Microbial transformations of alpha-santonin

Fungal biotransformations of alpha-santonin (1) were conducted with Mucor plumbeus (ATCC 4740), Cunninghamella bainieri (ATCC 9244), Cunninghamella echinulata (ATCC 9245), Curvularia lunata (ATCC 12017) and Rhizopus stolonifer (ATCC 10404). Rhizopus stolonifer (ATCC 10404) metabolized compound 1 to afford 3,4-epoxy-alpha-santonin (2) and 4,5-dihydro-alpha-santonin (3) while Cunninghamella bainieri (ATCC 9244), Cunninghamella echinulata (ATCC 9245) and Mucor plumbeus (ATCC 4740) were capable of metabolizing compound 1 to give a reported metabolite, 1,2-dihydro-alpha-santonin (4). The structures of these transformed metabolites were established with the aid of extensive spectroscopic studies. These fungi regiospecifically reduced the carbon-carbon double bond in ring A of alpha-santonin.     

Characterization of Genes Involved in D-Sorbitol Oxidation in Thermotolerant Gluconobacter frateurii

A microbial screening indicated that two fungal strains, Beauveria bassiana DSM 1344=ATCC 7159 and Cunninghamella elegans DSM 1908=ATCC 9245, as well as four bacterial strains belonging to the genus Streptomyces were able to hydroxylate 4,5-dianilinophthalimide (DAPH, CGP52411) to 4-(4′-hydroxyanilino)-5-anilinophthalimide. Cunninghamella elegans DSM 1908 turned out to be the most active biocatalyst and was also able to form the dihydroxy derivative, 4,5-bis(4′-hydroxyanilino)phthalimide. The reaction for the monohydroxylated biotransformation product was carried out on a preparative scale, and the culture conditions for the formation of 4-(4′-hydroxy- anilino)-5-anilinophthalimide with this strain were op-timized.     

Regiospecific synthesis of isoapocodeine from 10,11-dimethoxyaporphine by using Cunninghamella elegans.

A preparative-scale regiospecific conversion of 10,11-dimethoxyaporphine to isoapocodeine was conducted with Cunninghamella elegans ATCC 9245. This biotransformation proceeded quantitatively in suspensions and was pH dependent. The influence of antioxidants on the conversion was studied. Attempts to preserve the activity of isolated C. elegans cells by a number of methods were unsuccessful.     

Transformation of Schizandrin into Gomisin A by Use of Microbial O-Demethylation and Chemical Reactions

The transformation of schizandrin (I) into gomisin A (II) was accomplished by use of a combination of biotransformation and chemical reactions. The biotransformation, microbial O-demethylation of I by Cuntiinghamella echinulata var. elegans (ATCC 9245) produced two novel metabolites [3-norschizandrin (IV) and 2-norschizandrin (VI)] and two known metabolites [gomisin T (III) and 13-norschizandrin (V)]. Among those metabolites, compound III was derived to II by the O-demethylation with a Lewis acid in the presence of an acid scavenger, followed by methylenation.     

Regiospecific synthesis of isoapocodeine from 10,11-dimethoxyaporphine by using Cunninghamella elegans.

A preparative-scale regiospecific conversion of 10,11-dimethoxyaporphine to isoapocodeine was conducted with Cunninghamella elegans ATCC 9245. This biotransformation proceeded quantitatively in suspensions and was pH dependent. The influence of antioxidants on the conversion was studied. Attempts to preserve the activity of isolated C. elegans cells by a number of methods were unsuccessful.     

Microbiological production of omeprazole metabolites by Cunninghamella elegans

Incubation of Cunninghamella elegans ATCC 9245 and the anti ulcer drug omeprazole allowed putative fungal metabolites to be isolated in sufficient quantities for structural elucidation. Three metabolites produced by the fungi were isolated using semi-preparative HPLC and their structures identified by a combination of LC/MS(n) and NMR experiments. These isolates will be used as reference standards in the confirmatory analysis of mammalian metabolites of this drug.     

Studies of the ribosome-associated vitamin B12S adenosylating enzyme of Lactobacillus leichmannii.

