Global gene expression of Prochlorococcus ecotypes in response to changes in nitrogen availability

Abstract

Nitrogen (N) often limits biological productivity in the oceanic gyres where Prochlorococcus is the most abundant photosynthetic organism. The Prochlorococcus community is composed of strains, such as MED4 and MIT9313, that have different N utilization capabilities and that belong to ecotypes with different depth distributions. An interstrain comparison of how Prochlorococcus responds to changes in ambient nitrogen is thus central to understanding its ecology. We quantified changes in MED4 and MIT9313 global mRNA expression, chlorophyll fluorescence, and photosystem II photochemical efficiency (Fv/Fm) along a time series of increasing N starvation. In addition, the global expression of both strains growing in ammonium-replete medium was compared to expression during growth on alternative N sources. There were interstrain similarities in N regulation such as the activation of a putative NtcA regulon during N stress. There were also important differences between the strains such as in the expression patterns of carbon metabolism genes, suggesting that the two strains integrate N and C metabolism in fundamentally different ways.

Figure 1

Effect of N starvation on culture fluorescence (A, B) and photochemical energy conversion efficiency F_v/F_m (C, D) in Prochlorococcus MED4 (A, C) and MIT9313 (B, D). Days −3, −2, and −1 correspond to culture growth prior to start of experiment. N-replete log-phase cells were spun down and resuspended in N-replete (+NH4, circles) and N-deficient (−N, triangles) media at t=0. The discontinuity in MED4 chlorophyll fluorescence at t=0 resulted from incomplete harvest of the cells before resuspension. Data points are means of replicates. Error bars show the range and are smaller than the symbols when not apparent.

Figure 2

Number of differentially expressed genes (q<0.01) in Prochlorococcus MED4 (A) and MIT9313 (B) genes after the onset of N deprivation at t=0 for the cultures shown in Figure 1. Solid lines with triangles show upregulated genes and dashed lines with circles show repressed genes.

Figure 3

Temporal patterns of differentially expressed MED4 (A) and MIT9313 (B) genes clustered using the K-means algorithm. Each data point is the log₂-transformed median expression of all genes in the cluster; bars show range from 25th to 75th percentile. Plots are colored to show the gene function category with the greatest enrichment: red=transport and binding, blue=regulatory, black=amino-acid synthesis, magenta=translation, green=photosynthesis and respiration, cyan=fatty acid/phospholipid/sterol metabolism. Legend shows P-values associated with functional enrichment of each cluster. Asterisks in legend denote significant enrichment for functional categories according to ‘stringent' (^**) or ‘permissive' (^*) significance thresholds (see Materials and methods).

Figure 4

Comparison of MED4 and MIT9313 expression patterns under N stress for selected genes: ntca (A), sigma factors (B), glnB cluster (C), glnA (D), hli genes (E), and rbcLS (F). Data points show log₂-transformed mean expression values of duplicate cultures; error bars show one standard deviation.

Figure 5

MIT9313 (A, B) and MED4 (C, D) differentially expressed genes on alternative N sources relative to ammonium. Plots show all genes differentially expressed (q<0.01) for MIT9313 on nitrite (A), MIT9313 on urea (B), MED4 on cyanate (C), or MED4 on urea (D) relative to expression on ammonium. A total of 69 MIT9313 genes were differentially expressed on urea; only those >4-fold changed are shown. Data points show log₂-transformed means of duplicate cultures; error bars show one standard deviation. Colored bars show genes that are differentially expressed on both N sources for a given strain.

Figure 6

Model of the MED4 (A) and MIT9313 (B) transcriptional response to N starvation focusing on the regulation of nitrogen metabolism and its integration with carbon metabolism. Shaded regions show general cell processes: regulators (pink), nitrogen transport/assimilation (green), carbon metabolism (yellow). Text for genes upregulated in response to N starvation are in red, repressed genes are in blue, and unchanged genes are in black. Genes unchanged in expression except at the final time point (t=48 h) are not labeled as differentially expressed. Arrows show proposed interactions between the proteins encoded by these genes. Amino-acid abbreviations: glutamate=E, glutamine=Q.