Corynebacterium rouxii sp. nov., a novel member of the diphtheriae species complex.

A group of six clinical isolates previously identified as Corynebacterium diphtheriae biovar Belfanti, isolated from human cutaneous or peritoneum infections and from one dog, were characterized by genomic sequencing, biochemical analysis and MALDI-TOF mass spectrometry. The six isolates were negative for the diphtheria toxin gene. Phylogenetic analyses showed that the six isolates (including FRC0190T) are clearly demarcated from C. diphtheriae, Corynebacterium belfantii, Corynebacterium ulcerans and Corynebacterium pseudotuberculosis. The average nucleotide identity of FRC0190T with C. diphtheriae NCTC11397T was 92.6%, and was 91.8% with C. belfantii FRC0043T. C. diphtheriae subsp. lausannense strain CHUV2995T appeared to be a later heterotypic synonym of C. belfantii (ANI, 99.3%). Phenotyping data revealed an atypical negative or heterogeneous intermediate maltose fermentation reaction for the six isolates. MALDI-TOF mass spectrometry differentiated the new group from the other Corynebacterium taxa by the presence of specific spectral peaks. rpoB sequences showed identity to atypical, maltose-negative C. diphtheriae biovar Belfanti isolates previously described from two cats in the USA. We propose the name Corynebacterium rouxii sp. nov. for the novel group, with FRC0190T (= CIP 111752T = DSM 110354T) as type strain.

Second-generation IL-2 receptor-targeted diphtheria fusion toxin exhibits antitumor activity and synergy with anti-PD-1 in melanoma.

Denileukin diftitox (DAB-IL-2, Ontak) is a diphtheria-toxin-based fusion protein that depletes CD25-positive cells including regulatory T cells and has been approved for the treatment of persistent or recurrent cutaneous T cell lymphoma. However, the clinical use of denileukin diftitox was limited by vascular leak toxicity and production issues related to drug aggregation and purity. We found that a single amino acid substitution (V6A) in a motif associated with vascular leak induction yields a fully active, second-generation biologic, s-DAB-IL-2(V6A), which elicits 50-fold less human umbilical vein endothelial cell monolayer permeation and is 3.7-fold less lethal to mice by LD50 analysis than s-DAB-IL-2. Additionally, to overcome aggregation problems, we developed a production method for the fusion toxin using Corynebacterium diphtheriae that secretes fully folded, biologically active, monomeric s-DAB-IL-2 into the culture medium. Using the poorly immunogenic mouse B16F10 melanoma model, we initiated treatment 7 days after tumor challenge and observed that, while both s-DAB-IL-2(V6A) and s-DAB-IL-2 are inhibitors of tumor growth, the capacity to treat with higher doses of s-DAB-IL-2(V6A) could provide a superior activity window. In a sequential dual-therapy study in tumors that have progressed for 10 days, both s-DAB-IL-2(V6A) and s-DAB-IL-2 given before checkpoint inhibition with anti-programmed cell death-1 (anti-PD-1) antibodies inhibited tumor growth, while either drug given as monotherapy had less effect. s-DAB-IL-2(V6A), a fully monomeric protein with reduced vascular leak, is a second-generation diphtheria-toxin-based fusion protein with promise as a cancer immunotherapeutic both alone and in conjunction with PD-1 blockade.

Hydrogen-Deuterium Exchange Epitope Mapping Reveals Distinct Neutralizing Mechanisms for Two Monoclonal Antibodies against Diphtheria Toxin.

The diphtheria toxoid (DT) antigen is one of the major components in pediatric and booster combination vaccines and is known to raise a protective humoral immune response upon vaccination. However, a structurally resolved analysis of diphtheria toxin (DTx) epitopes with underlying molecular mechanisms of antibody neutralization has not yet been reported. Using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and Biolayer Interferometry (BLI) assays, we have characterized two neutralizing anti-DTx monoclonal antibodies (mAbs), 2-25 and 2-18, by identifying the specific epitopes on the diphtheria toxin responsible for antibody binding. Our results show that both epitopes are conformational, and mechanistically distinct. Monoclonal antibody 2-25 binds selectively to the B-subunit (translocation and receptor domain) of DTx, blocking the heparin-binding EGF-like growth factor (HBEGF) binding site. In contrast, mAb 2-18 binds to the A-subunit (catalytic domain), partially covering the catalytic loop region that shuttles NAD during catalysis. The results are discussed in the context of antigen neutralization mechanisms and can ultimately help to reveal the underlying factors that contribute to Diptheria vaccine efficacy.

