Prof David Lloyd - PhD, DSc
Biology of the flagellate fish parasite, Spironucleus vortens
In collaboration with , Dr J. Cable, Dr Mike Coogan (Chemistry) and Dr D. Williams (NEEM Biotech).
The control of energy metabolism; oscillations, rhythms and clocks
Studies on the carbon and energy metabolism of colourless algae led to the conclusion that mitochondrial energy generation is as efficient in these organisms as it is in mammalian liver, but in some respects is more versatile. Thus, growth with propionate leads to the induction de novo of a mitochondrial pathway of short chain fatty acid oxidation. In other lower eukaryotes, (protists and yeasts), the development of cyanide-resistant electron transport can provide a functional by-pass of cytochrome c oxidase; we showed that in Acanthamoeba castellanii this mechanism is required for survival in the presence of sulphide, (eg. in water-logged soils). Some unique features of microbial mitochondria were discovered, eg. the anomalous cytochrome a620 of Tetrahymena pyriformis. A variety of other protozoal CO-reacting haemoproteins, previously thought to represent alternative terminal oxidases, were shown not to be functional oxidases, (by high resolution photochemical action spectra obtained using a liquid dye laser). It now seems likely that these pigments are protozoal haemoglobins.
Biogenesis of normal mitochondria in Tetraymena pyriformis was disrupted by growth in the presence of chloramphenicol. As well as becoming respiratory deficient, perturbation of nucleo-cytoplasmic control circuits resulted in the dissociation of coupling between cycles of mitochondrial division and cell division so as to produce large numbers of very small mitochondria per organism. Investigation of this relationship using cultures showing synchronous growth and division led to the discovery of oscillatory energy metabolism in a variety of yeasts and protists. The discovery of in vivo mitochondrial respiratory control introduced a new concept, that of energy generation and utilization as a pair of oscillatory subsystems coupled by the adenine nucleotides. Thus, mitochondrial electron transport and energy production, acts as an enslaved system; its slow dynamics, (cycling between alternating energized and de-energized states, eg. once every 90 minutes in A. castellanii) is determined by the periodic demands of cellular biosynthesis. This model, (periodic turnover), first emphasized the importance of degradative phases of protein turnover, in a rapidly growing organism, an heretical viewpoint at a time when growth control was thought to be entirely mediated by synthetic processes. Recent recognition of the widespread and pervasive importance, both of oscillatory cellular dynamics and of proteolysis, confirm these early discoveries. That these oscillators have temperature-compensated periods led to the exciting observation that they are governed by a cellular clock. Thus, through studying the "epigenetic" oscillations, we had been led to the discovery of a device analogous with the circadian clock responsible for biological timekeeping on a daily basis. The ultradian clock, (so-called because of its higher than daily frequencies), has a period of about an hour. We have now shown that this clock operates in ten different species of lower eukaryotes, (and also that in each, this clock has a characteristic period indicating its genetic basis). There is now growing evidence for the ultradian clock in mammalian cells. Whereas the circadian clock is necessary for synchronization of the organism with its periodic environment, the ultradian clock is concerned with intracellular co-ordination. It has many outputs, but one of its most important functions is in timekeeping for convergent processes required for successful completion of the cell division cycle. The ultradian clock can "gate" cell division to give incremental increases in doubling times, (quantization of cell cycle times), as temperature is reduced. Work with the fission yeast Schizosaccharomyces pombe has recently shown that the ultradian clock modulates the passage of cells from G2 into mitosis at the cdc 2 control network. We have formulated a mathematical model whereby mitotic oscillator and ultradian clock interact and which can explain observed dispersion and quantization of cell division times. We have shown that for certain parameter values, the model gives physiologically realistic chaotic (deterministic) solutions. This demonstration helps reconcile the long-standing controversy of the probabilistic versus deterministic nature of cell cycle controls. An extension of these ideas has led to the concept of the organism as a self-tuneable multi-oscillator in which optimization of function is achieved by self-adaptation. A plausible basis for these principles comes from recent demonstrations of the advantages of controlled chaos and consequent emergent properties in a number of different physical and chemical systems.
