Fructose utilization in Lactococcus lactis as 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.

FIG. 1.

(A) Genetic organization of the L. lactis IL1403 fructose operon. fruR encodes a protein homologous to a transcriptional regulator, fruC, a 1-phosphofructokinase, and fruA, a fructose-specific enzyme II (EIIABC components). The transcriptional start point (+1) determined by 5′-RACE and the corresponding −10 and −35 boxes are indicated and underlined. A putative rho-independent terminator is indicated by a circle. A putative CRE box is shown in bold. The primers lacR1 and fruA2 are shown by arrows. (B) Northern blot with fruR (lacR2-lacR3) and fruCA (lacC1-fruA2) probes and, right panel, PCRs on the fructose gene transcripts using primers complementary to fruR (lacR2) and fruA (fruA2). RNAs were prepared from IL1403 cells grown in CDM-glucose (lane 1), CDM-fructose (lanes 2, A, and B), and CDM-glucose-fructose (lane 3). Lane L, Smart ladder (Promega), sizes of which are indicated in the left margin; lane A, PCR on 500 ng of RNA; lane B, RT-PCR on 500 ng of RNA diluted 40-fold. The asterisks on the left of the Northern blot indicate the positions of 16S and 23S RNAs that produced slightly artifactual bands. Arrows to the right of the panel indicate the position of the expected band corresponding to the fruRCA transcript (3.7 kb) and RT-PCR product (3 kb). (C) Schematic representation of inserted constructions in the fru operon and the corresponding strains used in this study. The promoter of the fru operon (P_fru) is indicated by an arrow. A putative rho-independent terminator is indicated by a circle. Genes of the fru operon are shown either in black when they are intact or in white when they are inactivated. The inserted genes are represented in gray. The pGEM-T plasmid is indicated by a line and a circle in either full features, when it is present in all the constructions, or in stippled features when it is not present in all the constructions.

FIG. 2.

Differential gene expression plots in the fruR (A) and the fruC (B) mutants compared to the wild-type strain. Relative signal intensities (mean normalized quanta) are plotted for genes as measured for the wild-type (x axis) and mutant (y axis) strains. The names of the genes that differ significantly (based on a threefold and a z-test [see Materials and Methods]) in the strains are indicated. Of these, only fruR, fruC, and fruA were induced more than threefold. In the fruR mutant their corresponding ratio and P values are 45 and 4 × 10⁻²⁹ for fruR, 38 and 3 × 10⁻²⁰ for fruC, and 21 and 8 × 10⁻²³ for fruA. In the fruC mutant their corresponding ratio and P values are 23 and 1 × 10⁻⁴⁹ for fruR, 41 and 8 × 10⁻⁶⁹ for fruC, and 5 and 4 × 10⁻¹⁴ for fruA.

FIG. 3.

Alignment of the promoter regions of L. lactis fruR (A) and lacR and lacA (B) with the corresponding homologues in Streptococcus agalactiae NEM316 (accession no. AL732656), Streptococcus pneumoniae R6 (accession no. AE007317), S. mutans UA159 (accession no. AE014133), Streptococcus pyogenes MGAS315 (accession no. AE014074), S. gordonii Challis NCTC 7868 (http://tigrblast.tigr.org), Staphylococcus aureus Mu50 (accession no. BA000017), Staphylococcus epidermidis ATCC 12228 (accession no. AE015929), Bacillus cereus ATCC 14579 (accession no. AE016877), B. subtilis 168 (accession no. AL009126), Listeria monocytogenes EGD-e (accession no. NC_003210), Enterococcus faecalis VE583 (accession no. AE016830), and Lactobacillus plantarum WCFS1 (accession no. AL935263). IIA indicates that the corresponding genes encode a PTS enzyme IIA component. The putative −10 and −35 regions are underlined. The FruR and LacR operators are boxed, and direct repeats are represented by horizontal arrows. Regions protected against DNase I cleavage by the LacR repressor in lacR and lacA genes of L. lactis are framed (29). Bases in bold are those conserved in the determined consensus sequence. Between panels A and B are indicated the potential consensus sequences recognized by FruR and LacR as determined by the alignments of the 48 and 30 presented repeats, respectively. The similarities and the significant differences are indicated by a colon and an X, respectively.

FIG. 4.

Schematic representation of the modified fru operon promoter sequences used to characterize the DNA-binding motif recognized by FruR. The promoter region inserted upstream of the fru operon is shown in the upper panel. The putative binding site of FruR is in bold. Strains were obtained by integration in the IL1403 chromosome of the plasmid pJIM2374 carrying pGEM-T, and the promoter regions amplified by the primers are indicated by arrows. Conserved sequences are shown by dotted lines, and TG→AA mutations in the M4 primer are indicated. The −10 and −35 regions are underlined. + or - at the right of the fragments indicate that the ratios of expression, as measured by QRT-PCR with lacC1 and lacC2 primers (complementary to the fruC gene) in cells grown in CDM with trehalose and fructose versus those grown in CDM-trehalose, are similar to that of the wild-type strain or close to 1, respectively.