The ribosomes of Lactobacillus leichmannii (ATCC 7830) are the loci of an enzyme system that converts vitamin B₁₂ (cyanocobalamin, CN-Cbl) to adenosylcobalamin (AdoCbl) in two steps, reduction of B_12a (Co III) to B_12s (Co I) by B_12a reductase and the adenosylation of B_12s to AdoCbl by an adenosylating enzyme. The vitamin B₁₂ reductase, as in other organisms, is unstable. Adenosylating enzyme, however, is readily demonstrable. Reported experiments deal primarily with that enzyme. Evidence of the association between ribosomes and adenosylating enzyme was found in sucrose density gradient analyses. Intact, washed ribosomes yielded an enzyme activity profile that coincided with the ultraviolet maximum of 70S reference ribosomes. When intact ribosomes were exposed to 2.5 m CsCl so that 70% of ribosomal protein was recoverable in the 144,000g supernatant fraction and >90% of RNA was in the pellet, adenosylating enzyme was found in the supernatant fraction. “Stripped” ribosomes had low levels of enzyme activity and could reassociate with free enzyme protein. Stripped ribosomes remained competent in protein synthesis. Hence, adenosylating enzyme is not an integral ribosome component. Partially purified ribosome-associated B_12s adenosylating enzyme has requirements for vitamin B_12s, ATP, and Mn²⁺, though Mn²⁺ could be partially replaced by Mg²⁺. Isotopic studies showed that ATP is the source of the adenosyl moiety of AdoCbl and that inorganic tripolyphosphate is a reaction product. Substantial adenosylating activity is associated with ribosomes only in the vitamin B₁₂-requiring lactobacilli, L. delbrueckii (ATCC 9649) and L. leichmannii. Surprisingly, L. casei (ATCC 9595) ribosomes displayed a measurable, if low, level of activity. L. acidophilus (ATCC 11506) ribosomes had no detectable activity. The bulk of the activity in Clostridium tetanomorphum (ATCC 3606) and Propionibacterium shermannii (ATCC 9614) is in the 144,000g supernatant fraction. Ribosomes from animal cells (liver, reticulocytes, and Ehrlich ascites tumor) were without detectable activity.     

Stereospecific microbiological 10-O-demethylation of R-(-)-10,11-dimethoxyaporphines

The microbiological O-dealkylation of (+) and (-) 10,11-dimethoxyaporphine and the corresponding N-n-propyl analog 10,11-dimethoxy-N-n-propylnoraporphine utilizing the fungus Cunninghamella elegans (ATCC 9245) was found to proceed with regioselectivity for the 10-position, and with a high degree of substrate stereospecificity for the 6a R(-)enantiomer. Only the (R) 10-demethylated products were isolated i.e. (R) iosapocodeine and (R) N-n-propylnor-isoapocodeine. The products were confirmed by comparison with their GC/MS spectra.     

Fungal biotransformation of the antihistamine azatadine by Cunninghamella elegans.

The metabolism of the antihistamine azatadine by the zygomycete fungus Cunninghamella elegans ATCC 9245 was investigated. Within 72 h from the addition of the drug to 48-h-old cultures grown in Sabouraud dextrose broth, 95% of azatadine was biotransformed. Two major metabolites, 7-hydroxyazatadine (25%) and 8-hydroxyazatadine (50%), and two minor metabolites, N-desmethylazatadine and 9-hydroxyazatadine, were isolated by high-performance liquid chromatography and characterized by mass spectrometric and proton nuclear magnetic resonance spectroscopic analyses.     

Metabolism of the ethanolamine-type antihistamine diphenhydramine (Benadryl)TM by the fungus Cunninghamella elegans

Two strains of the filamentous fungus Cunninghamella elegans (ATCC 9245 and ATCC 36112) were grown in Sabouraud dextrose broth and screened for the ability to metabolize the ethanolamine-type antihistamine diphenhydramine. Based on the amount of parent drug recovered after 7 days incubation, both C. elegans strains metabolized approximately 74% of the diphenhydramine, 58% of this being identified as organic extractable metabolites. The organic extractable metabolites were isolated by reversed-phase high-performance liquid chromatography and identified by analyzing their mass and nuclear magnetic resonance spectra. Desorption chemical ionization mass spectrometry (DCIMS) with deuterated ammonia was used to differentiate possible isobaric diphenhydramine metabolites and to probe the mechanisms of ion formation under ammonia DCIMS conditions. C. elegans transformed diphenhydramine by demethylation, oxidation, and N-acetylation. The major metabolites observed were diphenhydramine-N-oxide (3%), N-desmethyldiphenhydramine (30%), N-acetyldidesmethyldiphenhydramine (13%), and N-acetyl-N-desmethyldiphenhydramine (12%). These compounds are known mammalian metabolites of diphenhydramine and may be useful for further toxicological studies. Received: 24 June 1999 / Revision received: 23 August 1999 / Accepted: 24 September 1999     