Since 1998 we have studied continuous aerobic cultures of yeast (Saccharomyces cerevisiae) growing under controlled conditions that lead to spontaneous self-organization to a highly synchronized state (800ml, 10 9 organisms per ml). Continuous non-invasive monitoring of respiration and H2S production (membrane inlet mass spectrometry) and intracellular redox state (NAD(P)H fluorescence) minute by minute for periods of months provides outputs. Discrete time samples are used for high-throughput transcriptomics, proteomics and metabolome analyses. A short period (~40min) ultradian clock provides the timebase for a massively-connected intracellular network that involves not only the reactions of central metabolism but also transcription, biosyntheses, organelle assembly, ribosome assembly, and macromolecular turnover. Timekeeping for the cell division cycle is also based on the ultradian clock. Cell-cell interactions necessary for population synchrony and the coherence of behaviour in biofilms involves H2S and acetaldehyde production, also under the control of the 40 min. clock. Temporal separation of H2S production and oxidation between cytosol and mitochondria echoes the ancestral origin of eukaryotic evolution that involved symbiotic liaison between Archeon and respiratory efficient bacterium.
The operation of a tuneable chaotic attractor has been observed directly in this system by three independent experimental approaches. Thus (i) period doubling in the respiratory rhythm results from perturbation with an A-type monamine oxidase inhibitor (phenelezine), (ii) stepwise lowering of culture pH, (iii)continuous monitoring of dissolved O2, CO2 and H2S in the culture over a period of 3 months (40,000 data points). These results provide the first unequivocal experimental demonstration of chaotic control in a microbiological system at a whole cell level.
Microbial physiology and ecology : New insights from mass spectrometric and other physical methods
The other major thrust of my research has been to introduce microbiologists to the advantages provided by non-invasive physical methods for studies of microbial physiology in laboratory cultures and in the natural environment. I have made extensive use of different physical techniques in conjunction or in combination. Examples are given of far-reaching significance in (a) Biomedical and pharmaceutical, and (b) Agricultural sciences, as well as in (c) Global ecology.
Several different combined methods have been devised; these include spectrophotometry, fluorimetry, bioluminescence, electron spin resonance, or 13C nuclear magnetic resonance, combined with membrane inlet mass spectrometry (MIMS) for the measurement of gases in solution. MIMS itself has proved an extremely powerful tool and has led to several new discoveries: it provides the ideal probe, (sensitive, specific, rapid, multi-species monitoring with continuous readout, etc.,).
Biomedical and pharmaceutical research
Simultaneous measurement of H2, CO2 and O2 in suspensions of the most common human parasite, Trichomonas vaginalis, together with consideration of its natural environment, has redefined this organism as a CO2-requiring microaerophile. ESR measurements of intracellular metronidazole radical anions, have established that metronidazole resistance occurs by radical quenching in strains incompetent at scavenging O2. Drug resistance in another important parasitic protozoan, Giardia lamblia, cannot be explained by this mechanism, as the electron transport chain is located in the plasma membrane and several redox components including the ferredoxin are different from those of trichomonads.
Flow cytometry has enabled studies of bacterial population heterogeneity (eg. the rapid discrimination of antibiotic-sensitive and resistant individuals in bacterial populations by using non-toxic membrane voltage-sensitive dyes, (eg. various oxonols). This demonstration now progresses towards a rapid and generally useful diagnostic test for clinical microbiologists.
Combined simultaneous monitoring of bacterial denitrification products, (N2 and N2O) with O2, has led to the discovery of aerobic denitrification as widespread phenomenon; diversion of this process so as to minimize the production of the greenhouse gas N2O is an important, but neglected topic.
We have used MIMS to monitor gases, (H2, CH4, CO2, O2), dissolved in rumen fluid in situ in fistulated sheep and goats; episodic appearance of low concentrations of O2 during intervals of starvation between feeds has profound effects, (decreased methanogenesis; increased H2). Using purified suspensions of single protozoal species, we have been able to determine the functions of the protozoa, (accounting for about a half of the biomass). Thus, we showed that Dasytricha ruminantium and at least eight other species have hydrogenosomes, organelles with a flavoprotein-iron sulphur electron transport chain specially adapted to produce H2 and to scavenge O2. 13C-nmr demonstrated that individual species have important metabolic differences, and that traces of O2 and high ambient CO2 concentrations control the balances of carbon fluxes. These findings could be related to results with crude rumen fluid and enable the conclusion that the protozoa are responsible for half of the O2 scavenging in the rumen, (approximately equal to the contribution of the rumen bacteria). In all this work, MIMS played a central part; there is as great an interest in reducing methanogenic carbon loss from the rumen ecosystem as of reducing sources of atmospheric methane.
Measurement by MIMS of H2, a key intermediate, (anaerobic interspecies H2 transfer), has enabled automatic control of laboratory-scale anaerobic digestion of farm wastes.
