Deletion of a cyclic AMP receptor protein homologue diminishes germination and affects morphological development of Streptomyces coelicolor.

Open reading frame SCO3571 of Streptomyces coelicolor encodes a protein of the cyclic AMP (cAMP) receptor protein (CRP) superfamily of regulatory proteins. A mutant revealed a dramatic defect in germination, followed by growth delay and earlier sporulation. This phenotype correlates with those of an adenylate cyclase (cya) mutant that cannot synthesize cAMP. This finding suggests that S. coelicolor may use a Cya-cAMP-CRP system to trigger complex physiological processes such as morphogenesis.

Site-directed mutagenesis of conserved inverted repeat sequences in the xylanase C promoter region from Streptomyces sp. EC3.

Streptomyces sp. EC3, a strain which was originally isolated from cattle manure compost, was shown to possess a strong xylanolytic activity. One of the genes responsible for this activity, xlnC, encodes a secreted xylanase. In the native strain, as in the heterologous host S. lividans, expression of xlnC was detectable in the presence of xylan but not in the presence of glucose. Induction by xylan was shown to take place at the transcriptional level. The transcriptional start site of xlnC was mapped and likely -35 (5'-TTGACA-3') and -10 (5'-GAGAAC-3') motifs were identified. In order to localise putative conserved regulatory sequences, the promoter regions of xylanase-encoding genes from various Streptomyces species were aligned. This alignment revealed the existence of three sets of quite well conserved palindromic AT rich sequences called boxes 1, 2 and 3. Box 3 (5'-CGAAA N TTTCG-3') is the farthest away from the promoter region (150-200 bp). A shorter version of this palindrome (5'-GAAA NN TTTC-3') or (5'-CGAAA-3') constitutes box 1, which is located just upstream of the putative -35 promoter sequence. Box 2, located 5-7 bp upstream of box 1, comprises a shorter palindrome than box 3, with inverted polarity [5'-(G/C)TTTC (N) GAAA(G/C)-3']. The putative regulatory role of the conserved inverted repeats in boxes 2 and 3 in the promoter region of the xlnC gene from Streptomyces sp. EC3, was assessed. These boxes were modified by site-directed mutagenesis, and the mutant promoter regions, as well as the wild-type promoter region, were separately fused to a beta-lactamase reporter gene. Analysis of the expression patterns of these fusions in cultures grown in the presence of glucose, xylan or both carbon sources demonstrated that these motifs were cis -acting negative regulatory elements, each playing a specific role in the regulation of xlnC expression. Box 3 was shown to be critical for the establishment of repression of xlnC expression by glucose, whereas box 2 was shown to play an important role in the induction of xlnC expression by xylan.

BlaB, a protein involved in the regulation of Streptomyces cacaoi beta-lactamases, is a penicillin-binding protein.

Streptomyces cacaoi beta-lactamase genes are controlled by two regulators named blaA and blaB. Whereas BlaA has been identified as a LysR-type activator, the function of BlaB is still unknown. Its primary structure is similar to that of the serine penicillin-recognizing enzymes (PREs). Indeed, the SXXK and KTG motifs are perfectly conserved in BlaB, whereas the common SXN element found in PREs is replaced by a SDG motif. Site-directed mutations were introduced in these motifs and they all disturb beta-lactamase regulation. A water-soluble form of BlaB was also overexpressed in the Streptomyces lividans TK24 cytoplasm and purified. To elucidate the activity of BlaB, several compounds recognized by PREs were tested. BlaB could be acylated by some of them, and it can therefore be considered as a penicillin-binding protein. BlaB is devoid of beta-lactamase, D-aminopeptidase, DD-carboxypeptidase or thiolesterase activity.

[Weill-Marchesani syndrome. Late athalamia following antiglaucomatous surgery]. / Síndrome de Weill-Marchesani. Atalamia tardía tras cirugía antiglaucomatosa.

