The significance of enterobacterial mutants for the chemical investigation of their cell-wall polysaccharides

The outer cell surface of Enterobacteriaceae, i.e. the cell wall, consist of a rigid structure (murein) on which additional proteins, lipids, and polysaccharides are deposited. In the bacterial wild types (S forms) the polysaccharide is species-specific and carries the serologically determinant groups of the respective O antigen. These specific polysaccharides often consist of a large number of monosaccharides; up to eight different monosaccharides may be involved. Bound to lipid A, the cell-wall lipopolysaccharides represent the endotoxins of the bacteria. During the past few years the structures of the enterobacterial cell-wall polysaccharides have been elucidated by chemical, immunochemical, biochemical, and genetic investigations, particularly in the Salmonella. Here the polysaccharides consists of a basal polysaccharide common to all species, to which (in the S form) are bound longer species-specific side chains, consisting of repeating oligosachharide units. Spontaneous S → R mutation leads to R forms which are deficient mutants of the wild types in regard to the biosynthesis of the complete cell-wall polysaccharide. In contrast to the multiplicity of the serological specificities of the somatic antigens of the S forms, only a few serological types were found among the R forms (R I, R II, etc.). These R polysaccharides correspond to intermediates in the biosynthesis of the wile-type polysaccharides. The S → R mutation leads to the loss (or block) of an enzymes (transferase, synthetase) participating in the synthesis of the S polysaccharides. Recently many deficient mutants have been isolated and analysed, and in this way numerous stages in the biosynthesis of the cell-wall polysaccharides, for example, of Salmonella minnesota, have been made accessible to direct analysis. According to these investigations, the cell-wall polysaccharides of Salmonella consist of a basic polyheptose phosphate core which is bound to lipid A via ketodeoxyoctonic acid. To the basic core are linked pentasaccharide units of (R II structure). All other R forms are structurally deficient mutants of R II. In the complete polysaccharides of Salmonella S forms (wild types), the repeating oligosaccharide units of the specific side, chain are bound to the terminal N-acetylglucosamine of the R II structure.     

An improved silver stain for the visualization of lipopolysaccharides on polyacrylamide gels

A sensitive, brief, and user-friendly silver stain to meet the needs in high-efficiency detection of lipopolysaccharides (LPS) on polyacrylamide gels is described. In this study, the most commonly used formaldehyde-based LPS silver stain, which is potentially hazardous to the operator, is replaced by ascorbic acid (Vc) in alkaline sodium thiosulfate solution. It takes only about 35 min to complete all the protocol, with a detection limit of 4 ng of total LPS. The results indicate that this user-friendly method could be a good choice for LPS visualization on polyacrylamide gels.     

Participation of ions in promoting intermolecular associations of cell wall polysaccharides

The physiological functions of the two ions, Ca²⁺ and H⁺, in controlling mechanical properties of plant cell wall are reviewed. The interactions of these ions with major cell wall polysaccharides during cell growth and development are described. Experimental results for Ca²⁺/H⁺-induced molecular associations of some polysaccharides in solutions are also given. This article aims to bridge the understandings of molecular associations in solutions (in vitro) with those occurring in cell wall matrix of high order structure (in vivo).     

Specific polysaccharides of the lipopolysaccharides of Gram-negative bacteria

This review generalizes the result of structural investigations of the polysaccharides of the O-specific side chains of the lipopolysaccharides (LPSs) of the most studied representations of the family Enterobacteriaceae. The O-specific polysaccharides are the serologically dominating part of the molecule that is responsible for the O-antigenic specificity of the LPSs. The structures of the specific polysacchrides from various Gram-positive bacteria are given. The serological specificity of the O-antigens is discussed and a connection is shown between the chemical structures of the polysaccharides and their serological affinity.Pacific Institute of Bioorganic Chemistry of the Far-Eastern Scientific Center of the USSR Academy of Sciences, Vladivostok. Translated from khimiya Prirodnykh Soedinenii, No. 5, pp. 532–547, September–October, 1986.     

Specific polysaccharides of the lipopolysaccharides of Gram-negative bacteria

This review generalizes the result of structural investigations of the polysaccharides of the O-specific side chains of the lipopolysaccharides (LPSs) of the most studied representations of the family Enterobacteriaceae. The O-specific polysaccharides are the serologically dominating part of the molecule that is responsible for the O-antigenic specificity of the LPSs. The structures of the specific polysacchrides from various Gram-positive bacteria are given. The serological specificity of the O-antigens is discussed and a connection is shown between the chemical structures of the polysaccharides and their serological affinity.     

Permeability of the outer membrane of bacteria.

The outer membrane of Gram-negative bacteria is an attractive system for the study of the structure-function relationships in biological membranes. This membrane has, inter alia, the task of regulating the inflow of nutrients and outflow of waste products. Investigations with the aid of mutants showed that there are at least two general pathways for the diffusion of small molecules across the outer membrane: one for hydrophobic and one for hydrophilic compounds. In the case of the “hydrophobic pathway” the hydrophobic compound dissolves in the interior of the membrane and then crosses the membrane in accordance with the partition coefficient. In wild-type forms of enteric bacteria this pathway cannot be used—presumably owing to the absence of regions with phospholipid bilayers. Small hydrophilic molecules, on the other hand, penetrate the membrane through water-filled pores.     

