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W Hendrickson

Publications and source records attributed to W Hendrickson.

At least 19 recordsLinked to original sources
PubMed2001

FlhD/FlhC-regulated promoters analyzed by gene array and lacZ gene fusions.

The Escherichia coli transcriptional regulatory complex FlhD/FlhC, initially identified as a flagella-specific activator, is a global regulator involved in many cellular processes. Using gene arrays, lacZ gene fusions and enzyme assays, eight new targets of FlhD/FlhC were recognized. These are the transporter for galactose (MglBAC), the rod-shape determination proteins (MreBCD), malate dehydrogenase, and several enzymes involved in anaerobic respiration (glycerol 3-phosphate dehydrogenase, GlpABC; periplasmic nitrate reductase, NapFAGHBC; nitrite reductase, NrfABCDEFG; dimethyl sulfoxide reductase, DmsABC; and the modulator for hydrogenases, HydNHypF).

Bacterial Proteins↗
PubMed2000

Clinical and environmental isolates of Burkholderia cepacia exhibit differential cytotoxicity towards macrophages and mast cells.

Burkholderia cepacia is an emerging opportunistic pathogen that causes fatal infections in patients suffering from cystic fibrosis (CF) and chronic granulomatous disease. Various environmental isolates of B. cepacia are, however, capable of degrading environmental pollutants, such as trichloroethylene, 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), etc., and are also highly effective in controlling plant diseases caused by nematodes and fungi. Such strains have therefore been proposed for environmental release to clean up toxic dump sites or as biopesticides. Various efforts to distinguish between clinical and environmental isolates of B. cepacia with regard to their virulence characteristics have produced ambiguous results, suggesting that newer methods are needed to test for the presence or absence of pathogenic potential in B. cepacia strains proposed for environmental release. We now report that several clinical strains of B. cepacia secrete cytotoxic factors that allow macrophage and mast cell death in the presence of external ATP. Several environmental strains had reduced activity in this regard. We also demonstrate that, while all the strains secrete enzymes that have nucleoside diphosphate kinase (Ndk), adenylate kinase (Ak) and 5'-nucleotidase activity, the level of secretion of the 5'-nucleotidase (and/or ATPase/phosphatase) appears to be lower in the environmental strains than in the clinical strains. The secretion of these enzymes is specifically activated in the presence of eukaryotic proteins such as alpha2-macroglobulin. As macrophage-or mast cell surface-associated P2Z receptors promote their cell death in the presence of mM concentrations of ATP, and as the secreted ATP-using enzymes generate various phosphorylated or non-phosphorylated adenine nucleotides that may even be better agonists than ATP in activating the P2Z receptors or may act through the activation of additional purinergic receptors, such enzymes may play an important role in allowing B. cepacia to evade host defence.

Adenosine Diphosphate↗
PubMed1999

Conformational changes of gp120 in epitopes near the CCR5 binding site are induced by CD4 and a CD4 miniprotein mimetic.

Binding of the T-cell antigen CD4 to human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 has been reported to induce conformational rearrangements in the envelope complex that facilitate recognition of the CCR5 coreceptor and consequent viral entry into cells. To better understand the mechanism of virus docking and cell fusion, we developed a three-component gp120-CD4-17b optical biosensor assay to visualize the CD4-induced conformational change of gp120 as seen through envelope binding to a neutralizing human antibody, 17b, which binds to epitopes overlapping the CCR5 binding site. The 17b Fab fragment was immobilized on a dextran sensor surface, and kinetics of gp120 binding were evaluated by both global and linear transformation analyses. Adding soluble CD4 (sCD4) increased the association rate of full-length JR-FL gp120 by 25-fold. This change is consistent with greater exposure of the 17b binding epitope on gp120 when CD4 is bound and correlates with CD4-induced conformational changes in gp120 leading to higher affinity binding to coreceptor. A smaller enhancement of 17b binding by sCD4 was observed with a mutant of gp120, DeltaJR-FL protein, which lacks V1 and V2 variable loops and N- and C-termini. Biosensor results for JR-FL and DeltaJR-FL argue that CD4-induced conformational changes in the equilibrium state of gp120 lead both to movement of V1/V2 loops and to conformational rearrangement in the gp120 core structure and that both of these lead to greater exposure of the coreceptor-binding epitope in gp120. A 17b binding enhancement effect on JR-FL also was observed with a 32-amino acid charybdotoxin miniprotein construct that contains an epitope predicted to mimic the Phe 43/Arg 59 region of CD4 and that competes with CD4 for gp120 binding. Results with this construct argue that CD4-mimicking molecules with surrogate structural elements for the Phe 43/Arg 59 components of CD4 are sufficient to elicit a similar gp120 conformational isomerization as expressed by CD4 itself.