PURPOSE/METHOD: A case of a patient with Weill-Marchesani syndrome who developed a secondary glaucoma due to synechiae in both eyes is described. As intraocular pressure (IOP) could not be controlled with medical treatment in the left eye (LE), the patient underwent glaucoma filtering surgery. IOP was controlled and no complications occurred. However, 15 months later, athalamia stage 1 was diagnosed in the LE, without any alterations in the posterior pole. To solve this complication, a vitrectomy with lens extraction and intraocular lens implantation in the LE was performed. Currently, IOP is 12 mmHg and the anterior chamber remains deep. RESULTS/CONCLUSIONS: The association of vitrectomy and lens surgery in those cases where there is a predisposition to forward movement of the lens, might reduce intra and postoperative complications.

Síndrome de Weill-Marchesani. Atalamia tardía tras cirugía antiglaucomatosa / Weill-Marchesani syndrome. Late athalamia following antiglaucomatous surgery

Objetivo/Método: Se presenta el caso clínico de una paciente diagnosticada de síndrome de Weill-Marchesani que desarrolló un glaucoma secundario por sinequias anteriores en ambos ojos (AO). Al no controlarse la presión intraocular (PIO) del ojo izquierdo (OI) con tratamiento médico, se realizó cirugía filtrante antiglaucomatosa. La evolución postoperatoria fue favorable y sin complicaciones. Sin embargo, a los 15 meses, se diagnosticó una atalamia grado I en OI, sin alteraciones en el segmento posterior. Para solucionar esta complicación, se llevó a cabo una vitrectomía, con extracción del cristalino e implantación de lente intraocular. Actualmente, la paciente mantiene una PIO de 12 mm Hg en OI, con una cámara anterior de profundidad normal. Resultados/Conclusiones: La asociación de vitrectomía y cirugía de cristalino en aquellos casos en los que existe predisposición al desplazamiento anterior del cristalino, puede reducir el número de complicaciones (AU)

Crystallographic analysis of family 11 endo-beta-1,4-xylanase Xyl1 from Streptomyces sp. S38.

Family 11 endo-beta-1,4-xylanases degrade xylan, the main constituent of plant hemicelluloses, and have many potential uses in biotechnology. The structure of Xyl1, a family 11 endo-xylanase from Streptomyces sp. S38, has been solved. The protein crystallized from ammonium sulfate in the trigonal space group P321, with unit-cell parameters a = b = 71.49, c = 130.30 A, gamma = 120.0 degrees. The structure was solved at 2.0 A by X-ray crystallography using the molecular-replacement method and refined to a final R factor of 18.5% (R(free) = 26.9%). Xyl1 has the overall fold characteristic of family 11 xylanases, with two highly twisted beta-sheets defining a long cleft containing the two catalytic residues Glu87 and Glu177.

The diversity, structure and regulation of beta-lactamases.

beta-Lactamase production is responsible for the appearance of a large number of pathogenic bacterial strains exhibiting a high degree of resistance to beta-lactam antibiotics. A large number of enzymes have been described with very diverse primary structures and catalytic profiles. Nevertheless, all known three-dimensional structures of active-site serine beta-lactamases exhibit a high degree of similarity with apparently equivalent chemical functionalities in the same strategic positions. These groups might not, however, play identical roles in the various classes of enzymes. Structural data have also been recently obtained for the zinc metallo-beta-lactamases, but the detailed catalytic mechanisms might also differ widely, depending on the enzyme studied. Similarly, the induction of the synthesis of beta-lactamases is now better understood, but many questions remain to be answered.

The two beta-lactamase genes of Streptomyces cacaoi, blaL and blaU, are under the control of the same regulatory system.

