With this method it was shown that the covalent binding of trypsin to CNBr-activated Sephadex sta... more With this method it was shown that the covalent binding of trypsin to CNBr-activated Sephadex stabilized the protein against the denaturing effect of concentrated urea solutions, in contradistinction to the similar agarose-bound trypsin, which behaved as the free trypsin. This agrees with activity measurements (Gabel et al., 1970), the latter derivative being inactive in 8M-urea, as is the free enzyme, whereas Sephadex-bound trypsin retains most of its activity even after long storage in urea solutions. The stabilization of the active surface of trypsin by Ca , found for the free enzyme (Sipos & Merkel, 1970), was not observed with the Sephadex-bound enzyme. The protein in solution unfolds at 60°C in the presence of Ca2+, as reflected by a red shift of the tryptophan fluorescence, whereas the fluorescence properties of Sephadex-bound trypsin did not change up to 70°C. At that temperature the fluorescence intensity increased, but no red shift was observable. This increase coincided with the loss of activity. It is suggested that the temperature-induced conforma-tional transitions that inactivate trypsin in the absence and in the presence of Ca2+, as well as the Ca2+_ dependent transition leading from native trypsin to a more compact structure (Sipos & Merkel, 1970), are not possible in Sephadex-bound trypsin owing to a more-or-less extensive cross-linking between the protein and the rigid carrier. Attempts were made to identify those c-amino groups that contribute to the stabilization of Sephadex-bound trypsin against denaturing influences.
Bacillus subtilis responds to phosphate starvation stress by inducing the PhoP and SigB regulons.... more Bacillus subtilis responds to phosphate starvation stress by inducing the PhoP and SigB regulons. While the PhoP regulon provides a specific response to phosphate starvation stress, maximizing the acquisition of phosphate (P i) from the environment and reducing the cellular requirement for this essential nutrient, the SigB regulon provides nonspecific resistance to stress by protecting essential cellular components, such as DNA and membranes. We have characterized the phosphate starvation stress response of B. subtilis at a genome-wide level using DNA macroarrays. A combination of outlier and cluster analyses identified putative new members of the PhoP regulon, namely, yfkN (2,3 cyclic nucleotide 2-phosphodiesterase), yurI (RNase), yjdB (unknown), and vpr (extracellular serine protease). YurI is thought to be responsible for the nonspecific degradation of RNA, while the activity of YfkN on various nucleotide phosphates suggests that it could act on substrates liberated by YurI, which produces 3 or 5 phosphoribonucleotides. The putative new PhoP regulon members are either known or predicted to be secreted and are likely to be important for the recovery of inorganic phosphate from a variety of organic sources of phosphate in the environment.
Applied and Environmental Microbiology, Feb 1, 2002
The bacteria on the surface of a farmhouse smear-ripened cheese at four stages of ripening (4, 16... more The bacteria on the surface of a farmhouse smear-ripened cheese at four stages of ripening (4, 16, 23, and 37 days) from inoculated (i.e., deliberately inoculated with Brevibacterium linens BL2) and noninoculated (not deliberately inoculated with B. linens BL2) cheese were investigated. The results show that, contrary to accepted belief, B. linens is not a significant member of the surface flora of smear cheese and no microbial succession of species occurred during the ripening of the cheeses. Of 400 isolates made, 390 were lactate-utilizing coryneforms and 10 were coagulase-negative Staphylococcus spp. A detailed analysis of the coryneforms was undertaken using phenotypic analysis, molecular fingerprinting, chemotaxonomic techniques, and 16S rRNA gene sequencing. DNA banding profiles (ramdom amplified polymorphic DNA [RAPD]-PCR) of all the coryneform isolates showed large numbers of clusters. However, pulsed-field gel electrophoresis (PFGE) of the isolates from the cheeses showed that all isolates within a cluster and in many contiguous clusters were the same. The inoculated and noninoculated cheeses were dominated by single clones of novel species of Corynebacterium casei (50.2% of isolates), Corynebacterium mooreparkense (26% of isolates), and Microbacterium gubbeenense (12.8% of isolates). In addition, five of the isolates from the inoculated cheese were Corynebacterium flavescens. Thirty-seven strains were not identified but many had similar PFGE patterns, indicating that they were the same species. C. mooreparkense and C. casei grew at pH values below 4.9 in the presence of 8% NaCl, while M. gubbeenense did not grow below pH 5.8 in the presence of 5 to 10% NaCl. B. linens BL2 was not recovered from the inoculated cheese because it was inhibited by all the Staphylococcus isolates and many of the coryneforms. It was concluded that within a particular batch of cheese there was significant bacterial diversity in the microflora on the surface.
