mannuronic acid and its C-5-epimer, ␣-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 (<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.

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confidence: 87%

“…All genes involved in bacterial alginate biosynthesis, except algC (20), are clustered on the chromosome and are transcribed in one or several units (6,8,34,36). Polymerization of the sugar monomers takes place in the cytoplasmic membrane, using GDP-mannuronic acid as a precursor.…”

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confidence: 99%

Identification and Characterization of anAzotobacter vinelandiiType I Secretion System Responsible for Export of the AlgE-Type Mannuronan C-5-Epimerases

et al. 2006

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“…The A. vinelandii biosynthetic pathway has been shown previously to be identical to that found in Pseudomonas aeruginosa and many of the genes are conserved (Fialho et al, 1990 ;Gacesa, 1998 ;Rehm et al, 1996). The transcriptional activation of the GDP-mannose dehydrogenase gene (algD) is a key point in pathway regulation (Campos et al, 1996) and is mediated by the alternative sigma factor AlgU, a homologue of RpoE (MartinezSalazar et al, 1996). Recently it was shown that A. vinelandii strain UW136 has a natural insertion element in the algU gene , which may explain the alginate-minus phenotype of strains derived from UW.…”

Section: -4101 # 2001 Sgmmentioning

confidence: 99%

Alginate formation in Azotobacter vinelandii UWD during stationary phase and the turnover of poly-β-hydroxybutyrate

et al. 2001

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Azotobacter vinelandii UWD is a mutant of strain UW that is defective in the respiratory oxidation of NADH. This mutation causes an overproduction of polyhydroxyalkanoates (PHAs), as polyester synthesis is used as an alternative electron sink. Since PHAs have potential for use as natural, biodegradable plastics, studies of physiology related to their production are of interest.Alginate production by this strain is limited to T 11 µg (mg cell protein) N1 , which permits high efficiency conversion of carbon source into PHA. However, Z 400 µg (mg cell protein) N1 was formed when UWD cells were oxygen-limited and in the stationary phase of growth. Alginate formation was fuelled by PHA turnover, which was coincident with the synthesis of alkyl resorcinols, under conditions of exogenous glucose limitation. However, alginate production was a phenotypic and reversible change. Alginate production was stopped by interruption of algD with Tn5lacZ. LacZ activity in UWD was shown to increase in stationary phase, while LacZ activity in a similarly constructed mutant of strain UW did not. Transcription of algD in strain UWD started from a previously identified RpoD promoter and not from the AlgU (RpoE) promoter. This is because strain UWD has a natural insertion element in algU. Differences between strain UW and UWD may reside in the defective respiratory oxidation of NADH, where the NADH surplus in strain UWD may act as a signal of stationary phase. Indeed, a backcross of UW DNA into UWD generated NADHoxidase-proficient cells that failed to form alginate in stationary phase. Evidence is also presented to show that the RpoD promoter may be recognized by the stationary phase sigma factor (RpoS), which may mediate alginate production in strain UWD.

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“…The mature cysts are surrounded by two capsule-like layers containing a high proportion of the exopolysaccharide alginate. This exopolysaccharide is essential for the encystment process, since nonmucoid strains fail to encyst (Campos, et al, 1996). In Azotobacter vinelandii, alginate protects nitrogenase from oxygen and increases nitrogen fixation (Sabra, et al, 2000).…”

Section: Exopolysaccharide Productionmentioning

confidence: 99%

Alginate biopolymer production by Azotobacter chroococcum from whey degradation

Khanafari

Sepahei

2007

Int. J. Environ. Sci. Technol.

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ABSTRACT:The potential of three Azotobacter chroococcum strains for whey degradation and alginate production were investigated. After dilution, samples were spread plated on isolation agar and Manitol agar and incubated at 30 °C for 24 h. Microorganisms were screened for their ability to whey degradation and alginate production based on colony morphology, negative and capsule staining, ability to decrease the apparent turbidity of the fermentation broths in batch and semi continuous culture by spectrophotometer assay at 400 nanometer and tensiometer assay. Of the three strains tested for whey degradation, only Azotobacter chroococcum 1723 produced significant apparent growth on whey broth and could decrease about 70 % of turbidity in fermentation broth during 6 days in batch culture. Colonies of this strain was characteristically yellow, large, moucoid and slimy on whey agar than Manitol agar after 24 h at 30 °C. Transmission electron microscopy assay and Carbazole reagent were used to recognize the alginate biopolymer. After optimizing environmental factors such as pH, salt concentration and temperature, this strain was able to produce exopolysaccharide greater than 5 mg/mL. Optimum results were obtained when using whey broth as a fermentation medium without extra salt, temperature at 35 °C and pH 7. Increasing inorganic and organic nitrogen sources (yeast extract and NH 4 NO 3 ) reduced whey degradation at least 30%. Transmission electron microscopy assay showed a net-structured polysaccharide capsule around the cells. Semi-continuous culture results demonstrated that, alginate production as well as whey degradation was decreased (1 mg/mL and 30 %).

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Characterization of the gene coding for GDP-mannose dehydrogenase (algD) from Azotobacter vinelandii

Cited by 83 publications

References 41 publications

Identification and Characterization of anAzotobacter vinelandiiType I Secretion System Responsible for Export of the AlgE-Type Mannuronan C-5-Epimerases

Identification and Characterization of anAzotobacter vinelandiiType I Secretion System Responsible for Export of the AlgE-Type Mannuronan C-5-Epimerases

Alginate formation in Azotobacter vinelandii UWD during stationary phase and the turnover of poly-β-hydroxybutyrate

Alginate biopolymer production by Azotobacter chroococcum from whey degradation

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