Jean Dusart

Subdivision of the helix-turn-helix GntR family of bacterial regulators in the FadR, HutC, MocR, and YtrA subfamilies

Haydon and Guest (Haydon, D. J, and Guest, J. R. (1991) FEMS Microbiol. Lett. 63, 291–295) first described the helix-turn-helix GntR family of bacterial regulators. They presented them as transcription factors sharing a similar N-terminal DNA-binding (d-b) domain, but they observed near-maximal divergence in the C-terminal effector-binding and oligomerization (E-b/O) domain. To elucidate this C-terminal heterogeneity, structural, phylogenetic, and functional analyses were performed on a family that now comprises about 270 members. Our comparative study first focused on the C-terminal E-b/O domains and next on DNA-binding domains and palindromic operator sequences, has classified the GntR members into four subfamilies that we called FadR, HutC, MocR, and YtrA. Among these subfamilies a degree of similarity of about 55% was observed throughout the entire sequence. Structure/function associations were highlighted although they were not absolutely stringent. The consensus sequences deduced for the DNA-binding domain were slightly different for each subfamily, suggesting that fusion between the D-b and E-b/O domains have occurred separately, with each subfamily having its own D-b domain ancestor. Moreover, the compilation of the known or predicted palindromiccis-acting elements has highlighted different operator sequences according to our subfamily subdivision. The observed C-terminal E-b/O domain heterogeneity was therefore reflected on the DNA-binding domain and on the cis-acting elements, suggesting the existence of a tight link between the three regions involved in the regulating process.

The diversity, structure and regulation of beta-lactamases.

Abstract.β-Lactamase production is responsible for the appearance of a large number of pathogenic bacterial strains exhibiting a high degree of resistance to β-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 β-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-β-lactamases, but the detailed catalytic mechanisms might also differ widely, depending on the enzyme studied. Similarly, the induction of the synthesis of β-lactamases is now better understood, but many questions remain to be answered.

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.

Cloning and amplified expression in Streptomyces lividans of a gene encoding extracellular β-lactamase from Streptomyces albus G

A 4.9-kb DNA fragment containing the bla gene for the extracellular beta-lactamase (BLA) of Streptomyces albus G was cloned in Streptomyces lividans using the conjugative, low-copy-number plasmid pIJ61 as vector. No expression of bla was observed when this DNA fragment was introduced into Escherichia coli HB101 on a plasmid vector. A 1.5-kb PstI-SstI fragment containing the bla gene was cloned in S. lividans on the nonconjugative, high-copy-number plasmid pIJ702. A tenfold higher yield of BLA was obtained from S. lividans carrying this plasmid than from S. albus G grown under optimal production conditions. The BLA from the clone reacts with beta-iodopenicillanate according to a branched pathway which is characteristic of the original S. albus G BLA enzyme.

Serine-Type D-Ala-D-Ala Peptidases and Penicillin-Binding Proteins

Publisher Summary This chapter describes serine-type D-Ala-D-Ala peptidases and penicillin-binding proteins. Penicillin is a suicide substrate. Because of the endocyclic nature of the scissile β-lactam amide bond, the leaving group of the enzyme acylation step remains part of the acyl enzyme. The first part only of the transfer cycle is achieved, leading to a long-lived; serine ester-linked acyl(penicilloyl)-enzyme and the enzyme behaves as a penicillin-binding protein (PBP). All bacteria possess an assortment of low and high molecular weight membrane-bound PBPs. The low molecular weight PBPs are single catalytic entities. The bulk of the protein is on the outer face of the plasma membrane and bears a carboxy-terminal extension, the end of which serves as membrane anchor. The low molecular weight PBPs helps to control the extent of wall peptidoglycan cross-linking throughout the life cycle of the cells. The high molecular weight PBPs involved in wall peptidoglycan assembly and cell morphogenesis are multimodule proteins. The bulk of the protein is on the outer face of the membrane and consists of an N-terminal module, fused to a C-terminal, penicillin-binding module.

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.

dd-Carboxypeptidase-Transpeptidase and Killing Site of β-Lactam Antibiotics in Streptomyces Strains R39, R61, and K11

Additional evidence is given that in Streptomyces strains R39, R61, and K11 the same enzyme performs dd-carboxypeptidase and transpeptidase activities and that this enzyme is the killing site of β-lactam antibiotics. With strain R61, it was found that the exocellular enzyme has a sensitivity towards some antibiotics different from that of the membrane-bound enzyme. Under the growth conditions used in the present investigations, β-lactamase activity was not involved in susceptibility to β-lactam antibiotics.

The penicillin receptor in Streptomyces

Penicillin kills bacteria by suppressing or decreasing the efficiency of the membrane-bound transpeptidase, which during the last steps of the wall synthesis catalyzes cross-linking between the peptide units of the nascent peptidoglycan and makes the polymer insoluble.l, 2 In addition to this specific receptor, other penicillin-binding sites also occur within the bacterial membrane~.3-~ These sites, or at least most of them, seem to be irrelevant as far as peptidoglycan synthesis is concerned.4 Although involved in antibiotic specificity, they do not appear to be the killing target of penicillin. At present, no transpeptidase has been isolated from bacterial membranes and characterized. For a long time, the technical limitation to such an achievement has resided in the lack of a suitable assay for transpeptidase activity. Cell-free particulate multienzyme preparations obtained from various bacteria were shown to catalyze the in vitro utilization of the nucleotide precursors UDP-N-acetylglucosamine and UDP-N-acetylmuramyl pentapeptide for the entire sequence of peptidoglycan synthesis, which includes the peptide crosslinking.g-15 These assays, however, were devised in such a way that they did not allow measurement of the transpeptidation reaction per se. Another approach was undertaken through a joint effort between our laboratories and was based on the development of well-defined systems of peptide donors and acceptors that could be used by bacterial transpeptidases for transpeptidation reactions and, hence, would allow these enzymes to be operative and tested independently of the preceding biosynthetic sequential reactions. Streptornyces sp were chosen as a model, because they had the property, probably unique in the bacterial world, of spontaneously excreting an enzyme that appeared to be a soluble form of the membrane-bound transpeptidase.

Cloning and nucleotide sequence of a xylanase-encoding gene from Sfrepfomyces 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.

Exocellular β-Lactamases of Streptomyces albus G and Strains R39 and K11

The β-lactamases excreted by the highly benzylpenicillin-susceptible Streptomyces strain R39 and the highly benzylpenicillin-resistant Streptomyces albus G were isolated and purified. Neither β-lactamase exhibited dd-carboxypeptidase activity. Both were anionic at pH 8.3, did not require metal ions, and were not sensitive to iodine, but were inhibited by Cu2+ and readily inactivated by heat. p-Chloromercuribenzoate, iodoacetate, p-aminobenzoate, and substrates and inhibitors of dd-carboxypeptidase had no effect on β-lactamase activity. The Km and Vmax values for β-lactamase activity were studied with 6-aminopenicillanic acid and with various penicillins and cephalosporins. The β-lactamase from the related strain K11 of Streptomyces, which is intermediate in its susceptibility to benzylpenicillin, was partially purified, and its activity was compared on the various substrates. Images