From lakes to lungs : assessing microbial activity in diverse environments

All major geochemical cycles on the Earth’s surface are mediated by microorganisms. Our understanding of how these microbes have interacted with their environments (and vice versa) throughout Earth's history, and how they will respond to changes in the future, is primarily based on studying their activity in different environments today. The overarching questions that motivate the research presented in the two parts of this thesis -- how do microorganisms shape their environment (and vice versa)? and how can we best study microbial activity in situ? -- have arisen from the ultimate goal of being able to predict microbial activity in response to changes within their environments both past and future.

Part one focuses on work related to microbial processes in iron-rich Lake Matano and, more broadly, microbial interactions with the biogeochemical cycling of iron. Primarily, we find that the chelation of ferrous iron by organic ligands can affect the role of iron in anoxic environmental systems, enabling photomixotrophic growth of anoxygenic microorganisms with ferrous iron, as well as catalyzing the oxidation of ferrous iron by denitrification intermediates. These results imply that the ability to grow photomixotrophically on ferrous iron might be more widespread than previously assumed, and that the co-occurrence of chemical and biological processes involved in the coupled biogeochemical cycling of iron and nitrogen likely dominate organic-rich environmental systems.

Part two switches focus to in situ measurements of growth activity and comprises work related to microbial processes in the Cystic Fibrosis lung, and more broadly, the physiology of slow growth. We introduce stable isotope labeling of microbial membrane fatty acids and whole cells with heavy water as a new technique to measure microbial activity in a wide range of environments, demonstrate its application in continuous culture in the laboratory at the population and single cell level, and apply the tool to measure the in situ activity of the opportunistic pathogen Staphylococcus aureus within the environment of expectorated mucus from cystic fibrosis patients. We find that the average in situ growth rates of S. aureus fall into a range of generation times between ~12 hours and ~4 days, with substantial heterogeneity at the single-cell level. These data illustrate the use of heavy water as a universal environmental tracer for microbial activity, and highlight the crucial importance of studying the physiology of slow growth in representative laboratory systems in order to understand the role of these microorganisms in their native environments.

Study of ion movements in isolated chicken retinas during spreading depression

Spreading depression (SD) is a phenomenon observed in several sections of vertebrate central nervous system. It can occur spontaneously or be evoked by a variety of stimuli, and consists of a wave of depression of the normal electrical activity of the nervous tissue which spreads slowly in all directions in the tissue. This wave of depression is accompanied by several concomitants including ion movements. All the concomitants of SD can be explained by an increase in the sodium permeability of the plasma membranes of cellular elements involved in this phenomenon.

In the chicken retina, SD is accompanied by a transparency change which can be detected with the naked eye. The isolated retina is a thin (0.1 mm) membrane in which the extracellular fluid quickly and completely equilibrates with the incubation solutions. This preparation was therefore used to study the ion movements during SD by measuring and comparing the ion contents and the extracellular space (ECS) of retinas incubated in various solutions of which some inhibited SD, whereas others allowed this phenomenon to occur.

The present study has shown that during SD there is a shift of extracellular sodium into the intracellular compartment of the retina, a release of intracellular K and a decrease in the magnitude of ECS. These results are in agreement with previous postulates about SD, although the in vitro experimental condition makes the ion movements appear larger and the loss of ECS smaller than observed in the intact cortical tissue. The movements of Na and K, in opposite directions, are reversible. The development and magnitudes of SD is very little affected by deprivation of the oxygen supply.

It was established that the inward sodium shift is not a consequence of an arrest of the Na-pump. It can be prevented, together with SD by the membrane stabilizers, magnesium and procaine. Spreading depression and the ion movements are incompletely inhibited by tetrodotoxin, which blocks the sodium influx into nerve fibers during the action potential. The replacement of Na in the bathing solution by Li does not prevent SD, which is accompanied by Li accumulation in the intracellular compartment. From these experiments and others it was concluded that the mechanism underlying SD and the ion shifts is an increase in the sodium permeability of cell membranes.