Adjuvant Activity of Mycobacterial Fractions

The addition of adjuvant (Bacillus Calmette–Guérin-cell wall skeleton; BCG-CWS) to the culture medium substantially increased the immune response of mouse spleen cells to immunization with heterologous erythrocytes and hapten–protein conjugate in vitro. Cell walls of mycobacteria, nocardia and corynebacteria and their cell wall constituents were used as adjuvants. In the present case, it was found that cell walls of mycobacteria, nocardia and corynebacteria markedly facilitated primary humoral response of mouse spleen cells to heterologous erythrocytes in vitro. The adjuvant effect of BCG-CWS was present only in the formation of 19 S antibody in both primary and secondary responses, but not in that of 7 S antibody in vitro. Primary antihapten response of mouse spleen cells against dinitrophenylated keyhole limpet hemocyanin (DNP-KLH) also succeeded when BCG-CWS was added to the culture medium, and it was found that BCG-CWS increased the helper activity of carrier specific helper T cells in vitro where a double chamber system, separated by a cell-impermeable nucleopore membrane, was used. This result suggested that BCG-CWS acts on T cells, resulting in the release of soluble factor(s) from T cells capable of exerting an adjuvant effect. Furthermore, mucopeptide moiety of BCG-CWS retained some adjuvant activity, but other cell wall constituents, such as mycolic acid, arabinose mycolate, and arabinogalactan, did not show any adjuvant effect in vitro. These results strongly imply that mucopeptide moiety of BCG-CWS plays an important role in the development of adjuvanticity.     

Serological Study of Nocardia by Using Mycobacterial Precipitation Reference Systems

Forty-six nocardia strains and ten strains labeled "Mycobacterium" were analyzed with the comparative immunodiffusion technique, employing as reference material nine mycobacterial precipitation systems, each one representing a different species. Three precipitinogens were common for many of the tested strains and the mycobacterial reference strains. The serological analyses indicated the possibility of differentiating two groups within Nocardia depending on the characteristics of one of the common precipitinogens. Five of eight tested Nocardia farcinica strains showed a serologically close relationship with the mycobacteria and might be regarded as belonging to this genus. The "M." pellegrino and "M." rhodochrous strains, however, did not react with the antimycobacterial sera to a higher degree than did the tested nocardia strains.     

Characterization and taxonomic implications of the rRNA genes of Mycobacterium leprae.

The number of rRNA genes of Mycobacterium leprae was determined by restriction analysis of M. leprae total chromosomal DNA. A single set of rRNA genes was found. This set was subcloned from a cosmid library of M. leprae DNA into pUC13 and was characterized by restriction analysis and hybridization with Escherichia coli rRNA genes. The 16S, 23S, and 5S genes of M. leprae were clustered on a 5.3-kilobase DNA fragment. On one hand, restriction analysis of the set of rRNA genes showed the uniqueness of M. leprae among mycobacteria, but on the other hand, it suggested that M. leprae strains of several origins are very much alike. Quantitative hybridization studies between M. leprae rDNA and total DNA of various bacteria demonstrated a close relatedness between M. leprae and corynebacteria, nocardia, and mycobacteria, especially Mycobacterium tuberculosis.     

Genetic Identification of Members of the Genus Corynebacterium at Genus and Species Levels with 16S rDNA-Targeted Probes

16S rRNA gene-targeted probes were designed for the identification of corynebacteria at the genus and species levels. The genus-specific probe hybridized all clinically important members of the genus Corynebacterium and could distinguish them from other coryneform bacteria and phylogenetically related high G + C% gram-positive bacteria, including Actinomyces, Rhodococcus, Gordona, Nocardia, Streptomyces, Brevibacterium and Mycobacterium. The species-specific probes for C. jeikeium and C. diphtheriae could differentiate these two species from other members of this genus. The probes were used to select corynebacteria among gram-positive clinical isolates which had been tentatively identified as corynebacteria by biochemical tests. We screened 59 strains with the genus-specific probe; 51 strains hybridized to the genus-specific probe, 8 did not. Of the 51 strains that hybridized to the genus-specific probe, 1 hybridized to the C. diphtheriae species probe and 13 hybridized to the C. jeikeium species probe. The 8 strains that did not hybridize to the genus probe were further characterized by analyzing cell wall diaminopimelic acid and partial 16S rRNA sequencing. The results indicated that these strains were distributed in the genera Arthrobacter and Brevibacterium.     

