The use of tannic acid for the ultrastructural visualization of hyaluronic acid

Summary This study describes a method, which makes use of tannic acid (2%) as a component of a paraformaldehyde-glutaraldehyde based fixative, to reveal the presence and ultrastructure of glycosaminoglycans in the extracellular matrix. The ultrastructure of the extracellular matrix in the stage 24 chick embryo wing is examined after fixation by several procedures. After fixation in the absence of tannic acid, the intercellular spaces contain little extracellular matrix, except for occasional fibrils (collagen?). On the other hand, when tannic acid is included in the primary fixative, the intercellular spaces contain considerable amounts extracellular matrix which includes 3±0.5 nm filaments, ±30 nm granules, as well as putative collagen fibrils. The 3±0.5 nm diameter fibrils are not observed when the limbs had been injected in ovo with Streptomyces hyaluronidase (specific for hyaluronic acid) prior to fixation. Furthermore, the 3±0.5 nm fibrils resemble authentic hyaluronic acid that had been fixed by the same procedure in the presence of tannic acid. Limbs treated with tannic acid after osmication contained only small amounts of extracellular material, which was confined largely to cell surfaces. These results demonstrate that the use of tannic acid in the primary fixative can serve as a useful method for the ultrastructural visualization of several extracellular matrix materials, including hyaluronic acid.This study was supported by NIH grant HD 05505     

Effects of tannic acid on antigenicity and membrane contrast in ultrastructural immunocytochemistry.

We have modified our previous method for immunogold staining of unosmicated, plastic-embedded tissue by addition of tannic acid as a post-fixative to increase membrane contrast. Overall cell ultrastructure and organelle membranes, in particular, appeared well preserved after this treatment. We evaluated quantitatively the effect of tannic acid on the antigenicity of several membrane proteins in rat liver and intestine. For all antigens tested, significant antigenicity was retained on both intracellular and plasma membranes. However, the level of antigenicity decreased with increased concentrations of tannic acid. This effect was most apparent on the apical and basolateral membranes of hepatocytes and on the apical membrane of enterocytes, surfaces that had been in direct contact with the tannic acid fixative. The results indicate that when low concentrations of tannic acid are employed, this method yields greatly enhanced membrane contrast while preserving sufficient antigenicity to facilitate the ultrastructural localization of many membrane and other antigens.     

Molecular diversity of tannic acid degrading bacteria isolated from tannery soil

AIMS: The aim of this study was to enrich and isolate bacteria from a tannery soil that were capable of utilizing tannic acid and gallic acid as sole source of carbon aerobically, and to characterize their diversity in order to identify efficient strains that can be used for tannin bioremediation. METHODS AND RESULTS: Bacterial strains were isolated after enrichment in minimal medium with tannic acid or gallic acid as sole carbon source. Polymerase chain reaction (PCR) restricted fragment length polymorphism of 16S rDNA [amplified ribosomal DNA restriction analysis (ARDRA)] and BOX-PCR was used to characterize their diversity. Two strains showing relatively high efficiency in degrading tannic acid and gallic acid were identified on the basis of carbon source utilization pattern (BIOLOG) and 16S rDNA sequence. CONCLUSIONS: Bacterial strains capable of degrading tannic acid and gallic acid could be grouped into six and seven clusters on the basis of ARDRA and BOX-PCR, respectively. On the basis of 16S rDNA sequence, the most efficient isolate degrading tannic acid belonged to Pseudomonas citronellolis, whereas the most efficient gallic acid degrader showed maximum phylogenetic relatedness to P. plecoglossicida. SIGNIFICANCE AND IMPACT OF THE STUDY: Aerobic tannic acid degraders such as the two strains isolated in this study can be used for tannin bioremediation, and in the study of genes involved in the production of tannase, an industrially important enzyme.     

