Aquabacterium gen. nov., with description of Aquabacterium citratiphilum sp. nov., Aquabacterium parvum sp. nov. and Aquabacterium commune sp. nov., three in situ dominant bacterial species from the Berlin drinking water system.

Three bacterial strains isolated from biofilms of the Berlin drinking water system were characterized with respect to their morphological and physiological properties and their taxonomic position. Phenotypically, the bacteria investigated were motile, Gram-negative rods, oxidase-positive and catalase-negative, and contained polyalkanoates and polyphosphate as storage polymers. They displayed a microaerophilic growth behaviour and used oxygen and nitrate as electron acceptors, but not nitrite, chlorate, sulfate or ferric iron. The substrates metabolized included a broad range of organic acids but no carbohydrates at all. The three species can be distinguished from each other by their substrate utilization, ability to hydrolyse urea and casein, cellular protein patterns and growth on nutrient-rich media as well as their temperature, pH and NaCl tolerances. Phylogenetic analysis, based on 16S rRNA gene sequence comparison, revealed that the isolates are affiliated to the beta 1-subclass of Proteobacteria. The isolates constitute three new species with internal levels of DNA relatedness ranging from 44.9 to 51.3%. It is proposed that a new genus, Aquabacterium gen. nov., should be created, including Aquabacterium citratiphilum sp. nov., Aquabacterium parvum sp. nov. and Aquabacterium commune sp. nov. The type species of the new genus is Aquabacterium commune. The type strain of A. citratiphilum is strain B4T (= DSM 11900T), the type strain of A. parvum is strain B6T (= DSM 11968T) and the type strain of A. commune is strain B8T (= DSM 11901T).

Isolation of new bacterial species from drinking water biofilms and proof of their in situ dominance with highly specific 16S rRNA probes.

A polyphasic approach involving cultivation, direct viable counts, rRNA-based phylogenetic classification, and in situ probing was applied for the characterization of the dominant microbial population in a municipal drinking water distribution system. A total of 234 bacterial strains cultivated on R2A medium were screened for bacteria affiliated with the in situ dominating beta subclass of Proteobacteria. The isolates were grouped according to common features of their cell and colony morphologies, and eight representative strains were used for 16S rRNA sequencing and the development of a suite of strain-specific oligonucleotide probes. Phylogenetic analysis indicated that all of the isolates were hitherto unknown bacteria. Three of them, strains B4, B6, and B8, formed a separate cluster of closely related organisms within the beta 1 subclass of Proteobacteria. In situ probing revealed that (i) 67 to 72% of total bacteria, corresponding to more than 80% of beta-subclass bacteria, could be encompassed with the strain-specific probes and (ii) the dominating bacterial species were culturable on R2A medium. Additionally, two-thirds of the autochthonous drinking water population could be shown to be in a viable but nonculturable (VBNC) state by using a direct viable count approach. The comparison of isolation frequencies with the in situ abundances of the eight investigated strains revealed differences in their culturability, indicating variable ratios of culturable to VBNC cells among the strains. The further characterization of biofilms throughout the distribution network demonstrated strains B6 and B8 to be dominant bacterial strains in groundwater and distribution system biofilms. The other strains could be found at various frequencies in the different parts of the distribution system; several strains appeared exclusively in drinking water biofilms obtained from a house installation system.

Immunohistochemical staining and enzyme activity measurements show myo-inositol-1-phosphate synthase to be localized in the vasculature of brain.

Rabbit anti-bovine myo-inositol-1-phosphate synthase was used to examine the distribution of that enzyme in perfused and immersion-fixed bovine brain and testis. In brain, intense and specific staining was found in the walls of all the vascular elements including cerebral capillaries. The remainder of brain parenchyma exhibited only low levels of background staining. In testis, an organ rich in the enzyme, blood vessels showed no specific staining. Instead, the enzyme was found in the seminiferous epithelium of the seminiferous tubules, perhaps localized in spermatozoa. To confirm the brain finding, the activity of myo-inositol-1-phosphate synthase was measured in bovine brain microvessel preparations and brain pial vessels. In these preparations the activity of the enzyme was found on average to be 7 and 22 times enriched over that in whole brain, respectively. The activities of two other enzymes of inositol metabolism, myo-inosose reductase and myo-inositol-1-phosphatase, were also examined for their distribution in brain. Those enzymes were found to be generally distributed. The surprising finding of a vascular localization of myo-inositol-1-phosphate synthase in brain raises new questions about the mechanism by which myo-inositol is concentrated to such high cellular levels in the principal substance of that organ.