Amino Acid Sequence↗
PubMed1998

Analysis of the interaction of antibodies with a conserved enzymatically deglycosylated core of the HIV type 1 envelope glycoprotein 120.

The binding of a panel of monoclonal antibodies to V1, V2, and V3 loop-deleted HIV-1 gp120 was studied by competition analysis. Most of the previously defined relationships between gp120 epitopes were preserved on the variable loop-deleted protein, although interactions between some epitopes were dependent on the presence of the V1, V2, and V3 loops. Enzymatic deglycosylation of the variable loop-deleted protein only minimally altered the binding of most antibodies examined. Thus, a carbohydrate-deficient, conserved HIV-1 gp120 core can be produced that has a structure closely approximating that of the full-length, correctly folded gp120 monomer.

Amino Acid Sequence↗
PubMed1998

Genes for 2,4,5-trichlorophenoxyacetic acid metabolism in Burkholderia cepacia AC1100: characterization of the tftC and tftD genes and locations of the tft operons on multiple replicons.

Burkholderia cepacia AC1100 uses the chlorinated aromatic compound 2, 4,5-trichlorophenoxyacetic acid (2,4,5-T) as a sole source of carbon and energy. The enzyme which converts the first intermediate in the pathway, 2,4,5-trichlorophenol, to 5-chlorohydroquinone has been purified and consists of two subunits of 58 and 22 kDa, encoded by the tftC and tftD genes (48). A degenerate primer was designed from the N terminus of the 58-kDa polypeptide and used to isolate a clone containing the tftC and tftD genes from a genomic library of AC1100. The derived amino acid sequences of tftC and tftD show significant homology to the two-component monooxygenases HadA of Burkholderia pickettii, HpaBC of Escherichia coli, and HpaAH of Klebsiella pneumonia. Expression of the tftC and tftD genes appeared to be induced when they were grown in the presence of 2,4,5-T, as shown by RNA slot blot and primer extension analyses. Three sets of cloned tft genes were used as probes to explore the genomic organization of the pathway. Pulsed-field gel electrophoresis analyses of whole chromosomes of B. cepacia AC1100 demonstrated that the genome is comprised of five replicons of 4.0, 2.7, 0.53, 0.34, and 0.15 Mbp, designated I to V, respectively. The tft genes are located on the smaller replicons: the tftAB cluster is on replicon IV, tftEFGH is on replicon III, and copies of the tftC and the tftCD operons are found on both replicons III and IV. When cells were grown in the absence of 2,4,5-T, the genes were lost at high frequency by chromosomal deletions and rearrangements to produce 2,4,5-T-negative mutants. In one mutant, the tftA and tftB genes translocated from one replicon to another, with the concomitant loss of tftEFGH and one copy of tftCD.

2,4,5-Trichlorophenoxyacetic Acid↗
PubMed1997

A fusion promoter created by a new insertion sequence, IS1490, activates transcription of 2,4,5-trichlorophenoxyacetic acid catabolic genes in Burkholderia cepacia AC1100.

Transposition and transcriptional activation by insertion sequences in Burkholderia cepacia AC1100 were investigated. Two closely related new elements, IS1413 and IS1490, were identified and characterized. These elements are not highly related to other insertion sequences identified in AC1100 or other B. cepacia isolates. Based on their structures and the sequences of the inverted terminal repeats and the putative transposase protein, the insertion elements (IS elements) are similar to IST2 of Thiobacillus ferrooxidans and several related elements. All the IS elements that have been identified in this strain are found in multiple copies (10 to 40), and they have high-level promoter activity capable of stimulating transcription from a distance up to 500 bp from a target gene. Strain AC1100 was originally isolated after prolonged selection for the ability to utilize the herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) as a sole carbon source. Three IS elements are located near the first gene of the 2,4,5-T catabolic pathway, tftA. IS1490 inserted 110 bp upstream of tftA and created a fusion promoter responsible for constitutive transcription of the gene. Our results confirm the hypothesis that IS elements play a central role in transcription of 2,4,5-T genes and likely have stimulated rapid evolution of the metabolic pathway.

2,4,5-Trichlorophenoxyacetic Acid↗
PubMed1996

Biodegradation of 2,4,5-trichlorophenoxyacetic acid by Burkholderia cepacia strain AC1100: evolutionary insight.

Many microorganisms in nature have evolved new genes which encode catabolic enzymes specific for chlorinated aromatic substrates, allowing them to utilize these compounds as sole sources of carbon and energy. An understanding of the evolutionary mechanisms involved in the acquisition of such genes may facilitate the development of microorganisms with enhanced capabilities of degrading highly chlorinated recalcitrant compounds. A number of studies have been based on microorganisms isolated from the environment which utilize simple chlorinated substrates. In our laboratory, a selective technique was used to isolate microorganisms capable of degrading highly chlorinated compounds, such as 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), as sole sources of carbon and energy. This article summarizes the genetic and biochemical information obtained regarding the pathway of degradation, the mechanism of recruitment of new genes, and the organization of the degradative genes. In addition, we discuss the potential practical application of such microorganisms in the environment.

