Biomedical subjects

E Z Ron

Publications and source records attributed to E Z Ron.

At least 19 recordsLinked to original sources

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↗

PubMed1999

High- and low-molecular-mass microbial surfactants.

Microorganisms synthesize a wide variety of high- and low-molecular-mass bioemulsifiers. The low-molecular-mass bioemulsifiers are generally glycolipids, such as trehalose lipids, sophorolipids and rhamnolipids, or lipopeptides, such as surfactin, gramicidin S and polymyxin. The high-molecular-mass bioemulsifiers are amphipathic polysaccharides, proteins, lipopolysaccharides, lipoproteins or complex mixtures of these biopolymers. The low-molecular-mass bioemulsifiers lower surface and interfacial tensions, whereas the higher-molecular-mass bioemulsifiers are more effective at stabilizing oil-in-water emulsions. Three natural roles for bioemulsifiers have been proposed: (i) increasing the surface area of hydrophobic water-insoluble growth substrates; (ii) increasing the bioavailability of hydrophobic substrates by increasing their apparent solubility or desorbing them from surfaces; (iii) regulating the attachment and detachment of microorganisms to and from surfaces. Bioemulsifiers have several important advantages over chemical surfactants, which should allow them to become prominent in industrial and environmental applications. The potential commercial applications of bioemulsifiers include bioremediation of oil-polluted soil and water, enhanced oil recovery, replacement of chlorinated solvents used in cleaning-up oil-contaminated pipes, vessels and machinery, use in the detergent industry, formulations of herbicides and pesticides and formation of stable oil-in-water emulsions for the food and cosmetic industries.

Glycolipids↗

PubMed1999

Enhancement of solubilization and biodegradation of polyaromatic hydrocarbons by the bioemulsifier alasan.

Alasan, a high-molecular-weight bioemulsifier complex of an anionic polysaccharide and proteins that is produced by Acinetobacter radioresistens KA53 (S. Navon-Venezia, Z. Zosim, A. Gottlieb, R. Legmann, S. Carmeli, E. Z. Ron, and E. Rosenberg, Appl. Environ. Microbiol. 61:3240-3244, 1995), enhanced the aqueous solubility and biodegradation rates of polyaromatic hydrocarbons (PAHs). In the presence of 500 microg of alasan ml-1, the apparent aqueous solubilities of phenanthrene, fluoranthene, and pyrene were increased 6.6-, 25.7-, and 19.8-fold, respectively. Physicochemical characterization of the solubilization activity suggested that alasan solubilizes PAHs by a physical interaction, most likely of a hydrophobic nature, and that this interaction is slowly reversible. Moreover, the increase in apparent aqueous solubility of PAHs does not depend on the conformation of alasan and is not affected by the formation of multimolecular aggregates of alasan above its saturation concentration. The presence of alasan more than doubled the rate of [14C]fluoranthene mineralization and significantly increased the rate of [14C]phenanthrene mineralization by Sphingomonas paucimobilis EPA505. The results suggest that alasan-enhanced solubility of hydrophobic compounds has potential applications in bioremediation.

Bacterial Proteins↗

PubMed1999

A nonessential signal peptidase II (Lsp) of Myxococcus xanthus might be involved in biosynthesis of the polyketide antibiotic TA.

Myxococcus xanthus is a gram-negative soil bacterium that produces the polyketide antibiotic TA. In this study, we describe the analysis of an M. xanthus gene which encodes a homologue of the prolipoprotein signal peptidase II (SPase II; lsp). Overexpression of the M. xanthus SPase II in Escherichia coli confers high levels of globomycin resistance, confirming its function as an SPase II. The M. xanthus gene encoding the lsp homologue is nonessential for growth, as determined by specific gene disruption. It has been mapped to the antibiotic TA gene cluster, and the disrupted mutants do not produce the antibiotic, indicating a probable involvement in TA production. These results suggest the existence of more than one SPase II protein in M. xanthus, where one is a system-specific SPase II (for TA biosynthesis).

Amino Acid Sequence↗

PubMed1999

Differential and independent roles of a sigma(32) homolog (RpoH) and an HrcA repressor in the heat shock response of Agrobacterium tumefaciens.

The heat shock response in alpha proteobacteria is unique in that a combination of two regulators is involved: a positive regulator, RpoH (sigma(32) homolog), found in the alpha, beta, and gamma proteobacteria, and a negative regulator, HrcA, widely distributed in eubacteria but not in the gamma proteobacteria. To assess the differential roles of the two regulators in these bacteria, we cloned the hrcA-grpE operon of Agrobacterium tumefaciens, analyzed its transcription, and constructed deletion mutants lacking RpoH and/or HrcA. The DeltarpoH mutant and DeltarpoH DeltahrcA double mutant were unable to grow above 30 degrees C. Whereas the synthesis of heat shock proteins (e.g., DnaK, GroEL, and ClpB) was transiently induced upon temperature upshift from 25 to 37 degrees C in the wild type, such induction was not observed in the DeltarpoH mutant, except that GroEL synthesis was still partially induced. By contrast, the DeltahrcA mutant grew normally and exhibited essentially normal heat induction except for a higher level of GroEL expression, especially before heat shock. The DeltarpoH DeltahrcA double mutant showed the combined phenotypes of each of the single mutants. The amounts of dnaK and groE transcripts before and after heat shock, as determined by primer extension, were consistent with those of the proteins synthesized. The cellular level of RpoH but not HrcA increased significantly upon heat shock. We conclude that RpoH plays a major and global role in the induction of most heat shock proteins, whereas HrcA plays a restricted role in repressing groE expression under nonstress conditions.

