Abstract
Shigella species, a major cause of shigellosis, remain a substantial global health issue and the emergence of antibiotic-resistant Shigella strains has aggravated the situation. Hence, four Shigella phages were investigated to provide insights into the evolutionary trajectories and genomic properties of Shigella-infecting bacteriophages using comparative genome analysis. Analysis shows that these four phages belong to the Tequatrovirus genus and include a considerable number of proteins for 'Tail' and "DNA, RNA and Nucleotide Metabolism," indicating their aptitude for specialized host interaction and replication efficiency. The identification of 10 tRNAs further support that, these phages have high replication efficiency. Thus, this study improves our understanding of phage evolution by exposing the genetic mechanisms that drive phage adaptability and host specificity. This also highlights the significance of phage genomic research in developing viable therapies for antibiotic-resistant Shigella infections.
Keywords: Anti-CRISPR, shigella phage, functional category, tRNA, antimicrobial resistance (AMR)
Background:
Shigella species are facultative intracellular, gram-negative bacteria that cause shigellosis, a highly contagious illness that is mostly characterized by acute gastroenteritis and diarrhoea [1], with more than 165 million cases and around 1 million fatalities every year. Shigella continues to pose a serious threat to world health, especially in low- and middle-income nations [2, 3]. Aside from its toxicity, Shigella's development of antibiotic-resistant strains has grown in importance as a public health issue, making treatment plans more difficult and emphasizing the need for alternate therapeutic approaches [4]. The use of viruses that specifically infect and destroy bacteria, known as bacteriophage treatment, has drawn a lot of attention as potential tactic to counteract Shigella strains that are resistant to multiple drugs [5-6]. Bacteriophages, often known as phage's, are a diverse and widespread group of viruses that highly selectively infect bacteria [7]. For a variety of bacterial illnesses, including those brought on by pathogens resistant to antibiotics like Shigella, they have been investigated as possible therapeutic agents [8- 9]. Targeting harmful bacteria without altering the human microbiome is one of phage therapy's many benefits, which makes it a good option when antibiotics haven't worked [10- 11]. The ability of Shigella-infecting phages to function as efficient bio-control agents has been demonstrated by recent research, particularly in light of the developing issue of Shigella's resistance to several antibiotic classes [12, 13]. Shigella phages have not yet reached their full therapeutic potential despite these encouraging uses, mainly because a better understanding of their genetic diversity, evolutionary trajectories and interactions with bacterial hosts [14]. Similar to their bacterial hosts, phages are influenced by evolutionary forces like bacterial resistance mechanisms and phage counter-defenses influence the co-evolutionary dynamics between Shigella and its infecting phages [15]. Shigella bacteria have evolved a number of defense mechanisms, including as receptor alterations, the acquisition of defense systems like CRISPER-Cas and the synthesis of anti-phage enzymes, to avoid phage invasion [16]. Phages respond by developing new strategies to get past these bacterial defenses, which leads to a continuous evolutionary "arms race." This co-evolution process is essential for determining the phages' long-term efficacy as therapeutic agents as well as for comprehending their biological success in natural settings [17]. The study of phage evolution is essential for the creation of phage-based treatments because of the dynamic nature of this interaction, which forces phages to continuously change in order to overcome novel bacterial resistance tactics [18, 19]. By investigating the genetic mechanisms that drive phage adaptability, we can find factors that influence phage efficacy, host specificity and resistance to bacterial defense systems [20]. Phages that evolve to escape bacterial CRISPR-Cas systems or other immune mechanisms may provide an extra benefit in phage therapy by allowing phages to infect and proliferate even in the presence of bacterial resistance [21]. Shigella phages, like all other bacteriophages, have a limited host range, infecting just certain strains of Shigella, whilst others have a broader host spectrum and these host specificity differences are shaped by the development of important genomic characteristics that govern phage-host interactions, such as the proteins involved in host recognition and genome injection, so comparative genomic studies of Shigella phages are thus critical for discovering conserved genetic components that may be significant for phage infectivity, as well as understanding the processes that contribute to phage population diversity [22]. The application of comparative genomics to phage evolution enables researchers to find evolutionary patterns that provide insights into how shigella phages adapt to their bacterial hosts. This genomic perspective also helps to explain how shigella phages evolve in the face of selective pressures from bacterial resistance mechanisms and immunological responses including the CRISPER-Cas system [23]. Some phages create anti-CRISPR proteins that disrupt bacteria's CRISPER-Cas defensive system, allowing the phage to successfully infect the host despite bacterial defenses. The discovery and characterization of anti-CRISPR genes in Shigella phages may provide important insights into how phages develop to overcome bacterial resistance. Furthermore, the presence of antimicrobial resistance (AMR) genes in phage genomes may suggest that phages have been exposed to antibiotic-resistant bacterial strains and it may even contribute to the horizontal transmission of resistance genes among bacteria [24, 25- 26]. Therefore, it is of interest to show that Shigella phage evolution is critical for enhancing our knowledge on phage therapy and better understanding of phage-bacteria interactions through comparative genomics analysis of Shigella-infecting bacteriophages.
Methodology:
Sequencing and assembly:
Isolation and quantitative analysis of DNA:
The four bacteriophages (ADG1, CDR3, AKR2 and TMC4) were isolated from lake water in Kolkata, India. The lake was chosen as a sampling site because it is exposed to both natural microbial ecosystems and probable human or animal waste, both of which are known repositories of phages infecting intestinal diseases Shigella. Following the initial screening and isolation processes, only these four phages were successfully recovered and propagated for further research. Isolating bacteriophages infecting Shigella from an environmental source is consistent with our study's purpose of investigating naturally existing phage diversity and its possible involvement in countering antibiotic-resistant Shigella strains. Their availability as viable isolates from the sampling effort made them ideal candidates for genetic analysis. These phages give an important picture of the genetic diversity, functional adaptations and evolutionary dynamics of Shigella-specific phages from an environmental reservoir. By characterizing these phages, we hope to provide light on their genomic features, host interaction mechanisms and therapeutic potential, as well as provide insights into the larger ecological and evolutionary background of Shigella-infecting phages. Samples were processed using CTAB DNA isolation method. DNA quantity was measured using Qubit® 4.0-fluorometer and DNA quality was analyzed on 1.0% agarose gel.
Preparation of library:
The paired-end sequencing library was prepared using Twist NGS Library Preparation Kits for Illumina® (CAT No. ID 104119). The library preparation process was initiated with 50 ng input. DNA was enzymatically sheared into smaller fragments by kit protocol and continuous step of end-repair and A-tailing where an 'A' is added to the 3' ends making the DNA fragments ready for adapter ligation. Following this step, illumine specific adapters are ligated to both ends of the DNA fragments. These adapters contain sequences essential for binding barcoded libraries to a flow cell for sequencing, allowing for PCR amplification of adapter-ligated fragments and binding standard Illumina sequencing primers. To ensure maximum yields from limited amounts of starting material, a high-fidelity amplification step was performed using HiFi PCR Master Mix.
