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.     

Construction of a chimeric gene cluster for the biosynthesis of apoemulsan with altered molecular weight

Acinetobacter venetianus RAG-1 produces an extracellular protein/high-molecular-weight (HMW) polysaccharide complex termed emulsan. As an emulsion stabilizer, emulsan has potential industrial applications. To control the molecular weight of the polymer, a stable chromosomal mutant was generated where RAG-1 wza, wzb, wzc genes were replaced by Escherichia coli homologs. The heterologous Wza, Wzb, Wzc proteins restored production of HMW polysaccharide. The polymer produced was of higher molecular weight than from the parent strain and with the cells exhibiting modified hydrophobicity. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

Unmasking of surface components by removal of cell-associated emulsan from Acinetobacter Sp. RAG-1

Summary Acinetobacter calcoaceticusRAG-1 cells lacking the emulsan capsule on the cell surface were obtained by two methods; a) by selecting for mutants that lack emulsan with a specific phage and b) by removal of the emulsan capsule from wild type cells with a specific emulsan depolymerase. Emulsan deficient cells obtained by either method become deficient in the adsorption of phage ap3 and sensitive to a newly isolated bacteriophage, nø. When RAG-1 cells were first treated with emulsan depolymerase and subsequently incubated without the enzyme, regeneration of the cell-associated emulsan was correlated with an increase in phage ap3 adsorption and an inhibition in phage nø adsorption. By partial regeneration of cell surface emulsan, a physiological state was obtained in which RAG-1 cells were sensitive to and efficiently adsorbed found phages. Enzyme-treated RAG-1 cells were found to be more adherent to hexadecane than the untreated RAG-1 cells. The data indicate that in addition to its function as the ap3 receptor, cell-associated emulsan masks the expression of other cell-surface determinant(s) which function(s) as: (i) receptor for bacteriophage nø, and (ii) cell-surface sites which enhance adherence to hydrophobic surfaces.Present address: Department of Applied Biological Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA     

Unidirectional polar growth of cells of Seliberia stellata and aquatic seliberia-like bacteria revealed by immunoferritin labeling

When grown in a complex peptone-yeast extract culture medium, Seliberia stellata and related morphologically similar aquatic bacterial strains typically divided asymmetrically, giving rise to a motile swarmer and a longer sessile rod. Indirect immunoferritin labeling of these bacteria, followed by incubation during which cell growth occurred, has provided evidence that antigenic cell-surface components are synthesized de novo in a sharply demarcated zone at one pole of the growing parent cells. Cell elongation occurred unidirectionally from the pole showing the de novo surface synthesis; it was this end of the elongating, helically sculptured (i.e., screw-like) rod that became the daughter swarmer cell. The daughter swarmers, produced after polar growth and division of the immunoferritinlabeled parent cells, were not labeled. The immunoferritin label remaining on the parent cell did not appear to be diluted or disturbed by the cell growth and division process. Under the cultural conditions used in this study, the growth and division events which led to production of swarmer cells in the seliberia strains examined met two major criteria of accepted definitions of budding (de novo cell surface synthesis and transverse asymmetry of division). However, the developing daughter cell was not initially narrower than the parent and thus did not increase in cell diameter during growth.In memory: R. Y. Stanier     

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.     

Agglutination Typing of Vibrio anguillarum Isolates from Diseased Fish and from the Environment

Agglutinating activity was widely distributed among 101 Vibrio anguillarum strains of different origin and three Vibrio ordalii strains from salmonids. The spectrum of cells which were agglutinated comprised yeast cells and human (type O), poultry, guinea pig, and trout erythrocytes, whereas ovine, bovine, and tanned bovine erythrocytes were not affected. Mannose-sensitive hemagglutination, mannose-resistant hemagglutination, and non-agglutinating strains were recognized. The three V. ordalii strains showed mannose-resistant hemagglutination, whereas V. anguillarum exhibited either mannose-sensitive hemagglutination or was non-agglutinating. Among V. anguillarum, sensitivity to d-galactose and l-fucose occurred sporadically. An agglutination typing scheme was developed for strains of V. anguillarum based on the agglutination pattern of human, poultry, guinea pig, and trout erythrocytes and yeast cells. Eight different agglutination types (A through H) were defined. The distribution of these types among fish pathogenic and environmental V. anguillarum strains were studied. The application of the typing scheme in ecological and epidemiological studies and for preventive medical purposes is discussed.     

