Chemoreception of sugars by an excitable liquid membrane

Studies were made on electrical oscillations across a liquid membrane consisting of an oil layer, nitrobenzene containing picric acid, between two aqueous layers. This system showed sustained rhythmic oscillations of electrical potential of 200-300 mV with intervals in the order of 1 min. It was found that the histogram of frequency of oscillations was characteristic depending on the structures of the sugars. The histograms of glucose, fructose, galactose and sorbose showed a single maximum whereas those of sorbitol and mannitol showed double maxima.     

Membrane studies of Streptococcus pyogenes and its L-form growing in hypertonic and physiologically isotonic media. An electron spin resonance spectroscopy approach.

Electron spin resonance spectroscopy (ESR) was used to compare the lipid organization, thermal stability and the physical state of the membrane of a human pathogen, Streptococcus pyogenes and its osmotically fragile L-form with this same L-form now adapted to grow under physiologically isotonic conditions (physiological L-form). Comparison of the hyperfine splittings of a derivative of 5-ketostearic acid spin label, I(12, 3), after incorporation into the membrane, revealed that the lipid chain rigidity of these membranes is in the order physiological L-form greater than osmotically fragile L-form greater than streptococcus. The signal intensity (of the center magnetic field line) versus temperature analysis showed two transitions for these membranes. The first with melting points of 45, 26 and 36 degrees C and second transition at 70, 63 and 60 degrees C for the physiological L-form, osmotically fragile L-form and streptococcal membranes, respectively. This same order of membrane lipid chain rigidity was seen from the cooperativities obtained for each of these systems from analysis based on the expression for an n-order reaction. The I(12, 3) and other probes with the paramagnetic group close to the methyl end of the molecule suggested that this difference in lipid chain rigidity between these organisms resides in the environment closer to the lipid head group region rather than in the hydrophobic lipid core. Another major finding was the binding of I(12, 3) at two or more different sites in each of the membranes examined. This change in lipid chain rigidity now provides an explanation to account for the survival of a previously osmotically fragile L-form in physiologically isotonic media by focusing on changes in the physical nature of its membrane. In so doing, it adds to and reinforces the speculation of the potential survival in vivo and involvement in pathogenesis of osmotically fragile aberrant forms of bacteria.     

Cell Wall Polysaccharide Biosynthesis by Membrane Fragments from Streptococcus pyogenes and Stabilized L-Form

The formation and composition of a cell wall rhamnose-containing polysaccharide by membrane fragments from Streptococcus pyogenes and its stabilized L-form were compared. Also, the effect of prior treatment on the ability of coccal whole-cell and membrane fragments to incorporate radioactivity from thymidine diphosphate-¹⁴C-rhamnose, and the results of subsequent attempts to remove labeled polysaccharide from such membranes are given. L-form membrane fragments were capable of only 10% uptake of ¹⁴C-rhamnose from this nucleotide as compared with streptococcal membranes. However, once bound, both membrane fragments polymerized rhamnose to the same extent. These findings tend to negate the almost complete lack of polymeric rhamnose within the intact L-form as being due to the absence of membrane enzymes necessary for the transfer of rhamnose from a suitable precursor to membrane acceptor sites or enzymes responsible for rhamnose polymerization. Degradation of labeled rhamnose polysaccharide after isolation from coccal membranes by mild acid hydrolysis showed muramic acid and glucosamine to be attached. This same polysaccharide from L-form membrane fragments was devoid of amino sugars. These data suggest the possible involvement of amino sugars in the attachment of cell wall polymeric rhamnose to the streptococcal cytoplasmic membrane. The absence of attached amino sugars to rhamnose polysaccharide from L-form membrane fragments is discussed in terms of this organism's continued inability for new cell wall formation. The isolation, from streptococcal membrane fragments, of a polysaccharide containing rhamnose and amino sugars common to at least two different streptococcal cell wall-type polymers was demonstrated.     