The human derma emits volatile compounds whose interaction with a receiver's olfactory sensory sy... more The human derma emits volatile compounds whose interaction with a receiver's olfactory sensory system may affect individual recognition and mating preferences. Studies suggest that both genes and environmental factors determine characteristic odor of an individual. Here, we use solid phase micro-extraction and GC-MS to identify 3-methylbutanal (3-MB) in human axillary odor, show that the abundance of this volatile compound varies significantly between individuals and demonstrate in-vitro that its formation may be influenced by interaction between human leukocyte antigen peptide and dermal microflora.
A culture-independent analysis of the bacterial assemblages in the sediments of a lugworm Arenico... more A culture-independent analysis of the bacterial assemblages in the sediments of a lugworm Arenicola marina L. bioturbated mesocosm was carried out using a molecular RNA-gene based approach. Clone libraries were constructed for 4 spatial locations in the mesocosm: deep yeast layer (DYL), mid sand (MS), new casts (NC) and old casts (OC) after 29 d of bioturbation. In total, 495 clones were analysed and grouped into 159 unique phylotypes. All libraries were dominated by Epsilonproteobacteria, with Alpha-, Delta-and Gammaproteobacteria, Verrucomicrobia and members of the Cytophaga-Flavobacteria-Bacteroides group also prevalent. Phyologenetic comparison of the sites with respect to evolutionary distance showed that the libraries fell into 2 groups: (1) DYL and MS and (2) NC and OC. There were significant differences in species composition between but not within the 2 groupings. Bacteria involved with sulphur cycling are most likely to be affected by lugworm bioturbation. An Epsilonproteobacterium phylotype with > 95% 16S similarity to Sulfurimonas denitrificans was abundant in the DYL sand layer but was not present in the egested sand (NC, OC). Promotion of the growth of sulphide-oxidising bacteria and their subsequent digestion would be consistent with the 'gardening' hypothesis and suggests that chemoautotrophic processes in shallow-water anaerobic sediments may be enhanced by the activities of the lugworm.
Large numbers of filamentous actinomycetes which formed distinctive red coloured colonies were is... more Large numbers of filamentous actinomycetes which formed distinctive red coloured colonies were isolated from three out of four composite soil samples using a medium designed to be selective for members of the Streptomyces violaceoruber clade, a taxon which includes the model organisms ''Streptomyces coelicolor'' A3(2) and ''Streptomyces lividans'' 66. The isolation medium, dextran-histidine-sodium chloride-mineral salts agar supplemented with antibacterial and antifungal antibiotics, also supported the growth of representatives of the S. violaceoruber clade. One hundred and ninety one representatives of the isolates that produced red colour colonies on the isolation medium were distributed into four colour groups based on their ability to form distinctive pigments and morphological properties typical of members of the S. violaceoruber clade, an assignment that was confirmed by corresponding 16S rRNA gene sequencing studies. The selective isolation and characterisation procedures used in the present investigation provide a practical means of determining the taxonomic diversity, geographical distribution and roles of representatives of the S. violaceoruber clade in natural habitats.
Filamentous bacteria belonging to the genus Thiothrix were detected in activated sludge samples u... more Filamentous bacteria belonging to the genus Thiothrix were detected in activated sludge samples using the fluorescent in situ hybridisation (FISH) technique. A 16S rRNA-targeted oligonucleotide probe was developed for the detection of members of the T. fructosivorans group, and the performance of probe TNI for the detection of Thiothrix nivea group was enhanced by using an unlabeled competitor. A set of 5 probes covering all phylogenetic groups of Thiothrix were used to examine samples taken from selected activated sludge plants treating paper and board mill wastes. Members of the T. eikelboomii group formed the predominant filamentous bacterial population in plants experiencing poor sludge settleability, whereas members of the T. nivea group were commonly found but not dominantly in the remaining plants. Members of the T. fructosivorans group were not detected at any significant level in any of the samples. The distribution of the main Thiothrix types remained unchanged throughout ...
