Received date: June 01, 2017; Accepted date: July 13, 2017; Published date: July 20, 2017
Citation: Pardini GMT, Silva BL, Soto LME (2017) Bacteriophage Genome Sequencing: A New Alternative to Understand Biochemical Interactions between Prokaryotic Cells and Phages. J Microb Biochem Technol 9:169-173. doi:10.4172/1948-5948.1000362
Copyright: © 2017 Pardini GMT, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
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Bacteriophages are viruses or semi-autonomous genetic entities that depend on prokaryotic cell’s metabolism to multiply. The use of lytic phages as biocontrol agents in many fields such as food preservation, disease control, agriculture production signifies the need of most appropriate and standard methods to insure application safety. Bacteriophage full genetic material sequencing is a new alternative to better understand phage encoded proteins and biomolecules (especially phage lytic enzymes) involved in the process of bacterial cell lysis and death. Thus, this short review aimed to discuss sequencing importance applied to bacteria-phage interactions in society commercial sectors, focusing in the food industry.
Bacteriophages were independently discovered by Frederick Twort in 1915 and Felix d’Herelle in 1917 in cell cultures of Staphylococcus aureus and Shigella, respectively . Phages were first used in medical field by d’Herelle in 1919. Despite promising results, antibiotic discovery in 1940 decreased researcher’s interest in the use of bacteriophages for human disease treatment due controversial public opinion [2,3]. Nowadays, researches concerning bacteriophage application have risen.
Until last decade, the main form to control bacteria human pathogenesis and prevent bacterial multiplication in many sectors was using chemical agents, such as antibiotics and sanitizers. However, indiscriminate use of these chemical agents resulted in bacterial multiresistance. In this way, bacteriophage technology and phage therapy emerged [4,5]. Phages are able to infect only specific species of prokaryote, or even strains within the same species . Bacteriophages are able to adhere on the microbial cell surface, intermediated by proteins and other accessory structures, and inject their genetic material into the host, and may present different cycles of infection: lytic cycle, lysogenic cycle, pseudo-lysogenic cycle or chronic infection. Regarding phage application, preferentially, phages who present lytic cycle of infection could be used as biocontrol tools. In this cycle, bacteriophages inject their genetic material into the cell to produce new viral particles, causing cellular lysis [5,6].
The first complete genome ever sequenced was the one of the bacteriophage øX174 in 1977. This advance was a breakthrough for science. The information contained in the genetic material, which varies widely among bacteriophages, is able to predict the proteins synthesized by the polynucleotide sequence genes and is used to taxonomically classify viruses in orders, families, subfamilies, genus and species [7,8]. Therefore, this mini review paper aimed to discuss bacteriophage sequencing importance encompassing different fields of application, focusing on phages specific for foodborne pathogens.
Phage genome sequencing
Ten families of bacteriophages have been already reported, according to the International committee on taxonomy of viruses (ICTV), phage classification in families and genera are based on capsid morphology, conserved genomic synteny and homology in amino acid sequences of phage genetic material encoded proteins . There are basically four types of genetic material comprising bacteriophages genome, single-stranded and double-stranded DNA and RNA (ssDNA, dsDNA, ssRNA, dsRNA) [10,11]. Genetic material varies widely among phages: genome length ranges from 3405 bp to 497513 bp, gene density ranges from 0.29 to 1.36 and number of encoded proteins ranges from 1 to 675. Until 2017 first semester, there were 7163 viruses’ genetic material sequences available online on NCBI genome bank (Table 1). Bacteriophages genome, which includes viruses’ specific for archaea and bacteria domains, represents about 31.6% of the total.
|Host||Number of available genomes *||Oldest deposited Genome||Most recent deposited genome||Minimal length
|Minimal encoded proteins||Maximal encoded proteins|
|Invertebrates and plants||66||1993||2017||3164||29339||1||14|
|Invertebrates and vertebrates||11||1987||2013||11088||15867||3||12|
|Invertebrates, vertebrates and humans||7||1987||2005||11088||11703||3||5|
|Vertebrates and humans||310||1982||2017||1682||235646||1||233|
|Vertebrates and invertebrates||157||1993||2017||4401||170101||1||152|
|Vertebrates, invertebrates and humans||124||1993||2017||6391||29210||1||13|
* The total does not equal 7163 because some genomes appear more than once in the table when infecting more than one specific host Source: Adapted from NCBI (https://www.ncbi.nlm.nih.gov/genome).
Table 1: Division of host’s virus’s genomic material available on NCBI genome bank.
