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P J Stephens

Publications and source records attributed to P J Stephens.

At least 37 records · Page 2Linked to original sources
PubMed2000

Analysis of bacterial function by multi-colour fluorescence flow cytometry and single cell sorting.

With the increased awareness of the problems associated with the growth dependent analysis of bacterial populations, direct optical detection methods such as flow cytometry have enjoyed increased popularity over the last few years. Among the analyses discussed here are: (1) Bacterial discrimination from other particles on the basis of nucleic acid staining, using sample disaggregation to provide fast reliable enumeration while minimizing data artefacts due to post sampling growth; (2) Determination of basic cell functions such as reproductive ability, metabolic activity and membrane integrity, to characterise the physiological state or degree of viability of bacteria; and (3) The use of single cell sorting onto agar plates, microscope slides or into multi-well plates to correlate viability as determined by cell growth with fluorescent labelling techniques. Simultaneous staining with different fluorochromes provides an extremely powerful way to demonstrate culture heterogeneity, and also to understand the functional differences revealed by each stain in practical applications. Analysis of bacterial fermentations showed a considerable drop (20%) in membrane potential and integrity during the latter stages of small scale (5L), well mixed fed-batch fermentations. These changes, not found in either batch or continuous culture fermentations, are probably due to the severe, steadily increasing stress associated with glucose limitation during the fed-batch process, suggesting 'on-line' flow cytometry could improve process control. Heat injured cells can already show up to 4 log of differences in recovery in different pre-enrichment media, thus contributing to the problem of viable but non-culturable cells (VBNC's). Cytometric cell sorting demonstrated decreasing recovery with increasing loss of membrane function. However, a new medium protecting the cells from intracellular and extracellular causes of oxidative stress improved recovery considerably. Actively respiring cells showed much higher recovery improvement than the other populations, demonstrating for the first time the contribution of oxidative respiration to intracellular causes of damage as a key part of the VBNC problem. Finally, absolute and relative frequencies of one species in a complex population were determined using immunofluorescent labelling in combination with the analysis of cell function. The detail and precision of multiparameter flow cytometric measurements of cell function at the single cell level now raise questions regarding the validity of classical, growth dependent viability assessment methods.

Bacteria↗
PubMed1999

Four deaths due to carbon monoxide poisoning in car washes.

In a period of 13 months, three separate incidents of lethal carbon monoxide (CO) poisoning in closed car wash bays resulted in the deaths of 4 white men aged 20 to 36 years. Each man appears to have been intoxicated with mind-altering substances, which may impair judgment, perception of outside conditions, and self-awareness. All four died in winter months. For three men, the deaths were ruled accidental, and for the remaining man, the previous deaths appear to have provided a model for suicide. Warning signs may not be effective to prevent future CO deaths in car washes because of the possible role of intoxication. Mechanical or electronic methods to prevent a bay door from closing completely may be preferable.

Adult↗
PubMed1998

Direct inoculation into media containing bile salts and antibiotics is unsuitable for the detection of acid/salt stressed Escherichia coli O157:H7.

The efficiency of selective enrichment broths for the recovery of low numbers of acid/salt stressed Escherichia coli O157:H7 was determined. Stressed cultures were diluted to low levels and recovered in tryptone soya broth with added bile salts, to make modified tryptone soya broth, and buffered peptone water with various combinations of antibiotic supplementation including novobiocin, acriflavine and a mixture of vancomycin, cefsulodin and cefixime (VCC) at 37 degrees C and 42 degrees C. Significantly fewer stressed cells, in some cases as little as 0.3% of the starting population, were recovered by all the selective enrichment broths containing bile salts or VCC antibiotics compared to the nonselective controls. The use of such enrichments to recover low numbers of stressed E. coli O157:H7 may result in failure to detect the organism. Parallels with salmonella methodology are made and the need for a non-selective pre-enrichment stage in E. coli O157:H7 methods discussed.

Animals↗
PubMed1998

An improved direct plate method for the enumeration of stressed Escherichia coli O157:H7 from food.