Membrane asymmetry and enhanced ultrastructural detail of sarcoplasmic reticulum revealed with use of tannic acid

Fixation of purified sarcoplasmic reticulum (SR) membrane vesicles, using glutaraldehyde supplemented with 1% tannic acid, reveals newly visualized ultrastructure in thin sections. The trilaminar appearance of the membrane is highly asymmetric; the outer electron-opaque layer is appreciably wider (70 A) than the inner layer (20 A). The asymmetry is not referable to lack of penetration of the tannic acid since: (a) SR vesicles made permeable with 1 mM EDTA, pH 8.5, show similar asymmetry; (b) treatment of SR with trypsin results in progressive loss in protein content and decrease in the thickness of the outer layer, until in the limit the trilayer has a symmetric appearance; (c) within the same muscle section, the SR membrane appears highly asymmetric whereas the sarcolemma has a more symmetric appearance; (d) reconstituted SR vesicles have a symmetric appearance with equally broad inner and outer layers (approximately 70 A); the symmetric structure is confirmed by freeze-fracture and negative staining electron microscopy. Heavy and light SR vesicles obtained by isopycnic density sedimentation of purified SR have the same asymmetric appearance of the membrane and seem to differ mainly in that the heavy vesicles contain internal contents consisting largely of Ca++-binding protein. The asymmetry of the SR membrane is referable mainly to the unidirectional alignment of the Ca++ pump protein, the major component (90% of the protein) of the membrane. The asymmetry of the SR membrane can be visualized now for the first time in situ in thin sections of muscle.     

Metabolism of o-phthalic acid by different gram-negative and gram-positive soil bacteria

Different bacteria, isolated from soil by the enrichment method, were able to grow on phthalic acid as carbon source. Protocatechuate was identified as intermediate in phthalate metabolism. All phthalategrown bacteria oxidized phthalate and protocatechuate rapidly without having a lag-period. Benzoic acid, terephthalic acid, protocatechuic acid, salicylic acid, di- and mono-butyl phthalate were also metabolized by some of the organisms, benzoic acid being degraded via catechol and terephthalic acid via protocatechuate as intermediate. All organisms tested cleaved protocatechuate or catechol, respectively, by the ortho fission, when grown on phthalate, terephthalate, or benzoate as carbon source. A characterization and tentative identification of the organisms is given.     

Acquisition of siderophores in gram-negative bacteria

The outer membrane of Gram-negative bacteria constitutes a permeability barrier that protects the cell from exterior hazards, but also complicates the uptake of nutrients. In the case of iron, the challenge is even greater, because of the scarcity of this indispensable element in the cell's surroundings. To solve this dilemma, bacteria have evolved sophisticated mechanisms whereby the concerted actions of receptor, transporter and energy-transducing proteins ensure that there is a sufficient supply of iron-containing compounds, such as siderophores.     

Use of a fluorescent redox probe for direct visualization of actively respiring bacteria.

The redox dye 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) was employed for direct epifluorescent microscopic enumeration of respiring bacteria in environmental samples. Oxidized CTC is nearly colorless and is nonfluorescent; however, the compound is readily reduced via electron transport activity to fluorescent, insoluble CTC-formazan, which accumulates intracellularly. Bacteria containing CTC-formazan were visualized by epifluorescence microscopy in wet-mount preparations, on polycarbonate membrane filter surfaces, or in biofilms associated with optically opaque surfaces. Counterstaining of CTC-treated samples with the DNA-specific fluorochrome 4',6-diamidino-2-phenylindole allowed enumeration of active and total bacterial subpopulations within the same preparation. Municipal wastewater, groundwater, and seawater samples supplied with exogenous nutrients yielded CTC counts that were generally lower than total 4',6-diamidino-2-phenylindole counts but typically equal to or greater than standard heterotrophic (aerobic) plate counts. In unsupplemented water samples, CTC counts were typically lower than those obtained with the heterotrophic plate count method. Reduction of CTC by planktonic or biofilm-associated bacteria was suppressed by formaldehyde, presumably because of inhibition of electron transport activity and other metabolic processes. Because of their bright red fluorescence (emission maximum, 602 nm), actively respiring bacteria were readily distinguishable from abiotic particles and other background substances, which typically fluoresced at shorter wavelengths. The use of CTC greatly facilitated microscopic detection and enumeration of metabolically active (i.e., respiring) bacteria in environmental samples.     

