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D E Taylor

Publications and source records attributed to D E Taylor.

At least 109 records · Page 6Linked to original sources
PubMed1994

In vivo complementation and site-specific mutagenesis of the tellurite resistance determinant kilAtelAB from IncP alpha plasmid RK2Ter.

The IncP alpha plasmid RK2 carries a cryptic tellurite resistance (Ter) determinant. This determinant from RK2Ter has been previously cloned into a pUC8 plasmid (pDT1558). The Ter determinant identified as the kilA locus comprises an operon of three genes: kilA, telA and telB [also referred to as klaA, klaB and klaC on RK2(Tes)]. Each of the genes was subcloned into the expression vector pJF118EH behind an inducible tac-promotor using PCR. The PCR primers were used to engineer an efficient ribosome-binding site and adjacent sequence to improve protein expression. Expression plasmids were modified by inclusion of different resistance markers for selection during complementation. The tellurite-resistance phenotype was studied with the overexpressing plasmids. The study provides further evidence that all three genes within the kilAtelAB operon are required for cells to show resistance to potassium tellurite. Additionally, site-directed mutagenesis was carried out on the two cysteine residues in TelB. Changing either Cys125 or Cys132 to a Ser or Ala residue decreased the resistance mediated by the operon. These mutants demonstrate the requirement of cysteine residues within TelB for expression of tellurite resistance.

Amino Acid Sequence↗
PubMed1994

Molecular analysis of RepHI1A, a minimal replicon of the IncHI1 plasmid R27.

R27, a large conjugative plasmid of the HI incompatibility group, was subjected to a subcloning analysis which revealed the presence of a Poll-independent replicon and determinants contributing to incompatibility within a 2.7 kb SalI/XbaI fragment. The DNA sequence of the minimal replicon revealed the presence of a large open reading frame (ORF) as well two sets of 19 bp repeated oligonucleotides (iterons), in addition to characteristic Escherichia coli origin elements. The protein encoded by the ORF possesses homology with replication initiator proteins encoded by a number of plasmids from different incompatibility groups. Deletion analysis suggested that the iterons are responsible for incompatibility reactions. Dissection of the replicon confirmed this and defined a minimal origin of 230 bp. The putative replication initiator was expressed in an in vitro transcription-translation system, and the 5' end of the mRNA encoding its synthesis was identified. Transcriptional fusion of the repA promoter to lacZ demonstrated an autoregulatory function of RepA. A series of iterons present downstream of the RepA coding sequence are dispensable but are responsible for copy-number control. The minimal replicon appears to be partition-defective.

Amino Acid Sequence↗
PubMed1994

Molecular characterization of a conserved 20-kilodalton membrane-associated lipoprotein antigen of Helicobacter pylori.

Antisera raised in rabbits to whole cells of Helicobacter pylori recognized as a major antigen a protein with an apparent molecular weight of 20,000. The antigen was purified by differential solubilization with N-octyl-beta-D-glucopyranoside, urea, and sodium dodecyl sulfate followed by molecular sieving. The mass of the protein, Lpp20, was 18,283 Da as determined by mass spectrometry. The lpp20 gene encoding this protein was cloned in Escherichia coli by using the vector lambda EMBL3, and plasmid subclones expressed the full-length protein from the native H. pylori promoter. lpp20 was mapped to the same 358-kb NruI fragment as flaB. DNA sequence analysis showed that the gene was 525 bp long and encoded a 175-amino-acid protein with a molecular weight of 19,094 containing a 21-residue typical lipoprotein signal peptide and consensus prolipoprotein processing site. The mass of the deduced 154-residue mature protein was 16,865 Da. Growth of E. coli cells expressing the cloned H. pylori lpp20 gene in the presence of [3H]palmitic acid resulted in radiolabelled Lpp20 while treatment of the E. coli cells with globomycin caused accumulation of unprocessed Lpp20, consistent with Lpp20 being a lipoprotein. Lpp20 cofractionated with the cytoplasmic membrane fraction, although a proportion of the protein was also found in the outer membrane. A mutant generated by mutant-allele exchange displayed normal viability, showing that Lpp20 belonged to the nonessential class of lipoproteins.

