Polyphosphate at the Streptomyces lividans cytoplasmic membrane is enhanced in the presence of the potassium channel KcsA.

The distribution of polyphosphate (polyP) within the cytoplasmic membrane of Streptomyces lividans hyphae or protoplasts has been determined at high spatial resolution by elemental mapping using energy-filtered electron microscopy (EFTEM). The results revealed that polyP was best traceable after its interaction with lead ions followed by their precipitation as lead sulphide. Concomitant studies of the S.lividans wildtype (WT) strain and its co-embedded mutant DeltaK (lacking a functional kcsA gene) were conducted by labelling as the surface matrix of either one was labelled by cationic colloidal thorium dioxide. Within the WT strain, additional polyP was found to accumulate distinctly at the inner face of the cytoplasmic membrane. After removal of the cell wall (within protoplasts), the polyP-derived lead-sulphide (PbS) precipitate formed clusters of fibrillar material extending up to 50 nm into the cytoplasm. This feature was absent in the DeltaK mutant strain. Together the results revealed that the presence of the KcsA channel and the structured polyP coincide.

Mutational analysis of the binding affinity and transport activity for N-acetylglucosamine of the novel ABC transporter Ngc in the chitin-degrader Streptomyces olivaceoviridis.

The highly differentiated bacterium Streptomyces olivaceoviridis efficiently hydrolyses chitin, a highly abundant natural polysaccharide, to low molecular weight products including N-acetylglucosamine (NAG) and N,N' -diacetylchitobiose (chitobiose). NAG is taken up by a PTS (phosphoenolpyruvate-dependent phosphotransferase system) which includes the PtsC2 protein, and via the ABC (ATP-binding cassette) transporter Ngc, which itself includes the substrate-binding protein NgcE. This is at present the only ABC transporter which is known to mediate specific uptake of NAG (K(m) 0.48 microM, V(max) 1.3 nmol/min/mg dry weight) and is competitively inhibited by chitobiose (K(i) 0.68 microM). The latter finding suggests that the Ngc system transports both NAG and chitobiose efficiently. To identify amino acid residues required for the function of NgcE, either the wild-type or one of several mutant forms of the ngcE gene was introduced into the strain S. olivaceoviridis DeltaNgcE/DeltaPtsC1/DeltaPtsC2, which lacks both functional transport systems for NAG, and chromosomal recombinants were selected. Based on the in vivo transport parameters of the recombinants, and the in vitro binding characteristics of the corresponding purified proteins, the following conclusions can be drawn. (1) Replacement of the C-terminally located residue Y396 by A (Y396A) has little effect on ligand-binding or transport parameters. The W395A mutation also induced little change in the substrate affinity in vitro, but it led in vivo to a marked increase (11 fold) in K(m), and enhanced V(max) (by 1.5 fold). (2) The amino acids Y201 and W280 both contribute (51% and 38%) to the ligand-binding capacity of NgcE. They are both very important for the in vivo function of the complete transport apparatus; strains expressing either Y201A or W280A show drastically (100 or 150 times) enhanced K(m) values. (3) The concomitant presence of either Y200 and W280 or Y201 and W280 is essential for the function of NgcE. (4) Y201 is located within a tyrosyl-rich motif. This has been found to share some features with the ligand-binding site of amelogenins (enamel matrix proteins), which interact with NAG residues in glycoconjugates. In addition, it is distantly related to the ligand-binding site(s) in the plant-lectins UDA ( Urtica dioicaagglutinin, specific for NAG and its oligomers) and WGA (wheat germ agglutinin, which recognises a motif comprising three consecutive NAG residues).

Amino acid residues involved in reversible thiol formation and zinc ion binding in the Streptomyces reticuli redox regulator FurS.

