Transcriptional regulation of the l-lactate permease gene lutP by the LutR repressor of Bacillus subtilis RO-NN-1

Abstract

The Bacillus subtilis lutABC operon encodes three iron-sulfur-containing proteins required for Llactate utilization and involved in biofilm formation. The transcriptional regulator LutR of the GntR family negatively controls lutABC expression. The lutP gene, which is situated immediately upstream of lutR, encodes an L-lactate permease. Here, we show that lutP expression can be strongly induced by L-lactate and is subject to partial catabolite repression by glucose. Disruption of the lutR gene led to a strong derepression of lutP and no further induction by L-lactate, suggesting that the LutR repressor can also negatively control lutP expression. Electrophoretic mobility shift assay revealed a LutR-binding site located downstream of the promoter of lutA or lutP and containing a consensus inverted repeat sequence 59-TCATC-N 1 -GATGA-39. Reporter gene analysis showed that deletion of each LutR-binding site caused a strong derepression of lutA or lutP. These results indicated that these two LutR-binding sites can function as operators in vivo. Moreover, deletion analysis identified a DNA segment upstream of the lutP promoter to be important for lutP expression. In contrast to the truncated LutR of laboratory strains 168 and PY79, the full-length LutR of the undomesticated strain RO-NN-1, and probably many other B. subtilis strains, can directly and negatively regulate lutP transcription. The absence or presence of the N-terminal 21 aa of the full-length LutR, which encompass a small part of the predicted winged helix-turn-helix DNA-binding motif, may probably alter the DNA-binding specificity or affinity of LutR.

Elucidating the Role and Regulation of a Lactate Permease as Lactate Transporter in Bacillus coagulans DSM1

“…This motif with an inverted repeat was reported to be the consensus binding site for LutR (12,26). For example, LutR in B. subtilis binds to the TCATC-N-GATGA motif in the promoter region of the lactate utilization operon (1). We noticed that in B. coagulans DSM1, there are 3 inverted repeat sequences in the intergenic region of lutR-lutP ( Fig.…”

“…Sequence alignment with the lactate transporter of B. subtilis (LutP, CAB12100.2) showed a 45% identity at the amino acid level (Table S1). The lutP gene from B. subtilis undergoes lactate-triggered transcription (1,12). B. coagulans DSM1 is an L-lactate producer, and the concentration of L-lactate increased gradually in the fermentation broth (19,22).…”

“…This family has been proven to regulate lactate utilization genes via binding with conserved inverted repeat sequences, such as TCATCnGATCA (n denotes any nucleotide) in Bacillus strains or AATTGGnnnCCAATT in Proteobacteria (Fig. 4B) (1,11,13,14). Although the effectors of various predicted transcription factors for lactate The threshold cycles (C T ) for each PCR with different concentrations of cDNA were determined and were compared with that for a standard DNA (the 16S rRNA gene) that was also analyzed at the same time.…”

“…Because lactate transport across the plasma membrane is the first step in lactate metabolism, lactate permease is a conserved component among a wide variety of lactate-utilizing strains (11,14). The lactate utilization operon is widely present in lactate-utilizing strains, including Pseudomonas strains, Escherichia coli, and Bacillus strains (1,11,13). It is composed of a lactate permease-encoding gene, lactate utilization genes (lldD, lldE, and lutABC), and an operon regulator-encoding gene (12).…”

“…The lactate permease gene, which forms an operon with NAD ϩ -independent lactate dehydrogenases (iLDH) genes and the putative lactate regulator, is widely distributed in microorganisms that utilize lactate as a carbon and energy source (9,10). Lactate permease is an influx protein that imports lactate into cells (1). iLDH genes, which are classified into D-iLDH and L-iLDH depending on their substrate specificities, are responsible for the oxidation of lactate into pyruvate (11).…”