The expression of nod genes of Rhizobium leguminosarum bv. viciae in nodules of Pisum sativum was... more The expression of nod genes of Rhizobium leguminosarum bv. viciae in nodules of Pisum sativum was investigated at both the translational and transcriptional levels. By using immunoblots, it was found that the levels of NodA, NodI, NodE, and NodO proteins were reduced at least 14-fold in bacteroids compared with cultured cells, whereas NodD protein was reduced only 3-fold. Northern (RNA) blot hybridization, RNase protection assays, and in situ RNA hybridization together showed that, except for the nodD transcript, none of the other nod gene transcripts were present in bacteroids. The amount of nodD transcript in bacteroids was reduced only two-to threefold compared with that in cultured cells. Identical results were found with a Rhizobium strain harboring multicopies of nodD and with a strain containing a NodD protein (NodD604) which is activated independently of flavonoids. Furthermore, it was found that mature pea nodules contain inhibitors of induced nod gene transcription but that NodD604 was insensitive to these compounds. In situ RNA hybridization on sections from P. sativum and Vicia hirsuta nodules showed that transcription of inducible nod genes is switched off before the bacteria differentiate into bacteroids. This is unlikely to be due to limiting amounts of NodD, the absence of inducing compounds, or the presence of anti-inducers. The observed switch off of transcription during the development of symbiosis is a general phenomenon and is apparently caused by a yet unknown negative regulation mechanism.
Copper-dependent lytic polysaccharide monooxygenases (LPMOs) are enzymes that oxidatively deconst... more Copper-dependent lytic polysaccharide monooxygenases (LPMOs) are enzymes that oxidatively deconstruct polysaccharides. The active site copper in LPMOs is coordinated by a histidine-brace. This utilises the amino group and side chain of the N-terminal His residue with the side chain of a second His residue to create a T-shaped arrangement of nitrogen ligands. We report a structural, kinetic and thermodynamic appraisal of copper binding to the histidine-brace in an auxiliary activity family 10 (AA10) LPMO from Streptomyces lividans (SliLPMO10E). Unexpectedly we discover the existence of two apo-SliLPMO10E species in solution that can each bind copper at a single site with distinct kinetic and thermodynamic (exothermic and endothermic) properties. The experimental EPR spectrum of copper-bound SliLPMO10E requires the simulation of two different line shapes, implying two different copper bound species, indicative of three and two nitrogen ligands coordinating the copper. Amino group coordination was probed through the creation of an N-terminal extension variant (SliLPMO10E-Ext). The kinetics and thermodynamics of copper binding to SliLPMO10E-Ext are in accord with copper binding to one of the apo-forms in the wild-type protein, suggesting that amino group coordination is absent in the two nitrogen coordinate form of SliLPMO10E. Copper binding to SliLPMO10B was also investigated, and again revealed the presence of two apo-forms with kinetics and stoichiometry of copper binding identical to that of SliLPMO10E. Our findings highlight that heterogeneity exists in the active site copper coordination sphere of LPMOs that may have implications for the mechanism of loading copper in the cell.
The genes for the b, b*, and seven sfactor subunits of RNA polymerase, for elongation factors EF-... more The genes for the b, b*, and seven sfactor subunits of RNA polymerase, for elongation factors EF-Tu1 and EF-Tu3, and for six rRNA operons were mapped on the combined genetic and physical map of theStreptomyces coelicolorchromosome. Like the previously mapped tRNA genes, the RNA polymerase and rRNA genes map to scattered positions. The lack of rRNA operons in the immediate
The filamentous bacterium Streptomyces lividans depends on the radical copper oxidase GlxA for th... more The filamentous bacterium Streptomyces lividans depends on the radical copper oxidase GlxA for the formation of reproductive aerial structures and, in liquid environments, for the formation of pellets. Incorporation of copper into the active site is essential for the formation of a cross-linked tyrosyl-cysteine cofactor, which is needed for enzymatic activity. In this study, we show a crucial link between GlxA maturation and a group of copper-related proteins including the chaperone Sco and a novel DyP-type peroxidase hereinafter called DtpA. Under copper-limiting conditions, the sco and dtpA deletion mutants are blocked in aerial growth and pellet formation, similarly to a glxA mutant. Western blot analysis showed that GlxA maturation is perturbed in the sco and dtpA mutants, but both maturation and morphology can by rescued by increasing the bioavailability of copper. DtpA acts as a peroxidase in the presence of GlxA and is a substrate for the twin-arginine translocation (Tat) tra...