Amino Acid Sequence↗
PubMed1994

Genetic and nucleotide sequence analysis of the gene htdA, which regulates conjugal transfer of IncHI plasmids.

IncHI plasmids are naturally repressed for conjugative transfer and do not allow efficient propagation of the IncH pilus-specific phage Hgal. Transposons Tn7, Tn5, and TnlacZ were inserted into IncHI plasmids R478, R477-1, and R27, respectively, leading to the isolation of several plasmid mutants which exhibited increased levels of transfer and also permitted good lysis with phage Hgal. A 4.3-kb HindIII fragment from R478 reversed both phenotypic effects of derepression for the R477-1::Tn5 and the R478::Tn7 derivatives, pKFW99 and pKFW100, respectively. Exonuclease III deletions of this fragment and nucleotide sequence analysis indicated that the gene responsible for transfer repression, named here htdA, encoded a polypeptide of 150 amino acids. Cloning and sequence analysis of pDT2454 (R27::TnlacZ) revealed that the transposon had inserted into an open reading frame (ORF) which had an 83% amino acid identity with the R478 htdA gene. Maxicell analysis showed both the R27 and R478 HtdA products had molecular masses of 19.9 kDa. Conjugation experiments showed that the cloned htdA determinants caused a significant reduction of the transfer frequencies of wild-type R478 and R27 plasmids. Examination of both R478 derepressed mutants, pKFW100 and pKFW101, indicated that both transposon insertions occurred upstream of the htdA ORF. The results suggest that HtdA is a regulatory component of IncH plasmid transfer and also show that the region upstream of the htdA ORF is involved in transfer repression. The locations of the htdA determinants were identified on the plasmid maps of R27 and R478.

Amino Acid Sequence↗
PubMed1994

Campylobacter fetus diarrhea in a Hutterite colony: epidemiological observations and typing of the causative organism.

Following a case of Campylobacter fetus sepsis and meningitis in a 4-month-old female member of a Hutterite colony, an epidemiological investigation revealed at least 18 cases of diarrhea in other members of the colony. C. fetus was isolated from 7 of 15 fecal samples submitted from affected persons. A case control study suggested that persons who worked in the abattoir were 2.03 times more likely to have had diarrhea, but none of the risk factors studied were significant. The epicurve of the outbreak was inconclusive as to the likely mode of spread of C. fetus. All of the C. fetus strains isolated from the blood of the infant and from the fecal samples were the same by biochemical and antibiotic susceptibility tests. Pulsed-field gel electrophoresis showed that all isolates produced identical restriction endonuclease patterns and differed from other nonepidemiologically related strains of C. fetus.

Adult↗
PubMed1993

Transformation of Helicobacter pylori by chromosomal metronidazole resistance and by a plasmid with a selectable chloramphenicol resistance marker.

Most strains of Helicobacter pylori are naturally competent for uptake of chromosomal DNA. Transformation frequencies for streptomycin resistance or rifampicin resistance markers ranged from 1 x 10(-4) to 1 x 10(-3) per viable cell using a plate transformation procedure. Transformation of a metronidazole resistance marker (MtrR) was demonstrated when either a laboratory-derived mutant or a MtrR clinical isolate were used as the source of donor DNA. MtrR was transformed at a frequency of 3 x 10(-5) per viable cell. All H. pylori strains tested produce large amounts of DNAase, which may reduce DNA available for transformation. Four H. pylori plasmids were isolated. DNA fragments from H. pylori plasmids were deleted or rearranged when cloned in pUC19 and propagated in Escherichia coli DH5 alpha. An H. pylori plasmid, pUOA26 which contained a chloramphenicol resistance determinant from Campylobacter coli, was constructed in H. pylori. This plasmid could be successfully introduced by natural transformation only into H. pylori recipients which contained a homologous resident plasmid. Transformation of pUOA26 into plasmid-free cells of H. pylori was achieved by electroporation. Transformation frequencies were 1 x 10(-4) transformants per viable cell when plasmid DNA was isolated from the same strain; however, introduction of pUOA26 DNA derived from H. pylori 8091 into a different H. pylori strain, NCTC 11639, resulted in transformation at much lower frequencies (< or = 1 x 10(-7) per viable cell).(ABSTRACT TRUNCATED AT 250 WORDS)

