Development of a biological test to evaluate the bioavailability of iron in culture media.

AIMS: To develop an easy-to-use and pathogen-free protocol giving reliable information on the bioavailability of iron in a medium. METHODS AND RESULTS: In aerobic conditions, iron bioavailability is very low, and most of its forms cannot be assimilated by micro-organisms. Media with similar iron contents can differ considerably in iron bioavailability, something that is not easily achieved using conventional physicochemical methods. The assay developed in the present work is based on a pyoverdin siderophore release by fluorescent Pseudomonas in response to iron stress. CONCLUSIONS: The test was applied to a complex medium used for the production of diphtheria toxin (DT). A significant difference between the bioavailable iron level and the total chemical concentrations contributed by the various compounds used to make the medium could thus be detected. This can be explained by the formation of salt complexes trapping the iron, which thus cannot be used directly by the micro-organism for its metabolism. SIGNIFICANCE AND IMPACT OF THE STUDY: The assay can easily be applied to any medium designed for the production of iron-regulated compounds. This is particularly useful when dealing with processes that use pathogenic strains as was shown in the case based on DT production.

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.

Cloning and amplified expression in Streptomyces lividans of the gene encoding the extracellular beta-lactamase from Streptomyces cacaoi.

A 19 kb SphI DNA fragment containing the gene for the extracellular active-site serine beta-lactamase of Streptomyces cacaoi KCC-SO352 was cloned in Streptomyces lividans TK24 using the high-copy-number plasmid pIJ702 as vector. A 30-fold higher yield of beta-lactamase was obtained from S. lividans strain ML1, carrying the recombinant plasmid pDML51, than from S. cacaoi grown under optimal production conditions. In all respects (molecular mass, isoelectric point, kinetics of inhibition by beta-iodopenicillanate) the overproduced S. lividans ML1 beta-lactamase was identical to the original S. cacaoi enzyme. A considerable reduction of beta-lactamase production was caused by elimination of a 12.8 kb portion of the 19 kb DNA fragment by cleavage at an internal SphI site located more than 3 kb upstream of the beta-lactamase structural gene. The beta-lactamase gene was located within a 1.8 NcoI-BclI fragment but when this fragment was cloned in S. lividans pIJ702, the resulting strain produced hardly any more beta-lactamase than the original S. cacaoi.

The crystal structure of the beta-lactamase of Streptomyces albus G at 0.3 nm resolution.

The crystal structure of the beta-lactamase of Streptomyces albus G has been solved at 0.3 nm resolution by X-ray-diffraction methods. The enzyme is a typical two-domain protein. One domain consists of five alpha-helices, and the other is five-stranded beta-sheet with alpha-helices on both sides of the sheet. The active-site serine residue (Ser-48) is within a cleft located between the two domains.

Nucleotide sequence of the gene encoding the Streptomyces albus G beta-lactamase precursor.

A 1400-base DNA fragment, which contains the gene encoding the extracellular active-site serine beta-lactamase of Streptomyces albus G previously cloned into Streptomyces lividans [Dehottay et al. (1986) Gene 42, 31-36], was sequenced. The gene codes for a 314-amino-acid precursor, the N-terminal region of which has the characteristics of a signal peptide. The beta-lactamase as excreted by the host strain S. lividans PD6 has a ragged N-terminus, indicating either the presence of a leader peptidase of poor specificity or the action of an aminopeptidase. The primary structure (as deduced from the nucleotide sequence) was confirmed by amino acid sequencing of a 16-residue stretch at the amino terminus of the protein, a 12-residue stretch containing the active-site serine [De Meester et al. (1987) Biochem. J. 244, 427-432] and a 23-residue stretch obtained by trypsin digestion of the protein. The beta-lactamase belongs to class A, has three half-cystine residues (one of which occurs on the amino side of the active-site serine) and is inactivated by thiol reagents. Putative ribosome binding site and terminator region were identified.

The active sites of the beta-lactamases of Streptomyces cacaoi and Streptomyces albus G.

The active-site serine of the extracellular beta-lactamases of Streptomyces cacaoi and Streptomyces albus G has been labelled with beta-iodopenicillanate. The determination of the sequence of the labelled peptides obtained after trypsin digestion of the denatured proteins indicate both enzymes to be class A beta-lactamases. Surprisingly the two Streptomyces enzymes do not appear to be especially homologous, and none of them exhibited a high degree of homology with the Streptomyces R61 DD-peptidase. Our data confirm that, as a family of homologous enzymes, class A is rather heterogeneous, with only a small number of conserved residues in all members of the class.

Cloning and amplified expression in Streptomyces lividans of a gene encoding extracellular beta-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.