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.     

Adherence of emulsan-producing Acinetobacter calcoaceticus to hydrophobic liquids

Summary The adherence of Acinetobacter calcoaceticus ATCC 31012 cells to hexadecane and perfluorocarbon FC-43 was measured using the Bacterial Adherence To Hydrocarbon (BATH) assay. In batch culture the adherence of cells to both hydrophobic liquids increased sharply during the exponential growth phase and remained high for the remainder of the culture period. No correlation was found between the surface emulsan concentration and the adherence to perfluorocarbon FC-43 and hexadecane. In continuous cultures, the production of cell-free emulsan was found to be growth-associated. The adherence to both hydrophobic liquids decreased with increasing dilution rate while the amount of surface emulsan increased. Furthermore, exogenously added emulsan decreased the adherence to hydrophobic liquids. Thus, the accumulation of surface emulsan does not appear to have a beneficial effect for cell adherence to hydrophobic liquids.     

G1 antigen: a cell-surface immunoprotective 96 kDa glycoprotein from the virulent fish pathogen Enterococcus seriolicida, its purification and characterization

Strains of the fish pathogen Enterococcus seriolicida were identified as agglutinating and non-agglutinating, according to their reaction with anti-serum raised against type strain YT-3 (ATCC49156). The non-agglutinating strains are highly pathogenic in contrast to agglutinating strains. A 96 kDa immunoprotective glycoprotein G1 antigen from non-agglutinating Ent. seriolicida strain SS91-014 (N) was purified and characterized. The purification procedure entailed extraction of antigen by glass bead agitation, 80% (NH4)(2)SO4 precipitation, gel filtration and electroelution. An immunofluorescence microscopy study using monoclonal antibody M3A5 raised against G1 antigen revealed that G1 antigen is present only on the cell surface of non-agglutinating strains. Therefore, the G1 antigen of virulent Ent. seriolicida could be a potential candidate for protective vaccine against enterococcosis in fish.     

Mutational changes in physiochemical cell surface properties of plant-growth-stimulating Pseudomonas spp. do not influence the attachment properties of the cells.

Bacteriophage-resistant mutant strains of the root-colonizing Pseudomonas strains WCS358 and WCS374 lack the O-antigenic side chain of the lipopolysaccharide, as was shown by the loss of the typical lipopolysaccharide ladder pattern after analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These strains differed from their parent strains in cell surface hydrophobicity and in cell surface charge. The observed variation in these physicochemical characteristics could be explained by the differences in sugar composition. The mutant strains had no altered properties of adherence to sterile potato roots compared with their parental strains, nor were differences observed in the firm adhesion to hydrophilic, lipophilic, negatively charged, or positively charged artificial surfaces. These results show that neither physicochemical cell surface properties nor the presence of the O-antigenic side chain plays a major role in the firm adhesion of these bacterial cells to solid surfaces, including potato roots.     

The detection of two antigenic groups among Renibacterium salmoninarum isolates

The analysis of the membrane proteins and their antigenic properties in a group of 14 geographically diverse strains of Renibacterium salmoninarum revealed the existence of antigenic diversity within this species. Eleven isolates, including the type strain ATCC 33209, shared a similar protein profile with a major component of 57 kDa whereas three strains showed a common pattern with a major protein of 30 kDa. The quantitative agglutination tests and Western blotting assays seem to indicate the existence of serological heterogeneity, with two distinct groups being detected.     

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.     

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 trinitrophenyl and H-2 antigens on trinitrophenyl-modified spleen cells. I. H-2 antigens on cells treated with trinitrobenzene sulfonic acid are derivatized.