Characterization of the second conserved domain in the heme uptake protein HtaA from Corynebacterium diphtheriae.

HtaA is a heme-binding protein that is part of the heme uptake system in Corynebacterium diphtheriae. HtaA contains two conserved regions (CR1 and CR2). It has been previously reported that both domains can bind heme; the CR2 domain binds hemoglobin more strongly than the CR1 domain. In this study, we report the biophysical characteristics of HtaA-CR2. UV-visible spectroscopy and resonance Raman experiments are consistent with this domain containing a single heme that is bound to the protein through an axial tyrosine ligand. Mutants of conserved tyrosine and histidine residues (Y361, H412, and Y490) have been studied. These mutants are isolated with very little heme (≤5%) in comparison to the wild-type protein (~20%). Reconstitution after removal of the heme with butanone gave an alternative form of the protein. The HtaA-CR2 fold is very stable; it was necessary to perform thermal denaturation experiments in the presence of guanidinium hydrochloride. HtaA-CR2 unfolds extremely slowly; even in 6.8M GdnHCl at 37°C, the half-life was 5h. In contrast, the apo forms of WT HtaA-CR2 and the aforementioned mutants unfolded at much lower concentrations of GdnHCl, indicating the role of heme in stabilizing the structure and implying that heme transfer is effected only to a partner protein in vivo.

CnaA domains in bacterial pili are efficient dissipaters of large mechanical shocks.

Pathogenic bacteria adhere despite severe mechanical perturbations induced by the host, such as coughing. In Gram-positive bacteria, extracellular protein appendages termed pili are necessary for adherence under mechanical stress. However, little is known about the behavior of Gram-positive pili under force. Here, we demonstrate a mechanism by which Gram-positive pili are able to dissipate mechanical energy through mechanical unfolding and refolding of isopeptide bond-delimited polypeptide loops present in Ig-type CnaA domains. Using single-molecule force spectroscopy, we find that these loops of the pilus subunit SpaA of the SpaA-type pilus from Corynebacterium diphtheriae and FimA of the type 2 pilus from Actinomyces oris unfold and extend at forces that are the highest yet reported for globular proteins. Loop refolding is limited by the hydrophobic collapse of the polypeptide and occurs in milliseconds. Remarkably, both SpaA and FimA initially refold to mechanically weaker intermediates that recover strength with time or ligand binding. Based on the high force extensibility, CnaA-containing pili can dissipate ∼28-fold as much energy compared with their inextensible counterparts before reaching forces sufficient to cleave covalent bonds. We propose that efficient mechanical energy dissipation is key for sustained bacterial attachment against mechanical perturbations.

EFFECT OF DIPHTHERIA TOXIN T-DOMAIN ON ENDOSOMAL pH.

A key step in the mode of cytotoxic action of diphtheria toxin (DT) is the transfer of its catalytic domain (Cd) from endosomes into the cytosol. The main activity in this process is performed by the transport domain (Td), but the molecular mechanism of its action remains unknown. We have previously shown that Td can have some influence on the endosomal transport of DT The aim of this work was to study the effect of diphtheria toxin on the toxin compartmentalization in the intracellular transporting pathway and endosomal pH. We used recombinant fragments of DT which differed only by the presence of Td in their structure, fused with fluorescent proteins. It was shown that the toxin fragment with Td moved slower by the pathway early-late endosomes-lysosomes, and had a slightly different pattern of colocalization with endosomal markers than DT fragment without Td. In addition, endosomes containing DT fragments with Td had a constant pH of about 6.5 from the 10th to 50th minute of observation, for the same time endosomes containing DT fragments without Td demonstrated a decrease in pH from 6.3 to 5.5. These results indicate that Td inhibits acidification of endosomal medium. One of possible explanations for this may be the effect of the ion channel formed by the T-domain on the process of the endosomal acidification. This property of Td may not only inhibit maturation of endosomes but also inhibit activation of endosomal pH-dependent proteases, and this promotes successful transport of Cd into the cell cytosol.

Structure of the response regulator ChrA in the haem-sensing two-component system of Corynebacterium diphtheriae.