Streptomyces lividans displays a distinct dependence on copper to fully initiate morphological de... more Streptomyces lividans displays a distinct dependence on copper to fully initiate morphological development. Evidence has accumulated to implicate the participation of an extracytoplasmic cuproenzyme in morphogenesis. In the present study, we show that GlxA fulfils all criteria to be that cuproenzyme. GlxA is membrane associated and has an active site consisting of a mononuclear copper and a cross-linked Y-C cofactor. The domain organization of the tertiary structure defines GlxA as a new structural member of the mono-copper oxidase family, with copper co-ordination geometry similar to, but spectroscopically distinct from fungal galactose oxidase (Gox). EPR spectroscopy reveals that the oxidation of cupric GlxA generates a protein radical residing on the Y-C cross-link. A variety of canonical Gox substrates (including D-galactose) were tested but none were readily turned over by GlxA. A glxA null-mutant leads to loss of glycan accumulation at hyphal tips and consequently a drasticall...
We have studied the regulation of the expression of tufA and tufB, the two genes encoding EF-Tu i... more We have studied the regulation of the expression of tufA and tufB, the two genes encoding EF-Tu in Escherichia coli. To this aim we have determined the intracellular concentrations of EF-TuA and EF-TuB under varying growth conditions by an immunological assay in mutants of E. coli constructed for this purpose. The data show that in wild-type cells the expression of tufA and tufB is regulated coordinately. This coordination is not restricted to steady-state growth conditions but is maintained throughout the life cycle of the cells up till the stationary phase. The ratio in which the two genes are expressed, however, may vary among cells with different genetic constitutions. Neither complete elimination of EF-TuB from the cell (by insertion of bacteriophage Mu DNA into tufB) nor elevation of the intracellular EF-TuB concentration (by transformation with plasmids harbouring tufB) has any effect on the expression of tufA. A specific single-site mutation of tufA, however, rendering EF-TuA resistant to the antibiotic kirromycin, disturbs the coordinate expression of tufA and tufB, enhancing tufB expression exclusively. These results have been interpreted by assuming that in wild-type cells the EF-Tu protein itself is involved in the regulation of the expression of tufB and that the mutant species of EF-Tu has lost this capacity either partially or completely. In agreement with this hypothesis are experiments performed in vitro with a coupled transcription/translation system programmed with DNA from a plasmid harbouring the entire tRNA-tufB transcriptional unit as a template. They show that addition to this system of EF-Tu in concentrations 2-5% of the endogenous amount results in strong inhibition of EF-Tu synthesis. We hypothesize that EF-Tu acts as an autogenous repressor, inhibiting tufB expression post-transcriptionally.
Recent discoveries of elongation factor-related proteins have considerably complicated the simple... more Recent discoveries of elongation factor-related proteins have considerably complicated the simple textbook scheme of the peptide chain elongation cycle. During growth and differentiation the cycle may be regulated not only by factor modification but also factor replacement. In addition, rare tRNAs may have their own rare factor proteins. A special case is the acquisition of resistance by bacteria to elongation factor-directed antibiotics. Pertinent data from the literature and our own work with Escherichia coli and Streptomyces are discussed. The GTP-binding domain of EF-Tu has been studied extensively, but little molecular detail is available on the interactions with its other ligands or effectors, or on the way they are affected by the GTPase switch signal. A growing number of EF-Tu mutants obtained by ourselves and others are helping us in testing current ideas. We have found a synergistic effect between EF-Tu and EF-G in their uncoupled GTPase reactions on empty ribosomes. Only the EF-G reaction is perturbed by fluoroaluminates.
Metal ion homeostasis in bacteria relies on metalloregulatory proteins to upregulate metal resist... more Metal ion homeostasis in bacteria relies on metalloregulatory proteins to upregulate metal resistance genes and enable the organism to preclude metal toxicity. The copper sensitive operon repressor (CsoR) family is widely distributed in bacteria and controls the expression of copper efflux systems. CsoR operator sites consist of G-tract containing pseudopalindromes of which the mechanism of operator binding is poorly understood. Here, we use a structurally characterized CsoR from Streptomyces lividans (CsoR(Sl)) together with three specific operator targets to reveal the salient features pertaining to the mechanism of DNA binding. We reveal that CsoR(Sl) binds to its operator site through a 2-fold axis of symmetry centred on a conserved 5'-TAC/GTA-3' inverted repeat. Operator recognition is stringently dependent not only on electropositive residues but also on a conserved polar glutamine residue. Thermodynamic and circular dichroic signatures of the CsoR(Sl)-DNA interaction ...