Streptomyces reticuli produces a mycelium-associated enzyme, CpeB, whose N-terminal and C-terminal portions mediate heme-dependent catalase-peroxidase and heme-independent manganese-peroxidase activities, respectively. The regulator FurS governs transcription of the furS- cpeB operon. The thiol form of FurS contains one zinc ion per monomer and binds in this state to its cognate operator. Oxidation of SH groups within FurS induces the release of the zinc ion. Substitution of the codons for the amino acids cysteine 96, histidine 92 and 93, and tyrosine 59 in furS disrupts the in vivo repressor activity of FurS and results in enhanced synthesis of CpeB in corresponding S. lividans transformants. Biochemical and footprinting studies with FurS and its mutant derivatives revealed that the cysteine residues 96 and 99 are involved in reversible S-S bond formation, while cysteine 96 and the histidine residues 92 and 93 are required for zinc coordination, and tyrosine 59 is necessary for the binding of FurS to DNA. On the basis of these data, functional predictions can be made for the mycobacterial regulator FurA, a close homologue of FurS.

Streptomyces olivaceoviridis possesses a phosphotransferase system that mediates specific, phosphoenolpyruvate-dependent uptake of N-acetylglucosamine.

We recently described the ABC transporter Ngc (encoded by the ncgEFG operon) from Streptomyces olivaceoviridis, the first of its kind to be shown to transport N-acetylglucosamine and N,N'-diacetylchitobiose (chitobiose). A chromosomal mutant carrying a disruption of the ngcE gene, which encodes the sugar binding protein, was still able to transport N-acetylglucosamine. This phenotype can now be attributed to a functional phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS). Two adjacent homologous genes, ptsC1 and ptsC2, were identified, and deduced to encode proteins which are 56% identical and can be predicted to contain eight transmembrane regions. PtsC1 (432 amino acids) and PtsC2 (403 residues) each correspond to a single EIIC domain; such domains are otherwise known only in several bacterial multidomain permeases for glucose/mannose or N-acetylglucosamine. The C-terminal sequences of PtsC1 and PtsC2 correspond to the motifs LKTPGREP and LPTRGRES, respectively. The ptsB gene located upstream of ptsC1 is predicted to encode a homologue of the EIIB domains usually found in bacterial multidomain permeases. Physiological and biochemical analyses of ngcE mutants carrying disruptive insertions in ptsC1 or ptsC2 or both revealed that, when grown on N-acetylglucosamine, the membrane component PtsC2, unlike PtsC1, mediates PEP-dependent, specific (K(m)=5 micro M) transport of N-acetylglucosamine, but not of other hexoses. Cross-complementation of membrane and cytoplasmic fractions from the various mutants led to the conclusion that S. olivaceoviridis also expresses the functional soluble components HPr, EI and EIIA of the PTS system. During growth on xylose, uptake of this pentose occurred if ptsC1 or ptsC2 was intact, but not in a mutant containing disrupted forms of both genes.

The novel Streptomyces olivaceoviridis ABC transporter Ngc mediates uptake of N-acetylglucosamine and N,N'-diacetylchitobiose.

During cultivation in the presence of N-acetylglucosamine or chitin, Streptomyces olivaceoviridis mycelium efficiently takes up [(14)C]-labelled N-acetylglucosamine. Uptake of the labelled compound can be completely inhibited by unlabelled N-acetylglucosamine and partially by chitobiose. After extraction of the membrane with Triton X-100, two forms of a protein that binds to N-acetylglucosamine and N, N'-diacetylchitobiose (chitobiose) were purified to homogeneity by two consecutive rounds of anionic exchange chromatography. The protein was named NgcE. Using surface plasmon resonance, its binding parameters were determined. It showed highest affinity for N-acetylglucosamine (K(D)=8.28 x 10(-9) M) and for chitobiose (K(D)=2.87 x 10(-8) M). Varying equilibrium dissociation constants in the micromolecular range were ascertained for chitotetraose (K(D)=4.5 x 10(-6) M), chitopentaose (K(D)=1.03 x 10(-6) M) and chitohexaose (K(D)=3.02 x 10(-6) M); the lowest value was measured for chitotriose (K(D)=19.4 x 10(-6) M). After having determined the sequences of several internal peptides from the binding protein by Edman degradation, the corresponding ngcE gene, which encodes a predicted lipid-anchored protein, was identified by reverse genetics. Using a genomic phage library of S. olivaceoviridis genes encoding two other membrane proteins (named NgcF and NgcG) were identified adjacent to ngcE. Each of these is predicted to have six membrane-spanning helices and a consensus motif for integral membrane proteins characteristic of ABC transporters. In addition, the gene for a predicted regulator protein (NgcR) was detected. The ngcEFG operon lacks a gene for an ATP-hydrolysing protein. NgcE is a new member of the CUT-1 family of ABC transporters for carbohydrates. Comparative studies of the wild-type and a mutant strain carrying an insertion within the ngc operon clearly demonstrate that the Ngc system mediates the uptake of N-acetylglucosamine and chitobiose in vivo.