ChrA is a response regulator (RR) in the two-component system involved in regulating the degradation and transport of haem (Fe-porphyrin) in the pathogen Corynebacterium diphtheriae. Here, the crystal structure of full-length ChrA is described at a resolution of 1.8 Å. ChrA consists of an N-terminal regulatory domain, a long linker region and a C-terminal DNA-binding domain. A structural comparison of ChrA with other RRs revealed substantial differences in the relative orientation of the two domains and the conformation of the linker region. The structural flexibility of the linker could be an important feature in rearrangement of the domain orientation to create a dimerization interface to bind DNA during haem-sensing signal transduction.

Structure of a DsbF homologue from Corynebacterium diphtheriae.

Disulfide-bond formation, mediated by the Dsb family of proteins, is important in the correct folding of secreted or extracellular proteins in bacteria. In Gram-negative bacteria, disulfide bonds are introduced into the folding proteins in the periplasm by DsbA. DsbE from Escherichia coli has been implicated in the reduction of disulfide bonds in the maturation of cytochrome c. The Gram-positive bacterium Mycobacterium tuberculosis encodes DsbE and its homologue DsbF, the structures of which have been determined. However, the two mycobacterial proteins are able to oxidatively fold a protein in vitro, unlike DsbE from E. coli. In this study, the crystal structure of a DsbE or DsbF homologue protein from Corynebacterium diphtheriae has been determined, which revealed a thioredoxin-like domain with a typical CXXC active site. Structural comparison with M. tuberculosis DsbF would help in understanding the function of the C. diphtheriae protein.

Tyrosine oxidation in heme oxygenase: examination of long-range proton-coupled electron transfer.

Heme oxygenase is responsible for the degradation of a histidine-ligated ferric protoporphyrin IX (Por) to biliverdin, CO, and the free ferrous ion. Described here are studies of tyrosyl radical formation reactions that occur after oxidizing Fe(III)(Por) to Fe(IV)=O(Por(·+)) in human heme oxygenase isoform-1 (hHO-1) and the structurally homologous protein from Corynebacterium diphtheriae (cdHO). Site-directed mutagenesis on hHO-1 probes the reduction of Fe(IV)=O(Por(·+)) by tyrosine residues within 11 Å of the prosthetic group. In hHO-1, Y58· is implicated as the most likely site of oxidation, based on the pH and pD dependent kinetics. The absence of solvent deuterium isotope effects in basic solutions of hHO-1 and cdHO contrasts with the behavior of these proteins in the acidic solution, suggesting that long-range proton-coupled electron transfer predominates over electron transfer.

[Mechanisms of formation of post-vaccinal immune response in children immunized with APDT and ADT-M preparations].

AIM: Study the mechanisms of formation of cell and humoral immunity against pertussis, diphtheria and tetanus in children immunized with immunobiological preparations (APDT vaccine and ADT anatoxin). MATERIALS AND METHODS: 30 practically healthy children (6 - 9 years of age) immunized with APDT and ADT-M preparations had TLR2, TLR4 expression determined in mononuclear cells (MNC). Vaccine preparations (APDT, ADT-M, AD-M, AT) and Corynebacterium diphtheriae gravis tox+, C. diphtheriae mitis tox- and Bordetella pertussis 345 were used as ligands. Cytokine production was determined in EIA. Content of anti-diphtheria, anti-tetanus and anti-pertussis antibodies--by PHA reaction and EIA. RESULTS: During stimulation with vaccines and B. pertussis 345 strain MNC were characterized by an increase (p < 0.05) of expression level of TLR2 and TLR4 and did not respond to stimulation with C. diphtheriae gravis tox+ and C. diphtheriae mitis tox- strains. Similar results were obtained during study of cytokine production (TNFalpha, IL-1, IL-6). A direct correlation between levels of antitoxic antibodies against diphtheria and tetanus (R = 0.486), antibacterial antibodies against pertussis and diphtheria was detected (R = 0.529). CONCLUSION: Analysis of cytokine production profile and determination of surface TLR expression can be used during evaluation of functional status of innate immunity cells and intensity of post-vaccinal immunity.

A slow-forming isopeptide bond in the structure of the major pilin SpaD from Corynebacterium diphtheriae has implications for pilus assembly.

