Sporulation as an effective strategy for the survival of bacteria in a biofilter with discontinuous operation

The microbial population of the biofilter developed for the treatment of air contaminated by solvent vapours was evaluated using plate count techniques. The number of total heterotrophic bacteria and bacteria utilizing contaminant as the only source of carbon and energy was estimated during the exchange of the filter-bed material and start-up of its operation. Spore-forming bacteria (Bacillus spp.) occupied a significant part of the filter-bed niche. It is proposed that sporulation helps bacteria to survive during the breaks in operation of the biofilter.     

Biochemical, crystallographic, and mutagenic characterization of hint, the AMP-lysine hydrolase, with novel substrates and inhibitors

Hint, histidine triad nucleotide-binding protein, is a universally conserved enzyme that hydrolyzes AMP linked to lysine and, in yeast, functions as a positive regulator of the RNA polymerase II C-terminal domain kinase, Kin28. To explore the biochemical and structural bases for the adenosine phosphoramidate hydrolase activity of rabbit Hint, we synthesized novel substrates linking a p-nitroaniline group to adenylate (AMP-pNA) and inhibitors that consist of an adenosine group and 5'-sulfamoyl (AdoOSO(2)NH(2)) or N-ethylsulfamoyl (AdoOSO(2)NHCH(2)CH(3)) group. AMP-pNA is a suitable substrate for Hint that allowed characterization of the inhibitors; titration of each inhibitor into AMP-pNA assays revealed their K(i) values. The N-ethylsulfamoyl derivative has a 13-fold binding advantage over the sulfamoyl adenosine. The 1.8-A cocrystal structure of rabbit Hint with N-ethylsulfamoyl adenosine revealed a binding site for the ethyl group against Trp-123, a residue that reaches across the Hint dimer interface to interact with the alkyl portion of the inhibitor and, presumably, the alkyl portion of a lysyl substrate. Ser-107 is positioned to donate a hydrogen bond to the leaving group nitrogen. Consistent with a role in acid-base catalysis, the Hint S107A mutant protein displayed depressed catalytic activity.     

Higher expression of Fab antibody fragments in a CHO cell line at reduced temperature

A chimeric Fab was expressed in Chinese hamster ovary cells under the control of the CMV promoter in a two-stage production process. Cells were first grown to 90% confluence at 37 degrees C in a proliferation phase, followed by a production phase at either 37 degrees C or 28 degrees C. Medium supplemented with serum and medium free from serum was tested in the production phase at both temperatures. Comparison of Fab expression revealed that reducing the temperature to 28 degrees C resulted in a 14-fold increase in product yield when cells were cultivated in serum-containing medium, and in a 38-fold increase in product yield when serum-free medium was applied.     

Mode of action of family 10 and 11 endoxylanases on water-unextractable arabinoxylan

Microbial endo-beta-1,4-xylanases (EXs, EC 3.2.1.8) belonging to glycanase families 10 and 11 differ in their action on water-unextractable arabinoxylan (WU-AX). WU-AX was incubated with different levels of a Thermoascus aurantiacus family 10 and a Sporotrichum thermophile family 11 endoxylanases. At 10 g l(-1) arabinoxylan, enzyme concentrations (KE values) needed to obtain half-maximal hydrolysis rates (V(max) values) were 4.4 nM for the xylanase from T. aurantiacus and 7.1 nM for the xylanase from S. thermophile. Determination of Vmax/KE revealed that the family 10 enzyme hydrolysed two times more efficiently WU-AX than the family 11 enzyme. Molecular weights of the products formed were assessed and separation of feruloyl-oligosaccharides was achieved by anion-exchange and size-exclusion chromatography (SEC). The main difference between the feruloylated products by xylanases of family 10 and 11 concerned the length of the products containing feruloyl-arabinosyl substitution. The xylanase from T. aurantiacus liberated from WU-AX a feruloyl arabinoxylodisaccharide (FAX2) as the shortest feruloylated fragment in contrast with the enzyme from S. thermophile, which liberated a feruloyl arabinoxylotrisaccharide (FAX3). These results indicated that different factors govern WU-AX breakdown by the two endoxylanases.     

