Two different primary oxidation mechanisms during biotransformation of thymol by gram-positive bacteria of the genera Nocardia and Mycobacterium

Thymol has antibacterial, antifungal, insecticidal, and antioxidative properties which are the basis for the wide use of this compound in the cosmetic, food, and pharmaceutical industries. Although thymol is a ubiquitously occurring substance in the environment, data about its degradation and detoxification by bacteria are sparse. Here, we show the existence of two different pathways for the biotransformation of thymol by Nocardia cyriacigeorgica and Mycobacterium neoaurum which were described for the first time for gram-positive bacteria. The first pathway starts with hydroxylation of thymol to thymohydroquinone (2-isopropyl-5-methylbenzene-1,4-diol) with subsequent oxidation to thymobenzoquinone (2-isopropyl-5-methyl-1,4-benzoquinone). The second pathway involves hydroxylation of the methyl group followed by oxidation to 3-hydroxy-4-isopropylbenzoic acid, possibly via the aldehyde 3-hydroxy-4-isopropylbenzaldehyde. It is noteworthy that the branched side chain of thymol was not oxidized. Similarities and differences of these oxidation processes with those of the gram-negative bacterium Pseudomonas putida, fungi, and plants are discussed and, in addition, the toxicity of thymol towards N. cyriacigeorgica and M. neoaurum was tested. The experiments showed a temporary growth inhibition with 0.025 % thymol. This was explained by degradation of thymol and the formation of products which are less toxic than thymol itself.     

Characterization of bacteria of the genera Mycobacterium, Rhodococcus and Nocardia isolated from environmental objects in the Republic of Daghestan

The bacteriological study of specimens taken from environmental objects, biomaterials from animals and sputa from tuberculosis patients revealed the ubiquity of Mycobacterium, Nocardia and rhodococci obtained from different kinds of environment in the Republic of Daghestan. In the identification 83.6% out of the isolated atypical Mycobacterium cultures were classified with Ranion groups II and IV. Among Nocardia, the most widespread organisms were N. asteroides (62.7%) and among rhodococci, R. erithropolis (61.9%).     

Effect of the C-2 hydroxyl group on the mesomorphism of alkyl glycosides: synthesis and thermotropic behavior of alkyl 2-deoxy-D-arabino-hexopyranosides

A homologous series of alkyl 2-deoxy-alpha-d-arabino-hexopyranosides and alkyl 2-deoxy-beta-d-arabino-hexopyranosides were synthesized, upon glycosylation of 1-alkanols (from C8 to C18 alkanols) with ethyl 2-deoxy-3,4,6-tri-O-acetyl-1-thio-d-arabino-hexopyranoside, followed by a deprotection. The thermotropic behavior of these new types of alkyl glycosides was investigated. It was observed that the beta-anomers of these alkyl glycosides, bearing nonyl to tetradecyl alkyl chain are mesomorphic, exhibiting monotropic smectic A phase. In contrast, the alpha-anomers are all non-mesomorphic. An effort to identify the liquid crystalline behavior of binary mixtures of the alpha- and beta-anomers was undertaken and it was found that mixtures containing equimolar amounts of the anomers exhibited mesomorphic behavior. A fine balance of the hydrophilic and hydrophobic components within the molecule is also found to be important for the alkyl 2-deoxy glycosides to form the mesophase.     

Characterization of Superoxide dismutase genes from Gram-positive bacteria by polymerase chain reaction using degenerate primers

Abstract An internal fragment representing approximately 85% of sod genes from seven Gram-positive bacteria was amplified by using degenerate primers in a polymerase chain reaction assay. The DNA sequences of sod polymerase chain reaction products from Clostridium perfringens, Enterococcus faecalis, Enterococcus faecium, Lactococcus lactis, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pneumoniae , and Streptococcus pyogenes were determined. Comparisons of their deduced amino acid sequences with those of the corresponding regions of the SOD proteins from Bacillus stearothermophilus, Listeria monocytogenes , and Streptococcus mutans revealed strong relatedness. Phylogenetic analysis of SOD peptides showed that members of the genera Streptococcus and those of the genera Enterococcus constitute two well-supported monophyletic groups. The method described in this study provides a means for easy recovery of sod genes and the construction of sod mutants of various Gram-positive pathogens.     

Mechanism of chlorpromazine binding by Gram-positive and Gram-negative bacteria

Chlorpromazine forms charge-transfer complexes with xanthene dyes in bacteria. These complexes permit the differentiation of Gram-positive and Gram-negative bacteria in both light and polarization microscopy. The birefringence induced by the charge-transfer complex might explain the molecular basis of bacterial staining.The charge-transfer complexes formed between chorpromazine and xanthene dyes accumulate in the bacterial cell, mainly inside the bacterial cell wall. The complexes give the cells a color, which depends on the chemical composition of the staining structure, and in particular the polysaccharides of the cell wall in bacteria.Metachromatic granules were seen inside Gram-positive bacteria after chlorpromazine and rose bengal staining. Although the nature of these granules remains unclear, this type of binding may have a role in the inhibition of biochemical processes in the bacterial cells.     

