The genome stability in Corynebacterium species due to lack of the recombinational repair system

Corynebacterium species are members of gram-positive bacteria closely related to Mycobacterium species, both of which are classified into the same taxonomic order Actinomycetales. Recently, three corynebacteria, Corynebacterium efficiens, Corynebacterium glutamicum, and Corynebacterium diphtheriae have been sequenced independently. We found that the order of orthologous genes in these species has been highly conserved though it has been disrupted in Mycobacterium species. This synteny suggests that corynebacteria have rarely undergone extensive genome rearrangements and have maintained ancestral genome structures even after the divergence of corynebacteria and mycobacteria. This is the first report that the genome structures have been conserved in free-living bacteria such as C. efficiens and C. glutamicum, although it has been reported that obligate parasites such as Mycoplasma and Chlamydia have the stable genomes. The comparison of recombinational repair systems among the three corynebacteria and Mycobacterium tuberculosis suggested that the absence of recBCD genes in corynebacteria be responsible for the suppression of genome shuffling in the species. The genome stability in Corynebacterium species will give us hints of the speciation mechanism with the non-shuffled genome, particularly the importance of horizontal gene transfer and nucleotide substitution in the genome.     

Ammonium assimilation and nitrogen control in Corynebacterium glutamicum and its relatives: an example for new regulatory mechanisms in actinomycetes

Nitrogen is an essential component of nearly all complex macromolecules in a bacterial cell, such as proteins, nucleic acids and cell wall components. Accordingly, most prokaryotes have developed elaborate control mechanisms to provide an optimal supply of nitrogen for cellular metabolism and to cope with situations of nitrogen limitation. In this review, recent advances in our knowledge of ammonium uptake, its assimilation, and related regulatory systems in Corynebacterium glutamicum, a Gram-positive soil bacterium used for the industrial production of amino acids, are summarized and discussed with respect to the situation in the bacterial model organisms, Escherichia coli and Bacillus subtilis, and in comparison to the situation in other actinomycetes, namely in mycobacteria and streptomycetes. The regulatory network of nitrogen control in C. glutamicum seems to be a patchwork of different elements. It includes proteins similar to the UTase/GlnK pathway of E. coli and expression regulation by a repressor protein as in B. subtilis, but it lacks an NtrB/NtrC two-component signal transduction system. Furthermore, the C. glutamicum regulation network has unique features, such as a new sensing mechanism. Based on its extremely well-investigated central metabolism, well-established molecular biology tools, a public genome sequence and a newly-established proteome project, C. glutamicum seems to be a suitable model organism for other corynebacteria, such as Corynebacterium diphtheriae and Corynebacterium efficiens.     

Plasmids of corynebacteria

Corynebacteria are pleomorphic, asporogenous, Gram-positive bacteria. Included in this group are nonpathogenic soil corynebacteria, which are widely used for the industrial production of amino acids and detergents, and in biotransformation of steroids. Other members of this group are plant and animal pathogens. This review summarizes the current information available about the plasmids of corynebacteria. The emphasis is mainly on the small plasmids, which have been used for construction of vectors for expression of genes in these bacteria. Moreover, considerable information is now available on their nucleotide sequence, gene organization and modes of replication, which would make it possible to further manipulate these plasmids. Other plasmid properties, such as incompatibility and host range, are also discussed. Finally, use of these plasmids as cloning vectors for the expression of heterologous proteins using corynebacteria as hosts is also summarized to highlight the potential of these bacteria as hosts for recombinant DNA.     

Contribution to the taxonomy of haemolytic corynebacteria

In an attempt to assess the taxonomic relationships among human (Corynebacterium haemolyticum), animal (Corynebacterium pyogenes bovis) haemolytio corynebacteria, typical corynebacteria (Corynebacterium diphteriae mitis, C. ovis, C. ulcerans) and group A and G streptococci, a number of biochemical parameters were established: the DNA content of G + C, the presence of the cytochrome system, composition of fatty acids in free lipids and production of carboxylic acids as end products of fermentation. It was found that according to the above criteria, streptococci differed significantly from the corynebacteria studied. In addition, it was possible to differentiate a subgroup of typically aerobic haemolytic corynebacteria (different from both human and animal corynebacteria), possessing a complete cytochrome system, producing propionic acid and having a different composition of fatty acids.     