Mutations stabilizing an open conformation within the external region of the permeation pathway of the potassium channel KcsA.

Four subunits of the bacterial Streptomyces lividans protein KcsA form a K+ channel which can be functionally reconstituted in vitro. Here we show that substitution of the tyrosine residue 82 by cysteine, valine or threonine, but not by glycine, led to functional channel types. Like the wild-type (WT) and an L81C channel, the mutant channels exhibit an internal pH-sensitive side and are cation selective. Based on the relative positions of the blocker tetraethylammonium within the electric field, the external entryways of the channels are concluded to have similar dimensions. For inward currents, the WT and the mutant channels vary in the occupancy of their subconductance states and concomitantly in their mean currents. Rectification properties are scarcely (L81C), little (Y82C) or considerably (Y82T and Y82V) altered. The data suggest that the amino acid type in position 82 stabilizes to varying degrees an open conformation within the external region of the permeation pathway.

Synthesis of the Streptomyces lividans maltodextrin ABC transporter depends on the presence of the regulator MalR.

During growth with maltotriose or amylose, Streptomyces lividans and Streptomyces coelicolor A3(2) synthesize a maltodextrin uptake system with highest specificity for maltotriose. The transport activity is absent in mutants of S. coelicolor A3(2) lacking a functional MalE binding protein. Cloning and sequencing data suggest that the mal operon of S. coelicolor A3(2) corresponds to the one of S. lividans and that the deduced S. lividans Reg1 amino acid sequence is identical to that of MalR from S. coelicolor A3(2). It can be concluded that both strains have the same ABC transport system for maltodextrins. The S. lividans malR was cloned in Escherichia coli in frame with six histidine-encoding codons. The resulting, purified 6HisMalR(SI) was shown to bind to two motifs within the S. lividans malR-malE intergenic region and to dissociate in the presence of maltopentaose.

Characteristics of a Streptomyces coelicolor A3(2) extracellular protein targeting chitin and chitosan.

Upstream of the Streptomyces coelicolor A3(2) chitinase G gene, a small gene (named chb3) is located whose deduced product shares 37% identical amino acids with the previously described CHB1 protein from Streptomyces olivaceoviridis. The chb3 gene and its upstream region were cloned in a multicopy vector and transformed into the plasmid-free Streptomyces lividans TK21 strain. The CHB3 protein (14.9 kDa) was secreted by the S. lividans TK21 transformant during growth in the presence of glucose, N-acetylglucosamine, yeast extract, and chitin. The protein was purified to homogeneity using anionic exchange, hydrophobic interaction chromatographies, and gel filtration. In contrast to CHB1, CHB3 targets alpha-chitin, beta-chitin, and chitosan at pH 6.0 but does so relatively loosely. The ecological implications of the divergence of substrate specificity of various types of chitin-binding proteins are described.

Recognition and degradation of chitin by streptomycetes.