Spleen cells were treated with TNBS in order to determine if cell surface H-2 antigens are derivatized with TNP. By labeling the cell membrane of the TNP-modified cells with 125I, followed by detergent lysis and immune precipitation with anti-TNP, it was determined that no H-2 antigenic activity remained in the supernatant. Further, by the use of an antibody-induced antigen redistribution assay it was found that previous exposure to TNP-modified cells to anti-TNP in the absence of complement rendered these cells resistant to lysis by anti-H-2 in the presence of complement. Together these data indicate that at the concentration of TNBS used for modification, H-2 antigens are derivatized with TNP. However, in addition to H-2, other proteins including immunoglobulin were also derivatized with TNP. Anti-TNP cytotoxic effector cells were blocked from their cytotoxic activity by anti-TNP antiserum. These data indicate that TNP directly couples to H-2 antigens on the cell surface of TNP-modified cells and that TNP is associated with the antigenic determinant that the cytotoxic T cell recognizes.     

Characterization of polyagglutinating and surface antigens in Pseudomonas aeruginosa

Mutants of Pseudomonas aeruginosa PAC1R (serotype O:3) which were resistant to bacteriophage D were isolated and shown to react with O:5d, O:9 and O:13 antisera as well as O:3. Antisera to the parent strain and to the three polyagglutinating (PA) mutants also showed cross-reactions. The mutants differed from the parent strain in their lipopolysaccharide (LPS) composition. The LPS from two of the three mutants yielded high molecular weight polysaccharide fractions. Although the high molecular weight fraction from one of the mutants contained the amino sugars and other components characteristic of the O:3 serotype strains, its mobility on Sephadex G75 was different from that of the parent strain. The high molecular weight material from the second mutant lacked the O-antigenic determinants but these were present in a semi-rough LPS fraction. The third mutant appeared rough and completely lacked the O-antigenic components. These three mutants were compared with the parent strain and with a non-agglutinating LPS-defective mutant which lacked both O-antigenic side chains and all neutral sugars in the outer core. Agglutination with absorbed sera and haemagglutination using purified LPS and ELISA detection suggested that wall components other than LPS were responsible for some of the cross-reactions observed. The components responsible were detected after SDS-PAGE of crude outer membrane fractions by a combination of Coomassie blue and silver-staining and antigenic components were detected by immunoelectrophoresis and ELISA-linked immunoblotting of the gels. The main antigenic determinants detected by antiserum to the parent strain were in the high molecular weight O-polysaccharide fractions and in the semirough fractions of the LPS, with some activity due to the H protein of the outer membrane. O:5d antisera reacted with unidentified high molecular weight polysaccharide fractions. Cross-reactions with the O:9 antiserum appeared to be due mainly to the F porin and, to a lesser extent, to the G and E proteins of the outer membrane. O:13 antiserum reacted with high molecular weight polysaccharide fractions but also with the rough core and F and H protein. Cross-reactivity of the other three mutant antisera could largely be interpreted in terms of the outer membrane components exposed in each strain. One reacted strongly with the F porin and the rough core, while the others reacted with a number of protein and LPS-derived fractions.(ABSTRACT TRUNCATED AT 400 WORDS)     

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)     

Regulated exopolysaccharide production in Myxococcus xanthus

Myxococcus xanthus fibrils are cell surface-associated structures composed of roughly equal amounts of polysaccharide and protein. The level of M. xanthus polysaccharide production under different conditions in the wild type and in several mutants known to have alterations in fibril production was investigated. Wild-type exopolysaccharide increased significantly as cells entered the stationary phase of growth or upon addition of Ca2+ to growing cells, and the polysaccharide-induced cells exhibited an enhanced capacity for cell-cell agglutination. The activity of the key gluconeogenic pathway enzyme phosphoenolpyruvate carboxykinase (Pck) also increased under these conditions. Most fibril-deficient mutants failed to produce polysaccharide in a stationary-phase- or Ca2+-dependent fashion. However, regulation of Pck activity was generally unimpaired in these mutant strains. In an stk mutant, which overproduces fibrils, polysaccharide production and Pck activity were constitutively high under the conditions tested. Polysaccharide production increased in most fibril-deficient strains when an stk mutant allele was present, indicating that these fibril-deficient mutants retained the basic cellular components required for fibril polysaccharide production. In contrast to other divalent cations tested, Sr2+ effectively replaced Ca2+ in stimulating polysaccharide production, and either Ca2+ or Sr2+ was required for fruiting-body formation by wild-type cells. By using transmission electron microscopy of freeze-substituted log-phase wild-type cells, fibril material was observed as a cell surface-associated layer of uniform thickness composed of filaments with an ordered structure.     

Role of the cell wall microenvironment in expression of a heterologous SpaP-S1 fusion protein by Streptococcus gordonii

The charge density in the cell wall microenvironment of Gram-positive bacteria is believed to influence the expression of heterologous proteins. To test this, the expression of a SpaP-S1 fusion protein, consisting of the surface protein SpaP of Streptococcus mutans and a pertussis toxin S1 fragment, was studied in the live vaccine candidate bacterium Streptococcus gordonii. Results showed that the parent strain PM14 expressed very low levels of SpaP-S1. By comparison, the dlt mutant strain, which has a mutation in the dlt operon preventing d-alanylation of the cell wall lipoteichoic acids, and another mutant strain, OB219(pPM14), which lacks the LPXTG major surface proteins SspA and SspB, expressed more SpaP-S1 than the parent. Both the dlt mutant and the OB219(pPM14) strain had a more negatively charged cell surface than PM14, suggesting that the negative charged cell wall played a role in the increase in SpaP-S1 production. Accordingly, the addition of Ca(2+), Mg(2+), and K(+), presumably increasing the positive charge of the cell wall, led to a reduction in SpaP-S1 production, while the addition of bicarbonate resulted in an increase in SpaP-S1 production. The level of SpaP-S1 production could be correlated with the level of PrsA, a peptidyl-prolyl cis/trans isomerase, in the cells. PrsA expression appears to be regulated by the cell envelope stress two-component regulatory system LiaSR. The results collectively indicate that the charge density of the cell wall microenvironment can modulate heterologous SpaP-S1 protein expression in S. gordonii and that this modulation is mediated by the level of PrsA, whose expression is regulated by the LiaSR two-component regulatory system.     

Binding of complement component C3b to glycoprotein gC of herpes simplex virus type 1: mapping of gC-binding sites and demonstration of conserved C3b binding in low-passage clinical isolates.

The sites on glycoprotein gC of herpes simplex virus type 1 (HSV-1) which bind complement component C3b were evaluated by using anti-gC monoclonal antibodies and mutants which have alterations at defined regions of the glycoprotein. Monoclonal antibodies were incubated with HSV-1-infected cells in a competitive assay to block C3b binding. Each of 12 different monoclonals, which recognize the four major antigenic sites of gC, completely inhibited C3b binding. With this approach, no one antigenic group on gC could be assigned as the C3b-binding region. Next, 21 gC mutants were evaluated for C3b binding, including 1 which failed to synthesize gC, 4 which synthesized truncated forms of the glycoprotein such that gC did not insert into the cell's membrane, and 16 which expressed gC on the cell's surface but which had mutations in various antigenic groups. Eleven strains did not bind C3b. This included the 1 strain which did not synthesize gC, the 4 strains which secreted gC without inserting the glycoprotein into the cell membrane, and 6 of 16 strains which expressed gC on the cell surface. In these six strains, the mutations were at three different antigenic sites. One hypothesis to explain these findings is that C3b binding is modified by changes in the conformation of gC which develop either after antibodies bind to gC or as a result of mutations in the gC gene. Attachment of C3b to gC was also evaluated in 31 low-passage clinical isolates of HSV-1. Binding was detected with each HSV-1 isolate, but not with nine HSV-2 isolates. Therefore, although mutants that lack C3b binding are readily selected in vitro, the C3b-binding function of gC is maintained in vivo. These results indicate that the sites on gC that bind C3b are different from those that bind monoclonal antibodies, that antibodies directed against all sites on gC block C3b binding, and that C3b binding is a conserved function of gC in vivo.