Molecular control mechanisms of lysine and threonine biosynthesis in amino acid-producing corynebacteria: Redirecting carbon flow

Abstract Threonine and lysine are two of the economically most important essential amino acids. They are produced industrially by species of the genera Corynebacterium and Brevibacterium . The branched biosynthetic pathway of these amino acids in corynebacteria is unusual in gene organization and in the control of key enzymatic steps with respect to other microorganisms. This article reviews the molecular control mechanisms of the biosynthetic pathways leading to threonine and lysine in corynebacteria, and their implications in the production of these amino acids. Carbon flux can be redirected at branch points by gene disruption of the competing pathways for lysine or threonine. Removal of bottlenecks has been achieved by amplification of genes which encode feedback resistant aspartokinase and homoserine dehydrogenase (obtained by in vitro directed mutagenesis).     

Evaluation of invertebrate infection models for pathogenic corynebacteria

For several pathogenic bacteria, model systems for host-pathogen interactions were developed, which provide the possibility of quick and cost-effective high throughput screening of mutant bacteria for genes involved in pathogenesis. A number of different model systems, including amoeba, nematodes, insects, and fish, have been introduced, and it was observed that different bacteria respond in different ways to putative surrogate hosts, and distinct model systems might be more or less suitable for a certain pathogen. The aim of this study was to develop a suitable invertebrate model for the human and animal pathogens Corynebacterium diphtheriae, Corynebacterium pseudotuberculosis, and Corynebacterium ulcerans. The results obtained in this study indicate that Acanthamoeba polyphaga is not optimal as surrogate host, while both Caenorhabtitis elegans and Galleria larvae seem to offer tractable models for rapid assessment of virulence between strains. Caenorhabtitis elegans gives more differentiated results and might be the best model system for pathogenic corynebacteria, given the tractability of bacteria and the range of mutant nematodes available to investigate the host response in combination with bacterial virulence. Nevertheless, Galleria will also be useful in respect to innate immune responses to pathogens because insects offer a more complex cell-based innate immune system compared with the simple innate immune system of C.?elegans.     

Identification of the emerging skin pathogen Corynebacterium amycolatum using PCR-amplification of the essential divIVA gene as a target

The actinomycete Corynebacterium amycolatum is a saprophytic bacterium usually associated with the human skin, but it is at present considered an emergent pathogen as it is isolated from nosocomial settings from samples of immunosuppressed patients. The conventional method to distinguish C. amycolatum from closely related species is mainly based on phenotypic or chemotaxonomic studies. We developed a molecular method to identify rapidly C. amycolatum based on the use of different primers for amplification of the cell division divIVA gene using conventional or real-time PCR. This technique was used for the first time to distinguish C. amycolatum from the closely related Corynebacterium striatum, Corynebacterium minutissimum and Corynebacterium xerosis, without the requirement of further molecular analysis. The suitability of the identification method was tested on 51 clinical isolates belonging to the nonlipophilic fermentative group of corynebacteria (cluster C. striatum/C. amycolatum), which were accurately characterized by sequencing a 0.8 kb fragment of the 16S rRNA gene.     

Infection et fertilité —Corynebacterium seminale: point de vue du bactériologiste

The authors present two independent studies designed to identify corynebacteria isolated from the semen of patients consulting for infertility. Corynebacteria were identified by conventional biochemical and physiological tests and by determination of volatile fatty acids. In the first study based on 420 patients, the commonest species were Corynebacterium seminale (synonym C. glucuronolyticum) found in 7.4% of specimens, CDC group G (5%) and C. amycolatum (3.8%). Of the 92 semen specimens with more than 103 cfu/ml, 44 were positive for corynebacteria, including 15 C. seminale strains, whereas streptococci, staphylococci and enterobacteriacae were found in 23, 18 and 6 of the 420 specimens, respectively. The presence of C. seminale was more frequently associated with a high bacteria count than the other corynebacteria (p<0.02). In the second study, we compared the presence of corynebacteria in the semen of 1,902 patients with semen indices. C. seminale was present at levels greater than 103 cfu/ml in 2.7% of these specimens, while several other species of corynebacteria were detected in 5.3% of cases. Normal motility was found in only 25.4% of semen specimens with a high C. seminale count in contrast with 45% of specimens containing similar counts of other corynebacteria. These studies demonstrate that the isolation rates from human genital specimens and their clinical implications are different according to the species isolated. Microbiologists should be aware of the need to accurately identify these corynebacteria for further in vitro or in vivo studies on genital infections.     