Mixture of periodontopathogenic bacteria influences interaction with KB cells

IntroductionThe purpose of this study was to investigate the adhesion and invasion of periodontopathogenic bacteria in varied mixed infections and the release of interleukins from an epithelial cell line (KB cells).MethodsKB cells were co-cultured with Porphyromonas gingivalis ATCC 33277 and M5-1-2, Tannerella forsythia ATCC 43037, Treponema denticola ATCC 35405 and Fusobacterium nucleatum ATCC 25586 in single and mixed infections. The numbers of adherent and internalized bacteria were determined up to 18 h after bacterial exposure. Additionally, the mRNA expression and concentrations of released interleukin (IL)-6 and IL-8 were measured.ResultsAll periodontopathogenic bacteria adhered and internalized in different numbers to KB cells, but individually without any evidence of co-aggregation also to F. nucleatum. High levels of epithelial mRNA of IL-6 and IL-8 were detectable after all bacterial challenges. After the mixed infection of P. gingivalis ATCC 33277 and F. nucleatum ATCC 25586 the highest levels of released interleukins were found. No IL-6 and IL-8 were detectable after the mixed infection of P. gingivalis M5-1-2 and F. nucleatum ATCC 25586 and the fourfold infection of P. gingivalis ATCC 33277, T. denticola ATCC 35405, T. forsythia ATCC 43037 and F. nucleatum ATCC 25586.ConclusionAnaerobic periodontopathogenic bacteria promote the release of IL-6 and IL-8 by epithelial cells. Despite a continuous epithelial expression of IL-8 mRNA by all bacterial infections these effects are temporary because of the time-dependent degradation of cytokines by bacterial proteases. Mixed infections have a stronger virulence potential than single bacteria. Further research is necessary to evaluate the role of mixed infections and biofilms in the pathogenesis of periodontitis.     

Response of Pathogenic Vibrio Species to High Hydrostatic Pressure

Vibrio parahaemolyticus ATCC 17802, Vibrio vulnificus ATCC 27562, Vibrio cholerae O:1 ATCC 14035, Vibrio cholerae non-O:1 ATCC 14547, Vibrio hollisae ATCC 33564, and Vibrio mimicus ATCC 33653 were treated with 200 to 300 MPa for 5 to 15 min at 25°C. High hydrostatic pressure inactivated all strains of pathogenic Vibrio without triggering a viable but nonculturable (VBNC) state; however, cells already existing in a VBNC state appeared to possess greater pressure resistance.     

Novel hybrids of fluconazole and furanones: design, synthesis and antifungal activity

During our efforts to develop new antifungal agents, a number of hybrid molecules containing furanones and fluconazole pharmacophores were designed and synthesized. The new chemical entities thus synthesized were tested for their potential as antifungal agents against various fungal strains and it was observed that the compounds with general structure 7 were potent inhibitors of Candida albicans ATCC 24433, Candida glabrata ATCC 90030, Candida tropicalis ATCC 750 and Candida neoformans ATCC 34664 while the fluconazole analogues 12 exhibited antifungal activity against Candida albicans ATCC 24433 and Candida glabrata ATCC 90030. The structure-activity relationship for these compounds is discussed. The synthetic strategies used in the present work have potential to prepare a large number of compounds for further refinement of structures to obtain molecules suitable for development as antifungal drugs.     

Fate of some types of bacteria in hydrocortisone acetate ointment

The survival of Pseudomonas aeruginosa ATCC 9027, Escherichia coli ATCC 8739, Staphylococcus aureus ATCC 6538 and Bacillus pumilus ATCC 14884 in hydrocortisone acetate ointment was investigated. When the ointment was contaminated and packed in metal tubes all bacteria except B. pumilus were killed in a matter of days, but when the contaminated ointment was packed in jars, all bacteria survived provided atmospheric oxygen was allowed at intervals to pass into the containers.     