Quantity and quality check (QC) of library on agilent tape station 4150:
The amplified libraries were analyzed on TapeStation 4150 (Agilent Technologies) using High Sensitivity D1000 ScreenTape® as per manufacturer's instructions.
Cluster generation and sequencing:
After obtaining the Qubit concentration for the library and the mean peak size from Tape Station profile, library will be loaded onto illumina Novaseq 6000 for cluster generation and sequencing. Paired-End sequencing allows the template fragments to be sequenced in both the forward and reverse directions. The library molecules will bind to complementary adapter oligos on paired-end flow cell. The adapters are designed to allow selective cleavage of the forward strands after re-synthesis of the reverse strand during sequencing. The copied reverse strand is then used to sequence from the opposite end of the fragment.
Genomic assembly and analysis:
After Gathering raw sequencing readings from a sequencer is the initial stage of our study. We use FastQC [27], a program that evaluates quality of raw reads. As low-quality readings might induce biases or inaccuracies in later studies, this quality check is essential. After quality control, we trim any low-quality areas from the reads and eliminate adapter sequences using Trimmomatic [28]. This reduces the possibility of inaccurate data and raises the assembly's overall correctness by guaranteeing that only high-quality sequences are used for downstream assembly. After that, we go on to de-novo assembly, which uses the trimmed reads to rebuild the genomes without a reference genome. We make use of four distinct assemblers: Megahit [29], Velvet [30], SPAdes [31] and SKESA [32]. The benefit of comparing several assemblies to choose the most accurate one is that each of these assemblers uses distinct genome assembly algorithms and techniques. In our case, SKESA produced the assembly with the highest N50, making it the best option for additional analysis. The resulting assemblies are evaluated for quality using QUAST [33], a tool that assesses multiple metrics, including the N50 value, which is crucial for determining the completeness of the assembly. Following the selection of the assembly, Prokka [34] is used for genome annotation, making predictions about the genes and proteins present in the phage genome. Prokka provides a thorough summary of the phage's gene composition and functional potential by identifying coding sequences, tRNAs and other genomic characteristics.
The GenBank accession numbers for four bacteriophage genome sequences are:
ADG1: PQ666539
AKR2: PQ666540
CRD3: PQ666541
TMC4:PQ666542
Functional classification:
We use the PHROGs database [35], an extensive resource that lists phage proteins and their functional annotations, to categorize the discovered proteins' functional roles. We find the best matches for each of our proteins by comparing our phage proteins with the PHROGS database using Blastp [36]. We use the Galaxy server's blast best Hit Identification Program to make sure we choose the most accurate functional classification. By using sequence similarity, this program lets us sift through the BLAST findings and find the most pertinent hits. Following the identification of the best matches, we categorize the proteins according to their matching PHROGs IDs, which offer information about the proteins' putative functional roles in relation to the phage's lifecycle, host interactions and other biological processes.
Core genome analysis:
Using Roary [37], a program intended to examine the variety of bacterial and viral genomes, we conduct pan-genome analysis after acquiring the gene annotation file from Prokka. We can identify the core genome the genes that all phages share and the accessory genome the genes that are found in some but not all phages with the aid of Roary's pan-genome analysis. In order to assess the genomic similarities and differences among the phages and get insight into their evolutionary history and functional variety, it is critical to comprehend the distribution of these genes. Additionally, this study aids in the identification of distinct genes that might be involved in particular traits, including virulence or host specificity.
Multivariate analysis:
For correspondence analysis we use CodonW [38], a program that enables us to examine Amino Acid Usage (AAU) patterns, to conduct multivariate analysis in order to investigate the evolutionary dynamics of the phages. CodonW looks for any notable variations or patterns in the amino acid composition of the genes across the phages. Since changes in amino acid utilization can reveal selection pressure, functional adaptability, or evolutionary restrictions acting on the phages, this approach is useful for identifying evolutionary patterns in the genome. We can learn more about the evolutionary forces that have influenced the phages' genetic composition by contrasting these trends between the core and auxiliary genomes.
Whole genome phylogenetic tree:
We create a full genome phylogenetic tree to comprehend the evolutionary relationships between our phages and other closely related phages. Using the NCBI database, which has a sizable number of bacteriophage genomes (as of October 2024), we start by running a BLAST search. A subset of 83 closely related phages is obtained by applying strict criteria to identify phages with at least 90% query coverage and 95% sequence identity. MAFFT, a tool that uses complex algorithms to provide precise multiple sequence alignments, is used to align the chosen genomes. RAxML [39], which is based on the GTR + G + I evolutionary model, is then used to build a maximum likelihood phylogenetic tree using the alignments. This model provides a strong and trustworthy phylogenetic tree that shows the evolutionary relationships among the phages and places them in the larger context of other phages in the NCBI database by taking into consideration the substitution rates and variability throughout the genome.
Anti-CRISPR and AMR gene identification:
The existence of anti-CRISPR and antimicrobial resistance (AMR) genes in phage genomes is a critical topic of research since these genes can influence the phages' capacity to avoid host immune systems and their possible role in antimicrobial resistance. To identify these genes, we use the AcrDB [40] and Anti-CRISPRdb [41] databases for anti-CRISPR gene sequences, as well as the CARD database [42] for AMR genes. We use BLASTP to match the anti-CRISPR and AMR gene sequences in these databases with the proteins from our phage genomes. In order to comprehend how the phages interact with bacterial hosts and contribute to the dynamics of antimicrobial resistance, it is essential that we discover any potential anti-CRISPR or AMR genes in our phages. The identification of these genes can also aid in assessing the phages' possible application in therapeutic contexts, where resistance gene modification and bacterial immune system evasion are crucial elements.
