The phosphate starvation stimulon of Corynebacterium glutamicum determined by DNA microarray analyses

Abstract

The phosphate (P(i)) starvation stimulon of Corynebacterium glutamicum was characterized by global gene expression analysis by using DNA microarrays. Hierarchical cluster analysis of the genes showing altered expression 10 to 180 min after a shift from P(i)-sufficient to P(i)-limiting conditions led to identification of five groups comprising 92 genes. Four of these groups included genes which are not directly involved in P metabolism and changed expression presumably due to the reduced growth rate observed after the shift or to the exchange of medium. One group, however, comprised 25 genes, most of which are obviously related to phosphorus (P) uptake and metabolism and exhibited 4- to >30-fold-greater expression after the shift to P(i) limitation. Among these genes, the RNA levels of the pstSCAB (ABC-type P(i) uptake system), glpQ (glycerophosphoryldiester phosphodiesterase), ugpAEBC (ABC-type sn-glycerol 3-phosphate uptake system), phoH (unknown function), nucH (extracellular nuclease), and Cgl0328 (5'-nucleotidase or related esterase) genes were increased, and pstSCAB exhibited a faster response than the other genes. Transcriptional fusion analyses revealed that elevated expression of pstSCAB and ugpAEBC was primarily due to transcriptional regulation. Several genes also involved in P uptake and metabolism were not affected by P(i) starvation; these included the genes encoding a PitA-like P(i) uptake system and a putative Na(+)-dependent P(i) transporter and the genes involved in the metabolism of pyrophosphate and polyphosphate. In summary, a global, time-resolved picture of the response of C. glutamicum to P(i) starvation was obtained.

FIG. 1.

Growth of C. glutamicum ATCC 13032 in CGXII minimum medium with 40 g of glucose per liter and different concentrations of P_i. Cells were precultured in CGXII medium with 0.13 mM potassium phosphate for 24 h before inoculation into CGXII medium containing 0.13 mM (▪), 0.26 mM (▴), 0.65 mM (×), 1.3 mM (∗), or 13 mM (•) potassium P_i. (Inset) Doubling times plotted against reciprocal P_i concentrations.

FIG. 2.

Hierarchical cluster analysis of gene expression changes during the response of C. glutamicum ATCC 13032 to P_i starvation. Gene expression data from 12 microarray experiments (columns) and 92 genes (lines) are represented. The microarray experiments included comparing gene expression of C. glutamicum ATCC 13032 before and 10, 30, 60, 90, 120, and 180 min after a shift from P_i-sufficient conditions to P_i-limiting conditions. Subclusters 1 to 5 are indicated by red, blue, pink, green, and brown, respectively. The scale bar indicates the color coding of the relative RNA levels.

FIG. 3.

Kinetics of changes in gene expression during the response of C. glutamicum ATCC 13032 to P_i starvation. Changes in gene expression 10, 30, 60, 90, 120, and 180 min after a shift from P_i-sufficient to P_i-limiting conditions compared to preshift conditions taken from Table 2 are shown for selected ORFs (see text for details). (A to D) Kinetics of changes in gene expression for subcluster 1 genes (Fig. 2): genes encoding the putative high-affinity phosphate transporter pstSCAB (ORFs 2871 to 2876) (A), the glycerophosphoryl diester phosphodiesterase and the sn-glycerol 3-phosphate transport system (glpQ and ugpAEBC, ORFs 2637 to 2641) (B), and the putative extracellular nuclease (ORF 2851, nucH), 5′-nucleotidase or a related esterase (Cgl0328, ORF 766), and the hypothetical proteins (ORFs 1086, 3081, and 3082) (C) and all other subcluster 1 genes (D). (E and F) Kinetics of changes in gene expression for the subcluster 2 genes (Fig. 2) except ORF 2831 (E) and for subcluster 3 genes and ORF 2831 (F).

FIG. 4.

Expression of the pstS-cat and ugpA-cat transcriptional fusions in C. glutamicum ATCC 13032(pET2-pst) and ATCC 13032(pET2-ugp) during the response to P_i starvation. The activities obtained from two or more independent cultivations varied less than 20% and were normalized to the activity before the shift to P_i starvation conditions. Symbols: •, pstS-cat fusion; ○, ugpA-cat fusion.