Twenty-five representative isolates from a collection of 54 indigenous actinomycetes isolated fro... more Twenty-five representative isolates from a collection of 54 indigenous actinomycetes isolated from coastal resources, were evaluated for inhibition against plant pathogenic fungi in vitro. Fusarium oxysporum f.sp. cubense (Foc) race 4 and Rhizoctonia solani exhibited higher resistance to the antifungal effects of the isolates when compared to the other test fungi. Seven isolates with potential pathogen-inhibitory capabilities belonged to the gray colour class of Streptomyces. Isolate g10 with a wide and strong antifungal spectrum against a variety of pathogenic fungi belonged to the Streptomyces violaceusniger cluster. This isolate could be a potential biological control agent.
Polyunsaturated fatty acids (PUFAs) are chemically present as esters, ethers, glycerides glycolip... more Polyunsaturated fatty acids (PUFAs) are chemically present as esters, ethers, glycerides glycolipids, phospholipids, phosphonolipids, glycosphingolipids, sulpholipids and lipoproteins in storage oils and membranes lipids. Marine microorganisms such as Shewanella frigidimarina are important sources of polyunsaturated fatty acids with promising biomedical applications, commercial value and the potential ability to be used in the bioremediation of environments contaminated with petroleum hydrocarbons. The growth media dependency of S. frigidimarina in terms of its growth behavior in response to modifications made to the media as well as its potential to produce PUFAs was evaluated. S. frigidimarina was cultured in conventional shake-flasks and controlled bioreactors with a batch-type procedure using different media compositions. The media used included artificial sea water, modified artificial sea water 1, modified artificial sea water 2, Luria Bertani, modified Luria Bertani, sodium p...
A screening method was established to detect inhibitors of the biosynthetic pathways of aromatic ... more A screening method was established to detect inhibitors of the biosynthetic pathways of aromatic amino acids and para-aminobenzoic acid, the precursor of folic acid, using an agar plate diffusion assay modified as an antagonism test. By this screening method, a family of three novel polycyclic polyketides named as abyssomicins was isolated from a marine strain of Verrucosispora. The main component abyssomicin C inhibits the pathway between chorismate and para-aminobenzoic acid and is strongly active against Gram-positive bacteria, including multi-resistant clinical isolates of Staphylococcus aureus.
Abstract This paper describes studies on the optimisation of ‘fed-batch’ bioreactors. Two bioreac... more Abstract This paper describes studies on the optimisation of ‘fed-batch’ bioreactors. Two bioreactors are considered: an industrial enzymatic reaction involving a racemic mixture and a combined microbial/enzymatic reaction taken from the literature. Both reactions are of the ‘fed-batch’ type and therefore require optimisation of feed profiles, as well as initial batch concentrations. The Chemotaxis optimisation algorithm is applied to both reactors to specify initial conditions and feed profiles in order to attempt to maximise a pre-specified profit function. This optimisation algorithm, together with the representation of feed profiles by polynomial equations, results in an extremely straightforward procedure for sub-optimal bioreactor operational specification. Although achieving a sub-optimal policy requires an accurate reaction model, the simplicity of the optimisation algorithm enables account to be taken of parameter uncertainty and thus reduced sensitivity to the model inaccuracies.
Abstract Implementation of advanced control strategies in bioprocesses is often hindered by the l... more Abstract Implementation of advanced control strategies in bioprocesses is often hindered by the lack of on-line measurements reflecting the physiological state of the culture. Although a number of techniques have been used to estimate key variables from data monitored on-line, these often do not explicitly take into account changes in physiological state and information on many aspects of physiological state may not be present in on-line data. This paper demonstrates that data obtained from chemical fingerprinting methods, such as Pyrolysis Mass Spectrometry, can be used to identify changes in physiological state during cultivation. This information can be utilised for estimation of the physiological state with on-line data that will enable physiological-state-specific model development for on-line bioprocess control.