Results:
Comparative genomic analysis:
The genomes of phages ADG1, CDR3, AKR2 and TMC4 have been sequenced and uploaded as supplementary files. The GenBank accession numbers are also provided in the 'Methodology' section. These phages have the following genome lengths: ADG1 (164,945 bp), CDR3 (165,220 bp), AKR2 (165,034 bp) and TMC4 (164,971 bp). Their average G+C content are: 35.16%, 35.52%, 35.49% and 35.35%, respectively. Figure 1 shows a schematic genomic map of the four phages developed using the Proksee server [43]. In this map, a visual representation of the genomic structures is provided where the inner ring represents coding sequences (CDS) in blue, whereas tRNA genes are highlighted in pink (Figure 1). An analysis of annotated open reading frames (ORFs) of the four phages indicates that the phage ADG1 encodes 285 proteins, CDR3 encodes 255 proteins, AKR2 encodes 254 proteins and TMC4 encodes 291 proteins. These include structural proteins, genome packaging proteins, lysis proteins, holins and tail proteins. Each of the phage genomes has 10 tRNA genes: tRNA-Arg(tct), tRNA-Asn(gtt), tRNA-Tyr(gta), tRNA-Met(cat), tRNA-Thr(tgt), tRNA-Ser(tga), tRNA-Pro(tgg), tRNA-Gly(tcc), tRNA-Leu(taa) and tRNA-Gln(ttg). These tRNAs are thought to give a high level of independence from the host's translational machinery, which is a well-known approach for improving phage protein production during infection [44]. Taxonomic classification put all four phages to the Tunavirus genus, suggesting their evolutionary link within this group. These findings show the four phages' share strong evolutionary relationship and common genomic characteristics, providing vital information for the study of bacteriophage genetics and taxonomy.
Figure 1.
Genomic map of four phages, where purple colored lines are CDS and yellow colored lines are tRNA. Where A, B, C, D is representing the phage ADG1, AKR2, CRD3 and TMC4 respectively.
Functional classification:
Functional classification divides phage proteins into nine categories based on their biological functions (Table 1). However, we did not consider two functional categories namely, 'Unknown function' and 'Others' in our analysis. The majority of phage proteins belong to the "Tail" and "DNA, RNA and Nucleotide Metabolism" categories (Table 1), indicating their role of the phage interaction with its host and replication inside the host. Phage tails are crucial for host recognition, attachment and genome injection, particularly in tailed bacteriophages and a proteins involved in DNA, RNA and Nucleotide Metabolism are essential for transcription as well as replication of the virus's genome in the host cell.
Table 1. Functional classification of phage proteins into nine categories based on their biological functions.
| Query | phrog | category |
| TMC4_169_head_closure_Shigella_phage_pSs-1 | 218 | connector |
| TMC4_189_head-tail_adaptor_Ad2_Shigella_phage_pSs-1 | 211 | connector |
| TMC4_191_head_closure_Hc2_Shigella_phage_pSs-1 | 679 | connector |
| TMC4_188_head-tail_adaptor_Ad2_Shigella_phage_pSs-1 | 211 | connector |
| TMC4_190_head_closure_Hc2_Shigella_phage_pSs-1 | 679 | connector |
| TMC4_025_exonuclease_Shigella_phage_pSs-1 | 255 | DNA, RNA and nucleotide metabolism |
| TMC4_044_nucleoside_triphosphate_pyrophosphohydrolase_Shigella_phage_pSs-1 | 173 | DNA, RNA and nucleotide metabolism |
| TMC4_065_DNA_polymerase_processivity_factor_Shigella_phage_pSs-1 | 223 | DNA, RNA and nucleotide metabolism |
| TMC4_252_dCMP_deaminase_Shigella_phage_pSs-1 | 174 | DNA, RNA and nucleotide metabolism |
| TMC4_003_DNA_topoisomerase_II_Shigella_phage_pSs-1 | 551 | DNA, RNA and nucleotide metabolism |
| TMC4_052_DnaB-like_replicative_helicase_Shigella_phage_pSs-1 | 19 | DNA, RNA and nucleotide metabolism |
| TMC4_062_clamp_loader_of_DNA_polymerase_Shigella_phage_pSs-1 | 225 | DNA, RNA and nucleotide metabolism |
| TMC4_089_anaerobic_ribonucleoside_reductase_large_subunit_Shigella_phage_pSs-1 | 4218 | DNA, RNA and nucleotide metabolism |
| TMC4_092_endonuclease_VII_Shigella_phage_pSs-1 | 423 | DNA, RNA and nucleotide metabolism |
| TMC4_239_DNA_ligase_Shigella_phage_pSs-1 | 114 | DNA, RNA and nucleotide metabolism |
| TMC4_272_NrdA-like_aerobic_NDP_reductase_large_subunit_Shigella_phage_pSs-1 | 84 | DNA, RNA and nucleotide metabolism |
| TMC4_002_DNA_topoisomerase_II_Shigella_phage_pSs-1 | 551 | DNA, RNA and nucleotide metabolism |
| TMC4_004_Ndd-like_nucleoid_disruption_protein_Shigella_phage_pSs-1 | 1102 | DNA, RNA and nucleotide metabolism |
| TMC4_017_DNA_topoisomerase_II_large_subunit_Shigella_phage_pSs-1 | 543 | DNA, RNA and nucleotide metabolism |
| TMC4_029_Dda-like_helicase_Shigella_phage_pSs-1 | 325 | DNA, RNA and nucleotide metabolism |
| TMC4_046_DNA_primase_Shigella_phage_pSs-1 | 47 | DNA, RNA and nucleotide metabolism |
| TMC4_051_Dmd_discriminator_of_mRNA_degradation_Shigella_phage_pSs-1 | 2102 | DNA, RNA and nucleotide metabolism |
| TMC4_057_thymidylate_synthase_Shigella_phage_pSs-1 | 160 | DNA, RNA and nucleotide metabolism |
| TMC4_064_clamp_loader_of_DNA_polymerase_Shigella_phage_pSs-1 | 168 | DNA, RNA and nucleotide metabolism |
| TMC4_068_SbcC-like_subunit_of_palindrome_specific_endonuclease_Shigella_phage_pSs-1 | 77 | DNA, RNA and nucleotide metabolism |
| TMC4_168_DNA_end_protector_Shigella_phage_pSs-1 | 429 | DNA, RNA and nucleotide metabolism |