Unusual monosaccharides in the O-factors of lipopolysaccharides of Gram-negative bacteria

Information is generalized on the structure of the O-specific polysaccharides forming components of the lipopolysaccharides of Gram-negative bacteria in which residues of unusual monosacchrides rarely found in nature have been identified. A chemical substantiation of the immunological specificity of the O-factors of specific chains is given. The role of the unusual monosaccharides in the manifestation of O-factor specificity is discussed.Pacific Ocean Institute of Bioorganic Chemistry, Far Eastern Branch, USSR Academy of Sciences, Vladivostok. Translated from Khimiya Prirodnykh Soedinenii, No. 6, pp. 743–763, November–December, 1989.     

Unusual monosaccharides in the O-factors of lipopolysaccharides of Gram-negative bacteria

Information is generalized on the structure of the O-specific polysaccharides forming components of the lipopolysaccharides of Gram-negative bacteria in which residues of unusual monosacchrides rarely found in nature have been identified. A chemical substantiation of the immunological specificity of the O-factors of specific chains is given. The role of the unusual monosaccharides in the manifestation of O-factor specificity is discussed.     

Accumulation of 2,2-difluoroethanol by bacteria

2,2-Difluoroethanol was accumulated by bacteria, giving difluoroacetic acid and so on. The conversion of 2,2,-difluoroethanol to difluoroacetic acid is unusual in a biological system.     

Recent chemistry and biochemistry of bile pigments

Bile pigments are not merely waste products of the degradation of hemoglobin, but have specific functions in plants and lower animals, in which they occur in the form of chromoproteins (biliproteins). Chromic acid degradation under controlled conditions is particularly suitable for structural investigations. In the case of the phycobilins (bile pigments of red and blue algae), not only have the structures been established, but the linkages with the proteins have also been located.     

Recent advances in the research of bacterial glucuronosyltransferases

Many bacterial extracellular polysaccharides, which play an important role in various biological processes, contain glucuronic acid (GlcA) as a major component. Glucuronosyltransferase (GlcA-T) is the enzyme responsible for GlcA attachment in the biosynthesis of these polysaccharides. GlcA-T has recently attracted significant attention because of its biological activities as well as its potential application to chemoenzymatic synthesis of GlcA-containing oligosaccharides and polysaccharides that are difficult to achieve chemically due to the problems related to glycosylation reactions with GlcA. At present, nine GlcA-Ts derived from Xanthomonas campestris, Pasteurella multocida, Escherichia coli, Sphingomonas paucimobilis, and Streptococci have been characterized. This article reviews the recent progresses made in understanding the structures, functions, physical, and chemical properties of reported GlcA-Ts.     

Detection of bacteria by surface-enhanced Raman spectroscopy

The detection and identification of dilute bacterial samples by surface-enhanced Raman spectroscopy has been explored by mixing aqueous suspensions of bacteria with a suspension of nanocolloidal silver particles. An estimate of the detection limit of E. coli was obtained by varying the concentration of bacteria. By correcting the Raman spectra for the broad librational OH band of water, reproducible spectra were obtained for E. coli concentrations as low as approximately 10³ cfu/mL. To aid in the assignment of Raman bands, spectra for E. coli in D₂O are also reported. Figure Light scattering apparatus used to detect bacteria     

Role of the cell-wall structure in the retention of bacteria by microfiltration membranes

This experimental study investigates the retention of bacteria by porous membranes. The transfer of bacteria larger than the nominal pore size of microfiltration track-etched membranes has been studied for several kinds of bacterial strains. This unexpected transfer does not correlate to the hydrophobicity, neither to the surface charge of the microorganism, as suggested in previous reports. We conclude that, in our conditions, the kind of bacteria (Gram-positive or Gram-negative) is finally the most important parameter. As the distinction between those two types of bacteria is related to the cell-wall structure, we provide an experimental evidence, via the action of an antibiotic, that the cell-wall flexibility triggers the transfer of the bacteria through artificial membranes, when the pores are smaller in size than the cell.     

Controlled assembly of bacteria on chemical patterns using soft lithography

Highly ordered arrays of single living bacteria were obtained by selective adsorption of bacteria onto chemical patterns with micrometric resolution. The chemically engineered template surfaces were prepared with the combination of microcontact printing process and a simple incubation technique. This methodology can be used for fundamental studies of bacterium's inner mechanisms and sub-cellular organization as well as for interfacing living bacteria with artificial microsystems.     

Gram-negative bacteria viable in ultrapure water: identification of bacteria isolated from ultrapure water and effect of temperature on their behavior

This study describes the effect of temperature on the behavior of bacteria viable in ultrapure water and the contamination of ultrapure water by bacteria. Three species of bacteria were isolated from ultrapure water (total organic carbon, 60 ppb and 5 ppb; effluent resistivity > 18 MΩ cm at 25°C) and identified by morphological and physiological characteristics. The three isolates were incubated in water for injection and PYG broth to check the growth profile at various temperatures. In PYG broth, temperature influenced the behavior of bacteria directly; however, it did not in water for injection. By checking both viable and non-viable bacterial numbers and endotoxin concentration in pure water, the water was found to be contaminated with non-viable bacteria and newly generated endotoxins besides viable bacteria. A column treatment, a mixed bed of fully regenerated strong acid cation exchange resin (SACER) and strong base anion exchange resin (SBAER), was used to remove bacteria from pure water. Bacteria could not grow on the surfaces of ion exchange resins in the mixed bed. The removal of bacteria was more effective as pure water was circulated through the mixed bed more rapidly.     

Separation of bacteria by capillary electrophoresis

Differences in the surface charges of bacteria can be exploited for their separation by capillary electrophoresis. Because of their low electrophoretic mobility, the separation is not always easy to perform, especially in the presence of the electroosmotic flow. Elimination of electroosmotic flow by capillary wall modification with γ‐(trimethoxysilyl)propyl methacrylate followed by acrylamide bonding permits separation over a distance of 8.5 cm.