The use of sorbitol MacConkey agar (SMAC) performed poorly in supporting growth of stressed Escherichia coli O157:H7 cells. Up to a 3-log difference was observed between counts on SMAC and tryptone soy agar (TSA). It is critical in the risk assessment of certain foods to be able to enumerate stressed and healthy E. coli O157:H7 in a background of potentially healthy competing bacteria. Investigations carried out to overcome the inhibitory effect of SMAC included the reduction of the selective agent concentration, inclusion of a recovery stage in broth prior to plating out, addition of recovery agents, and delayed exposure to the selective agent. The only successful approach was delayed exposure to the selective agent. This was achieved by resuscitating the stressed cells on a membrane placed on the surface of a TSA plate and, after a defined time period sufficient for full resuscitation, transferring the membrane to the surface of a SMAC plate. The choice of membrane material was critical for maintaining the positive sorbitol color change used to identify wild-type E. coli. Track-etched polycarbonate membranes allowed the typical color reactions to be visualized, whereas cellulose acetate did not. The method was validated with E. coli O157:H7 cells stressed by low pH and high salt conditions, whereby all cells that would previously be undetectable on direct inoculation of SMAC were countable.

Animals↗
PubMed1997

The use of an automated growth analyser to measure recovery times of single heat-injured Salmonella cells.

A new approach to the study of recovery times of single heat-injured Salmonella cells is described. It comprises the generation of a standard heat-injured culture, serial dilution of this culture to near extinction, inoculation of the serial dilutions across many microtitre plates and measurement of the subsequent recovery and growth using an automated turbidometric analyser. Lag times for individual cells were estimated from turbidity data using a model that accurately extrapolated the growth curve back to the starting inoculum level. Lag times were compared using a number of different commercially available pre-enrichment media. The most typical result was a very broad distribution of lag times at the single cell inoculum level, with many values in excess of 20 h. Even at an inoculum level 10-fold higher, lag times for some injured cells were estimated to be > 10 h. More significantly, it was found that some media recovered more injured cells than others and vice versa. Between the worst and best media there were as many as 3 log10 cycles difference in the number of cells recoverable. No trends were apparent linking choice of medium with performance. The implications of these findings, in relation to traditional and rapid methodology, are discussed.

Automation↗
PubMed1996

Effect of pre- and post-heat shock temperature on the persistence of thermotolerance and heat shock-induced proteins in Listeria monocytogenes.

The effect of incubation temperature, before and after a heat shock, on thermotolerance of Listeria monocytogenes at 58 degrees C was investigated. Exposing cells grown at 10 degrees C and 30 degrees C to a heat shock resulted in similar rises in thermotolerance while the increase was significantly higher when cells were grown at 4 degrees C prior to the heat shock. Cells held at 4 degrees C and 10 degrees C after heat shock maintained heat shock-induced thermotolerance for longer than cells held at 30 degrees C. The growth temperature prior to inactivation had negligible effect on the persistence of heat shock-induced thermotolerance. Concurrent with measurements of thermotolerance were measurements of the levels of heat shock-induced proteins. Major proteins showing increased synthesis upon the heat shock had approximate molecular weights of 84, 74, 63, 25 and 19 kDa. There was little correlation between the loss of thermotolerance after the heat shock and the levels of these proteins. Thermotolerance of heat shocked and non-heat shocked cells was described by traditional log-linear kinetics and a model describing a sigmoidal death curve (logistic model). Employing log-linear kinetics resulted in a poor fit to a major part of the data whereas a good fit was achieved by the use of a logistic model.

Bacterial Proteins↗
PubMed1995

Site-directed mutagenesis of Azotobacter vinelandii ferredoxin I: cysteine ligation of the [4Fe-4S] cluster with protein rearrangement is preferred over serine ligation.

The [4Fe-4S] cluster of Azotobacter vinelandii ferredoxin I receives three of its four ligands from a Cys-Xaa-Xaa-Cys-Xaa-Xaa-Cys sequence at positions 39-45 while the fourth ligand, Cys20, is provided by a distal portion of the sequence. Previously we reported that the site-directed mutation of Cys20 to Ala (C20A protein) resulted in the formation of a new [4Fe-4S] cluster that obtained its fourth ligand from Cys24, a free cysteine in the native structure. That ligand exchange required significant protein rearrangement. Here we report the conversion of Cys20 to Ser (C20S protein), which gives the protein the opportunity either to retain the native structure and use the Ser20 O gamma as a ligand or to rearrange and use Cys24. X-ray crystallography demonstrates that the cluster does not use the Ser20 O gamma as a ligand; rather it rearranges to use Cys24. In the C20S protein the [4Fe-4S] cluster has altered stability and redox properties relative to either C20A or the native protein.

Amino Acid Sequence↗
PubMed1994

Mössbauer and EPR studies of Azotobacter vinelandii ferredoxin I.

