Claude Frehel

Methylophaga marina gen. nov., sp. nov. and Methylophaga thalassica sp. nov., marine methylotrophs

After enrichment in a medium containing seawater and methanol, 42 methylotrophic strains were isolated. All of these strains were gram-negative, strictly aerobic, motile, rod-shaped organisms that required vitamin B12. None grew on methane or on complex nutrient media supplemented or not supplemented with NaCI. All but 2 strains grew on methanol, methylamine, and fructose, 17 strains grew on dimethylamine, and 10 strains grew on trimethylamine. Fructose was the only multicarbon compound tested that was used as a growth substrate. All 11 strains tested used the ribulose monophosphate pathway of carbon assimilation. Depending on the strain, methylamine was oxidized either through a methylamine dehydrogenase or through a methylglut-amate dehydrogenase. The mean guanine-plus-cytosine content of 33 strains was 43 mol%. Based on deoxyribonucleic acid-deoxyribonucleic acid hybridization, two related groups were identified among 11 strains examined. We propose a new genus, Methylophaga, with two species, Methylophaga marina (type species) and Methylophaga thalassica. There was no significant deoxyribonucleic acid hybridization between Methylophaga and the terrestrial obligate methanol utilizers tested. The type strains of M. marina and M. thalassica are strains ATCC 35842 (= NCMB 2244) and ATCC 33146 (= NCMB 2163), respectively.

Triple-layered structure of mycobacterial cell wall: evidence for the existence of a polysaccharide-rich outer layer in 18 mycobacterial species

An additional outer wall layer, composed mainly of acidic polysaccharides, was revealed in 18 species of mycobacteria by ruthenium red staining in electron microscopy. This report deals with the description of this additional layer. The ultrastructural implications at the level of the mycobacterial cell wall model and also the possible physiological role of this layer are briefly discussed.

The electron transparent zone in phagocytized Mycobacterium avium and other Mycobacteria Formation, persistence and role in bacterial survival

After phagocytosis by bone-marrow macrophages, Mycobacterium avium was surrounded by a thick electron-transparent zone (ETZ). The use of various fixation and embedding procedures showed that ETZ did not seem to be an artifactual structure. A quantitative assessment of ETZ frequency was performed at different times after infection of macrophages with SmD and SmT colony variants of M. avium. For SmT-variant-infected macrophages, a higher percentage of ETZ+ bacilli paralleled a higher percentage of intact bacilli than was the case for SmD-infected macrophages. Macrophages were also infected with bacteria killed with UV or gamma rays, H2O2, heat or glutaraldehyde. About 50% of bacilli killed with any of these treatments were found ETZ+ instead of 80-85% with live bacteria. Unlike live bacilli, for which the percentage of ETZ frequency remained stable throughout incubation time, ETZ frequency for killed bacilli decreased with time. ETZ assessment performed on M. tuberculosis H37 Rv for comparison showed that, despite a very low ETZ frequency (8-15%), the percentage of intact bacteria was identical to that observed with M. avium. In contrast, three rapidly growing non-pathogenic species (M. smegmatis, M. phlei and M. fallax) presented a low ETZ frequency after phagocytosis and were rapidly degraded. The process of ETZ formation and its role in bacterial survival are discussed.

Nutritionally variant streptococci develop ultrastructural abnormalities during experimental endocarditis

Nutritionally variant streptococci (NVS) are fastidious micro-organisms responsible for most of the so-called negative blood culture endocarditis. In patients, the relative resistance of these bacteria to antibiotic treatment may be relevant to bacterial alterations in infected tissues. We used here a rabbit experimental model of endocarditis in order to examine, under scanning and transmission electron microscope, the NVS ultrastructure inside cardiac vegetations and to follow alterations at the different stages of the disease. In the early phase (day 7) of endocarditis, NVS were found dispersed inside vegetations and exhibited a typical streptococcal morphology similar to that observed during an in vitro balanced growth. In contrast, on day 11 and day 18, bacteria were found gathered as large and numerous clusters, in which they exhibited abnormal ultrasturctural features similar to those previously described during in vitro unbalanced growth. At this stage, ruthenium red staining revealed a large amount of exopolysaccharide surrounding the bacteria. None of these bacterial alterations were observed inside the vegetations of rabbits treated from day 7 to day 11 with penicillin or vancomycin. These abnormalities might be mainly related to nutrient limitation inside the clusters and could contribute to the pathogenicity of these micro-organisms.

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.

