[CORYNEBACTERIUM: FEATURES OF THE STRUCTURE OF THE BACTERIAL CELL].

In a review of the features of the bacterial cells are Corynebacterium structure: characterized by an upper layer, highly organized cell wall, cytoplasmic membrane, cytoplasm, nucleoid. Described in detail the structure of the upper layer containing pili (fimbriae), microcapsule surface proteins - PS-2, DIP1281, 67-72r protein (hemagglutinin), porins, sialidase (neuraminidase). These components are the ability to initiate a serial of Corynebacterium work with the host cell, followed by colonization. It submitted a detailed description .of the structure and functions of cell wall structures - cord factor, which is a second barrier permeability; arabinogalactan, peptidoglycan, lipomannan and lipoarabinomannan. The structure and function of the cytoplasmic membrane as the main diffusion barrier cell cytoplasm and the genome of Corynebacterium. Presented differ- ent molecular genetic methods for the identification and differentiation of closely related species of Corynebacterium.

Colonization of human epithelial cell lines by Corynebacterium ulcerans from human and animal sources.

Corynebacterium ulcerans is an emerging pathogen transmitted by a zoonotic pathway to humans. Despite rising numbers of infections and potentially fatal outcomes, data on the colonization of the human host are lacking up to now. In this study, adhesion of two C. ulcerans isolates to human epithelial cells, invasion of host cells and the function of two putative virulence factors with respect to these processes were investigated. C. ulcerans strains BR-AD22 and 809 were able to adhere to Detroit562 and HeLa cells, and invade these epithelial cell lines with a rate comparable to other pathogens as shown by scanning electron microscopy, fluorescence microscopy and replication assays. Infection led to detrimental effects on the cells as deduced from measurements of transepithelial resistance. Mutant strains of putative virulence factors phospholipase D and DIP0733 homologue CULC22_00609 generated in this study showed no influence on colonization under the experimental conditions tested. The data presented here indicate a high infectious potential of this emerging pathogen.

Biofilm production by multiresistant Corynebacterium striatum associated with nosocomial outbreak.

Corynebacterium striatum is a potentially pathogenic microorganism that causes nosocomial outbreaks. However, little is known about its virulence factors that may contribute to healthcare-associated infections (HAIs). We investigated the biofilm production on abiotic surfaces of multidrug-resistant (MDR) and multidrug-susceptible (MDS) strains of C. striatum of pulsed-field gel electrophoresis types I-MDR, II-MDR, III-MDS and IV-MDS isolated during a nosocomial outbreak in Rio de Janeiro, Brazil. The results showed that C. striatum was able to adhere to hydrophilic and hydrophobic abiotic surfaces. The C. striatum 1987/I-MDR strain, predominantly isolated from patients undergoing endotracheal intubation procedures, showed the greatest ability to adhere to all surfaces. C. striatum bound fibrinogen to its surface, which contributed to biofilm formation. Scanning electron microscopy showed the production of mature biofilms on polyurethane catheters by all pulsotypes. In conclusion, biofilm production may contribute to the establishment of HAIs caused by C. striatum.

[THE COMPARATIVE ANALYSIS OF TECHNIQUES OF IDENTIFICATION OF CORYNEBACTERIUM NON DIPHTHERIAE].

The comparative analysis was carried out concerning effectiveness of three techniques of identification of Corynebacterium non diphtheriae: bacteriological, molecular genetic (sequenation on 16SpRNA) andmass-spectrometric (MALDI-ToFMS). The analysis covered 49 strains of Corynebacterium non diphtheriae (C.pseudodiphheriticum, C.amycolatum, C.propinquum, C.falsenii) and 2 strains of Corynebacterium diphtheriae isolated under various pathology form urogenital tract and upper respiratory ways. The corinbacteria were identified using bacteriologic technique, sequenation on 16SpRNA and mass-spectrometric technique (MALDIToF MS). The full concordance of results of species' identification was marked in 26 (51%) of strains of Corynebacterium non diphtheriae at using three analysis techniques; in 43 (84.3%) strains--at comparison of bacteriologic technique with sequenation on 16S pRNA and in 29 (57%)--at mass-spectrometric analysis and sequenation on 16S pRNA. The bacteriologic technique is effective for identification of Corynebacterium diphtheriae. The precise establishment of species belonging of corynebacteria with variable biochemical characteristics the molecular genetic technique of analysis is to be applied. The mass-spectrometric technique (MALDI-ToF MS) requires further renewal of data bases for identifying larger spectrum of representatives of genus Corynebacterium.

Physiological and biochemical consequences of host-plasmid interaction--a case study with Corynebacterium renale, a multiple cryptic plasmid containing strain.

