Coaggregation between Porphyromonas gingivalis and Mutans Streptococci

Coaggregation occurred between Porphyromonas gingivalis and mutans streptococci. The coaggregation was completely inhibited by l -arginine, Nα-p-tosyl-l -lysine chloromethyl ketone (TLCK), and a trypsin inhibitor, and weakly inhibited by l -lysine, N-ethylmaleimide, lysozyme, and human whole saliva. The results of heat and proteinase K treatment suggested that a heat-labile proteinaceous substance of P. gingivalis and a heat-stable substance of mutans streptococci may play a role in the coaggregation. Mutans streptococci also aggregated in the presence of the heat-labile factor in the supernatant of P. gingivalis. The aggregation was also inhibited by l -arginine, TLCK, and a trypsin inhibitor.     

Interspecies differences in the enantioselectivity of epoxide hydrolases in Cryptococcus laurentii (Kufferath) C.E. Skinner and Cryptococcus podzolicus (Bab'jeva & Reshetova) Golubev

Isolates representing Cryptococcus laurentii and Cryptococcus podzolicus, originating from soil of a heathland indigenous to South Africa, were screened for the presence of enantioselective epoxide hydrolases for 2,2-disubstituted epoxides. Epoxide hydrolase activity for the 2,2-disubstituted epoxide (+/-)-2-methyl-2-pentyl oxirane was found to be abundantly present in all isolates. The stereochemistry of the products formed by the epoxide hydrolase enzymes from isolates belonging to the two species (11 isolates representing C. laurentii and 23 isolates representing C. podzolicus) was investigated. The enantiopreferences of the epoxide hydrolases for 2,2-disubstituted epoxides of these two species were found to be opposite. All strains of C. laurentii preferentially hydrolysed the (S)-epoxides while all C. podzolicus isolates preferentially hydrolysed the (R)-epoxides of (+/-)-2,2-disubstituted epoxides. These findings indicate that the stereochemistry of the products formed from 2,2-disubstituted epoxides by the epoxide hydrolase enzymes of these yeasts should be evaluated as additional taxonomic criterion within the genus Cryptococcus. Also, the selectivity of some epoxide hydrolases originating from isolates of C. podzolicus was high enough to be considered for application in biotransformations for the synthesis of enantiopure epoxides and vicinal diols.     

Multi-copy expression and fed-batch production of Rhodotorula araucariae epoxide hydrolase in Yarrowia lipolytica

Epoxide hydrolases (EHs) of fungal origin have the ability to catalyze the enantioselective hydrolysis of epoxides to their corresponding diols. However, wild type fungal EHs are limited in substrate range and enantioselectivity. Additionally, the production of fungal epoxide hydrolase (EH) by wild-type strains is typically very low. In the present study, the EH-encoding gene from Rhodotorula araucariae was functionally expressed in Yarrowia lipolytica, under the control of a growth phase inducible hp4d promoter, in a multi-copy expression cassette. The transformation experiments yielded a positive transformant, with a final EH activity of 220 U/g dw in shake-flask cultures. Evaluation of this transformant in batch fermentations resulted in ~ 7-fold improvement in EH activity over the flask scale. Different constant specific feed rates were tested in fed-batch fermentations, resulting in an EH activity of 1,750 U/g dw at a specific feed rate of ~ 0.1 g/g/h, in comparison to enzyme production levels of 0.3 U/g dw for the wild type R. araucariae and 52 U/g dw for an Escherichia coli recombinant strain expressing the same gene. The expression of EH in Y. lipolytica using a multi-copy cassette demonstrates potential for commercial application.     

A cel44C-man26A gene of endophytic Paenibacillus polymyxa GS01 has multi-glycosyl hydrolases in two catalytic domains

A bacterial strain Paenibacillus polymyxa GS01 was isolated from the interior of the roots of Korean cultivars of ginseng (Panax ginseng C. A. Meyer). The cel44C-man26A gene was cloned from this endophytic strain. This 4,056-bp gene encodes for a 1,352-aa protein which, based on BLAST search homologies, contains a glycosyl hydrolase family 44 (GH44) catalytic domain, a fibronectin domain type 3, a glycosyl hydrolase family 26 (GH26) catalytic domain, and a cellulose-binding module type 3. The multifunctional enzyme domain GH44 possesses cellulase, xylanase, and lichenase activities, while the enzyme domain GH26 possesses mannanase activity. The Cel44C enzyme expressed in and purified from Escherichia coli has an optimum pH of 7.0 for cellulase and lichenase activities, but is at an optimum pH of 5.0 for xylanase and mannanase activities. The optimum temperature for enzymatic activity was 50°C for all substrates. No detectable enzymatic activity was detected for the Cel44C-Man26A mutants E91A and E222A. These results suggest that the amino acid residues Glu₉₁ and Glu₂₂₂ may play an important role in the glycosyl hydrolases activity of Cel44C-Man26A.     

