Biomedical subjects

A B Oppenheim

Publications and source records attributed to A B Oppenheim.

At least 37 records · Page 2Linked to original sources

PubMed1997

Molecular cloning, structural analysis, and expression in Escherichia coli of a chitinase gene from Enterobacter agglomerans.

The gene chiA, which codes for endochitinase, was cloned from a soilborne Enterobacter agglomerans. Its complete sequence was determined, and the deduced amino acid sequence of the enzyme designated Chia_Entag yielded an open reading frame coding for 562 amino acids of a 61-kDa precursor protein with a putative leader peptide at its N terminus. The nucleotide and polypeptide sequences of Chia_Entag showed 86.8 and 87.7% identity with the corresponding gene and enzyme, Chia_Serma, of Serratia marcescens, respectively. Homology modeling of Chia_Entag's three-dimensional structure demonstrated that most amino acid substitutions are at solvent-accessible sites. Escherichia coli JM109 carrying the E. agglomerans chiA gene produced and secreted Chia_Entag. The antifungal activity of the secreted endochitinase was demonstrated in vitro by inhibition of Fusarium oxysporum spore germination. The transformed strain inhibited Rhizoctonia solani growth on plates and the root rot disease caused by this fungus in cotton seedlings under greenhouse conditions.

Amino Acid Sequence↗

PubMed1997

Stability of CII is a key element in the cold stress response of bacteriophage lambda infection.

Bacteria are known to adapt to environmental changes such as temperature fluctuations. It was found that temperature affects the lysis-lysogeny decision of lambda such that at body temperature (37 degrees C) the phage can select between the lytic and lysogenic pathways, while at ambient temperature (20 degrees C) the lytic pathway is blocked. This temperature-dependent discriminatory developmental pathway is governed mainly by the phage CII activity as a transcriptional activator. Mutations in cII or point mutations at the pRE promoter lead to an over-1,000-fold increase in mature-phage production at low temperature while mutations in cI cause a smaller increase in phage production. Interference with CII activity can restore lytic growth at low temperature. We found that at low temperature the stability of CII in vivo is greatly increased. It was also found that phage DNA replication is blocked at 20 degrees C but can be restored by supplying O and P in trans. It is proposed that CII hampers transcription of the rightward pR promoter, thus reducing the levels of the lambda O and P proteins, which are necessary for phage DNA replication. Our results implicate CII itself or host proteins affecting CII stability as a "molecular thermometer".

Bacteriophage lambda↗

PubMed1996

Identification of an UP element within the IHF binding site at the PL1-PL2 tandem promoter of bacteriophage lambda.

An UP element defines a supplementary promoter element located upstream of the -35 region that stimulates transcription by interacting with the C-terminal domain of the RNA polymerase alpha subunit (alpha CTD). The alpha CTD also responds to various transcription activators, including the integration host factor protein, IHF, in the stimulation of the bacteriophage lambda PL promoter. PL consists of the tandem PL1-PL2 promoters where PL1 is stimulated and PL2 is repressed by IHF. We identified a functional UP element that binds the alpha subunit of RNA polymerase and is located in the region from -36 to -60 relative to the PL2 start site. PL2 expression requires the presence of the UP element and requires an intact alpha CTD. The UP element is nested within the DNA region protected by IHF against DNase I digestion. We used mutational analysis to identify the IHF recognition sequence which was found to be located downstream of the UP element, overlapping the -35 region of PL2. The possible function of the complex structure of the PL promoter is discussed.

Amino Acid Sequence↗

PubMed1996

Analysis of the shut-off of ribosomal RNA promoters in Escherichia coli upon entering the stationary phase of growth.

Most bacterial RNA consists of stable RNA which is composed of rRNA and tRNA. We have followed by primer extension analysis the level of ribosomal RNA synthesis along the growth phases of a cell culture. A sharp drop in rRNA synthesis was observed upon the transition from the exponential to the stationary phase of growth. Our results demonstrate that an effective shut-off of rRNA synthesis occurs also in the absence of ppGpp. Mutations in the host factors Fis and H-NS, which are known to regulate rrn P1 promoters, did not affect the shut-off process of ribosomal RNA promoters. We also tested the effect of RpoS, the sigma factor known to induce a number of genes in the stationary phase. It was shown that the host factors Fis, H-NS and RpoS do not play a major role in the regulation of the shut-off process of rRNA synthesis. The results presented demonstrate that the rate of rRNA synthesis provides a sensitive measure of the growth phase of the bacterial culture.

Bacterial Proteins↗

PubMed1996

Differential mRNA stability of the cspA gene in the cold-shock response of Escherichia coli.