| TMC4_213_DNA_helicase_Shigella_phage_pSs-1 | 16 | DNA, RNA and nucleotide metabolism |
| TMC4_214_DNA_helicase_Shigella_phage_pSs-1 | 1143 | DNA, RNA and nucleotide metabolism |
| TMC4_217_UvsY-like_recombination_mediator_Shigella_phage_pSs-1 | 231 | DNA, RNA and nucleotide metabolism |
| TMC4_238_DNA_ligase_Shigella_phage_pSs-1 | 114 | DNA, RNA and nucleotide metabolism |
| TMC4_270_endonuclease_Shigella_phage_pSs-1 | 1111 | DNA, RNA and nucleotide metabolism |
| TMC4_276_thymidylate_synthase_Shigella_phage_pSs-1 | 160 | DNA, RNA and nucleotide metabolism |
| TMC4_284_single_strand_DNA_binding_protein_Shigella_phage_pSs-1 | 224 | DNA, RNA and nucleotide metabolism |
| TMC4_285_DNA_helicase_loader_Shigella_phage_pSs-1 | 269 | DNA, RNA and nucleotide metabolism |
| TMC4_287_hypothetical_protein_Shigella_phage_pSs-1 | 107 | DNA, RNA and nucleotide metabolism |
| TMC4_011_DenB-like_DNA_endonuclease_IV_Shigella_phage_pSs-1 | 1360 | DNA, RNA and nucleotide metabolism |
| TMC4_032_RNA_polymerase_ADP-ribosylase_Shigella_phage_pSs-1 | 832 | DNA, RNA and nucleotide metabolism |
| TMC4_033_RNA_polymerase_ADP-ribosylase_Shigella_phage_pSs-1 | 832 | DNA, RNA and nucleotide metabolism |
| TMC4_034_RNA_polymerase_ADP-ribosylase_Shigella_phage_pSs-1 | 832 | DNA, RNA and nucleotide metabolism |
| TMC4_035_RNA_polymerase_ADP-ribosylase_Shigella_phage_pSs-1 | 832 | DNA, RNA and nucleotide metabolism |
| TMC4_059_DNA_polymerase_Shigella_phage_pSs-1 | 262 | DNA, RNA and nucleotide metabolism |
| TMC4_060_DNA_polymerase_Shigella_phage_pSs-1 | 262 | DNA, RNA and nucleotide metabolism |
| TMC4_063_clamp_loader_of_DNA_polymerase_Shigella_phage_pSs-1 | 225 | DNA, RNA and nucleotide metabolism |
| TMC4_066_RNA_polymerase_binding_Shigella_phage_pSs-1 | 1285 | DNA, RNA and nucleotide metabolism |
| TMC4_071_SbcD-like_subunit_of_palindrome_specific_endonuclease_Shigella_phage_pSs-1 | 100 | DNA, RNA and nucleotide metabolism |
| TMC4_072_SbcD-like_subunit_of_palindrome_specific_endonuclease_Shigella_phage_pSs-1 | 100 | DNA, RNA and nucleotide metabolism |
| TMC4_087_anaerobic_ribonucleotide_reductase_small_subunit_Shigella_phage_pSs-1 | 626 | DNA, RNA and nucleotide metabolism |
| TMC4_088_anaerobic_ribonucleoside_reductase_large_subunit_Shigella_phage_pSs-1 | 4218 | DNA, RNA and nucleotide metabolism |
| TMC4_091_endonuclease_VII_Shigella_phage_pSs-1 | 24351 | DNA, RNA and nucleotide metabolism |
| TMC4_095_ribonucleotide_reductase_Shigella_phage_pSs-1 | 2294 | DNA, RNA and nucleotide metabolism |
| TMC4_098_NrdC_thioredoxin_Shigella_phage_pSs-1 | 22 | DNA, RNA and nucleotide metabolism |
| TMC4_110_hypothetical_protein | 388 | DNA, RNA and nucleotide metabolism |
| TMC4_128_valyl_tRNA_synthetase_modifier_Shigella_phage_pSs-1 | 1256 | DNA, RNA and nucleotide metabolism |
| TMC4_130_endoribonuclease_Shigella_phage_pSs-1 | 1323 | DNA, RNA and nucleotide metabolism |
| TMC4_164_RNA_ligase_Shigella_phage_pSs-1 | 755 | DNA, RNA and nucleotide metabolism |
| TMC4_207_RNA_ligase_Shigella_phage_pSs-1 | 548 | DNA, RNA and nucleotide metabolism |
| TMC4_208_RNA_ligase_Shigella_phage_pSs-1 | 548 | DNA, RNA and nucleotide metabolism |
| TMC4_212_DNA_helicase_Shigella_phage_pSs-1 | 16 | DNA, RNA and nucleotide metabolism |
| TMC4_271_ribonucleotide_reductase_class_Ia_beta_subunit_Shigella_phage_pSs-1 | 86 | DNA, RNA and nucleotide metabolism |
| TMC4_273_NrdA-like_aerobic_NDP_reductase_large_subunit_Shigella_phage_pSs-1 | 3987 | DNA, RNA and nucleotide metabolism |
| TMC4_277_thymidylate_synthase_Shigella_phage_pSs-1 | 160 | DNA, RNA and nucleotide metabolism |
| TMC4_279_dihydrofolate_reductase_Shigella_phage_pSs-1 | 316 | DNA, RNA and nucleotide metabolism |
| TMC4_163_internal_head_protein_Shigella_phage_pSs-1 | 3499 | head and packaging |
| TMC4_194_terminase_small_subunit_Shigella_phage_pSs-1 | 735 | head and packaging |
| TMC4_206_capsid_vertex_protein_Shigella_phage_pSs-1 | 138 | head and packaging |
| TMC4_043_virion_structural_protein_Shigella_phage_pSs-1 | 1715 | head and packaging |
| TMC4_196_terminase_large_subunit_Shigella_phage_pSs-1 | 2 | head and packaging |
| TMC4_210_Hoc-like_head_decoration_Shigella_phage_pSs-1 | 1149 | head and packaging |
| TMC4_053_head_vertex_assembly_chaperone_Shigella_phage_pSs-1 | 999 | head and packaging |
| TMC4_137_internal_virion_protein_Shigella_phage_pSs-1 | 3155 | head and packaging |
| TMC4_200_portal_protein_Shigella_phage_pSs-1 | 213 | head and packaging |
| TMC4_203_head_maturation_protease_Shigella_phage_pSs-1 | 207 | head and packaging |
| TMC4_204_head_scaffolding_protein_Shigella_phage_pSs-1 | 237 | head and packaging |
| TMC4_205_major_head_protein_Shigella_phage_pSs-1 | 138 | head and packaging |
| TMC4_211_minor_head_protein_inhibitor_of_protease_Shigella_phage_pSs-1 | 1051 | head and packaging |
| TMC4_249_head_morphogenesis_Shigella_phage_pSs-1 | 931 | head and packaging |
| TMC4_195_terminase_small_subunit_Shigella_phage_pSs-1 | 735 | head and packaging |
| TMC4_201_hypothetical_protein | 1064 | head and packaging |
| TMC4_202_head_scaffolding_protein_Shigella_phage_pSs-1 | 1049 | head and packaging |
| TMC4_121_lysis_inhibition_Shigella_phage_pSs-1 | 1246 | lysis |
| TMC4_295_holin_Shigella_phage_pSs-1 | 860 | lysis |
| TMC4_013_RIIB_lysis_inhibitor_Shigella_phage_pSs-1 | 609 | lysis |
| TMC4_014_RIIA_lysis_inhibitor_Shigella_phage_pSs-1 | 612 | lysis |
| TMC4_139_glycoside_hydrolase_family_protein_Shigella_phage_pSs-1 | 7 | lysis |