Attenuation of blood parameters in smokers and non-smokers after intake of a complex food additive

This report describes an intervention study with healthy volunteers (20 smokers, 28 non-smokers) taking a food additive mainly containing vitamin C (ascorbic acid), vitamin E (alpha-tocopherol), ubiquinone (Q10), vitamin A and zinkoxide for four weeks in a double blind, randomized and placebo controlled manner. Before and after the intervention blood was withdrawn and general blood parameters were analyzed. In addition, lipid soluble antioxidants were analyzed in blood plasma by HPLC and the water soluble antioxidative properties were tested with the enzymic xanthin/xanthinoxidase-reaction. In summary the results show that the smoker-verum group exhibit a significant down regulation of the leukocyte counts. The test for antioxidants show the following significant differences after intervention: Smokers exhibit an increase of both vitamin E and coenzyme Q10 and an attenuation of their (before intervention) clearly increased water soluble-antioxidative potential, non-smokers showed only an increase of vitamin E and trends of an increase of Q10 and water soluble-antioxidative potential. These results may contribute to the discussion of the intrinsic deficiency brought about by smoking and the possible attenuation of part of these deficiency by increasing the intake of certain vitamins or food additives.     

Biochemical function of female-lethal (2)D/Wilms' tumor suppressor-1-associated proteins in alternative pre-mRNA splicing

Genetic and molecular data have implicated the Drosophila gene female-lethal (2)d (fl (2)d) in alternative splicing regulation of genes involved in sexual determination. Sex-specific splicing is under the control of the female-specific regulatory protein sex-lethal (SXL). Co-immunoprecipitation and mass spectrometry results indicate that SXL and FL (2)D form a complex and that the protein VIRILIZER and a Ran-binding protein implicated in protein nuclear import are also present in complexes containing FL (2)D. A human homolog of FL (2)D was identified and cloned. Interestingly, this gene encodes a protein (WTAP) that was previously found to interact with the Wilms' tumor suppressor-1 (WT1), an isoform of which binds to and co-localizes with splicing factors. Alternative splicing of transformer pre-mRNA, a target of SXL regulation, was affected by immunodepletion of hFL (2)D/WTAP from HeLa nuclear extracts, thus arguing for a biochemical function of FL (2)D/WTAP proteins in splicing regulation.     

Imprinting and disease

Deregulation of imprinted genes has been observed in a number of human diseases such as Beckwith-Wiedemann syndrome, Prader-Willi/Angelman syndromes and cancer. Imprinting diseases are characterised by complex patterns of mutations and associated phenotypes affecting pre- and postnatal growth and neurological functions. Regulation of imprinted gene expression is mediated by allele-specific epigenetic modifications of DNA and chromatin. These modifications preferentially affect central regulatory elements that control in cis over long distances allele-specific expression of several neighbouring genes. Investigations of imprinting diseases have a strong impact on biomedical research and provide interesting models for function and mechanisms of epigenetic gene control.     

Staphylococcal alpha-toxin provokes neutrophil-dependent cardiac dysfunction: role of ICAM-1 and cys-leukotrienes

The role of polymorphonuclear neutrophils (PMN) in septic myocardial dysfunction is presently unknown. Staphylococcus aureus infections are frequently associated with septic sequelae. Therefore, we perfused isolated rat hearts with low doses of alpha-toxin, the major staphylococcal exotoxin, followed by application of human PMN, N-formyl-methionyl-leucyl-phenylalanine, and arachidonic acid. In contrast to sham-perfused hearts (no alpha-toxin), a rise in coronary perfusion pressure (CPP) and a reduction of contractile function were noted, and cardiac expression of intercellular adhesion molecule (ICAM)-1 was detected by immunohistochemical methods and real-time PCR. Histological analysis and myeloperoxidase activity indicated cardiac PMN accumulation in alpha-toxin-challenged hearts. Major quantities of cysteinyl (cys)-leukotrienes (LT), LTB4, and 5-hydroxyeicosatetraenoic acid (5-HETE) were found in the perfusate of alpha-toxin-exposed hearts. With an anti-ICAM-1 antibody, neutrophil accumulation, leukotriene (LT) synthesis, coronary vasoconstriction, and the accompanying cardiodepression were suppressed. Similarly, the lipoxygenase inhibitor MK-886 blocked LT synthesis and maintained cardiac function. We conclude that low-dose alpha-toxin provokes coronary endothelial ICAM-1 expression and neutrophil accumulation, with subsequent synthesis of cys-LTs, LTB4, and 5-HETE under conditions of appropriate stimulation. This response is linked with coronary vasoconstriction and contractile dysfunction, with cys-LT synthesis and maldistribution of perfusion offered as likely underlying mechanisms.     

