The Active Component of the Bioemulsifier Alasan from Acinetobacter radioresistens KA53 Is an OmpA-Like Protein

ABSTRACT The bioemulsifier of Acinetobacter radioresistens KA53, referred to as alasan, is a high-molecular-weight complex of polysaccharide and protein. Recently, one of the alasan proteins, with an apparent molecular mass of 45 kDa, was purified and shown to constitute most of the emulsifying activity. The N-terminal sequence of the 45-kDa protein showed high homology to an OmpA-like protein from Acinetobacter spp. In the research described here the gene coding for the 45-kDa protein was cloned, sequenced, and expressed in Escherichia coli. Recombinant protein AlnA (35.77 kDa without the leader sequence) had an amino acid sequence homologous to that of E. coli OmpA and contained 70% of the specific (hydrocarbon-in-water) emulsifying activity of the native 45-kDa protein and 2.4 times that of the alasan complex. In addition to their emulsifying activity, both the native 45-kDa protein and the recombinant AlnA were highly effective in solubilizing phenanthrene, ca. 80 μg per mg of protein, corresponding to 15 to 19 molecules of phenanthrene per molecule of protein. E. coli OmpA had no significant emulsifying or phenanthrene-solubilizing activity. The production of a recombinant surface-active protein (emulsification and solubilization of hydrocarbons in water) from a defined gene makes possible for the first time structure-function studies of a bioemulsan.

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Acetylacetone-cleaving enzyme Dke1: a novel C-C-bond-cleaving enzyme from Acinetobacter johnsonii

Biochemical Journal ◽

10.1042/bj20021047 ◽

2003 ◽

Vol 369 (3) ◽

pp. 573-581 ◽

Cited By ~ 76

Author(s):

Grit D. STRAGANZ ◽

Anton GLIEDER ◽

Lothar BRECKER ◽

Douglas W. RIBBONS ◽

Walter STEINER

Keyword(s):

Metabolic Pathway ◽

Metabolic Pathways ◽

Active Protein ◽

Acinetobacter Johnsonii ◽

High Affinity ◽

Gene Coding ◽

High Yields ◽

Relationship Of ◽

First Time

The toxicity of acetylacetone has been demonstrated in various studies. Little is known, however, about metabolic pathways for its detoxification or mineralization. Data presented here describe for the first time the microbial degradation of acetylacetone and the characterization of a novel enzyme that initiates the metabolic pathway. From an Acinetobacter johnsonii strain that grew with acetylacetone as the sole carbon source, an inducible acetylacetone-cleaving enzyme was purified to homogeneity. The corresponding gene, coding for a 153 amino acid sequence that does not show any significant relationship to other known protein sequences, was cloned and overexpressed in Escherichia coli and gave high yields of active enzyme. The enzyme cleaves acetylacetone to equimolar amounts of methylglyoxal and acetate, consuming one equivalent of molecular oxygen. No exogenous cofactor is required, but Fe2+ is bound to the active protein and essential for its catalytic activity. The enzyme has a high affinity for acetylacetone with a Km of 9.1μM and a kcat of 8.5s-1. A metabolic pathway for acetylacetone degradation and the putative relationship of this novel enzyme to previously described dioxygenases are discussed.

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Rhamnolipid Biosurfactants Produced by Renibacterium salmoninarum 27BN During Growth on n-Hexadecane

Zeitschrift für Naturforschung C ◽

10.1515/znc-2004-1-215 ◽

2004 ◽

Vol 59 (1-2) ◽

pp. 70-74 ◽

Cited By ~ 46

Author(s):

Nelly Christova ◽

Borjana Tuleva ◽

Zdravko Lalchev ◽

Albena Jordanova ◽

Bojidar Jordanov

Keyword(s):