Similarity of rpoB gene sequences of sucrose-fermenting and non-fermenting Corynebacterium diphtheriae strains

During the last decades, the majority of Brazilian Corynebacterium diphtheriae isolates were shown to be capable to metabolize sucrose, sometimes leading to erroneous identification as a non-diphtheric Corynebacterium species. The sequencing of the polymorphic region of the RNA polymerase beta subunit-encoding gene (rpoB) is an important taxonomic tool for identification of corynebacteria. The present study aimed to investigate the rpoB gene polymorphic features of sucrose-fermenting and non sucrose-fermenting strains. The results showed that sucrose-fermenting strains presented rpoB gene polymorphic regions with more than 98% similarity with the sequences deposited in the gene bank corresponding to non sucrose-fermenting strains. Data indicate that sucrose-fermenting isolates may act as a variant of C. diphtheriae biotype mitis. In addition we alert that sucrose-fermenting strains should not be discarded as contaminants mainly in countries where the possibility of isolation of this variant is higher.     

Relationship between group JK corynebacteria and the biotypes of Corynebacterium genitalium and Corynebacterium pseudogenitalium

Twenty-six strains of group JK corynebacteria had the same colonial morphology and biological reactions as the biotypes of the biovars of Corynebacterium genitalium and C. pseudogenitalium. Therefore, group JK corynebacteria can be assigned to the biovars of C. genitalium or C. pseudogenitalium. Although the strains differed in sensitivity to 16 antibiotics tested by Sensi-Discs or by the Micro-Media technique, they are uniformly sensitive to 4-5 micrograms/mL of vancomycin. Medium containing 10 micrograms vancomycin/mL was bactericidal and the killing time was dependent on the concentration. The rate of mutation to resistance to 10 micrograms vancomycin was greater than 1 in 10(10) corynebacteria. Therefore, vancomycin sensitivity is a stable characteristic of these corynebacteria which also indicates that group JK corynebacteria are strains of either C. genitalium or C. pseudogenitalium. Since group JK corynebacteria are considered pathogens, this finding supports the belief that C. genitalium is a pathogen and suggests that some biotypes of the commensal C. pseudogenitalium may infect compromised hosts.     

Complete genome sequence and analysis of the multiresistant nosocomial pathogen Corynebacterium jeikeium K411, a lipid-requiring bacterium of the human skin flora

Corynebacterium jeikeium is a "lipophilic" and multidrug-resistant bacterial species of the human skin flora that has been recognized with increasing frequency as a serious nosocomial pathogen. Here we report the genome sequence of the clinical isolate C. jeikeium K411, which was initially recovered from the axilla of a bone marrow transplant patient. The genome of C. jeikeium K411 consists of a circular chromosome of 2,462,499 bp and the 14,323-bp bacteriocin-producing plasmid pKW4. The chromosome of C. jeikeium K411 contains 2,104 predicted coding sequences, 52% of which were considered to be orthologous with genes in the Corynebacterium glutamicum, Corynebacterium efficiens, and Corynebacterium diphtheriae genomes. These genes apparently represent the chromosomal backbone that is conserved between the four corynebacteria. Among the genes that lack an ortholog in the known corynebacterial genomes, many are located close to transposable elements or revealed an atypical G+C content, indicating that horizontal gene transfer played an important role in the acquisition of genes involved in iron and manganese homeostasis, in multidrug resistance, in bacterium-host interaction, and in virulence. Metabolic analyses of the genome sequence indicated that the "lipophilic" phenotype of C. jeikeium most likely originates from the absence of fatty acid synthase and thus represents a fatty acid auxotrophy. Accordingly, both the complete gene repertoire and the deduced lifestyle of C. jeikeium K411 largely reflect the strict dependence of growth on the presence of exogenous fatty acids. The predicted virulence factors of C. jeikeium K411 are apparently involved in ensuring the availability of exogenous fatty acids by damaging the host tissue.     

Novel expression system for Corynebacterium acetoacidophilum and Escherichia coli based on the T7 RNA polymerase-dependent promoter

The industrially important species of corynebacteria viz. Corynebacterium acetoacidophilum appear to be alternative hosts for recombinant protein production; despite many efforts, a strong promoter-based system in corynebacteria has not been established so far. Described here is a T7 promoter-based expression system which was functional in both gram-positive C. acetoacidophilum and gram-negative Escherichia coli in an external inducer independent manner. This is the very first report of a T7 expression system for Corynebacterium sp. Also, it is a useful addition in the existing T7 expression systems of E. coli.     

Distribution of neuraminidase and n-acetylneuraminate lyase activities among corynebacteria, mycobacteria, and nocardias

In Corynebacterium diphtheriae and closely related neuraminidase-producing corynebacteria, we have found an N-acetylneuraminate (NAN) lyase activity which cleaves NAN into N-acetyl-d-mannosamine and, presumably, pyruvate. In vitro, these lyases can be shown to synthesize NAN. A survey of representative corynebacteria, "plant pathogenic corynebacteria," mycobacteria, and nocardias revealed that only those corynebacteria closely related to C. diphtheriae exhibited both neuraminidase and NAN lyase activities.