Binding, Competitive↗
PubMed1993

Effect of mutational alteration of Asn-128 in the putative GTP-binding domain of tetracycline resistance determinant Tet(O) from Campylobacter jejuni.

The deduced amino acid sequence of Campylobacter jejuni Tet(O), cloned in Escherichia coli, has shown that it contains the five highly conserved sequences of the GTP-binding domain found in other GTPases. Asn-128 belongs to the G4 motif of such a domain and is involved in hydrogen bonding with the guanine ring of the nucleotide. Substitution of Asn-128 by 11 other amino acids resulted in a decrease in tetracycline resistance, indicating that tetracycline resistance conferred by Tet(O) is related to GTP binding. The effect of the mutations on the GTP-binding domain is discussed with the EF-Tu-GDP complex as a model.

Amino Acid Sequence↗
PubMed1993

Cloning and nucleotide sequence of the Campylobacter jejuni gyrA gene and characterization of quinolone resistance mutations.

The gyrA gene of Campylobacter jejuni UA580, which encodes the A subunit of DNA gyrase, was cloned and its nucleotide sequence was determined. An open reading frame of 2,589 nucleotides was identified, which could code for a polypeptide of 863 amino acids with a M(r) of 97 kDa. Both the nucleotide sequence and the putative amino acid sequence show ca. 50% identity with those of other gyrA genes from gram-positive and gram-negative bacteria. The locations of the gyrA gene on genome maps of both C. jejuni UA580 and Campylobacter coli UA417 were determined. Six nalidixic acid-resistant isolates of C. jejuni were shown to carry mutations in gyrA. Three clinical isolates had Thr-86-to-Ile substitutions. Three laboratory mutants had substitutions of Thr-86 to Ile, Asp-90 to Ala, and Ala-70 to Thr, respectively. The mutation at Thr-86, which is homologous to Ser-83 in Escherichia coli, was associated with high-level resistance to ciprofloxacin in C. jejuni.

4-Quinolones↗
PubMed1993

H-pilus assembly kinetics determined by electron microscopy.

The kinetics of pilus outgrowth were examined for Escherichia coli containing pDT1942, a TnlacZ insertion derivative of the IncHI1 plasmid R27, which was derepressed for transfer. IncHI1 plasmids are thermosensitive for transfer. The pili specified by pDT1942 were examined by transmission electron microscopy after the pilus had been labeled with the H-pilus-specific bacteriophage Hgal, which had been inactivated with RNase A. H pili were extended by extrusion from the cell surface and not by the addition of pilin subunits to the pilus tip. After pili were removed by vortexing, the outgrowth of full-length pili (2 microns long) required 20 min. H pili expressed at the transfer optimal temperature (27 degrees C) remained stable after incubation at the transfer inhibitory temperature (37 degrees C), but the formation of mating aggregates was inhibited at 37 degrees C. Within 1 min of exposure of the host cell to a heat stimulus of 50 degrees C, pili vanished. Pili were observed in straight and flexible forms with a field emission scanning electron microscope, which may indicate a dynamic role for the pilus in conjugation.

Escherichia coli↗
PubMed1993

Value of whole-cell antigen extracts for serologic detection of Helicobacter pylori.

Whole-cell protein extracts of Helicobacter pylori strains were evaluated by enzyme-linked immunosorbent assay to detect immunoglobulin G antibody against H. pylori in 113 patients with upper gastrointestinal complaints. These antigen preparations were of value for detecting infection by H. pylori in patients with high antibody titers (> or = 12,800), whereas for patients with lower titers, the results were inconclusive.

