Sulfur and nitrogen limitation in Escherichia coli K-12: specific homeostatic responses

Abstract

We determined global transcriptional responses of Escherichia coli K-12 to sulfur (S)- or nitrogen (N)-limited growth in adapted batch cultures and cultures subjected to nutrient shifts. Using two limitations helped to distinguish between nutrient-specific changes in mRNA levels and common changes related to the growth rate. Both homeostatic and slow growth responses were amplified upon shifts. This made detection of these responses more reliable and increased the number of genes that were differentially expressed. We analyzed microarray data in several ways: by determining expression changes after use of a statistical normalization algorithm, by hierarchical and k-means clustering, and by visual inspection of aligned genome images. Using these tools, we confirmed known homeostatic responses to global S limitation, which are controlled by the activators CysB and Cbl, and found that S limitation propagated into methionine metabolism, synthesis of FeS clusters, and oxidative stress. In addition, we identified several open reading frames likely to respond specifically to S availability. As predicted from the fact that the ddp operon is activated by NtrC, synthesis of cross-links between diaminopimelate residues in the murein layer was increased under N-limiting conditions, as was the proportion of tripeptides. Both of these effects may allow increased scavenging of N from the dipeptide D-alanine-D-alanine, the substrate of the Ddp system.

FIG.1.

Four aligned genome images showing transcriptional responses of strain NCM3722 (or its Eut⁺ derivative NCM3954) to N or S limitation in adapted batch cultures. Row 1 (NCM3954), cells grown on ethanolamine (Cy5 label, red) versus cells grown on ammonium (Cy3 label, green); row 2, cells grown on arginine (red) versus cells grown on ammonium (green); row 3, cells grown on taurine (red) versus cells grown on sulfate (green); row 4, cells grown on glutathione (red) versus cells grown on sulfate (green). Glass slide DNA microarrays were probed with mixtures of appropriately labeled cDNAs as described in Materials and Methods. Spots from fluorescent scanning images of microarrays were arranged in genome order to generate the genome images (60, 61). The b number groups are indicated on the left. Blanks represent b numbers that either do not correspond to ORFs or no longer exist. Easily visible red spots are discussed in the text, and green spots will be discussed elsewhere. The eut operon extends from b2437 through b2462 but is interrupted by b2442 to b2450 (3, 44). Spots for b2438, b2440, b2455, and b2458 were problematic (44).

FIG. 2.

Four aligned genome images showing transcriptional responses of strain NCM3722 after an S downshift (rows 1 and 2) or upshift (rows 3 and 4). Rows 1 and 2, shift from sulfate (green) to glutathione (red); rows 3 and 4, shift from glutathione (green) to sulfate (red). Samples were taken before the shift and 30 min after the shift and were treated as described in the legend to Fig. 1. In each case two prints from the same slide are shown. Easily visible red and green spots are discussed in the text. Note the opposite responses to the S downshift and upshift.

FIG. 3.

Venn diagram summarizing the numerical analysis of the effects of N and S availability on transcription (mRNA levels) in strain NCM3722 (and Eut⁺ strain NCM3954 for comparison of ethanolamine to ammonium). The numbers in all panels indicate the numbers of genes with higher expression under nutrient-limiting conditions than under nutrient-excess conditions, and the numbers in panel C also include the numbers of genes with lower expression after a nutrient upshift (determined as described in Materials and Methods). The numbers in yellow ovals are the numbers of genes in Table 1 (S-related genes), the numbers in green ovals are the numbers of NtrC/Nac-regulated genes (61), and the numbers in blue ovals are the numbers of known RpoS-controlled genes (26). (A) Comparison of taurine (Tau) to sulfate (left circle) and comparison of ethanolamine (EtNH₂) to ammonium (right circle). (B) Comparison of glutathione (GSH) to sulfate (left circle) and comparison of arginine (Arg) to ammonium (right circle). (C) Comparison of S down- and upshift (S shift) to preshift samples (left circle) and comparison of N down- and upshift to preshift samples (right circle). The large circles and regions of overlap between them are not drawn to scale. The common gene in yellow and green ovals in panel A is cbl (b1987). The common genes in the green oval in panel B are ompF (b0929), yeaG (b1783), and ygiG (b3073). The gene in the green oval for glutathione is cbl, which is also included in the genes in the yellow oval. This gene failed to make the cutoff for a common gene in panel B because the average red/green ratio on arginine versus ammonium was 1.6. The red/green ratio for cbl in the experiment shown in Fig. 1, row D, was 3.2. The common gene in the yellow oval in panel C is cbl; the common genes in the green oval are cbl, nac (b1988), glnH (b0811), oppA (b1243), and yeaG. The gene in the green oval for S shifts is ompF.

FIG. 4.

Venn diagram summarizing various means of analysis of S shift experiments. The numbers indicate the numbers of genes with higher expression (mRNA levels) after a downshift and/or lower expression after an upshift. As indicated, these genes were detected numerically (see Materials and Methods), by k-means clustering, or by agglomerative hierarchical clustering (HCA) (see text). The numbers in grey ovals indicate the numbers of genes in Table 1. The circles and regions of overlap are not drawn to scale.

FIG. 5.

k-means clusters containing NtrC/Nac-regulated genes. Clusters were obtained by analysis of data from adapted batch cultures of NCM3722 (18 genome prints) (A and B) or cultures subjected to nutrient shifts (16 genome prints) (C and D). The experiments are indicated on the x axis, and the log ratios of expression are indicated on the y axis. The green lines show data for individual genes, and the pink and black lines show averages. The blue lines in panel B show data for eut genes (see text). In panels A and B, the log ratios are values for a comparison of nutrient-limiting conditions to excess conditions. In panels C and D, the log ratios are values for a comparison of data obtained after the shift with data obtained before the shift. The numbers on the x axis indicate the times (in minutes) after the shift at which the samples were taken.

FIG. 6.

k-means clusters containing S-related genes (see text). For an explanation of the analysis see the legend to Fig. 5. (A) Adapted cultures. (B) Cultures subjected to a shift.

FIG. 7.

Reclustering of genes assessed numerically to be upregulated (A to F) or downregulated (G to L) under nutrient-limiting conditions (see text). The y axis indicates log ratios of expression in the same units used for Fig. 5 and 6. The experiments indicated on the x axis are those shown in Fig. 5 followed by those shown in Fig. 6. The thick lines show the average log ratios of expression.

FIG. 8.

Homeostatic responses to S limitation (A) and generation of 1C units from serine and glycine (46) (B). (A) Pathways responding to S limitation are indicated schematically along with the regulators controlling them (ovals). Details of several of the pathways and ligands for the regulators are discussed in the text (also see Table 1). (B) 3-Phosphoglycerate (3PGA) is the central metabolite. The 1C units are transferred to tetrahydrofolate, yielding N⁵,N¹⁰-tetrahydrofolate. The gene products that catalyze the reactions are indicated above the arrows.