Reconstitution of emulsifying activity of Acinetobacter calcoaceticus BD4 emulsan by using pure polysaccharide and protein.

Acinetobacter calcoaceticus BD4 and BD413 produce extracellular emulsifying agents when grown on 2% ethanol medium. For emulsifying activity, both polysaccharide and protein fractions were required, as demonstrated by selective digestion of the polysaccharide with a specific bacteriophage-borne polysaccharide depolymerase, deproteinization of the extracellular emulsifying complex with hot phenol, and reconstitution of emulsifier activity with pure polysaccharide and a polysaccharide-free protein fraction. Chemical modification of the carboxyl groups in the polysaccharide resulted in a loss of activity. The protein required for reconstitution of emulsifying activity was purified sevenfold. The BD4 emulsan apparently derives its amphipathic properties from the association of an anionic hydrophilic polysaccharide with proteins.     

Relationship between emulsifying activity and carbohydrate backbone structure of emulsan from Acinetobacter calcoaceticus RAG-1

Various emulsan samples with the different degrees of branching of the carbohydrate backbone were obtained from Acinetobacter calcoaceticus under different culture conditions. The emulsifying activity of emulsan had a linear correlation to the branching degrees of the carbohydrate backbone (r²= 0.930) suggesting that the structure of carbohydrate backbone was an important factor influencing emulsifying activity. This revised version was published online in November 2006 with corrections to the Cover Date.     

Structural studies of the capsular polysaccharide of Acinetobacter calcoaceticus BD4

Compositional analysis of the intact and carboxyl-reduced capsular polysaccharide of Acinetobacter calcoaceticus BD4 (PS-4) showed it to consist of L-rhamnose, D-glucose, D-glucuronic and D-mannose in molar ratios of 4:1:1:1. 13C-nuclear magnetic resonance spectroscopy, methylation analysis, oligosaccharide analysis and base-catalyzed beta-elimination were used to elucidate the primary structure. Oligosaccharides were obtained by enzymatic depolymerization with a specific bacteriophage-induced depolymerase and by partial acid hydrolysis. Form the results it is concluded that PS-4 consists of repeating units of the heptasaccharide (Formula: see text). The bacteriophage-induced depolymerase was found to be an endo-beta-D-glucosidase that hydrolyzed the bond beta-D-Glc-(1----3)-L-Rha to generate a heptasaccharide in 40% yield.     

Drag reduction by Acinetobacter calcoaceticus BD4

The encapsulated bacterium Acinetobacter calcoaceticus BD4 at a density of 3.6 X 10(9) cells per ml reduced the friction of turbulent water in a narrow pipe by 55%. This drag reduction was due to the tightly bound polysaccharide capsules (0.4 mg per ml) of culture. Capsule-deficient mutants of BD4 failed to reduce drag. The cell-bound polysaccharide demonstrated a threefold-higher drag-reducing activity than the polymer which was free in solution.     

Drag reduction by Acinetobacter calcoaceticus BD4.

The encapsulated bacterium Acinetobacter calcoaceticus BD4 at a density of 3.6 X 10(9) cells per ml reduced the friction of turbulent water in a narrow pipe by 55%. This drag reduction was due to the tightly bound polysaccharide capsules (0.4 mg per ml) of culture. Capsule-deficient mutants of BD4 failed to reduce drag. The cell-bound polysaccharide demonstrated a threefold-higher drag-reducing activity than the polymer which was free in solution.     

Exopolysaccharide Distribution of and Bioemulsifier Production by Acinetobacter calcoaceticus BD4 and BD413

The heavily encapsulated Acinetobacter calcoaceticus BD4 and the “miniencapsulated” single-step mutant A. calcoaceticus BD413 produced extracellular polysaccharides in addition to the capsular material. The molar ratio of rhamnose to glucose (3:1) in the extracellular BD413 polysaccharide fraction was similar to the composition of the capsular material. In both strains, the increase in capsular polysaccharide was parallel to cell growth and remained constant in stationary phase. The extracellular polysaccharides were detected starting from mid-logarithmic phase and continued to accumulate in the growth medium for 5 to 8 h after the onset of stationary phase. Strain BD413 produced one-fourth the total rhamnose exopolysaccharide per cell that strain BD4 did. Depending on the growth medium, 32 to 63% of the rhamnose polysaccharide produced by strain BD413 was extracellular, whereas in strain BD4 only 7 to 14% was extracellular. In all cases, strain BD413 produced more extracellular rhamnose polysaccharide than strain BD4 did. In glucose medium, strain BD413 also produced approximately 10 times more extracellular emulsifying activity than strain BD4 did. The isolated capsular polysaccharide obtained after shearing of BD4 cells showed no emulsifying activity. Thus, strain BD413 either produces a modified extracellular polysaccharide or excretes an additional substance(s) that is responsible for the emulsifying activity. Emulsions induced by the ammonium sulfate-precipitated BD413 extracellular emulsifier require the presence of magnesium ion and a mixture of an aliphatic and an aromatic hydrocarbon.     

