Biomedical subjects

E Z Ron

Publications and source records attributed to E Z Ron.

At least 19 recordsLinked to original sources

PubMed2005

Myxobacteria isolated in Israel as potential source of new anti-infectives.

AIMS: To evaluate the patterns of the production of antimicrobial compounds by Israeli myxobacteria newly isolated from soil samples and barks by a battery of isolation and purification methods. METHODS AND RESULTS: A total of 100 myxobacteria belonging to five of the 12 described genera, were isolated from 48 soil and 45 tree bark samples collected in different areas inside the State of Israel. Four isolation methods based on the peculiar metabolic and cell cycle aspects of myxobacteria, were combined with purification procedures and optimization of cultivation conditions. Ninety-seven strains were fermented and screened for antimicrobial activities. Production of antimicrobial activities was detected in 62 isolates. More than 50% of the collection (54 strains) was able to inhibit Escherichia coli growth. CONCLUSIONS: The results of this study support the idea that myxobacterial strains can be isolated from particular habitats and then cultivated and screened for their capacity to produce secondary metabolites endowed with antibacterial and antifungal activities. Myxovirescin, a typical poliketide myxobacterial antibiotic, has been identified in one Israeli isolate. Althiomycin, a thiazolyl peptide, which inhibits prokaryotic protein synthesis, usually produced by actinomycetes, was detected in three strains selected in this study. SIGNIFICANCE AND IMPACT OF THE STUDY: The results confirm that myxobacteria are prolific producers of a variety of bioactive secondary metabolites including antibacterial and antifungal compounds, being their high frequency of anti-Gram-negative activities particularly appealing for the current anti-infective research. So far their screening has often been hampered because their isolation is time-consuming and are quite difficult to handle and cultivate. In this paper we demonstrate that a proper combination of isolation, purification and cultivation methods allow their pharmaceutical exploitation.

Anti-Bacterial Agents↗

PubMed2004

The AlnB protein of the bioemulsan alasan is a peroxiredoxin.

The bioemulsifier of Acinetobacter radioresistens KA53, referred to as alasan, is a high molecular weight complex of a polysaccharide and three proteins (AlnA, AlnB and AlnC). AlnA has previously been shown to be an OmpA-like protein that is largely responsible for the emulsifying activity of alasan. To further elucidate the nature of alasan, the gene coding for AlnB was cloned, sequenced and overexpressed in Escherichia coli. The overall 561 bp sequence of the hypothetical AlnB showed strong homology, including all conserved regions and residues known to be essential for enzymatic activity, to the ubiquitous family of thiol-specific antioxidant enzymes known as peroxiredoxins. Transgenic E. coli overexpressing AlnB exhibited increased resistance to cumene hydroperoxide both in liquid culture and on agar medium. Recombinant AlnB had no emulsifying activity but stabilized oil-in-water emulsion generated by AlnA.

Acinetobacter↗

PubMed2003

Combined phage typing and amperometric detection of released enzymatic activity for the specific identification and quantification of bacteria.

Here, we describe a novel electrochemical method for the rapid identification and quantification of pathogenic and polluting bacteria. The design incorporates a bacteriophage, a virus that recognizes, infects, and lyses only one bacterial species among mixed populations, thereby releasing intracellular enzymes that can be monitored by the amperometic measurement of enzymatic activity. As a model system, we used virulent phage typing and cell-marker enzyme activity (beta-D-galactosidase), a combination that is specific for the bacterial strain Escherichia coli (K-12, MG1655). Filtration and preincubation before infecting the bacteria with the phage enabled amperometric detection at a wide range of concentrations, reaching as low as 1 colony-forming unit/100 mL within 6-8 h. In principle, this electrochemical method can be applied to any type of bacterium-phage combination by measuring the enzymatic marker released by the lytic cycle of a specific phage.

Bacteria↗

PubMed2002

Solubilization of polyaromatic hydrocarbons by recombinant bioemulsifier AlnA.