Teichoic acid of a stabilized L-form of Streptococcus pyogenes

A stabilized L-form of Streptococcus pyogenes continues to synthesize glycerol teichoic acid. This polymer was obtained from S. pyogenes and its L-form, treated in identical fashion, and compared. Highly purified glycerol teichoic acid from only the L-form was found to be devoid of d-alanine and to have a shorter chain length. Otherwise, the glycerol teichoic acid from these two organisms was found to be a 1,3-phosphodiester-linked glycerophosphate polymer substituted with d-glucose. Evidence is presented that most, if not all, of the glycerol teichoic acid in this streptococcus lies between the wall and membrane. A possible need for the continued synthesis of a minute amount of glycerol teichoic acid by this L-form for survival is discussed in terms of the known function of teichoic acids in bacteria.     

Opposing actions of cGMP and calcium on the conductance of the F(0) subunit c pore

Subunit c of ATP synthase can be purified from neuronal plasma membrane and from the inner mitochondrial membrane. In the latter location the hydrophobic 75 amino acid protein is one component of the F(1) F(0) ATP synthase complex but in the former it is alone as a pore that is capable of generating spontaneous electrical oscillations. Pure mammalian subunit c when reconstituted in lipid bilayers and voltage clamped, yields a voltage sensitive pore that conducts a cation current regulated by calcium. The current is here found to be activated by cGMP with a K(M) ranging from 14 nM to 19 microM depending on calcium and temperature. It is sensitively inhibited by a number of ligands. The K(I) for calcium ranges from 100 nM to 100 microM depending on cGMP and temperature. DCCD inhibits with a K(app) of 100 nM. The polyamine nicotine inhibits at 84 nM. The pore has properties that would allow it to deliver sodium or calcium through the cell membrane in a controlled manner while maintaining membrane polarization.     

Membrane Studies of Streptococcus pyogenes and its L-form growing in hypertonic and physiologically isotonic media. An electron spin resonance spectroscopy approach

Electron spin resonance spectroscopy (ESR) was used to compare the lipid organization, thermal stability and the physical state of the membrane of a human pathogen, Streptococcus pyogenes and its osmotically fragile L-form with this same L-form now adapted to grow under physiologically isotonic conditions (physiological L-form). Comparison of the hyperfine splittings of a derivative of 5-ketostearic acid spin label, I(1 2, 3), after incorporation into the membrane, revealed that the lipid chain rigidity of these membranes is in the order physiological L-form > osmotically fragile L-form > streptococcus. The signal intensity (of the center magnetic field line) versus temperature analysis showed two transitions for these membranes. The first with melting points of 45, 26 and 36 °C and second transition at 70, 63 and 60 °C for the physiological L-form, osmotically fragile L-form and streptococcal membranes, respectively. This same order of membrane lipid chain rigidity was seen from the cooperativities obtained for each of these systems from analysis based on the expression for an $\text{n-}\text{order}$ reaction. The I(12,3) and other probes with the paramagnetic group close to the methyl end of the molecule suggested that this difference in lipid chain rigidity between these organisms resides in the environment closer to the lipid head group region rather than in the hydrophobic lipid core. Another major finding was the binding of I(12, 3) at two or more different sites in each of the membranes examined. This change in lipid chain rigidity now provides an explanation to account for the survival of a previously osmotically fragile L-form in physiologically isotonic media by focusing on changes in the physical nature of its membrane. In so doing, it adds to and reinforces the speculation of the potential survival in vivo and involvement in pathogenesis of osmotically fragile aberrant forms of bacteria.     

Oscillation of electrical potential in a porous-membrane doped with triolein induced by an Na+/K+ concentration gradient

The electrical potential across a fine pore membrane doped with trioleoyl glyceride (triolein) and separating aqueous solutions of 0.5M NaCl and 0.5M KCl, respectively, was studied. It was found that this system showed rhythmic and sustained oscillations of electrical potential between the two aqueous solutions. These oscillations were attributed to the change of permeability of Na+ and K+ ions across the membrane, which originated from the phase-transition of triolein molecules within the fine pores.     

