scholarly journals Fluorescein Diacetate Hydrolysis Using the Whole Biofilm as a Sensitive Tool to Evaluate the Physiological State of Immobilized Bacterial Cells

Catalysts ◽  
2018 ◽  
Vol 8 (10) ◽  
pp. 434 ◽  
Author(s):  
Anna Dzionek ◽  
Jolanta Dzik ◽  
Danuta Wojcieszyńska ◽  
Urszula Guzik
Keyword(s):  
Enzymatic Activity ◽  
Physiological State ◽  
Dry Mass ◽  
Original Method ◽  
Fluorescein Diacetate ◽  
Bacterial Cells ◽  
Reliable Quantification ◽  
Fda Hydrolysis ◽  
Orbital Shaker ◽  
Slow Injection

Due to the increasing interest and the use of immobilized biocatalysts in bioremediation studies, there is a need for the development of an assay for quick and reliable measurements of their overall enzymatic activity. Fluorescein diacetate (FDA) hydrolysis is a widely used assay for measuring total enzymatic activity (TEA) in various environmental samples or in monoculture researches. However, standard FDA assays for TEA measurements in immobilized samples include performing an assay on cells detached from the carrier. This causes an error, because it is not possible to release all cells from the carrier without affecting their metabolic activity. In this study, we developed and optimized a procedure for TEA quantification in the whole biofilm formed on the carrier without disturbing it. The optimized method involves pre-incubation of immobilized carrier in phosphate buffer (pH 7.6) on the orbital shaker for 15 min, slow injection of FDA directly into the middle of the immobilized carrier, and incubation on the orbital shaker (130 rpm, 30 °C) for 1 h. Biofilm dry mass was obtained by comparing the dried weight of the immobilized carrier with that of the unimmobilized carrier. The improved protocol provides a simple, quick, and more reliable quantification of TEA during the development of immobilized biocatalysts compared to the original method.

scholarly journals Glutamate Dehydrogenase from Thermus thermophilus Is Activated by AMP and Leucine as a Complex with Catalytically Inactive Adenine Phosphoribosyltransferase Homolog

2019 ◽  
Vol 201 (14) ◽  
Author(s):  
Takeo Tomita ◽  
Hajime Matsushita ◽  
Ayako Yoshida ◽  
Saori Kosono ◽  
Minoru Yoshida ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Glutamate Dehydrogenase ◽  
Ternary Complex ◽  
Thermus Thermophilus ◽  
Thermophilic Bacterium ◽  
Regulatory Subunit ◽  
Binary Complex ◽  
Bacterial Cells ◽  
Adenine Phosphoribosyltransferase ◽  
Content Type

ABSTRACT Glutamate dehydrogenase (GDH) from a thermophilic bacterium, Thermus thermophilus, is composed of two heterologous subunits, GdhA and GdhB. In the heterocomplex, GdhB acts as the catalytic subunit, whereas GdhA lacks enzymatic activity and acts as the regulatory subunit for activation by leucine. In the present study, we performed a pulldown assay using recombinant T. thermophilus, producing GdhA fused with a His tag at the N terminus, and found that TTC1249 (APRTh), which is annotated as adenine phosphoribosyltransferase but lacks the enzymatic activity, was copurified with GdhA. When GdhA, GdhB, and APRTh were coproduced in Escherichia coli cells, they were purified as a ternary complex. The ternary complex exhibited GDH activity that was activated by leucine, as observed for the GdhA-GdhB binary complex. Furthermore, AMP activated GDH activity of the ternary complex, whereas such activation was not observed for the GdhA-GdhB binary complex. This suggests that APRTh mediates the allosteric activation of GDH by AMP. The present study demonstrates the presence of complicated regulatory mechanisms of GDH mediated by multiple compounds to control the carbon-nitrogen balance in bacterial cells. IMPORTANCE GDH, which catalyzes the synthesis and degradation of glutamate using NAD(P)(H), is a widely distributed enzyme among all domains of life. Mammalian GDH is regulated allosterically by multiple metabolites, in which the antenna helix plays a key role to transmit the allosteric signals. In contrast, bacterial GDH was believed not to be regulated allosterically because it lacks the antenna helix. We previously reported that GDH from Thermus thermophilus (TtGDH), which is composed of two heterologous subunits, is activated by leucine. In the present study, we found that AMP activates TtGDH using a catalytically inactive APRTh as the sensory subunit. This suggests that T. thermophilus possesses a complicated regulatory mechanism of GDH to control carbon and nitrogen metabolism.

scholarly journals A Simple Protocol for the Determination of Lysostaphin Enzymatic Activity