Development and Characterization of a Gene Expression Reporter System for Clostridium acetobutylicum ATCC 824

A gene expression reporter system (pHT3) for Clostridium acetobutylicum ATCC 824 was developed by using the lacZ gene from Thermoanaerobacterium thermosulfurogenes EM1 as the reporter gene. In order to test the reporter system, promoters of three key metabolic pathway genes, ptb (coding for phosphotransbutyrylase), thl (coding for thiolase), and adc (coding for acetoacetate decarboxylase), were cloned upstream of the reporter gene in pHT3 in order to construct vectors pHT4, pHT5, and pHTA, respectively. Detection of β-galactosidase activity in time course studies performed with strains ATCC 824(pHT4), ATCC 824(pHT5), and ATCC 824(pHTA) demonstrated that the reporter gene produced a functional β-galactosidase in C. acetobutylicum. In addition, time course studies revealed differences in the β-galactosidase specific activity profiles of strains ATCC 824(pHT4), ATCC 824(pHT5), and ATCC 824(pHTA), suggesting that the reporter system developed in this study is able to effectively distinguish between different promoters. The stability of the β-galactosidase produced by the reporter gene was also examined with strains ATCC 824(pHT4) and ATCC 824(pHT5) by using chloramphenicol treatment to inhibit protein synthesis. The data indicated that the β-galactosidase produced by the lacZ gene from T. thermosulfurogenes EM1 was stable in the exponential phase of growth. In pH-controlled fermentations of ATCC 824(pHT4), the kinetics of β-galactosidase formation from the ptb promoter and phosphotransbutyrylase formation from its own autologous promoter were found to be similar.     

Deoxyribonucleic acid hybridization among strains of lactobacilli

Hybridization of deoxyribonucleic acid (DNA) from Lactobacillus bulgaricus (ATCC 11842) with DNA of L. lactis (ATCC 12315), L. helveticus (ATCC 15009), and L. jugurt (ATCC 521) showed 86.0% reassociation with L. lactis, 4.8% with L. helveticus, and none with L. jugurt.     

Antimicrobial activity of invertebrate-pathogenic fungi in the genera Akanthomyces and Gibellula

Infectious disease caused by antibiotic resistant microorganisms is a global public health problem. There is a need to search for new bioactive compounds from new sources. In this study, we focused on invertebrate-pathogenic fungi infecting spiders. One hundred and sixty-five crude extracts from Akanthomyces (n = 45) and Gibellula (n = 10) were screened for their antimicrobial activity against nine human pathogens. Twenty-one extracts out of 165 (12.73%) from 16 (29.09%) isolates exhibited antimicrobial activity against at least one test strain. The most activity was against Staphylococcus aureus American Type Culture Collection (ATCC 25923) (8.48%) followed by Cryptococcus neoformans ATCC 90112 (3.03%), C. neoformans ATCC 90113 (2.42%), methicillin-resistant Staphylococcus aureus (MRSA) SK-1 (2.42%), Penicillium marneffei (2.42%), Microsporum gypseum (1.21%), Candida albicans ATCC 90028 (1.21%), Pseudomonas aeruginosa ATCC 27853 (0.61%) and Escherichia coli ATCC 25922 (0.61%), respectively. The ethyl acetate extract of mycelia from Gibellula pulchra EPF083 had the strongest broad spectrum antimicrobial activity with a minimum inhibitory concentration (MIC) value of 16 μg/ml against S. aureus ATCC 25923, MRSA SK-1, C. neoformans (ATCC 90112 and ATCC 90113) and P. marneffei and exhibited fungicidal activity against C. neoformans ATCC 90112 and P. marneffei with minimum fungicidal concentration (MFC) values of 16 and 32 μg/ml, respectively. These preliminary data show that invertebrate-pathogenic fungi could be a potential source of antimicrobial agents.     

