An essential saccharide binding domain for the mAb 2C7 established for Neisseria gonorrhoeae LOS by ES-MS and MSn

A study of bacterial surface oligosaccharides were investigated among different strains of Neisseria gonorrhoeae to correlate structural features essential for binding to the MAb 2C7. This epitope is widely expressed and conserved in gonococcal isolates, characteristics essential to an effective candidate vaccine antigen. Sample lipooligosaccharides (LOS), was prepared by a modification of the hot phenol-water method from which de-O-acetylated LOS and oligosaccharide (OS) components were analyzed by ES-MS-CID-MS and ES-MSnin a triple quadrupole and an ion trap mass spectrometer, respectively. Previously documented natural heterogeneity was apparent from both LOS and OS preparations which was admixed with fragments induced by hydrazine and mild acid treatment. Natural heterogeneity was limited to phosphorylation and antenni extensions to the alpha-chain. Mild acid hydrolysis to release OS also hydrolyzed the beta(1-->6) glycosidic linkage of lipid A. OS structures were determined by collisional and resonance excitation combined with MS and multistep MSn which provided sequence information from both neutral loss, and nonreducing terminal fragments. A comparison of OS structures, with earlier knowledge of MAb binding, enzyme treatment, and partial acid hydrolysis indicates a generic overlapping domain for 2C7 binding. Reoccurring structural features include a Hepalpha(1-->3)Hepbeta(1-->5)KDO trisaccharide core branched on the nonreducing terminus (Hep-2) with an alpha(1-->2) linked GlcNAc (gamma-chain), and an alpha-linked lactose (beta-chain) residue. From the central heptose (Hep-1), a beta(1-->4) linked lactose (alpha-chain), moiety is required although extensions to this residue appear unnecessary.      

Sporulation of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> in binary cultures with ultramicrobacteria

The effect of ultramicrobacterial epibionts of the genera Kaistia (strain NF1), Chryseobacterium (strain NF4), and Stenotrophomonas (strain FM3) on the process of sporulation of Bacillus subtilis ATCC 6633 was studied. The investigated strains of ultramicrobacteria (UMB) were found to inhibit the sporulation process of B. subtilis ATCC 6633 in binary mixed cultures, exhibiting a 3-day delay of the onset of sporulation compared to the control one, an extended period of the prospore maturation, formation of the fraction of immature spores, and development of ultrastructural defects in many endospores. Thus, investigation of binary mixed cultures of B. subtilis and UMB revealed that, apart from suppression of reproduction and lysis of host vegetative cells, inhibition of spore formation and destruction of endospores was yet another feature of intermicrobial parasitism. The UMB parasites of the studied genera are assumed to participate in the regulation of development and reproduction of B. subtilis in natural habitats of this spore-forming bacterium.     

Structural and functional rearrangements in the cells of actinobacteria <Emphasis Type="Italic">Microbacterium foliorum</Emphasis> BN52 during transition from vegetative growth to a dormant state and during germination of dormant forms

Cellular organization of the cystlike cells (CLC) of non-spore-forming heterotrophic actinobacteria isolated from soils contaminated with chemical plant waste (Bereznyaki, Russia) and identified as Microbacterium foliorum BN52 was studied. CLC were obtained in laboratory conditions in limited or starving cultures. Two morphotypes of CLC capable of reversion to the vegetative growth were revealed and characterized in detail. The morphological, ultrastructural, and physiological peculiarities of germination and transition to the vegetative growth were studied at first for the dormant forms of M. foliorum BN52. Germination of morphotype II CLC was associated with gradual recovery of the cell shape and subcellular structures. In contrast to the germination of morphotype II CLC, during the first hours of germination of morphotype I CLC cell volume increased significantly with the subsequent formation of very large cell with sizes several times larger than the typical vegetative cells of the strain. The obtained cells were characterized by polynucleoidity, being polyploids undergoing fission at the next stage of germination, resulting in formation of numerous small and ultrasmall viable cell forms. Formation of cyst-like dormant cells, germinating in the form of polyploid cells is assumed to be the basis of survival and adaptation strategies of heterotrophic bacteria, which are incapable of toxicant degradation in natural habitats, under the conditions of the toxic pressure.     

Morphological,physiological, and biochemical characteristics of a benzoate-degrading strain <Emphasis Type="Italic">Rhodococcus opacus</Emphasis> 1CP under stress conditions

Ability of actinobacteria Rhodococcus opacus 1CP to survive under unfavorable conditions and retain its biodegradation activity was assessed. The morphological and ultrastructural features of R. opacus 1CP cells degrading benzoate in the presence of oxidants and stress-protecting agents were investigated. The cells of R. opacus 1CP were resistant to oxidative stress caused by up to 100 mM H₂O₂ or up to 25 μM juglone (5-oxy-1,4-naphthoquinone). After 2 h of stress impact, changes in the fatty acid composition, increased activity of antioxidant enzymes, and changes in cell morphology and ultrastructure were observed. The strain retained its ability to degrade benzoate. Quercetin had a protective effect on benzoate-degrading cells of R. opacus 1CP. The strategy for cells survival under unfavorable conditions was formulated, which included decreased cell size/volume and formation of densely-packed cell conglomerates, in which the cells are embedded into a common matrix. Formation of conglomerates may probably be considered as a means for protecting the cells against aggressive environmental factors. The multicellular conglomerate structure and the matrix material impede the penetration of toxic substances into the conglomerates, promoting survival of the cells located inside.     