Cell envelope architectures of leprosy-derived corynebacteria,Mycobacterium leprae,and related organisms: a comparative study

Cell-envelope architectures of three strains of leprosy-derived corynebacteria (LDC) named Kim, FPSA, and 43LL were compared withMycobacterium leprae andM. avium as well as with related organisms (Nocardia asteroides andCorynebacterium psuedotuberculosis). Cytochemical studies were performed at the ultrastructural level after the lead citrate, silver proteinate, acidic phosphotungstic, and ruthenium red colorations. This study showed that, while the organisms belonging to theCorynebacterium-Mycobacterium-Nocardia (CMN) group had only the cytoplasmic membrane but not the cell wall reacting with the silver proteinate coloration, the LDC organisms had both the cell wall and the cytoplasmic membrane reacting with this coloration method. Moreover, the three strains of the LDC organisms differed from one another at the level of their exopolymer content.Mycobacterium leprae, on the other hand, gave a cytochemical response common to other mycobacteria and the members of the CMN group studied. Consequently, the envelopes of the LDC organisms were not identical toM. leprae, neither morphologically nor cytochemically.     

The cell wall porin of Nocardia farcinica: biochemical identification of the channel-forming protein and biophysical characterization of the channel properties

A channel-forming protein was identified in cell wall extracts of the Gram-positive, strictly aerobic bacterium Nocardia farcinica . The cell wall porin was purified to homogeneity and had an apparent molecular mass of about 87 kDa on tricine-containing SDS–PAGE. When the 87 kDa protein was boiled for a longer time in sodium dodecylsulphate (SDS) it dissociated into two subunits with molecular masses of about 19 and 23 kDa. The 87 kDa form of the protein was able to increase the specific conductance of artificial lipid bilayer membranes from phosphatidylcholine (PC) phosphatidylserine (PS) mixtures by the formation of ion-permeable channels. The channels had on average a single-channel conductance of 3.0 nS in 1 M KCl, 10 mM Tris-HCl, pH 8, and were found to be cation selective. Asymmetric addition of the cell wall porin to lipid bilayer membranes resulted in an asymmetric voltage dependence. The single-channel conductance was only moderately dependent on the bulk aqueous KCl concentration, which indicated point charge effects on the channel properties. The analysis of the single-channel conductance data in different salt solutions using the Renkin correction factor, and the effect of negative charges on channel conductance suggested that the diameter of the cell wall porin is about 1.4–1.6 nm. Channel-forming properties of the cell wall porin of N. farcinica were compared with those of mycobacteria and corynebacteria. The cell wall porins of these members of the order Actinomycetales share common features because they form large and water-filled channels that contain negative point charges.     

Numerical Classification of Bacteria

Sixty three organisms selected from 12 genera of bacteria were subjected to numerical analysis. The purpose of this work is to examine the relationships among 38 coryneform bacteria included in the test organisms by two coding methods—Sneath’s and Lockhart’s systems—, and to compare the results with conventional classification. In both cases of codification, five groups and one or two single item(s) were found in the resultant classifications. Different codings brought, however, a few distinct differences in some groups, especially in a group of sporogenic bacilli or lactic-acid bacteria. So far as the present work concerns, the result obtained on Lockhart’s coding rather than that obtained on Sneath’s coding resembled the conventional classification. The taxonomic positions of corynebacteria were quite different from those of the conventional classification, regardless of which coding method was applied.

Though animal corynebacteria have conventionally been considered to occupy the taxonomic position neighboring to genera Arthrobacter and Cellulomonas and regarded to be the nucleus of so-called “coryneform bacteria,’ the present work showed that many of the corynebacteria are akin to certain mycobacteria rather than to the organisms belonging to the above two genera.     

Bacteriophages That Lyse Mycobacteria and Corynebacteria,and Show Cytopathogenic Effect on Tissue Cultures of Renal Cells of Cercopithecus Aethiops: A Preliminary Communication

Bacteriophages isolated from sputum and resection specimens of patients suffering from carcinoma of the lung were found to lyse corynebacteria and mycobacteria, and to produce a cytopathogenic effect on certain cells in tissue cultures. From the same and other patients with neoplastic disease, bacteria were isolated and described as coryne-mycobacteria because of bacteriological features they shared with both species. These bacteria, which either were sensitive to mycobacteriophages and corynebacteriophages or were phage-immune lysogenic bacteria, could be induced to produce lytic particles with phagolytic activity on corynebacteria and mycobacteria and a cytopathogenic effect on HeLa cells and on the renal cells of Cercopithecus.     

Primary structure of the wall peptidoglycan of leprosy-derived corynebacteria.

The cell walls isolated from axenically grown leprosy-derived corynebacteria were submitted to various chemical and enzymatic degradations. The glycan strands of the wall peptidoglycan are essentially composed of N-acetylglycosaminyl-N-acetylmuramic acid disaccharide units. Small amounts of N-acetylglycosaminyl-N-glycolylmuramic acid (less than 10%) were also detected. The muramic acid residues of adjacent glycan strands are substituted by amidated tetrapeptide units which, in turn, are cross-linked through direct linkages extending between the C-terminal D-alanine residue of one tetrapeptide and the mesodiaminopimelic acid residue of another tetrapeptide. Such a structure is very similar to that of the wall peptidoglycan found in the taxonomically related microorganisms of the Corynebacterium, Mycobacterium, and Nocardia groups.     