Biotransformation of anisole and phenetole by aerobic hydrocarbonoxidizing bacteria

Wild type, mutant, and recombinant bacterial strains capable of oxidizing aromatic hydrocarbons were screened for their ability to oxidize anisole (methoxybenzene) and phenetole (ethoxybenzene). Toluene-induced cells ofPseudomonas putida F39/D transformed anisole to a compound tentatively identified ascis-1,2-dihydroxy-3-methoxyclohexa-3,5-diene (anisole-2,3-dihydrodiol), 2-methoxyphenol, catechol, and trace amounts of phenol while phenetole was converted primarily tocis-1,2-dihydroxy-3-ethoxycyclohexa-3,5-diene (phenetole-2,3-dihydrodiol) and 2-ethoxyphenol. Induced cells ofPseudomonas sp. NCIB 9816/11 andBeijerinckia sp. B8/36 transformed anisole to phenol, and phenetole to phenol and ethenyloxybenzene. Toluene-induced cells ofP. putida BG1 converted anisole to phenol but did not oxidize phenetole. In contrast, toluene-induced cells ofP. mendocina KR1, which oxidize toluene via monooxygenation at thepara position, transformed anisole to 4-methoxyphenol, and phenetole to 2-, 3- and 4-ethoxyphenol. The involvement of toluene and naphthalene dioxygenases in the reactions catalyzed by strains F39/D and NCIB 9816/11, respectively, was confirmed with recombinantE. coli strains expressing the cloned dioxygenase genes. The results show that the oxygenases from differentPseudomonas strains oxidize anisole and phenetole to different hydroxylated products.     

The ESAT-6 gene cluster of Mycobacterium tuberculosis and other high G+C Gram-positive bacteria

Background  

The genome of Mycobacterium tuberculosis H37Rv has five copies of a cluster of genes known as the ESAT-6 loci. These clusters contain members of the CFP-10 (lhp) and ESAT-6 (esat-6) gene families (encoding secreted T-cell antigens that lack detectable secretion signals) as well as genes encoding secreted, cell-wall-associated subtilisin-like serine proteases, putative ABC transporters, ATP-binding proteins and other membrane-associated proteins. These membrane-associated and energy-providing proteins may function to secrete members of the ESAT-6 and CFP-10 protein families, and the proteases may be involved in processing the secreted peptide.     

Modulating the activity of avian pancreatic lipases by an alkyl chain reacting with an accessible sulfhydryl group

Both turkey (TPL) and chicken (CPL) pancreatic lipases possess only one exposed sulfhydryl residue (Cystein114). After preincubation with the lipase, the sulfhydryl reagent C12 -TNB was found to be a powerful inhibitor of TPL whereas it had no effect on the CPL activity. Based on the 3D structure modelling and the molecular dynamics, the bulky dodecyl chain might hamper the lid movement of the TPL leading to the lipase inhibition upon reaction with C12 -TNB. Meanwhile, the predicted position of the C12 chain linked to Cystein114 of CPL could not block the lid opening mechanism which explains the absence of inhibition by C12 -TNB. Surprisingly, when added during the substrate hydrolysis, C12 -TNB activated the TPL but not the CPL that was slightly inhibited under these conditions. The 3D structure model generated for the open forms of C12 -TPL and C12 -CPL complexes showed that Cystein114 is still accessible and might react with C12 -TNB. Our models clearly explain the activation of TPL and the partial inhibition of CPL after the binding of the C12 chain to the enzyme.     

Differential expression of CYP102 in Bacillus megaterium by 17-beta-estradiol and 4-sec-butylphenol

Previously we have reported the induction of CYP102 in Bacillus megaterium by 17beta-estradiol (E2) and 4-sec-butylphenol (4-sBP). Electrophoretic mobility shift assay analyses demonstrated that E2 and 4-sBP both cause a dose-dependent disassociation of the Bm3R1 repressor protein from its binding site on the operator sequence of the CYP102 gene. Equimolar combinations of E2 and 4-sBP demonstrated additive induction of CYP102 compared to equivalent samples of E2 and 4-sBP added alone. Two gene constructs were used in this investigation. One construct designated BMC143 contained the entire regulatory region of CYP102. The other gene construct, designated BMA45, had the "Barbie box" sequence deleted. While the induction of CYP102 by 4-sBP was much higher in the BMC 143 construct, E2 induced CYP102 in both constructs to the same extent. This difference in induction of CYP102 by these two inducers indicates that they act at different sites, either on the Bm3R1 repressor protein or on positive regulatory sites, or that they act, in part, through different mechanisms.     