A specific binding site for a fragment of the B-loop of epidermal growth factor and related peptides

Fragments of the B-loop of the epidermal growth factor family of peptides are reported to have mitogenic and angiogenic properties but appear to fail to compete with radioiodinated EGF in receptor binding. In this study, 11 analogs of a fragment of the B-loop of EGF-related peptides from several species were synthesized to study binding to A431 human epidermoid carcinoma using both 125I-EGF and [3'4'-3H-Tyr(22,29), Abu(20,31)]EGF(20-31)-NH(2). Specific binding sites were found for the human fragment and 8 analogs at a density five times higher than that of the EGF receptors. Analogs did not compete with 125I-EGF for binding to the EGF receptor. The novel binding site may mediate the biological effects of the fragments. The primary rather than secondary structure of the fragments appears to determine affinity.     

Isolation of a Bacterial Strain with the Ability To Utilize the Sulfonated Azo Compound 4-Carboxy-4′-Sulfoazobenzene as the Sole Source of Carbon and Energy

A bacterial strain (strain S5) which grows aerobically with the sulfonated azo compound 4-carboxy-4′-sulfoazobenzene as the sole source of carbon and energy was isolated. This strain was obtained by continuous adaptation of “Hydrogenophaga palleronii” S1, which has the ability to grow aerobically with 4-aminobenzenesulfonate. Strain S5 probably cleaves 4-carboxy-4′-sulfoazobenzene reductively under aerobic conditions to 4-aminobenzoate and 4-aminobenzene-sulfonate, which are mineralized by previously established degradation pathways.It is generally assumed that sulfonated azo dyes are not degraded under aerobic conditions (14). Nevertheless, there have been some reports which suggest a conversion of certain sulfonated azo dyes under aerobic conditions (3, 7, 8, 13, 15). Furthermore, certain carboxylated analogs of sulfonated azo compounds are utilized aerobically as the sole source of carbon and energy by specifically adapted bacteria (11, 12, 16, 17). However, unequivocal evidence for the productive mineralization of a sulfonated azo compound by bacteria is lacking. In the present article the first observation of the utilization of a sulfonated azo compound as the sole source of carbon and energy by a bacterial strain is reported.Previously, a mixed bacterial culture which mineralizes sulfanilate (4-aminobenzenesulfonate) was isolated. This coculture consisted of the strains “Hydrogenophaga palleronii” S1 and Agrobacterium radiobacter S2 (4, 5). Because sulfanilate occurs as an azoaryl structural element in many azo dyes, it was of interest whether this mixed culture could adopt the ability to reduce azo bonds and release sulfanilate as growth substrate. Therefore, the model sulfonated azo compound 4-carboxy-4′-sulfoazobenzene (CSAB) was synthesized by nitro-amine condensation starting with sulfanilic acid and 4-nitrobenzoic acid (1). The precipitated CSAB was separated from the reaction mixture by filtration and purified by repeated dissolution in alkali and precipitation with acid. The identity and purity of the bright orange product were analyzed by UV-visible light spectroscopy, elementary analysis, and high-pressure liquid chromatography (HPLC). For the solid material obtained, molar extinction coefficients of 23.74 and 1.13 mM⁻¹ cm⁻¹ in water were determined at the wavelengths of 326 and 434 nm, respectively. The elementary analytic results were consistent with the structure of CSAB. The purity of the preparation was tested by HPLC with a reversed-phase column and a solvent gradient from 1 to 90% (vol/vol) methanol and 0.3% (vol/vol) H₃PO₄. A single band which showed absorbance at a wavelength of 326 nm was eluted. At 210 nm a minor contaminant (about 15% of the signal intensity of CSAB) was detected. This compound was clearly different from either 4-nitrobenzoate or sulfanilate.The mixed culture was grown in repeated batch cultures in a mineral medium with sulfanilate (5 mM). About every 2 weeks the culture was transferred (1:10 [vol/vol]) to fresh medium, in which the sulfanilate concentration was subsequently reduced and the CSAB concentration increased (±0.5 mM each). The color of the azo dye disappeared after 2 months. The culture was transferred to a solid mineral medium with CSAB as the sole source of carbon. From this culture was obtained strain S5, which grew aerobically with the sulfonated azo compound CSAB as its sole source of carbon and energy and with a doubling time of 9.5 h (Fig. ​(Fig.1).1). The complete disappearance of the dye was demonstrated by the loss of the orange color from the medium and by HPLC