With this method it was shown that the covalent binding of trypsin to CNBr-activated Sephadex sta... more With this method it was shown that the covalent binding of trypsin to CNBr-activated Sephadex stabilized the protein against the denaturing effect of concentrated urea solutions, in contradistinction to the similar agarose-bound trypsin, which behaved as the free trypsin. This agrees with activity measurements (Gabel et al., 1970), the latter derivative being inactive in 8M-urea, as is the free enzyme, whereas Sephadex-bound trypsin retains most of its activity even after long storage in urea solutions. The stabilization of the active surface of trypsin by Ca , found for the free enzyme (Sipos & Merkel, 1970), was not observed with the Sephadex-bound enzyme. The protein in solution unfolds at 60°C in the presence of Ca2+, as reflected by a red shift of the tryptophan fluorescence, whereas the fluorescence properties of Sephadex-bound trypsin did not change up to 70°C. At that temperature the fluorescence intensity increased, but no red shift was observable. This increase coincided with the loss of activity. It is suggested that the temperature-induced conforma-tional transitions that inactivate trypsin in the absence and in the presence of Ca2+, as well as the Ca2+_ dependent transition leading from native trypsin to a more compact structure (Sipos & Merkel, 1970), are not possible in Sephadex-bound trypsin owing to a more-or-less extensive cross-linking between the protein and the rigid carrier. Attempts were made to identify those c-amino groups that contribute to the stabilization of Sephadex-bound trypsin against denaturing influences.
Bacillus subtilis responds to phosphate starvation stress by inducing the PhoP and SigB regulons.... more Bacillus subtilis responds to phosphate starvation stress by inducing the PhoP and SigB regulons. While the PhoP regulon provides a specific response to phosphate starvation stress, maximizing the acquisition of phosphate (P i) from the environment and reducing the cellular requirement for this essential nutrient, the SigB regulon provides nonspecific resistance to stress by protecting essential cellular components, such as DNA and membranes. We have characterized the phosphate starvation stress response of B. subtilis at a genome-wide level using DNA macroarrays. A combination of outlier and cluster analyses identified putative new members of the PhoP regulon, namely, yfkN (2,3 cyclic nucleotide 2-phosphodiesterase), yurI (RNase), yjdB (unknown), and vpr (extracellular serine protease). YurI is thought to be responsible for the nonspecific degradation of RNA, while the activity of YfkN on various nucleotide phosphates suggests that it could act on substrates liberated by YurI, which produces 3 or 5 phosphoribonucleotides. The putative new PhoP regulon members are either known or predicted to be secreted and are likely to be important for the recovery of inorganic phosphate from a variety of organic sources of phosphate in the environment.
Applied and Environmental Microbiology, Feb 1, 2002
The bacteria on the surface of a farmhouse smear-ripened cheese at four stages of ripening (4, 16... more The bacteria on the surface of a farmhouse smear-ripened cheese at four stages of ripening (4, 16, 23, and 37 days) from inoculated (i.e., deliberately inoculated with Brevibacterium linens BL2) and noninoculated (not deliberately inoculated with B. linens BL2) cheese were investigated. The results show that, contrary to accepted belief, B. linens is not a significant member of the surface flora of smear cheese and no microbial succession of species occurred during the ripening of the cheeses. Of 400 isolates made, 390 were lactate-utilizing coryneforms and 10 were coagulase-negative Staphylococcus spp. A detailed analysis of the coryneforms was undertaken using phenotypic analysis, molecular fingerprinting, chemotaxonomic techniques, and 16S rRNA gene sequencing. DNA banding profiles (ramdom amplified polymorphic DNA [RAPD]-PCR) of all the coryneform isolates showed large numbers of clusters. However, pulsed-field gel electrophoresis (PFGE) of the isolates from the cheeses showed that all isolates within a cluster and in many contiguous clusters were the same. The inoculated and noninoculated cheeses were dominated by single clones of novel species of Corynebacterium casei (50.2% of isolates), Corynebacterium mooreparkense (26% of isolates), and Microbacterium gubbeenense (12.8% of isolates). In addition, five of the isolates from the inoculated cheese were Corynebacterium flavescens. Thirty-seven strains were not identified but many had similar PFGE patterns, indicating that they were the same species. C. mooreparkense and C. casei grew at pH values below 4.9 in the presence of 8% NaCl, while M. gubbeenense did not grow below pH 5.8 in the presence of 5 to 10% NaCl. B. linens BL2 was not recovered from the inoculated cheese because it was inhibited by all the Staphylococcus isolates and many of the coryneforms. It was concluded that within a particular batch of cheese there was significant bacterial diversity in the microflora on the surface.