Ultramicrobacteria: Formation of the concept and contribution of ultramicrobacteria to biology

Ultramicrobacteria (UMB) are species of the domain Bacteria characterized by very small sizes of proliferating cells (less than 0.1 μm³ in volume) and small genomes (3.2 to 0.58 Mb). Some authors use the term nanobacteria as a synonym of UMB. Several tens of UMB species have been isolated from various natural habitats: sea water, soil, silt, Greenland ice sheet, permafrost soils, and intestines of humans and insects. Under laboratory conditions, they are cultivated on different nutrient media. In the second prokaryotic domain, the Archaea, ultrasmall forms (ultramicroarchaea) have also been described, including nanoarchaea (members of the genus Nanoarchaeum) with a cell volume of less than 0.1 μm³. The term nanobacteria is used in the literature also to denote ultrasmall bacterium-like particles occurring in rocks, sands, soils, deep sub-surface layers, meteorites, and clinical samples. The systematic position and the capacity for self-reproduction of these particles are still unclear. The cultured UMB forms are characterized by highly diverse morphology, ultrastructural organization, physiology, biochemistry, and ecology. UMB form three groups according to the type of cell wall structure and the reaction to Gram staining: (1) gram-negative, (2) gram-positive, and (3) cell wall-lacking. Their cells divide by constriction, septation, or budding. The unique processes performed by UMB are dehalorespiration and obligate or facultative epibiotic parasitism. The UMB that synthesize organic compounds in ocean waters with the involvement of proteorhodopsin play a great role in the biosphere. UMB have been found in seven large phylogenetic groups of prokaryotes, where their closest relatives are organisms with larger cells typical of bacteria, which is evidence of the polyphyletic origin of the currently known UMB species and the reductive mode of their evolution.     

Cytophysiological Characteristics of the Vegetative and Dormant Cells of <Emphasis Type="Italic">Stenotrophomonas</Emphasis> sp. Strain FM3, a Bacterium Isolated from the Skin of a <Emphasis Type="Italic">Xenopus laevis</Emphasis> Frog

A bacterial strain (FM3) that is closely related to Stenotrophomonas acidaminiphila and S. maltophilia, was isolated from the skin surface of the frog Xenopus laevis. Cytophysiological studies on vegetative cells and cyst-like cells (CLCs) that were obtained in model experiments addressed the dynamics of transition of vegetative cells to the dormant state and their reversion to vegetative growth. The ultrastructural organization of the vegetative and dormant cells of strain FM3 possesses unique properties. Cultures that developed after inoculating vegetative cells were characterized by: (1) resistance to physical factors and sterilization procedures; (2) high antimicrobial activity with respect to some gram-positive and gram-negative bacteria; (3) resistance to polypeptide antibiotics; (4) the presence of an easily detaching S-layer on the cell surface; (5) the ability to secret outer membrane vesicles into the intercellular space; and (6) formation of S-layerderived tubular structures associated with outer membrane vesicles that are regularly arranged within the tubes. Dormant cells were characterized by: (1) resistance to dehydration; (2) resistance to high temperatures; and (3) the preservation of the S-layer on the surface of cystlike cells (CLCs). Depending on experimental conditions, strain FM3 formed three CLC morphotypes, which differed in their abundance and ultrastructural organization. The experimental conditions used for CLC formation approximated those under which bacteria survive in hospitals. A model of intermicrobial parasitism is suggested that applies to motile FM3 cells during the development of their populations (cultures).     

Study of ectoparasitism of ultramicrobacteria of the genus Kaistia, strains NF1 and NF3 by electron and fluorescence microscopy

Transmission electron and fluorescence microscopy was used to study the character of the interaction of free-living ultramicrobacterial (UMB) strains NF1 and NF3, affiliated with the genus Kaistia, and seven species of gram-positive and gram-negative heterotrophic bacteria. Strains NF1 and NF3 were found to exhibit parasitic activity against gram-positive Bacillus subtilis and gram-negative Acidovorax delafildii. UMB cells are tightly attached to the envelopes of the victim cells and induce their lysis, thus demonstrating the features of typical ectoparasitism. The selectivity of parasitism of the studied UMB to the victim bacteria has been shown: only two soil microorganisms of the seven test objects, B. subtilis ATCC 6633 and an aerobic gram-negative bacterium A. delafildii 39, were found to be sensitive to UMB attack. Other bacteria (Micrococcus luteus VKM Ac-2230, Staphylococcus aureus 209-P, Pseudomonas putida BS394, Escherichia coli C 600, and Pantoea agglomerans ATCC 27155) were not attacked by UMB. It was established for the first time that free-living UMB may be facultative parasites not only of phototrophic bacteria, as we have previously demonstrated, but of heterotrophic bacteria as well. The UMB under study seem to play an important role in the regulation of the quantity of microorganisms and in the functioning of microbial communities in some natural ecotopes.