The production of beta-lactamase in Streptomyces cacaoi, which contains two beta-lactamase-encoding genes, blaL and blaU, is inducible by beta-lactam compounds. The two genes have been cloned independently in S. lividans TK24, a beta-lactamase-negative species. The blaU clone did not respond to the presence of beta-lactams, whereas the blaL clone appeared to be inducible in S. lividans. The latter clone contains two open reading frames, blaA and blaB, located just upstream of but transcribed divergently from blaL, which were shown to be required for the production as well as the induction of BlaL. The deduced BlaA protein belongs to the LysR family of transcription regulators. In order to examine the role of BlaA in regulation, we here report on over-expression of a GST-BlaA fusion protein in Escherichia coli and its use for antibody preparation. The GST-BlaA fusion protein was partially purified and bandshift assays showed that it bound the 197-bp blaL-blaA intergenic region. The BlaA DNA binding-site was further restricted to a 30-bp sequence containing a T-N11-A motif, a characteristic of LysR-type promoters. Another T-N11-A motif upstream of the blaU gene was also shown to bind BlaA. The affinities of these two T-N11-A motifs in BlaA binding were comparable. A plasmid bearing the blaU structural gene and the blaA-blaB regulatory region was constructed and shown to confer on an S. lividans host the capacity to produce inducible beta-lactamase. It can thus be concluded that the S. cacaoi blaL and blaU genes are controlled by the same regulatory system.

Electroporation of intact cells of Streptomyces parvulus and Streptomyces vinaceus.

Various assays of classical PEG-assisted transformation as well as electrotransformation of Streptomyces parvulus IMET41380 and Streptomyces vinaceus NCIB8852 are described. Contrary to the so far reported assays of electrotransforming Streptomyces strains, electroporation in S. parvulus and S. vinaceus was carried out on intact cells, without any lysozyme treatment. In these two strains, the classical PEG-assisted transformation of protoplasts does not work efficiently (10(3) to 10(4) transformants per micrograms of pIJ702 DNA) and electrotransformation gives 10 to 100 times higher yields (10(5) transformants per micrograms of pIJ702 DNA). The electroporation method described here is not applicable to other Streptomyces strains (S. lividans or S. coelicolor).

Use of an automatic DNA sequencer for S1 mapping: transcriptional analysis of the Streptomyces coelicolor A3(2) dnaK operon.

The transcription start point of the dnaK operon of Streptomyces coelicolor A3(2) has been determined by S1 mapping, using the EMBL automated fluorescent DNA sequencer. The -35 and -10 hexamers correspond to a sigma 70-type promoter. This promoter responds to heat shock and involves an inverted repeat different from the CIRCE sequence characteristic of the Gram-positive heat-shock promoters.

A sequence-specific DNA-binding protein interacts with the xlnC upstream region of Streptomyces sp. strain EC3.

The alignment of the promoter region of several Streptomyces xylanases shows three conserved sequences which could be involved in gene regulation. By electromobility shift assays these specific sequences, present only in Streptomyces xylanolytic strains, were identified as protein-binding sites. The sequence required for efficient recognition by the retarding protein appeared to be a 4-bp inverted repeat: 5'-CTTT-Nx-AAAG-3'. The DNA-protein affinity was influenced by the culture conditions.

Cloning and nucleotide sequence of a xylanase-encoding gene from Streptomyces sp. strain EC3.

Using the p1J702 vector, a xylanase-encoding gene (xin) of Streptomyces sp. EC3 has been cloned by functional complementation of a mutant of Streptomyces lividans TK24, producing xylanase at a very low level. Normal level of xylanase synthesis was restored in at least three clones, containing the same 3802 bp Sstl DNA fragment. In this fragment, several open reading frames (ORFs) have been identified, one of which coded for a xylanase; the products of the other ORFs did not show homology with any of the already known proteins. The complete nucleotide sequence of the 3802 bp Ssti insert has been determined on both strands. Xylanase is very probably synthesized as a 240 amino acid (aa) precursor (25949 Da) including a long (49 aa) signal sequence presenting significant similarity with the signal sequences of other Streptomyces xylanase genes. The xylanase aa sequence showed a clear homology with the aa sequences of other xylanases of the glycanase G family. The xln gene has been introduced into Streptomyces parvulus, a naturally xylanase-negative species. In contrast with its expression in Streptomyces sp. EC3, in S. parvulus, xln was expressed constitutively, a probable consequence of the absence of a regulatory system.