Chitin is the second most abundant renewable polysaccharide, as it is a component of the exoskeleton of many organisms and of the cell walls of numerous fungi. Most streptomycetes secrete a number of chitinases, hydrolyzing chitin to oligomers, chitobiose or N-acetylglucosamine which can be utilized as carbon or nitrogen source. The chitinases of several streptomycetes have been shown to have a modular arrangement comprising catalytic, substrate binding as well as linker domains. Moreover, during growth in the presence of chitin-containing substrates, many Streptomyces strains have been shown to secrete formerly unknown, small (about 200 aa) chitin binding proteins (CHBs) which lack enzymatic activity and specifically target and invade chitin. Several motifs, including the relative location and spacing of four tryptophan residues, are conserved in the investigated CHB types, CHB1 and CHB2. The affinity of CHB1 to crab shell chitin is two times higher than that of CHB2. Comparative studies of various generated mutant CHB1 proteins led to the conclusion that it is one of the exposed tryptophan residues that directly contributes to the interaction with chitin. On the basis of immunological, biochemical and physiological studies, it can be concluded that the CHBs act like a glue with which streptomycetes target chitin-containing samples or organisms. The ecological implications of these findings are discussed.

The DNA-binding characteristics of the Streptomyces reticuli regulator FurS depend on the redox state of its cysteine residues.

Streptomyces reticuli produces a mycelium-associated enzyme (CpeB) which exhibits heme-dependent catalase and peroxidase activity, as well as heme-independent manganese-peroxidase activity. The cpeB gene does not have a promoter of its own. It is co-transcribed together with the adjacent furS gene from at least one promoter, the position of which was deduced on the basis of high-resolution S1 mapping of transcriptional start sites. Physiological and transcriptional studies suggested that FurS acts as a transcriptional repressor in the presence of Mn2+ and Fe2+ ions. A FurS fusion protein was purified, after cloning of the corresponding gene, either from Escherichia coli or Streptomyces lividans transformants. The fusion protein from each host strain can be converted into a form that exhibits reduced electrophoretic mobility following treatment with thiol-reducing agents; in the presence of diamide, in contrast, the mobility of the protein is enhanced. Additional immunological studies have shown that the native S. reticuli FurS also shows these properties, which are due to the presence of redox-sensitive cysteine residues. As revealed by gel-shift and in vitro footprinting studies, only the reduced form of the FurS fusion protein and the reduced FurS protein (partially purified from S. reticuli) is able to bind to a motif upstream of the furS gene. In the absence of first-row divalent ions, the binding site encompasses 22 bp. In the presence of Mn2+, Fe2+, Co2+, Cu2+ or Zn2+, however, the region bound is extended by 18 bp. It is noteworthy that the region upstream of the furA gene in several mycobacteria contains a very similar motif. The predicted mycobacterial FurA shares a high degree of sequence identity with FurS, and the furA gene is linked to one that encodes a catalase-peroxidase (KatG). The implications of these findings are discussed.

Binding characteristics of CebR, the regulator of the ceb operon required for cellobiose/cellotriose uptake in Streptomyces reticuli.

The Streptomyces reticuli Avicelase (cellulase, Cell) hydrolyzes crystalline cellulose to cellooligomers, cellobiose and cellotriose which are taken up by mycelia via an ABC transport system (Ceb) induced during growth with cellobiose or cellulose. The cebR gene located upstream of the cebEFG operon was cloned in Escherichia coli in frame with six histidine-encoding codons. The resulting purified fusion protein was shown to bind to a motif of 23 bp, including a perfect 18-bp palindrome situated upstream of the cebEFG. Cytoplasmic extracts of induced, but not of uninduced S. reticuli protected the same DNA motif. Release of the CebR regulator from its operator occurs upon addition of cellopentaose which can be assumed to act as inducer within the mycelia.

Solution structure and conformational changes of the Streptomyces chitin-binding protein (CHB1).

The shape and overall dimensions of the recently discovered Streptomyces alpha-chitin-binding protein, CHB1, were investigated by synchrotron radiation X-ray solution scattering. The radius of gyration and the maximum size of CHB1 were determined to be 1.75 +/- 0.03 nm and 6.0 +/- 0.2 nm, respectively. Using two independent ab initio approaches the low-resolution shape of the protein was found to consist of two domains, an elongated main globule with a length of about 4 nm and a foot-like domain of about 2 nm width. The structural and functional properties of CHB1 depend strongly on the presence of disulfide bonds; upon their reduction, the protein loses its affinity to chitin.