Oscillation of electrical potential in a porous membrane doped with glycerol alpha-monooleate induced by an Na+/K+ concentration gradient

The electrical potential across a fine-pore membrane doped with glycerol alpha-monooleate and separating aqueous solutions of 0.5 M NaCl and 0.5 M KCl was studied. It was found that this system showed rhythmic and sustained oscillations of electrical potential. These oscillations may be due to the phase transition of glycerol alpha-monooleate molecules within the fine pores. In relation to this, it is shown here that Na+ and K+ have different effects on the aggregation of glycerol alpha-monooleate. This oscillatory phenomenon is very interesting because in biological nervous membrane an Na+/K+ concentration difference across the membrane is essential for excitability.     

Characterization of a Stable L-Form of Bacillus subtilis 168

A stable L-form of Bacillus subtilis 168 (sal-1) has been isolated which grows and divides logarithmically in liquid medium with a generation time of 60 min. This mutant does not synthesize cell wall as evidenced by chemical, biochemical, and morphological analyses. Antibiotics which specifically inhibit cell wall biosynthesis do not affect the growth of the L-form. Significant differences exist between the membrane proteins of the bacillary form and the L-form. The relative profile of membrane proteins varies with the salt concentration of the medium in both the L-form and the bacillary form.     

Fourier analysis of potential oscillations of a liquid membrane for the discrimination of taste substances

Electrical potential oscillations were obtained across a liquid membrane composed of nitrobenzene/picric acid placed between two aqueous phases in the presence of various taste (i.e. salty, sweet and bitter) substances. The influence of these compounds on electrical oscillations was studied using Fourier analysis to establish a "fingerprint" of the substance that can be correlated with its taste index. Various concentrations of each substance were tested to obtain a Fourier spectrum with discrete peaks which can be further processed. The electrical oscillations consisted of a number of weak damped oscillators, and the Fourier spectra of these signals were found to have a number of discrete peaks of decreasing amplitude at low frequencies (0-0.5 Hz). A correlation of the frequency of the first peak of the Fourier spectrum with the taste index was found for bitter substances, whereas for salty substances the amplitude of the first two peaks of the spectrum was correlated with the taste index.     

Exhibition of persistent and drug-tolerant L-form habit of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> during infection in rats

A model for studying mycobacterial L-form formation in vivo was established to demonstrate the ability of M. tuberculosis to behave as a drug-tolerant L-form persister. Rats were infected by intranasal (i.n.) and intraperitoneal (i.p.) routes with 1×108 cells/ml of M. tuberculosis. At weekly intervals during a period of five weeks, samples from lung, spleen, liver, kidney, mesenterial and inguinal lymph nodes, broncho-alveolar and peritoneal lavage liquid were plated simultaneously on Löwenstein-Jensen (LJ) medium or inoculated into specially supplemented for L-forms Dubos broth (drug-free and drug-containing variants). The use of liquid media enabled isolation of mycobacterial L-form cultures during the whole period of experiment including the last two weeks, when tubercle bacilli were not isolated on LJ medium. An unique feature of mycobacterial L-forms was their ability to grow faster than the classical tubercle bacilli. Isolation and growth of L-form cultures in primary drug-containing media demonstrated their drug-tolerant properties. Electron microscopy of liquid media isolates showed that they consisted of morphologically heterogenous populations of membrane-bound and of variable sized L-bodies that completely lack cell walls. The identity of the isolated non-acid fast and morphologically modified L-forms as M. tuberculosis was verified by specific spoligotyping test. The results contribute to special aspects concerning the importance of mycobacterial L-form phenomenon for persistence and latency in tuberculosis, phenotypic drug tolerance, as well as for diagnosis of difficult to identify morphologically changed tubercle bacilli which are often mistaken for contaminants.     