| TMC4_248_lysis_inhibition;_accessory_protein_Shigella_phage_pSs-1 | 1457 | lysis |
| TMC4_264_Rz-like_spanin_Shigella_phage_pSs-1 | 812 | lysis |
| TMC4_265_Rz-like_spanin_Shigella_phage_pSs-1 | 739 | lysis |
| TMC4_015_RIIA_lysis_inhibitor_Shigella_phage_pSs-1 | 612 | lysis |
| TMC4_138_glycoside_hydrolase_family_protein_Shigella_phage_pSs-1 | 7 | lysis |
| TMC4_233_Alt-like_RNA_polymerase_ADP-ribosyltransferase_Shigella_phage_pSs-1 | 802 | moron, auxiliary metabolic gene and host takeover |
| TMC4_234_Alt-like_RNA_polymerase_ADP-ribosyltransferase_Shigella_phage_pSs-1 | 802 | moron, auxiliary metabolic gene and host takeover |
| TMC4_096_antitoxin_from_a_toxin-antitoxin_system_Shigella_phage_pSs-1 | 3402 | moron, auxiliary metabolic gene and host takeover |
| TMC4_111_hypothetical_protein_Shigella_phage_pSs-1 | 944 | moron, auxiliary metabolic gene and host takeover |
| TMC4_141_hypothetical_protein_Shigella_phage_pSs-1 | 2203 | moron, auxiliary metabolic gene and host takeover |
| TMC4_235_Alt-like_RNA_polymerase_ADP-ribosyltransferase_Shigella_phage_pSs-1 | 802 | moron, auxiliary metabolic gene and host takeover |
| TMC4_021_cef_modifier_of_supressor_tRNAs_Shigella_phage_pSs-1 | 1354 | moron, auxiliary metabolic gene and host takeover |
| TMC4_173_PAAR_motif_of_membran_proteins_Shigella_phage_pSs-1 | 281 | moron, auxiliary metabolic gene and host takeover |
| TMC4_031_Srd_anti-sigma_factor_Shigella_phage_pSs-1 | 1400 | moron, auxiliary metabolic gene and host takeover |
| TMC4_039_decoy_of_host_sigma32_Shigella_phage_pSs-1 | 2441 | moron, auxiliary metabolic gene and host takeover |
| TMC4_090_anaerobic_ribonucleoside_reductase_large_subunit_Shigella_phage_pSs-1 | 487 | moron, auxiliary metabolic gene and host takeover |
| TMC4_231_Alt-like_RNA_polymerase_ADP-ribosyltransferase_Shigella_phage_pSs-1 | 802 | moron, auxiliary metabolic gene and host takeover |
| TMC4_232_Alt-like_RNA_polymerase_ADP-ribosyltransferase_Shigella_phage_pSs-1 | 802 | moron, auxiliary metabolic gene and host takeover |
| TMC4_236_Alt-like_RNA_polymerase_ADP-ribosyltransferase_Shigella_phage_pSs-1 | 802 | moron, auxiliary metabolic gene and host takeover |
| TMC4_056_beta-glucosyl-HMC-alpha-glucosyltransferase_Shigella_phage_pSs-1 | 842 | other |
| TMC4_122_thymidine_kinase_Shigella_phage_pSs-1 | 592 | other |
| TMC4_127_phosphatase_Shigella_phage_pSs-1 | 335 | other |
| TMC4_140_nudix_hydrolase_Shigella_phage_pSs-1 | 1185 | other |
| TMC4_260_hypothetical_protein | 505 | other |
| TMC4_007_periplasmic_protein_Shigella_phage_pSs-1 | 2401 | other |
| TMC4_049_spackle_periplasmic_Shigella_phage_pSs-1 | 1586 | other |
| TMC4_054_recombinase_Shigella_phage_pSs-1 | 97 | other |
| TMC4_061_translation_repressor_Shigella_phage_pSs-1 | 242 | other |
| TMC4_073_alpha-glucosyltransferase_Shigella_phage_pSs-1 | 2888 | other |
| TMC4_009_hypothetical_protein | 5011 | other |
| TMC4_055_beta-glucosyl-HMC-alpha-glucosyltransferase_Shigella_phage_pSs-1 | 842 | other |
| TMC4_094_inhibitor_of_host_Lon_protease_Shigella_phage_pSs-1 | 2167 | other |
| TMC4_166_deoxynucleoside_monophosphate_kinase_Shigella_phage_pSs-1 | 139 | other |
| TMC4_261_polynucleotide_kinase_Shigella_phage_pSs-1 | 505 | other |
| TMC4_165_tail_fiber_chaperone_Shigella_phage_pSs-1 | 1002 | tail |
| TMC4_174_baseplate_wedge_subunit_Shigella_phage_pSs-1 | 219 | tail |
| TMC4_180_baseplate_wedge_subunit_Shigella_phage_pSs-1 | 977 | tail |
| TMC4_199_tail_protein_Shigella_phage_pSs-1 | 45 | tail |
| TMC4_290_hinge_connector_of_long_tail_fiber_protein_distal_connector_Shigella_phage_pSs-1 | 1425 | tail |
| TMC4_291_long_tail_fiber_protein_distal_subunit_Shigella_phage_pSs-1 | 1699 | tail |
| TMC4_167_tail_protein_Shigella_phage_pSs-1 | 45 | tail |
| TMC4_176_baseplate_wedge_subunit_Shigella_phage_pSs-1 | 964 | tail |
| TMC4_183_baseplate_wedge_subunit_Shigella_phage_pSs-1 | 958 | tail |
| TMC4_184_baseplate_wedge_subunit_Shigella_phage_pSs-1 | 963 | tail |
| TMC4_193_tail_sheath_stabilizer_Shigella_phage_pSs-1 | 227 | tail |
| TMC4_227_baseplate_tail_tube_cap_Shigella_phage_pSs-1 | 230 | tail |
| TMC4_170_baseplate_wedge_subunit_Shigella_phage_pSs-1 | 232 | tail |
| TMC4_171_baseplate_hub_subunit_and_tail_lysozyme_Shigella_phage_pSs-1 | 430 | tail |
| TMC4_177_baseplate_wedge_subunit_Shigella_phage_pSs-1 | 964 | tail |
| TMC4_181_baseplate_wedge_tail_fiber_protein_connector_Shigella_phage_pSs-1 | 967 | tail |
| TMC4_185_tail_collar_fiber_protein_Shigella_phage_pSs-1 | 910 | tail |
| TMC4_218_baseplate_wedge_subunit_Shigella_phage_pSs-1 | 261 | tail |
| TMC4_219_baseplate_hub_Shigella_phage_pSs-1 | 150 | tail |
| TMC4_221_baseplate_hub_assembly_catalyst_Shigella_phage_pSs-1 | 150 | tail |
| TMC4_222_baseplate_hub_Shigella_phage_pSs-1 | 1135 | tail |
| TMC4_226_baseplate_hub_subunit_and_tail_length_Shigella_phage_pSs-1 | 1283 | tail |
| TMC4_229_tail_tube_Shigella_phage_pSs-1 | 45 | tail |
| TMC4_267_RNA_ligase_and_tail_fiber_protein_attachment_catalyst_Shigella_phage_pSs-1 | 562 | tail |
| TMC4_289_long_tail_fiber_protein_proximal_connector_Shigella_phage_pSs-1 | 1154 | tail |
| TMC4_294_tail_fiber_protein;_host_specificity_Shigella_phage_pSs-1 | 2056 | tail |
| TMC4_175_baseplate_wedge_subunit_Shigella_phage_pSs-1 | 219 | tail |
| TMC4_178_baseplate_wedge_subunit_Shigella_phage_pSs-1 | 964 | tail |
| TMC4_179_baseplate_wedge_subunit_Shigella_phage_pSs-1 | 964 | tail |
