Search

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.

The ability of Acinetobacter calcoaceticus RAG-1 to adhere to human epithelial cells was investigated and compared with its ability to adhere to a test hydrocarbon (hexadecane). RAG-1, a microorganism originally isolated for growth on hydrocarbon, adhered to epithelial cells when grown under conditions which promote its adherence to hexadecane; similarly, RAG-1 cells adhered poorly to epithelial cells when grown under conditions which cause the cells to possess low affinity towards hexadecane. A mutant derived from RAG-1, MR-481, deficient in its ability to adhere to hydrocarbon, was similarly unable to adhere to epithelial cells. RAG-1 adherence to epithelial cells was not blocked by a number of sugars tested. Streptococcus pyogenes, whose adherence to epithelial cells has been previously attributed to hydrophobic interactions, was also able to adhere to hexadecane. Results suggest that hydrophobic interactions mediate adherence of the strains studied to both epithelial cells and hydrocarbon.

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.

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.

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.

Transport properties of membrane vesicles isolated from two adenosine triphosphatase-deficient mutants of Escherichia coli, NR70 and DL54, were compared with those of vesicles prepared from the corresponding parental strains. As reported previously (Rosen, 1973; Altendorf et al., 1974), vesicles prepared from these mutants grown under aerobic conditions exhibited defective amino acid transport, and activity was restored after treatment with dicyclohexylcarbodiimide. In sharp contrast, however, vesicles isolated from the same mutants grown anaerobically in the presence of nitrate exhibited completely normal transport activity when assayed under either anaerobic or aerobic conditions. Suppression of the transport defect was not due to the manner by which the vesicles were prepared, and the adenosine triphosphatase deficiency was not ameliorated by anaerobic growth in the presence of nitrite. Finally, the transport activity of vesicles prepared from the mutants grown under aerobic conditions was relatively resistant to the effect of 1.0 M guanidine hydrochloride extraction, whereas the activity of vesicles prepared from mutants grown anaerobically was totally refractory to the effect of the chaotrope.

A new phosphorus-containing compound (1) was detected by ³¹P NMR spectroscopy in Streptomyces sp. A50. Compound 1, 1(α)-O-methyl-2-(N-acetyl)glucoseamine-6-O-phosphate-1(α)-2-(N-acetyl)glucosamine, exhibited a pKa value around zero. The compound was found both in the extracellular culture broth and in the cells. While very low concentrations of 1 were found in the culture broth of other species of Streptomyces, its presence in high concentrations was specific to Streptomyces sp. A50. The highly acidic compound was isolated from the broth, and its structure was elucidated by a combination of 1D-, 2D-homonuclear, and inverse heteronuclear NMR techniques and mass spectroscopy.

Synthetic phylloquinone was resolved into cisand transisomers by thin-layer chromatography. The two isomers had identical ultraviolet spectra characteristic of vitamin K1and were differentiated by nuclear-magnetic-resonance spectroscopy on the basis of the displacement of the peak corresponding to the olefinic methyl group in the naphthoquinone side chain. Studies on the restoration of electron transport coupled to phosphorylation in irradiated preparations of Mycobacterium phleishowed that only the transisomer was active with substrates linked to nicotinamide-adenine dinucleotide. The purified transphylloquinone was enzymatically converted to the cisisomer. Under similar conditions, cisvitamin K1gave rise to the trans-naphthoquinone. The natural naphthoquinone of M. phleivitamin MK9(II-H) was similarly resolved into cisand transisomers.