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ARS Home » Pacific West Area » Albany, California » Western Regional Research Center » Produce Safety and Microbiology Research » Research » Publications at this Location » Publication #350602

Research Project: Ecology and Detection of Human Pathogens in the Produce Production Continuum

Location: Produce Safety and Microbiology Research

Title: Identification of novel bacteriophage peptides using a combination of gene sequence LC-MS-MS analysis and BLASTP

item Harden, Leslie - Les
item Liao, Yen-Te
item Wu, Vivian

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 3/6/2018
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: Introduction: In an effort to characterize novel bacteriophage with lytic activity against pathogenic E.coli associated with foodborne illness, gene sequencing and mass spectrometry have been used to identify expressed peptides which differentiate isolated bacteriophage from other known phage. Here, we report on two bacteriophage isolated from distinctly different sources. One from fresh surface water, the other from a municipal compost operation approximately 60 miles distant. LC-MS-MS analysis of digested proteins was searched against an unannotated nucleotide sequence generated in house. Subsequent BLASTP searches of identified peptides revealed major capsid peptides which are novel to our isolates when compared to peptide sequences in the public domain. Methods: Bacteriophage were isolated using an agar overlay method with O145 Shigatoxin producing E. coli as target hosts. Plaques were subcultured in broth containing the target host strains. Two bacteriophage from different sources (water and municipal compost) were concentrated by PEG precipitation and isolated with a Cesium Chloride gradient. Phage isolates were treated with ß-mercaptoethanol in Laemmli buffer prior to 1D SDS PAGE gel electrophoresis. Visible bands were excised from the stained gel and subjected to trypsin digestion. Digests were subjected to reverse phase LC-MS-MS analysis with an Orbitrap Elite Mass Spectrometer. Mascot was used to compare mass spectral data to an in house generated nucleotide assembly. BLASTP was used to identify similar peptides in the NCBI protein database. Preliminary Data: Mascot search of our LC-MS-MS data against public domain protein databases identified multiple peptides with homology to known Escherichia bacteriophage K1’s putative major capsid proteins. Sequence coverage of 31% for the compost isolate and 52% for the water isolate were obtained. The use of in house generated gene sequence allowed us to identify additional peptides not in the NCBI protein database. Subsequent BLAST searches of our newly found peptides showed similarities to other phage active against the O145 hosts. More importantly, the BLASTP searches revealed amino acid sequence differences novel to our phage isolates. Five sites of sequence variation were identified in the major capsid protein when compared to results from the NCBI protein database. Of the five sites of variation, three were common to both our isolates, while two were found in only one of the two isolates. Additional work is ongoing to identify novel characteristics in other proteins expressed in our isolates. Novel Aspects: Combined use of Gene sequence, LC-MS-MS analysis and BLASTP to identify novelty in expressed peptides.