The Gram-positive organism Corynebacterium diphtheriae, the cause of diphtheria in humans, expresses pili on its surface which it uses for adhesion and colonization of its host. These pili are covalent protein polymers composed of three types of pilin subunit that are assembled by specific sortase enzymes. A structural analysis of the major pilin SpaD, which forms the polymeric backbone of one of the three types of pilus expressed by C. diphtheriae, is reported. Mass-spectral and crystallographic analysis shows that SpaD contains three internal Lys-Asn isopeptide bonds. One of these, shown by mass spectrometry to be located in the N-terminal D1 domain of the protein, only forms slowly, implying an energy barrier to bond formation. Two crystal structures, of the full-length three-domain protein at 2.5 Å resolution and of a two-domain (D2-D3) construct at 1.87 Å resolution, show that each of the three Ig-like domains contains a single Lys-Asn isopeptide-bond cross-link, assumed to give mechanical stability as in other such pili. Additional stabilizing features include a disulfide bond in the D3 domain and a calcium-binding loop in D2. The N-terminal D1 domain is more flexible than the others and, by analogy with other major pilins of this type, the slow formation of its isopeptide bond can be attributed to its location adjacent to the lysine used in sortase-mediated polymerization during pilus assembly.

Structures of the substrate-free and product-bound forms of HmuO, a heme oxygenase from corynebacterium diphtheriae: x-ray crystallography and molecular dynamics investigation.

Heme oxygenase catalyzes the degradation of heme to biliverdin, iron, and carbon monoxide. Here, we present crystal structures of the substrate-free, Fe(3+)-biliverdin-bound, and biliverdin-bound forms of HmuO, a heme oxygenase from Corynebacterium diphtheriae, refined to 1.80, 1.90, and 1.85 Å resolution, respectively. In the substrate-free structure, the proximal and distal helices, which tightly bracket the substrate heme in the substrate-bound heme complex, move apart, and the proximal helix is partially unwound. These features are supported by the molecular dynamic simulations. The structure implies that the heme binding fixes the enzyme active site structure, including the water hydrogen bond network critical for heme degradation. The biliverdin groups assume the helical conformation and are located in the heme pocket in the crystal structures of the Fe(3+)-biliverdin-bound and the biliverdin-bound HmuO, prepared by in situ heme oxygenase reaction from the heme complex crystals. The proximal His serves as the Fe(3+)-biliverdin axial ligand in the former complex and forms a hydrogen bond through a bridging water molecule with the biliverdin pyrrole nitrogen atoms in the latter complex. In both structures, salt bridges between one of the biliverdin propionate groups and the Arg and Lys residues further stabilize biliverdin at the HmuO heme pocket. Additionally, the crystal structure of a mixture of two intermediates between the Fe(3+)-biliverdin and biliverdin complexes has been determined at 1.70 Å resolution, implying a possible route for iron exit.

[Macrophage apoptosis as a mechanism of pathogenic effect of diphtheria infectious agent].

AIM: Study of the apoptogenic effect of Corynebacterium diphtheriae toxigenic strains on mice peritoneal macrophages in vitro. MATERIALS AND METHODS: Evaluation ofapoptosis induced by Corynebacterium diphtheriae, Corynebacterium pseudodiphtheriticum, Staphylococcus aureus, Streptococcus pyogenes strains was performed by characteristic morphological changes in macrophages in smears stained by azure eosin by Romanovsky-Giemsa. RESULTS: Apoptogenic activity of diphtheria infectious agent was established to be determined by diphtheria exotoxin at early (after 1 hour) and surface structures and pathogenicity enzymes at later (3 hours) stages of effect. CONCLUSION: The ability of diphtheria infectious agent to cause macrophage apoptosis is one of the mechanisms of realization of its pathogenic properties determined by the effect of diphtheria exotoxin, as well as its surface structures and pathogenicity enzymes. The increase of apoptogenic activity of toxigenic strains of C. diphtheriae in association with S. pyogenes may be a pathogenetic base of formation of prolonged forms of bacteria carriage against the background of chronic ENT pathology.

Cell surface display of minor pilin adhesins in the form of a simple heterodimeric assembly in Corynebacterium diphtheriae.

Pilus assembly in Gram-positive bacteria occurs by a two-step mechanism, whereby pilins are polymerized and then covalently anchored to the cell wall. In Corynebacterium diphtheriae, the pilin-specific sortase SrtA catalyses polymerization of the SpaA-type pilus, consisting of the shaft pilin SpaA, tip pilin SpaC and minor pilin SpaB. Cell wall anchoring of the SpaA polymers is triggered when SrtA incorporates SpaB into the pilus base via lysine-mediated transpeptidation; anchoring to the cell wall peptidoglycan is subsequently catalysed by the housekeeping sortase SrtF. Here we show that SpaB and SpaC formed a heterodimer independent of SpaA polymerization. SrtA was absolutely required for the formation of the SpaBC heterodimer, while SrtF facilitated the optimal cell wall anchoring of this heterodimer. Alanine substitution of the SpaB lysine residue K139 or truncation of the SpaB cell wall-sorting signal (CWSS) abolished assembly of the SpaBC heterodimer, hence underscoring SpaB function in transpeptidation and cell wall linkage. Importantly, sortase specificity for the cell wall-anchoring step was found to be dependent on the LAFTG motif within the SpaB CWSS. Thus, C. diphtheriae employs a common sortase-catalysed mechanism involving lysine-mediated transpeptidation to generate both adhesive pilus and simple heterodimeric structures on the bacterial the cell wall.