Cloning and sequencing of the dnaK locus in Streptomyces coelicolor A3(2).

The dnaK operon of Streptomyces coelicolor A3(2) was cloned by the DNA-probing method using synthetic oligonucleotides designed on the basis of two of the most conserved regions in 30 different DnaK proteins (HSP70). The isolated insert-a BamHI 5.6-kb fragment-was sequenced and shown to contain three open-reading frames organized in an operon and coding for proteins analogous to DnaK, GrpE and DnaJ, successively.

Regulation of the beta-lactamase BlaL of Streptomyces cacaoi: the product of the blaB regulatory gene is an internal membrane-bound protein.

The beta-lactamase-encoding gene blaL, cloned from Streptomyces cacaoi in Streptomyces lividans, is inducible by beta-lactam compounds. This regulation has been shown to depend on the products of two open reading frames, ORF1 (blaA) and ORF2 (blaB) [Lenzini, Magdalena, Fraipont, Joris, Matagne and Dusart (1992) Mol. Gen. Genet. 235, 41-48]. BlaA belongs to the LysR family of transcription activators, whereas BlaB shares some features with the penicillin-recognizing proteins. BlaB has now been overexpressed in Escherichia coli, purified and used for antibody preparation. Immunoblotting of cell-fractionated materials from S. cacaoi showed that BlaB is attached to the internal face of the cytoplasmic membrane. It could not be released by high salt concentrations or EDTA, but only by protease treatment. Under the assay conditions, BlaB did not act as a penicillin-binding protein, a beta-lactamase, a D-amino-peptidase or a target in a phosphorylation step.

Cloning and nucleotide sequence of a region of the Kibdelosporangium aridum genome homologous to polyketide biosynthetic genes.

The actinomycete Kibdelosporangium aridum naturally produces ardacin, a new glycopeptide antibiotic, the biosynthetic pathway of which should involve the participation of a polyketide synthase (PKS). A K. aridum 2.9 kb BamHI genomic fragment homologous to actI (a locus of the PKS cluster catalyzing polyketide chain assembly for actinorhodin biosynthesis in Streptomyces coelicolor) was isolated by shotgun cloning. This DNA fragment, called ardI, was sequenced and the deduced protein products were compared with those of other polyketide synthase genes, revealing similarities ranging from 50 to 80%. ardI was further used to probe a cosmid library of the K. aridum genome. Three hybridizing cosmids were obtained which contain overlapping inserts, together covering a 50 kb region, and including, 15 kb away from ardI, a fragment homologous to actIII, which codes for the ketoreductase of the actinorhodin PKS of S. coelicolor. All these findings indicate that at least part of a polyketide biosynthetic gene cluster has been isolated from the genome of the ardacin producer K. aridum.

Secretion by overexpression and purification of the water-soluble Streptomyces K15 DD-transpeptidase/penicillin-binding protein.

Though synthesized with a cleavable signal peptide and devoid of membrane anchors, the 262-amino-acid-residue Streptomyces K15 DD-transpeptidase/penicillin-binding protein is membrane-bound. Overexpression in Streptomyces lividans resulted in the export of an appreciable amount of the synthesized protein (4 mg/litre of culture supernatant). The water-soluble enzyme was purified close to protein homogeneity with a yield of 75%. It requires the presence of 0.5 M-NaCl to remain soluble. It is indistinguishable from the detergent-extract wild-type enzyme with respect to molecular mass, thermostability, transpeptidase activity and penicillin-binding capacity.

Two different beta-lactamase genes are present in Streptomyces cacaoi.

Two beta-lactamase genes called blaL and blaU have been cloned independently in Liège and in Umeå, from Streptomyces cacaoi. Genes blaL and blaU were found to differ largely in their nucleotide sequences, although the encoded proteins both belonged to the class A of beta-lactamases (active-site serine penicillinases). DNA-hybridization and polymerase chain reaction assays have now demonstrated that both blaL and blaU genes were present in the S. cacaoi strains used in Liège and in Umeå.