Fatty Acid Composition of L-Forms of Streptococcus faecalis Cultured at Different Osmolalities

The fatty acid composition of the membranes of three different penicillin-produced L-forms of Streptococcus faecalis was determined: (i) a stable (nonreverting) L-form (T(53)) cultured in brain heart infusion (BHI) with 0.5 M sucrose; (ii) a stable L-form (T(531)) cultured in BHI without sucrose; and (iii) an unstable L-form (T(9)) cultured in BHI with 0.5 M sucrose and 1,000 U of penicillin per ml. L-forms were obtained by centrifugation and lysed by washing in 1 mM tris(hydroxymethyl)aminomethane-hydrochloride buffer. The parent S. faecalis was also cultured in BHI and BHI containing 0.5 M sucrose, and washed with buffer. The fatty acid composition of L-forms of S. faecalis cultured in BHI without sucrose (370 mosmol) had higher C(18:1) and lower C(18) than L-forms cultured in the same media with added 0.5 M sucrose (950 mosmol) in both exponential and stationary cultures. In the stationary phase of growth, C(19) was reduced in the L-forms cultured without sucrose. Similar changes were seen in the parent S. faecalis cultured in the two types of media. These changes in membrane fatty acids may relate to osmo-regulation of the L-forms.     

The interaction of lymphocyte membrane proteins with the lymphocyte cytoskeletal matrix

Lymphocyte membrane proteins are important in the transduction of signals across the plasma membrane. Visual and biophysical studies have shown that after ligand binding, membrane proteins may become immobile in the plane of the membrane and may cap. In intact cells, binding of cross-linking ligands to surface immunoglobulin converts it to a detergent-insoluble state (77% insoluble). This conversion is positively correlated with the transmission of a mitogenic signal. Class II histocompatibility proteins (Ia) and thy-1 remain predominantly detergent soluble (60 to 97% soluble). Insolubilized membrane proteins may be solubilized by incubating the detergent insoluble cytoskeletons with 0.34 M sucrose, 0.5 mM ATP, 0.5 mM dithiothreitol, 1 mM EDTA, or 3 X 10(-5) M DNAase I, 1 mM EDTA. To determine if the membrane-associated cytoskeleton contains the sufficient components for ligand-induced receptor insolubilization, experiments were done with a crude plasma membrane fraction. The results with whole cells or crude plasma membranes were comparable. These studies support the view that ligand-induced insolubilization of membrane proteins is due to their interaction with cytoskeletal structures.     

Comparative Electrophoretic and Amino Acid Analyses of Isolated Membranes from Streptococcus pyogenes and Stabilized L-Form

Major quantitative, but not qualitative, differences in the various species of proteins in purified membranes from Streptococcus pyogenes and its stabilized L-form have been demonstrated by acidic and alkaline disc gel electrophoresis with and without urea. The fact that no significant differences in the amino acid content or composition between these two membranes could be demonstrated emphasizes that these results are probably due to changes in the relative amounts of the various species of proteins in this subcellular component. The possibility of these protein changes in the L-form membrane being related to its inability to synthesize a rigid cell wall is discussed. Finally, phage-associated lysin, routinely used for removal of the group A streptococcal cell wall, does not appear to affect the protein profile or amino acid composition of the membrane either metabolically or nonmetabolically.     

Selective association of the chromosome with membrane in a stable L-form of Bacillus subtilis.

A stalbe L-form (Sal-1) of Bacillus subtilis was found to have retained a markedly modified chromosome-membrane association when compared to intact cells. The membrane-deoxyribonucleic acid complex of the L-form was similar to that of its parental strain in quantity and stability. Genetic analysis of the L-form membrane-deoxyribonucleic acid complex revealed enrichment for markers close to the replication origin, but not for internal markers, indicating preferential attachment of the origin of chromosomal replication to the membrane. These results are in close agreement with those found for the parental bacterial form. In contrast, the replication termius region was not preferentially attached to the membrane of the L-form, even though it is enriched in the bacterial form. The association of the chromosome with the membrane at the replication terminus does not appear to be necessary for cell growth and separation, but because the L-form divides aberrantly, it may be one of the factors required for normal deoxyribonucleic acid segregation and septation.     