Effect of cultivation time and growth medium on the coaggregation capability of <Emphasis Type="Italic">Acinetobacter johnsonii</Emphasis> S35

The effects of different cultivation periods and growth media on the cell surface hydrophobicity and coaggregation ability of Acinetobacter johnsonii S35 with Oligotropha carboxidovorans S23 (both sewage sludge isolates) were examined. It was observed that when cultivated in polypeptone medium, A. johnsonii S35 shows high hydrophobicity and strong coaggregation (98% aggregation index) with O. carboxidovorans S23, which remain unaltered after a 1-day, 3-day or 7-day cultivation period. When A. johnsonii S35 was cultivated with different carbon substrates, large coaggregates and a high aggregation index were observed using acetate-grown cells (96%), followed by ethanol-(83%) and vegetable oil-grown cells (72%). Coaggregates were small and narrow in ethanol-grown cells and still smaller in case of the vegetable oil-grown cells. Although no direct correlation could be observed, more hydrophobic cells showed a higher aggregation index. None of the culture supernatants of A. johnsonii S35 showed emulsification of hexadecane or a significant ability to aggregate O. carboxidovorans S23 cells, indicating that the coaggregation factor of A. johnsonii S35 is cell-bound. These results demonstrate that the coaggregation ability of A. johnsonii S35 is constitutive and is not lost under diverse cultivation conditions.     

Intracellular peptidoglycan hydrolases of the bacterium Xanthomonas campestris XL-1

A system of intracellular peptidoglycan hydrolases of Xanthomonas campestris XL-1 comprises about 10 enzymes of different localization and substrate specificity. Seven enzymes (A₁-A₇) are localized in cytosol, one enzyme (A₈) in periplasm, and two enzymes (A₉, A₁₀) were found in the fraction of cell walls and membranes. While the culture is entering the logarithmic growth stage from the stationary stage, a change occurs in the activity of the cytosolic enzymes: A₁ significantly increases, and A₅ and A₆ decrease. The spectrum of cytosolic enzymes also depends on the growth medium composition. The enzyme A₇ present in cells secreting extracellular enzymes (medium 5/5) was not found in non-secreting cells (LB medium). Unlike extracellular enzymes, intracellular peptidoglycan hydrolases are primarily acidic proteins. The data indicate that the system of intracellular peptidoglycan hydrolases of X. campestris is under complex and strict regulation.     

Genetic analysis of adherence by oral streptococci

Streptococci are one of the most successful bacterial colonizers of the human body and are major components of oral biofilms. The bacterial cells express multiple cell-surface adhesins that are responsible for the ability of streptococci to adhere to a wide range of substrates which include salivary and serous proteins, epithelial cells and other bacterial cells. Analysis of adherence-defective mutants has indicated the importance of high molecular mass wall-associated polypeptides and of enzymes catalyzing extracellular glucan polysaccharide synthesis to the adherence and accumulation of oral streptococci. The analysis of isogenic mutants of streptococci, generated through insertional inactivation (or allelic exchange), has confirmed the essential roles of specific surface polypeptides both to adhesive processes and to correct assembly of the cell wall layers.     

Identification of mutans streptococci with monoclonal antibodies

Mutans streptococci have been correlated with dental caries. The identification of the species within this group is still a problem. The characterization of a monoclonal antibody (Mab) OMVU10 against S. sobrinus as well as the isolation and characterization of Mabs against S. mutans (OMVU30 and OMVU31), S. cricetus (OMVU40) and mutans streptococci (OMVU2) is described. The epitope specificity for OMVU10 and OMVU31 was cell-wall antigen B in both cases although both Mabs recognized different species-specific epitopes. OMVU40 was cross reactive with Steptococcus sanguis taxon 3. All other Mabs were specific for one species. Using these Mabs, a key to the identification of mutans streptococci is developed. This key was tested for 85 wild type isolates of mutans streptococci and proved to be highly reliable and easy to perform.     

Coaggregation and biofilm formation of Leptospira with Staphylococcus aureus

It is not known how Leptospira react to wound or a cut infected with microbes, such as pathogenic Staphylococcus, or their common habitat on oral or nasal mucosal membranes. In the present study, Staphylococcus aureus MTCC‐737 showed strong co‐aggregation with leptospiral strains (>75%, visual score of + 4) in vitro. All tested strains of Leptospira were able to form biofilm with S. aureus. Scanning electron microscopy analysis revealed intertwined networks of attached cells of L. interrogans and S. aureus, thus providing evidence of a matrix‐like structure. This phenomenon may have implications in Leptospira infection, which occurs via cuts and wounds of the skin.