analysis, whereas CSAB was not degraded in a sterile control flask. Based on its colony morphology and the results obtained with the commercial identification system Biolog GN, this strain strongly resembled “H. palleronii” S1. Recently, it was demonstrated that, in the presence of low concentrations of biotin, cyanocobalamin, and 4-aminobenzoate, strain S1 also grows in axenic culture with sulfanilate (2). Therefore the adaptation experiment was repeated in the presence of these three substances with a pure culture of strain S1. This experiment also resulted in the isolation of a strain which grew in axenic culture with CSAB as the sole source of carbon and energy. Open in a separate windowFIG. 1Aerobic growth of strain S5 with CSAB as the sole source of carbon and energy. The growth was determined photometrically (OD₅₄₆), and the turnover of CSAB was measured by HPLC with a reversed-phase column and a solvent gradient consisting of H₂O, methanol, and 0.3% H₃PO₄ with increasing concentrations of methanol (1 to 90%). An OD₅₄₆ of 1 corresponded to 0.33 mg of protein ml⁻¹.To ensure that the genetic backgrounds of strains S5 and S1 were identical, the genes for the 16S rRNAs were amplified by PCR with different universal primers (6) and sequenced in comparison to the corresponding gene from the type strain, H. palleronii DSM 63. It was found that the sequences from strains S1 and S5 were > 99.8% identical (there were only two discrepancies between the two sequences), but they showed only 97.7 to 97.9% identity with the 16S rRNA gene from H. palleronii DSM 63. It was therefore concluded that strain S5 was derived from strain S1 and that the strains do not belong to the species H. palleronii.A reductive cleavage of the azo bond of CSAB would result in the formation of 4-aminobenzoate and sulfanilate. Like the parent strain, S1, strain S5 grew in the presence of sulfanilate, 4-aminobenzoate, and 4-sulfocatechol. The doubling times with these compounds were 6.2 to 6.4 h. We therefore investigated whether reductive cleavage of CSAB by strain S5 occurs. Strain S5 was grown aerobically with 5 mM CSAB, and cell extracts were prepared (10) in different buffers. These cell extracts were incubated aerobically in cuvettes containing 50 mM Tris-HCl buffer (pH 8.0), 0.5 mM CSAB, 1 mM NADH, or 1 mM NADPH and with various mixtures of possible cofactors. The enzyme activity was measured spectrophotometrically at the absorption maximum for CSAB (at a wavelength of 434 nm), but no significant decrease in absorbance was observed. Neither addition of a membrane fraction nor performing the enzyme assays under anaerobic conditions (9) improved the turnover of CSAB in the cell-free system. Furthermore, there was no significant increase in azo reductase activity when harvested cells were resuspended in the culture supernatant instead of Tris-HCl buffer.The maximal enzyme activities observed for cell extracts were only about 30% of the activities found for intact cells. This suggested that during the disruption of the cells some important components of the azo reductase system were destroyed or some cofactors were present in only limiting quantities.Because it was difficult to obtain reproducible enzyme activities with cell extracts, the turnover of CSAB by resting cells was investigated. Cells of strain S5 were grown with CSAB (5 mM), harvested by centrifugation, resuspended in Tris-HCl at an optical density at 546 nm (OD₅₄₆) of 5.3, and incubated in a water bath shaker (140 rpm; 30°C) with 0.5 mM CSAB (Fig. ​(Fig.2).2). Thus, the transient accumulation of two metabolites in the supernatants was observed by reversed-phase HPLC (column size, 250 by 4.6 mm) (SIL 100; Grom, Herrenberg, Germany). The solvent system consisted of a solvent gradient with increasing concentrations of methanol, starting with 1% (vol/vol) methanol, 98.9% (vol/vol) water, and 0.1% H₃PO₄. The flow rate was 0.7 ml min⁻¹. The metabolites formed were identified as sulfanilate and 4-sulfocatechol by comparison of their retention times and in situ UV-visible light-spectra with authentic standards. Surprisingly, the concentration of 4-sulfocatechol in the medium increased (and decreased) during the experiment more rapidly than the concentration of sulfanilate (Fig. ​(Fig.2).2). 