Melatonin inhibits Gram-negative pathogens by targeting citrate synthase

Citrate synthase has been purified to homogeneity from a strain of the Gram-negative aerobic bacterium Acinetobacter anitratum in a form which retains its sensitivity to the allosteric inhibitor NADH. In subunit size, amino acid composition, and antigenic reactivity the enzyme shows a marked structural resemblance to the citrate synthase of the Gram-negative facultative anaerobe Escherichia coli. Whereas the E. coli enzyme is subject to a strong, hyperbolic inhibition by NADH (Hill's number n = 1.0, K1 = 2 μM), the A. anitratum enzyme shows a weak, sigmoid response (n = 1.6, I0.5 = 140 μM) to this nucleotide. With E. coli, NADH inhibition is competitive with acetyl-CoA, and noncompetitive with oxaloacetate; with A. anitratum, NADH is noncompetitive with both substrates. Acinetobacter anitratum citrate synthase shows hyperbolic saturation with acetyl-CoA (n = 1.8). The finding of Weitzman and Jones (Nature (London) 219, 270 (1968)) that NADH inhibition of the enzyme from Acinetobacter spp. is reversible by AMP, while that from E. coli is not, is explained by the much greater affinity of the E. coli enzyme for NADH. Unlike E. coli citrate synthase, the A. anitratum enzyme does not react with the sulfhydryl reagent 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) in the absence of denaturation. With a second sulfhydryl reagent, 4,4′-dithiodipyridine (4,4′-PDS), the A. anitratum enzyme reacts with 1 equiv. of subunit; this modification induces a partial activity loss (attributable to a rise in the Km for acetyl-CoA) and an increase in the sensitivity to NADH. With the E. coli enzyme, 4,4′-PDS causes complete inactivation. Acinetobacter anitratum citrate synthase is much more resistant to urea denaturation than the E. coli enzyme is; the resistance of both enzymes to urea is greatly improved in the presence of 1 M KCl. It is suggested that the amino acid sequences of the subunits of the citrate synthases of these two bacteria are about 90% homologous, and that the 10% differences are in key residues, perhaps largely in the subunit contact regions, which account for the differences in allosteric properties.

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Activities of citrate synthase and other enzymes of Acetobacter xylinum in situ and in vitro

Biochemical Journal ◽

10.1042/bj1530499 ◽

1976 ◽

Vol 153 (2) ◽

pp. 499-501 ◽

Cited By ~ 15

Author(s):

M Swissa ◽

H Weinhouse ◽

M Benziman

Keyword(s):

Carbohydrate Metabolism ◽

Citrate Synthase ◽

Acetobacter Xylinum ◽

Kinetic Properties ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Permeabilized Cells

The activities of a number of enzymes, extracted from Acetobacter xylinum, that are involved in carbohydrate metabolism may be accounted for in situ in permeabilized cells. The kinetic properties of citrate synthase and glycerokinase observed in vitro are also retained in situ. So is the regulatory sensitivity of these enzymes. Both in vitro and in situ, (a) citrate synthase, in contrast with the enzyme for other Gram-negative bacteria, is inhibited by ATP and is insensitive to NADH, and (b) glycerokinase is inhibited by fructose diphosphate and the ratio of its activities towards glycerol and dihydroxyacetone is the same.

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A Freeze-Etch Study of Acinetobacter SP. Grown on a Hydrocarbon Substrate

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100050883 ◽

1974 ◽

Vol 32 ◽

pp. 174-175

Author(s):

C. L. Scott ◽

W. R. Finnerty

Keyword(s):

Membrane System ◽

Structural Distortion ◽

Intracytoplasmic Membrane ◽

Gram Negative ◽

Sectioned Material ◽

Acinetobacter Sp

Acinetobacter sp. HO-1-N, a gram-negative hydrocarbon oxidizing bacterium previously designated Micrococcus cerificans, has been shown to sequester the hydrocarbon into intracytoplasmic pools as a result of growth on this substrate. In hydrocarbon grown cells, an intracytoplasmic membrane system was also observed along with a doubling of cellular phospholipids (Z). However, using conventional dehydration and embedding procedures in preparing thin sectioned material, the hydrocarbon is extracted from the cells. This may lead to structural distortion, consequently, the freeze-etch technique was applied to preserve the integrity of the cell.

