The LacI-Type Transcriptional Regulator AraR Acts as an l -Arabinose-Responsive Repressor of l -Arabinose Utilization Genes in Corynebacterium glutamicum ATCC 31831

Abstract

bThe Corynebacterium glutamicum ATCC 31831 araBDA operon consists of three L-arabinose catabolic genes, upstream of which the galM, araR, and araE genes are located in opposite orientation. araR encodes a LacI-type transcriptional regulator that negatively regulates the L-arabinose-inducible expression of araBDA and araE (encoding an L-arabinose transporter), through a mechanism that has yet to be identified. Here we show that the AraR protein binds in vitro to three sites: one upstream of araBDA and two upstream of araE. We verify that a 16-bp consensus palindromic sequence is essential for binding of AraR, using a series of mutations introduced upstream of araB in electrophoretic mobility shift assays. Moreover, the DNA-binding activity of AraR is reduced by L-arabinose. We employ quantitative reverse transcription-PCR (qRT-PCR) analyses using various mutant strains deficient in L-arabinose utilization genes to demonstrate that the prominent upregulation of araBDA and araE within 5 min of L-arabinose supplementation is dependent on the uptake but independent of the catabolism of L-arabinose. Similar expression patterns, together with the upregulation by araR disruption without L-arabinose, are evident with the apparent galM-araR operon, although attendant changes in expression levels are much smaller than those realized with the expression of araBDA and araE. The AraR-binding site upstream of araB overlaps the ؊10 region of the divergent galM promoter. These observations indicate that AraR acts as a transcriptional repressor of araBDA, araE, and galM-araR and that L-arabinose acts as an intracellular negative effector of the AraR-dependent regulation.

AraR, an l -Arabinose-Responsive Transcriptional Regulator in Corynebacterium glutamicum ATCC 31831, Exerts Different Degrees of Repression Depending on the Location of Its Binding Sites within the Three Target Promoter Regions

“…Synthesized cDNA is also used as a template for quantitative RT-PCR analysis as described previously (24). The primers used are listed in Table S2 in the supplemental material.…”

“…Total RNA was extracted from C. glutamicum cells using an RNeasy minikit (Qiagen) as described previously (24). For reverse transcription-PCR (RT-PCR) analysis, single-stranded cDNA was synthesized from total RNA using PrimeScript reverse transcriptase (TaKaRa) with hexadeoxyribonucleotide mixture as a primer.…”

“…The resulting cDNA was used as a template for PCR with a primer set for the region of interest (see Table S2 in the supplemental material). C. glutamicum chromosomal DNA, prepared as described previously (24), was also used as a template for a control PCR with the same primer set. The reaction mixture was electrophoresed on an agarose gel, and the amplified DNA was detected with ethidium bromide.…”

“…For genetic manipulation, E. coli strains were grown at 37°C in Luria-Bertani medium. C. glutamicum strains were grown at 33°C in nutrient-rich A medium [2% (wt/vol) yeast extract, 7% (wt/vol) Casamino Acids, 2% (wt/vol) (NH 2 ) 2 Table S1 in the supplemental material; ⌬araA) was described previously (24).…”

“…However, their C-terminal effector-binding domains are homologous to the LacI family proteins. DNA binding activity of the C. glutamicum AraR protein is indicated to be decreased in the presence of L-arabinose, resulting in derepression of araBDA and araE (24). A slight increase in the Table S2 in the supplemental material) are as follows: 1-F/1-R (lanes 1), 2-F/2-R (lanes 2), 3-F/3-R (lanes 3), 4-F/4-R (lanes 4), 5-F/5-R (lanes 5), 6-F/6-R (lanes 6).…”