Characterization and Genome Study of a Newly Isolated Temperate Phage Belonging to a New Genus Targeting Alicyclobacillus acidoterrestris

Abstract

The spoilage of juices by Alicyclobacillus spp. remains a serious problem in industry and leads to economic losses. Compounds such as guaiacol and halophenols, which are produced by Alicyclobacillus, create undesirable flavors and odors and, thus, decrease the quality of juices. The inactivation of Alicyclobacillus spp. constitutes a challenge because it is resistant to environmental factors, such as high temperatures, and active acidity. However, the use of bacteriophages seems to be a promising approach. In this study, we aimed to isolate and comprehensively characterize a novel bacteriophage targeting Alicyclobacillus spp. The Alicyclobacillus phage strain KKP 3916 was isolated from orchard soil against the Alicyclobacillus acidoterrestris strain KKP 3133. The bacterial host’s range and the effect of phage addition at different rates of multiplicity of infections (MOIs) on the host’s growth kinetics were determined using a Bioscreen C Pro growth analyzer. The Alicyclobacillus phage strain KKP 3916, retained its activity in a wide range of temperatures (from 4 °C to 30 °C) and active acidity values (pH from 3 to 11). At 70 °C, the activity of the phage decreased by 99.9%. In turn, at 80 °C, no activity against the bacterial host was observed. Thirty minutes of exposure to UV reduced the activity of the phages by almost 99.99%. Based on transmission-electron microscopy (TEM) and whole-genome sequencing (WGS) analyses, the Alicyclobacillus phage strain KKP 3916 was classified as a tailed bacteriophage. The genomic sequencing revealed that the newly isolated phage had linear double-stranded DNA (dsDNA) with sizes of 120 bp and 131 bp and 40.3% G+C content. Of the 204 predicted proteins, 134 were of unknown function, while the remainder were annotated as structural, replication, and lysis proteins. No genes associated with antibiotic resistance were found in the genome of the newly isolated phage. However, several regions, including four associated with integration into the bacterial host genome and excisionase, were identified, which indicates the temperate (lysogenic) life cycle of the bacteriophage. Due to the risk of its potential involvement in horizontal gene transfer, this phage is not an appropriate candidate for further research on its use in food biocontrol. To the best of our knowledge, this is the first article on the isolation and whole-genome analysis of the Alicyclobacillus-specific phage.

Biological Characterization and Genomic Analysis of Three Novel Serratia- and Enterobacter-Specific Virulent Phages

“…showed activity after 24 h of incubation at 85 • C [58]. Different results were observed in a study conducted by Shymialevich and co-workers [37]. The lysogenic Alicyclobacillus phage KKP 3916 retained high activity in the temperature range from 4 • C to 30 • C and pH from 3 to 11 [37].…”

“…Different results were observed in a study conducted by Shymialevich and co-workers [37]. The lysogenic Alicyclobacillus phage KKP 3916 retained high activity in the temperature range from 4 • C to 30 • C and pH from 3 to 11 [37]. While A. acidoterrestris grows in a strongly acidic environment and its optimal growth temperature is 42-53 • C, it may also range 35-55 • C [59,60].…”

“…The difficulty in eliminating S. liquefaciens , S. marcescens and E. cloacae lies in the ability of these bacteria to secrete extracellular polymers, which allows the creation of dense biofilms that protect bacterial cells against environmental factors and hinders their elimination from raw materials and surfaces of food production lines [ 21 , 35 , 36 ]. A promising technique for extending the shelf life of food is the use of strictly lytic bacteriophages or their enzymes, which do not affect the nutritional value of the product and do not change the sensory characteristics [ 37 , 38 , 39 ]. Before practical application in biocontrol or phage therapy, a comprehensive characterization of bacteriophages is needed.…”