Immunochemistry of the Group-Specific Polysaccharide of Nocardia brasiliensis.

Estrada-Parra, Sergio (Escuela Nacional de Ciencias Biológicas, México, D.F., México), Abel Zamora, and L. F. Bojalil. Immunochemistry of the group-specific polysaccharide of Nocardia brasiliensis. J. Bacteriol. 90:571-574. 1965.-The group-specific polysaccharide of Nocardia brasiliensis was further purified, yielding an amorphous white material with the following characteristics: [alpha](D) (20) = + 48; nitrogen, 0.5%; phosphorus, 0.1%; and ash as sodium, 0.8%. The polymer is made of d-arabinose and d-galactose in a molar ratio of 3:1, and no other sugars were detected. Mild hydrolysis liberates mainly arabinose. The polysaccharide consumes 3.46 mumoles of periodate per mg of polymer in 15 days at 4 C (this value remains constant after 4 more days). Oxidation results in destruction of two of the arabinose, with the formation of two glycerols after borohydride reduction and hydrolysis. The polysaccharide oxidized by periodate and reduced under mild acid hydrolysis at 20 C yields glycerol and a polymer formed by galactose and arabinose (in a ratio of 1:1) which is resistant to a second oxidation. Therefore, the polysaccharide is probably formed by a main chain of glactose linked 1,3 and arabinose linked 1,2 or 1,3 or both, and nonreducing side chains of arabofuranose residues. The intact polysaccharide cross-reacts with sera from patients with active tuberculosis, and this, as well as the homologous reaction, is abolished by oxidation with periodate.     

Ultrastructure of “Gordona aurantiaca” Tsukamura 1971

Ultrastructure of Gordona aurantiaca^* M 296 (8128) was studied after the lead citrate coloration, whereas the cell envelope architecture was investigated by ruthenium red staining for outer wall acidic polysaccharides and the periodic-acid-thiocarbohydrazide-silver-proteinate cytochemical procedure (Thiéry method) for the detection of 1-2 glycol bond containing polysaccharides. The ultrastructural morphology of bacteria was distinct from both the mycobacteria and nocardia. The bacilli had a typical gram-positive cell wall that contained a thin, uniformly distributed, polysaccharide outer layer (POL) at its surface. The Thiéry cytochemical method stained only the cytoplasmic membrane, but not the cell wall, a feature that is common to the mycolic acid containing theCorynebacterium-Mycobacterium-Nocardia (CMN) group of organisms. The negative staining of the unfixed preparations of bacilli showed ribbonlike surface structures, common to the CMN group of organisms. The electron-microscopic preparations showed numerous lysing bacilli with bacteriophages indicating that the strain used was lysogenic.     

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.     

Lipid Composition in the Classification of Nocardiae and Mycobacteria

Ninety-six strains of aerobic actinomycetes with a type IV cell wall (major amounts of meso-diaminopimelic acid, arabinose, and galactose) were analyzed for the presence of mycolic acids and nocardomycolic acids. The method used was comparatively simple and permits the separation of these organisms into two groups: the mycobacteria and the nocardiae. In general, strains received as mycobacteria contained mycolic acids, confirming the generic assignment made by other methods. On the basis of nocardomycolic acid content, Mycobacterium brevicale, M. rhodochrous, and M. thamnopheos should be placed in the genus Nocardia, and on the basis of mycolic acid content, strains recently isolated from bovine farcy should be placed in the genus Mycobacterium. Nocardia farcinica should be considered a nomen dubium and N. asteroides should be considered the type species of the genus.     

Proteomics of corynebacteria: From biotechnology workhorses to pathogens

Corynebacteria belong to the high G+C Gram-positive bacteria (Actinobacteria) and are closely related to Mycobacterium and Nocardia species. The best investigated member of this group of almost seventy species is Corynebacterium glutamicum, a soil bacterium isolated in 1957, which is used for the industrial production of more than two million tons of amino acids per year. This review focuses on the technical advances made in proteomics approaches during the last years and summarizes applications of these techniques with respect to C. glutamicum metabolic pathways and stress response. Additionally, selected proteome applications for other biotechnologically important or pathogenic corynebacteria are described.     

Characterization of 6-deoxy-D-altritol in the cell-wall polysaccharide of Nocardia asteroides R 399

A polyol, found in the cell-wall of Nocardia asteroides R 399 as a component of a neutral polysaccharide mainly composed of D-arabinose and D-galactose, was identified by mass spectrometry, paper chromatography, thin-layer chromatography, and gas chromatography as 6-deoxy-D-altritol.     

