Cysteine-Mediated Gene Expression and Characterization of the CmbR Regulon in Streptococcus pneumoniae

Abstract

In this study, we investigated the transcriptomic response of Streptococcus pneumoniae D39 to cysteine. Transcriptome comparison of the D39 wild-type grown at a restricted concentration of cysteine (0.03 mM) to one grown at a high concentration of cysteine (50 mM) in chemically-defined medium (CDM) revealed elevated expression of various genes/operons, i.e., spd-0150, metQ, spd-0431, metEF, gshT, spd-0618, fhs, tcyB, metB-csd, metA, spd-1898, yvdE, and cysK, likely to be involved in the transport and utilization of cysteine and/or methionine. Microarray-based data were further confirmed by quantitative RT-PCR. Promoter lacZ-fusion studies and quantitative RT-PCR data showed that the transcriptional regulator CmbR acts as a transcriptional repressor of spd-0150, metEF, gshT, spd-0618, tcyB, metA, and yvdE, putatively involved in cysteine uptake and utilization. The operator site of CmbR in the promoter regions of CmbR-regulated genes is predicted and confirmed by mutating or deleting CmbR operator sites from the promoter regions of these genes.

Keywords: CmbR; Cysteine; MetA; MetE; pneumococcus.

FIGURE 1

The relative increase in the expression of spd-0150, metQ, spd-0431, metEF, gshT, spd-0618, fhs, tcyB, metB-csd, metA, spd-1898, yvdE, and cysK in S. pneumoniae D39 wild-type grown in CDM with 0.03 mM cysteine compared to that grown in CDM with 50 mM cysteine. The expression of these genes was normalized with housekeeping gene gyrA.

FIGURE 2

Identification of the CmbR operator site. (A) Weight matrix of the identified CmbR operator site in the promoter region of spd-0150, metEF, gshT, spd-0618, tcyB, metA, and yvdE. (B) Position of the CmbR operator site in the promoter region of spd-0150, metEF, gshT, spd-0618, tcyB, metA, and yvdE. Translational start sites are italic and putative CmbR operator sites are bold and underlined.

FIGURE 3

A phylogenetic tree of CmbR in different streptococci showing conservation of CmbR in these streptococci.

FIGURE 4

Expression levels (in Miller units) of Pspd-0150-lacZ, PmetE-lacZ, PgshT-lacZ, Pspd-0618-lacZ, PtcyB-lacZ, PmetA-lacZ, and PyvdE-lacZ in CDM with 50 mM cysteine in S. pneumoniae D39 wild-type and D39 ΔcmbR.

FIGURE 5

The relative increase in the expression of spd-0150, metEF, gshT, spd-0618, tcyB, metA, and yvdE in S. pneumoniae D39 ΔcmbR compared to D39 wild-type grown in CDM with 50 mM cysteine. The expression of these genes was normalized with that of housekeeping gene gyrA.

FIGURE 6

Expression levels (in Miller units) of mutated/terminated and non-mutated CmbR sites in Pspd-0150-lacZ, PmetEF-lacZ, Pspd-0618-lacZ, and PmetA-TER-lacZ in S. pneumoniae D39 wild-type grown in CDM with 50 mM cysteine.