AlnA is the protein responsible for the emulsifying and solubilizing activity of the Acinetobacter radioresistens KA53 bioemulsifier alasan. AlnA was produced in Escherichia coli, purified to homogeneity and then used to measure the enhanced solubility of 12 polyaromatic hydrocarbons (PAHs). The amount of PAH solubilized was directly proportional to AlnA concentration. The ratio of PAH solubilized by 40 micro g/ml AlnA compared to that soluble in the aqueous buffer varied greatly, from 4 (fluorene) to 81 (hexylbenzylcyclosilane). Calculations of moles PAH solubilized per mole AlnA yielded values from 4.3 (hexylphenylbenzene) to 55.8 (1,10-phenanthrolene). There was no obvious relationship between the amount of PAH solubilized and its molecular weight or intrinsic solubility. Native gel electrophoresis indicated that AlnA formed hexamers in the presence of PAHs. With molar ratios of fluorene to AlnA of 0.75 or less, only the monomer was observed, whereas at ratios of 7.5 or higher, only the hexamer was detected. At an intermediate molar ratio of 2.6, both monomer and hexamer appeared. The data indicate that PAHs are initially solubilized by binding to the monomeric form of AlnA, and as the amount bound increases above one molecule PAH per AlnA, the protein aggregates to form a specific oligomer of 5-8 monomers which allows for the binding and solubilization of more PAH.

Acinetobacter↗

PubMed2001

An unusual beta-ketoacyl:acyl carrier protein synthase and acyltransferase motifs in TaK, a putative protein required for biosynthesis of the antibiotic TA in Myxococcus xanthus.

The antibiotic TA of Myxococcus xanthus is produced by a type-I polyketide synthase mechanism. Previous studies have indicated that TA genes are clustered within a 36-kb region. The chemical structure of TA indicates the need for several post-modification steps, which are introduced to form the final bioactive molecule. These include three C-methylations, an O-methylation and a specific hydroxylation. In this study, we describe the genetic analysis of taK, encoding a specific polyketide beta-ketoacyl:acyl carrier protein synthase, which contains an unusual beta-ketoacyl synthase and acyltransferase motifs and is likely to be involved in antibiotic TA post-modification. Functional analysis of this beta-ketoacyl:acyl carrier protein synthase by specific gene disruption suggests that it is essential for the production of an active TA molecule.

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗

PubMed2001

Yersinia HPI in septicemic Escherichia coli strains isolated from diverse hosts.

High pathogenicity islands (HPIs), first identified in various Yersinia species, encode an iron uptake system. We have studied the occurrence of HPIs in septicemic strains of Escherichia coli isolated from a variety of hosts. The results presented in this communication indicate that most septicemic strains tested contained HPI sequences even though they already have the aerobactin encoding genes. We have also observed two types of HPI deletions, suggesting genetic instability of this element. Notable exceptions are several strains isolated from septicemia in sheep that lacked both iron acquisition systems.

Animals↗

PubMed2001

Natural roles of biosurfactants.

Microorganisms produce a variety of surface-active agents (or surfactants). These can be divided into low-molecular-weight molecules that lower surface and interfacial tensions efficiently and high-molecular-weight polymers that bind tightly to surfaces. These surfactants, produced by a wide variety of microorganisms, have very different chemical structures and surface properties. It is therefore reasonable to assume that different groups of biosurfactants have different natural roles in the growth of the producing microorganisms. Moreover, as their chemical structures and surface properties are so different, each emulsifier probably provides advantages in a particular ecological niche. Several bioemulsifiers have antibacterial or antifungal activities. Other bioemulsifiers enhance the growth of bacteria on hydrophobic water-insoluble substrates by increasing their bioavailability, presumably by increasing their surface area, desorbing them from surfaces and increasing their apparent solubility. Bioemulsifiers also play an important role in regulating the attachment-detachment of microorganisms to and from surfaces. In addition, emulsifiers are involved in bacterial pathogenesis, quorum sensing and biofilm formation. Recent experiments indicate that a high-molecular-weight bioemulsifier that coats the bacterial surface can be transferred horizontally to other bacteria, thereby changing their surface properties and interactions with the environment.

Adhesiveness↗

PubMed2001

Emulsifying activities of purified Alasan proteins from Acinetobacter radioresistens KA53.

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.

Acinetobacter↗

PubMed2001

Curli fibers mediate internalization of Escherichia coli by eukaryotic cells.

Curli fibers are adhesive surface fibers expressed by Escherichia coli and Salmonella enterica that bind several host extracellular matrix and contact phase proteins and were assumed to have a role in pathogenesis. The results presented here suggest that one such role is internalization into host cells. An E. coli K-12 strain transformed with a low-copy vector containing the gene cluster encoding curli fibers (csg operon) was internalized by several lines of eukaryotic cells. The internalization could be correlated with a high level of curli fiber expression and was abolished by disruption of the csg operon. The ability to be internalized by eukaryotic cells could be conferred even by the curli fiber gene cluster of a noninvasive K-12 strain, but the homologous csg cluster from a virulent septicemic E. coli isolate mediated a higher level of internalization. The finding that curli fibers promote bacterial internalization indicates a new role for curli fibers in pathogenesis.