An L-Form of Staphylococcus aureus Adapted to a Brain Heart Infusion Medium without Osmotic Stabilizers

An L-form derived from halotolerant Staphylococcus aureus Tasaki was adapted to growth in a brain heart infusion medium without any supplemental osmotically protective solutes (360 mOsm/kg). This L-form had no chemically detectable peptidoglycan residues on its surface. Electron microscopic observations confirmed morphologically the absence of the structures and also of other osmotically protective polymers within or exterior to the cytoplasmic membrane. The osmotic stability and susceptibility to bacitracin, d-cycloserine, and vancomycin of the L-form adapted to growth in 360 mOsm osmotically unprotective medium was higher than that of the L-form grown in 1,950 mOsm supplemented with 4.5% NaCl. The adapted L-form tended to be more sensitive to almost all of the antibiotics examined, other than the inhibitors for cell wall-synthesis, than the original L-form strain requiring osmotic protection for growth. Chemical analysis of the membrane of the adapted L-form indicated 16.3% total lipids and 20.6% proteins by dry weight of the membrane, and it contained larger amounts of lipid phosphorus (20.0 μ/mg).     

Reconstitution of M13 bacteriophage coat protein. A new strategy to analyze configuration of the protein in the membrane

The configuration of M13 bacteriophage coat protein in a model membrane was analyzed using protease digestion followed by gel permeation chromatography on Fractogel TSK in formic acid/ethanol. Important information is contained in the chromatographic patterns of the membrane-bound fragments, as well as of the fragments released by the digestion. A new reconstitution was thereby developed which involves adding a small volume of a concentrated solution of cholate-solubilized coat protein to preformed vesicles (with the amount of detergent added being less than that required to solubilize the vesicles), freezing in liquid nitrogen, thawing, followed by dialysis to remove excess detergent. The coat protein is incorporated with high efficiency (95 percent) making subsequent fractionation unnecessary. In addition, the incorporated protein is not aggregated, and is incorporated with most molecules spanning the membrane, oriented in the same manner as in vivo (N-terminus outwards). Two previously described reconstitutions, using sonication or cholate dialysis, are analyzed and found to be less satisfactory.     

Role of phenylalanine in the biosynthesis of fluorescent pigment in Pseudomonas putida bacteria]

The contemporary data of the participation of phenylalanine in the biosynthesis of fluorescent pigment pyoverdine PM of Pseudomonas putida strain M are presented. Using aro1phu1 mutant of this strain, it has been shown that one of the precursors of the dihydroxyquinoline moiety of the pyoverdine PM is phenylalanine in the D- or L-form. These results were confirmed in experiments with 14C-phenylalanine incorporation. Pyoverdine PM that was synthesized by aro1phu1 mutant from exogenous phenylalanine is identical with the pigment from wild type cells.     

L-型细菌蛋白表达系统

L-型细胞由于缺乏细胞壁以及具有的其它特殊生物学特性,通过连接合适的信号肽,可以用于许多外源蛋白的可溶性,功能活性形式的分泌表达,表达产物在培养基中,表达的产量依赖于不同的基因序列,载体,宿主和诱导表达条件等因素,此外,L-型细菌还能将具有功能活性的蛋白展示在胞浆膜表面。     

Membranes of the protoplast L-form of Proteus mirabilis

Isolated membranes of the cell wall-less stable protoplast L-form of Proteus mirabilis were characterized by density gradient centrifugation and by assay for their major chemical constituents, proteins, phospholipids and lipopolysaccharide, and for some specific marker enzymes of the cytoplasmic membrane. In most of the analyzed properties the L-form protoplast membrane resembled the bacterial cytoplasmic membrane, with some notable modifications. considerable amounts of lipopolysaccharide, normally an exclusive constituent of the outer membrane, were found. Furthermore, the L-form membranes contained the functions of the reduced nicotinamide adenine dinucleotide oxidase system, of d-lactate dehydrogenase (EC 1.1.1.28) and of succinate dehydrogenase (EC 1.3.99.1) at specific activities comparable to, or in some cases considerably higher than, those present in cytoplasmic membranes of the bacterial form. Of two peptidoglycan DD-carboxypetidase/transpeptidases (EC 3.4.17.8 and EC 2.3.2.10), which are normally present in the cytoplasmic membrane of the bacterial form of P. mirabilis, the membrane of the protoplast L-form contained only one. Electron microscopy of thin sectioned L-form protoplasts showed extensive heterogeneity of membraneous structures. In addition to the single membraneous integument, internal membrane-bounded vesicles and multiple stacks of membranes were present, as the result of unbalanced growth and membrane synthesis in the L-form state.