Pan-genome analysis of the genus Finegoldia identifies two distinct clades, strain-specific heterogeneity, and putative virulence factors.

Finegoldia magna, a Gram-positive anaerobic coccus, is an opportunistic pathogen, associated with medical device-related infections. F. magna is the only described species of the genus Finegoldia. We report the analysis of 17 genomes of Finegoldia isolates. Phylogenomic analyses showed that the Finegoldia population can be divided into two distinct clades, with an average nucleotide identity of 90.7%. One clade contains strains of F. magna, whereas the other clade includes more heterogeneous strains, hereafter tentatively named "Finegoldia nericia". The latter species appears to be more abundant in the human microbiome. Surface structure differences between strains of F. magna and "F. nericia" were detected by microscopy. Strain-specific heterogeneity is high and previously identified host-interacting factors are present only in subsets of "F. nericia" and F. magna strains. However, all genomes encode multiple host factor-binding proteins such as albumin-, collagen-, and immunoglobulin-binding proteins, and two to four copies of CAMP (Christie-Atkins-Munch-Petersen) factors; in accordance, most strains show a positive CAMP reaction for co-hemolysis. Our work sheds new light of the genus Finegoldia and its ability to bind host components. Future research should explore if the genomic differences identified here affect the potential of different Finegoldia species and strains to cause opportunistic infections.

Bacteria-based controlled assembly of metal chalcogenide hollow nanostructures with enhanced light-harvesting and photocatalytic properties.

Herein, a general bottom-up approach is proposed for the controlled assembly of metal chalcogenide nanoparticles into biomorphic porous hollow nanostructures by a sonochemical method using bacteria as morph-biotemplates. Biomorphic PbS and ZnS hollow nanostructures have been successfully synthesized with two species of bacteria cocci and bacillus as morph-templates. The biomorphic hollow assemblies possess shape-controllable, size-tunable and shell-thickness-adjustable characteristics. Thus, the structure and morphology of the hollow assemblies may be varied in a controllable way to tailor their properties over a broad range. A preliminary study on the light-harvesting properties of PbS and ZnS hollow spheres revealed that the hollow and porous structure is clearly far more favorable for the absorption of light than solid counterparts, which accounts for both multiple scattering effects at the large voids (hollow cavities) and Rayleigh scattering by nanovoids of the exterior shells. Furthermore, photocatalytic studies of ZnS nanostructures by degradation of acid fuchsine under solar irradiation have proved that the hollow structures possess superior photocatalytic activity to the corresponding solid counterparts. This versatile approach provides an effective route for the further extensive study of the distinct properties imparted by hollow nanostructures and extends their application potentials in photocatalysis and solar energy storage/conversion.

Characterization of intact subcellular bodies in whole bacteria by cryo-electron tomography and spectroscopic imaging.

We illustrate the combined use of cryo-electron tomography and spectroscopic difference imaging in the study of subcellular structure and subcellular bodies in whole bacteria. We limited our goal and focus to bodies with a distinct elemental composition that was in a sufficiently high concentration to provide the necessary signal-to-noise level at the relatively large sample thicknesses of the intact cell. This combination proved very powerful, as demonstrated by the identification of a phosphorus-rich body in Caulobacter crescentus. We also confirmed the presence of a body rich in carbon, demonstrated that these two types of bodies are readily recognized and distinguished from each other, and provided, for the first time to our knowledge, structural information about them in their intact state. In addition, we also showed the presence of a similar type of phosphorus-rich body in Deinococcus grandis, a member of a completely unrelated bacteria genus. Cryo-electron microscopy and tomography allowed the study of the biogenesis and morphology of these bodies at resolutions better than 10 nm, whereas spectroscopic difference imaging provided a direct identification of their chemical composition.

Target affinities of faropenem to and its impact on the morphology of gram-positive and gram-negative bacteria.