Development and maturation of central nervous system myelin: comparison of immunohistochemical localization of proteolipid protein and basic protein in myelin and oligodendrocytes.

The immunohistochemical localization of two myelin specific proteins-basic protein (BP) and proteolipid protein (PLP)-was compared during the process of myelination. Although both proteins were present in oligodendrocytes, (i) neither protein was observed in oligodendrocytes not already closely associated with nerve fibers exhibiting a fluorescent coating; (ii) in any discrete anatomical area oligodendrocytes were positive for BP before PLP was visible; and (iii) as myelination progressed, immunoreactivity for BP in oligodendrocytes appeared to decrease and simultaneously PLP immunofluorescence became visible in this cell type. During the period of active myelination, fibers exhibited a distinct varicose appearance. As myelination progressed, the myelin sheath increased in thickness and these varicosities became less prominent, eventually completely disappearing. Therefore, the nature and the appearance of varicosities can be used as an index of the relative stage of maturation of myelin in an individual fiber. In general, PLP appeared in fibers at a later stage of maturation than did BP based on the above criteria. However, in a relatively small number of fine fibers PLP was observed at a very early stage. In fully mature myelin, very large fibers were frequently more intensely fluorescent for BP than PLP, whereas fine myelinated fibers were more intensely stained for PLP. These observations are consistent with the following interpretations. (i) Substantial differentiation of oligodendrocytes occurs prior to appearance of either of these proteins by immunofluorescence. (ii) BP is added to the myelin sheath prior to PLP and there appears to be a shift in priority of synthesis from BP to PLP in individual oligodendrocytes during the process of myelination. (iii) Very small fibers often contain low concentrations of BP relative to PLP, and conversely, very large fibers may contain a high concentration of BP relative to PLP. Thus, the relative concentration of these proteins in myelin appears not to be constant but may vary as a function of the size of the myelinated fiber.

A comparative study of the immunohistochemical localization of basic protein to myelin and oligodendrocytes in rat and chicken brain.

Antisera to highly purified basic protein (BP) from rat and chicken brain were prepared and their purity and specificity demonstrated by double immunodiffusion and cross-immunoadsorption. These antisera were used for immunohistochemical localization of BP in the brains of adult and developing rat and chick. Myelin basic protein was exclusively localized to myelin or the myelin forming elements of the CNS. It was present in high concentrations in white matter and absent in areas free of myelin. Neuronal parikarya and dendrites were negative as were axons cut in cross section and at Nodes of Ranvier. The latter was best observed in cross sections of human spinal cord demonstrating also the immunoreactivity of the antibodies with human BP. The internodal distance in a fine (1.5 micrometer) rat cortical fiber was determined to be approximately 45 micrometers. Myelin basic protein was shown to extend into cranial roots, in contrast to myelin proteolipid protein which abruptly lose fluorescence as the nerves emerged from the brain. During development, BP was first observed on the fourteenth day of incubation in chick and at birth in the rat. The protein appeared in oligodendrocytes and in association with fibers near these cells. Fluorescent processes were frequently observed connecting the oligodendrocytes with the fibers. As myelination progressed, the intensity of the immunohistochemical reaction decreased in the oligodendrocytes while the brightness in fibers increase. Eventually, the oligodendrocytes became undetectable. Fibers with immature myelin exhibited a beaded or varicosed appearance with the highest concentration of immunofluorescence in the outer portion of the varicosities. The varicosities were postulated to represent dilations in the newly forming sheath between intervals of compaction along the axon undergoin myelination. These dilations might represent areas of increased cytoplasmic volume which could serve as channels for transport and/or storage sites for myelin proteins prior to incorporation into the membrane. The varicosities became less prominent with the thickening of the myelin sheath and mature myelinated fibers became smooth. The process of synthesis of BP, transport of the protein to the varicosed fibers, and maturation of the myelin sheath was seen to progress in a more or less caudal to rostral direction as myelination of the CNS takes place. In the rat, this was accomplished over approximately a 30-day period starting near the time of birth. In the chick, most of the myelination was accomplished in the three or four days immediately before hatching. At this time, innumerable oligodendrocytes were observed producing BP simultaneously in the major white fiber tracts. It is postulated that in chick some degree of oligodendrocytic cell death occurs normally during myelination.