Exposure of bacterial cells to temperature changes induces the synthesis of a set proteins. We investigated the control of expression of the cspA gene, coding for the major cold-shock protein of Escherichia coli. This protein was shown to be transiently induced upon shift to low temperature. We demonstrated that the cspA mRNA is extremely unstable at 37 degrees C with a half-life of approx. 10 s. Upon shift to 15 degrees C cspA mRNA becomes highly stable. This mRNA stability is transient and is lost once the cells are adapted to the low temperature. Transcription fusions of lacZ containing part or most of the cspA gene do not show the rapid degradation at high temperature. Our results suggest that mRNA stability plays a major role in the control of the cspA gene. The expression of cspA is also regulated, to a smaller extent, by the relative increase in transcription after transfer to low temperature. A model by which cspA mRNA is regulated in response to temperature shift is discussed.

Bacterial Proteins↗

PubMed1996

Structure and function of the Pseudomonas putida integration host factor.

Integration host factor (IHF) is a DNA-binding and -bending protein that has been found in a number of gram-negative bacteria. Here we describe the cloning, sequencing, and functional analysis of the genes coding for the two subunits of IHF from Pseudomonas putida. Both the ihfA and ihfB genes of P. putida code for 100-amino-acid-residue polypeptides that are 1 and 6 residues longer than the Escherichia coli IHF subunits, respectively. The P. putida ihfA and ihfB genes can effectively complement E. coli ihf mutants, suggesting that the P. putida IHF subunits can form functional heterodimers with the IHF subunits of E. coli. Analysis of the amino acid differences between the E. coli and P. putida protein sequences suggests that in the evolution of IHF, amino acid changes were mainly restricted to the N-terminal domains and to the extreme C termini. These changes do not interfere with dimer formation or with DNA recognition. We constructed a P. putida mutant strain carrying an ihfA gene knockout and demonstrated that IHF is essential for the expression of the P(U) promoter of the xyl operon of the upper pathway of toluene degradation. It was further shown that the ihfA P. putida mutant strain carrying the TOL plasmid was defective in the degradation of the aromatic model compound benzyl alcohol, proving the unique role of IHF in xyl operon promoter regulation.

Amino Acid Sequence↗

PubMed1995

Escherichia coli FtsH is a membrane-bound, ATP-dependent protease which degrades the heat-shock transcription factor sigma 32.

Escherichia coli FtsH is an essential integral membrane protein that has an AAA-type ATPase domain at its C-terminal cytoplasmic part, which is homologous to at least three ATPase subunits of the eukaryotic 26S proteasome. We report here that FtsH is involved in degradation of the heat-shock transcription factor sigma 32, a key element in the regulation of the E. coli heat-shock response. In the temperature-sensitive ftsH1 mutant, the amount of sigma 32 at a non-permissive temperature was higher than in the wild-type under certain conditions due to a reduced rate of degradation. In an in vitro system with purified components, FtsH catalyzed ATP-dependent degradation of biologically active histidine-tagged sigma 32. FtsH has a zinc-binding motif similar to the active site of zinc-metalloproteases. Protease activity of FtsH for histidine-tagged sigma 32 was stimulated by Zn2+ and strongly inhibited by the heavy metal chelating agent o-phenanthroline. We conclude that FtsH is a novel membrane-bound, ATP-dependent metalloprotease with activity for sigma 32. These findings indicate a new mechanism of gene regulation in E. coli.

ATP-Dependent Proteases↗

PubMed1995

Cloning and primary sequence of the rpoH gene from Pseudomonas aeruginosa.

A DNA fragment from Pseudomonas aeruginosa containing the rpoH gene encoding the heat-shock sigma factor sigma 32 has been cloned and sequenced. The gene is expressed in Escherichia coli and complements an rpoH- strain. An open reading frame encoding 284 amino acids shows 61% identity and 78% similarity to the RpoH protein of Escherichia coli.

Amino Acid Sequence↗

PubMed1995

Enhanced activity of the bacteriophage lambda PL promoter at low temperature.

The response of the early phage lambda PL promoter to temperature was investigated. Experiments with lacZ reporter gene fusions demonstrated that the activity of the phage lambda PL promoter is inversely dependent on temperature. The bacterial DNA-binding protein integration host factor (IHF) further enhances lambda PL promoter activity at low temperature, although no apparent changes in the cellular level of IHF protein were observed at the different temperatures. IHF protein binds DNA in vitro more avidly at low temperatures. In vitro transcription assays further revealed that the temperature response of PL is the result of an intrinsic property of the promoter as well as its activation by IHF.