Pseudomonas Aeruginosa ◽

Thin Layer ◽

Surface Pressure ◽

Emulsifying Activity ◽

Renibacterium Salmoninarum ◽

Pressure Lowering ◽

Surface Active ◽

First Time ◽

Layer Chromatography ◽

Active Substances

A new strain Renibacterium salmoninarum 27BN was isolated for its capacity to utilize nhexadecane as sole substrate. Growth on n-hexadecane was accompanied with the production of glycolipid surface active substances detected by surface pressure lowering and emulsifying activity. Glycolipid detection by thin layer chromatography and infrared spectra analyses showed for the first time that Renibacterium salmoninarum 27BN secretes the two rhamnolipids RLL and RRLL typical for Pseudomonas aeruginosa. Growth of Renibacterium salmoninarum 27BN on n-hexadecane depended on the bioavailability of the substrate and the secreted rhamnolipids appeared to be efficient in increasing hexadecane availability for the cells.

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Emulsifying Activities of Purified Alasan Proteins from Acinetobacter radioresistens KA53

Applied and Environmental Microbiology ◽

10.1128/aem.67.3.1102-1106.2001 ◽

2001 ◽

Vol 67 (3) ◽

pp. 1102-1106 ◽

Cited By ~ 80

Author(s):

Amir Toren ◽

Shiri Navon-Venezia ◽

Eliora Z. Ron ◽

Eugene Rosenberg

Keyword(s):

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Fold Increase ◽

Emulsifying Activity ◽

Active Components ◽

High Molecular Weight Complex ◽

Water Emulsion ◽

Chromatography Analysis ◽

Acinetobacter Radioresistens ◽

Oil In Water Emulsion

ABSTRACT The bioemulsifier of Acinetobacter radioresistens KA53, referred to as alasan, is a high-molecular-weight complex of polysaccharide and protein. The emulsifying activity of the purified polysaccharide (apo-alasan) is very low. Three of the alasan proteins were purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had apparent molecular masses of 16, 31, and 45 kDa. Emulsification assays using the isolated alasan proteins demonstrated that the active components of the alasan complex are the proteins. The 45-kDa protein had the highest specific emulsifying activity, 11% higher than the intact alasan complex. The 16- and 31-kDa proteins gave relatively low emulsifying activities, but they were significantly higher than that of apo-alasan. The addition of the purified 16- and 31-kDa proteins to the 45-kDa protein resulted in a 1.8-fold increase in the specific emulsifying activity and increased stability of the oil-in-water emulsion. Fast-performance liquid chromatography analysis indicated that the 45-kDa protein forms a dimer in nondenaturing conditions and interacts with the 16- and 31-kDa proteins to form a high-molecular-mass complex. The 45-kDa protein and the three-protein complex had substrate specificities for emulsification and a range of pH activities similar to that of alasan. The fact that the purified proteins are active emulsifiers should simplify structure-function studies and advance our understanding of their biological roles.

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Efficacy of compressed sodium chloride (CSC) against E. coli and Candida auris in minutes and methods improvement for testing

Scientific Reports ◽

10.1038/s41598-020-79212-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Lan N. Truong ◽

Brayden D. Whitlock

Keyword(s):

Sodium Chloride ◽

Improved Method ◽

Candida Auris ◽

Antimicrobial Surfaces ◽

E Coli ◽

Route Of Transmission ◽

The World ◽

Short Time ◽

First Time ◽

Time Point

AbstractControlling infections has become one of the biggest problems in the world, whether measured in lives lost or money spent. This is worsening as pathogens continue becoming resistant to therapeutics. Antimicrobial surfaces are one strategy being investigated in an attempt to decrease the spread of infections through the most common route of transmission: surfaces, including hands. Regulators have chosen two hours as the time point at which efficacy should be measured. The objectives of this study were to characterize the new antimicrobial surface compressed sodium chloride (CSC) so that its action may be understood at timepoints more relevant to real-time infection control, under two minutes; to develop a sensitive method to test efficacy at short time points; and to investigate antifungal properties for the first time. E. coli and Candida auris are added to surfaces, and the surfaces are monitored by contact plate, or by washing into collection vats. An improved method of testing antimicrobial efficacy is reported. Antimicrobial CSC achieves at least 99.9% reduction of E. coli in the first two minutes of contact, and at least 99% reduction of C. auris in one minute.

