The ftsH gene of the wine bacterium Oenococcus oeni is involved in protection against environmental stress

Abstract

The wine bacterium Oenococcus oeni has to cope with harsh environmental conditions, including an acidic pH, a high alcoholic content, nonoptimal growth temperatures, and growth-inhibitory compounds such as fatty acids, phenolic acids, and tannins. We describe the characterization and cloning of the O. oeni ftsH gene, encoding a protease belonging to the ATP binding cassette protein superfamily. The O. oeni FtsH protein is closest in sequence similarity to the FtsH homologue of Lactococcus lactis. The O. oeni ftsH gene proved to be stress-responsive, since its expression increased at high temperatures or under osmotic shock. O. oeni FtsH protein function was tested in an Escherichia coli ftsH mutant strain, and consistent with the O. oeni ftsH gene expression pattern, the O. oeni FtsH protein provided protection for the E. coli ftsH mutant against heat shock. O. oeni and Bradyrhizobium japonicum FtsH proteins also triggered E. coli resistance to wine toxicity. Genes homologous to O. oeni ftsH were detected in many other lactic acid bacteria found in wine, suggesting that this type of gene constitutes a well-conserved stress-protective molecular device.

FIG. 1.

Restriction mapping of the O. oeni ftsH gene. (A) Southern blots were hybridized with either the ftsH probe (left and middle panels) or probe 2 (right panel). The O. oeni genomic DNA was digested with the following enzymes: lane 1, EcoRI; lane 2, BamHI; lane 3, HindIII; lane 4, EcoRI and BamHI; lane 5, EcoRI and HindIII; lanes 6, SalI; lanes 7, EcoRV; lanes 8, EcoRI and SalI. Lanes M, molecular size markers (in base pairs). (B) Physical map of the O. oeni ftsH gene. Restriction enzyme sites: EI, EcoRI; EV, EcoRV; H, HindIII; S, SalI. The location of the ftsH probe is indicated by a grey box above the ftsH gene; the location of probe 2 is indicated by a hatched box below the ftsH gene. Distances between restriction sites are indicated.

FIG. 2.

Comparison of the amino acid sequences of FtsH proteins from O. oeni, L. lactis, B. japonicum, and E. coli. The first residue in each row is numbered. A asterisk above the sequences indicates a perfect consensus between the four protein sequences. A cross indicates three matching residues among the four sequences. The predicted transmembrane α-helices of FtsH proteins are underlined. The Walker A and B motifs are indicated. The SRH motif is indicated and appears in boldface. The boxed HEAGH domain is the zinc binding motif. Dashes indicate gaps introduced to optimize the alignment.

FIG. 2.

Comparison of the amino acid sequences of FtsH proteins from O. oeni, L. lactis, B. japonicum, and E. coli. The first residue in each row is numbered. A asterisk above the sequences indicates a perfect consensus between the four protein sequences. A cross indicates three matching residues among the four sequences. The predicted transmembrane α-helices of FtsH proteins are underlined. The Walker A and B motifs are indicated. The SRH motif is indicated and appears in boldface. The boxed HEAGH domain is the zinc binding motif. Dashes indicate gaps introduced to optimize the alignment.

FIG. 3.

The O. oeni ftsH gene is a stress-responsive gene. The induction of O. oeni ftsH gene expression after heat or osmotic shocks was analyzed by RT-PCR. The RT-PCR patterns were obtained with cDNA from the RNA of O. oeni IOEB 8406 extracted after 3 h of application of the indicated thermal or osmotic stress. The gene-specific primers that were used are indicated on the bottoms of the panels.

FIG. 4.

Growth complementation of an E. coli ΔftsH mutant by the O. oeni ftsH gene. The E. coli ΔftsH strain AR3291 transformed with pCR-XL-TOPO (diamonds) or with pJPB65 (squares) was grown in LB medium at either 25°C (open symbols) or 37°C (closed symbols). OD₆₀₀, optical density at 600 nm.

FIG. 5.

The O. oeni and B. japonicum ftsH genes protect E. coli from wine toxicity. The E. coli ΔftsH strain AR3291 was transformed with control plasmids (pCR-XL-TOPO [A] or pBAD18-Cm [B]) (light-gray bars) and O. oeni or B. japonicum ftsH gene-containing plasmids (pJPB65 [A] and pRJ5188 [B], respectively) (dark-gray bars). After 3 h of incubation at 25°C with the indicated concentrations of wine, the bacterial cultures were serially diluted and plated. Colonies were counted, and the survival ratios (ratios of CFU observed at a given concentration of wine to those observed without added toxic compounds) were calculated.

FIG. 6.

ftsH homologues in other wine lactic acid bacteria. Southern hybridization of the ftsH probe to chromosomal DNAs from various bacteria is shown. The genomic DNAs were digested before transfer with either the EcoRI (lanes E) or HindIII (lanes H) enzymes. Lanes M, molecular size markers (in kilobase pairs). Lanes: 1, Lactobacillus fructivorans ATCC 8288; 2, P. pentosaceus ATCC 33326; 3, P. dextrinicus ATCC 33087; 4, Lactobacillus plantarum IOEB 9106; 5, P. parvulus ATCC 19371; 6, Lactobacillus buchneri ATCC 11305; 7, P. damnosus ATCC 25248; 8, Lactobacillus hilgardii IOEB 9101; 9, Lactobacillus brevis ATCC 14869; 10, Lactobacillus delbrueckii ATCC 9649; 11, Leuconostoc mesenteroides IOEB 8293.

FIG.7.

PCR amplification of sequences from various lactic acid bacteria, homologous to the ABC domain of ftsH. The amplified sequences were aligned and compared. oeni, O. oeni; mesen, Leuconostoc mesenteroides; delbru, Lactobacillus delbrueckii; buch, Lactobacillus buchneri; hilg, Lactobacillus hilgardii; acidi, P. acidilactici ATCC 8042; pento, P. pentosaceus; damno, P. damnosus; fruct, Lactobacillus fructivorans; plant, Lactobacillus plantarum; saliv, Lactobacillus salivarius ATCC 11740; dextri, P. dextrinicus; lactis, L. lactis. An asterisk below the sequences indicates a perfect consensus between the protein sequences. Dots indicate conservative substitutions. The Walker B motif and the SRH domain are indicated. The consensus SRH sequence is outlined above the black bar. Dashes indicate gaps introduced to optimize the alignment.