Mutants of Rhodobacter (Rba.) sphaeroides are described which were designed to study electron tra... more Mutants of Rhodobacter (Rba.) sphaeroides are described which were designed to study electron transfer along the so-called B-branch of reaction center (RC) cofactors. Combining the mutation L(M214)H, which results in the incorporation of a bacteriochlorophyll, beta, for H(A) [Kirmaier et al. (1991) Science 251: 922-927] with two mutations, G(M203)D and Y(M210)W, near B(A), we have created a double and a triple mutant with long lifetimes of the excited state P(*) of the primary donor P, viz. 80 and 160 ps at room temperature, respectively. The yield of P(+)Q(A) (-) formation in these mutants is reduced to 50 and 30%, respectively, of that in wildtype RCs. For both mutants, the quantum yield of P(+)H(B) (-) formation was less than 10%, in contrast to the 15% B-branch electron transfer demonstrated in RCs of a similar mutant of Rba. capsulatus with a P(*) lifetime of 15 ps [Heller et al. (1995) Science 269: 940-945]. We conclude that the lifetime of P(*) is not a governing factor in sw...
Journal of molecular microbiology and biotechnology, 2000
The onset of morphological differentiation in Streptomyces lividans is intrinsically delayed in c... more The onset of morphological differentiation in Streptomyces lividans is intrinsically delayed in comparison to Streptomyces coelicolor, but can be advanced by adding extra copper to the medium. Copper-specific chelators block aerial hyphae formation in both strains illustrating the crucial role of copper in morphogenesis. The S. coelicolor ram cluster was isolated as a clone that complements the copper-dependent differentiation of S. lividans. The S. lividans ram cluster was cloned and shown to be 99.6% identical to the S. coelicolor clone. The difference in development between S. lividans and S. coelicolor could neither be related to functional differences between the two ram clusters nor to differences in the transcription level. In both strains the low level of ramAB transcription correlated with aerial mycelium formation and was coupled to the upstream ORF ramS. An increased ramAB expression level in S. lividans by the introduction of an extra copy of ram stimulated the developme...
The expression of nod genes of Rhizobium leguminosarum bv. viciae in nodules of Pisum sativum was... more The expression of nod genes of Rhizobium leguminosarum bv. viciae in nodules of Pisum sativum was investigated at both the translational and transcriptional levels. By using immunoblots, it was found that the levels of NodA, NodI, NodE, and NodO proteins were reduced at least 14-fold in bacteroids compared with cultured cells, whereas NodD protein was reduced only 3-fold. Northern (RNA) blot hybridization, RNase protection assays, and in situ RNA hybridization together showed that, except for the nodD transcript, none of the other nod gene transcripts were present in bacteroids. The amount of nodD transcript in bacteroids was reduced only two-to threefold compared with that in cultured cells. Identical results were found with a Rhizobium strain harboring multicopies of nodD and with a strain containing a NodD protein (NodD604) which is activated independently of flavonoids. Furthermore, it was found that mature pea nodules contain inhibitors of induced nod gene transcription but that NodD604 was insensitive to these compounds. In situ RNA hybridization on sections from P. sativum and Vicia hirsuta nodules showed that transcription of inducible nod genes is switched off before the bacteria differentiate into bacteroids. This is unlikely to be due to limiting amounts of NodD, the absence of inducing compounds, or the presence of anti-inducers. The observed switch off of transcription during the development of symbiosis is a general phenomenon and is apparently caused by a yet unknown negative regulation mechanism.
Copper-dependent lytic polysaccharide monooxygenases (LPMOs) are enzymes that oxidatively deconst... more Copper-dependent lytic polysaccharide monooxygenases (LPMOs) are enzymes that oxidatively deconstruct polysaccharides. The active site copper in LPMOs is coordinated by a histidine-brace. This utilises the amino group and side chain of the N-terminal His residue with the side chain of a second His residue to create a T-shaped arrangement of nitrogen ligands. We report a structural, kinetic and thermodynamic appraisal of copper binding to the histidine-brace in an auxiliary activity family 10 (AA10) LPMO from Streptomyces lividans (SliLPMO10E). Unexpectedly we discover the existence of two apo-SliLPMO10E species in solution that can each bind copper at a single site with distinct kinetic and thermodynamic (exothermic and endothermic) properties. The experimental EPR spectrum of copper-bound SliLPMO10E requires the simulation of two different line shapes, implying two different copper bound species, indicative of three and two nitrogen ligands coordinating the copper. Amino group coordination was probed through the creation of an N-terminal extension variant (SliLPMO10E-Ext). The kinetics and thermodynamics of copper binding to SliLPMO10E-Ext are in accord with copper binding to one of the apo-forms in the wild-type protein, suggesting that amino group coordination is absent in the two nitrogen coordinate form of SliLPMO10E. Copper binding to SliLPMO10B was also investigated, and again revealed the presence of two apo-forms with kinetics and stoichiometry of copper binding identical to that of SliLPMO10E. Our findings highlight that heterogeneity exists in the active site copper coordination sphere of LPMOs that may have implications for the mechanism of loading copper in the cell.
