Date of Award

5-2011

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Microbiology

Major Professor

Erik R. Zinser

Committee Members

Alison Buchan, Steven Wilhelm, Gladys Alexandre

Abstract

The unicellular cyanobacterium Prochlorococcus is the numerically dominant photosynthetic organism throughout the temperate and tropical open oceans, but it is difficult to grow in pure cultures. We developed a system for rendering spontaneous streptomycin-resistant mutants of Prochlorococcus axenic by diluting them to extinction in the presence of “helper” heterotrophic bacteria, allowing them to grow to high cell concentrations, and then killing the helpers with streptomycin. Using axenic strains obtained in this fashion, we demonstrated that Prochlorococcus experiences a number of growth defects in dilute axenic culture, including reduced growth rate, inability to form colonies on solid media, and higher incidence of mortality (i.e., catastrophic failure of liquid cultures). These defects were eliminated when Prochlorococcus was grown in co-culture with a phylogenetically diverse array of helper bacteria. The primary mechanism of helping was enzymatic removal of hydrogen peroxide (HOOH) from the culture medium. Axenic Prochlorococcus cultures were profoundly sensitive to HOOH additions in comparison with reported tolerance levels for all other wild-type aerobic bacteria, but in co-culture their resistance was similar to that of the helpers. Neither is dependence on helpers limited to the laboratory. Sterile-filtered seawater exposed to sunlight accumulated enough HOOH in 24h to kill ecologically relevant cell concentrations of Prochlorococcus. We also refined a method for delivering HOOH at a defined, steady rate using the buffer HEPES to more accurately simulate the steady accumulation of HOOH in natural waters. Even at the lowest production rates that could sustain the in situ HOOH concentration in the ocean, HEPES-generated HOOH was lethal to Prochlorococcus; again, co-culture with helpers prevented this effect. We speculate on the ecological consequences of Prochlorococcus’ dependency on other organisms for survival, as well as the evolutionary forces that have led to this lack of self-sufficiency.

Supplemental File 1.fasta (2984 kB)
JCVI-CMR Catalase Database (FASTA format)

Supplemental File 2.fasta (548 kB)
JCVI-CMR rpsL Database (FASTA format)

Supplemental File 3.csv (2630 kB)
Taxonomy of GOS catalase/rpsL hits

Supplemental File 4.csv (335 kB)
GOS catalase hits with metadata (CSV format)

Supplemental File 5.csv (1504 kB)
GOS rpsL hits with metadata (CSV format)

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