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Antimicrobial and Immunomodulating Activities of Two Endemic Nepeta Species and Their Major Iridoids Isolated from Natural Sources

Pharmaceuticals ◽

10.3390/ph14050414 ◽

2021 ◽

Vol 14 (5) ◽

pp. 414

Author(s):

Neda Aničić ◽

Uroš Gašić ◽

Feng Lu ◽

Ana Ćirić ◽

Marija Ivanov ◽

...

Keyword(s):

Biofilm Formation ◽

Food Industry ◽

Balkan Peninsula ◽

Antimicrobial Activities ◽

Natural Sources ◽

E Coli ◽

Food Borne ◽

Metabolite Profiles ◽

Aspergillus Sp ◽

First Time

Two Balkan Peninsula endemics, Nepeta rtanjensis and N. argolica subsp. argolica, both characterized by specialized metabolite profiles predominated by iridoids and phenolics, are differentiated according to the stereochemistry of major iridoid aglycone nepetalactone (NL). For the first time, the present study provides a comparative analysis of antimicrobial and immunomodulating activities of the two Nepeta species and their major iridoids isolated from natural sources—cis,trans-NL, trans,cis-NL, and 1,5,9-epideoxyloganic acid (1,5,9-eDLA), as well as of phenolic acid rosmarinic acid (RA). Methanol extracts and pure iridoids displayed excellent antimicrobial activity against eight strains of bacteria and seven strains of fungi. They were especially potent against food-borne pathogens such as L. monocytogenes, E. coli, S. aureus, Penicillium sp., and Aspergillus sp. Targeted iridoids were efficient agents in preventing biofilm formation of resistant P. aeruginosa strain, and they displayed additive antimicrobial interaction. Iridoids are, to a great extent, responsible for the prominent antimicrobial activities of the two Nepeta species, although are probably minor contributors to the moderate immunomodulatory effects. The analyzed iridoids and RA, individually or in mixtures, have the potential to be used in the pharmaceutical industry as potent antimicrobials, and in the food industry to increase the shelf life and safety of food products.

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Radical Dehalogenation and Purine Nucleoside Phosphorylase E. coli: How Does an Admixture of 2′,3′-Anhydroinosine Hinder 2-fluoro-cordycepin Synthesis

Biomolecules ◽

10.3390/biom11040539 ◽

2021 ◽

Vol 11 (4) ◽

pp. 539

Author(s):

Alexey L. Kayushin ◽

Julia A. Tokunova ◽

Ilja V. Fateev ◽

Alexandra O. Arnautova ◽

Maria Ya. Berzina ◽

...

Keyword(s):

Nmr Spectroscopy ◽

Chemical Synthesis ◽

Purine Nucleoside Phosphorylase ◽

Purine Nucleoside ◽

Preparative Synthesis ◽

Nucleoside Phosphorylase ◽

E Coli ◽

First Time

During the preparative synthesis of 2-fluorocordycepin from 2-fluoroadenosine and 3′-deoxyinosine catalyzed by E. coli purine nucleoside phosphorylase, a slowdown of the reaction and decrease of yield down to 5% were encountered. An unknown nucleoside was found in the reaction mixture and its structure was established. This nucleoside is formed from the admixture of 2′,3′-anhydroinosine, a byproduct in the preparation of 3-′deoxyinosine. Moreover, 2′,3′-anhydroinosine forms during radical dehalogenation of 9-(2′,5′-di-O-acetyl-3′-bromo- -3′-deoxyxylofuranosyl)hypoxanthine, a precursor of 3′-deoxyinosine in chemical synthesis. The products of 2′,3′-anhydroinosine hydrolysis inhibit the formation of 1-phospho-3-deoxyribose during the synthesis of 2-fluorocordycepin. The progress of 2′,3′-anhydroinosine hydrolysis was investigated. The reactions were performed in D2O instead of H2O; this allowed accumulating intermediate substances in sufficient quantities. Two intermediates were isolated and their structures were confirmed by mass and NMR spectroscopy. A mechanism of 2′,3′-anhydroinosine hydrolysis in D2O is fully determined for the first time.

