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. 2016 Dec 1:7:1929.
doi: 10.3389/fmicb.2016.01929. eCollection 2016.

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

Muhammad Afzal  1 , Irfan Manzoor  1 , Oscar P Kuipers  2 , Sulman Shafeeq  3
Affiliations

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

Muhammad Afzal et al. Front Microbiol. .

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.

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Figures

FIGURE 1
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
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
FIGURE 3
A phylogenetic tree of CmbR in different streptococci showing conservation of CmbR in these streptococci.
FIGURE 4
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
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
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.
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