Global ecological research
We are currently investigating control of methanogenesis and fermentation in peatlands. A mass spectrometer microprobe enables detailed determinations of gas profiles, (CH4, CO2, O2), as well as measurement of fluxes into the atmosphere. We have been astonished by the temperature sensitivity of the system, (Q10 values up to 3), and by the discovery that even in the middle of the Scottish winter, and where plant diversity is limited to grasses, sedges and heathers, the most active portion of the ecosystem, (the surface and layers down to 25 cm), is plant- rather than microbe-driven. Photosynthetically-produced root exudates are rapidly utilized by fermentative and methanogenic organisms. This is evident from the diurnal enhancement of fluxes of both CO2 and CH4 in the light and circadian control of CO2 concentrations down to 25 cm depth.
Land-atmosphere exchanges have been studied along a transect from NE Greenland, over Iceland, N. Sweden, N. Finland to W. Siberia. Soil temperature is the best predictor of CH4 emissions on the large scale. The influence of vascular plants is highly species specific. The region is a net source of greenhouse gas emission throughout the year, despite strong mid-summer uptake of CO2. A recent study indicates diurnal cycling of soil gases (O2, CO2, CH4 and N2).
Work supported by EU Funding, NERC, BBSRC, Wellcome, The Royal Society
University of Pennsylvania, 1967; 1969; 1971; 1975; 1978; 1979;
Rockefeller University 1978: Odense University 1977; 1978; 1980; 1981; 1983; 1986; 1990; 1994; 1996;
Soviet Academy of Science 1979: Moscow State University 1979;
INRA, Bordeaux 1984;
ATOMKI, Debrecen 1984;
TATA Institute for Fundamental Research, Bombay 1989; 1991; 1993; 1995; 1997; 1998;
University of Kebansang, Kuala Lumpur 1989;
University of New South Wales, Sydney 1997;
National Institute for Biosciences/Human Technology, Tsukuba Science City 1997; 1998;
INTECH, Buenos Aires 1998; Sabbatical at University of New South Wales 1999;
Johns Hopkins Medical School, Molecular Cardiobiology, Baltimore 2002 to present.
Industrial collaboration :
With more than 50 companies and institutes.
Professor Marc Roussel (on sabbatical leave from Lethbridge, Alberta) used membrane inlet mass spectrometry to reveal the strange attractor that underlies the respiratory dynamics of yeast in continuous culture. This required simultaneous monitoring of dissolved H2S, CO2 and O2; 40,000 points at 15s intervals were acquired in a 3 month continuous culture experiment. This is the first unequivocal demonstration of chaotic control in a biological system at whole cell level.
Dr Katey Lemar established mechanisms for the anti-candidal properties of selected garlic components, diallyldisulphide and allyl alcohol produce apoptotic cell death in this organism without being too toxic to humans. This affords a new way of tackling antibiotic resistance; there are no reported incidents of microorganisms becoming resistant to the garlic compounds.
Photo of David Lloyd and his Associates
Recent Former Associates
Dr Stefanie Scheerer and Dr Francisco Gomez have established a stable continuous culture of Photobacterium fisheri. This can be used as an on-line monitor for toxic compounds (e.g. biocides) in environmental samples including water supplies. Miniaturization will lead to the development of personal protection systems. Effects of microwaves were studied.
Dr. Simon Cottrell showed that freeze dried garlic powder is toxic to MRSA, and that synergistic affects in combination with oxycillin may provide a new chemotherapeutic strategy.
Dr Victoria Gray has shown that the morphology of Salmonella typhimurium and poona species is continually dependent on the tyrosine content of the growth medium. Various sources of the peptone used in the complex diagnostic media may be quite unsuitable on account of their tyrosine deficiency. Aflagellate organisms, unrecognisable as the pathogen, result if the medium is unsuitable.
Dr Kristina Harris investigated the effects of difluoro methylornithine on the growth, structure and function of Trichomonas vaginalis. Defective hydrogenosemes are one consequence of this ornithine decarboxylase inhibitor.
Dr Jonathan Wood investigated the application of garlic as an antibacterial specifically against MRSA. Resolution of effects required separation of the main toxic constituents; synergy with penicillin derivatives was researched.
Dr Coralie Millet investigated protozoal fish parasites Hexamita and Spironucleus spp. Market for viticulture of aquarium fish is $7b/an. Pathogenicity and invasiveness; biodiversity was studied and compared with that of free living species.