The genes for the b, b*, and seven sfactor subunits of RNA polymerase, for elongation factors EF-... more The genes for the b, b*, and seven sfactor subunits of RNA polymerase, for elongation factors EF-Tu1 and EF-Tu3, and for six rRNA operons were mapped on the combined genetic and physical map of theStreptomyces coelicolorchromosome. Like the previously mapped tRNA genes, the RNA polymerase and rRNA genes map to scattered positions. The lack of rRNA operons in the immediate
The filamentous bacterium Streptomyces lividans depends on the radical copper oxidase GlxA for th... more The filamentous bacterium Streptomyces lividans depends on the radical copper oxidase GlxA for the formation of reproductive aerial structures and, in liquid environments, for the formation of pellets. Incorporation of copper into the active site is essential for the formation of a cross-linked tyrosyl-cysteine cofactor, which is needed for enzymatic activity. In this study, we show a crucial link between GlxA maturation and a group of copper-related proteins including the chaperone Sco and a novel DyP-type peroxidase hereinafter called DtpA. Under copper-limiting conditions, the sco and dtpA deletion mutants are blocked in aerial growth and pellet formation, similarly to a glxA mutant. Western blot analysis showed that GlxA maturation is perturbed in the sco and dtpA mutants, but both maturation and morphology can by rescued by increasing the bioavailability of copper. DtpA acts as a peroxidase in the presence of GlxA and is a substrate for the twin-arginine translocation (Tat) tra...
Streptomyces lividans displays a distinct dependence on copper to fully initiate morphological de... more Streptomyces lividans displays a distinct dependence on copper to fully initiate morphological development. Evidence has accumulated to implicate the participation of an extracytoplasmic cuproenzyme in morphogenesis. In the present study, we show that GlxA fulfils all criteria to be that cuproenzyme. GlxA is membrane associated and has an active site consisting of a mononuclear copper and a cross-linked Y-C cofactor. The domain organization of the tertiary structure defines GlxA as a new structural member of the mono-copper oxidase family, with copper co-ordination geometry similar to, but spectroscopically distinct from fungal galactose oxidase (Gox). EPR spectroscopy reveals that the oxidation of cupric GlxA generates a protein radical residing on the Y-C cross-link. A variety of canonical Gox substrates (including D-galactose) were tested but none were readily turned over by GlxA. A glxA null-mutant leads to loss of glycan accumulation at hyphal tips and consequently a drasticall...
We have studied the regulation of the expression of tufA and tufB, the two genes encoding EF-Tu i... more We have studied the regulation of the expression of tufA and tufB, the two genes encoding EF-Tu in Escherichia coli. To this aim we have determined the intracellular concentrations of EF-TuA and EF-TuB under varying growth conditions by an immunological assay in mutants of E. coli constructed for this purpose. The data show that in wild-type cells the expression of tufA and tufB is regulated coordinately. This coordination is not restricted to steady-state growth conditions but is maintained throughout the life cycle of the cells up till the stationary phase. The ratio in which the two genes are expressed, however, may vary among cells with different genetic constitutions. Neither complete elimination of EF-TuB from the cell (by insertion of bacteriophage Mu DNA into tufB) nor elevation of the intracellular EF-TuB concentration (by transformation with plasmids harbouring tufB) has any effect on the expression of tufA. A specific single-site mutation of tufA, however, rendering EF-TuA resistant to the antibiotic kirromycin, disturbs the coordinate expression of tufA and tufB, enhancing tufB expression exclusively. These results have been interpreted by assuming that in wild-type cells the EF-Tu protein itself is involved in the regulation of the expression of tufB and that the mutant species of EF-Tu has lost this capacity either partially or completely. In agreement with this hypothesis are experiments performed in vitro with a coupled transcription/translation system programmed with DNA from a plasmid harbouring the entire tRNA-tufB transcriptional unit as a template. They show that addition to this system of EF-Tu in concentrations 2-5% of the endogenous amount results in strong inhibition of EF-Tu synthesis. We hypothesize that EF-Tu acts as an autogenous repressor, inhibiting tufB expression post-transcriptionally.