4-Sulfocatechol also temporarily accumulated when resting cells of strain S1 were incubated with sulfanilate (4, 5). This suggested that in the resting-cell assay the initial activity of the sulfanilate-converting enzyme was higher than the activity of the 4-sulfocatechol-oxidizing enzyme protocatechuate-3,4-dioxygenase type II. Presumably, the activity of the sulfanilate-converting enzyme decreased during the experiment more rapidly than the activity of protocatechuate-3,4-dioxygenase type II. No accumulation of 4-aminobenzoate or protocatechuate was found by HPLC analysis during the experiment. In a control experiment with cells of strain S1 grown with 4-aminobenzenesulfonate, no turnover of CSAB was observed by HPLC analysis. Open in a separate windowFIG. 2Conversion of CSAB (•) to sulfanilate (▪) and 4-sulfocatechol (□) by resting cells of strain S5. Strain S5 was grown in a mineral medium with CSAB as the sole source of carbon and energy, and resting cells were prepared as described in the text.The detection of sulfanilate derived from CSAB suggested a reductive cleavage of CSAB, yielding sulfanilate as one of the reduction products. This reaction should also proceed in the absence of oxygen. Therefore, resting cells were incubated under anaerobic conditions with CSAB. Surprisingly, the rate of CSAB turnover under anaerobic conditions was <2% of the turnover rate under aerobic conditions.A further indication of a reductive cleavage of CSAB into sulfanilate and 4-aminobenzoate was obtained by growing strain S5 with CSAB or a complex medium (HPG medium) (4). When the cells were grown in a mineral medium with CSAB and the turnover of the substrates was analyzed by HPLC, it was found that resting cells converted CSAB, 4-aminobenzoate, or 4-aminobenzenesulfonate with specific activities of 0.012, 0.026, and 0.011 μmol min⁻¹ mg of protein⁻¹, respectively. In contrast, after growth of the cells in HPG medium, these activities were only 0.007, 0.010, and 0.003 μmol min⁻¹ mg of protein⁻¹, respectively. Incubation of resting cells with CSAB and different potential inhibitors of ring cleavage dioxygenases showed that the turnover of CSAB was almost completely inhibited by the addition of 8-hydroxyquinoline or 2,2′-bipyridyl (1 mM each). The presence of 4-nitrocatechol (0.25 mM) also resulted in a pronounced reduction of the rate of CSAB turnover (6% of the rate in the absence of the inhibitor). In this system as well the formation of 4-sulfocatechol was observed.The degradation of sulfanilate and 4-aminobenzoate by strain S1 has been previously studied (5). The proposed degradation pathway for CSAB and its reduction products is shown in Fig. ​Fig.3.3. Open in a separate windowFIG. 3Proposed pathway for the degradation of CSAB by strain S5. 4AB, 4-aminobenzoate; 4ABS, 4-aminobenzenesulfonate (sulfanilate); 3,4DHB, 3,4-dihydroxybenzoate (protocatechuate); 4SC, 4-sulfocatechol; 2H4CMSA, 2-hydroxy-4-carboxymuconic semialdehyde; 3SM, 3-sulfomuconate; 4SL, 4-carboxymethyl-4-sulfobut-2-en-4-olide (4-sulfolactone); MA, maleylacetate; 3OA, 3-oxoadipate; TCC, tricarboxylic acid cycle.To obtain some information about the substrate specificity, resting cells were incubated with CSAB, 4,4′-dicarboxyazobenzene (DCAB), 4-hydroxy-4′-sulfoazobenzene, methyl orange [4-(N,N-dimethyl)-4′-sulfoazobenzene; color index (C.I.) 13025], orange II {4-[(2-hydroxy-1-naphthalenyl)azo]-benzenesulfonic acid; C.I. 15510}, or sunset yellow FCF {6-hydroxy-5-[(4-sulfophenyl)azo]-2-naphthol-6-sulfonic acid; FD&C no. 6; C.I. 15985}. Of these compounds, only CSAB and DCAB were converted by resting cells. DCAB was also utilized by strain S5 as the sole source of carbon and energy. Furthermore, no growth of strain S5 was found with acid black 24 and 52, acid blue 113, acid red 1, amaranth, direct red 81, direct yellow 4 and 50, mordant yellow 3, and naphthol blue black.The results presented in this study suggest that bacterial cultures with the ability to aerobically degrade simple sulfonated azo dyes may be obtained after preadaptation to sulfonated aminoaromatics and/or when reductive cleavage of the azo bond gives rise to an aerobically assimilable aminoaromatic structure, like 4-aminobenzoate. This selection scheme circumvents the problems observed during attempts to adapt bacteria with the ability to degrade carboxylated azo compounds for the degradation of sulfonated azo compounds (12). The ability of strain S5 to mineralize CSAB suggests that it is possible to degrade sulfonated azo dyes under aerobic conditions if biological systems which can grow and can mineralize the reduction products are available.

Nucleotide sequence accession number.

The nucleotide sequences for the 16S rRNAs from strains S5 and S1 have been deposited in the GenBank data library under accession no. {"type":"entrez-nucleotide","attrs":{"text":"AF019037","term_id":"3004497","term_text":"AF019037"}}AF019037 and {"type":"entrez-nucleotide","attrs":{"text":"AF019073","term_id":"3004495","term_text":"AF019073"}}AF019073, respectively.