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. 2017 Mar 9:7:70.
doi: 10.3389/fcimb.2017.00070. eCollection 2017.

Niacin-mediated Gene Expression and Role of NiaR as a Transcriptional Repressor of niaX, nadC, and pnuC in Streptococcus pneumoniae

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

Niacin-mediated Gene Expression and Role of NiaR as a Transcriptional Repressor of niaX, nadC, and pnuC in Streptococcus pneumoniae

Muhammad Afzal et al. Front Cell Infect Microbiol. .

Abstract

NAD (Nicotinamide Adenine Dinucleotide) biosynthesis is vital for bacterial physiology and plays an important role in cellular metabolism. A naturally occurring vitamin B complex, niacin (nicotinic acid), is a precursor of coenzymes NAD and NADP. Here, we study the impact of niacin on global gene expression of Streptococcus pneumoniae D39 and elucidate the role of NiaR as a transcriptional regulator of niaX, nadC, and pnuC. Transcriptome comparison of the D39 wild-type grown in chemically defined medium (CDM) with 0 to 10 mM niacin revealed elevated expression of various genes, including niaX, nadC, pnuC, fba, rex, gapN, pncB, gap, adhE, and adhB2 that are putatively involved in the transport and utilization of niacin. Niacin-dependent expression of these genes is confirmed by promoter lacZ-fusion studies. Moreover, the role of transcriptional regulator NiaR in the regulation of these genes is explored by DNA microarray analysis. Our transcriptomic comparison of D39 ΔniaR to D39 wild-type revealed that the transcriptional regulator NiaR acts as a transcriptional repressor of niaX, pnuC, and nadC. NiaR-dependent regulation of niaX, nadC, and pnuC is further confirmed by promoter lacZ-fusion studies. The putative operator site of NiaR (5'-TACWRGTGTMTWKACASYTRWAW-3') in the promoter regions of niaX, nadC, and pnuC is predicted and further confirmed by promoter mutational experiments.

Keywords: NiaR; Pneumococcus; nadC; niaX; niacin; pnuC.

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Figures

Figure 1
Figure 1
Expression levels (in Miller units) of PniaX-lacZ, PpnuC-lacZ, PnadC-lacZ, Pfba-lacZ, Prex-lacZ, PpncB-lacZ, Pgap-lacZ, PadhE-lacZ, PadhB2-lacZ, and PgapN-lacZ in CDM with 0 and 10 mM niacin in S. pneumoniae D39 wild-type. Standard deviations of three independent experiments are indicated in bars. Statistical significance of the differences in the expression levels was determined by one-way ANOVA (NS, not significant, **P < 0.001, and ***P < 0.0001).
Figure 2
Figure 2
Expression levels (in Miller units) of PniaX-lacZ, PpnuC-lacZ, and PnadC-lacZ in complete CDM in S. pneumoniae D39 wild-type and D39 ΔniaR. Standard deviations of three independent experiments are indicated in bars. Statistical significance of the differences in the expression levels was determined by one-way ANOVA (NS, not significant, **P < 0.001, and ***P < 0.0001).
Figure 3
Figure 3
Organization of the NiaR-regulated genes in S. pneumoniae D39. Putative NiaR operator sequences are rectangle and translational initiation sites are italicized, whereas the lollipop structures represent the putative transcriptional terminators. See text for further details.
Figure 4
Figure 4
Identification of the NiaR operator site. (A) Weight matrix of the identified NiaR operator site in the promoter regions of niaX, nadC, and pnuC. (B) Position of the NiaR operator site in the promoter region of niaX, nadC, and pnuC in different streptococci. Putative NiaR operator sites are bold and underlined. SP, S. pneumoniae; SI, Streptococcus mitis; SA, Streptococcus agalactiae; SD, Streptococcus dysgalactiae; SE, Streptococcus equi; SG, Streptococcus gallolyticus; SN, Streptococcus gordonii; SM, Streptococcus mutans; SO, Streptococcus pyogenes; SS, Streptococcus sanguinis; SU, Streptococcus suis; ST, Streptococcus thermophiles; and SB, Streptococcus uberis.
Figure 5
Figure 5
Expression levels (in Miller units) of PniaX-lacZ, PpnuC-lacZ, and PnadC-lacZ with mutated and non-mutated NiaR operator sites in S. pneumoniae D39 wild-type grown in complete CDM. Standard deviations of three independent experiments are indicated in bars. Statistical significance of the differences in the expression levels was determined by one-way ANOVA (NS, not significant, **P < 0.001, and ***P < 0.0001).
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