Agrobacterium tumefaciens↗

PubMed1998

An Escherichia coli gene responsive to heavy metals.

Using in vivo translational gene fusion in Escherichia coli K-12 we identified a gene that is specifically induced by heavy metals, cadmium, mercury and zinc, at nmolar concentrations. This gene was identified by homology to known zinc and cadmium transporters. We created a disruption of the gene that resulted only in a minor increase in sensitivity to cadmium, suggesting that the fusion, which is at the carboxy-terminal end of the molecule, probably allows for at least partial activity of the protein.

Adenosine Triphosphatases↗

PubMed1998

Regulation of heat-shock response in bacteria.

Stress response in bacteria is essential for effective adaptation to changes in the environment, as well as to the changes in the physiological state of the bacterial culture itself. This response is mediated by global regulatory mechanisms affecting several pathways. It now appears that these regulatory mechanisms operate by transcriptional control, translational control, and proteolysis. One example to be discussed extensively is the heat-shock response. In Escherichia coli, where it has been studied initially and most extensively, the expression of the heat-shock operon is transcriptionally controlled by the employment of the heat-shock transcription factor sigma 32, that recognizes specific heat-shock promoters. Later studies indicated that in most bacteria the control of the major heat-shock genes is much more complicated, and involves additional--or alternative--control channels. These regulatory elements will be reviewed looking at the groE and dnaK operons. These operons, coding for the bacterial equivalent of Hsp10+60 and Hsp70, respectively, contain in many bacteria a conserved regulatory inverted repeat (IR = CIRCE), and are transcribed either by the vegetative sigma factor--sigma 70--or by a sigma 32-like factor. The IR functions at the DNA level as a repressor binding site and also controls the half life of the transcript. In addition, in Agrobacterium tumefaciens there also exists a system for mRNA processing that involves a temperature-controlled cleavage of the groE transcript.

Bacterial Proteins↗

PubMed1997

An Escherichia coli gene divergently transcribed from a promoter overlapping the metA promoter.

The upstream region of the metA gene in Escherichia coli contains two promoters. We have identified by lacZ fusion an additional promoter in this region, and showed that it is transcribed in the opposite orientation from the metA gene. The putative translation product corresponds to a peptide of 147 amino acids-ORF19 by molecular mass. This peptide is probably not essential for growth, as an insertion mutant is viable.

Acyltransferases↗

PubMed1997

Virulence patterns from septicemic Escherichia coli O78 strains.

Several septicemic Escherichia coli O78 strains, isolated from different sources, were characterized phenotypically and genotypically. Two avian isolates, one of which is known to carry the AC/I fimbriae, hybridized with the sfa determinant in colony dot-blot assay. Southern hybridizations with specific sfa probes, following pulsed-field gel electrophoresis (PFGE), showed positive hybridization to the same fragment in each of these strains. Determination of the N-terminal amino acid sequence of the AC/I major subunit gene revealed high similarity to the sequence of the SfaA-II protein. These data suggest that the adhesin gene cluster, coding for AC/I fimbriae, belongs to the S-fimbrial adhesin family.

Adhesins, Bacterial↗

PubMed1997

Bioemulsans: microbial polymeric emulsifiers.

Bioemulsans are amphipathic proteins and/or polysaccharides that stabilize oil-in-water emulsions. Bioemulsans are produced by a wide diversity of microorganisms and have potential applications in the food, paper, paint, bioremediation, agriculture, detergent and cosmetics industries. The production of the RAG-1 emulsan has been studied in batch-fed fermentors via self-cycling fermentation and with immobilized cells using a Celite support matrix. Bioemulsans have several advantages over lower molecular weight emulsifiers presently used in industry. The last few years have seen a number of new bioemulsans described with commercial applications.

Azotobacter↗

PubMed1996

Oil bioremediation using insoluble nitrogen source.