Antibiotics ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 917
Author(s):  
Alexander V. Grishin ◽  
Svetlana V. Konstantinova ◽  
Irina V. Vasina ◽  
Nikita V. Shestak ◽  
Anna S. Karyagina ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Catalytic Efficiency ◽  
Bacterial Cells ◽  
Binding Domains ◽  
Simple Protocol ◽  
Specialized Equipment ◽  
Osmotic Lysis ◽  
Cross Bridges ◽  
Bacterial Peptidoglycan

Antibacterial lysins are enzymes that hydrolyze bacterial peptidoglycan, which results in the rapid death of bacterial cells due to osmotic lysis. Lysostaphin is one of the most potent and well-studied lysins active against important nosocomial pathogen Staphylococcus aureus. Similarly to most other lysins, lysostaphin is composed of enzymatic and peptidoglycan-binding domains, and both domains influence its antibacterial activity. It is thus desirable to be able to study the activity of both domains independently. Lysostaphin cleaves pentaglycine cross-bridges within the staphylococcal peptidoglycan. Here, we report the protocol to study the catalytic activity of lysostaphin on the isolated pentaglycine peptide that is based on the chromogenic reaction of peptide amino groups with ninhydrin. Unlike previously reported assays, this protocol does not require in-house chemical synthesis or specialized equipment and can be readily performed in most laboratories. We demonstrate the use of this protocol to study the effect of EDTA treatment on the lysostaphin enzymatic activity. We further used this protocol to determine the catalytic efficiency of lysostaphin on the isolated pentaglycine and compared it to the apparent catalytic efficiency on the whole staphylococcal cells. These results highlight the relative impact of enzymatic and peptidoglycan-binding domains of lysostaphin on its bacteriolytic activity.

Microbial development in distillers wet grains produced during fuel ethanol production from corn (Zea mays)

2007 ◽  
Vol 53 (9) ◽  
pp. 1046-1052 ◽  
Author(s):  
R. Michael Lehman ◽  
Kurt A. Rosentrater
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Heterotrophic Bacteria ◽  
Micrococcus Luteus ◽  
Phospholipid Fatty Acid ◽  
Dry Mass ◽  
Fuel Ethanol ◽  
Fatty Acid Analysis ◽  
Yeast Cells ◽  
Bacterial Cells

Distillers grains are coproduced with ethanol and carbon dioxide during the production of fuel ethanol from the dry milling and fermentation of corn grain, yet there is little basic microbiological information on these materials. We undertook a replicated field study of the microbiology of distillers wet grains (DWG) over a 9 day period following their production at an industrial fuel ethanol plant. Freshly produced DWG had a pH of about 4.4, a moisture content of about 53.5% (wet mass basis), and 4 × 105 total yeast cells/g dry mass, of which about 0.1% were viable. Total bacterial cells were initially below detection limits (ca. 106 cells/g dry mass) and then were estimated to be ∼5 × 107 cells/g dry mass during the first 4 days following production. Culturable aerobic heterotrophic organisms (fungi plus bacteria) ranged between 104 and 105 CFU/g dry mass during the initial 4 day period, and lactic acid bacteria increased from 36 to 103 CFU/g dry mass over this same period. At 9 days, total viable bacteria and yeasts and (or) molds topped 108 CFU/g dry mass and lactic acid bacteria approached 106 CFU/g dry mass. Community phospholipid fatty acid analysis indicated a stable microbial community over the first 4 days of storage. Thirteen morphologically distinct isolates were recovered, of which 10 were yeasts and molds from 6 different genera, 2 were strains of the lactic-acid-producing Pediococcus pentosaceus and only one was an aerobic heterotrophic bacteria, Micrococcus luteus . The microbiology of DWG is fundamental to the assessment of spoilage, deleterious effects (e.g., toxins), or beneficial effects (e.g., probiotics) in its use as feed or in alternative applications.

Determination of Bacterial Cell Dry Mass by Transmission Electron Microscopy and Densitometric Image Analysis

1998 ◽  
Vol 64 (2) ◽  
pp. 688-694 ◽  
Author(s):  
M. Loferer-Krößbacher ◽  
J. Klima ◽  
R. Psenner
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Image Analysis ◽  
Bacterial Biomass ◽  
Dry Weight ◽  
Dry Mass ◽  
Bacterial Cells ◽  
E Coli ◽  
Transmission Electron ◽  
Bacterial Assemblages

