Alessandro Cestaro

Andrea Goss

Andrea Mancini

Andrea Merz

Elena Franciosi

Nicola Cologna

Paolo Sonego

Stefano Piazza

English

Archivio istituzionale della ricerca - Fondazione Edmund Mach

Introduction	 Methods	Discussion	 Conclusion	Acknowledgments and funding	Contact: andrea.mancini@fmach.it	Trentingrana, a Protected Designation of Origin (PDO) hard Grana-like cheese produced in the north of Italy, is characterized by the use of a natural whey starter culture (NWS) in the cheese making process. These NWSs greatly influence the peculiarity and organoleptic characteristics of this cheese and are mainly characterized by thermophilic lactic acid bacterias (LABs) as Lactobacillus helveticus. The presence of phage in NWSs could determine a loss of acidification activity inducing as a consequence slow and/or incomplete fermentation, incomplete whey purge from the curd, growth of pathogenic or spoilage bacterias, all factors that negatively affect the quality or the yield of the final product.  Only few studies have characterized phages derived from Grana-like cheeses. Herein, seven Lactobacillus helveticus phages, isolated from Trentingrana NWS over one year of cheese production, were deeply characterized by focusing on their host range and morphology, as well as their genomic comparison.	Phages in Trentingrana cheeses’ natural whey starters: new insights from characterization and genomic comparison Andrea Mancini1, Alessandro Cestaro2, Paolo Sonego2, Stefano Piazza3, Nicola Cologna4, Andrea Goss4, Andrea Merz4, Elena Franciosi1  1 Biotechnology of Natural Products Unit, Research and Innovation Centre, Fondazione Edmund Mach, San Michele all’Adige, Trento, Italy 2 Computational Biology Unit, Research and Innovation Centre, Fondazione Edmund Mach, San Michele all’Adige, Trento, Italy 3 Plant Biotechnology Unit, Research and Innovation Centre, Fondazione Edmund Mach, San Michele all’Adige, Trento, Italy 4 Trentingrana Consorzio dei Caseifici Sociali Trentini s.c.a., Trento, Italy Considered phage φCR28, φCR191, φP185, φCV244, φS16, φS193 and φT280, were isolated from five dairy implants in the Trentino province    host range determination aginst 67 L. helveticus strains  phenotipic characterization Transmission Electron Microscophy genome sequencing  (Oxford Nanopore Tecnologies) gene annotation using PHAROKKA and UniProt phylogeny based on ANI and ViPTree ORFs determination 3 Mitomycin C lisogeny induction assay 1 2 Results	2 According to the new virus classification, genomic sequence information classified these phages as part of the Caudoviricetes class (myovirus phage type), with an average genome size of 36.792 bp and an overall average G+C content of 36.98%. For each of our phages, the first hit of genome similarity through BLASTn concerned L. helveticus phage φAQ113 (NC_019782.1), taken as reference genome in this study.  A	phage_CR28 [39,536 nt] B	The host range of these phages was narrow and limited to sensitive bacilli. Bacterial hosts of the isolated phages seems to be particularly sensitive to phage infection, as a majority of phage host ranges was observed towards them, with the exception of L. heveticus CR191 and S193.  By TEM phages have been assigned to the Caudoviricetes class, linked to the myovirus (φCR28 [A], φCR191 [B], φP185 [C], φS16 [D], φS193 [E], φT180 [F]) and siphovirus (φCV244 [G]) phage type. Genomes contained ∼60 putative ORFs (with ∼19 functional CDSs and ∼38 hypothetical proteins) that were divided based on the protein function into: host lysis (∼3 ORFs), structural (∼11 ORFs), DNA packaging and replication (∼7 ORFs), unknown function (∼38 ORFs). 7 NWS phages  RefSeq: Lactobacillus helveticus phage ΦAQ113 (NC_019782.1) unknown	proteins	DNA	metabolism	proteins	phage	structure	protein	host	cell	lysys	proteins	A	 B	 C	 D	E	 F	 G	Note. Strains susceptibility to phage infection are indicated as degree of “-” to “+++”. “+++” indicates a completely clear zone; “++” indicates a center clear zone with a blurred ring; “+” indicates a generally clear zone; “-“ indicates no lysis with light grey cells; black cells refers to complete lysis in respective phages host cells.  	