Biotransformation of furfural and 5-hydroxymethyl furfural by enteric bacteria

Summary A survey was conducted with seventeen enteric bacterial strains (including the generaKlebsiella, Enterobacter, Escherichia, Citrobacter, Edwardsiella andProteus) to examine their ability to transform furfural and 5-hydroxymethyl furfural (5-MHF). The enteric bacteria were able to convert furfural to furfuryl alcohol under both aerobic and anaerobic conditions in a relatively short incubation time of 8 h. 5-HMF was transformed by all the enteric bacteria studied to an unidentified compound postulated to be 5-hydroxymethyl furfuryl alcohol, which had an absorbance maximum of 222 nm. These bacteria did not transform furfuryl alcohol or 2-furoic acid. The enteric bacteria did not use furfural, 5-HMF, furfuryl alcohol or 2-furoic acid as sole source of carbon and energy. Biotransformation of furfural and 5-HMF was accomplished by co-metabolism in the presence of glucose and peptone as main substrates. The rate of transformation was similar under both aerobic and anaerobic conditions. These transformations are likely to be of value in the detoxification of furfurals, and in their ultimate conversion to methane and CO₂ by anaerobic digestion.     

Cometabolic ring fission of dibenzofuran by Gram-negative and Gram-positive biphenyl-utilizing bacteria

Thirty-five strains of soil bacteria were grown with biphenyl (BP) and tested for their capacity to cooxidize dibenzofuran (DBF). During metabolism of DBF, the culture medium of 17 strains changed from colorless to orange, indicating a meta-cleavage pathway of DBF degradation. The ring cleavage product of these isolates was shown to be 2-hydroxy-4-(3'-oxo-3' H-benzofuran-2'-yliden)but-2-enoic acid (HOBB). The strain SBUG 271, studied in detail and identified as Rhodococcus erythropolis, degraded DBF via 1,2-dihydroxydibenzofuran. The ensuing meta-cleavage yielded HOBB and salicylic acid. In addition, the four monohydroxylated monomers of DBF and two metabolites, which were not further characterized, were detected. Thus, our results demonstrate that the metabolic mechanism involves lateral dioxygenation of DBF followed by meta-cleavage and occurs in Gram-negative as well as in Gram-positive BP-degrading bacteria.     

Acceleration of phospholipid flip-flop in the erythrocyte membrane by detergents differing in polar head group and alkyl chain length

The detergents, alkyltrimethylammonium bromide, N-alkyl-N, N-dimethyl-3-ammonio-1-propanesulfonate (zwittergent), alkane sulfonate, alkylsulfate, alkyl-beta-D-glucopyranoside, alkyl-beta-D-maltoside, dodecanoyl-N-methylglucamide, polyethylene glycol monoalkyl ether and Triton X-100, all produce a concentration-dependent acceleration of the slow passive transbilayer movement of NBD-labeled phosphatidylcholine in the human erythrocyte membrane. Above a threshold concentration, which was well below the CMC and characteristic for each detergent, the flip rate increases exponentially upon an increase of the detergent concentration in the medium. The detergent-induced flip correlates with reported membrane-expanding effects of the detergents at antihemolytic concentrations. From the dependence of the detergent concentration required for a defined flip acceleration on the estimated membrane volume, membrane/water partition coefficients for the detergents could be determined and effective detergent concentrations in the membrane calculated. The effective membrane concentrations are similar for most types of detergents but are 10-fold lower for octaethylene glycol monoalkyl ether and Triton X-100. The effectiveness of a given type of detergent is rather independent of its alkyl chain length. Since detergents do not reduce the high temperature dependence of the flip process the detergent-induced flip is proposed to be due to an enhanced probability of formation of transient hydrophobic structural defects in the membrane barrier which may result from perturbation of the interfacial region of the bilayer by inserted detergent molecules.     

Adsorption of single chain zwitterionic phosphocholine surfactants: effects of length of alkyl chain and head group linker