Use of a fluorescent redox probe for direct visualization of actively respiring bacteria.

The redox dye 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) was employed for direct epifluorescent microscopic enumeration of respiring bacteria in environmental samples. Oxidized CTC is nearly colorless and is nonfluorescent; however, the compound is readily reduced via electron transport activity to fluorescent, insoluble CTC-formazan, which accumulates intracellularly. Bacteria containing CTC-formazan were visualized by epifluorescence microscopy in wet-mount preparations, on polycarbonate membrane filter surfaces, or in biofilms associated with optically opaque surfaces. Counterstaining of CTC-treated samples with the DNA-specific fluorochrome 4',6-diamidino-2-phenylindole allowed enumeration of active and total bacterial subpopulations within the same preparation. Municipal wastewater, groundwater, and seawater samples supplied with exogenous nutrients yielded CTC counts that were generally lower than total 4',6-diamidino-2-phenylindole counts but typically equal to or greater than standard heterotrophic (aerobic) plate counts. In unsupplemented water samples, CTC counts were typically lower than those obtained with the heterotrophic plate count method. Reduction of CTC by planktonic or biofilm-associated bacteria was suppressed by formaldehyde, presumably because of inhibition of electron transport activity and other metabolic processes. Because of their bright red fluorescence (emission maximum, 602 nm), actively respiring bacteria were readily distinguishable from abiotic particles and other background substances, which typically fluoresced at shorter wavelengths. The use of CTC greatly facilitated microscopic detection and enumeration of metabolically active (i.e., respiring) bacteria in environmental samples.     

Efficient subfractionation of gram-negative bacteria for proteomics studies

Proteomics studies of pathogenic bacteria are an important basis for biomarker discovery and for the development of antimicrobial drugs and vaccines. Especially where vaccines are concerned, it is of great interest to explore which bacterial factors are exposed on the bacterial cell surface and thus can be directly accessed by the immune system. One crucial step in proteomics studies of bacteria is an efficient subfractionation of their cellular compartments. We set out to compare and improve different protocols for the fractionation of proteins from Gram-negative bacteria into outer membrane, cytoplasmic membrane, periplasmic, and cytosolic fractions, with a focus on the outer membrane. Overall, five methods were compared, three methods for the fast isolation of outer membrane proteins and two methods for the fractionation of each cellular compartment, using Escherichia coli BL21 as a model organism. Proteins from the different fractions were prepared for further mass spectrometric analysis by SDS gel electrophoresis and consecutive in-gel tryptic digestion. Most published subfractionation protocols were not explicitly developed for proteomics applications. Thus, we evaluated not only the separation quality of the five methods but also the suitability of the samples for mass spectrometric analysis. We could obtain high quality mass spectrometry data from one-dimensional SDS-PAGE, which greatly reduces experimental time and sample amount compared to two-dimensional electrophoresis methods. We then applied the most specific fractionation technique to different Gram-negative pathogens, showing that it is efficient in separating the subcellular proteomes independent of the species and that it is capable of producing high-quality proteomics data in electrospray ionization mass spectrometry.     

Anhydrobiotic engineering of gram-negative bacteria

Anhydrobiotic engineering aims to improve desiccation tolerance in living organisms by adopting the strategies of anhydrobiosis. This was achieved for Escherichia coli and Pseudomonas putida by osmotic induction of intracellular trehalose synthesis and by drying from trehalose solutions, resulting in long-term viability in the dried state.     

Plasmid-borne streptothricin resistance in gram-negative bacteria

Plasmids, which code for a new type of drug resistance due to the acetylation of streptothricin and belonging to various incompatibility groups (I1, I2, W3, FII, S, X, and N) were identified in gram-negative bacteria. The gene for the acetyltransferase is closely linked to a gene for the streptomycin/spectinomycin adenylyltransferase AAD-3″ on a novel type of a transposon, designated Tn1825. This is related to Tn7.     