Adolescent↗
PubMed1993

Host range and transfer efficiency of incompatibility group HI plasmids.

HI plasmids are distinguished by their thermosensitive mode of conjugation (transfer efficiency is optimal at 22-30 degrees C) and their capacity to encode multiple antibiotic resistance. These traits have implicated HI plasmids as potential vectors in the dissemination of antibiotic resistance among pathogenic and indigenous bacterial species in water and soil environments. We compared the transfer efficiency of HI plasmids with that of plasmids from 13 other incompatibility groups at 37, 24, and 14 degrees C in intragenic conjugations between laboratory strains of Escherichia coli K-12 under in vitro conditions. Only the HI plasmids and a representative plasmid from incompatibility groups M, N, P alpha, T, and W were observed to be transmissible at 14 degrees C. These plasmids, along with HI plasmids and the related HII representative, were tested for their host range and transfer proficiency to Enterobacteria species and some other Gram-negative organisms of environmental significance at 24 and 14 degrees C. Notable differences in the host range of HI plasmids compared with plasmid representatives from the other enterobacterial groups were not evident at 24 degrees C. At 14 degrees C, R478 (HI2) displayed the broadest host range and transfer proficiency among the test plasmids. The ability of several plasmid groups, including HI, to transfer at 14-24 degrees C to Vibrio cholerae non 01, Salmonella typhi, and the fish pathogens Aeromonas salmonicida, Vibrio anguillarum, and Yersinia ruckeri needs to be corroborated by in situ studies.

Conjugation, Genetic↗
PubMed1993

Current concepts in the treatment of common compartment syndromes in athletes.

Compartment syndromes of the leg, and to a lesser extent the forearm and other areas, occur in both acute and chronic exertional forms. Similar inciting events may precipitate either form, with different presentations, prognoses and implications for treatment. A complete knowledge of the anatomy and pathophysiology of these syndromes is essential for diagnosis. Measurement of compartment pressures is a valuable tool in the diagnosis of chronic exertional compartment syndrome. In our experience, athletes who desire to return to the same level of exercise will require fascial release, although symptoms may improve if the intensity or duration of the activity is decreased. Fascial release is the treatment of choice in acute compartment syndrome and in chronic exertional compartment syndrome unresponsive to nonoperative treatment.

Athletic Injuries↗
PubMed1992

Attitudes to specialist recertification: results of a national survey among pathologists.

AIMS: to evaluate attitudes and preferred mechanisms in the establishment of specialist recertification for pathologists. METHODS: a national survey was conducted amongst the membership of the New Zealand Society of Pathologists. RESULTS: responses were received from 54 (62%) financial members of the society. The majority of respondents agreed with the concept of compulsory periodic recertification. The ideal recertification period should be 10 years and the system should be supervised by the New Zealand Society of Pathologists. The favoured recertification mechanism was by continuing medical education which should be funded by the individual candidate. The majority of respondents supported the establishment of voluntary recertification on a trial basis. CONCLUSIONS: the survey suggested a favourable attribute amongst pathologists to the introduction of continuing education based periodic recertification.

Attitude of Health Personnel↗
PubMed1992

Use of diethyldithiocarbamate for quantitative determination of tellurite uptake by bacteria.

We have developed a simple method for the quantitative determination of tellurite in biological media. This assay is suitable for studying tellurite uptake in bacteria and overcomes the problems of older techniques which are time consuming and labor intensive. In earlier protocols diethyldithiocarbamate was reacted with tellurite and the resulting complex was extracted into organic solvents before spectrophotometric determination. In this study, diethyldithiocarbamate was incubated with tellurite at neutral pH to form a yellow colloidal solution. The absorbance of the aqueous yellow sol was used to determine tellurite concentrations in the range of 1 to 50 micrograms/ml (4 to 200 microM) without the need for solvent extraction.

Arsenic↗