ABSTRACT We applied transmission electron microscopy and densitometric image analysis to measure the cell volume (V) and dry weight (DW) of single bacterial cells. The system was applied to measure the DW ofEscherichia coli DSM 613 at different growth phases and of natural bacterial assemblages of two lakes, Piburger See and Gossenköllesee. We found a functional allometric relationship between DW (in femtograms) and V (in cubic micrometers) of bacteria (DW = 435 · V 0.86); i.e., smaller bacteria had a higher ratio of DW to V than larger cells. The measured DW of E. coli cells ranged from 83 to 1,172 fg, and V ranged from 0.1 to 3.5 μm3(n = 678). Bacterial cells from Piburger See and Gossenköllesee (n = 465) had DWs from 3 fg (V = 0.003 μm3) to 1,177 fg (V = 3.5 μm3). Between 40 and 50% of the cells had a DW of less than 20 fg. By assuming that carbon comprises 50% of the DW, the ratio of carbon content to Vof individual cells varied from 466 fg of C μm−3 forVs of 0.001 to 0.01 μm3 to 397 fg of C μm−3 (0.01 to 0.1 μm3) and 288 fg of C μm−3 (0.1 to 1 μm3). Exponentially growing and stationary cells of E. coli DSM 613 showed conversion factors of 254 fg of C μm−3 (0.1 to 1 μm3) and 211 fg of C μm−3 (1 to 4 μm3), respectively. Our data suggest that bacterial biomass in aquatic environments is higher and more variable than previously assumed from volume-based measurements.

Simultaneous Determination of Gene Expression and Enzymatic Activity in Individual Bacterial Cells in Microdroplet Compartments

2009 ◽  
Vol 131 (42) ◽  
pp. 15251-15256 ◽  
Author(s):  
Jung-uk Shim ◽  
Luis F. Olguin ◽  
Graeme Whyte ◽  
Duncan Scott ◽  
Ann Babtie ◽  
...  
Keyword(s):  
Gene Expression ◽  
Enzymatic Activity ◽  
Simultaneous Determination ◽  
Bacterial Cells

Understanding the cryotolerance of lactic acid bacteria using combined synchrotron infrared and fluorescence microscopies

The Analyst ◽  
2015 ◽  
Vol 140 (17) ◽  
pp. 5920-5928 ◽  
Author(s):  
Stéphanie Passot ◽  
Julie Gautier ◽  
Frédéric Jamme ◽  
Stéphanie Cenard ◽  
Paul Dumas ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Fluorescence Microscopy ◽  
Physiological State ◽  
Bacterial Cells ◽  
Ftir Microspectroscopy ◽  
Simultaneous Assessment

Combining synchrotron-FTIR microspectroscopy and fluorescence microscopy made it possible the simultaneous assessment of biochemistry and physiological state of small bacterial cells for better understanding the mechanisms of cell cryotolerance.

scholarly journals Enzymatic Activity as a Measure of Total Microbial Activity on Historical Stone

Heritage ◽  
2020 ◽  
Vol 3 (3) ◽  
pp. 671-681
Author(s):  
Elif Sırt Çıplak ◽  
Kiraz Göze Akoğlu
Keyword(s):  
Enzymatic Activity ◽  
Microbial Activity ◽  
Plate Count ◽  
Biological Factors ◽  
Bacterial Counts ◽  
Hydrolysis Method ◽  
Deterioration Process ◽  
Plate Count Method ◽  
Fda Hydrolysis

Stones of historical monuments exposed to the open air deteriorate over the course of time depending on physical, chemical, and biological factors acting in co-association. Among the biological factors, microorganisms play a key role in the deterioration process of stones. Detecting the level of microbial activity on stones is an essential step in diagnostic and monitoring studies of stone biodeterioration, and aids in controlling the performance of treatments applied to the stones. Therefore, this study aimed to develop a practical and rapid method for the determination of microbial activity on historical stones and use this method on the Mount Nemrut monuments (MNMs) (Adiyaman, Turkey). For that purpose, the fluorescein diacetate (FDA) hydrolysis method, frequently employed for soil environments, was adapted for the estimation and assessment of total microbial activity to understand whether microorganisms posed a potential risk for the biodeterioration of the limestones and sandstones of the MNMs. The traditional plate count method was also applied simultaneously to the same stone samples to compare and assist in the interpretation of the results of the FDA hydrolysis method, which relies on the quantitative determination of bacterial and fungal colonies in nutrient agar and malt extract agar medium, respectively. The results of the FDA hydrolysis and plate count methods showed consistency. The total microbial activity determined by the FDA hydrolysis method was low for both types of stone samples. In addition, the plate count method showed low bacterial and fungal counts on all of the samples. This revealed that microbial activity did not play an important role in the stone deterioration process on the MNMs, although different lichen species were frequently observed on both the sandstones and the limestones. Hence, further investigation must be undertaken for determination of their long-term behavior and effects on the stones of the MNMs. On the other hand, the results of the FDA hydrolysis and plate count methods showed correlation. Lower bacterial counts were observed when lower enzymatic activity was observed in the stone samples, and likewise, higher bacterial counts were observed when higher enzymatic activity was observed. Consequently, the application of the FDA hydrolysis method was determined to be reliable for the estimation of total microbial activity on historical stones. The method had obvious advantages in terms of its rapid measurement rate and sensitivity, even on small samples.