Bacteriophage DNA sequencing still presents difficulties even with new sequencing techniques development, which is the bottleneck for phage functional genomic studies [12,13]. The main obstacles are: i) obtaining pure phage genomic material, ii) PCR amplification, and iii) complex nature of its genetic material due to intrinsic characteristics, such as methylated bases and repetition zones, which are intrinsically difficult to sequence and organize .
From a technological point of view, bacteriophage sequencing is still essential for any study of functional genomics, as well as for approval and release of bacteriophages use or derived products by regulatory agencies such as the Food and Drug Administration (FDA). As biocontrol tools in the food industry and in the medical field, genetic studies are necessary, once it is known that some viruses are capable of enhancing bacteria pathogenicity [14,15]. In this way, a few companies already market products based on bacteriophages, including products used to control of foodborne pathogens. These products are considered safe for the consumer and are approved by FDA, such as ListShieldTM and SalmFresTM, used to control Listeria monocytogenes and Salmonella on food processing surfaces, respectively .
Genome sequencing presents one of the most complete technique to study phage encoded proteins, however, genetic material only shows potential predicted proteins, not showing each one of these proteins are actually expressed in the process of host infection [17-20]. In this way, other omic approaches like transcriptomics, proteomics and metabolomics could be used in combination with phage genome sequencing to completely understand phage-bacteria interactions [21-24].
Phage genome sequencing focusing on economic applications in the food industry
In Table 2 is presented some of the phage genome sequences focusing on phage application in the food industry. As shown, phage genetics vary among phages of different hosts, even in an interlinked supply chain like the industry of food processing [25-28]. The main bottleneck for functional overall genomics, including phage genomics, is the low number of available genomes and described genes (open reading frames or ORFs). Many of phage predicted proteins represent “hypothetical proteins” with homology among phages, but with none described function. This scenario shows how low we know about genome and demonstrates the need of much more studies concerning genome sequencing [29,30].
|Phage identification||Host species||Accession number||Main founds and highlights||References|
|UFV-AREG1||Escherichia coli O157:H7||KX009778||Genome size: 170788 bpÂ
ORFs encoding hypothetical proteins: 134
Gene density: 1.60
G+C content: 35.3%
Phage specific to control E. coli causing food poisoning and more severe symptoms. Similar genome organization and encoded proteins among other enteric phages, providing further information especially for E. coli specific phages. Presence of both holin and endolysin conserved genes.
|UFV-P2||Pseudomonas fluorescens||JX863101||Genome size: 45517 bp
ORFs encoding hypothetical proteins: 51
Gene density: 1.65
G+C content: 51.5%
Pioneer study regarding food safety applications to control spoilage microorganisms in the food industry. Presence of holin independent endolysins as lytic enzymes to rupture host membrane.
|KU878088||Genome size: 251042 bp
ORFs encoding hypothetical proteins: 150
Gene density: 1.16
G+C content: 27.8%
Presence of an endolysin, with the conserved N-terminal glycoside hydrolase domain and a C-terminal cell wall binding domain. In this study it was proposed some holing candidates possessing transmembrane domains.
|clP1||Pediococcus damnosus||JN051154||Genome size: 38013 bp
ORFs encoding hypothetical proteins: 35
Gene density: 1.50
G+C content: 47.6%
Phage specific to control spoilage Pediococcus in beer. This phage possesses putative endolysin and holing, conserved in Lactobacillus phages.
|PLgT-1||Lactococcus garvieae||KU892558||Genome size: 40273 bp
ORFs encoding hypothetical proteins: 42
Gene density: 1.63
G+C content: 35.4%
This genome showed high similarity among other Lactococcus genomes. This phage presented two probable holin genes but no endolysin or similar was predicted, which could explain the lysogenic behavior of this phage.
|FCD6356||Clostridium difficile||GU949551||Genome size: 37664 bp
ORFs encoding hypothetical proteins: 38
Gene density: 1.57
G+C content: 28.4%
This bacteriophage presented a genome more similar to others Siphoviridaephages than among C. difficile previously described phages. The endolysin described in this study was similar to the ones of C. difficile strains, but no conserved domain of holing was observed.
|LDG||Leuconostoc sp.||KX555527||Genome size: 26500 to 28900 bp
ORFs: 35 to 50
G+C content: 36 to 39%
All four phages presented similar genome size, G+C content and number of ORFs. However, phylogenetic clustering showed more similarities among previously sequenced phages. Endolysins encoded by these presented two different versions of putative domains. Theses bacteriophages presented conserved holin.