:	                         Phage    Strain  Dairy  implant Period  of isolation φCR28 φCR191 φCV244 φP185 φS16 φS193 φT280 L. helveticus_ CR62 CR February - - - - - - - L. helveticus_ CR67 CR February - - - - - - - L. helveticus_ CR72 CR February - - - - - - - L. helveticus_ CR80 CR February - - - - - - - L. helveticus_ CR81 CR February - - - - - - - L. helveticus_ CR93 CR May - - - - - - - L. helveticus_ CR100 CR May - - - - - - - L. helveticus_ CR102 CR May - - - - - - - L. helveticus_ CR104 CR May - - - - - - - L. helveticus_ CR106 CR September - - - - - - - L. helveticus_ CR108 CR September - - - - - - - L. helveticus_ CR119 CR December ++ - - - - - -           L. helveticus_ CR113 CR December - - - - - - - L. helveticus_ S348 S December - - - - - - - L. helveticus_ T173 T September - - - - - - - L. helveticus_ T195 T September - - - - - - - L. helveticus_ T221 T September + + + + + + ++ L. helveticus_ T260 T December - - - - - - - L. helveticus_CR28 CR September +++ - - +++ - -  - L. helveticus_CR191 CR September  + +++  - +   - -  - L. helveticus_CV244 CV September - - +++ ++ - - ++ L. helveticus_P185 P September +++ - - +++ - - +++ L. helveticus_S16 S February ++ - - +++ +++ - - L. helveticus_S193 S May  + -  -  ++ +  +++ -  L. helveticus_T280 T December - - - - - - +++           	                      Phage    Strain  Dairy  implant Period  of isolation φCR28 φCR191 φCV244 φP185 φS16 φS193 φT280 L. helveticus_ CR62 CR February - - - - - - - . l ti  7  r r  - - - - - - - . l ti  72  r r  - - - - - - - . l ti  80  r r  - - - - - - - . l ti  1  r r  - - - - - - - . l ti  93  May - - - - - - - . l ti  100   - - - - - - - . l ti  2   - - - - - - - . l ti  4   - - - - - - - . l ti  6  September - - - - - - - . l ti  8  t r - - - - - - - . l ti  19  D cember ++ - - - - - -           L. helveticus_ CR113 CR December - - - - - - - . l ti  S348 S r - - - - - - - . l ti  T173 T S ptember - - - - - - - . l ti  95  t r - - - - - - - . l ti  221  t r + + + + + + ++ L. helveticus_ T260 T December - - - - - - - . l ti CR28 CR S ptember +++ - - +++ - -  - . l ti 191  t r   +++  -    - -  - . l ti V244 V t r - - +++ + - - ++ . l ti P185 P t r +++ - - +++ - - +++ . l ti S 6 S F bruary ++ - -  +++ - - . l ti 93  May   -  -     +++ -  L. helveticus_T280 T December - - - - - - +++           	:	:	:	:	:	:	:	:	:	:	:	:	:	:	:	:	:	:	:	:	1 Integrase genes were present in φCR191 (ORF 21), φP185 (ORF 5), φS16 (ORF 41) and φS193 (ORF 53). Other genes potentially involved in phage genome integration/prophage induction, were found in phages φCR28 (ORF 23, ORF 15, ORF 31), φCV244 (ORF 7, ORF 18, ORF 40) and φT280 (ORF 7, ORF 50). Any lysogenic behavior was observed over MitC+ induction, after L. helveticus hosts exposition to the relatives phages. Based on ViPTree global protein content comparison, phages φCR28, φCR191, φP185, φS16 and φS193 were clustered with phage φAQ113 in the myovirus phage-type group of the tree, while φCV244 was grouped with phage phig1e (NC_004305.1), as part of the siphovirus phage-type group.  25[64]	16[49]	16[35]	32[84]	23[80]	22[77]	22[90]	Total number of shared proteins; in squared brackets, total number of shares.  Hypothetical/unknown proteins present singularly or in multiple copies between genomes. In particular, we counted 40 ORFs for φCR28, 34 ORFs for φCR191, 40 ORFs for φCV244, 42 ORFs for φP185, 35 ORFs for φS16, 36 ORFs for φS193 and 39 ORFs for φT280. Half of these hypothetical/unknown proteins (n=44) were conserved and shared between genomes.  Phylogenetic tree based on ANI (A) and proteomic trees computed with ViPTree (B), respectively between considered phages and relative proteomic database. Representative growth curves (OD600nm) of the prophage induction test on the seven Lactobacillus helveticus strains and phages considered in this work. Mitomycin C [1µg/mL];  Genome comparison showed high homology (over 70%) for all the studied phages, particularly for ORF encoding phage structural proteins (∼14ORFs) and the type involved in host lysis (∼2ORFs). Besides, a lesser sequence homology was observed for ORFs related to DNA metabolism (∼9 ORFs) and hypothetical proteins (∼14 ORFs). The phage genome with the highest homology to φAQ113 was φCR28.  