Exocellular esterase and emulsan release from the cell surface of Acinetobacter calcoaceticus

An esterase activity has been found, both in the cell-free growth medium and on the cell surface of the hydrocarbon-degrading Acinetobacter calcoaceticus RAG-1. The enzyme catalyzed the hydrolysis of acetyl and other acyl groups from triglycerides and aryl and alkyl esters. Emulsan, the extracellular heteropolysaccharide bioemulsifier produced by strain RAG-1, was also a substrate for the enzyme. Gel filtration showed that the cell-free enzyme was released from the cell surface either emulsan free or associated with the bioemulsifier. The partially purified enzyme was found to interact specifically with the esterified fully active emulsan, but not with the deesterified polymer. A role for esterase in emulsan release from the cell surface was indicated when the enzyme was preferentially depleted from the cell surface under conditions in which emulsan was not released. Such cells lost the capacity to release the biopolymer.     

An exocellular protein from the oil-degrading microbe Acinetobacter venetianus RAG-1 enhances the emulsifying activity of the polymeric bioemulsifier emulsan

The oil-degrading microorganism Acinetobacter venetianus RAG-1 produces an extracellular polyanionic, heteropolysaccharide bioemulsifier termed emulsan. Emulsan forms and stabilizes oil-water emulsions with a variety of hydrophobic substrates. Removal of the protein fraction yields a product, apoemulsan, which exhibits much lower emulsifying activity on hydrophobic substrates such as n-hexadecane. One of the key proteins associated with the emulsan complex is a cell surface esterase. The esterase (molecular mass, 34.5 kDa) was cloned and overexpressed in Escherichia coli BL21(DE3) behind the phage T7 promoter with the His tag system. After overexpression, about 80 to 90% of the protein was found in inclusion bodies. The overexpressed esterase was recovered from the inclusion bodies by solubilization with deoxycholate and, after slow dialysis, was purified by metal chelation affinity chromatography. Mixtures containing apoemulsan and either the catalytically active soluble form of the recombinant esterase isolated from cell extracts or the solubilized inactive form of the enzyme recovered from the inclusion bodies formed stable oil-water emulsions with very hydrophobic substrates such as hexadecane under conditions in which emulsan itself was ineffective. Similarly, a series of esterase-defective mutants were generated by site-directed mutagenesis, cloned, and overexpressed in E. coli. Mutant proteins defective in catalytic activity as well as others apparently affected in protein conformation were also active in enhancing the apoemulsan-mediated emulsifying activity. Other proteins, including a His-tagged overexpressed esterase from the related organism Acinetobacter calcoaceticus BD4, showed no enhancement.     

Variation in composition and yield of exopolysaccharides produced by Klebsiella sp. strain K32 and Acinetobacter calcoaceticus BD4

The exopolysaccharides produced by Klebsiella sp. strain K32 and Acinetobacter calcoaceticus BD4 under different growth conditions have been analyzed for sugar composition. The first use of ion chromatography for the quantitative determination of microbial exopolysaccharide composition is reported. Klebsiella sp. strain K32 produced a polymer composed of rhamnose, galactose, and mannose early in its fermentation. The composition of the polymer varied markedly depending on the growth stage of the organism. Klebsiella sp. strain K32 grown in a fermentor produced a polymer which was rich in mannose during early exponential growth in a complex medium, but in the late stationary phase it did not contain detectable levels of mannose. The rhamnose present in the polymer increased from 12 to 55% over the course of growth, whereas galactose decreased from 63 to 45%. A. calcoaceticus BD4 produced a polymer containing rhamnose, glucose, mannose throughout its growth and stationary phase. Klebsiella sp. strain K32 and A. calcoaceticus BD4 were grown on various carbon sources in shake flasks. The polymer yield and composition from both organisms were found to vary with the carbon source. The exopolysaccharide with the highest mannose composition was obtained by using rhamnose as a carbon source for both organisms. These and other data suggest that regulatory changes caused by growth on different substrates result in either the production of a different distribution of polymers or a change in exopolysaccharide structure.     