|vB_YenP_AP5||Yersinia enterocolitica||KM253764||Genome size: 38646 bp
ORFs encoding hypothetical proteins: 16
Gene density: 1.16
G+C content: 50.7%
This phage genome was similar to other T7like phages, which includes other bacteriophages specific for enteric pathogens like Salmonella. This phage DNA also encoded both endolysins and holing genes, with putative domains conserved and observed in some Yersinia previously sequenced phages.
|SA11||Staphylococcus aureus||JX194239||Genome size: 136326 bp
ORFs encoding hypothetical proteins: 171
Gene density: 1.36
G+C content: 30.0%
Bioinformatics analysis showed that this phage genome showed low similarity to other S. aureus phages found in literature, once only 19 of the predicted 186 ORFs were identified. This phage genome also encoded conserved endolysins with putative catalytic and binding domains, but not holing genes were predicted.
|SS3e||Salmonella||AY730274||Genome size: 40793 bp
ORFs encoding hypothetical proteins: 56
Gene density: 1.45
G+C content: 50.0%
SS3 bacteriophage showed high similarity to other Siphoviridae salmonella phages. It was not observed any endolysin or holin conserved genes in this phage genome, which shows the need of further studies to identify proteins responsible for Salmonella cell lysis.
|phiE142||Escherichia coli O157:H7 and Salmonella enterica||KU255730||Genome size: 121442 bp
ORFs encoding hypothetical proteins: 115
Gene density: 1.65
G+C content: 37.4%
This phage genome showed high similarity to E. coli, Enterobacteria and Shigellaphages, including endolysin and holin genes.
|vB_LmoS_188||Listeria monocytogenes||KP399677||Genome size: 38392 bp and 40759 bp
ORFs: 60 and 72
ORFs encoding hypothetical proteins: 32 and 39
Gene density: 1.56 and 1.77
G+C content: 35.9% and 36.9%
Genome organization and clustering of both of these phages were similar to other previously described Listeria bacteriophages, but genome identity was slightly low in the group of Listeriaphages. Both phages presented conserved domains of both holin and endolysin genes.
|pSf-1||Shigella||KC710998||Genome size: 51821 bp
ORFs encoding hypothetical proteins: 94
Gene density: 1.81
G+C content: 44.0%
pSf-1 genome sequence showed high similarity with other Shigellaphages, but showing low gene homology, especially regarding some genome translocation a no identification of lytic enzyme genes.
|CP21||Campylobacter jejuni and Campylobacter coli||HE815464||Genome size: 182833 bp
ORFs encoding hypothetical proteins: 124
Gene density: 1.42
CP21 genome showed high homology with other three Campylobacter phage genomes, in the genus T4-like of bacteriophages. No obvious lytic enzymes were observed in theses phage genome.
Table 2: Comparative analysis of some sequenced bacteriophages specific for foodborne bacteria and main results found in which research.
However, the main focus of the phage genomic studies target the identification of lytic enzymes, and these enzymes show some homology and putative domains among species of bacteriophages . Studies regarding these enzymes description have grown exponentially over the years compared to other genetic fields.
Depending on specificity of the catalytic domain of the lytic enzyme, phage endolysins can be classified in putative groups, showing similar mode of action. Until further discoveries, there are six classes of lytic endolysins described in literature: i) N-acetyl-β-D-glucosaminidase; ii) N-acetyl-β-D-muramidase and iii) lytic transglycosylase; all responsible for breaking the sugar bonds of β-(1,4) N-acetylglucosamine and N-acetylmuramic acid; iv) N-acetylmuramoyl-L-alanine amidase, responsible for cleave the bond between L-alanine and N-acetylmuramic acid; v) L-alanoyl-D-glutamate endopeptidase, which cleaves the chemical bond between L-alanine and D-glutamic acid; and vi) D-alanyl-glycyl endopeptidase, which breaks the peptide bond between the 5-glycine inter-bridge between glycine and L-alanine in Gram-positive bacteria (Figure 1) [31,32].
In this way, phages and phage enzymes have advantages of usage related to bacterial resistance. Once endolysins present some specificity among different genera or species, the probability of the bacteria acquiring mechanisms of resistance is low. This is explained by the theory of phage-bacteria co-evolution, which postulates that to ensure phage multiplication and survival in the environment, phage and its endolysins were naturally selected, which difficult bacterial resistance.
Genome sequencing is one of the most effective technologies to understand phage-bacteria interactions during phage infection cycle, however, compared to the number of isolated phages, phage genomes available online are limited. In this way, it is widely necessary more researches concerning genomics and phage sequencing, including other omic approaches, which in combination can provide additional information to complement bacteriophage studies in the biological fields and insure phage application and diffusion in many society sectors.