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Bacterial-Mucosal Cell Junctional Complexes

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100050731 ◽

1974 ◽

Vol 32 ◽

pp. 144-145

Author(s):

Roger C. Wagner

Keyword(s):

Intestinal Wall ◽

Rat Colon ◽

Mucosal Cell ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Mucosal Cells ◽

Mucosal Lining ◽

Degenerative Alterations ◽

Junctional Complexes ◽

Intestinal Mucosal Cells

Bacteria exhibit the ability to adhere to the apical surfaces of intestinal mucosal cells. These attachments either precede invasion of the intestinal wall by the bacteria with accompanying inflammation and degeneration of the mucosa or represent permanent anchoring sites where the bacteria never totally penetrate the mucosal cells.Endemic gram negative bacteria were found attached to the surface of mucosal cells lining the walls of crypts in the rat colon. The bacteria did not intrude deeper than 0.5 urn into the mucosal cells and no degenerative alterations were detectable in the mucosal lining.

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Microanatomy of the Periplasm of Bacillus Licheniformis

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100065365 ◽

1971 ◽

Vol 29 ◽

pp. 262-263

Author(s):

B.K. Ghosh

Keyword(s):

Cell Wall ◽

Plasma Membrane ◽

Bacillus Licheniformis ◽

External Environment ◽

Permeability Barrier ◽

Periplasmic Space ◽

Modified Technique ◽

Gram Positive ◽

Gram Negative ◽

Gram Positive Bacteria

Periplasm of bacteria is the space outside the permeability barrier of plasma membrane but enclosed by the cell wall. The contents of this special milieu exterior could be regulated by the plasma membrane from the internal, and by the cell wall from the external environment of the cell. Unlike the gram-negative organism, the presence of this space in gram-positive bacteria is still controversial because it cannot be clearly demonstrated. We have shown the importance of some periplasmic bodies in the secretion of penicillinase from Bacillus licheniformis.In negatively stained specimens prepared by a modified technique (Figs. 1 and 2), periplasmic space (PS) contained two kinds of structures: (i) fibrils (F, 100 Å) running perpendicular to the cell wall from the protoplast and (ii) an array of vesicles of various sizes (V), which seem to have evaginated from the protoplast.

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Rapid demonstration of septic or infectious arthritis due to Gram-negative bacteria

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100123830 ◽

1992 ◽

Vol 50 (1) ◽

pp. 686-687

Author(s):

Jacob S. Hanker ◽

Paul R. Gross ◽

Beverly L. Giammara

Keyword(s):

Chronic Arthritis ◽

Blood Cultures ◽

Gram Negative Bacteria ◽

Infectious Arthritis ◽

Bacterial Arthritis ◽

Gram Positive ◽

Gram Negative ◽

Gram Positive Bacteria

Blood cultures are positive in approximately only 50 per cent of the patients with nongonococcal bacterial infectious arthritis and about 20 per cent of those with gonococcal arthritis. But the concept that gram-negative bacteria could be involved even in chronic arthritis is well-supported. Gram stains are more definitive in staphylococcal arthritis caused by gram-positive bacteria than in bacterial arthritis due to gram-negative bacteria. In the latter situation where gram-negative bacilli are the problem, Gram stains are helpful for 50% of the patients; they are only helpful for 25% of the patients, however, where gram-negative gonococci are the problem. In arthritis due to gram-positive Staphylococci. Gramstained smears are positive for 75% of the patients.

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Ultrastructure of periplasmic bodies in gram-negative bacteria

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100160753 ◽

1990 ◽

Vol 48 (3) ◽

pp. 640-641

Author(s):

Xie Nianming ◽

Ding Shaoqing ◽

Wang Luping ◽

Yuan Zenglin ◽

Zhan Guolai ◽

...

Keyword(s):

Electron Microscope ◽

Campylobacter Jejuni ◽

Proteus Mirabilis ◽

Shigella Flexneri ◽

Morphological Description ◽

Gram Negative Bacteria ◽

Periplasmic Space ◽

Clostridium Tetani ◽

Gram Negative ◽

Brucella Canis

Perhaps the data about periplasmic enzymes are obtained through biochemical methods but lack of morphological description. We have proved the existence of periplasmic bodies by electron microscope and described their ultrastructures. We hope this report may draw the attention of biochemists and mrophologists to collaborate on researches in periplasmic enzymes or periplasmic bodies with each other.One or more independent bodies may be seen in the periplasmic space between outer and inner membranes of Gram-negative bacteria, which we called periplasmic bodies. The periplasmic bodies have been found in seven species of bacteria at least, including the Pseudomonas aeroginosa. Shigella flexneri, Echerichia coli. Yersinia pestis, Campylobacter jejuni, Proteus mirabilis, Clostridium tetani. Vibrio cholerae and Brucella canis.