Faropenem is a new oral beta-lactam antibiotic unique from carbapenems and other available beta-lactams. Determinants of the in vitro activity of beta-lactam antibiotics include affinity to penicillin-binding proteins (PBPs) and beta-lactamase stability. In this study, the binding affinity of faropenem to various PBPs and its impact on the morphology of Staphylococcus aureus and Escherichia coli were evaluated. In general, faropenem demonstrated high binding affinity to high-molecular-weight PBPs but low affinity to low-molecular-weight PBPs. In S. aureus and Streptococcus pneumoniae, faropenem exhibited high binding affinity to PBP1, followed by PBP3 and PBP2. In E. coli, faropenem showed the highest affinity for PBP2, followed by PBP1A, PBP1B, PBP3 and PBP4. In Proteus vulgaris, binding was highest to PBP4, followed by PBP1A, PBP2 and PBP3. In Serratia marcescens, faropenem bound preferentially to PBP2 and PBP4. Exposure of S. aureus to faropenem at minimum inhibitory concentrations (MICs) of 1/8 or 1/4 resulted in irregular septum formation. At 1x MIC or higher, a larger number of lysed cells were observed. Exposure of E. coli to 1/8x MIC or 1/4x MIC also induced changes in cellular shape; the normal rod-shaped form changed to a spherical form in a time-dependent manner. After exposure of E. coli to 1x MIC for 2 h, bulging-shaped E. coli cells were observed and after 4 h of exposure cell lysis was demonstrated. In the presence of 4x MIC, spheroplast-like forms and cell lysis were observed. The morphological changes triggered by faropenem are in agreement with the PBP binding affinities reported. Thus, the high binding affinities of faropenem to PBPs from gram-negative and gram-positive bacteria are mirrored by its pronounced and concentration-dependent bactericidal effect.

Ultrastructural analysis of an infected collagen-coated vascular graft.

The incidence of infection following arterial reconstruction using synthetic graft materials varies from less than 1 to 5%. One of three mechanisms is thought to be responsible: 1. intraoperative contamination, 2. extension from adjacent infected or colonized tissue, or 3. hematogenous or lymphogenous seeding. We present ultrastructural data of a patient with a polymicrobial graft infection due to a prostheto-enteric fistula 16 years after reconstruction of an aortobifemoral graft. The polymer surface showed signs of biodegradation and was completely covered with a layer of plasma proteins. Disrupted fibroblasts on the intersegmental graft surface were surrounded by bundles of collagen. Gram-negative rods and grampositive cocci were embedded in an extracellular EPS matrix. Bacterial culture confirmed growth of Eikenella corrodens, Fusobacterium nucleatum and Peptostreptococcus species. Fibrin and granulation tissue from the neoadventitia started to mark off the inflammatory process. Transmission electron microscopy is a valuable tool for the investigation of alloplastic arterial devices. After 16 years of implantation the graft shows different signs of biodegradation.

Hemagglutinin of unusual specificity from Helcococcus kunzii.

Helcococcus kunzii is a gram-positive, catalase-negative opportunist. The organism has been isolated from the lower extremities and breast masses of several patients. A clinical isolate of Helcococcus kunzii was shown to possess a hemagglutinin-lectin with a specificity for N-acetylglucosamine and lactose, two structurally unrelated carbohydrates. The lectin is sensitive to protease, heat and mutanolysin. Electron microscopy failed to reveal fimbriae or fibrillae, suggesting that the lectin is associated with peptidoglycan or the cytoplasmic membrane. It is likely that the lectin is involved in adhesion and colonization of H. kunzii.

High resolution structure of the large ribosomal subunit from a mesophilic eubacterium.

We describe the high resolution structure of the large ribosomal subunit from Deinococcus radiodurans (D50S), a gram-positive mesophile suitable for binding of antibiotics and functionally relevant ligands. The over-all structure of D50S is similar to that from the archae bacterium Haloarcula marismortui (H50S); however, a detailed comparison revealed significant differences, for example, in the orientation of nucleotides in peptidyl transferase center and in the structures of many ribosomal proteins. Analysis of ribosomal features involved in dynamic aspects of protein biosynthesis that are partially or fully disordered in H50S revealed the conformations of intersubunit bridges in unbound subunits, suggesting how they may change upon subunit association and how movements of the L1-stalk may facilitate the exit of tRNA.

Subcellular distribution of glycanases and related components in Ruminococcus albus SY3 and their role in cell adhesion to cellulose.

AIMS: To compare the subcellular distribution of glycanase-related components between wild-type Ruminococcus albus SY3 and an adhesion-defective mutant, to identify their possible contribution to the adhesion process, and to determine their association with cellulosome-like complexes. METHODS AND RESULTS: Cell fractionation revealed that most of the cellulases and xylanases were associated with capsular and cell-wall fractions. SDS-PAGE and gel filtration indicated that most of the bacterial enzyme activity was not integrated into cellulosome-like complexes. The adhesion-defective mutant produced significantly less (5- to 10-fold) overall glycanase activity, and the 'true cellulase activity' appeared to be entirely confined to the cell membrane fractions. Antibodies specific for the cellulosomal scaffoldin of Clostridium thermocellum recognized a single 240 kDa band in R. albus SY3. CONCLUSIONS: The adhesion-defective mutant appeared to be blocked in exocellular transport of enzymes involved in true cellulase activity. A potential cellulosomal scaffoldin candidate was identified in R. albus SY3. SIGNIFICANCE AND IMPACT OF THE STUDY: Several glycanase-related proteins and more than one mechanism appear to be involved in the adhesion of R. albus SY3 to cellulose.

