Roles of Glucitol in the GutR-mediated Transcription Activation Process in Bacillus subtilis

Abstract

GutR is a 95-kDa glucitol-dependent transcription activator that mediates the expression of the Bacillus subtilis glucitol operon. Glucitol allows GutR to bind tightly to its binding site located upstream of the gut promoter. In this study, a second functional role of glucitol is identified. Glucitol induces GutR to change its conformation and triggers GutR to bind ATP efficiently. After sequential binding of glucitol and ATP to GutR, GutR adopts a new conformation by forming a compact structure that is resistant to trypsin digestion. Under this condition, the ATP⅐glucitiol⅐GutR complex can dissociate slowly from the gutR-binding site (t1 ⁄2 ‫؍‬ 274 min). Interestingly, if ATP in the ATP⅐glucitiol⅐GutR complex is replaced by ADP, GutR adopts another conformation and can dissociate from the gutR-binding site even faster (t1 ⁄2 ‫؍‬ 82 min). In all these GutR-DNA binding studies in the presence of different ligands (glucitol, ATP, or ADP), only the off-rate is affected. The vital role of ATP in the GutRmediated transcription activation process is reflected by the poor transcription from the gut promoter with GutR(D285A) which has a mutation in the motif B of the putative ATP-binding site. A working model for this transcription activation process is presented.

STAND, a Class of P-Loop NTPases Including Animal and Plant Regulators of Programmed Cell Death: Multiple, Complex Domain Architectures, Unusual Phyletic Patterns, and Evolution by Horizontal Gene Transfer

“…Consistent with these observations, the size of the products of limited proteolysis of two STAND ATPases, GutR and MalT, was compatible with cleavage occurring between the GxP module and the C-terminal (predicted) helical domain. 32,33 Thus, the GxP module in most STAND NTPases appears to be followed by a distinct helical domain, which we named the helical third domain of STAND proteins (HETHS) domain ( Figure 4). Database searches with the HETHS domain PSSM did not detect significant similarity to any other known protein domains.…”

“…The red arrowhead indicates the site of a tryptic digestion site in GutR. 32 Sequences are identified with protein name and an organism name abbreviation. Open reading frames have been labeled with the identifier from the /gene, /allele, or /locus_tag field in the GenBank sequence record (where present).…”

“…Among the bacterial AP-ATPases, AfsR is a transcription factor involved in both regulation of secondary metabolism and in morphological differentiation in Streptomyces, 43,44 whereas the Bacillus subtilis GutR protein regulates expression of the glucitol dehydrogenase gene GutB. 32,45 Prokaryotic AP-ATPases are represented mostly in Actinobacteria and Cyanobacteria, and sporadically in other bacterial and archaeal lineages ( Table 2). Among eukaryotes, APATPases are found in animals and plants, and in fungi with large genomes, such as Neurospora, Aspergillus and Magnaporthe.…”

Insights from the architecture of the bacterial transcription apparatus

“…They typically contain superstructure-forming repeat domains, such as the WD and TPR domains, which may serve as surfaces for the assembly of multi-protein complexes (Leipe et al, 2004). The archetypal members of the architectural class combining a DNA-binding HTH and STAND NTPases are the E. coli MalT (Larquet et al, 2004; Marquenet and Richet, 2010), B. subtilis GutR (Poon et al, 2001) and Streptomyces AfsR proteins (Lee et al, 2002). The DNA-binding HTH domains in these proteins are of several distinct types.…”

Sequence — Evolution — Function

“…Functional information on bacterial homologs of eukaryotic apoptotic proteins is scarce. For the AP-ATPase homologs, the only available data point to a role of some of these proteins, such as GutR from B. subtilis and AfsR from S. coelicolor, in transcription regulation [233,681]. The function of GutR, which is a regulator of the glucitol operon, has been shown to be ATP-dependent [681].…”