Characterization of Lactobacillus coryniformis DSM 20001(T) Surface Protein Cpf Mediating Coaggregation with and Aggregation among Pathogens

Phenotypic characterization of aggregation phenotypes of Lactobacillus coryniformis revealed that strain DSM 20001(T) coaggregated with Escherichia coli K88, Campylobacter coli, and Campylobacter jejuni but not with other human pathogens. In addition, cells of these pathogens aggregated in the presence of the spent culture supernatant (SCS) of strain DSM 20001(T). Cells of E. coli K88 remained viable in the coaggregates and aggregates for up to 24 h. Both coaggregation and aggregation (co/aggregation) occurred at pH 3.5 to 7.5 and was sensitive to heat (85°C for 15 min) and proteinase K. The co/aggregation-promoting factor (Cpf) was purified, and the gene was identified by PCR with degenerate primers derived from internal amino acid sequences. The cpf gene encoded a 19.9-kDa preprotein with a sec-dependent leader and an isoelectric point of 4.4. The amino acid sequence had no significant similarity to proteins with known functions. Northern analysis revealed not only major transcription from the promoter of cpf but also major transcription from the promoter of the preceding insertion element, ISLco1 belonging to the IS3 family. Recombinant Cpf produced in E. coli mediated aggregation of pathogens comparable to the aggregation obtained with purified Cpf or SCS of strain DSM 20001(T). Cpf could be removed from cells of strain DSM 20001(T) by treatment with 5 M LiCl and could be subsequently reattached to the cell surface by using SCS or recombinant Cpf, which resulted in restoration of the co/aggregation property. These results together with those of the amino acid sequence analysis suggest that Cpf is a novel surface protein of L. coryniformis that mediates co/aggregation of some pathogens

Identification and Population Dynamics of Yeasts in Sourdough Fermentation Processes by PCR-Denaturing Gradient Gel Electrophoresis

Four sourdoughs (A to D) were produced under practical conditions, using a starter obtained from a mixture of three commercially available sourdough starters and baker's yeast. The doughs were continuously propagated until the composition of the microbiota remained stable. A fungi-specific PCR-denaturing gradient gel electrophoresis (DGGE) system was established to monitor the development of the yeast biota. The analysis of the starter mixture revealed the presence of Candida humilis, Debaryomyces hansenii, Saccharomyces cerevisiae, and Saccharomyces uvarum. In sourdough A (traditional process with rye flour), C. humilis dominated under the prevailing fermentation conditions. In rye flour sourdoughs B and C, fermented at 30 and 40°C, respectively, S. cerevisiae became predominant in sourdough B, whereas in sourdough C the yeast counts decreased within a few propagation steps below the detection limit. In sourdough D, which corresponded to sourdough C in temperature but was produced with rye bran, Candida krusei became dominant. Isolates identified as C. humilis and S. cerevisiae were shown by randomly amplified polymorphic DNA-PCR analysis to originate from the commercial starters and the baker's yeast, respectively. The yeast species isolated from the sourdoughs were also detected by PCR-DGGE. However, in the gel, additional bands were visible. Because sequencing of these PCR fragments from the gel failed, cloning experiments with 28S rRNA amplicons obtained from rye flour were performed, which revealed Cladosporium sp., Saccharomyces servazii, S. uvarum, an unculturable ascomycete, Dekkera bruxellensis, Epicoccum nigrum, and S. cerevisiae. The last four species were also detected in sourdoughs A, B, and C