Azotobacter vinelandii ferredoxin I (FdI) is a small protein that contains one Fe4S4 cluster and one Fe3S4 cluster. Previous studies of FdI have shown that the redox potential of the Fe3S4 cluster and the MCD and CD spectra of the reduced Fe3S4 cluster are pH-dependent. Using Mössbauer and EPR spectroscopy, we have studied FdI in different oxidation states and at different pH values. Here, we report the spin Hamiltonian parameters of the oxidized (S = 1/2) Fe3S4 cluster at pH 7.4 and the reduced (S = 2) Fe3S4 cluster at pH 6.0 and 8.5. The pH dependence observed by MCD is also evident in the Mössbauer spectra which show a change of the magnetic hyperfine tensor for one Fe site of the valence-delocalized pair. The Fe4S4 cluster is ligated by cysteines 20, 39, 42, and 45, but not by the adjacent cysteine 24. Treatment of FdI with 3 equiv of ferricyanide alters the Fe4S4 cluster, yielding a new species, [Fe4S4]'. The S = 1/2 EPR signal of [Fe4S4]' has previously been attributed to the formation of a cysteine disulfide radical from Cys24 and cluster sulfide. Here we show that the EPR signal is broadened by 57Fe, indicating that the electronic spin is significantly coupled to the cluster iron. Consistent with this, substantial magnetic hyperfine interactions are observed by Mössbauer spectroscopy. In addition, the average isomer shift of the four Fe sites is smaller for [Fe4S4]' than for [Fe4S4]2+, indicating that the oxidation is iron-based to at least some extent. Incubation of FdI with excess ferricyanide destroys the Fe4S4 cluster but leaves the Fe3S4 cluster intact. Our studies of (3Fe)FdI show that the S = 1/2 spin of the Fe3S4 cluster interacts with another paramagnet, presumably a radical generated at the site left vacant by the removal of the Fe4S4 cluster.

Azotobacter vinelandii↗
PubMed1994

Calculation of the redox potentials of iron-sulfur proteins: the 2-/3-couple of [Fe4S*4Cys4] clusters in Peptococcus aerogenes ferredoxin, Azotobacter vinelandii ferredoxin I, and Chromatium vinosum high-potential iron protein.

Calculations of the redox potentials of the 2-/3-couples of [Fe4S*4Cys4] clusters in the iron-sulfur proteins Peptococcus aerogenes ferredoxin (PaFd), Azotobacter vinelandii ferredoxin I (AvFdI) and Chromatium vinosum high potential iron protein (CvHiPIP) based on the Protein Dipoles Langevin Dipoles (PDLD) method are reported. The structures of these proteins have been determined by X-ray crystallography; in the case of PaFd the structure has recently been revised due to a change in the sequence close to Cluster II. The large differences between the potentials of the [Fe4S*4Cys4] clusters of PaFd and AvFdI and the potential of the [Fe4S*4Cys4] cluster of CvHiPIP are successfully modeled and originate principally in differences in the configuration of main-chain amide groups near the clusters. The small difference between the potentials of PaFd and AvFdI is also satisfactorily modeled in the case of Cluster I of PaFd. Solvent dipoles close to the cluster in PaFd are an important contributor to its higher potential. The two X-ray structures of PaFd yield similar results for Cluster I of PaFd. In contrast, the results for Cluster II differ substantially; for reasons not yet clear, the recently revised structure leads to results in worse agreement with experiment.

Amino Acid Sequence↗
PubMed1994

Azotobacter vinelandii ferredoxin I. Alteration of individual surface charges and the [4FE-4S]2+/+ cluster reduction potential.

The structures of Azotobacter vinelandii ferredoxin I (AvFdI) and Peptococcus aerogenes ferredoxin (PaFd), near their analogous [4e-4S]2+/+ clusters, are highly conserved (Backes, G., Mino, Y., Loehr, T.M., Meyer, T.E., Cusanovich, M.A., Sweeney, W.V., Adman, E.T., and Sanders-Loehr, J. (1991) J. Am. Chem. Soc. 11, 2055-2064). Despite these similarities, the reduction potential (E0') of the AvFdI [4Fe-4S]2+/+ cluster is more than 200 mV more negative than that of PaFd. We have tested the contribution that individual amino acid residues make to the control of E0' by converting residues in AvFdI into the corresponding residue in PaFd. Four mutations involved substitutions of negatively charged surface residues with neutral residues and two involved substitution of buried hydrophobic residues. All AvFdI variants were characterized by x-ray crystallography, absorption, CD, EPR, and 1H NMR spectroscopies and by electrochemical methods. For the F25I mutation, significant structural changes occurred that affected the EPR and 1H NMR spectroscopic properties of AvFdI and had a minor influence on E0'. For all other mutations there were no changes in reduction potential. Thus we conclude, that variations in charged surface residues do not account for the observed differences in E0' between the analogous [4Fe-4S]2+/+ cluster of PaFd and AvFdI. These differences are therefore most likely to be due to differences in solvent accessibility.