The human derma emits volatile compounds whose interaction with a receiver's olfactory sensory sy... more The human derma emits volatile compounds whose interaction with a receiver's olfactory sensory system may affect individual recognition and mating preferences. Studies suggest that both genes and environmental factors determine characteristic odor of an individual. Here, we use solid phase micro-extraction and GC-MS to identify 3-methylbutanal (3-MB) in human axillary odor, show that the abundance of this volatile compound varies significantly between individuals and demonstrate in-vitro that its formation may be influenced by interaction between human leukocyte antigen peptide and dermal microflora.
A culture-independent analysis of the bacterial assemblages in the sediments of a lugworm Arenico... more A culture-independent analysis of the bacterial assemblages in the sediments of a lugworm Arenicola marina L. bioturbated mesocosm was carried out using a molecular RNA-gene based approach. Clone libraries were constructed for 4 spatial locations in the mesocosm: deep yeast layer (DYL), mid sand (MS), new casts (NC) and old casts (OC) after 29 d of bioturbation. In total, 495 clones were analysed and grouped into 159 unique phylotypes. All libraries were dominated by Epsilonproteobacteria, with Alpha-, Delta-and Gammaproteobacteria, Verrucomicrobia and members of the Cytophaga-Flavobacteria-Bacteroides group also prevalent. Phyologenetic comparison of the sites with respect to evolutionary distance showed that the libraries fell into 2 groups: (1) DYL and MS and (2) NC and OC. There were significant differences in species composition between but not within the 2 groupings. Bacteria involved with sulphur cycling are most likely to be affected by lugworm bioturbation. An Epsilonproteobacterium phylotype with > 95% 16S similarity to Sulfurimonas denitrificans was abundant in the DYL sand layer but was not present in the egested sand (NC, OC). Promotion of the growth of sulphide-oxidising bacteria and their subsequent digestion would be consistent with the 'gardening' hypothesis and suggests that chemoautotrophic processes in shallow-water anaerobic sediments may be enhanced by the activities of the lugworm.
Large numbers of filamentous actinomycetes which formed distinctive red coloured colonies were is... more Large numbers of filamentous actinomycetes which formed distinctive red coloured colonies were isolated from three out of four composite soil samples using a medium designed to be selective for members of the Streptomyces violaceoruber clade, a taxon which includes the model organisms ''Streptomyces coelicolor'' A3(2) and ''Streptomyces lividans'' 66. The isolation medium, dextran-histidine-sodium chloride-mineral salts agar supplemented with antibacterial and antifungal antibiotics, also supported the growth of representatives of the S. violaceoruber clade. One hundred and ninety one representatives of the isolates that produced red colour colonies on the isolation medium were distributed into four colour groups based on their ability to form distinctive pigments and morphological properties typical of members of the S. violaceoruber clade, an assignment that was confirmed by corresponding 16S rRNA gene sequencing studies. The selective isolation and characterisation procedures used in the present investigation provide a practical means of determining the taxonomic diversity, geographical distribution and roles of representatives of the S. violaceoruber clade in natural habitats.
Filamentous bacteria belonging to the genus Thiothrix were detected in activated sludge samples u... more Filamentous bacteria belonging to the genus Thiothrix were detected in activated sludge samples using the fluorescent in situ hybridisation (FISH) technique. A 16S rRNA-targeted oligonucleotide probe was developed for the detection of members of the T. fructosivorans group, and the performance of probe TNI for the detection of Thiothrix nivea group was enhanced by using an unlabeled competitor. A set of 5 probes covering all phylogenetic groups of Thiothrix were used to examine samples taken from selected activated sludge plants treating paper and board mill wastes. Members of the T. eikelboomii group formed the predominant filamentous bacterial population in plants experiencing poor sludge settleability, whereas members of the T. nivea group were commonly found but not dominantly in the remaining plants. Members of the T. fructosivorans group were not detected at any significant level in any of the samples. The distribution of the main Thiothrix types remained unchanged throughout ...
