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Title: Sequence analysis of Leuconostoc mesenteroides bacteriophage (phi)1-A4 isolated from industrial vegetable fermentation

Author
item LU, ZHONGJING - Kennesaw State University
item ALTERMANN, ERIC - Agresearch
item Breidt, Frederick
item KOZYAVKIN, SERGEI - Fidelity Systems

Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/20/2010
Publication Date: 3/1/2010
Citation: Lu, Z., Altermann, E., Breidt, F., Kozyavkin, S. 2010. Sequence analysis of Leuconostoc mesenteroides bacteriophage (phi)1-A4 isolated from industrial vegetable fermentation. Applied and Environmental Microbiology. 76(6):1955-1966.

Interpretive Summary: Some disease causing bacteria have been found to be very acid resistant. This would allow the bacteria to survive for extended periods in some foods that are preserved by vinegar and other food acids, if the disease causing bacteria were present. To understand more about these acid resistant disease causing bacteria, we did a survey of acid resistance with isolates of these microorganisms taken from a variety of sources. We examined different aspects of acid resistance with many similar strains, and also examined the genetic relatedness of the strains using DNA fingerprinting mechanisms. We found that strains isolated from beef and cow manure were more acid resistant than similar bacterial strains isolated from rivers and streams, food outbreaks of disease and other sources. This work will help us understand how pathogenic bacteria can threaten the safety of foods protected by vinegar and related food acids.

Technical Abstract: Vegetable fermentations rely on the proper succession of a variety of lactic acid bacteria (LAB) including Leuconostoc mesenteroides. L. mesenteroides initiates the fermentation, producing lactic and acetic acids, CO2, and many flavor compounds. As the fermentation proceeds, L. mesenteroides dies off and other LAB complete the fermentation. Phage infecting L. mesenteroides may significantly influence the die-off of L. mesenteroides. However, no L. mesenteroides phages have been previously sequenced and genetically characterized. Knowledge of more phage genome sequences may provide new insights into phage genomics and phage-host interactions. We have determined the complete genome sequence of L. mesenteroides phage (phi)1-A4, which was 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. Based on N-terminal amino acid sequencing and bioinformatic analyses, putative functions were assigned to 26 ORFs (52%), including 5 ORFs of structural proteins. The phage genome was found to be modularly organized and consist of DNA replication, DNA packaging, head-and-tail morphogenesis, cell lysis, and DNA regulation/modification modules. In silico analyses supported the observation that (phi)1-A4 is a unique lytic phage. A large scale genome inversion (~30% of the genome) was identified by comparison with other phages. The genome inversion encompassed the lysis module, part of the structural protein module and a putative cos-site. Promoter structures were identified that may initiate the transcription of the inverted genome region. Interestingly, the lysin gene was found to be flanked by two holin genes. The tail morphogenesis module was interspersed by cell lysis genes and other genes with unknown functions. The predicted amino acid sequences of the phage proteins showed little similarity with other phages, but functional analyses showed that (phi)1-A4 clusters with several Lactococcus phages. To our knowledge, (phi)1-A4 is the first genetically characterized Leuconostoc mesenteroides phage.