Sequence Analysis of Leuconostoc mesenteroides Bacteriophage Φ1-A4 Isolated from an Industrial Vegetable Fermentation

Abstract

Vegetable fermentations rely on the proper succession of a variety of lactic acid bacteria (LAB). Leuconostoc mesenteroides initiates fermentation. As fermentation proceeds, L. mesenteroides dies off and other LAB complete the fermentation. Phages infecting L. mesenteroides may significantly influence the die-off of L. mesenteroides. However, no L. mesenteroides phages have been previously genetically characterized. Knowledge of more phage genome sequences may provide new insights into phage genomics, phage evolution, and phage-host interactions. We have determined the complete genome sequence of L. mesenteroides phage ⌽1-A4, isolated from an industrial sauerkraut fermentation. The phage possesses a linear, double-stranded DNA genome consisting of 29,508 bp with a G؉C content of 36%. Fifty open reading frames (ORFs) were predicted. Putative functions were assigned to 26 ORFs (52%), including 5 ORFs of structural proteins. The phage genome was modularly organized, containing DNA replication, DNA-packaging, head and tail morphogenesis, cell lysis, and DNA regulation/modification modules. In silico analyses showed that ⌽1-A4 is a unique lytic phage with a large-scale genome inversion (ϳ30% of the genome). The genome inversion encompassed the lysis module, part of the structural protein module, and a cos site. The endolysin gene was flanked by two holin genes. The tail morphogenesis module was interspersed with cell lysis genes and other genes with unknown functions. The predicted amino acid sequences of the phage proteins showed little similarity to other phages, but functional analyses showed that ⌽1-A4 clusters with several Lactococcus phages. To our knowledge, ⌽1-A4 is the first genetically characterized L. mesenteroides phage.Bacteriophages are the most abundant biological entities (estimated to be on the order of Ն10 31 ) on the planet (9, 18). Phages are ubiquitous in nature and can influence the microbial ecology and genetics of bacteria. Because of their small (usually Ͻ60 kb) genomes, phages can provide an excellent model system for studying many biological processes, including DNA replication and genetic evolution. Despite this, many phages remain uncharacterized. Very little is known about phage diversity and phage-host interactions owing to the small number of sequenced phages. Furthermore, the existing phage sequence database is highly biased toward a limited spectrum of phage hosts, namely, Enterobacteriaceae, Bacillus, Staphylococcus, Pseudomonas, Vibrio cholerae, Lactococcus, Streptococcus thermophilus, and S. pyogenes. The majority of host species for sequenced phages are either pathogenic or dairy-related bacteria. Most of the newly sequenced phage genes have no assigned functions or matches in the GenBank database (7).Vegetable fermentations rely on a variety of lactic acid bacteria (LAB). The proper succession of LAB directly determines the quality and safety of the final fermentation products. Leuconostoc mesenteroides initiates most vegetable fermentations. It converts the sugars in vegetables...

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