Evidence for taxonomic utility of periodic acid-thiocarbohydrazide-silver proteinate cytochemical staining for electron microscopy

Various species of bacteria belonging to taxonomically separate genera were studied by using electron microscopy and different cytochemical staining methods. Judging from our results and those reported previously in the literature, we concluded that the periodic acid-thiocarbohydrazide-silver proteinate reaction (Thiery method) separates the bacteria into four large groups according to the production of silver grains at the site of the cell wall or the cytoplasmic membrane or both. In the Corynebacterium-Mycobacterium-Nocardia group only the cytoplasmic membrane reacts; in Bacillus and Brucella only the cell wall reacts; in Lactobacillus and Microbacterium both the cell wall and the cytoplasmic membrane react; and in Escherichia and Clostridium neither the cell wall nor the cytoplasmic membrane reacts. Moreover, this staining method clearly separated Mycobacterium leprae from certain leprosy-derived coryneform bacteria.

Electron microscopic cytochemical study of cell-wall polysaccharides in Bacillus subtilis and two strains of Bacillus megaterium

Thin sections of Bacillus subtilis and B. megaterium cells were treated by two cytochemical techniques revealing polysaccharides: the phosphotungstic-acid (PTA) and the silver-proteinate (SP) stainings. These procedures were applied to exponentially growing cells as well as cells treated with chloramphenicol (CMP) or cells regrown after antibiotic elimination. It appeared that both stains mainly deposited on the cell wall, but did not give the same staining patterns. In vegetative and CMP-treated cells, PTA gave a homogenous dark deposit, whereas with SP a deposit was only observed on the outer and inner wall faces. During cell-wall thinning, PTA-stained walls remained rather homogeneous, whereas the silver deposit spread throughout the cell-wall thickness. The meaning of these differences in relation to cell-wall polymer organization is discussed.

Relationship between biochemical and cytochemical results obtained on Bacillus megaterium and Bacillus subtilis cell-wall polysaccharides.

Two different polysaccharide stains, phosphotungstic acid (PTA) and silver proteinate (SP), were applied to isolated walls of Bacillus subtilis 168 and B. megaterium MA and MB. To get a better insight into their staining specificity for the cell-wall polymers, these procedures were carried out after extraction of nonpeptidoglycan polymers or after peptidoglycan degradation. It appeared that in the bacterial cell wall, PTA stains peptidoglycan and SP stains nonpeptidoglycan polymers. In B. subtilis the SP-positive polymer is teichoic acid, in B. megaterium MB another kind of polysaccharide; in strain MA teichoic acid and another polysaccharide were probably responsible for the SP reaction. A partial analysis of the cell-wall sugar and phosphorus content confirmed these findings. The nature of the anionic sites present in the cell wall was also investigated by using cationized ferritin.

Evidence that coating of Mycobacterium leprae surface antigens reduces its ability to hinder host microbicidal functions.

Mycobacterium leprae extracted and purified from experimentally infected armadillo was coated with rabbit sera raised against the total antigens of the following species of mycobacteria: M. leprae, M. avium, M. bovis BCG, and M. fallax. In addition, the bacteria were also coated either with serum from a lepromatous (LL), or a tuberculoid (TT) leprosy patient. The effectiveness of surface coating was verified by electron microscopy, with the aid of gold immunolabelling. The coated bacilli were phagocytized by mice bone marrow-derived macrophages, and the phagosome-lysosome fusions (PLF) were assessed during phagocytosis using acid-phosphatase (AcPase) cytochemistry. As compared to control preparations (like-wise treated with non-immune serum), significant but partial reversion of PLF inhibition was observed in all cases except when bacteria had been incubated with M. fallax antiserum (rapidly growing, non-pathogenic species). The results obtained suggest that some of the antimycobacterial antibodies may offer partial protection to the host during early events of infection by reverting the usual pattern of inhibition of PLF in infected macrophages.

Ultrastructural evidence for the accumulation of a polysaccharide-like substance during mycobacteriophage D29 replication in Mycobacterium smegmatis

Summary Mycobacterium smegmatis was infected with phage D 29 and five previously characterized temperature-sensitive (ts) mutants. Electron microscopy revealed an accumulation of large amounts of an electron-transparent material immediately after infection of the bacteria. This material appeared in the form of intracellular plages following lead citrate coloration. After silver proteinate staining, the bacteria were found to accumulate a polysaccharide-like substance (PLS) rich in α-1-2-glycol bonds. After bacterial lysis, this PLS disappeared, probably solubilizing in the surrounding medium. In contrast, certain ts mutants which did not cause lysis of the bacteria at the restrictive temperature resulted in bacilli which were heavily loaded with PLS.