Amino Acid Sequence↗
PubMed1994

Effect of heating rate on the thermal inactivation of Listeria monocytogenes.

In order to quantify the effect of heating rate on the thermal inactivation of Listeria monocytogenes an accurate means of describing the inactivation kinetics at near instantaneous heating was used. Survivor curves for L. monocytogenes, at near instantaneous heating, were obtained over the temperature range 50-64 degrees C. The use of a linear function to describe the data would have given only a poor approximation of the true inactivation kinetics. With a model based on a logistic algorithm extremely accurate descriptions were made. In processes which had rates of heating < or = 5.0 degrees C min-1, significant deviations of real kill from predicted kill were observed. Predicted kill assumed that heating rate did not affect the inactivation kinetics of a thermal process. At rates of heating between 5.0 and 0.7 degrees C min-1 the deviation greatly increased as the rate of heating decreased; approximately a 1.7 x 10(5)-fold difference at 0.7 degrees C min-1. Maximum thermotolerance was induced at rates of heating < or = 0.7 degrees C min-1. The increased thermotolerance during slow rates of heating was analogous to the induction of the heat-shock response. The models described in this work allow for confident assessments of safety to be made not only at near instantaneous heating but also when the heating rate varies.

Colony Count, Microbial↗
PubMed1993

Azotobacter vinelandii ferredoxin I. Aspartate 15 facilitates proton transfer to the reduced [3Fe-4S] cluster.

The [3Fe-4S]+/0 cluster of Azotobacter vinelandii ferredoxin I (AvFdI) has an unusually low and strongly pH-dependent reduction potential (E'0). The reduced cluster exists in two forms, depending upon pH, that exhibit substantially different magnetic circular dichroism (MCD) spectra. Recent studies have established that the MCD changes observed on decreasing the pH from 8.3 (alkaline form) to 6.0 (acid form) cannot be explained either by a change in spin state of the cluster (Stephens, P.J., Jensen, G.M., Devlin, F.J., Morgan, T.V., Stout, C. D., Martin, A.E., and Burgess, B.K. (1991) Biochemistry 30, 3200-3209) or by a major structural change (e.g. ligand exchange) (Stout, C.D. (1993) J. Biol. Chem. 268, 25920-25927). Here, we have examined the influence of aspartate 15 on the pH dependence of the spectroscopic and electrochemical properties of AvFdI by construction of a D15N mutant. Aspartate 15, which is salt-bridged to lysine 84 at the protein surface, is the closest ionizable residue to the [3Fe-4S] cluster. The results show that replacement of aspartate by asparagine results in an approximately 20-mV increase in E'0 for the [3Fe-4S]+/0 cluster at high pH concomitant with an approximately 0.8-pH unit decrease in the pK of the reduced form. The major pH dependence of E'0 is preserved as is the effect observed by MCD. These data eliminate the possibility that the MCD change is due to the presence of Asp-15 and support the conclusion that it originates in direct protonation of the [3Fe-4S]0 cluster, probably on a sulfide ion. Voltammetric studies show that interconversion between [3Fe-4S]+ and [3Fe-4S]0 at acidic pH involves rapid electron transfer followed by proton transfer (for reduction) and then proton transfer followed by electron transfer (for oxidation). Ionized aspartate 15 facilitates proton transfer. Thus, protonation and deprotonation are much slower for D15N relative to the native protein at pH > 5.5. Proton transfer reactions necessary for further reduction of the [3Fe-4S]0 cluster to the [3Fe-4S]- and [3Fe-4S]2- states are also retarded in D15N. The results suggest that the carboxylate-ammonium salt bridge afforded by Asp-15-Lys-84 conducts protons between the cluster and solvent H2O molecules. Overproduction of D15N FdI, but not native FdI, in A. vinelandii has a negative effect on the growth rate of the organism, suggesting that the rate of protonation or deprotonation of the [3Fe-4S]0 cluster may be important in vivo.