Cyclic AMP stimulates the protein tyrosine kinase activity of Acinetobacter calcoaceticus

The protein tyrosine kinase activity of Acinetobacter calcoaceticus was analyzed in vitro through the specific phosphorylation of an endogenous protein which is modified exclusively at tyrosine residues. A strong stimulation of this activity by cyclic AMP was observed. This finding represents the first example of a protein tyrosine kinase, in prokaryotes as well as in eukaryotes, whose functioning is cyclic nucleotide-dependent.     

Variation in composition and yield of exopolysaccharides produced by Klebsiella sp. strain K32 and Acinetobacter calcoaceticus BD4.

The exopolysaccharides produced by Klebsiella sp. strain K32 and Acinetobacter calcoaceticus BD4 under different growth conditions have been analyzed for sugar composition. The first use of ion chromatography for the quantitative determination of microbial exopolysaccharide composition is reported. Klebsiella sp. strain K32 produced a polymer composed of rhamnose, galactose, and mannose early in its fermentation. The composition of the polymer varied markedly depending on the growth stage of the organism. Klebsiella sp. strain K32 grown in a fermentor produced a polymer which was rich in mannose during early exponential growth in a complex medium, but in the late stationary phase it did not contain detectable levels of mannose. The rhamnose present in the polymer increased from 12 to 55% over the course of growth, whereas galactose decreased from 63 to 45%. A. calcoaceticus BD4 produced a polymer containing rhamnose, glucose, mannose throughout its growth and stationary phase. Klebsiella sp. strain K32 and A. calcoaceticus BD4 were grown on various carbon sources in shake flasks. The polymer yield and composition from both organisms were found to vary with the carbon source. The exopolysaccharide with the highest mannose composition was obtained by using rhamnose as a carbon source for both organisms. These and other data suggest that regulatory changes caused by growth on different substrates result in either the production of a different distribution of polymers or a change in exopolysaccharide structure.     

Enhanced emulsan production in mutants of Acinetobacter calcoaceticus RAG-1 selected for resistance to cetyltrimethylammonium bromide

Mutants of Acinetobacter calcoaceticus RAG-1 that produced elevated levels of the polymeric bioemulsifier emulsan were isolated on the basis of their resistance to the cationic surfactant cetyltrimethylammonium bromide (CTAB). Such mutants showed maximum enhancement in both overall yield and specific productivity of some two- to threefold over that of the wild type. In addition, the effect was also observed in a resting cell system in the presence of chloramphenicol, indicating that the mutation is not simply the result of faster growth. When CTAB-tolerant mutants were subjected together with the sensitive parent to the detergent under growing conditions, only the mutants were found to grow. The results suggest that the mutation for CTAB resistance leads to enhanced capsule production. This was confirmed quantitatively by a specific enzyme-linked immunosorbent assay for the cell-bound emulsan minicapsule.     

Tolerance of Acinetobacter calcoaceticus RAG-1 to the cationic surfactant cetyltrimethylammonium bromide: role of the bioemulsifier emulsan

Emulsan, the polyanionic heteropolysaccharide bioemulsifier produced by Acinetobacter calcoaceticus RAG-1, was found to enhance the tolerance of RAG-1 cells to the toxic effects of the cationic detergent cetyltrimethylammonium bromide (CTAB). Emulsan-mediated tolerance was obtained with the purified deproteinated apoemulsan; ca. 9 micrograms of apoemulsan neutralized 1 microgram of CTAB. Deesterified apoemulsan was only half as effective in protecting the cells from CTAB toxicity. Tolerance was also mediated by the cell-associated emulsan minicapsule. Mutants lacking this capsule were more sensitive to CTAB than the corresponding parent. The growth of mutants and parent cells in mixed-culture experiments demonstrated that the cell-associated polymer mediates CTAB tolerance in the early stages of growth. Once sufficient cell-free polymer has been released into the aqueous medium (ca. 0.5 micrograms/ml), this extracellular emulsan also plays a role in CTAB tolerance.     

Enhanced emulsan production in mutants of Acinetobacter calcoaceticus RAG-1 selected for resistance to cetyltrimethylammonium bromide.

Mutants of Acinetobacter calcoaceticus RAG-1 that produced elevated levels of the polymeric bioemulsifier emulsan were isolated on the basis of their resistance to the cationic surfactant cetyltrimethylammonium bromide (CTAB). Such mutants showed maximum enhancement in both overall yield and specific productivity of some two- to threefold over that of the wild type. In addition, the effect was also observed in a resting cell system in the presence of chloramphenicol, indicating that the mutation is not simply the result of faster growth. When CTAB-tolerant mutants were subjected together with the sensitive parent to the detergent under growing conditions, only the mutants were found to grow. The results suggest that the mutation for CTAB resistance leads to enhanced capsule production. This was confirmed quantitatively by a specific enzyme-linked immunosorbent assay for the cell-bound emulsan minicapsule.     

