Purification and properties of the myo-inositol-binding protein from a Pseudomonas sp.

Emulsan, the extracellular polyanionic emulsifying agent produced by Acinetobacter calcoaceticus RAG-1, has been implicated as a receptor for a specific virulent RAG-1 bacteriophage, ap3. Aqueous solutions of emulsan did not interfere with phage ap3 adsorption to RAG-1 cells. However, binding of phage ap3 occurred at the interfaces of hexadecane-in-water emulsions specifically stabilized by emulsan polymers. Binding of ap3 to emulsions was inhibited either in the presence of anti-emulsan antibodies or in the presence of a specific emulsan depolymerase. Moreover, when the phage was first bound to emulsan-stabilized emulsions and the emulsions subsequently treated with emulsan depolymerase, viable phage was released, indicating that phage ap3 DNA ejection was not triggered by binding. The results indicate that emulsan functions as the ap3 receptor and suggest that to function as a receptor, emulsan assumes a specific conformation conferred on it by its specific interaction with hydrophobic surfaces.     

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.     

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.     

Localization of emulsan-like polymers associated with the cell surface of acinetobacter calcoaceticus

Various immunochemical techniques were employed to probe the relationship between the extracellular emulsifying agent (emulsan) and the cell-associated form of the polymer in Acinetobacter calcoaceticus RAG-1. Using an emulsan-specific antibody preparation, immunocytochemical labeling revealed that an emulsan-like antigen is a major component of the 125-nm minicapsule which envelopes the exponential-phase cell of the parent strain. The marked reduction of this capsule in stationary-phase cells was correlated with the production of extracellular emulsifying activity. Crossed immunoelectrophoresis techniques demonstrated that the major antigenic component (S1) of the culture supernatant fluid is immunochemically identical to purified emulsan, yet electrophoretically distinct. The characteristics of the parent strain were compared with those of two phage-resistant mutant strains which are defective in extracellular emulsan production. One of these mutants, termed TR3, lacked both the emulsan-like capsule on the cell surface and the extracellular S1 component. A second phage-resistant emulsan-defective mutant (TL4) was characterized by an antigenically altered and inactive form of extracellular emulsan. A relatively small amount of emulsan-like capsular material was consistently demonstrated on the cell surface of this mutant. The correlation between phage sensitivity and extracellular emulsan production was strengthened by the fact that emulsan-specific antibodies inhibited both emulsification activity and phage adsortion onto cells of the parent strain.     

Enzymatic depolymerization of emulsan

Emulsan, the polyanionic emulsifying agent synthesized by Acinetobacter calcoaceticus RAG-1, was depolymerized by an enzyme obtained from a soil bacterium YUV-1. The extracellular emulsan depolymerase was produced when strains RAG-1 and YUV-1 were grown together on agar medium. The enzyme was extracted from the agar and concentrated by ultrafiltration and ammonium sulfate precipitation. The molecular weight of the enzyme was estimated to be 89,000. Emulsan depolymerase activity was due to an eliminase reaction which split glycosidic linkages within the heteropolysaccharide backbone of emulsan to generate reducing groups and alpha, beta-unsaturated uronides with an absorbance maximum of 233 nm. Deesterified emulsan was degraded by emulsan depolymerase at only 27% of the rate of the native polymer. The treatment of emulsan solutions with emulsan depolymerase for brief periods caused a rapid and parallel drop in viscosity and emulsifying activity. More than 75% of the viscosity and emulsifying activity was lost at a time when less than 0.5% of the glycosidic linkages were broken. These data indicate that (i) emulsan depolymerase is an endoglycosidase and (ii) the higher the molecular weight of emulsan, the greater its emulsifying activity. Exhaustive digestion of emulsan with emulsan depolymerase produced oligosaccharides with a number average molecular weight of about 3,000. The fractionation of the digest on Bio-Gel P-6 yielded four broad peaks. The pooled fractions from each of the peaks contained the same relative amounts of reducing sugar and had an absorbance at 233 nm. The molar ratio of esterified sugar to reducing groups was close to 2 in each fraction.     