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Antimicrobial Activity of L-Lysine and Poly-L-Lysine with Pulsed Electric Fields

Applied Sciences ◽

10.3390/app11062708 ◽

2021 ◽

Vol 11 (6) ◽

pp. 2708

Author(s):

Jurgita Švedienė ◽

Vitalij Novickij ◽

Rokas Žalnėravičius ◽

Vita Raudonienė ◽

Svetlana Markovskaja ◽

...

Keyword(s):

Electric Fields ◽

Pulsed Electric Fields ◽

Antimicrobial Treatment ◽

Treatment Development ◽

E Coli ◽

Yeast Fungi ◽

New Treatment ◽

First Time ◽

Resistant Microorganism ◽

Susceptibility Patterns

For the first time, the possibility to use L-lysine (Lys) and poly-L-lysine (PLL) as additives with pulsed electric fields (PEF) for antimicrobial treatment is reported. The antimicrobial efficacy of Lys and PLL for Escherichia coli, Staphylococcus aureus, Trichophyton rubrum and Candida albicans was determined. Inactivation of microorganisms was also studied by combining Lys and PLL with PEF of 15 and 30 kV/cm. For PEF treatment, pulses of 0.5, 1, 10 or 100 μs were applied in a sequence of 10 to 5000 at 1 kHz frequency. The obtained results showed that 100 μs pulses were the most effective in combination with Lys and PLL for all microorganisms. Equivalent energy PEF bursts with a shorter duration of the pulse were less effective independently on PEF amplitude. Additionally, various treatment susceptibility patterns of microorganisms were determined and reported. In this study, the Gram-negative E. coli was the most treatment-resistant microorganism. Nevertheless, inactivation rates exceeding 2 log viability reduction were achieved for all analyzed yeast, fungi, and bacteria. This methodology could be used for drug-resistant microorganism’s new treatment development.

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Surface active properties at the air–water interface of β-casein and its fragments derived by plasmin proteolysis

Journal of Dairy Research ◽

10.1017/s0022029900028971 ◽

1989 ◽

Vol 56 (3) ◽

pp. 487-494 ◽

Cited By ~ 8

Author(s):

Michael Wilson ◽

Daniel M. Mulvihill ◽

William J. Donnelly ◽

Brian P. Gill

Keyword(s):

Surface Tension ◽

Water Interface ◽

Active Protein ◽

Drop Volume ◽

V Protein ◽

Proteose Peptone ◽

Rate Determining Step ◽

Surface Active Properties ◽

Air Water Interface ◽

Surface Active

Summaryβ-Casein, was enzymically modified by incubation with plasmin to yield γ-caseins and proteose peptones. Whole γ-, γ1-, γ2/γ3-caseins and whole proteose peptone (pp) were isolated from the hydrolysate mixture. The time dependence of surface tension at the air-water interface of solutions of β-casein and its plasmin derived fragments, at concentrations of 10−1 to 10−4% (w/v) protein, pH 7.0, was determined, at 25 °C, using a drop volume apparatus. The ranking of the proteins with respect to rate of reduction of surface tension, during the first rate determining step, at 10-2% (w/v) protein, was γ2/γ3 ≫ pp > whole γ- > γ1- > β-casein. The ranking of the proteins with respect to surface pressures attained after 40 min (π40) was concentration dependent. γ2/γ3-Caseins were found to be very surface active, decreasing surface tension rapidly and giving a high π40. γ1 Casein decreased surface activity somewhat faster than β-casein, but generally reached a lower π40. Whole γ-casein reflected the properties of both γ1 and γ2/γ3-caseins. Proteose peptone was found to decrease surface tension rapidly during the initial rate determining step; it gave a relatively high π40 at a bulk phase concentration of 10−3% (w/v) protein, but, it was the least surface active protein at 10−1 and 10−2% (w/v) protein.