Structural elucidation of the predominant motifs of the major cell wall arabinogalactan antigens from the borderline species Tsukamurella paurometabolum and Mycobacterium fallax

Tsukamurella paurometabolum and Mycobacterium fallax are members of the suprageneric actinomycete group Corynebacterineae that possesses a cell wall skeleton composed of a peptidoglycan to which an arabinogalactan is covalently attached. This polysaccharide is further modified by esterification with C60-C80 mycolic acid residues in mycobacteria and T. paurometabolum. However, M. fallax and T. paurometabolum produce polyenoic (up to six double bonds) mycolic acids whereas the most common type of mycobacterial mycolates, called alpha-mycolates, are mono- and di-enoic or -cyclopropanated mycolic acids. To determine whether this difference also applied to the structures of cell wall arabinogalactans, competitive inhibition experiments using antibodies raised against the cell wall from Mycobacterium bovis and the arabinogalactans from T. paurometabolum and M. fallax were performed. They demonstrated the structural identity between the polysaccharide of M. fallax and those of mycobacteria and showed a strong similarity between the latter polysaccharides and that of T. paurometabolum. Structural analyses of the per-O-alkylated alditol fragments derived from the polysaccharides by gas chromatography-mass spectrometry (GC-MS) and 13C nuclear magnetic resonance (NMR) spectroscopy of the intact solubilized polysaccharides demonstrated that the polysaccharides from the two species analyzed contained all the major structural features previously characterized in mycobacterial arabinogalactans. These include (1) the homogalactan of alterning 5-linked galactofuranosyl (Galf) and 6-linked Galf residues, (2) a linear 5-linked arabino furanosyl (Araf), (3) a beta-Araf-(1-->2)-alpha-Araf disaccharide branched on both position 3 and position 5 of an alpha-Araf unit, and (4) a 5-linked-alpha-Araf unit branched on both position 3 and position 5 of an alpha-Araf residue. The polysaccharide from T. paurometabolum possesses additional structural domains composed of a terminal (t) Araf directly linked to either a 5-linked-alpha-Araf or to both position 3 and position 5 of a 3,5-linked alpha-Araf unit. Both the remarkable similarity of arabinogalactans from Corynebacterineae and their genus- and/or species-specificities are reflected in their 13C NMR spectra that may be used as a valuable help in the identification of members of the actinomycete group.     

Cell polarity and the control of apical growth in Streptomyces

Streptomyces cells grow by building cell wall at one pole-the hyphal tip. Although analogous to hyphal growth in fungi, this is achieved in a prokaryote, without any of the well-known eukaryotic cell polarity proteins, and it is also unique among bacterial cases of cell polarity. Further, polar growth of Streptomyces and the related mycobacteria and corynebacteria is independent of the MreB cytoskeleton and involves a number of coiled-coil proteins, including the polarity determinant DivIVA. Recent progress sheds light on targeting of DivIVA to hyphal tips and highlight protein phosphorylation in the regulation of actinobacterial growth. Furthermore, cell polarity affects not only cell envelope biogenesis in Streptomyces, but apparently also assembly of fimbriae, conjugation and migration of nucleoids.     

Detailed structural and quantitative analysis reveals the spatial organization of the cell walls of in vivo grown Mycobacterium leprae and in vitro grown Mycobacterium tuberculosis

The cell wall of mycobacteria consists of an outer membrane, analogous to that of gram-negative bacteria, attached to the peptidoglycan (PG) via a connecting polysaccharide arabinogalactan (AG). Although the primary structure of these components is fairly well deciphered, issues such as the coverage of the PG layer by covalently attached mycolates in the outer membrane and the spatial details of the mycolic acid attachment to the arabinan have remained unknown. It is also not understood how these components work together to lead to the classical acid-fast staining of mycobacteria. Because the majority of Mycobacterium tuberculosis bacteria in established experimental animal infections are acid-fast negative, clearly cell wall changes are occurring. To address both the spatial properties of mycobacterial cell walls and to begin to study the differences between bacteria grown in animals and cultures, the cell walls of Mycobacterium leprae grown in armadillos was characterized and compared with that of M. tuberculosis grown in culture. Most fundamentally, it was determined that the cell wall of M. leprae contained significantly more mycolic acids attached to PG than that of in vitro grown M. tuberculosis (mycolate:PG ratios of 21:10 versus 16:10, respectively). In keeping with this difference, more arabinogalactan (AG) molecules, linking the mycolic acids to PG, were found. Differences in the structures of the AG were also found; the AG of M. leprae is smaller than that of M. tuberculosis, although the same basic structural motifs are retained.