| TMC4_182_baseplate_wedge_subunit_Shigella_phage_pSs-1 | 958 | tail |
| TMC4_186_tail_collar_fiber_protein_Shigella_phage_pSs-1 | 910 | tail |
| TMC4_187_fibritin_neck_whisker_Shigella_phage_pSs-1 | 1056 | tail |
| TMC4_192_tail_sheath_stabilizer_Shigella_phage_pSs-1 | 227 | tail |
| TMC4_197_tail_sheath_Shigella_phage_pSs-1 | 23 | tail |
| TMC4_220_baseplate_hub_Shigella_phage_pSs-1 | 150 | tail |
| TMC4_223_baseplate_hub_Shigella_phage_pSs-1 | 1135 | tail |
| TMC4_224_Baseplate_hub_assembly_protein_gp28 | 1140 | tail |
| TMC4_225_baseplate_hub_distal_subunit_Shigella_phage_pSs-1 | 1140 | tail |
| TMC4_228_baseplate_tail_tube_cap_Shigella_phage_pSs-1 | 230 | tail |
| TMC4_268_RNA_ligase_and_tail_fiber_protein_attachment_catalyst_Shigella_phage_pSs-1 | 562 | tail |
| TMC4_269_RNA_ligase_and_tail_fiber_protein_attachment_catalyst_Shigella_phage_pSs-1 | 562 | tail |
| TMC4_288_tail_fiber_protein_proximal_subunit_Shigella_phage_pSs-1 | 972 | tail |
| TMC4_292_long_tail_fiber_protein_distal_subunit_Shigella_phage_pSs-1 | 1699 | tail |
| TMC4_293_long_tail_fiber_protein_distal_subunit_Shigella_phage_pSs-1 | 1699 | tail |
| TMC4_022_MotB-like_transcriptional_regulator_Shigella_phage_pSs-1 | 1671 | transcription regulation |
| TMC4_078_RNA_polymerase_sigma_factor_Shigella_phage_pSs-1 | 234 | transcription regulation |
| TMC4_286_late_promoter_transcriptional_regulator_Shigella_phage_pSs-1 | 254 | transcription regulation |
| TMC4_301_MotA-like_activator_of_middle_period_transcription_Shigella_phage_pSs-1 | 1345 | transcription regulation |
| TMC4_037_Mrh_transcription_modulator_under_heat_shock_Shigella_phage_pSs-1 | 1234 | transcription regulation |
| TMC4_040_Mrh_transcription_modulator_under_heat_shock_Shigella_phage_pSs-1 | 1234 | transcription regulation |
| TMC4_120_starvation-inducible_transcriptional_regulator_Shigella_phage_pSs-1 | 858 | transcription regulation |
| TMC4_266_inhibitor_of_host_transcription_Shigella_phage_pSs-1 | 1253 | transcription regulation |
| TMC4_300_MotA-like_activator_of_middle_period_transcription_Shigella_phage_pSs-1 | 1345 | transcription regulation |
| TMC4_250_SH3_beta-barrel_fold-containing_protein_Shigella_phage_pSs-1 | 1086 | unknown function |
| TMC4_023_hypothetical_protein_Shigella_phage_pSs-1 | 2333 | unknown function |
| TMC4_027_dextranase_Shigella_phage_pSs-1 | 1700 | unknown function |
| TMC4_080_gp78_Shigella_phage_pSs-1 | 1475 | unknown function |
| TMC4_082_gp80_Shigella_phage_pSs-1 | 2084 | unknown function |
| TMC4_086_gp86_Shigella_phage_pSs-1 | 1930 | unknown function |
| TMC4_124_hypothetical_protein_Shigella_phage_pSs-1 | 3452 | unknown function |
| TMC4_134_autonomous_glycyl_radical_cofactor_GrcA_Shigella_phage_pSs-1 | 1209 | unknown function |
| TMC4_136_hypothetical_protein_Shigella_phage_pSs-1 | 1488 | unknown function |
| TMC4_143_hypothetical_protein_Shigella_phage_pSs-1 | 2604 | unknown function |
| TMC4_243_hypothetical_protein_Shigella_phage_pSs-1 | 2042 | unknown function |
| TMC4_244_hypothetical_protein_Shigella_phage_pSs-1 | 2153 | unknown function |
| TMC4_254_hypothetical_protein_Shigella_phage_pSs-1 | 1504 | unknown function |
| TMC4_263_hypothetical_protein_Shigella_phage_pSs-1 | 2347 | unknown function |
| TMC4_274_hypothetical_protein | 464 | unknown function |
| TMC4_278_hypothetical_protein_Shigella_phage_pSs-1 | 9550 | unknown function |
| TMC4_298_hypothetical_protein_Shigella_phage_pSs-1 | 839 | unknown function |
| TMC4_016_gp17_Shigella_phage_pSs-1 | 1683 | unknown function |
| TMC4_067_protein_GP45.2_Shigella_phage_pSs-1 | 1045 | unknown function |
| TMC4_245_hypothetical_protein_Shigella_phage_pSs-1 | 1582 | unknown function |
| TMC4_001_gp1_Shigella_phage_pSs-1 | 3487 | unknown function |
| TMC4_005_hypothetical_protein_pSs1_006_Shigella_phage_pSs-1 | 2183 | unknown function |
| TMC4_006_hypothetical_protein | 4135 | unknown function |
| TMC4_008_gp8_Shigella_phage_pSs-1 | 3098 | unknown function |
| TMC4_010_gp11_Shigella_phage_pSs-1 | 2390 | unknown function |
| TMC4_012_gp14_Shigella_phage_pSs-1 | 774 | unknown function |
| TMC4_018_gp19_Shigella_phage_pSs-1 | 902 | unknown function |
| TMC4_019_hypothetical_protein_Shigella_phage_pSs-1 | 622 | unknown function |
| TMC4_020_hypothetical_protein | 622 | unknown function |
| TMC4_024_hypothetical_protein_Shigella_phage_pSs-1 | 1106 | unknown function |
| TMC4_026_gp28_Shigella_phage_pSs-1 | 1700 | unknown function |
| TMC4_028_gp30_Shigella_phage_pSs-1 | 4967 | unknown function |
| TMC4_030_gp32_Shigella_phage_pSs-1 | 1079 | unknown function |
| TMC4_036_gp36_Shigella_phage_pSs-1 | 1647 | unknown function |
| TMC4_038_gp38_Shigella_phage_pSs-1 | 2681 | unknown function |
| TMC4_041_gp41_Shigella_phage_pSs-1 | 2031 | unknown function |
| TMC4_042_gp42_Shigella_phage_pSs-1 | 1873 | unknown function |
| TMC4_045_gp45_Shigella_phage_pSs-1 | 2024 | unknown function |
| TMC4_047_gp47_Shigella_phage_pSs-1 | 1823 | unknown function |
| TMC4_048_gp48_Shigella_phage_pSs-1 | 4122 | unknown function |
| TMC4_050_gp50_Shigella_phage_pSs-1 | 2375 | unknown function |
| TMC4_058_gp59_Shigella_phage_pSs-1 | 1754 | unknown function |
| TMC4_069_gp68_Shigella_phage_pSs-1 | 2327 | unknown function |
| TMC4_070_gp69_Shigella_phage_pSs-1 | 2192 | unknown function |
| TMC4_074_gp72_Shigella_phage_pSs-1 | 2140 | unknown function |
| TMC4_075_gp73_Shigella_phage_pSs-1 | 1648 | unknown function |