Corynebacterium renale harbors four small cryptic plasmids, pCR1, pCR2, pCR3 and pCR4, and can be a good system for understanding host-plasmid interactions. In the present study, effect of plasmid loss and their subsequent introduction on various properties of the host was evaluated. Loss of plasmids caused a reduction in bacterial size and also slowed down their growth rate, µ, and respiratory rate, r. Both µ and r values were partially recovered in C. renale R, obtained by retransformation of the cured strain with all the four cryptic plasmids. Further delineation revealed that a 3153bp plasmid pCR2 alone is sufficient for the observed increase in µ in C. renale R. The advantages conferred by the remaining three plasmids may be are two subtle to be seen under laboratory conditions. Overall, the observations point to the gross metabolic crisis in the host partly as a result of loss of plasmids. Based on the findings, a mutualistic relationship between the host and the plasmids resulting from their coevolution is proposed.

Disclosure of the mycobacterial outer membrane: cryo-electron tomography and vitreous sections reveal the lipid bilayer structure.

The cell walls of mycobacteria form an exceptional permeability barrier, and they are essential for virulence. They contain extractable lipids and long-chain mycolic acids that are covalently linked to peptidoglycan via an arabinogalactan network. The lipids were thought to form an asymmetrical bilayer of considerable thickness, but this could never be proven directly by microscopy or other means. Cryo-electron tomography of unperturbed or detergent-treated cells of Mycobacterium smegmatis embedded in vitreous ice now reveals the native organization of the cell envelope and its delineation into several distinct layers. The 3D data and the investigation of ultrathin frozen-hydrated cryosections of M. smegmatis, Myobacterium bovis bacillus Calmette-Guérin, and Corynebacterium glutamicum identified the outermost layer as a morphologically symmetrical lipid bilayer. The structure of the mycobacterial outer membrane necessitates considerable revision of the current view of its architecture. Conceivable models are proposed and discussed. These results are crucial for the investigation and understanding of transport processes across the mycobacterial cell wall, and they are of particular medical relevance in the case of pathogenic mycobacteria.

Direct visualization of the outer membrane of mycobacteria and corynebacteria in their native state.

The cell envelope of mycobacteria, which include the causative agents of tuberculosis and leprosy, is crucial for their success as pathogens. Despite a continued strong emphasis on identifying the multiple chemical components of this envelope, it has proven difficult to combine its components into a comprehensive structural model, primarily because the available ultrastructural data rely on conventional electron microscopy embedding and sectioning, which are known to induce artifacts. The existence of an outer membrane bilayer has long been postulated but has never been directly observed by electron microscopy of ultrathin sections. Here we have used cryo-electron microscopy of vitreous sections (CEMOVIS) to perform a detailed ultrastructural analysis of three species belonging to the Corynebacterineae suborder, namely, Mycobacterium bovis BCG, Mycobacterium smegmatis, and Corynebacterium glutamicum, in their native state. We provide new information that accurately describes the different layers of the mycobacterial cell envelope and challenges current models of the organization of its components. We show a direct visualization of an outer membrane, analogous to that found in gram-negative bacteria, in the three bacterial species examined. Furthermore, we demonstrate that mycolic acids, the hallmark of mycobacteria and related genera, are essential for the formation of this outer membrane. In addition, a granular layer and a low-density zone typifying the periplasmic space of gram-positive bacteria are apparent in CEMOVIS images of mycobacteria and corynebacteria. Based on our observations, a model of the organization of the lipids in the outer membrane is proposed. The architecture we describe should serve as a reference for future studies to relate the structure of the mycobacterial cell envelope to its function.

Pilus biogenesis of Gram-positive bacteria: Roles of sortases and implications for assembly.

Successful adherence, colonization, and survival of Gram-positive bacteria require surface proteins, and multiprotein assemblies called pili. These surface appendages are attractive pharmacotherapeutic targets and understanding their assembly mechanisms is essential for identifying a new class of 'anti-infectives' that do not elicit microbial resistance. Molecular details of the Gram-negative pilus assembly are available indepth, but the Gram-positive pilus biogenesis is still an emerging field and investigations continue to reveal novel insights into this process. Pilus biogenesis in Gram-positive bacteria is a biphasic process that requires enzymes called pilus-sortases for assembly and a housekeeping sortase for covalent attachment of the assembled pilus to the peptidoglycan cell wall. Emerging structural and functional data indicate that there are at least two groups of Gram-positive pili, which require either the Class C sortase or Class B sortase in conjunction with LepA/SipA protein for major pilin polymerization. This observation suggests two distinct modes of sortase-mediated pilus biogenesis in Gram-positive bacteria. Here we review the structural and functional biology of the pilus-sortases from select streptococcal pilus systems and their role in Gram-positive pilus assembly.

Molecular biology of S-layers.

