The ecology and genetics of bacteriophages in commercial vegetable fermentations

by Lu, Zhongjing.

LU, ZHONGJING. The ecology and genetics of bacteriophages in commercial vegetable fermentations. (Under the direction of Drs. Henry P. Fleming and Fred Breidt, Jr.) The objectives of this study were to investigate the ecology of bacteriophages (phages) infecting lactic acid bacteria (LAB) in commercial sauerkraut fermentations, to explore the possible role phages may play in microbial succession during the fermentations, to characterize predominant phages in commercial sauerkraut fermentations, to isolate a phage infecting a starter culture, Lactobacillus plantarum MU45, from a commercial cucumber fermentation, and to determine and analyze the complete genome sequence of the L. plantarum phage. A total of 171 independent phage isolates, including at least 26 distinct phages, were obtained from four commercial sauerkraut fermentations (90-ton fermentation tanks) in 2000 and 2001. Host range and the temporal sequence of occurrence of these phages were determined. Twenty-eight distinct bacterial hosts, including Leuconostoc, Lactobacillus, and Weissella species, were identified by ITS restriction and 16S rDNA sequence analyses. It was found that there were two phage-host systems in the fermentations, with the dividing line occurring between day 3 and day 7 after the start of the fermentations, corresponding to the population shift from heterofermentative to homofermentative LAB. The data strongly suggested that phages may play an important role in the microbial ecology and the succession of LAB species in vegetable fermentations. Eight phage isolates which were independently obtained two or more times were further characterized. They belonged to the Myoviridae or Siphoviridae family, and showed distinct host ranges and DNA fingerprints. These results demonstrated for the first time the complex phage ecology present in commercial sauerkraut fermentations, providing new insights into the bioprocess of vegetable fermentations. More research is needed to evaluate the impact of these phages on vegetable fermentations. Additionally, a virulent phage ?JL-1, active against L. plantarum starter cultures, was isolated from a commercial cucumber fermentation. The phage has an isometric head and a long non-contractile tail, and belongs to morphotype B1 within the Siphoviridae family. The host range of phage ?JL-1 was limited to two related strains of L. plantarum, MU45 and BI7. Using L. plantarum MU45 as a host, the phage had an average burst size of 22 and a latent period of 35 min. The phage possesses a linear double-stranded DNA genome consisting of 36,674 bp with a G+C content of 39.4%. Forty-six possible open reading frames (ORFs) were identified. According to the N-terminal amino acid sequencing and bioinformatic analyses, proven or putative functions were assigned to 17 ORFs (39%), including 6 structural protein genes. It was found that functionally related genes were clustered together in the ?JL-1 genome, resulting in a modular genome structure: DNA packaging, head and tail morphogenesis, lysis, DNA replication, and transcriptional regulation modules. This genomic organization was similar to several other phages infecting lactic acid bacteria. An attempt was made to experimentally determine the anti-receptors of phage ?JL-1 via phage display technology. The results from this study indicate that phage infection is common in vegetable fermentations, suggesting that a phage-control strategy will be needed in any vegetable fermentations relying on use of starter cultures. The genetic information obtained from phage ?JL-1 and other phages is fundamental to the understanding of the phage-host interaction in vegetable fermentations and to the development of phage-control strategies for high and consistent quality of fermented vegetable products.