Correlation of the capsular phenotype in Propionibacterium freudenreichii with the level of expression of gtf, a unique polysaccharide synthase-encoding gene

Abstract

Many food-grade bacteria produce exopolysaccharides (EPS) that affect the texture of fermented food products and that may be involved in probiotic properties. Propionibacterium freudenreichii is a Gram-positive food-grade bacterium with reported probiotic capabilities that is widely used as starter in Swiss-type cheese. In this study, 68 strains of P. freudenreichii were screened for the beta-glucan capsular phenotype by immunoagglutination with a specific antibody and for the presence of the gtf gene coding for polysaccharide synthase. All strains were positive for PCR amplification with gtf gene-specific primers, but the presence of beta-glucan capsular EPS was detected for only 35% of the strains studied. Disruption of gtf in P. freudenreichii revealed that gtf is a unique gene involved in beta-glucan capsular EPS production in P. freudenreichii. The gtf gene was transferred into and expressed in Lactococcus lactis, in which it conferred an agglutination-positive phenotype. Expression of the gtf gene was measured by performing quantitative reverse transcription-PCR assays with RNA from four capsular and three noncapsular strains. A positive correlation was found between the beta-glucan capsular phenotype and gtf gene expression. Sequencing of the region upstream of the gtf open reading frame revealed the presence of an insertion element (IS element) in this upstream region in the four strains with the beta-glucan capsular phenotype. The role of the IS element in the expression of neighboring genes and its impact on interstrain variability of the P. freudenreichii capsule phenotype remain to be elucidated.

FIG. 1.

Microscopic observations of the agglutination-positive TL 176 and agglutination-negative TL 20 strains of P. freudenreichii. (A and B) P. freudenreichii TL 20 (A) and TL 176 (B) suspended in UF retentate of skim milk and observed by light microscopy. The white halo surrounding cells corresponds to capsular EPS. (C and D) Strains TL 20 (C) and TL 176 (D) analyzed by transmission electron microscopy. (E and F) Strains TL 20 (E) and TL 176 (F) examined by scanning electron microscopy.

FIG. 2.

Immunoagglutination test performed with S. pneumoniae type 37-specific antiserum. The agglutination test was performed with P. freudenreichii TL 34 (A) in the absence of the antiserum and (B) in the presence of the antiserum and with the P. freudenreichii TL 34 Δgtf mutant (C) in the presence of the antiserum and (D) in the absence of the antiserum. The agglutination test was also performed with L. lactis IL1403(pMG36C:gtf) before addition of the antiserum (E) and after addition of the antiserum (F) and with L. lactis IL1403(pMG36C) after the addition of the antiserum (G).

FIG. 3.

Levels of expression of the gtf gene in agglutination-positive and agglutination-negative strains of P. freudenreichii. The levels of expression in both the exponential and stationary growth phases are indicated by gray and black bars, respectively. Strain numbers are indicated on the x axis, and the agglutination-positive strains are underlined. (A) Absolute levels of expression expressed as copy number for 100 ng of RNA; (B) relative levels of expression after geNorm normalization using groL1 and groL2 as normalization genes.

FIG. 4.

Organization of the upstream region of the gtf CDS in agglutination-negative (A) and agglutination-positive (B) strains of P. freudenreichii. The gtf gene is indicated by a black arrow. The position of the start codon (ATG) is indicated. Transposase genes are indicated by gray arrows. Regions conserved in all the strains analyzed are indicated by cross-hatched boxes and are linked with lines.