Transcriptional and translational regulatory responses to iron limitation in the globally distributed marine bacterium Candidatus pelagibacter ubique

Abstract

Iron is recognized as an important micronutrient that limits microbial plankton productivity over vast regions of the oceans. We investigated the gene expression responses of Candidatus Pelagibacter ubique cultures to iron limitation in natural seawater media supplemented with a siderophore to chelate iron. Microarray data indicated transcription of the periplasmic iron binding protein sfuC increased by 16-fold, and iron transporter subunits, iron-sulfur center assembly genes, and the putative ferroxidase rubrerythrin transcripts increased to a lesser extent. Quantitative peptide mass spectrometry revealed that sfuC protein abundance increased 27-fold, despite an average decrease of 59% across the global proteome. Thus, we propose sfuC as a marker gene for indicating iron limitation in marine metatranscriptomic and metaproteomic ecological surveys. The marked proteome reduction was not directly correlated to changes in the transcriptome, implicating post-transcriptional regulatory mechanisms as modulators of protein expression. Two RNA-binding proteins, CspE and CspL, correlated well with iron availability, suggesting that they may contribute to the observed differences between the transcriptome and proteome. We propose a model in which the RNA-binding activity of CspE and CspL selectively enables protein synthesis of the iron acquisition protein SfuC during transient growth-limiting episodes of iron scarcity.

Figure 1. Growth of Candidatus Pelagibacter ubique cells was arrested by iron-sequestering siderophores.

(A) Cell densities observed during a pilot experiment to test the effect of the two siderophores ferrichrome and deferoxamine mesylate salt at varying concentrations on the growth of Candidatus Pelagibacter ubique HTCC1062. The first arrow indicates the introduction of siderophore/iron as described by the legend. The second arrow indicates the delayed 1 µM iron additions parenthetically noted in the legend. (B) Cultures for harvesting were grown in six 20 L carboys. The first arrow indicates the introduction of siderophore/iron as described by the legend. Proteins and mRNA were analyzed on the dates indicated by the unfilled arrows: microarray samples were taken from cultures on days 17, 18, and 28; proteomic samples were taken on days 18 and 28.

Figure 2. Genes transcribed during iron limitation were different from stationary phase genes.

The four clusters indicate up-regulation of similar condition-specific mRNA. Symbols for each microarray sample (open circles) were manually positioned on a circle according to each sample's iron availability and growth rate. Genes were “attracted” to the samples in which they were most abundant. Larger points indicate genes with larger condition-to-condition variation; a key for the 10 largest points in each cluster is provided. The complete list of gene locations for this graph can be found in Supplementary Table S1.

Figure 3. Protein abundances were largely decoupled from transcript abundances.

The change in protein abundance versus the change in mRNA abundance was plotted for all Ca. Pelagibacter ubique genes that showed a significant (P< = 0.05) change in either measurement. Each color represents a different comparison between treatments or timepoints, with R² values of 0.11, 0.08, 0.09, and 0.02 respective to the legend's ordering. Large ellipses indicate clusters of the same colored points. Histograms on the low end of each axis further define the distribution of points. Points represented by a diamond are discussed at length in the text.

Figure 4. Translation of Ca. Pelagibacter ubique's cold shock and iron-binding genes are influenced by iron availability.

The abundance of two Ca. Pelagibacter ubique cold shock proteins, CspE and CspL, and the iron-binding protein SfuC, appear to be correlated with iron availability (p-value of .02, .08, and 3e-79, respectively).