2,4,5-Trichlorophenoxyacetic Acid↗
PubMed1996

Genomic complexity and plasticity of Burkholderia cepacia.

Burkholderia cepacia has attracted attention because of its extraordinary degradative abilities and its potential as a pathogen for plants and for humans. This bacterium was formerly considered to belong to the genus Pseudomonas in the gamma-subclass of the Proteobacteria, but recently has been assigned to the beta-subclass is based on rrn gene sequence analyses and other key phenotypic characteristics. The B. cepacia genome is comprised of multiple chromosomes and is rich in insertion sequences. These two features may have played a key role in the evolution of novel degradative functions and the unusual adaptability of this bacterium.

Biodegradation, Environmental↗
PubMed1992

AraC protein contacts asymmetric sites in the Escherichia coli araFGH promoter.

AraC protein regulates the transcription of arabinose transport and catabolic operons in Escherichia coli through interaction with specific DNA sequences in the promoter regions of the operons. The interaction of AraC protein with two binding sites in the araFGH promoter was determined and compared to previously studied AraC binding sites in the araBAD promoter. Methylation and ethylation interference assays show that AraC protein binds along one side of the DNA to four adjacent major groove regions at each of the araFG1 and araFG2 sites. Mutations within any of the four regions of araFG1 greatly reduce protein binding in vitro. The promoter function in vivo is also greatly reduced, indicating that all four regions of the binding site are required. The chemical interference and genetic data, combined with the consensus sequence for AraC protein binding to ara promoters, support a binding motif in which two directly repeated units each span two adjacent turns of the DNA helix. The function of the two AraC binding sites was also examined. The proximal araFG1 site is required for promoter activation, whereas the distal araFG2 site has only a slight effect on the promoter activity.

Alkylation↗
PubMed1992

Sequence elements in the Escherichia coli araFGH promoter.

The Escherichia coli araFGH operon codes for proteins involved in the L-arabinose high-affinity transport system. Transcriptional regulation of the operon was studied by creating point mutations and deletions in the control region cloned into a GalK expression vector. The transcription start site was confirmed by RNA sequencing of transcripts. The sequences essential for polymerase function were localized by deletions and point mutations. Surprisingly, only a weak -10 consensus sequence, and no -35 sequence is required. Mutation of a guanosine at position -12 greatly reduced promoter activity, which suggests important polymerase interactions with DNA between the usual -10 and -35 positions. A double mutation toward the consensus in the -10 region was required to create a promoter capable of significant AraC-independent transcription. These results show that the araFGH promoter structure is similar to that of the galP1 promoter and is substantially different from that of the araBAD promoter. The effects of 11 mutations within the DNA region thought to bind the cyclic AMP receptor protein correlate well with the CRP consensus binding sequence and confirm that this region is responsible for cyclic AMP regulation. Deletion of the AraC binding site nearest the promoter, araFG1, eliminates arabinose regulation, whereas deletion of the upstream AraC binding site, araFG2, has only a slight effect on promoter activity.

Arabinose↗
PubMed1992

Alginate synthesis in Pseudomonas aeruginosa: environmental regulation of the algC promoter.

The exopolysaccharide alginate is a major virulence factor of Pseudomonas aeruginosa strains that infect the lungs of cystic fibrosis patients. The synthesis of alginate is almost uniquely associated with the pathogenicity of P. aeruginosa within the environment of the cystic fibrosis lung. The gene algC is one of the essential alginate biosynthetic genes and codes for the enzyme phosphomannomutase. In this report, we present data on the transcriptional regulation of algC expression. The activity of the algC promoter is modulated by the response regulator, AlgR1, a member of the two-component signal transduction protein family, which also regulates other alginate-specific promoters. In both mucoid (alginate-positive) and nonmucoid (alginate-negative) P. aeruginosa strains, transcriptional activation of algC increased with the osmolarity of the culture medium. This osmolarity-induced activation was found to be dependent on AlgR1. AlgR1 was found to interact directly with the algC promoter. Deletion mapping, in conjunction with mobility shift assays, showed that AlgR1 specifically bound with two regions of algC upstream DNA. A fragment spanning nucleotide positions -378 to -73 showed strong specific binding, while a fragment located between positions -73 and +187 interacted relatively weakly with AlgR1. Phosphorylation of the AlgR1 protein resulted in the stimulation of its in vitro ability to bind to the algC promoter region (a fragment spanning nucleotides -378 to -73). Transcription from the algC promoter, which has significant homology with the RNA polymerase sigma-54 (RpoN) recognition sequence, decreased in an rpoN mutant of P. aeruginosa.