The adsorption of a range of single chain zwitterionic phosphocholine surfactants (C(n)P(m)C) at the air/liquid interface has been studied by a combination of surface tension and neutron reflectivity. The critical micellar concentration (CMC) for C(n)PC (or C(n)P(2)C), where n varied from 12, 14 to 16, was found to be 0.91, 0.14, and 1.2 x 10(-2) mM respectively, and followed the same trend as observed for other zwitterionic and non-ionic surfactants. The area per molecule at the CMC, A(cmc), for C(n)PC was found to remain constant between 50 and 53 A(2), indicating that the increase in the alkyl chain length had little effect on A(cmc) at the interface. The neutron reflection measurement also showed an almost constant layer thickness (tau) of 20+/-2 A from all the alkyl chain deuterated PC surfactants (dC(n)hPC) in null reflecting water (NRW), suggesting that the alkyl chains of the surfactant responded to changes in either chain length or solution concentration by varying their angle of tilt. In contrast, increasing the length of head group linker between P and N atoms in C(12)P(m)C, where m=2, 4, to 6, resulted in a much slower decrease of CMC from 0.91, 0.7, to 0.5 mM, consistent with a different contribution to the free energy of micellization. A(cmc) for C(12)P(m)C did not vary when m was increased from 2 to 4, and this observation together with the thickness of the head group region indicated an almost perpendicular projection of the head group in C(12)P(2)C and C(12)P(4)C. A further increase in m to 6 resulted in an A(cmc) of 70 A(2). This increase in A(cmc) however did not result in any change in either the total layer thickness or the fraction of the head group region submerged in the aqueous subphase, suggesting that the head group in C(12)P(6)C was bent away from the surface normal direction. Both increase in temperature from 25 to 40 degrees C and the addition of 0.1 M NaCl had little effect on the area per molecule or the thickness of C(12)P(m)C surfactant layer, showing that the C(12)P(m)C series behaved like C(n)P(2)C series. The main conclusion from this study is that for all the C(n)P(m)C surfactants studied, change in m or n has little effect on the total thickness, the thickness of the alkyl chain or that of the head group region.     

Design and synthesis of novel antimicrobials with activity against Gram-positive bacteria and mycobacterial species,including M. tuberculosis

The alarming increase in bacterial resistance over the last decade along with a dramatic decrease in new treatments for infections has led to problems in the healthcare industry. Tuberculosis (TB) is caused mainly by Mycobacterium tuberculosis which is responsible for 1.4 million deaths per year. A world-wide threat with HIV co-infected with multi and extensively drug-resistant strains of TB has emerged. In this regard, herein, novel acrylic acid ethyl ester derivatives were synthesized in simple, efficient routes and evaluated as potential agents against several Mycobacterium species. These were synthesized via a stereospecific process for structure activity relationship (SAR) studies. Minimum inhibitory concentration (MIC) assays indicated that esters 12, 13, and 20 exhibited greater in vitro activity against Mycobacterium smegmatis than rifampin, one of the current, first-line anti-mycobacterial chemotherapeutic agents. Based on these studies the acrylic ester 20 has been developed as a potential lead compound which was found to have an MIC value of 0.4 μg/mL against Mycobacterium tuberculosis. The SAR and biological activity of this series is presented; a Michael-acceptor mechanism appears to be important for potent activity of this series of analogs.     

Chlamydomonas reinhardtii-expressed multimer of ToAMP4 inhibits the growth of bacteria of both Gram-positive and Gram-negative

The antimicrobial peptide ToAMP4 was isolated from Taraxacum officinale Wigg. flowers. Bacterial-expressed ToAMP4 showed antifungal activity but was inactive against bacteria. In this study, Chlamydomonas reinhardtii was used to express a C-terminal hemagglutinin (HA) and 6×His double tagged three repeats of ToAMP4 (3×ToAMP4)and the 3×ToAMP4-expressing cells were screened to express 3×ToAMP4 stably following continuous passaging for six months. Finally, a yield of 0.32 % of total soluble protein was achieved for 3×ToAMP4, which inhibits the growth of bacteria of both Gram-positive and Gram-negative at a minimum inhibitory concentration between 40 and 50 μg/ml. C. reinhardtii-derived 3×ToAMP4 maintained high stability in a wide range of temperature and pHs, tolerated to protease digestion at different extents and showed low hemolytic activity and cytotoxicity. Moreover, C. reinhardtii-expressed 3×ToAMP4 effectively caused damage on the membrane of targeted bacterial cells. In conclusion, our data show that C. reinhardtii has the potential to be used as a platform for the production of bioactive ToAMP4.     

Preparation of the pentasaccharide hapten of the GPL of Mycobacterium avium serovar 19 by achieving the glycosylation of a tertiary hydroxyl group

The chemical synthesis of the glycopeptidolipid-type pentasaccharide hapten of Mycobacterium avium serovar 19 with a trifluoroacetamido spacer at the reducing end is described. The spacer-armed pentasaccharide 31, when conjugated to an immunogenic protein, can be applied to the serodiagnosis of mycobacterial infections. The questionable structure of the penultimate monosaccharide unit was clarified as 6-deoxy-3-C-methyl-2,4-di-O-methyl-L-mannopyranose. The occurrence of the 6-deoxy-3-C-methyl-2,4-di-O-methyl-L-talopyranose could be excluded by the presence of the large H-1'-H-2' coupling constant, which proves the 4C1 (L) conformation as the favoured one.