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.     

Bacterial degradation of emulsan

Emulsan is a polyanionic heteropolysaccharide bioemulsifier produced by Acinetobacter calcoaceticus RAG-1. A mixed bacterial population was obtained by enrichment culture that was capable of degrading emulsan and using it as a carbon source. From this mixed culture, an emulsan-degrading bacterium, termed YUV-1, was isolated. Strain YUV-1 is an aerobic, gram-negative, non-spore-forming, rod-shaped bacterium which grows best in media containing yeast extract. When placed on preformed lawns of A. calcoaceticus RAG-1, strain YUV-1 produced translucent plaques which grew in size until the entire plate was covered. Plaque formation was due to solubilization of the emulsan capsule of RAG-1. Plaque formation was not observed on emulsan-negative mutants of RAG-1. As a consequence of the solubilization of the emulsan capsule, RAG-1 cells became more hydrophobic, as determined by adherence to hexadecane. Growth of YUV-1 on a medium containing yeast extract and emulsan was biphasic. During the initial 24 h, cell concentration increased 10-fold, but emulsan was not degraded; during the lag in growth (24 to 48 h), emulsan was inactivated and depolymerized but not consumed; during the second growth phase (48 to 70 h) the depolymerized emulsan products were consumed.     

Bacterial degradation of emulsan.

Emulsan is a polyanionic heteropolysaccharide bioemulsifier produced by Acinetobacter calcoaceticus RAG-1. A mixed bacterial population was obtained by enrichment culture that was capable of degrading emulsan and using it as a carbon source. From this mixed culture, an emulsan-degrading bacterium, termed YUV-1, was isolated. Strain YUV-1 is an aerobic, gram-negative, non-spore-forming, rod-shaped bacterium which grows best in media containing yeast extract. When placed on preformed lawns of A. calcoaceticus RAG-1, strain YUV-1 produced translucent plaques which grew in size until the entire plate was covered. Plaque formation was due to solubilization of the emulsan capsule of RAG-1. Plaque formation was not observed on emulsan-negative mutants of RAG-1. As a consequence of the solubilization of the emulsan capsule, RAG-1 cells became more hydrophobic, as determined by adherence to hexadecane. Growth of YUV-1 on a medium containing yeast extract and emulsan was biphasic. During the initial 24 h, cell concentration increased 10-fold, but emulsan was not degraded; during the lag in growth (24 to 48 h), emulsan was inactivated and depolymerized but not consumed; during the second growth phase (48 to 70 h) the depolymerized emulsan products were consumed.     

Role for Emulsan in Growth of Acinetobacter calcoaceticus RAG-1 on Crude Oil

When Acinetobacter calcoaceticus RAG-1 was grown together with an emulsan-deficient mutant on crude oil, only the emulsan-producing RAG-1 was found to grow, regardless of whether the medium was supplemented with emulsan. The results suggested that the cell-associated form of the bioemulsifier is the biologically active species required for growth on crude oil. A revertant of an emulsan-deficient strain was isolated which simultaneously regained the ability to produce both cell-associated and cell-free emulsan as well as the ability to grow on crude oil.     

Emulsan production by Acinetobacter calcoaceticus in the presence of chloramphenicol.

When exponentially growing cultures of Acinetobacter calcoaceticus RAG-1 or RAG-92 were either treated with inhibitors of protein synthesis or starved for a required amino acid, there was a stimulation in the production of emulsan, an extracellular polyanionic emulsifier. Emulsan synthesis in the presence of chloramphenicol was dependent on utilizable sources of carbon and nitrogen and was inhibited by cyanide or azide or anaerobic conditions. Radioactive tracer experiments indicated that the enhanced production of emulsan after the addition of chloramphenicol was due to both the release of material synthesized before the addition of the antibiotic (40%) and de novo synthesis of the polymer (60%). Chemical analysis of RAG-1 cells demonstrated large amounts of polymeric amino sugars; it was estimated that cell-associated emulsan comprised about 15% of the dry weight of growing cells. The data are consistent with the hypothesis that a polymeric precursor of emulsan accumulates on the cell surface during the exponential growth phase; in the stationary phase or during inhibition of protein synthesis, the polymer is released as a potent emulsifier.     