Twenty-five representative isolates from a collection of 54 indigenous actinomycetes isolated fro... more Twenty-five representative isolates from a collection of 54 indigenous actinomycetes isolated from coastal resources, were evaluated for inhibition against plant pathogenic fungi in vitro. Fusarium oxysporum f.sp. cubense (Foc) race 4 and Rhizoctonia solani exhibited higher resistance to the antifungal effects of the isolates when compared to the other test fungi. Seven isolates with potential pathogen-inhibitory capabilities belonged to the gray colour class of Streptomyces. Isolate g10 with a wide and strong antifungal spectrum against a variety of pathogenic fungi belonged to the Streptomyces violaceusniger cluster. This isolate could be a potential biological control agent.
Polyunsaturated fatty acids (PUFAs) are chemically present as esters, ethers, glycerides glycolip... more Polyunsaturated fatty acids (PUFAs) are chemically present as esters, ethers, glycerides glycolipids, phospholipids, phosphonolipids, glycosphingolipids, sulpholipids and lipoproteins in storage oils and membranes lipids. Marine microorganisms such as Shewanella frigidimarina are important sources of polyunsaturated fatty acids with promising biomedical applications, commercial value and the potential ability to be used in the bioremediation of environments contaminated with petroleum hydrocarbons. The growth media dependency of S. frigidimarina in terms of its growth behavior in response to modifications made to the media as well as its potential to produce PUFAs was evaluated. S. frigidimarina was cultured in conventional shake-flasks and controlled bioreactors with a batch-type procedure using different media compositions. The media used included artificial sea water, modified artificial sea water 1, modified artificial sea water 2, Luria Bertani, modified Luria Bertani, sodium p...
A screening method was established to detect inhibitors of the biosynthetic pathways of aromatic ... more A screening method was established to detect inhibitors of the biosynthetic pathways of aromatic amino acids and para-aminobenzoic acid, the precursor of folic acid, using an agar plate diffusion assay modified as an antagonism test. By this screening method, a family of three novel polycyclic polyketides named as abyssomicins was isolated from a marine strain of Verrucosispora. The main component abyssomicin C inhibits the pathway between chorismate and para-aminobenzoic acid and is strongly active against Gram-positive bacteria, including multi-resistant clinical isolates of Staphylococcus aureus.
Abstract This paper describes studies on the optimisation of ‘fed-batch’ bioreactors. Two bioreac... more Abstract This paper describes studies on the optimisation of ‘fed-batch’ bioreactors. Two bioreactors are considered: an industrial enzymatic reaction involving a racemic mixture and a combined microbial/enzymatic reaction taken from the literature. Both reactions are of the ‘fed-batch’ type and therefore require optimisation of feed profiles, as well as initial batch concentrations. The Chemotaxis optimisation algorithm is applied to both reactors to specify initial conditions and feed profiles in order to attempt to maximise a pre-specified profit function. This optimisation algorithm, together with the representation of feed profiles by polynomial equations, results in an extremely straightforward procedure for sub-optimal bioreactor operational specification. Although achieving a sub-optimal policy requires an accurate reaction model, the simplicity of the optimisation algorithm enables account to be taken of parameter uncertainty and thus reduced sensitivity to the model inaccuracies.
Abstract Implementation of advanced control strategies in bioprocesses is often hindered by the l... more Abstract Implementation of advanced control strategies in bioprocesses is often hindered by the lack of on-line measurements reflecting the physiological state of the culture. Although a number of techniques have been used to estimate key variables from data monitored on-line, these often do not explicitly take into account changes in physiological state and information on many aspects of physiological state may not be present in on-line data. This paper demonstrates that data obtained from chemical fingerprinting methods, such as Pyrolysis Mass Spectrometry, can be used to identify changes in physiological state during cultivation. This information can be utilised for estimation of the physiological state with on-line data that will enable physiological-state-specific model development for on-line bioprocess control.
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Papers by Alan Ward