In this chapter we report on the molecular biology of crystalline surface layers of different bacterial groups. The limited information indicates that there are many variations on a common theme. Sequence variety, antigenic diversity, gene expression, rearrangements, influence of environmental factors and applied aspects are addressed. There is considerable variety in the S-layer composition, which was elucidated by sequence analysis of the corresponding genes. In Corynebacterium glutamicum one major cell wall protein is responsible for the formation of a highly ordered, hexagonal array. In contrast, two abundant surface proteins from the S-layer of Bacillus anthracis. Each protein possesses three S-layer homology motifs and one protein could be a virulence factor. The antigenic diversity and ABC transporters are important features, which have been studied in methanogenic archaea. The expression of the S-layer components is controlled by three genes in the case of Thermus thermophilus. One has repressor activity on the S-layer gene promoter, the second codes for the S-layer protein. The rearrangement by reciprocal recombination was investigated in Campylobacter fetus. 7-8 S-layer proteins with a high degree of homology at the 5' and 3' ends were found. Environmental changes influence the surface properties of Bacillus stearothermophilus. Depending on oxygen supply, this species produces different S-layer proteins. Finally, the molecular bases for some applications are discussed. Recombinant S-layer fusion proteins have been designed for biotechnology.

Characterization of energetically functional inverted membrane vesicles from Corynebacterium glutamicum.

We show that inverted membrane vesicles from Corynebacterium glutamicum, a Gram-positive bacterium, are able to generate and maintain an electrochemical gradient of protons in response to the addition of NADH. This result indicates that the respiratory chain is intact and that the vesicles are reasonably impermeable to protons. These membrane vesicles may be the starting point for in vitro translocation studies of proteins in Gram-positive bacteria.

Cell wall-deficient bacterial variant cultural surveillance. A useful laboratory aid.

A cell wall-deficient variant of Corynebacterium species was isolated from the blood of a patient with a chronic febrille illness of unkown etiology. He was given antimicrobial therapy, to which both variant and reverted bacterial forms of this organism were sensitive, and he recovered. Cultural surveillance for cell wall-deficient organisms was not only helpful in confirming the presence of infection in our patient but also in following his clinical course once antimicrobial therapy had been instituted.

Corynebacterium group D2 ("Corynebacterium urealyticum") constitutes a new genomic species.

Twenty-one Corynebacterium group D2 ("C. urealyticum") strains were found to constitute a tight DNA hybridization group distinct from named Corynebacterium species. The strains of Corynebacterium group D2 had cell wall component type IV, short chain mycolic acids and G+C content of DNA of 65-66 mol %. Corynebacterium group D2 constitutes a genomic species which can be identified by phenotypic tests.

Mycomembrane and S-layer: two important structures of Corynebacterium glutamicum cell envelope with promising biotechnology applications.

Corynebacteria belong to a distinct Gram-positive group of bacteria including mycobacteria and nocardia, which are characterized by the presence of mycolic acids in their cell wall. These bacteria share the property of having an unusual cell envelope structural organization close to Gram-negative bacteria. In addition to the inner membrane, the cell envelope is constituted of a thick arabinogalactan-peptidoglycan polymer covalently linked to an outer lipid layer, which is mainly composed of mycolic acids and probably organized in an outer membrane like structure. In some species, the cell is covered by a crystalline surface layer composed of a single protein species, which is anchored in the outer membrane like barrier. An increasing number of reports have led to a better understanding of the structure of the cell wall of Corynebacterium glutamicum. These works included the characterization of several cell wall proteins like S-layer protein and porins, genetic and biochemical characterization of mycolic acids biosynthesis, ultrastructural description of the cell envelope, and chemical analysis of its constituents. All these data address new aspects regarding cell wall permeability towards macromolecules and amino acids but also open new opportunities for biotechnology applications.

Comparison of surface hydrophobicity of piliated and non-piliated clones of Corynebacterium renale and Corynebacterium pilosum.

Piliated (P+) and non-piliated (P-) clones of Corynebacterium renale and C. pilosum were similar in hydrophobicity as measured by hydrophobic interaction chromatography, bacterial adherence to hydrocarbons and the salt aggregation test. Therefore, the previously reported adherence of P+ clone to various cells, which is more effective than that of P- clone, may be uncorrelated with the degree of hydrophobicity of both clones of these bacteria. Hydrophobicity of P+ and P- clones was found to be high when measured by hydrophobic interaction chromatography and bacterial adherence to hydrocarbons, but low when measured by the salt aggregation test.

Isolation and characterization of monoclonal antibodies against pili of Corynebacterium renale and Corynebacterium pilosum.