Alternate hydrophobic sites on the cell surface of Acinetobacter calcoaceticus RAG-1

Abstract Mutants of A. calcoaceticus RAG-1 lacking thin fimbriae (35 Å) do not adhere to hydrophobic surfaces [5], or grow on hydrocarbons under conditions of weak agitation and small inocula. Emulsan-deficient derivatives of such mutants, isolated in the present study, (i) lacked cell-surface emulsan, (ii) adhered avidly to hydrocarbons, (iii) lacked thin fimbriae, and (iv) regained the capacity to grow on hydrocarbons. The results show that emulsan masks an alternate hydrophobic site(s) on the cell surface of RAG-1.     

Involvement of a protein tyrosine kinase in production of the polymeric bioemulsifier emulsan from the oil-degrading strain Acinetobacter lwoffii RAG-1

The genes associated with the biosynthesis of the polymeric bioemulsifier emulsan, produced by the oil-degrading Acinetobacter lwoffii RAG-1 are clustered within a 27-kbp region termed the wee cluster. This report demonstrates the involvement of two genes of the wee cluster of RAG-1, wzb and wzc, in emulsan biosynthesis. The two gene products, Wzc and Wzb were overexpressed and purified. Wzc exhibited ATP-dependent autophosphorylating protein tyrosine kinase activity. Wzb was found to be a protein tyrosine phosphatase capable of dephosphorylating the phosphorylated Wzc. Using the synthetic substrate p-nitrophenyl phosphate (PNPP) Wzb exhibited a V(max) of 12 micromol of PNPP min(-1) mg(-1) and a K(m) of 8 mM PNPP at 30 degrees C. The emulsifying activity of mutants lacking either wzb or wzc was 16 and 15% of RAG-1 activity, respectively, suggesting a role for the two enzymes in emulsan production. Phosphorylation of Wzc was found to occur within a cluster of five tyrosine residues at the C terminus. Colonies from a mutant in which these five tyrosine residues were replaced by five phenylalanine residues along with those of a second mutant, which also lacked Wzb, exhibited a highly viscous colony consistency. Emulsan activity of these mutants was 25 and 24% of that of RAG-1, respectively. Neither of these mutants contained cell-associated emulsan. However, they did produce an extracellular high-molecular-mass galactosamine-containing polysaccharide. A model is proposed in which subunit polymerization, translocation and release of emulsan are all associated and coregulated by tyrosine phosphorylation.     

Escherichia coli capsule bacteriophages. IV. Free capsule depolymerase 29.

The free host capsule depolymerase, induced by Escherichia coli capsule bacteriophage no. 29, and causing the formation of haloes around its plaques, has been purified to homogeneity. As judged from the following facts, this "enzyme" consists of free phage 29 spikes. (i) Detached phage organelles and depolymerase 29 particles exhibit the same molecular weight (about 245,000, as determined from the sedimentation equilibrium), contain polypeptide chains of the same two sizes (57,000 plus or minus 3,000 and 29,500 plus or minus 2,000, as determined by SDS-PAA gel electrophoresis), and have (within experimental error) the same sedimentation coefficient, isoelectric point, and amino acid composition. (ii) Isolated depolymerase and phage spikes in situ both catalyze the hydrolysis of glucosidic bonds in host capsular polysaccharide, leading ultimately to the formation of oligosaccharide fragments of one, two, and three hexasaccharide repeating units. (iii) Depolymerase 29 and phage 29 spikes have roughly the same electron optical dimensions. As tentatively estimated from the total and the virus-associated capsule depolymerase activity in the lysates, phage 29 infection seems to produce eight to seventeen times more free than incorporated spikes.     

Localization and functional role of the pseudomonas bacteriophage 2 depolymerase.

