Fructose Utilization inLactococcus lactisas a Model for Low-GC Gram-Positive Bacteria: Its Regulator, Signal, and DNA-Binding Site

Abstract

In addition to its role as carbon and energy source, fructose metabolism was reported to affect other cellular processes, such as biofilm formation by streptococci and bacterial pathogenicity in plants. Fructose genes encoding a 1-phosphofructokinase and a phosphotransferase system (PTS) fructose-specific enzyme IIABC component reside commonly in a gene cluster with a DeoR family regulator in various gram-positive bacteria. We present a comprehensive study of fructose metabolism in Lactococcus lactis, including a systematic study of fru mutants, global messenger analysis, and a molecular characterization of its regulation. The fru operon is regulated at the transcriptional level by both FruR and CcpA and at the metabolic level by inducer exclusion. The FruR effector is fructose-1-phosphate (F1P), as shown by combined analysis of transcription and measurements of the intracellular F1P pools in mutants either unable to produce this metabolite or accumulating it. The regulation of the fru operon by FruR requires four adjacent 10-bp direct repeats. The well-conserved organization of the fru promoter region in various low-GC gram-positive bacteria, including CRE boxes as well as the newly defined FruR motif, suggests that the regulation scheme defined in L. lactis could be applied to these bacteria. Transcriptome profiling of fruR and fruC mutants revealed that the effect of F1P and FruR regulation is limited to the fru operon in L. lactis. This result is enforced by the fact that no other targets for FruR were found in the available low-GC gram-positive bacteria genomes, suggesting that additional phenotypical effects due to fructose metabolism do not rely directly on FruR control, but rather on metabolism.Carbohydrate utilization systems are of particular importance to provide carbon and energy to bacteria. Among them, glucose and lactose systems have been widely studied, while other sugar utilization systems have been studied mostly with regard to their implication in targeted processes. Considering fructose availability in most ecosystems associated with plants, fructose metabolism and its regulation received little attention to date. The utilization of fructose is best documented in Escherichia coli, with the existence of three routes. In the main route, fructose is taken up via the membrane-spanning protein FruA and concomitantly phosphorylated to fructose-1-phosphate. Phosphorylation takes place by transfer of the phosphate group from phosphoenolpyruvate to fructose, which involves concerted action of two cytoplasmic proteins, EI of the phosphotransferase system (PTS) and a membrane-associated diphosphoryl transfer protein (FruB). The fructose-1-phosphate thus formed is further phosphorylated by ATP and 1-phosphofructokinase (FruK) to fructose-1,6-bisphosphate (17). The fruBKA operon of enteric bacteria is regulated at the transcriptional level primarily by the catabolite repressor-activator Cra (previously designated FruR) and by the cyclic AMP-CRP complex, which plays a secondary role (9,25).Although non-PTS...

GlpR Represses Fructose and Glucose Metabolic Enzymes at the Level of Transcription in the HaloarchaeonHaloferax volcanii

“…S1A in the supplemental material). The DeoR/GlpR protein family is widespread among bacteria, and its members often serve as transcriptional repressors (3,16,28,34,41,44) or activators (14) of either sugar or nucleoside metabolism. DeoR/GlpR-type repressors are composed of approximately 250 amino acids, possess a helix-turn-helix DNA-binding motif near the amino terminus, and generally bind a sugar phosphate effector molecule of the relevant metabolic pathway in the C-terminal portion of the protein, which can also contain oligomerization domains (43).…”

“…SD sites are indicated by a line above the DNA sequence, and the translational start codon is double underlined. An inverted hexameric repeat with a consensus sequence, TCSnCn (3)(4) SSnGGA (where S is C or G and n is any nucleotide base), was found common to the glpR-pfkB and kdgK1 promoter regions. Inverted repeats are indicated, with arrows displaying directionality.…”

“…Desalted oligonucleotides were from Integrated DNA Technologies (Coralville, IA). 2Ј-Deoxyuridine-5Ј-triphosphate coupled by an 11-atom spacer to digoxigenin (DIG) (DIG-11-dUTP), alkaline phosphataseconjugated antibody raised against DIG, disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2Ј-(5Ј-chloro)tricyclo[3.3.1.1 3,7 ]decan}-4-yl) phenyl phosphate (CSPD), and the other DIG-related biochemicals were from Roche Molecular Biochemicals (Indianapolis, IN). Positively charged membranes for Southern hybridization were from Ambion (Austin, TX).…”

“…Next, inverted repeats located near these elements were aligned to identify the potential GlpR binding site(s). With this approach, an inverted hexameric repeat, TCSnCn (3)(4) SSnGGA (where S is FIG. 4.…”

Lactose and β-Glucosides Metabolism and Its Regulation in Lactococcus lactis: A Review

“…It exerts its regulatory role in carbon catabolite repression (CCR) by binding to DNA sites called cres, which occur in the vicinity of CcpAregulated genes (Weickert & Chambliss, 1990). In L. lactis the known targets of CcpA are the gal operon for galactose utilization (Luesink et al, 1998), the fru operon for fructose utilization (Barrière et al, 2005), the ptcABC operon for cellobiose utilization , and cel-lac genes for cellobiose and lactose utilization (Aleksandrzak-Piekarczyk et al, 2011). Thus, one could speculate that in L. lactis IL1403 cellobiose-inducible chromosomal alternative lactose utilization genes are under the negative control of CcpA, and, therefore, inactivation of the ccpA gene could result in their derepression and ability to assimilate lactose by the IL1403 ccpA mutant.…”