Graduate Thesis Or Dissertation

Genome-enabled investigation of the minimal growth requirements andphosphate metabolism for Pelagibacter marine bacteria

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  • Members of the SAR11 clade of heterotrophic α-proteobacteria are ubiquitous and abundant in the world's oceans where they are thought to play a pivotal role in the global carbon cycle. The first SAR11 bacterium cultivated in vitro, 'Candidatus Pelagibacter ubique' HTCC1062 (Ca. P. ubique), was isolated by dilution into sterile natural seawater, from which undefined dissolved organic carbon supplied nutrients for heterotrophic growth. However, variation in the composition of such native dissolved organic matter hindered efforts to identify the specific nutrient requirements of Ca. P. ubique and elucidate its metabolism. For this dissertation work, genome-enabled metabolic reconstruction was used to develop a defined artificial seawater medium for Ca. P. ubique that consisted of inorganic salts, defined types and amounts of organic carbon, reduced sulfur and vitamins. Subsequent in vitro experimentation was used to show that Ca. P. ubique requires simultaneous additions of glycine and methionine to meet cellular requirements for glycine and sulfur, respectively. A new requirement for pyruvate was identified and linked to the production of alanine. We found that pyruvate could be replaced by additions of glucose or oxaloacetate and that glycine could be replaced with serine or glycine betaine. Interestingly, glycolate partially fulfilled the glycine requirement of Ca. P. ubique, likely because of dual use as a carbon and glycine source. Once these major organic nutrient requirements were established, the defined medium was used to identify specific trace requirements of Ca. P. ubique for vitamins or vitamin precursors. Previously, analysis of the Ca. P. ubique genome did not identify complete biosynthetic pathways for thiamine (vitamin B₁), pantothenate (vitamin B₅), pyridoxine (vitamin B₆), biotin (vitamin B₇) or cobalamin (vitamin B₁₂), suggesting that these vitamins cannot be synthesized de novo. We describe requirements for the thiamine-pyrimidine precursor 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP) and vitamin B₇ as determined from laboratory cultures. We determined in situ (the Sargasso Sea) concentrations of HMP by liquid chromatography coupled tandem mass spectrometry. Our measurements show that in situ HMP concentrations are ample for the native SAR11 population. Genomic and experimental evidence that vitamins B₅ and B₆ are synthesized de novo is presented and genomic evidence that vitamin B₁₂ is not required by Ca. P. ubique is discussed. A second Pelagibacter isolate, Pelagibacter sp. str. HTCC7211 was also grown on a defined medium. Pelagibacter sp. str. HTCC7211 encodes a suite of genes, absent in Ca. P. ubique, dedicated to the acquisition and storage of inorganic phosphate and the utilization of organic phosphorus. On a defined medium, the growth of both Ca. P. ubique and Pelagibacter sp. str. HTCC7211 was limited by excluding inorganic phosphate (Pi) and we show that Ca. P. ubique has an apparent requirement for 10.2 attomoles P[subscript i] cell⁻¹ and Pelagibacter sp. str. HTCC7211 has an apparent requirement for 45.7 attomoles P[subscript i] cell⁻¹. Discrete phosphorus utilization physiotypes were evident, whereby Pelagibacter sp. str. HTCC7211, but not Ca. P. ubique, utilized assorted P[subscript i]-esters and phosphonates in place of P[subscript i] to meet its cellular P-requirement. When grown on methylphosphonic acid (Mpn), the apparent P-requirement decreased in Pelagibacter sp. str. HTCC7211 to 11.4 attomoles cell⁻¹. We investigated the underlying transcriptional dynamics that give rise to the observed physiotypes using DNA microarrays. Ca. P. ubique responded rapidly to P-limitation by upregulating a high-affinity transport system operon (pstSCAB-phoUB) and stress response genes suggestive of a stringent response. Conversely, Pelagibacter sp. str. HTCC7211 responded slowly to the onset of P-limitation; no transcripts were differentially regulated for 68 hours, after which, phosphonate transport and utilization genes showed the greatest increases in expression. Collectively, these experiments exemplify the utility of growing Ca. P. ubique (and other Pelagibacter isolates) on a defined medium and have opened the door for additional experiments that were previously impossible to conduct on natural seawater. This work also represents an important step towards developing Ca. P. ubique into a model system that can be used to understand the cellular adaptations that make oligotrophy a successful life strategy in the sea.
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