The adsorption apparatus of phage 2 consits of a symmetrical base plate of snowflake appearance, composed of six droplike spikes 7.0 to 7.5 nm in length with a maximum diameter of 4.5 to 5.0 nm. The spikes are attached by their narrow ends to a central ring 7.0 to 7.5 nm in diameter. Phage 2 deopolymerase, a phage 2-induced hydrolytic enzyme, was found to be a structural protein of phage 2 or in close association with the base plate. Pdp1, a phage 2 mutant, possesses a polypeptide that is antigenically similar to the depolymerase, but devoid of hydrolytic activity. This polypeptide was found to be located in the region of the base plate of pdp1. Treatment of intact cells of strain BI with purified phage 2 depolymerase inhibited the adsorption of phage 2. When phage receptor-containing fractions of slime glycolipoprotein and lipopolysaccharide were hydrolyzed by the depolymerase, amino sugars were released, and the phage-inactivating activities of these fractions were lost. The depolymerase was also observed to induce the lysis of strain BI cells in hypotenic medium. The phage 2 depolymerase appears to play a role in adsorption and release of phage.     

Escherichia coli capsule bacteriophages. VIII. Fragments of bacteriophage 28-1.

As described previously, a host capsule depolymerase activity is associated with the particles of Escherichia coli capsule bacteriophage 28-1. This is a large virus with a long, contractile tail terminating in a base plate with spikes. In the present work, isolated virions were exposed to a variety of dissociative reagents and conditions. They were then tested for residual infectivity and depolymerase activity, as well as inspected under an electron microscope. Very mild acid treatment (10 to 15 min at pH 4.0 and 37 C) was found to cause a specific detachment of some phage spikes, together with a moderate drop in both infectivity and depolymerase activity. Large batches of viruses were fragmented in this manner, and the detached spikes were isolated. The host capsule depolymerase activity was found to be associated with these organelles. In negatively stained preparations, the spikes exhibited a length of approximately 18 nm and a thickness of about 5 nm. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, they were found to contain polypeptides with molecular weights of 80,000 and 145, 000.     

Influence of environmental conditions on infection of Klebsiella pneumoniae by two different types of bacteriophages

The adsorption and efficiency of plating of bacteriophages FC3-1 and FC3-9 on Klebsiella pneumoniae C3 (serotype O1:K66) cells grown at different pHs and temperatures were quantitated. Bacteriophage FC3-1, with lipopolysaccharide as its bacterial receptor, showed a large decrease in efficiency of plating on bacteria grown at low pH or low temperature. Under the same conditions, no significant decrease in efficiency of plating was found for bacteriophage FC3-9, a phage requiring capsule and lipopolysaccharide for its adsorption and carrying capsule-depolymerizing activity. We demonstrate that K. pneumoniae C3 cells grown at low pH or low temperature have less lipopolysaccharide exposed on their surface. We conclude that this is why lipopolysaccharide-specific phage FC3-1 less efficiently infects bacterial cells grown under those conditions. We propose that bacteriophage FC3-9 efficiently infects bacterial cells grown at low pH or low temperature because its enzymatic activity on the capsule makes lipopolysaccharide available to this phage.     

Production of exopolysaccharides by Acinetobacter strains in a controlled fed-batch fermentation process using soap stock oil (SSO) as carbon source