| TMC4_076_a-gt.4_family_protein_Shigella_phage_pSs-1 | 951 | unknown function |
| TMC4_077_hypothetical_protein | 1169 | unknown function |
| TMC4_079_gp77_Shigella_phage_pSs-1 | 1822 | unknown function |
| TMC4_081_gp79_Shigella_phage_pSs-1 | 1475 | unknown function |
| TMC4_083_gp81_Shigella_phage_pSs-1 | 24953 | unknown function |
| TMC4_084_gp82_Shigella_phage_pSs-1 | 1774 | unknown function |
| TMC4_085_gp83_Shigella_phage_pSs-1 | 2283 | unknown function |
| TMC4_093_gp91_Shigella_phage_pSs-1 | 3272 | unknown function |
| TMC4_097_gp96_Shigella_phage_pSs-1 | 2112 | unknown function |
| TMC4_099_hypothetical_protein_Shigella_phage_pSs-1 | 2052 | unknown function |
| TMC4_100_hypothetical_protein_Shigella_phage_pSs-1 | 1474 | unknown function |
| TMC4_101_hypothetical_protein_Shigella_phage_pSs-1 | 1435 | unknown function |
| TMC4_102_hypothetical_protein_Shigella_phage_pSs-1 | 1435 | unknown function |
| TMC4_103_hypothetical_protein_Shigella_phage_pSs-1 | 2351 | unknown function |
| TMC4_104_hypothetical_protein_Shigella_phage_pSs-1 | 2257 | unknown function |
| TMC4_105_hypothetical_protein_Shigella_phage_pSs-1 | 2547 | unknown function |
| TMC4_106_hypothetical_protein_Shigella_phage_pSs-1 | 2274 | unknown function |
| TMC4_107_hypothetical_protein_Shigella_phage_pSs-1 | 2274 | unknown function |
| TMC4_108_hypothetical_protein_Shigella_phage_pSs-1 | 1931 | unknown function |
| TMC4_109_hypothetical_protein_Shigella_phage_pSs-1 | 1250 | unknown function |
| TMC4_112_hypothetical_protein_Shigella_phage_pSs-1 | 1502 | unknown function |
| TMC4_113_molybdopterin-guanine_dinucleotide_biosynthesis_protein_MobD_Shigella_phage_pSs-1 | 1502 | unknown function |
| TMC4_114_hypothetical_protein_Shigella_phage_pSs-1 | 3215 | unknown function |
| TMC4_115_hypothetical_protein_Shigella_phage_pSs-1 | 3515 | unknown function |
| TMC4_116_hypothetical_protein_Shigella_phage_pSs-1 | 2758 | unknown function |
| TMC4_117_hypothetical_protein_Shigella_phage_pSs-1 | 653 | unknown function |
| TMC4_118_hypothetical_protein_Shigella_phage_pSs-1 | 653 | unknown function |
| TMC4_119_hypothetical_protein_Shigella_phage_pSs-1 | 3024 | unknown function |
| TMC4_123_hypothetical_protein_Shigella_phage_pSs-1 | 653 | unknown function |
| TMC4_125_hypothetical_protein_Shigella_phage_pSs-1 | 2306 | unknown function |
| TMC4_126_hypothetical_protein_Shigella_phage_pSs-1 | 2008 | unknown function |
| TMC4_129_hypothetical_protein_Shigella_phage_pSs-1 | 600 | unknown function |
| TMC4_131_hypothetical_protein_Shigella_phage_pSs-1 | 2037 | unknown function |
| TMC4_132_hypothetical_protein_Shigella_phage_pSs-1 | 717 | unknown function |
| TMC4_133_hypothetical_protein_Shigella_phage_pSs-1 | 1986 | unknown function |
| TMC4_135_hypothetical_protein_Shigella_phage_pSs-1 | 2049 | unknown function |
| TMC4_142_hypothetical_protein_Shigella_phage_pSs-1 | 2330 | unknown function |
| TMC4_144_hypothetical_protein_Shigella_phage_pSs-1 | 2607 | unknown function |
| TMC4_145_hypothetical_protein_Shigella_phage_pSs-1 | 1203 | unknown function |
| TMC4_146_hypothetical_protein_Shigella_phage_pSs-1 | 6869 | unknown function |
| TMC4_147_hypothetical_protein_Shigella_phage_pSs-1 | 2678 | unknown function |
| TMC4_148_hypothetical_protein_Shigella_phage_pSs-1 | 2280 | unknown function |
| TMC4_149_hypothetical_protein_Shigella_phage_pSs-1 | 2280 | unknown function |
| TMC4_160_hypothetical_protein_Shigella_phage_pSs-1 | 2023 | unknown function |
| TMC4_161_hypothetical_protein_Shigella_phage_pSs-1 | 1411 | unknown function |
| TMC4_162_hypothetical_protein_Shigella_phage_pSs-1 | 1692 | unknown function |
| TMC4_172_hypothetical_protein_Shigella_phage_pSs-1 | 1168 | unknown function |
| TMC4_198_hypothetical_protein_Shigella_phage_pSs-1 | 5104 | unknown function |
| TMC4_209_hypothetical_protein | 1566 | unknown function |
| TMC4_215_hypothetical_protein_Shigella_phage_pSs-1 | 1043 | unknown function |
| TMC4_216_hypothetical_protein_Shigella_phage_pSs-1 | 1541 | unknown function |
| TMC4_230_hypothetical_protein_Shigella_phage_pSs-1 | 1739 | unknown function |
| TMC4_237_hypothetical_protein_Shigella_phage_pSs-1 | 1976 | unknown function |
| TMC4_240_hypothetical_protein | 2054 | unknown function |
| TMC4_241_hypothetical_protein_Shigella_phage_pSs-1 | 420 | unknown function |
| TMC4_242_hypothetical_protein_Shigella_phage_pSs-1 | 652 | unknown function |
| TMC4_246_hypothetical_protein_Shigella_phage_pSs-1 | 1507 | unknown function |
| TMC4_247_hypothetical_protein | 1427 | unknown function |
| TMC4_251_hypothetical_protein_Shigella_phage_pSs-1 | 2010 | unknown function |
| TMC4_253_hypothetical_protein_Shigella_phage_pSs-1 | 2063 | unknown function |
| TMC4_255_hypothetical_protein_Shigella_phage_pSs-1 | 3710 | unknown function |
| TMC4_256_hypothetical_protein_Shigella_phage_pSs-1 | 3710 | unknown function |
| TMC4_257_hypothetical_protein_Shigella_phage_pSs-1 | 1695 | unknown function |
| TMC4_258_hypothetical_protein | 2360 | unknown function |
| TMC4_259_hypothetical_protein_Shigella_phage_pSs-1 | 2430 | unknown function |
| TMC4_262_hypothetical_protein_Shigella_phage_pSs-1 | 2364 | unknown function |
| TMC4_275_hypothetical_protein_Shigella_phage_pSs-1 | 2458 | unknown function |
| TMC4_280_hypothetical_protein_Shigella_phage_pSs-1 | 3183 | unknown function |
| TMC4_281_hypothetical_protein | 1254 | unknown function |