Relationship between phage resistance and emulsan production,interaction of phages with the cell-surface of Acinetobacter calcoaceticus RAG-1

The hydrocarbon-degrading strain Acinetobacter calcoaceticus RAG-1 produces an extracellular emulsifying agent capable of forming stable oil-in-water emulsions. The bioemulsifier, termed emulsan, is a polyanionic heteropolysaccharide (M.W. 10⁶) composed mainly of N-acyl D-galactosamine and an N-acyl hexosamine uronic acid. In order to probe the interaction of emulsan with the cell surface prior to its release into the growth medium, two new virulent bacteriophages for A. calcoaceticus RAG-1 were isolated from sewage and the properties of phage resistant mutants were studied. The two phages, ap-2 and ap-3, were differentiated on the basis of plaque morphology, electron microscopy and buoyant density. Isolated mutants of A. calcoaceticus RAG-1 which were resistant to one of the two phages retained sensitivity to the other phage. Resistance to phage ap-3 was accompanied by a severe drop in emulsan production. Independently isolated derivatives of A. calcoaceticus RAG-1 with a defect in emulsan production also turned out to be resistant towards phage ap-3. Antibodies prepared against purified emulsan specifically inhibited phage ap-3 adsorption to the cell surface of the parental strain.     

Multiple forms of carboxylesterase activity in Acinetobacter calcoaceticus

A carboxylesterase activity (E.C.3.1.1) was found in all four strains of Acinetobacter calcoaceticus tested. The activity was present in both 2 X 10(4) gav h-1 supernatant and bacterial wall-membrane fractions. The activity in the supernatant was in two molecular weight forms, the predominant form with a Mr of about 10(3) K and a minor form Mr approximately 600 K. The activity was inhibited by phenylmethylsulfonyl fluoride. SDS-PAGE showed that in A. calcoaceticus NCIB 8250 the activity was composed of three components of Mr 43, 40 and 38 K. The individual components showed different activities with 1- or 2-naphthyl esters. Of the other strains used, one showed a nearly identical pattern of component activities, while the other two showed only two component activities.     

Uptake of acetate by Acinetobacter calcoaceticus

The uptake of acetate by intact nongrowing cells of Acinetobacter calcoaceticus was studied in dependence on the C-source (acetate, n-alcanes, yeast extract, succinate, L-malate) and the growth phase. Single kinetic parameters of acetate uptake were determined. The best acetate uptake was observed with cells cultivated with acetate as the only C-source. Bacteria in the early growth phase were found to transfer acetate twice as fast as cells of the late logarithmic growth phase. The uptake of acetate can be described by a biphasic saturation kinetics with 2 Km values: the Km value for the first phase being 1.10(-5) M, and for the second one, 1.8 .10(-4) M. The corresponding maximal uptake rates are 8 and 37 mM/min/mg dry weight, respectively. Alpha-ketoglutarate, fumarate, L-malate, and oxalacetate inhibit the initial uptake of acetate. Uranylacetate, inhibitors of the respiratory chain and proton conductors in part completely inhibit the uptake of acetate.     

Lipase activity and gene cloning of Acinetobacter calcoaceticus LP009

The production of lipase from Acinetobacter calcoaceticus LP009, a bacterium isolated from raw milk, was found to be best induced by Tween-80 at 1.0% concentration. It was efficiently secreted, and only a minute amount of activity was detected at the cell surface and intracellularly. A. calcoaceticus LP009 lipase exhibited maximum activity at pH 7.0 and 50 degrees C, and was relatively stable upon storage at pH 5.0 to 7.0 and at 4, 30, or 37 degrees C. The enzyme was found to be inactivated by EDTA suggesting that it was a metalloenzyme. Its activity was reduced by less than 20% with the addition of various ions to reaction mixtures, but long storage with them caused approximately 50% reduction in subsequent reactions under standard conditions. By contrast, the addition of Fe(3+) enhanced activity. The enzyme was highly stable upon storage with 0.1% of Triton X-100, Tween-80 or Tween-20, but highly unstable with various organic solvents tested. PMSF, a serine enzyme inhibitor, and 2-mercaptoethanol, a reducing agent, did not affect enzyme activity. After extraction and transfer, the lipase gene was efficiently expressed in recombinant Aeromonas sobria. This recombinant strain was shown to have increased hydrolyzing efficiency and have high potential for lipid-rich wastewater treatment.