Bacterial Proteins↗

PubMed1995

Enhanced activity of the bacteriophage lambda PL promoter at low temperature.

We found that the activity of the phage lambda PL promoter is inversely dependent on temperature. Both in vivo and in vitro transcription assays revealed that the rather unique temperature response of PL is a sum of an intrinsic property of the promoter and its activation by integration host factor. We also found that at low temperature, phage lambda can lysogenize efficiently but cannot complete the lytic cycle. We hypothesize that by sensing the low environmental temperature the PL promoter plays a role in determining the direction of phage lambda development.

Bacterial Proteins↗

PubMed1995

Multidrug resistance in Candida albicans: disruption of the BENr gene.

The BENr gene of Candida albicans, which confers resistance on susceptible strains of Saccharomyces cerevisiae to six structurally and functionally unrelated drugs, was described recently (R. Ben-Yaacov, S. Knoller, G. Caldwell, J. M. Becker, and Y. Koltin, Antimicrob. Agents Chemother. 38:648-652, 1994). This gene bears similarity to membrane proteins encoding antibiotic resistance in prokaryotes and eukaryotes. The effect of disruption of this gene on viability and drug susceptibility was determined. The results indicate that the gene is not essential but its inactivation leads to susceptibility to three of the four drugs tested. Inactivation of this gene did not increase the susceptibility of the mutant to benomyl, suggesting that C. albicans has other mechanisms of resistance, some of which may be additional efflux pumps that confer resistance to this tubulin-destabilizing agent.

Benomyl↗

PubMed1995

Involvement of IHF protein in expression of the Ps promoter of the Pseudomonas putida TOL plasmid.

Regulation of the xyl gene operons of the Pseudomonas putida TOL plasmid is mediated by the products of the downstream clustered and divergently oriented xylR and xylS regulatory genes. The xylR-xylS intergenic region contains the xylR and xylS promoters Pr and Ps, respectively. A binding site for the XylR activator protein is located upstream of Ps and overlapping Pr. DNase I footprint experiments showed that one of these sites, which overlaps the recognition site for XylR activator, as well as an AT-rich region comprising the Ps promoter consensus were protected by integration host factor (IHF). IHF was found to act negatively in the in vivo activation of the Ps promoter, since the activity of a Ps promoter::lacZ fusion was elevated in an Escherichia coli mutant lacking IHF. In contrast, no alteration in the synthesis of XylR protein in the E. coli IHF-deficient mutant was detected.

Bacterial Proteins↗

PubMed1995

Cloning and primary structure of the chiA gene from Aeromonas caviae.

The chiA gene from Aeromonas caviae encodes an extracellular chitinase, 865 amino acids long, that shows a high degree of similarity to chitinase A of Serratia marcescens. Expression in Escherichia coli yielded an enzymatically active protein from which a leader sequence was removed, presumably during transport of the enzyme across the cell membrane.

Aeromonas↗

PubMed1994

Crystal structure of a bacterial chitinase at 2.3 A resolution.

BACKGROUND: Chitinases cleave the beta-1-4-glycosidic bond between the N-acetyl-D-glucosamine units of which chitin is comprised. Chitinases are present in plants, bacteria and fungi, but whereas structures are available for two prototypic plant enzymes, no structure is available for a bacterial or fungal chitinase. RESULTS: To redress this imbalance, the structure of native chitinase A from Serratia marcescens has been solved by multiple isomorphous replacement and refined at 2.3 A resolution, resulting in a crystallographic R-factor of 16.2%. The enzyme comprises three domains: an all beta-strand amino-terminal domain, a catalytic alpha/beta-barrel domain, and a small alpha+beta-fold domain. There are several residues with unusual geometries in the structure. Structure determination of chitinase A in complex with N,N',N",N"'-tetra-acetylo-chitotetraose, together with biochemical and sequence analysis data, enabled the positions of the active-site and catalytic residues to be proposed. CONCLUSIONS: The reaction mechanism seems to be similar to that of lysozyme and most other glycosylhydrolases, i.e. general acid-base catalysis. The role of the amino-terminal domain could not be identified, but it has similarities to the fibronectin III domain. This domain may possibly facilitate the interaction of chitinase A with chitin.

Amino Acid Sequence↗

PubMed1994

The chitinase encoding Tn7-based chiA gene endows Pseudomonas fluorescens with the capacity to control plant pathogens in soil.