Oil bioremediation is limited by the availability of nitrogen and phosphorous, which are needed by the bacteria and not present in sufficient amounts in hydrocarbons. The supply of these two essential elements as water-soluble salts presents several problems. These include the rapid dilution of the salts in the large volumes of polluted land or water and their utilization by other bacteria that do not degrade oil. In addition, increasing the concentration of mobile nitrogen creates further environmental problems. The use of hydrophobic sources of nitrogen and phosphorous that have a low water solubility can overcome these problems. We have studied one such compound. F-1, that is not used by most bacteria but serves as a good nitrogen and phosphorous source for those bacterial strains that are capable of utilizing it. We have shown that bacteria using F-1 do not cross-feed other bacterial strains. Moreover, when the concentration of the pollutant is sufficiently reduced, the multiplication of the bacteria slows down until they become a negligible fraction of the bacterial population. Chemical analysis indicated that following a 28-day treatment of Alaskan crude oil, most of the hydrocarbons, including polycyclic aromatics, are degraded to undetectable levels. The C34 and C35 components were also degraded, although their degradation was not completed within this time period. In treatment of a sandy beach that was accidentally polluted with crude heavy oil, about 90% degradation was obtained within about 4 months at an outside average temperature of 5 -10 degrees C.

Biodegradation, Environmental↗

PubMed1996

Regulation and organization of the groE and dnaK operons in Eubacteria.

groEL and dnaK are the most highly conserved protein-coding genes known. Most groEL operons and several dnaK and dnaJ operons contain a highly conserved inverted repeat (IR) sequence in their regulatory region. So far, this IR has been found only as part of the groE, dnaK and dnaJ operons and genes. In most cases, the IR is part of the operon transcript, and is involved in the regulation of expression at both the DNA and mRNA levels. A detailed analysis of groE and dnaK operons indicates that the organization of the groE operons is highly conserved. They contain only the groES and groEL genes and always in the same order. In contrast, the organization of the dnaK operons has changed during evolution: genes have been added and deleted from it, and the gene order within the operon is variable.

Amino Acid Sequence↗

PubMed1996

Cadmium binding by bacteria: screening and characterization of new isolates and mutants.

A fast and simple methodology was developed that enables screening of microbial strains for their ability to bind cadmium. It is based on the use of a radioisotope of cadmium (109Cd) for screening colonies and for evaluation of cadmium binding. The methods described here can be used to screen new environmental isolates or to obtain mutants with altered ability to bind cadmium. Examples for the two uses are described in the paper.

Bacteriological Techniques↗

PubMed1996

Heat shock activation of the groESL operon of Agrobacterium tumefaciens and the regulatory roles of the inverted repeat.

Deletions were constructed in the conserved inverted repeat (IR) found in the groESL operon of Agrobacterium tumefaciens and in many other groE and dnaK operons and genes in eubacteria. These deletions affected the level of expression of the operon and the magnitude of its heat shock activation. The IR seems to operate at the DNA level, probably as an operator site that binds a repressor under non-heat shock conditions. The IR was also found to function at the mRNA level, since under non-heat shock conditions transcripts containing deletions of one side of the IR had longer half-lives than did transcripts containing the wild-type IR. Under heat shock conditions, the half-life of the mRNA was unaffected by this deletion because of heat shock-dependent cleavage. However, the groESL operon was found to be heat shock activated even after most of the IR was deleted. This observation, together with the fact that the groESL operon of A. tumefaciens was heat shock activated in Escherichia coli and vice versa, suggests that a heat shock promoter regulates the heat shock activation of this operon. The primary role of the IR appears to be in reducing the MRNA levels from this promoter under non-heat shock conditions.

Agrobacterium tumefaciens↗

PubMed1995

A cluster of cell division genes maps to the terC region of the chromosome of Escherichia coli K-12.

Thirty-nine cell division mutants were isolated in Escherichia coli K-12 and were mapped in the terminus region of the chromosome, between 33.5 and 36 min. They were obtained by two different approaches involving specific mutagenesis of the terC region. The mutants could be divided into eight classes (I to VIII) based on their map position and phenotype at the restrictive temperature, and constitute a new cell division gene cluster.

Cell Division↗

PubMed1995

ppGpp-mediated regulation of DNA replication and cell division in Escherichia coli.

ppGpp serves as an alarmon in prokaryotes, distributing and coordinating different cellular processes according to the nutritional potential of the growth medium. This work is interpreted as favoring the view that, in addition to its previously documented role in regulating the rate of ribosome synthesis, ppGpp participates in coordinating DNA replication and cell division. We studied the effects of ppGpp on the cell division cycle, using cells containing plasmid pSM11 that codes for the 55-kDa truncated RelA protein under the inducible Ptac promoter. In this system it was found that the rate of initiation of new rounds of DNA replication is inversely correlated with the intracellular level of ppGpp. Furthermore, ppGpp levels similar to those found during the activation of stringent control inhibited replication initiation, in a manner comparable to that resulting from inhibition of protein synthesis by amino acid starvation or by chloramphenicol addition. However, in contrast to chloramphenicol treatment, elevated ppGpp levels did not block septum formation, and, in fact, there is some evidence for enhanced septation. As a result, the residual cell division following elevation in ppGpp levels was higher than after chloramphenicol treatment, resulting in cells with a size similar to that of stationary phase cells.

Cell Division↗