Amino Acid Sequence↗

PubMed2001

S-Fimbria-encoding determinant sfa(I) is located on pathogenicity island III(536) of uropathogenic Escherichia coli strain 536.

The sfa(I) determinant encoding the S-fimbrial adhesin of uropathogenic Escherichia coli strains was found to be located on a pathogenicity island of uropathogenic E. coli strain 536. This pathogenicity island, designated PAI III(536), is located at 5.6 min of the E. coli chromosome and covers a region of at least 37 kb between the tRNA locus thrW and yagU. As far as it has been determined, PAI III(536) also contains genes which code for components of a putative enterochelin siderophore system of E. coli and Salmonella spp. as well as for colicin V immunity. Several intact or nonfunctional mobility genes of bacteriophages and insertion sequence elements such as transposases and integrases are present on PAI III(536). The presence of known PAI III(536) sequences has been investigated in several wild-type E. coli isolates. The results demonstrate that the determinants of the members of the S-family of fimbrial adhesins may be located on a common pathogenicity island which, in E. coli strain 536, replaces a 40-kb DNA region which represents an E. coli K-12-specific genomic island.

Adhesins, Escherichia coli↗

PubMed2000

Effect of the adhesive antibiotic TA on adhesion and initial growth of E. coli on silicone rubber.

Catheter-associated urinary tract infection is the most common nosocomial infection, and contributes to patient morbidity and mortality. We investigated the effect that the TA adhesive antibiotic had on adhesion and initial growth in urine of Escherichia coli on silicone rubber. The TA antibiotic had reduced adhesion, and inhibited initial growth of the bacteria on the surface. Since adhesion and initial growth on the surface are an essential part of biofilm formation and subsequent infection, we speculate that the TA antibiotic coating might decrease the infection rate associated with indwelling urinary catheter.

Anti-Bacterial Agents↗

PubMed2000

Control of methionine biosynthesis in Escherichia coli by proteolysis.

Most bacterial proteins are stable, with half-lives considerably longer than the generation time. In Escherichia coli, the few exceptions are unstable regulatory proteins. The results presented here indicate that the first enzyme in methionine biosynthesis - homoserine trans-succinylase (HTS) - is unstable and subject to energy-dependent proteolysis. The enzyme is stable in triple mutants defective in Lon-, HslVU- and ClpP-dependent proteases. The instability of the protein is determined by the amino-terminal part of the protein, and its removal or substitution by the N-terminal part of beta-galactosidase confers stability. The effect of the amino-terminal segment is not caused by the N-end rule, as substitution of the first amino acid does not affect the stability of the protein. HTS is the first biosynthetic E. coli enzyme shown to have a short half-life and may represent a group of biosynthetic enzymes whose expression is controlled by proteolysis. Alternatively, the proteolytic processing of HTS may be unique to this enzyme and could reflect its central role in regulating bacterial growth, especially at elevated temperatures.

Acyltransferases↗

PubMed2000

Online and in situ monitoring of environmental pollutants: electrochemical biosensing of cadmium.

Online sensitive monitoring of gene expression is essential for understanding microbial life and microbial communities, especially under stress-inducing conditions, such as the presence of environmental pollutants. We describe here a novel use of promoter-based electrochemical biosensing for online and in situ monitoring of gene expression in response to pollutants. As a model system, we used a cadmium-responsive promoter from Escherichia coil fused to a promoterless lacZ gene, which was monitored using an electrochemical assay of beta-galactosidase activity. This whole-cell biosensor could detect, within minutes, nanomolar concentrations of cadmium in water, sea water and soil samples, and it can be used for continuous online and in situ monitoring.

Adenosine Triphosphatases↗

PubMed2000

Horizontal transfer of an exopolymer complex from one bacterial species to another.

