YrxA is the transcriptional regulator that represses de novo NAD biosynthesis in Bacillus subtilis

Abstract

The first genetic, in vivo, and in vitro evidences that YrxA is the regulator of NAD de novo biosynthesis in Bacillus subtilis are hereby reported. The protein is essential to the transcription repression of the divergent operons nadBCA and nifS-yrxA in the presence of nicotinic acid and binds to their shared operator-promoter region.

FIG. 1.

Selection of Ery^r (0.3 μg/ml erythromycin) colonies using DNA of PB1934 (ΩpncB′::pMUTIN4) to transform PB168 (wt) competent cells on MM (5) in the presence or absence of IPTG (1 mM) and NA (50 μg/ml). Only sporadic spontaneous Ery^r mutants can be observed on the bottom left in MM with NA. Very small colonies can be observed in the absence of NA (top left) after 48 h of incubation at 37°C. When IPTG is added to the medium, the colonies observed in the presence of NA are smaller (bottom right) than the ones observed in absence of NA (top right).

FIG. 2.

Selection of Ery^r transformants (0.3 μg/ml erythromycin) on TBAB medium with or without IPTG (1 mM): in the top part of the figure, transformation of PB168 (wt) competent cells with chromosomal DNA of PB1934 (ΩpncB′::pMUTIN4), colonies are visible only on plates containing IPTG. When the same DNA was used to transform PB1932 (ΔyrxA) competent cells, the same number of colonies grew on TBAB plates with or without IPTG (lower part of the figure).

FIG. 3.

RT-PCR experiments on the transcripts of the nifS/iscS and nadB genes in the PB1932 (ΔyrxA::cat) mutant and PB168 (wt) strains, grown in MM medium with or without NA (50 μg/ml). S, Mass Ruler DNA ladder (low range; Fermentas). ΔyrxA, RT-PCR on RNA extracted from PB1932 cells; WT, RT-PCR on RNA extracted from PB168 cells. A product of 297 bp indicates the presence of nadBCA operon transcripts, and a product of 480 bp indicates the presence of nifS/iscS transcripts.

FIG. 4.

Electrophoresis mobility shift assays. Panel A: band shift induced by YrxA. Increasing concentrations of the protein (0.003, 0.034, 0.34, 0.77, and 3.4 μM) induce an increasing retardation of the ³²P OP DNA-labeled fragment (1.7 nM) migration in the gel. Panel B: competition in EMSA between ³²P-labeled 87-bp OP fragment and cold specific SC₁ DNA (a 170-bp fragment spanning from just upstream to just downstream OP, including the 87-bp fragment) or nonspecific DNA (AC₁, a 318-bp fragment covering a region in nadB outside OP; and AC₂, a 330-bp fragment in nifS). The YrxA concentration was 50 nM, NA was 150 μM, and the increasing concentrations of competitors were, respectively, 2, 5, and 10ng in 20 μl. Panel C: band shift and “supershift” in the presence of NA and analogues, intermediates, or end products of NAD synthesis (added at a concentration of 300 μM). Lanes: 1, no cofactors, no YrxA; 2, only YrxA; 3, 150 μM NA; 4, 300 μM NA; 5, nicotinamide; 6, quinolinic acid; 7, isoniazide; 8, nicotinic acid hydrazide; 9: pyrazynamide; 10, nicotinamide mononucleotide; 11, NAD; 12, NADH. A plus sign indicates the presence of 3.4 μM YrxA.