Five monoclonal antibodies against pili of Corynebacterium renale 115 P+ (piliated clone) and two monoclonal antibodies against pili of C. pilosum 92 P+ (piliated clone) were produced. These antibodies bound to pili of the homologous strain in in enzyme-linked immunosorbent assay (ELISA) and agglutinated P+ but not P- (non-piliated clone) of each homologous strain. The five monoclonal antibodies against C. renale 115 P+ pili were divided into 2 groups, comprising 16/5, 160/1 and 32/6 and 13/4 and B20/3, based on the results of a competitive binding assay. The results may indicate the presence of at least 2 distinct antigenic areas on the pilus of C. renale 115 P+. The monoclonal antibodies of the first group inhibited adhesion of C. renale 115 P+ bacteria to the epithelial cells of bovine vulva, while the second group did not. Two monoclonal antibodies against C. pilosum 92 P+ pili recognized the same area on the pilus of C. pilosum 92 P+, and inhibited the adhesion of C. pilosum 92 P+ bacteria to the epithelial cells of bovine vulva. The adhesion of these bacteria was inhibited by the monoclonal antibodies in the form of IgG as well as by the Fab fragment. The strains of C. renale and C. pilosum which reacted with each of the anti-C. renale 115 P+ pili and anti-C. pilosum 92 P+ pili monoclonal antibodies were small in number and of restricted distribution.

Hygromycin and epihygromycin from a bacterium, Corynebacterium equi No. 2841.

A newly isolated bacterium which produces antibacterial substances as below was taxonomically characterized and identified with Corynebacterium equi. The major antibiotic produced was determined to be hygromycin by direct comparison. The minor component, newly named epihygromycin was proved to be an epimer of hygromycin by spectrosocopic evidences and by chemical conversions. The presence of epihygromycin in the metabolites of Streptomyces noboritoensis (reported to be a hygromycin producer) was also observed.

Production of intracellular enzyme by Corynebacterium glutamicum T6-13 protoplasts immobilized in Ca-alginate gels.

The glutamate dehydrogenase (GDH) (EC 1.4.1.4) productivity of the immobilized Corynebacterium glutamicum T6-13 protoplasts in Ca-alginate gels was investigated. GDH in Corynebacterium glutamicum T6-13 cells is an intracellular enzyme. The cells pretreated with 0.5 U/l-1 penicillin G were used for the preparation of protoplasts. Protoplasts were prepared by treating these cells with lysozyme at 30 degrees C for 14 h in 0.5 M NaCl solution and separating the protoplasts. Protoplasts were directly immobilized in 3% Ca-alginate gels (method I). The immobilized protoplasts could be also prepared by treating the immobilized whole cells with lysozyme (method II); this method was more convenient than method I. The GDH productivity of the immobilized protoplasts amounted to 205% of that of the free cells (intracellular). The immobilized protoplasts could be repeatedly used for at least 6 batches (18 days) and had good storage stability.

Attachment of Corynebacterium renale to tissue culture cells by the pili.

One or more cells of Corynebacterium renale strains (serologic types, I, II and III), which possessed numerous pili, frequently were attached to BHK-21 cells, primary dog kidney cells, and primary rabbit kidney cells. The percentage of the cultured cells to which C renal cells were attached was about 70%. The percentage was less with cells of C renale possessing fewer pili, around 30%. After C renale was treated with the homologous anti-pili serum, the percentage of BHK-21 cells to which bacterial cells were attached was even less (22%). In electron micrographs, the pili of C renale were observed to attach themselves to the membranes of BHK-21 cells. The adhesive property of the pili of C renale to tissue culture cells was thus demonstrated.

Pili-mediated attachment of Corynebacterium renale to mucous membrane of urinary bladder of mice.

Corynebacterium renal strain 115 with numerous pili became attached in vivo to the mucous membrane of the urinary bladder of mice 10 to 30 times more frequently than did that of C renale American Type Culture Collection 19412, which showed few pili. Antipili serum-treated C renale strain 115 was not recovered from the membrane in as large amounts as was untreated bacteria. Antisomatic serum-treated strain 115, on the other hand, was recovered from the membrane in amounts similar to untreated bacteria. Untreated organisms became attached to the mucous membrane of the urinary bladder more effectively than did the antipili serum-treated bacteria, as seen on scanning electron micrographs. It may be concluded that C renale strain 115 attaches itself to the mucous membrane of the urinary bladder om mice by the pili.

[Heteromorphism of corynebacteria. II. Microcells]. / Geteromorfizm korinebakterii. Soobshchenie II. Mikrokletki.

The formation of microcells is one of the manifestations of the heteromorphism of Corynebacterium. It can occur in 6 ways, of these, 3 are possible during septation (chaotic septation of gigantic macroforms, irregular multiseptation of elongated organisms, and microcells appearing as inserts at the sites of the septum formation), and 3 in undividing cells (microcells at the edge segments of the protoplast, exogemmation, endogemmation). The analysis of the ultrastructure of microcells indicated the possibility of their independent existence for a certain period of time.