Alasan, the exocellular polymeric emulsifier produced by Acinetobacter radioresistens KA53 was shown to bind to the surface of Sphingomonas paucimobilis EPA505 and Acinetobacter calcoaceticus RAG-1. The presence of alasan on the surface of S. paucimobilis EPA505 and A. calcoaceticus RAG-1 caused a decrease in their cell-surface hydrophobicities. Binding was proportional to the concentration of recipient cells and input alasan. At the highest concentration of A. calcoaceticus RAG-1 (4 x 10(9) ml(-1)) and alasan (20 microg ml(-1)) tested, 75% of the alasan was cell bound. Alasan binding was measured by the loss of emulsifying activity and alasan protein and polysaccharide from the aqueous phase after incubation of alasan with the recipient cells. In addition, alasan was visualized on the surface of the recipient cells by staining with anti-alasan antibodies and rhodamine-labelled secondary antibodies. Moreover, when the alasan-producing A. radioresistens KA53 was grown together with A. calcoaceticus RAG-1, alasan was released from the producing strain and became bound to the recipient RAG-1 cells, as demonstrated by fluorescence microscopy. This horizontal transfer of exopolymers from one bacterial species to another has significant implications in natural microbial communities, coaggregation and biofilms.

Acinetobacter↗

PubMed2000

New fimbrial gene cluster of S-fimbrial adhesin family.

Fimbrial adhesins that mediate attachment to host cells are produced by most virulent Escherichia coli isolates. These virulence factors play an important role in the initial stages of bacterial colonization and also in determination of the host and tissue specificity. Isolates belonging to serotype O78 are known to cause a large variety of clinical syndromes in farm animals and humans and have been shown to produce several types of adherence fimbriae. We studied the fimbrial adhesin from an avian septicemic E. coli isolate of serotype O78. Analysis of the genetic organization of the fac (fimbria of avian E. coli) gene cluster indicates that it belongs to the S-fimbrial adhesin family. Seven open reading frames coding for major and minor structural subunits were identified, and most of them showed a high degree of homology to the corresponding Sfa and Foc determinants. The least-conserved open reading frame was facS, encoding a protein known to play an important role in determining adherence specificity in other S-fimbrial gene clusters.

Adhesins, Escherichia coli↗

PubMed1999

Cloning and characterization of a Myxococcus xanthus cytochrome P-450 hydroxylase required for biosynthesis of the polyketide antibiotic TA.

The antibiotic TA, a complex macrocyclic polyketide of Myxococcus xanthus, is produced, like many other polyketides, through successive condensations of acetate by a type I polyketide synthase (PKS) mechanism. The chemical structure of this antibiotic and the mechanism by which it is synthesized indicate the need for several post-modification steps, such as a specific hydroxylation at C-20. Previous studies have shown that several genes, essential for TA biosynthesis, are clustered in a region of at least 36kb, which was subsequently cloned and analyzed. In this study, we report the analysis of a DNA fragment, containing a specific cytochrome P-450 hydroxylase, presumably responsible for the sole non-PKS hydroxylation at position C-20. Functional analysis of the cytochrome P-450 hydroxylase gene through specific gene disruption confirms that it is essential for the production of an active TA molecule.

Amino Acid Sequence↗

PubMed1999

The first gene in the biosynthesis of the polyketide antibiotic TA of Myxococcus xanthus codes for a unique PKS module coupled to a peptide synthetase.

The polyketide antibiotic TA is synthesized by the Gram negative bacterium Myxococcus xanthus in a multi-step process in which a unique glycine-derived molecule is used as a starter unit and elongated through the condensation of 11 acetate molecules by polyketide synthases (PKSs). Analysis of a 7.2 kb DNA fragment, encoding the protein that carries out the first condensation step, revealed that the fragment constitutes a single open reading frame, referred to as Ta1, which lacks the 5' and 3' ends and displays two regions of similarity to other proteins. The first 1020 amino acid residues at the N terminus of the polypeptide are similar to sequences of the large family of enzymes encoding peptide synthetases. They are followed by a second region displaying a high degree of similarity to type I PKS genes. The genetic analysis of this open reading frame is compatible with the proposed chemical structure of TA. The data indicate that the genes encoding TA have a modular gene organization, typical of a type I PKS system. The unusual feature of Ta1 is that the first PKS module of TA resides on the same polypeptide as the peptide synthetase functional unit.

Acetates↗

PubMed1999

A NusG-like transcription anti-terminator is involved in the biosynthesis of the polyketide antibiotic TA of Myxococcus xanthus.

The antibiotic TA of Myxococcus xanthus is synthesized through a type I polyketide synthase mechanism. Previous studies have indicated that several genes essential for TA production are clustered within a 40-kb region and are transcriptionally co-regulated. In this study, we report the genetic analysis of the first gene in the TA gene cluster, identified as a NusG-like transcription anti-terminator. Functional analysis of this NusG-like anti-terminator gene by specific gene disruption confirms that it is essential for TA production but not for normal growth and development.

Amino Acid Sequence↗