Amino Acid Sequence↗
PubMed1993

Reduced ribosomal thermal denaturation in Listeria monocytogenes following osmotic and heat shocks.

Increased thermotolerance of Listeria monocytogenes induced by exposure to a high NaCl concentration or a sublethal heat shock was concurrent with increased thermal stability of the 30S ribosomal subunit as measured by differential scanning calorimetry. It is proposed that protection of the 30S subunit is a critical mechanism for increased thermotolerance.

Calorimetry, Differential Scanning↗
PubMed1992

Protein control of iron-sulfur cluster redox potentials.

The relationship between the three-dimensional structures of iron-sulfur proteins and the redox potentials of their iron-sulfur clusters is of fundamental importance. We report calculations of the redox potentials of the [Fe4S4(S-cys)4]-2/-3 couple in four crystallographically characterized proteins: Azotobacter vinelandii ferredoxin I, Peptococcus aerogenes ferredoxin, Bacillus thermoproteolyticus ferredoxin, and Chromatium vinosum high potential iron protein (HiPIP). Our calculations use the "protein dipoles Langevin dipoles" microscopic electrostatic model, which includes both protein and solvent water. The variations in calculated redox potentials are in excellent agreement with experimental data. In particular, our results confirm the important role of amide groups close to the cluster in separating the potential of C. vinosum HiPIP from those of the other three proteins. However, the potentials of these latter exhibit a substantial range despite extremely similar amide group environments of their clusters. Our results show that the potentials in these proteins are tuned in part by varying the access of solvent water to the neighborhood of the cluster. Our calculations provide the first successful quantitative modeling of the protein control of iron-sulfur cluster redox potentials.

Azotobacter vinelandii↗
PubMed1992

On the structure of the nickel/iron/sulfur center of the carbon monoxide dehydrogenase from Rhodospirillum rubrum: an x-ray absorption spectroscopy study.

The nickel/iron/sulfur center of the carbon monoxide dehydrogenase (carbon monoxide:(acceptor)oxidoreductase; EC 1.2.99.2) enzyme from Rhodospirillum rubrum (Rr-CODH) was studied by x-ray absorption spectroscopy at the Ni K edge. Extended x-ray absorption fine structure data show that the first Ni coordination shell consists of 2 S atoms at 2.23 A and 2-3 N/O atoms at 1.87 A. The edge structure indicates a distorted tetrahedral or five-coordinate Ni environment in both oxidized and reduced Rr-CODH. By comparing second-shell extended x-ray absorption fine structure data of Rr-CODH to that of (Et4N)3[NiFe3S4(SEt)4], a cubane-type cluster, it was clearly established that Ni in the Rr-CODH center is not involved in the core of a NiFe3S4 cubane cluster. One model consistent with the results is a mononuclear Ni2+ site, bridged by S-Cys or sulfide to one or both of the Fe4S4 clusters of the enzyme, with the remaining coordination sites occupied by additional S-Cys or N/O-liganding amino acid residues.

Absorptiometry, Photon↗
PubMed1992

The effects of ethanol on intracellular potassium and the membrane potential of an identified crab motor axon.

1. Observations were made on the fast bender excitor axon in autotomized crab walking limbs bathed in normal crab saline, and in salines made up with 0.2 M sucrose or 0.2 M ethanol. Microelectrode techniques were used to measure the resting membrane potential and the intracellular level of potassium. 2. Sucrose-saline had little effect on the membrane potential or the intracellular level of potassium. Ethanol-saline hyperpolarized the membrane potential by about 3 mV and increased the level of intracellular potassium. 3. The ethanol-induced changes in intracellular potassium levels and membrane potential took place with the same time course. Further, the changes in membrane potential could be accounted for by changes in the equilibrium potential for potassium, Ek as predicted by the Nernst equation.

Action Potentials↗
PubMed1992

The use of Apple Macintosh computers and Hypercard in teaching physiology laboratories.

A network of Apple Macintosh computers has been integrated into existing undergraduate physiology laboratories. Hypercard software programs replace the traditional laboratory handout; the text contains a number of buttons linked to cards that provide additional information on a particular subject. This additional information includes theory, the use of various instruments, expected results, and details on how to perform data analysis. This system of information layering allows students to work independently, at their own pace, and allows them to learn as much or as little as they consider necessary. Each computer is interfaced with a MacLab/4 data-acquisition unit, which permits the computer screen to display a four-channel chart recorder or a two-channel oscilloscope.

Computer Communication Networks↗