The genome sequence of Bacillus cereus ATCC 10987 reveals metabolic adaptations and a large plasmid related to Bacillus anthracis pXO1

We sequenced the complete genome of Bacillus cereus ATCC 10987, a non-lethal dairy isolate in the same genetic subgroup as Bacillus anthracis. Comparison of the chromosomes demonstrated that B.cereus ATCC 10987 was more similar to B.anthracis Ames than B.cereus ATCC 14579, while containing a number of unique metabolic capabilities such as urease and xylose utilization and lacking the ability to utilize nitrate and nitrite. Additionally, genetic mechanisms for variation of capsule carbohydrate and flagella surface structures were identified. Bacillus cereus ATCC 10987 contains a single large plasmid (pBc10987), of ~208 kb, that is similar in gene content and organization to B.anthracis pXO1 but is lacking the pathogenicity-associated island containing the anthrax lethal and edema toxin complex genes. The chromosomal similarity of B.cereus ATCC 10987 to B.anthracis Ames, as well as the fact that it contains a large pXO1-like plasmid, may make it a possible model for studying B.anthracis plasmid biology and regulatory cross-talk.     

Suppression of stop codon UGA in acrB can contribute to antibiotic resistance in Klebsiella pneumoniae ATCC10031

We previously reported that Klebsiella pneumoniae MGH78578 exhibited higher resistance against various antimicrobials than K. pneumoniae ATCC10031. In this study, we showed that the plasmid, pKPN5, in K. pneumoniae MGH78578 played an important role in resistance against aminoglycosides, ampicillin, tetracycline, and chloramphenicol, while genome-derived β-lactamases and drug efflux pumps appeared to be more important in resistance to cloxacillin. acrAB, encoding a potent multidrug efflux pump, was cloned from K. pneumoniae MGH78578 and ATCC10031, to investigate reasons for the high drug resistance of K. pneumoniae MGH78578, and the results revealed that AcrAB from K. pneumoniae ATCC10031 conferred weaker drug resistance than AcrAB from K. pneumoniae MGH78578. DNA sequencing revealed that acrB from K. pneumoniae ATCC10031 carried the nonsense mutation, UGA, which was not found in acrB from K. pneumoniae MGH78578. However, acrB from K. pneumoniae ATCC10031 conferred slightly elevated resistant levels to several antimicrobials. The intact length of AcrB was detected in K. pneumoniae ATCC10031 by Western blot analysis, even though its quantity was small. Therefore, the stop codon UGA in acrB was thought to be overcome to some extent in this strain. We artificially introduced the nonsense mutation, UGA to the cat gene on pACYC184, and the plasmid also elevated the MIC of chloramphenicol in K. pneumoniae ATCC10031. These results suggest that a mechanism to overcome the nonsense mutation in acrB sustained resistance against a few β-lactams, dyes, and cholic acid in K. pneumoniae ATCC10031.     

Total phenolic content, flavonoid concentration, antioxidant and antimicrobial activity of methanol extracts from three Seseli L. taxa

The present study describes the total phenolic content, concentrations of flavonoids and in vitro antioxidant and antimicrobial activity of methanol extracts from Seseli pallasii Besser, S. libanotis (L.) Koch ssp. libanotis and S. libanotis (L.) Koch ssp. intermedium (Rupr.) P. W. Ball, growing wild in Serbia. The total phenolic content in the extracts was determined using Folin-Ciocalteu reagent and their amounts ranged between 84.04 to 87.52 mg GA (gallic acid)/g. The concentrations of flavonoids in the extracts varied from 4.75 to 19.37 mg Qu (quercetin)/g. Antioxidant activity was analyzed using DPPH reagent. Antioxidant activity ranged from 0.46 to 4.63 IC₅₀ (mg/ml) and from 1.98 to 2.19 mg VitC (vitamin C)/g when tested with the DPPH and ABTS reagents, respectively, using BHA and VitC as controls. The antimicrobial activity of the extracts was investigated using a micro-well dilution assay for the most common human gastrointestinal pathogenic bacterial strains: Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 9027, Salmonella enteritidis ATCC 13076, Bacillus cereus ATCC 10876, Listeria monocytogenes ATCC15313, Staphylococcus aureus ATCC 25923 and Candida albicans ATCC 10231. This finding suggests that Seseli species may be considered as a natural source of antioxidants and antimicrobial agents.