Transmission electron microscope study of bacterial morphotypes on the anterior dorsal surface of human tongues.

The human tongue has been the subject of many cytological and histological studies. When a literature search disclosed no reports of the ultrastructure of the morphotypes of bacteria residing on the tongue's surface, a transmission electron microscope study of ultrathin sections of bacteria obtained by scraping eight human tongues was undertaken. The scrapings from the anterior dorsal tongue surfaces, processed conventionally for electron microscope study, revealed 33-35 different bacterial morphotypes. Several of the morphotypes were unique to a tongue. Morphotype differences were also related to donor characteristics such as smoking, tongue site, location in centrifuge pellet, diet, and medications. The predominant morphotypes were Gram-positive cocci. These preliminary findings suggest that the microbiota of the human tongue and variations in that microbiota, related to physical condition, lifestyle, medications, and dietary preferences, merit more attention from anatomists.

Adhesion to cellulose by Ruminococcus albus: a combination of cellulosomes and Pil-proteins?

An obligatory step in cellulose degradation by anaerobic bacteria is the adhesion of the bacterium to the polysaccharide. In many anaerobic bacteria the adhesion protein, and the enzymes required for extensive polysaccharide hydrolysis, are organized into a complex and interesting structure called the cellulosome. The Gram-positive anaerobe Ruminococcus albus also produces a cellulosome-like complex, but the bacterium appears to possess other mechanism(s) for adhesion to plant surfaces and genes encoding functions relevant to growth on cellulose are conditionally expressed, as suggested by a combination of functional proteomics, differential display reverse-transcriptase PCR, and mutational analysis. A novel form of cellulose-binding protein has been identified and shown to belong to the Pil-protein family, being most similar to the type 4 fimbrial proteins of Gram-negative, pathogenic bacteria. These studies have provided new insights into the adhesion of bacteria to plant surfaces, and call attention to the likely existence of genetically analogous adhesion determinants in both pathogenic and non-pathogenic bacteria.

Ultracytochemical localization of hydrogen peroxide production by dental plaque bacteria.

This study looked for evidence of in vitro hydrogen peroxide (HP) synthesis in human dental plaque, using an ultracytochemical technique that included incubation in a CeCl3-rich medium. Supragingival dental plaque was obtained from periodontally healthy individuals and subgingival dental plaque from human periodontal disease sites. Specificity of the cytochemical reaction was demonstrated using catalase (as HP scavenger). HP production was indicated by an electron-dense precipitate localized at the cell envelope of unidentified gram-positive and gram-negative bacteria in both supra- and subgingival samples. The ultracytochemical reaction localized the HP production primarily to the plasma membrane and periplasmic space.

Deinococcus geothermalis sp. nov. and Deinococcus murrayi sp. nov., two extremely radiation-resistant and slightly thermophilic species from hot springs.

Strains of Deinococcus geothermalis sp. nov. were isolated from the hot spring and runoff at Agnano, Naples, Italy, and from the hot spring at São Pedro do Sul in central Portugal, while strains of Deinococcus murrayi sp. nov. were isolated from the hot springs at São Pedro do Sul, São Gemil, and Alcafache in central Portugal. The strains of D. geothermalis and D. murrayi produce orange-pigmented colonies and have an optimum growth temperature of about 45 to 50 degrees C. The type strains of the two new species are extremely gamma radiation resistant. The fatty acids of these new species are primarily branched-chain fatty acids. The two new species can be distinguished from each other by the lower pH range of D. geothermalis than of D. murrayi, by their fatty acid compositions, and by several biochemical parameters, including the ability of D. geothermalis to grow in minimal medium without yeast extract. 16S rRNA gene sequencing also showed that the isolates constitute two species and that these species are distinct from the other species of the genus Deinococcus. The type strain of D. geothermalis is AG-3a (= DSM 11300), and the type strain of D. murrayi is ALT-1b (= DSM 11303).

Successful cultivation of a potentially pathogenic coccoid organism with trophism for gastric mucin.