3 600nm OD measurement based test With a constant income of new viruses carried by the raw milk, NWSs represent an ideal niche of interaction between bacteria and phages that may impact on the technological parameters for cheese production. Here we characterized seven phages isolated from Trentingrana NWSs and able to infect Lactobacillus helveticus. Classified as part of the tailed-phages groups of myovirus and siphovirus, they harbor most of the needed genes for a complete lytic life in Lactobacillus spp, while the founded lysogeny-associated genes could represent remnants of a temperate origin. Interestingly 57% of gene could not be annotated with specific functions, so was possible to speculate the potential presence here of a set of new phage-genes well conserved between these genomes and shared a total of 479 times. The presence of these phages, without affecting the starter acidification activity, is opening new interesting insights into the beneficial functions for the host. A deep characterization of phage genomic information would allow a better understanding of their role in NWSs, to maintain the excellence in Grana cheese production, e.g. for Trentingrana  0 2 4 6 8 10 12 14 16 18 20 22 240.00.20.40.60.81.01.21.41.61.82.0Time (h)OD600nmL. helveticus CR28 vs ϕCR280 2 4 6 8 10 12 14 16 18 20 22 240.00.20.40.60.81.01.21.41.61.82.0Time (h)OD600nmL. helveticus S16 vs ϕS160 2 4 6 8 10 12 14 16 18 20 22 240.00.20.40.60.81.01.21.41.61.82.0Time (h)OD600nmL. helveticus CR191 vs ϕCR1910 2 4 6 8 10 12 14 16 18 20 22 240.00.20.40.60.81.01.21.41.61.82.0Time (h)OD600nmL. helveticus S193 vs ϕS1930 2 4 6 8 10 12 14 16 18 20 22 240.00.20.40.60.81.01.21.41.61.82.0Time (h)OD600nmL. helveticus CV244 vs ϕCV2440 2 4 6 8 10 12 14 16 18 20 22 240.00.20.40.60.81.01.21.41.61.82.0Time (h)OD600nmL. helveticus T280 vs ϕT2800 2 4 6 8 10 12 14 16 18 20 22 240.00.20.40.60.81.01.21.41.61.82.0Time (h)OD600nmL. helveticus P185 vs ϕP185L. helveticusL. helveticus + Mitomycin CL. helveticus + phage L. helveticus + phage (washed at OD 0.2) + Mitomycin C L. helveticus + phage (washed at OD 0.2) 0 2 4 6 8 10 12 14 16 18 20 22 240.00.20.40.60.81.01.21.41.61.82.0Time (h)OD600nmL. helveticus CR28 vs ϕCR280 2 4 6 8 10 12 14 16 18 20 22 240.00.20.40.60.81.01.21.41.61.82.0Time (h)OD600nmL. helveticus S16 vs ϕS160 2 4 6 8 10 12 14 16 18 20 22 240.00.20.40.60.81.01.21.41.61.82.0Time (h)OD600nmL. helveticus CR191 vs ϕCR1910 2 4 6 8 10 12 14 16 18 20 22 240.00.20.40.60.81.01.21.41.61.82.0Time (h)OD600nmL. helveticus S193 vs ϕS1930 2 4 6 8 10 12 14 16 18 20 22 240.00.20.40.60.81.01.21.41.61.82.0Time (h)OD600nmL. helveticus CV244 vs ϕCV2440 2 4 6 8 10 12 14 16 18 20 22 240.00.20.40.60.81.01.21.41.61.82.0Time (h)OD600nmL. helveticus T280 vs ϕT2800 2 4 6 8 10 12 14 16 18 20 22 240.00.20.40.60.81.01.21.41.61.82.0Time (h)OD600nmL. helveticus P185 vs ϕP185L. helveticusL. helveticus + Mitomycin CL. helveticus + phage L. helveticus + phage (washed at OD 0.2) + Mitomycin C L. helveticus + phage (washed at OD 0.2) GenBank Accession Numbers φCR28 - OQ627802.1 φCR191 - OQ627803.1 φCV244 - OQ627804.1 φP185 - OQ627805.1 φS16 - OQ627806.1 φS193 - OQ627807.1 φT280 - OQ627808.1 The authors would like to thank all the operators of the dairy facilities for providing precious information about the NWS production. This work was funded by the Trento province Region Rural Development Program (PSR). 

Phages in Trentingrana cheeses’ natural whey starters: new insights from characterization and genomic comparison

https://openpub.fmach.it/bitstream/10449/82435/1/MD23_ManciniA.pdf

Phages in Trentingrana cheeses’ natural whey starters: new insights from characterization and genomic comparison

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