| TMC4_282_hypothetical_protein_Shigella_phage_pSs-1 | 622 | unknown function |
| TMC4_283_hypothetical_protein_Shigella_phage_pSs-1 | 1764 | unknown function |
| TMC4_296_hypothetical_protein_Shigella_phage_pSs-1 | 1359 | unknown function |
| TMC4_297_hypothetical_protein_Shigella_phage_pSs-1 | 1844 | unknown function |
| TMC4_299_hypothetical_protein_Shigella_phage_pSs-1 | 1594 | unknown function |
Correspondence analysis on amino acid usage of Shigella bacteriophage:
We performed Correspondence analysis on amino acid usage of the four newly sequenced Shigella bacteriophage genomes taken together to ascertain if there exists any difference in amino acid usage among the four bacteriophages. Figure 2 clearly shows that genes from four bacteriophages are completely overlapped on each other indicating identical amino acid usage of four bacteriophages. Later, we performed Correspondence analysis on amino acid usage by taking the one of the four bacteriophages and its host, (i.e.,) Shigella flexneri. Here, 10% of Shigella flexneri genes are overlapping with the 17.5% of bacteriophage genes (Figure 3). More than 80% of the preferred amino acids are perfectly matching between the bacteriophage and its host.
Figure 2.
Amino acid usage of shigella bacterium with one of the four bacteriophages. Green colored points represent Shigella and yellow colored points represent Phage.
Figure 3.
Similarity in amino acid usage of four bacteriophages. Green, yellow, purple and blue colored points represent each of the four phages
Whole genome tree and core genome construction:
A complete genome tree (Figure 4) demonstrates a strong evolutionary link among the phages, particularly with Shigella and Escherichia phages. Shigella phage KNP5 and Shigella phage pSs-1 have been observed to be the closest relative with the four newly sequenced phages. This observation highlights the phages' shared evolutionary history and genetic similarities. We also built core genomes from the newly sequenced four phages. This core genome provides a unified framework of conserved genetic components throughout the bacteriophages studied. The core genome also depicts similar amino acid usage patterns among the four phages, indicating similarity of amino acid composition in the core genome.
Figure 4.
Phylogenetic tree of isolated four phages with other phage species. The four isolated phages are highlighted with yellow gradient box.
Anti-CRISPR and AMR gene identification:
We identified Rz-like spanin, belong to the lysis functional category and the SbcC-like subunit of palindrome-specific endonuclease and belong to DNA, RNA and nucleotide metabolism. Both proteins have the ability to demonstrate anti-CRISPR activity, implying that they are involved in countering host CRISPR-Cas systems during infections. Additionally, we also searched for antimicrobial resistance (AMR) genes in these phages and found one protein called dihydrofolate reductase, belong to the DNA, RNA and nucleotide metabolism group. Further analysis revealed that this protein is orthologous to the known AMR proteins dfrA9, dfrA10 and dfrA26, with more than 70% sequence identity. These findings indicate that, while this protein may serve a natural role in phage biology, its similarity to AMR proteins warrants further investigation into its possible impact.
Discussion:
The four phages show high genetic conservation, particularly with respect to important functional and structural proteins. These phages also demonstrate distinct adaptive strategies to represent the dynamic interaction between phages and their bacterial hosts as is evident from correspondence analysis on amino acid usage. The four phages have a similar genome length (~165 kb) and G+C content (~35%), which is consistent with Tunavirus phage features. This consistency highlights the evolutionary forces that drive genomic stability within Shigella-infecting phages. Structural proteins, such as head-tail adaptors, tail sheath proteins and receptor-binding proteins (RBPs), are highly conserved, implying common processes of host recognition, attachment and genome transport. For example, RBP is closely related to those found in Shigella phage JK23 and Escherichia phage BYEP02, highlighting the evolutionary requirement of preserving host receptor recognition [45]. Packaging proteins, such as the portal protein in our phages, share similarity with related phages, indicating a common role in effective DNA entry and departure during assembly and infection [46]. These conserved structural traits are consistent with previous research on Enterobacteriaceae phages, supporting the fact that these phages have evolved robust mechanisms to enable effective infection and multiplication in similar bacterial environments [47]. Beyond structural proteins, these phages have remarkable adaptive traits that improve infectivity and fitness. The presence of auxiliary metabolic genes, such as thymidylate synthase in our phages and dihydrofolate reductase in our phages, demonstrates how horizontal gene transfer (HGT) drives their evolution. These genes, which are essential for nucleotide metabolism, allow phages to avoid host metabolic limitations, resulting in faster replication. The identification of these genes highlights the significance of HGT in increasing phage-host compatibility, stressing bacteriophages' evolutionary flexibility in adapting to host metabolic pathways [48- 49]. