Performance analyses of a neutralizing agent combination strategy for the production of succinic acid by Actinobacillus succinogenes ATCC 55618.

A neutralizing agent combination strategy was developed to enhance the succinic acid production by Actinobacillus succinogenes ATCC 55618. First, a maximal succinic acid production of 48.2 g/L was obtained at a culture pH of 7.5. Second, NaOH and KOH were screened to identify the optimal neutralizing agent for pH control. However, the production of succinic acid did not increase, and severe cell flocculation was observed due to a high concentration of metal ions when only one neutralizing agent was used to control pH. Finally, a neutralizing agent combination strategy was developed with a supply of neutralizing agents with OH(-) and carbonate. The cell flocculation was eliminated, and a maximum succinic acid production of 59.2 g/L was obtained with 5 M NaOH and 40 g/L of MgCO(3); this production was 27.9% higher than that obtained with NaOH alone. The results obtained in this study may be useful for the large-scale industrial production of succinic acid.

Purification of a fibroblast-inhibitory factor from Actinobacillus actinomycetemcomitans Y4.

A factor showing inhibitory activity against human gingival fibroblasts was extracted from the cytosol fraction of Actinobacillus actinomycetemocimitans Y4. The activity markedly inhibited the proliferation of human gingival fibroblasts, but had no effect on cell viability or gross morphology. No such activity was found in cytosol fractions from either Porphyromonas gingivalis 381 or Escherichia coli HB101. The extract from A. actinomycetemocomitans Y4 was then purified by anion-exchange chromatography, hydroxyapatite chromatography and gel-filtration chromatography to give a single band on SDS-PAGE with an apparent molecular mass of 65 kDa. The purification ratio was 183-fold with a recovery rate of 5% compared with the crude extract (starting material) when the activity was assessed by direct cell counts.

Characterization of Aqx and its operon: the hemolytic RTX determinant of Actinobacillus equuli.

Actinobacillus equuli, a member of the family Pasteurellaceae is the etiologic agent of a frequently lethal septicemia in neonatal foals as well as other more chronic diseases like arthritis, pleuritis, pneumonia or peritonitis. It may also be isolated from the oral cavity of healthy horses. Hemolytic isolates of A. equuli are known but so far no virulence determinants have been described for this bacterial species. By screening hemolytic A. equuli strains with specific gene probes, a hemolysin, designated Aqx (A. equuli RTX (repeats in the structural toxin)) was identified. This hemolysin was shown to be an RTX type of toxin by characterization of the aqxCABD operon. All hemolytic A. equuli isolates contained a functional aqxCABD operon and expressed the Aqx hemolysin as shown by genetic and phenotypic assays. The structural toxin AqxA is the hemolysin of A. equuli as shown by expression of recombinant aqx constructs in E. coli. Its hemolytic activity can be inhibited by specific antibodies raised against AqxA. Sequence analysis of the 16S rRNA gene (rrs) of the taxonomically diffuse group of A. equuli and related strains defined two phylogenetically distinct groups. The presence of the Aqx operon is not correlated with this phylogenetic grouping. The operon is found in both groups of A. equuli strains where it specifies the hemolytic activity and is supposedly to be a determinative virulence factor. The aqx operon was not found in closely related members of the Pasteurellaceae family. The description of the Aqx hemolysin will open new ways for studying the pathogenesis of A. equuli.

The first description of a (1--> 6)-beta-D-glucan in prokaryotes: (1 --> 6)-beta-D-glucan is a common component of actinobacillus suis and is the basis for a serotyping system.

The chemical and antigenic properties of the cell-surface lipopolysaccharides (LPSs) and capsular polysaccharides (CPSs) of seven representative strains of Actinobacillus suis from healthy and diseased pigs were investigated. Four strains produced a linear (1 --> 6)-beta-D-glucan homopolymer, beta-D-Glcp-(1-[ --> 6)-beta-D-Glcp-(1-]n -->, as a LPS-O-chain (O1) and as a CPS (K1). Polyclonal antisera prepared against a (1 --> 6)-beta-D-glucan-containing strain showed a positive reaction against both LPSs and CPSs derived from the above strains (designated serotype O1/K1). One strain carried the (1 --> 6)-beta-D-glucan solely as a LPS-O-chain (serotype O1) and two strains did not express the (1 --> 6)-beta-D-glucan, but, instead, produced a different O-chain (designated serotype 02); these three strains expressed their own characteristic CPSs. (1 --> 6)-beta-D-Glucan structures are common cell wall components of yeast, fungi and lichens, but, to our knowledge, this is the first time a (1 --> 6)-beta-D-glucan has been described in a prokaryotic organism. Conformational and nuclear magnetic resonance analyses showed that the beta-D-Glcp-(1 --> 6)-beta-D-Glcp linkage was flexible and two distinct glycosidic conformers are described. Cross-reactive antibodies to the A. suis (1 --> 6)-beta-D-glucan could be detected in sera from a variety of species and in sera from specific pathogen free pigs. This cross-reactivity may arise from immuno-stimulation of organisms present in the surrounding environment that contain (1 --> 6)-beta-D-glucan, which may also explain the high incidence of false positive results in previous serological tests for A. suis. In addition, these (1 --> 6)-beta-D-glucan background antibodies may be protective against A. suis infection. The characterization herein of (1 --> 6)-beta-D-glucan is the foundation for the development of a serotyping system for A. suis.

[Cloning and expression of the apx III A gene of Actinobacillus pleuropneumonie].

The apx III A gene of Actinobacillus pleuropneumonie (App) was amplified by PCR. The amplified DNA fragment 3 466bp was cloned into pMD18-T. After R.E. analysis and sequencing, the apx III A gene in pMD18-T was ligated into pBluescrip II SK(+), the recombinant expression plasmid pET-28b/apx III A was constructed and analysed with R. E., the protein of apx III A gene expressed in E. coli BL21 was detected by Western blotting. Based on expressed apx III A protein as antigen, empty expression vector as control, the ELISA to detect antibody against apx III A was developed and was primarily used to detect serum samples.

External and internal glucose mass transfers in succinic acid fermentation with stirred bed of immobilized Actinobacillus succinogenes under substrate and product inhibitions.

This paper is dedicated to the study on the external and internal mass transfers of glucose for succinic acid fermentation under substrate and product inhibitions using a bioreactor with stirred bed of immobilized Actinobacillus succinogenes cells. By means of the substrate mass balance for a single particle of biocatalysts, considering the kinetic model adapted for both inhibitory effects, specific mathematical models were developed for describing the profiles of the substrate concentration in the outer and inner regions of biocatalysts and for estimating the substrate mass flows in the liquid boundary layer surrounding the particle and inside the particle. The values of the mass flows were significantly influenced by the internal diffusion velocity and rate of the biochemical reaction of substrate consumption. These cumulated influences led to the appearance of a biological inactive region near the particle center, its magnitude varying from 0 to 5.3% of the overall volume of particles.

pH neutralization while succinic acid adsorption onto anion-exchange resins.

Succinic acid is a useful chemical and its purification from fermentation broth by ion-exchange resins has widely drawn attention. In this study, pH neutralization in the process of adsorption of succinic acid from model solutions and fermentation broth by anion-exchange resin NERCB 04 has been tested. Adsorption capacity of NERCB 04 was about 0.41 g succinic acid/g resin at concentrations of succinic acid in the range of 10-50 g/L in packed column. In the process of succinic acid removal, pH of the system could also be neutralized. The neutralizing ability of the resin as a neutralizing agent has also been studied in the model cycle system and in the real fermentation cycle process. It was found that NERCB 04 showed stable adsorption capacity and pH neutralization ability after each regeneration. A certain amount of anion-exchange resin could neutralize the low pH values (pH 2-5) and maintain the system around pH 7.0. This means the anion-exchange resins have the function of neutralizing reagent in the process of adsorbing succinic acid.

Distribution of diaminopropane, putrescine and cadaverine in Haemophilus and Actinobacillus.

Cellular levels of diaminopropane, putrescine and cadaverine, and decarboxylase activities to produce these diamines in six species (16 strains) of Haemophilus and four species (5 strains) of Actinobacillus belonging to the family Pasteurellaceae of the gamma subclass of the class Proteobacteria, were determined by high performance liquid chromatography (HPLC). Diaminopropane was ubiquitously distributed within all Haemophilus and Actinobacillus species, and L-2,4-diaminobutyric acid decarboxylase activity was detected in them. Putrescine and ornithine decarboxylase activity were found in H. aphrophilus, H. parainfluenzae and H. influenzae (type a, b, d, e and f except for type c) but not detected in H. aegyptius, H. parahaemolyticus, H. ducreyi and Actinobacillus species. Cadaverine occurred in H. aphrophilus, H. aegyptius, H. influenzae, H. parainfluenzae, A. actinomycetemcomitans, A. equuli and A. lignieresii, whereas their lysine decarboxylase activity was scarcely detected. Cadaverine was not found in H. parahaemolyticus, H. ducreyi and A. suis. The diamine profile serves as a phenotypic marker for the chemotaxonomic classification of the family Pasteurellaceae.

Whole cell protein profiling of actinobacillus-like strains classified as taxon 2 and taxon 3 according to Bisgaard.

Thirty-nine strains representing all biovars established within the taxa 2 and 3 complex of Bisgaard and two strains belonging to the avian [P.] haemolytical[A.] salpingitidis complex were characterized by one-dimensional SDS-PAGE of cellular proteins. The protein patterns, which contained 40 to 45 discrete protein bands, were highly reproducible. Numerical analysis of the background protein patterns obtained resulted in six major and twelve minor groups (phena). Comparison of the phena defined by protein profiling with species/groups previously established by DNA: DNA hybridization, chemotyping and "biotyping" showed that the best correlation existed between DNA:DNA hybridization and "biotyping". A correlation between results obtained from DNA:DNA hybridization and protein profiling was not obtained. With a few exceptions, a connection was demonstrated between protein profiles and hosts from which the strains belonging to the respective phena originated.

Investigation of the structure of lipid A from Actinobacillus actinomycetemcomitans strain Y4 and human clinical isolate PO 1021-7.

The lipopolysaccharides of Actinobacillus actinomycetemcomitans strain Y4 and a human clinical isolate PO 1021-7 were examined by SDS/PAGE, deoxycholate/PAGE and mass spectrometry. PAGE analysis revealed an electrophoretic pattern similar to the SR-type lipopolysaccharide (LPS) of Salmonella. Deoxycholate/PAGE indicated the LPS of A. actinomycetemcomitans to consist of short sugar chains. Chemical analysis revealed the presence of thiobarbituric-acid-positive material (3-deoxy-D-manno-octulosonic acid equivalents) and four neutral sugars: glucose, galactose, D-glycero-D-manno-heptose and L-glycero-D-manno-heptose. Phosphate, glucosamine, glycine, and the fatty acids, 3-hydroxymyristic acid, myristic acid and palmitic acid, comprised the remainder of the molecule. The structure of the free lipid A revealed it to consist of a 1,6-glucosamine disaccharide esterified at C4' by a phosphomonoester. The hydroxyl group at C3 and the amide group of the non-reducing glucosamine were both acylated by 3-myristoylmyristic acid; analogous sites on the reducing glucosamine were acylated by 3-hydroxymyristic acid. Hydroxyl groups at C4 and C6' in the free lipid A were unsubstituted, with C6 being the proposed attachment site of the polysaccharide moiety. Chemical analysis revealed the presence of glycine in the intact LPS; its exact location in the A. actinomycetemcomitans LPS is still to be determined. Both intact LPS and free lipid A were highly lethal to galactosamine-sensitized mice, comparable to that of Salmonella.

Characterization of the family Pasteurellaceae on the basis of cellular lipids and carbohydrates.

Selected strains representing established and newly described taxa in the family Pasteurellaceae were investigated for their cellular lipid and carbohydrate composition to clarify the taxonomic significance of such features. Methylated cellular fatty acids and acetylated derivatives of the cellular carbohydrates were determined by capillary gas chromatography using a flame ionization detector. In part the carbohydrates were identified by mass spectrometry. Phospholipids were determined by thin layer chromatography, the lipoquinones by high pressure liquid chromatography. The cellular fatty acid patterns proved to be uniform with minor variations, but the separation from the Neisseriaceae and from Moraxella was possible. Also the distribution of the phospholipids was uniform within the family. The lipoquinone contents were useful for the discrimination of groups within the family not necessarily reflecting the degree of genomic relatedness. The analysis of the cellular carbohydrates resulted in a common sugar pattern with all members of the family and characteristic carbohydrate profiles discriminating groups, often to the species level. All of the cytochemical features considered were useful for the characterization of the family Pasteurellaceae.

Purification of secreted leukotoxin (LtxA) from Actinobacillus actinomycetemcomitans.

The RTX (repeats in toxin) family of toxins is important in the pathogenesis of many Gram-negative bacteria. The oral and systemic human pathogen Actinobacillus actinomycetemcomitans produces a member of this family known as leukotoxin (LtxA). Previously, we found that LtxA is secreted into culture supernatants of A. actinomycetemcomitans and that this protein is abundant and relatively pure. Here, we report a large-scale method for the isolation and purification of LtxA from culture supernatants of A. actinomycetemcomitans strain JP2. The purification scheme involves ammonium sulfate precipitation of culture supernatants, dialysis, and ultrafiltration to concentrate LtxA to approximately 10mg/ml. We found that LtxA remained soluble in buffer that contained at least 250mM NaCl. Purified LtxA was >98% pure and the final preparations were active against HL-60 cells. The entire purification protocol can be completed within 2 days. The ability to readily obtain a large amount of purified leukotoxin should accelerate investigations into the structure and biology of this important virulence factor.