Expression, purification, crystallization and preliminary crystallographic analysis of SpaA, a major pilin from Corynebacterium diphtheriae.

Bacterial pili are cell-surface organelles that are critically involved in adhesion to host cells, leading to the colonization of host tissues and the establishment of infections. Whereas the pili of Gram-negative bacteria have been extensively studied, those of Gram-positive bacteria came to light only recently after the discovery and characterization of Corynebacterium diphtheriae pili. These newly discovered pili are formed by the covalent polymerization of pilin subunits catalyzed by sortase enzymes, making them fundamentally different from the noncovalent pilin assemblies of Gram-negative bacteria. Here, the expression, crystallization and preliminary crystallographic analysis of SpaA, which forms the shaft of one of the three types of pili expressed by C. diphtheriae, are reported. SpaA(53-486) crystals diffracted to 1.6 A resolution and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 34.9, b = 64.1, c = 198.7 A, alpha = beta = gamma = 90 degrees .

Novel lipoarabinomannan-like lipoglycan (CdiLAM) contributes to the adherence of Corynebacterium diphtheriae to epithelial cells.

The genus Corynebacterium is part of the phylogenetic group nocardioform actinomycetes. Members of this group have a characteristic cell envelope structure composed primarily of branched long-chain lipids, termed mycolic acids, and a rich number of lipoglycans such as lipoarabinomanans (LAM) and lipomannans. In this study, we identified a novel LAM variant isolated from Corynebacterium diphtheriae named CdiLAM. The key structural features of CdiLAM are a linear alpha-1-->6-mannan with side chains containing 2-linked alpha-D-Manp and 4-linked alpha-D-Araf residues. The polysaccharide backbone is linked to a phosphatidylinositol anchor. In contrast to the LAMs of other members of actinomycetales, CdiLAM presents an unusual substitution at position 4 of alpha-1-->6-mannan backbone by alpha-D-Araf. Unlike the non-fimbrial adhesin 62-72p, CdiLAM did not function as a hemagglutinin to human red blood cells. Experimental evidences pointed to CdiLAM as an adhesin of C. diphtheriae to human respiratory epithelial cells, thereby, contributing to the pathogenesis of diphtheria.

Mechanism of metal ion activation of the diphtheria toxin repressor DtxR.

The diphtheria toxin repressor (DtxR) is a metal ion-activated transcriptional regulator that has been linked to the virulence of Corynebacterium diphtheriae. Structure determination has shown that there are two metal ion binding sites per repressor monomer, and site-directed mutagenesis has demonstrated that binding site 2 (primary) is essential for recognition of the target DNA repressor, leaving the role of binding site 1 (ancillary) unclear. Calorimetric techniques have demonstrated that although binding site 1 (ancillary) has high affinity for metal ion with a binding constant of 2 x 10(-7), binding site 2 (primary) is a low-affinity binding site with a binding constant of 6.3 x 10(-4). These two binding sites act in an independent fashion, and their contribution can be easily dissected by traditional mutational analysis. Our results clearly demonstrate that binding site 1 (ancillary) is the first one to be occupied during metal ion activation, playing a critical role in stabilization of the repressor. In addition, structural data obtained for the mutants Ni-DtxR(H79A,C102D), reported here, and the previously reported DtxR(H79A) have allowed us to propose a mechanism of metal activation for DtxR.

Analysis of a DtxR-like metalloregulatory protein, MntR, from Corynebacterium diphtheriae that controls expression of an ABC metal transporter by an Mn(2+)-dependent mechanism.