Recent discoveries of elongation factor-related proteins have considerably complicated the simple... more Recent discoveries of elongation factor-related proteins have considerably complicated the simple textbook scheme of the peptide chain elongation cycle. During growth and differentiation the cycle may be regulated not only by factor modification but also factor replacement. In addition, rare tRNAs may have their own rare factor proteins. A special case is the acquisition of resistance by bacteria to elongation factor-directed antibiotics. Pertinent data from the literature and our own work with Escherichia coli and Streptomyces are discussed. The GTP-binding domain of EF-Tu has been studied extensively, but little molecular detail is available on the interactions with its other ligands or effectors, or on the way they are affected by the GTPase switch signal. A growing number of EF-Tu mutants obtained by ourselves and others are helping us in testing current ideas. We have found a synergistic effect between EF-Tu and EF-G in their uncoupled GTPase reactions on empty ribosomes. Only the EF-G reaction is perturbed by fluoroaluminates.
Metal ion homeostasis in bacteria relies on metalloregulatory proteins to upregulate metal resist... more Metal ion homeostasis in bacteria relies on metalloregulatory proteins to upregulate metal resistance genes and enable the organism to preclude metal toxicity. The copper sensitive operon repressor (CsoR) family is widely distributed in bacteria and controls the expression of copper efflux systems. CsoR operator sites consist of G-tract containing pseudopalindromes of which the mechanism of operator binding is poorly understood. Here, we use a structurally characterized CsoR from Streptomyces lividans (CsoR(Sl)) together with three specific operator targets to reveal the salient features pertaining to the mechanism of DNA binding. We reveal that CsoR(Sl) binds to its operator site through a 2-fold axis of symmetry centred on a conserved 5'-TAC/GTA-3' inverted repeat. Operator recognition is stringently dependent not only on electropositive residues but also on a conserved polar glutamine residue. Thermodynamic and circular dichroic signatures of the CsoR(Sl)-DNA interaction ...
Mutants of Rhodobacter (Rba.) sphaeroides are described which were designed to study electron tra... more Mutants of Rhodobacter (Rba.) sphaeroides are described which were designed to study electron transfer along the so-called B-branch of reaction center (RC) cofactors. Combining the mutation L(M214)H, which results in the incorporation of a bacteriochlorophyll, beta, for H(A) [Kirmaier et al. (1991) Science 251: 922-927] with two mutations, G(M203)D and Y(M210)W, near B(A), we have created a double and a triple mutant with long lifetimes of the excited state P(*) of the primary donor P, viz. 80 and 160 ps at room temperature, respectively. The yield of P(+)Q(A) (-) formation in these mutants is reduced to 50 and 30%, respectively, of that in wildtype RCs. For both mutants, the quantum yield of P(+)H(B) (-) formation was less than 10%, in contrast to the 15% B-branch electron transfer demonstrated in RCs of a similar mutant of Rba. capsulatus with a P(*) lifetime of 15 ps [Heller et al. (1995) Science 269: 940-945]. We conclude that the lifetime of P(*) is not a governing factor in sw...
Journal of molecular microbiology and biotechnology, 2000
The onset of morphological differentiation in Streptomyces lividans is intrinsically delayed in c... more The onset of morphological differentiation in Streptomyces lividans is intrinsically delayed in comparison to Streptomyces coelicolor, but can be advanced by adding extra copper to the medium. Copper-specific chelators block aerial hyphae formation in both strains illustrating the crucial role of copper in morphogenesis. The S. coelicolor ram cluster was isolated as a clone that complements the copper-dependent differentiation of S. lividans. The S. lividans ram cluster was cloned and shown to be 99.6% identical to the S. coelicolor clone. The difference in development between S. lividans and S. coelicolor could neither be related to functional differences between the two ram clusters nor to differences in the transcription level. In both strains the low level of ramAB transcription correlated with aerial mycelium formation and was coupled to the upstream ORF ramS. An increased ramAB expression level in S. lividans by the introduction of an extra copy of ram stimulated the developme...
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Papers by Erik Vijgenboom