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Expression of Heterologous Cellulases inThermotogasp. Strain RQ2

BioMed Research International ◽

10.1155/2015/304523 ◽

2015 ◽

Vol 2015 ◽

pp. 1-11 ◽

Cited By ~ 8

Author(s):

Hui Xu ◽

Dongmei Han ◽

Zhaohui Xu

Keyword(s):

Enzyme Activities ◽

Bacterial Cells ◽

Recombinant Enzymes ◽

Caldicellulosiruptor Saccharolyticus ◽

Shuttle Vectors ◽

E Coli ◽

Chimeric Enzymes ◽

Coding Regions ◽

First Time ◽

Culture Supernatants

The ability ofThermotogaspp. to degrade cellulose is limited due to a lack of exoglucanases. To address this deficiency, cellulase genes Csac_1076 (celA) and Csac_1078 (celB) fromCaldicellulosiruptor saccharolyticuswere cloned intoT.sp. strain RQ2 for heterologous overexpression. Coding regions of Csac_1076 and Csac_1078 were fused to the signal peptide of TM1840 (amyA) and TM0070 (xynB), resulting in three chimeric enzymes, namely, TM1840-Csac_1078, TM0070-Csac_1078, and TM0070-Csac_1076, which were carried byThermotoga-E. colishuttle vectors pHX02, pHX04, and pHX07, respectively. All three recombinant enzymes were successfully expressed inE. coliDH5αandT.sp. strain RQ2, rendering the hosts with increased endo- and/or exoglucanase activities. InE. coli, the recombinant enzymes were mainly bound to the bacterial cells, whereas inT.sp. strain RQ2, about half of the enzyme activities were observed in the culture supernatants. However, the cellulase activities were lost inT.sp. strain RQ2 after three consecutive transfers. Nevertheless, this is the first time heterologous genes bigger than 1 kb (up to 5.3 kb in this study) have ever been expressed inThermotoga, demonstrating the feasibility of using engineeredThermotogaspp. for efficient cellulose utilization.

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About the Results of Monitoring Researches of Ballast Waters and Data of Identification of the Vibrioes Strains Selected During the Ships Researches in Russian Seaports in 2018

Journal of microbiology epidemiology immunobiology ◽

10.36233/0372-9311-2020-97-1-55-61 ◽

2020 ◽

Vol 97 (1) ◽

pp. 55-61

Author(s):

S. Yu. Vodyanitskаyа ◽

O. V. Sergienko ◽

N. G. Ivanova ◽

V. V. Balachnova ◽

I. V. Arkhangelskаya ◽

...

Keyword(s):

Russian Federation ◽

Ballast Water ◽

International Convention ◽

International Standard ◽

E Coli ◽

Ballast Waters ◽

Rostov Region ◽

Intestinal Infections ◽

The Russian Federation ◽

First Time

Relevance. In September, 2017 the International Convention on Control of Ships’ Ballast Waters and sediments, in which the Russian Federation takes part, came into effect.Aim of article is to cover the results of implementation of the Convention in Russia, regarding selection and analysis of ballast waters tests for compliance with the international standard.Material and methods. The materials for work were data on ship arrivals at the international seaports of the Russian Federation, provided by sanitary and quarantine departments of the Russian ports, and monitoring researches of ballast waters in seaports of some regions of the Russian Federation. Analytical, bacteriological, molecular methods were applied.Results. The studies of ballast waters in the Leningrad and Kaliningrad regions have been conducted for the first time in 2018, and in the Rostov region the study lasts since 2010. The laboratory researches of ships’ ballast showed that E. coli, Enterococcus spp. were in norm, V. cholerae O1 and O139 in ballast were absent. 12 of 21 ballast water tests investigated by specialists of the Rostov region laboratories contained V. cholerae non-O1/non-O139, ballast was taken on the ships which arrived from Romania and Turkey.Сonclusion. The results of the molecular and genetic researches suggest that there is a probability of V. сholeraе introduction brougth with ship ballast. Management decisions are demanded to ensure biological safety of shipping and to decrease intestinal infections incidence in residents of the seaside cities.

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