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Dual stain kit for the microscopic gram classification of ocular infection bacteria

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100147089 ◽

1993 ◽

Vol 51 ◽

pp. 248-249

Author(s):

Jacob S. Hanker ◽

Dale N. Holdren ◽

Kenneth L. Cohen ◽

Beverly L. Giammara

Keyword(s):

Contact Lenses ◽

Ocular Infection ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Gram Staining ◽

Ocular Infections ◽

Different Types ◽

Culture Positive ◽

Increased Susceptibility

Keratitis and conjunctivitis (infections of the cornea or conjunctiva) are ocular infections caused by various bacteria, fungi, viruses or parasites; bacteria, however, are usually prominent. Systemic conditions such as alcoholism, diabetes, debilitating disease, AIDS and immunosuppressive therapy can lead to increased susceptibility but trauma and contact lens use are very important factors. Gram-negative bacteria are most frequently cultured in these situations and Pseudomonas aeruginosa is most usually isolated from culture-positive ulcers of patients using contact lenses. Smears for staining can be obtained with a special swab or spatula and Gram staining frequently guides choice of a therapeutic rinse prior to the report of the culture results upon which specific antibiotic therapy is based. In some cases staining of the direct smear may be diagnostic in situations where the culture will not grow. In these cases different types of stains occasionally assist in guiding therapy.

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Neutrophil pseudoplatelets in subgingival plaque and crevicular fluid samples from periodontal diseased sites

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100156298 ◽

1989 ◽

Vol 47 ◽

pp. 862-863 ◽

Cited By ~ 1

Author(s):

J Hanker ◽

E.J. Burkes ◽

G. Greco ◽

R. Scruggs ◽

B. Giammara

Keyword(s):

Electron Microscopy ◽

Bone Marrow ◽

Peripheral Blood ◽

Gingival Crevicular Fluid ◽

Light And Electron Microscopy ◽

Subgingival Plaque ◽

Staining Reaction ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Crevicular Fluid

The mature neutrophil with a segmented nucleus (usually having 3 or 4 lobes) is generally considered to be the end-stage cell of the neutrophil series. It is usually found as such in the bone marrow and peripheral blood where it normally is the most abundant leukocyte. Neutrophils, however, must frequently leave the peripheral blood and migrate into areas of infection to combat microorganisms. It is in such areas that neutrophils were first observed to fragment to form platelet-size particles some of which have a nuclear lobe. These neutrophil pseudoplatelets (NPP) can readily be distinguished from true platelets because they stain for neutrophil myeloperoxidase. True platelets are not positive in this staining reaction because their peroxidase Is inhibited by glutaraldehyde. Neutrophil pseudoplatelets, as well as neutrophils budding to form NPP, could frequently be observed in peripheral blood or bone marrow samples of leukemia patients. They are much more prominent, however, in smears of inflammatory exudates that contain gram-negative bacteria and in gingival crevicular fluid samples from periodontal disease sites. In some of these samples macrophages ingesting, or which contained, pseudoplatelets could be observed. The myeloperoxidase in the ingested pseudoplatelets was frequently active. Despite these earlier observations we did not expect to find many NPP in subgingival plaque smears from diseased sites. They were first seen by light microscopy (Figs. 1, 3-5) in smears on coverslips stained with the PATS reaction, a variation of the PAS reaction which deposits silver for light and electron microscopy. After drying replicate PATS-stained coverslips with hexamethyldisilazane, they were sputter coated with gold and then examined by the SEI and BEI modes of scanning electron microscopy (Fig. 2). Unstained replicate coverslips were fixed, and stained for the demonstration of myeloperoxidase in budding neutrophils and NPP. Neutrophils, activated macrophages and spirochetes as well as other gram-negative bacteria were also prominent in the PATS stained samples. In replicate subgingival plaque smears stained with our procedure for granulocyte peroxidases only neutrophils, budding neutrophils or NPP were readily observed (Fig. 6).

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