We have devised a procedure that permits the cultivation of a gram-positive coccoid species from biopsy material obtained from the antrum of the stomachs of patients with gastric disorders. Antibodies directed against surface proteins obtained from the coccoid isolates were detected in all patients with gastric disorders examined in this study, including both Helicobacter pylori-infected and H. pylori-uninfected patients. Several of these isolates, including a prototype designated strain SL100, have been characterized in some detail. Strain SL100 exhibits urease and exceptionally high catalase activities and assumes a variety of spherical morphologies as detected by electron microscopy. This isolate expresses an adhesin that binds to gastric mucin. The adhesin activity was detected only after the isolate was exposed to an acidic pH, suggesting that in the natural process of infection, the low pH of the stomach unmasks a cell surface component with adhesin activity. Strain SL100 grows best under a microaerophilic conditions (10% CO2, 5% O2, 85% N2), but it also grows quite well under aerobic conditions. Thus, this organism would be expected to proliferate outside of the human host as well as in the gastric mucosa. Oral infection of newborn piglets resulted in colonization of the gastric antrum and growth retardation. Preliminary taxonomic classification indicates similarity to the Staphylococcus DNA homology groups containing S. cohnii and S. xylosus. One of us (C.K.) apparently became infected with this organism as indicated by gastric symptoms and the subsequent presence of strain-specific antisera not present in other workers in the laboratory.

Luteococcus japonicus gen. nov., sp. nov., a new gram-positive coccus with LL-diaminopimelic acid in the cell wall.

A new gram-positive, nonmotile coccus is described. Strains IFO 12422T (T = type strain) and IFO 15385 in the Institute for Fermentation, Osaka, culture collection, which were isolated from soil and water, respectively, have the following chemotaxonomic characteristics: menaquinone MK-9(H4); G + C content of DNA of 67 mol%; and LL-diaminopimelic acid, alanine, glycine, and glutamic acid in a molar ratio of ca. 1:2:1:1 (type A3 gamma). Mycolic acids are not present. The taxonomic characteristics of these organisms are different from those of previously described gram-positive, high-G + C-content cocci. The partial 16S rRNA sequence indicated that IFO 12422T represents a distinct line of descent among gram-positive bacteria with a high G + C content. The name Luteococcus japonicus gen. nov., sp. nov. is proposed. The type strain is strain IFO 12422.

Platinum/iridium/carbon: a high-resolution shadowing material for TEM, STM and SEM of biological macromolecular structures.

Thin Pt/Ir/C coating films (1.5 nm) show a fine granularity and provide a high structural resolution in the transmission electron microscope (TEM) when applied to freeze-dried biological macromolecules. They keep their structure when exposed to atmospheric conditions, without the need of an additional stabilizing carbon layer, in contrast to conventional high-resolution shadowing materials such as Ta/W and Pt/C. However, the correct ratio of the components has turned out to be crucial. When evaporating Pt/Ir/C from the source electrode in an electron-beam-heated evaporator, the ratio of the three elements changes progressively, and, consequently, the properties of such films depend strongly on the mass that has been pre-evaporated. In this paper we present a quantitative analysis of the composition of Pt/Ir/C films by wavelength-dispersive X-ray analysis (WDX) undertaken in association with TEM experiments. We applied Pt/Ir/C shadowing to two regular biological test specimens, the phage T4 type III polyhead and the HPI-layer of Deinococcus radiodurans. It turns out that Pt/Ir/C films containing at least 25% C are three-dimensionally stable on the freeze-dried macromolecular samples. By the dramatically improved resolution power of the latest scanning electron microscopes (SEM) and the invention of the scanning tunnelling microscope (STM), two new surface-sensitive tools for the investigation of biological macromolecular structures became available. The Pt/Ir/C coating has proved to be well suited for STM and SEM imaging of freeze-dried biological structures because of its good electrical conductivity and its direct three-dimensional stability. We compare STM, SEM and TEM images of freeze-dried and Pt/Ir/C-coated polyheads.

Atomic force microscopy of a hydrated bacterial surface protein.

The protein surface layer of the bacterium Deinococcus radiodurans (HPI layer) was examined with an atomic force microscope (AFM). The measurements on the air-dried, but still hydrated layer were performed in the attractive imaging mode in which the forces between tip and sample are much smaller than in AFM in the repulsive mode or in scanning tunnelling microscopy (STM). The results are compared with STM and transmission electron microscopy (TEM) data.

Characterization of a halotolerant-psychroloterant bacterium from dry valley Antarctic soil.

The saline soils of the ice free dry valleys of Victoria Land, Antarctica may provide the closest analog on Earth to Martian conditions. We have initiated a study aimed at examining microbial adaptations to the harsh environment of these dry valley soils. In this report we describe the characterization of one bacterium, strain A4a, isolated from Taylor Valley soil. Strain A4a was an obligately aerobic, orange-pigmented, Gram-positive coccus that grew over wide ranges of both temperature (0 degrees C-40 degrees C) and sodium chloride concentration (0-2.0M). The optimal temperature for growth at all NaCl concentrations was 25 degrees C. Phospholipid composition and guanine plus cytosine content of the DNA of the isolate indicate a close relation to the genus Planococcus.