The heme-independent manganese-peroxidase activity depends on the presence of the C-terminal domain within the Streptomyces reticuli catalase-peroxidase CpeB.

Streptomyces reticuli produces a heme-containing homodimeric enzyme (160 kDa), the catalase-peroxidase CpeB, which is processed to the enzyme CpeC during prolonged growth. CpeC contains four subunits of 60 kDa each that do not include the C-terminal portion of the progenitor subunits. A genetically engineered cpeB gene encodes a truncated subunit lacking 195 of the C-terminal amino acids; four of these subunits assemble to form the enzyme CpeD. Heme binds most strongly in CpeB, least in CpeD. The catalase-peroxidase CpeB and its apo-form (obtained after extraction of heme) catalyze the peroxidation of Mn(II) to Mn(III), independent of the presence or absence of the heme inhibitor KCN. CpeC and CpeD, in contrast, do not exhibit manganese-peroxidase activity. The data show for the first time that a bacterial catalase-peroxidase has a heme-independent manganese-peroxidase activity, which depends on the presence of the C-terminal domain.

Architecture of the Streptomyces lividans DnaA protein-replication origin complexes.

The Streptomyces oriC region contains two clusters of 19 DnaA boxes separated by a spacer (134 bp). The Streptomyces DnaA protein consists, like all other DnaA proteins, of four domains: domain III and the carboxyterminal part (domain IV) are responsible for binding of ATP and DNA, respectively. Binding of the DnaA protein to the entire oriC region analysed by electron microscopy showed that the DnaA protein forms separate complexes at each of the clusters of DnaA boxes, but not at the spacer separating them. In vivo mutational analysis revealed that the number of DnaA boxes and the presence of the spacer linking both groups of DnaA boxes seem to be important for a functional Streptomyces origin. We suggest that the arrangement of DnaA boxes allows the DNA-bound DnaA protein to induce bending and looping of the oriC region. As it was shown by electrophoretic mobility shift assay and "one hybrid system", two domains, I and III, facilitate interactions between DnaA molecules. We postulate that domain I and domain III could be involved in cooperativity at distant and at closely spaced DnaA boxes, respectively. The long domain II extends the range over which N termini (domain I) of DNA-bound DnaA protein can form dimers. Thus, interactions between DnaA molecules may bring two clusters of DnaA boxes separated by the spacer into functional contact by loop formation. Removal of the spacer region or deletion of domains I and II resulted, respectively, in nucleoprotein complexes which are not fully developed, or huge nucleoprotein aggregates.

Pore mutations affecting tetrameric assembly and functioning of the potassium channel KcsA from Streptomyces lividans.

Designed mutations within the Streptomyces lividans kcsA gene resulted in a set of mutant proteins, which were characterized in respect to their assembly and channel activities. (i) The amino acid residue leucine 81 located at the external side of KcsA was found to be exchangeable by a cysteine residue without affecting the channel characteristics. (ii) Substitution of the first glycine (G77) residue within the GYG motif by an alanine or substitution of the tyrosine (Y) residue 78 by a phenylalanine (F) led to mutant proteins which form tetramers of reduced stability. In contrast to the AYG mutant protein, GFG functions as an active K(+) channel whose characteristics correspond to those of the wild-type KcsA channel. (iii) The investigated mutant proteins, which carry different mutations (T72A, T72C, V76A, V76E, G77E, Y78A, G79A, G79D, G79E) within the signature sequence of the pore region, do not at all or only to a very small degree assemble as tetramers and lack channel activity.

Structure and regulation of the dnaA promoter region in three Streptomyces species.

The regulatory region of the Streptomyces dnaA gene comprises a single promoter and two DnaA boxes that are located upstream of the promoter. Comparative analysis of the dnaA promoter region from S. chrysomallus, S. lividans and S. reticuli revealed that the location, spacing and orientation of the DnaA boxes are conserved. In vitro studies demonstrated that efficient binding of the Streptomyces DnaA protein to DNA requires the presence of two DnaA boxes. In vivo analysis of dnaA promoter mutants deleted for one or both DnaA boxes indicated that the dnaA gene is autoregulated. However, the degree of derepression observed is relatively modest.