scholarly journals Integrated Approach for Detection of Nonculturable Cells of Ralstonia solanacearum in Asymptomatic Pelargonium spp. Cuttings

2008 ◽  
Vol 98 (8) ◽  
pp. 949-955 ◽  
Author(s):  
E. Marco-Noales ◽  
E. Bertolini ◽  
C. Morente ◽  
M. M. López
Keyword(s):  
Ralstonia Solanacearum ◽  
Physiological State ◽  
Integrated Approach ◽  
Screening Tests ◽  
Physiological Status ◽  
Bacterial Cells ◽  
Tomato Plants ◽  
Solid Media ◽  
Latently Infected ◽  
Nonculturable Cells

Ralstonia solanacearum (biovar 2, race 3) is a soil and water-borne pathogen that causes serious diseases in several solanaceous hosts. It can also infect geranium plants, posing an important threat to their culture when latently infected cuttings are imported from countries where the pathogen is endemic. R. solanacearum can be present in very low numbers in asymptomatic geranium cuttings, and/or in a particular stressed physiological state that escapes direct isolation on the solid media usually employed. Consequently, an integrated protocol has been developed to analyze asymptomatic geranium cuttings routinely. The first screening tests include isolation and co-operational-polymerase chain reaction (Co-PCR), based on the simultaneous and co-operational action of three primers from 16S rRNA of R. solanacearum. This method was selected as the most sensitive one, able to detect only 1 cell/ml including nonculturable cells. When isolation is negative but Co-PCR is positive, the bioassay in tomato plants is proposed, since stressed bacterial cells or those present in low numbers that do not grow on solid media can be recovered from inoculated tomato plants and retain pathogenicity. This methodology has been demonstrated to be useful and has allowed us to assess the relevance of the physiological status of bacterial cells and its implications in detection. It also reveals the risk of introducing R. solanacearum through asymptomatic geranium material when relying only on bacterial isolation.

scholarly journals PRELIMINARY STUDIES ON THE HISTOLOGICAL DEMONSTRATION OF DESOXYRIBONUCLEASE II BY ADAPTATION OF THE GOMORI ACID PHOSPHATASE METHOD

1958 ◽  
Vol 6 (4) ◽  
pp. 255-259 ◽  
Author(s):  
J. ARONSON ◽  
L. H. HEMPELMANN ◽  
S. OKADA
Keyword(s):  
Acid Phosphatase ◽  
Enzymatic Activity ◽  
Intestinal Mucosa ◽  
Kupffer Cells ◽  
Intracellular Localization ◽  
Original Method ◽  
Kidney Cortex ◽  
Nucleotidase Activity ◽  
Red Pulp

An attempt has been made to adapt the acid phosphatase method of Gomori to the demonstration of desoxyribonuclease II activity. The procedure as used is subject to the disadvantages of the original method. The enzymatic activity may be demonstrable only when desoxyribonuclease and nucleotidase activity occur in the same types of cells. Although the staining occurred mainly within discrete cells, the intracellular localization apparent under these conditions of study may well be artifact. In the spleen, the stainning occurs mainly in the red pulp and in a ring of cells, possibly macrophages, surrounding the lymphatic nodules. Hepatic and Kupffer cells stain as do cells in the tubules of the kidney cortex and in the intestinal mucosa.

Survival and inoculum potential of conidia and chlamydospores of Fusarium oxysporum f.sp. lini in soil

1990 ◽  
Vol 36 (8) ◽  
pp. 551-556 ◽  
Author(s):  
Yvonne Couteaudier ◽  
C. Alabouvette
Keyword(s):  
Enzymatic Activity ◽  
Fusarium Oxysporum ◽  
Key Words ◽  
Inoculum Density ◽  
Fluorescein Diacetate ◽  
Natural Soil ◽  
Inoculum Potential ◽  
Kinetics Of ◽  
Grown Culture

The kinetics of survival and inoculum potential of Fusarium oxysporum f. sp. lini were studied in soil. Two types of inoculum were compared: microconidia freshly harvested from a laboratory-grown culture and microchlamydospores produced in sterilized soil. Introduced at the same inoculum densities into a natural soil, the two types of inoculum showed similar behaviour; the inoculum densities changed little with time, at least during 100 days. However, the two types of inoculum did differ in disease potential. A higher percentage of microchlamydospores than microconidia germinated in the rhizosphere of flax seedlings, and the heterotrophic fluorescein diacetate hydrolysing activity of the microchlamydospores was 100 times higher than that of microconidia. Moreover, the microchlamydospores produced more disease on flax than the microconidia even at a much lower inoculum density. Key words: survival, inoculum potential, enzymatic activity, conidia, chlamydospores, Fusarium oxysporum, soil.

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