A transposon system based on Tn7 carrying the chiA gene (encoding chitinase) of Serratia marcescens was established. The transposon was introduced by conjugation into Pseudomonas fluorescens as a stable integrate expressing and secreting the active chitinase enzyme. The resulting strain was shown to control the plant pathogen Rhizoctonia solani in soil.

Chitinases↗

PubMed1994

Genetic and biochemical analysis of IHF/HU hybrid proteins.

Integration host factor (IHF) is a small heterodimeric DNA-binding protein of E coli composed of two subunits, alpha and beta, encoded by the himA and hip genes, respectively. IHF binds to DNA at a consensus sequence and bends DNA. HU protein, encoded by the hupA and hupB genes, is similar to IHF except that it does not bind to a specific DNA sequence. To investigate the protein determinants for IHF specificity we exchanged progressively longer segments from the C-terminus of Hip with those of HupA, and followed the activity in vivo and in vitro of four such IHF/HU hybrids. Replacement of 11 residues from the C-terminal alpha helix of Hip by the complementary eight residues of HupA (hybrid 1), had only minor effects on the DNA binding activity of the protein. As progressively longer segments of Hip were replaced by HupA, a precipitous decrease in IHF activity was observed. The hybrid with the longest substitution, hybrid 4, was totally inactive in vivo and could not be purified. None of the hybrid proteins could complement HU activity. Comparing the activities of hybrid 1, hybrid 2 and IHF point mutants, led us to conclude that the structural integrity of the C-terminal alpha helix and its spatial position, but not its amino acid sequence, are important for DNA binding specificity. We favor the hypothesis that alpha helices 3 of both IHF subunits interact with the body of IHF so as to anchor the arms. This interaction stabilizes the arms to permit DNA binding specificity. Thus the C-termini of IHF influence, in an indirect way, the recognition of specific sites on DNA.

Amino Acid Sequence↗

PubMed1994

Expression of the genes coding for the Escherichia coli integration host factor are controlled by growth phase, rpoS, ppGpp and by autoregulation.

Transcriptional control of the himA and the himD/hip genes coding for the two subunits of the integration host factor (IHF) was investigated. The promoters for the two genes were identified by the use of primer extension and S1 analysis. Expression from both promoters was found to increase as the cells enter stationary phase. Mutation in rpoS, known to be induced upon entry to stationary phase, dramatically reduced the growth-phase response of the himA P4 promoter but had only a small effect on the induction of the himD/hip promoter. The increased activity of both promoters required the presence of the relA and spoT genes, suggesting that ppGpp plays a major role in the response to stationary phase. An artificial increase in ppGpp in exponentially growing cells induced a rapid increase in himA P4 and himD/hip mRNA levels. Experiments with a mutant defective in rpoS showed that the response of the himA P4 promoter to high ppGpp levels was greatly reduced while that of himD/hip was only slightly affected. Therefore, it seems that different mechanisms involving RpoS and ppGpp regulate the growth-phase response of the two promoters. We propose that the effect of ppGpp on himA P4 is mediated via RpoS whereas the himD/hip promoter is affected by ppGpp independently of RpoS. Expression of the himD/hip and himA genes was found to be subject to negative autoregulation. IHF-binding sites, implicated in autoregulation, were found to overlap both the himD/hip and himA P4 promoters. An additional IHF-binding site was found upstream of the himD/hip promoter. All three sites show low binding affinity to IHF suggesting that autoregulation can take place only after sufficiently high levels of IHF accumulate in the cell.

Bacterial Proteins↗

PubMed1993

Genetic and biochemical analysis of the integration host factor of Escherichia coli.

Integration host factor (IHF) is a small, heterodimeric DNA-binding protein of Escherichia coli composed of two subunits, alpha and beta, encoded by the himA and hip genes, respectively. IHF binds to the minor groove at a consensus sequence and bends DNA. We mutagenized the hip gene and studied the activity of the mutant IHF proteins in vivo and in vitro. Substitutions at the C-terminal alpha-helix (alpha-helix 3) reduced IHF activity and relaxed the specificity to DNA without abolishing the ability of IHF to bend DNA. These results indicate that the C-terminal region of Hip participates in determining IHF specificity. Alanine substitutions in beta-strands 2 and 3 generally had no effect on IHF activity in vivo suggesting that individually, many of these residues make only small contributions to the binding of IHF to DNA. Replacing the single amino acid of Hip that differs from HU in a highly conserved region of the arm did not affect IHF activity. This finding led us to conclude that this region of Hip does not contribute to specific DNA recognition by IHF. The binding of IHF to DNA is probably not restricted to one domain, but requires the co-operative participation of a number of regions of the protein.

Bacterial Proteins↗