The production of two extracellular capsular heteropolysaccharides by two different Acinetobacter strains has been studied in separate controlled fermentation processes with a view to their industrial applications as specific dispersing agents. The first, emulsan, is an extracellular polyanionic amphipathic heteropolysaccharide (MW 10(6) D) made by A. calcoaceticus RAG-1. It forms and stabilizes oil in water emulsions. The other, biodispersan (PS-A2), is another extracellular zwitterionic heteropolysaccharide (MW 51 kD) made by A. calcoaceticus A2. This polysaccharide disperses big solid limestone granules forming micron-size water suspension. Both polysaccharides are synthesized within the cells, exported to their outer surface to form an extracellular cell-associated capsule and released subsequently into the growth medium. The polymers were produced in a computer-controlled fed-batch intensively aerated fermentation process. A commercially available and cheap fatty acids mixture (soap stock oil) served as the carbon source, and was fed in coordination with the required nitrogen. The coordinated feed of carbon and nitrogen was operated on the basis of two metabolic correlations: The first correlation related the cell protein produced and the ammonium nitrogen consumed with the outcoming coeffients of 24 and 21 mM NH3/g protein for the emulsan and the biodispersan fermentations respectively. The second correlation linked the consumption of the fatty acids with that of the nitrogen source dictating the appropriate C/N ratio of the feed into the operating fermentor. These ratios were 7.7 g C/g N for the emulsan fermentation and 8.5 gC/g N in the case of the biodispersan production process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Escherichia coli capsule bacteriophages. V. Lysozyme 29.

In addition to the spike-associated host capsule depolymerase, infection by Escherichia coli capsule bacteriophage no. 29 also induces the synthesis of a large bacteriolytic enzyme which has been purified to homogeneity. On incubation of isolated host murein sacculi with this enzyme, no amino groups but reducing sugar groups were liberated, and muraminitol, but no glucosaminitol, was found in the degraded sacculi after subsequent reduction with NaBH4. The bacteriolytic enzyme is thus another lysozyme (mucopeptide N-acetylmuramylhydrolase; EC 3.2.1.17). Electron optical visualization of negatively stained lysozyme specimens showed oblong particles of roughly 4.5 to 5.5 nm in diameter and 15 to 19 nm in length. Although the material tended to dissociate, a crude estimate of its molecular weight (270,000 plus or minus 30,000) could be obtained from these dimensions, from its sedimentation equilibrium, and from its behavior in gel chromatography. After disintegration of homogeneous lysozyme 29 by heating in solution with sodium dodecyl sulfate and dithiothreitol, polypeptides of one size only (about 46,000 dalton, probably six copies per molecule) were found in sodium dodecyl sulfate-polyacrylamide electrophoresis. The amino acid analysis of the enzyme accounted for more than 90% of its dry weight. One percent or less of the bacteriolytic activity in phage 29 lysates was found to be associated with the intact or disrupted virus particles, and a polypeptide of 46,000 daltons was not detected in the virions. These results strongly suggest that, in contrast to the host capsule depolymerase also induced by the same phage, and in spite of its comparatively large size, "lysozyme 29" does not constitute an integral part also of the homologous bacteriophage particles.     

Role of adherence in growth of Acinetobacter calcoaceticus RAG-1 on hexadecane.

The high affinity of Acinetobacter calcoaceticus RAG-1 for liquid hydrocarbons permitted the isolation of a spontaneous nonadherent mutant, MR-481. Strain MR-481 exhibited no significant affinity for three test hydrocarbons, yet resembled the wild type in many properties, including production of the extracellular emulsifying agent emulsan. To study the role of adherence in growth on hydrocarbons, RAG-1 and MR-481 were compared for growth on hexadecane under conditions of limited agitation and at low initial cell densities. Adherent RAG-1 cells were able to grow rapidly under these conditions, whereas nonadherent MR-481 cells failed to grow for at least 54 h. However, the addition of emulsan either initially or at various times after inoculation enabled the nonadherent MR-481 cells to grow on hexadecane. Growth was not the result of reversion of MR-481 from nonadherent to adherent cells. The data demonstrate that adherence is a crucial factor in the growth of A. calcoaceticus RAG-1 on hexadecane in the absence of extracellular emulsification of the substrate.     

Emulsan quantitation by Nile red quenching fluorescence assay

A Nile red fluorescent technique to quantify 20–200 g ml^–1 of emulsan was developed. Nile red dissolved in DMSO showed an adsorption peak at 552 nm, and emission peak at 636 nm, with molar extinction coefficient of 19,600 cm^–1 M^–1. Nile red fluorescence in DMSO was proportionally quenched by emulsan and the quenching was time-dependent. The assay was used to follow the production of emulsan by cultures of Acinetobacter venetianus RAG-1.