The DtxR protein is a global iron-dependent repressor in Corynebacterium diphtheriae that regulates transcription from multiple promoters. A search of the partially completed C. diphtheriae genome identified a gene, mntR, whose predicted product has significant homology with the DtxR repressor protein. The mntR gene is the terminal gene in a five-gene operon that also carries the mntABCD genes, whose predicted products are homologous to ABC metal transporters. Transcription of this genetic system, as measured by expression of an mntA-lacZ reporter fusion, is strongly repressed by Mn(2+). The divalent metals Fe(2+), Cu(2+), and Zn(2+) did not repress expression of the mntA-lacZ construct. A mutation in the mntR gene abolished Mn(2+)-dependent repression of the mntA-lacZ fusion, demonstrating that MntR is essential for the Mn(2+)-dependent regulation of this promoter. Footprinting experiments showed that MntR protects from DNase I digestion an approximately 73-bp AT-rich region that includes the entire mntA promoter. This large region protected from DNase I suggests that as many as three MntR dimer pairs may bind to this region. Binding studies also revealed that DtxR failed to bind to the MntR binding site and that MntR exhibited weak and diffuse binding at the DtxR binding site at the tox promoter. A C. diphtheriae mntA mutant grew as well as the wild type in a low-Mn(2+) medium, which suggests that the mntABCD metal transporter is not required for growth in a low-Mn(2+) medium and that additional Mn(2+) transport systems may be present in C. diphtheriae. This study reports the characterization of MntR, a Mn(2+)-dependent repressor, and the second member of the family of DtxR-like metalloregulatory proteins to be identified in C. diphtheriae.

Corynebactin and a serine trilactone based analogue: chirality and molecular modeling of ferric complexes.

Because the hydrolysis of ferric ion makes it very insoluble in aerobic, near neutral pH environments, most species of bacteria produce siderophores to acquire iron, an essential nutrient. The chirality of the ferric siderophore complex plays an important role in cell recognition, uptake, and utilization. Corynebactin, isolated from Gram-positive bacteria, is structurally similar to enterobactin, a well-known siderophore first isolated from Gram-negative bacteria, but contains L-threonine instead of L-serine in the trilactone backbone. Corynebactin also contains a glycine spacer unit in each of the chelating arms. A hybrid analogue (serine-corynebactin) has been prepared which has the trilactone ring of enterobactin and the glycine spacer of corynebactin. The chirality and relative conformational stability of the three ferric complexes of enterobactin, corynebactin, and the hybrid have been investigated by molecular modeling (including MM3 and pBP86/DN density functional theory calculations) and circular dichroism spectra. While enterobactin forms a Delta-ferric complex, corynebactin is Lambda. The hybrid serine-corynebactin forms a nearly racemic mixture, with the Lambda-conformer in slight excess. Each ferric complex has four possible isomers depending on the metal chirality and the conformation of the trilactone ring. For corynebactin, the energy difference between the two possible Lambda conformations is 2.3 kcal/mol. In contrast, only 1.5 kcal/mol separates the inverted Lambda- and normal Delta-configuration for serine-corynebactin. The small energy difference of the two lowest energy configurations is the likely cause for the racemic mixture found in the CD spectra. Both the addition of a glycine spacer and methylation of the trilactone ring (serine to threonine) favor the Lambda-conformation. These structural changes suffice to change the chirality from all Delta (enterobactin) to all Lambda (corynebactin). The single change (glycine spacer) of the hybrid ferric serine-corynebactin gives a mixture of Delta and Lambda, with the Lambda in slight excess.

Efficient electrotransformation of corynebacterium diphtheriae with a mini-replicon derived from the Corynebacterium glutamicum plasmid pGA1.

Efficient transformation of the human pathogen Corynebacterium diphtheriae was achieved with novel cloning vectors consisting of a mini-replicon from the cryptic C. glutamicum plasmid pGA1 as well as of the aph(3')-IIa or tetA(Z ) antibiotic resistance genes. Plasmid-containing transformants of C. diphtheriae were recovered at frequencies ranging from 1.3 x 10(5) to 4.8 x 10(6) colony forming units (cfu)/microg of plasmid DNA. Vector DNA was directly transferred from Escherichia coli into C. diphtheriae with frequencies up to 5.6 x 10(5) cfu/microg of plasmid DNA. On the basis of the pGA1 mini-replicon, an expression vector system was established for C. diphtheriae by means of the P(tac) promoter and the green fluorescent reporter protein. In addition, other commonly used vector systems from C. glutamicum, including the pBL1 and pHM1519 replicons, and the sacB conditionally lethal selection marker from Bacillus subtilis, were shown to be functional in C. diphtheriae. Thus, the ability to apply the standard methods of C. glutamicum recombinant DNA technology will greatly facilitate the functional analysis of the recently completed C. diphtheriae genome sequence.