Effects of quinolone carboxylic acid derivatives on the ultrastructure of gram-positive and gram-negative bacteria

The impact of oxolinic acid, pefloxacin and ciprofloxacin on the structure of Staphylococcus aureus, Pseudomonas aeruginosa and Serratia marcescens was studied. The concentration dependent effects were revealed. Comparative estimation of the action of the compounds by the content of normal, dead and changed cells in the cultures showed that ciprofloxacin had advantages. The results of the pefloxacin use were close. The time course of the oxolinic acid effect was analogous whereas the changes in the cells induced by the acid were less pronounced. Investigation of the ultrastructural changes demonstrated that the response of the gram-negative bacteria to the action of the compounds was of one type. There was noted formation of filamentous structures and vacuolized cells at low concentrations and large egg-shaped cells at high concentrations of the compounds. In the gram-positive bacteria there were detected large cells with much thicker cell walls at low concentrations of the compounds while at the high concentrations the cell walls were on the contrary thinner, the cell stiffness was lost and the cells acquired a festoon-like shape. The compounds induced significant changes in the nucleoids, cell walls and membranes. Along with insignificant changes in the nucleoid structure there were constantly detected coarse impairments of the cell walls and membranes which should be considered in estimating the effect of the quinolone carboxylic acid derivatives.     

Type I secretion in gram-negative bacteria

In gram-negative bacteria, type I secretion is carried out by a translocator made up of three proteins that span the cell envelope. One of these proteins is a specific outer membrane protein (OMP) and the other two are cytoplasmic membrane proteins: an ATP-binding cassette (ABC) and the so-called membrane fusion or adaptor protein (MFP). Type I secretion is sec-independent and bypasses the periplasm. This widespread pathway allows the secretion of proteins of diverse sizes and functions via a C-terminal uncleaved secretion signal. This C-terminal secretion signal specifically recognizes the ABC protein, triggering the assembly of the functional trans-envelope complex. This report will mainly deal will recent data concerning the structure and assembly of the secretion complex as well as the effects and role of substrate folding on secretion by this pathway.     

Replication of plasmids in gram-negative bacteria.

Replication of plasmid deoxyribonucleic acid (DNA) is dependent on three stages: initiation, elongation, and termination. The first stage, initiation, depends on plasmid-encoded properties such as the replication origin and, in most cases, the replication initiation protein (Rep protein). In recent years the understanding of initiation and regulation of plasmid replication in Escherichia coli has increased considerably, but it is only for the ColE1-type plasmids that significant biochemical data about the initial priming reaction of DNA synthesis exist. Detailed models have been developed for the initiation and regulation of ColE1 replication. For other plasmids, such as pSC101, some hypotheses for priming mechanisms and replication initiation are presented. These hypotheses are based on experimental evidence and speculative comparisons with other systems, e.g., the chromosomal origin of E. coli. In most cases, knowledge concerning plasmid replication is limited to regulation mechanisms. These mechanisms coordinate plasmid replication to the host cell cycle, and they also seem to determine the host range of a plasmid. Most plasmids studied exhibit a narrow host range, limited to E. coli and related bacteria. In contrast, some others, such as the IncP plasmid RK2 and the IncQ plasmid RSF1010, are able to replicate in nearly all gram-negative bacteria. This broad host range may depend on the correct expression of the essential rep genes, which may be mediated by a complex regulatory mechanism (RK2) or by the use of different promoters (RSF1010). Alternatively or additionally, owing to the structure of their origin and/or to different forms of their replication initiation proteins, broad-host-range plasmids may adapt better to the host enzymes that participate in initiation. Furthermore, a broad host range can result when replication initiation is independent of host proteins, as is found in the priming reaction of RSF1010.     

The oxidation of methanol in gram-negative bacteria

Abstract This is a brief review of the structure and interaction of methanol dehydrogenase and its electron acceptor cytochrome c _L , their regulation by modifier protein, and the synthesis and assembly of the 'methanol oxidase' system.