Efficient membrane assembly of the KcsA potassium channel in Escherichia coli requires the protonmotive force.

Very little is known about the biogenesis and assembly of oligomeric membrane proteins. In this study, the biogenesis of KcsA, a prokaryotic homotetrameric potassium channel, is investigated. Using in vivo pulse-chase experiments, both the monomeric and tetrameric form could be identified. The conversion of monomers into a tetramer is found to be a highly efficient process that occurs in the Escherichia coli inner membrane. KcsA does not require ATP hydrolysis by SecA for insertion or tetramerization. The presence of the proton-motive force (pmf) is not necessary for transmembrane insertion of KcsA; however, the pmf proved to be essential for the efficiency of oligomerization. From in vivo and in vitro experiments it is concluded that the electrical component, deltapsi, is the main determinant for this effect. These results demonstrate a new role of the pmf in membrane protein biogenesis.

Characterization of the binding protein-dependent cellobiose and cellotriose transport system of the cellulose degrader Streptomyces reticuli.

Streptomyces reticuli has an inducible ATP-dependent uptake system specific for cellobiose and cellotriose. By reversed genetics a gene cluster encoding components of a binding protein-dependent cellobiose and cellotriose ABC transporter was cloned and sequenced. The deduced gene products comprise a regulatory protein (CebR), a cellobiose binding lipoprotein (CebE), two integral membrane proteins (CebF and CebG), and the NH2-terminal part of an intracellular beta-glucosidase (BglC). The gene for the ATP binding protein MsiK is not linked to the ceb operon. We have shown earlier that MsiK is part of two different ABC transport systems, one for maltose and one for cellobiose and cellotriose, in S. reticuli and Streptomyces lividans. Transcription of polycistronic cebEFG and bglC mRNAs is induced by cellobiose, whereas the cebR gene is transcribed independently. Immunological experiments showed that CebE is synthesized during growth with cellobiose and that MsiK is produced in the presence of several sugars at high or moderate levels. The described ABC transporter is the first one of its kind and is the only specific cellobiose/cellotriose uptake system of S. reticuli, since insertional inactivation of the cebE gene prevents high-affinity uptake of cellobiose.

Interactions of the Streptomyces lividans initiator protein DnaA with its target.

The Streptomyces lividans DnaA protein (73 kDa) consists, like other bacterial DnaA proteins, of four domains; it binds to 19 DnaA boxes in the complex oriC region. The S. lividans DnaA protein differs from others in that it contains an additional stretch of 120 predominantly acidic amino acids within domain II. Interactions between the DnaA protein and the two DnaA boxes derived from the promoter region of the S. lividans dnaA gene were analysed in vitro using three independent methods: Dnase-I-footprinting experiments, mobility-shift assay and surface plasmon resonance (SPR). The Dnase-I-footprinting analysis showed that the wild-type DnaA protein binds to both DnaA boxes. Thus, as in Escherichia coli and Bacillus subtilis, the S. lividans dnaA gene may be autoregulated. SPR analysis showed that the affinity of the DnaA protein for a DNA fragment containing both DnaA boxes from the dnaA promoter region (KD = 1.25 nM) is 10 times higher than its affinity for the single 'strong' DnaA box (KD = 12.0 nM). The mobility-shift assay suggests the presence of at least two classes of complex containing different numbers of bound DnaA molecules. The above data reveal that the DnaA protein binds to the two DnaA boxes in a cooperative manner. To deduce structural features of the Streptomyces domain II of DnaA protein, the amino acid DnaA sequences of three Streptomyces species were compared. However, according to the secondary structure prediction, Streptomyces domain II does not contain any common relevant secondary structural element(s). It can be assumed that domain II of DnaA protein can play a role as a flexible protein spacer between the N-terminal domain I and the highly conserved C-terminal part of DnaA protein containing ATP-binding domain III and DNA-binding domain IV.