Alginates↗
PubMed1991

Environmentally regulated algD promoter is responsive to the cAMP receptor protein in Escherichia coli.

The environmentally activated algD promoter of Pseudomonas aeruginosa has been shown to be influenced by DNA supercoiling. It is believed that protein-induced bending or looping is required for this activation. We studied the role of Escherichia coli cAMP-CRP on algD promoter activation in E. coli and show that a functional CRP is required for this activation. We also demonstrate that the algD promoter is sensitive to glucose repression both in E. coli and P. aeruginosa. Deletion of a putative consensus CRP binding sequence upstream of the algD promoter renders the promoter non-responsive to glucose repression. The involvement of cAMP-CRP complex in the activation of the algD promoter in E. coli has been demonstrated directly through binding of a 255 base pair DNA fragment containing the putative consensus CRP binding sequence. Other fragments, upstream or downstream but without any consensus CRP binding sequence, did not show any binding with CRP. A CRP-like analogue, similar to that in Xanthomonas campestris, but capable of activating genes without forming a complex with cAMP, is believed to allow glucose repression in P. aeruginosa.

Bacterial Proteins↗
PubMed1990

Characterization of the Escherichia coli araFGH and araJ promoters.

The identities of two cloned, arabinose-inducible promoters were tested by hybridizing promoter DNA fragments with restriction digests of chromosomal DNA containing Mudlac phage inserted in either araFGH or in araE transport operons. One promoter, thought to be araE, is within 10(3) base-pairs of a Mudlac insertion in the araE gene. The second promoter was not found within several thousand base-pairs of either of the known transport genes. This promoter is now named araPJ (araJ). The DNA sequence of the fragment containing the araFGH promoter was determined. The start site of transcription in vivo was located to within +/- 1 base-pair (bp) by S1 nuclease mapping. DNase 1 footprinting revealed that, in comparison with the araBAD and araE promoters, the locations of the AraC and cyclic AMP receptor protein (CRP) binding sites are reversed with CRP lying between AraC and RNA polymerase. The central location of the CRP binding site may explain why the araFGH promoter is more catabolite sensitive than the other ara promoters. AraC and CRP were both required for maximal transcription in vitro, although a low level of transcription was detected with CRP alone. S1 nuclease mapping of mRNA-DNA hybrids from the araJ promoter located the transcription start point to within #/- 3 bp, and demonstrates that the promoter is dependent upon AraC protein and CRP in vivo. DNase footprinting showed that the location of the AraC protein binding site on araJ is adjacent to the RNA polymerase site, as seen at the araBAD and araE promoters. Two CRP sites were observed; one is upstream from the AraC site and one is downstream from the transcription start site.

Amino Acid Sequence↗
PubMed1986

Transcription of Escherichia coli ara in vitro. The cyclic AMP receptor protein requirement for PBAD induction that depends on the presence and orientation of the araO2 site.

The mechanism by which the cyclic AMP receptor protein, CRP, stimulates transcription of the Escherichia coli araBAD promoter was studied in vitro. Under one set of conditions, CRP stimulated by eightfold the rate of RNA polymerase open complex formation on supercoiled DNA template containing the normal wild-type araBAD regulatory region. Since previous studies in vivo had identified an upstream site termed araO2 that is involved in both repression and in the CRP requirement for PBAD induction, we performed similar experiments in vitro. Deletion of araO2 or alterations of its orientation with respect to the araI site by half integral numbers of turns greatly reduced the CRP requirement for induction of PBAD. Linearizing the DNA has the same effect as deleting araO2 from the supercoiled DNA template. The similarity of conditions that relieve the classical repression of PBAD in vivo and the conditions that eliminate the requirement for CRP for maximal activity in vitro suggest a close relationship between repression in the ara system and the role of CRP. At lower concentrations of AraC protein and slightly different conditions than those used in the above-mentioned experiments, CRP does stimulate transcription from linear or supercoiled templates lacking araO2. On linear DNA under these conditions, one dimer of AraC protein binds to linear araPBAD DNA, but is incapable of stimulating transcription without the additional binding of CRP. The responses of the ara system under the second set of conditions are unlike its behavior in vivo.

AraC Transcription Factor↗
PubMed1985

Altered DNA contacts made by a mutant AraC protein.

Mutant AraC proteins were selected for their ability to induce but not to repress, or their ability to repress but not to induce the araBAD operon. One such unusual mutant is able to bind to the araI site with an affinity only two to three-fold weaker than the wild type AraC protein, but the mutant protein was shown, both in crude extracts and when purified, to contact only two of the three major groove regions of the DNA that are contacted by the wild type protein.

AraC Transcription Factor↗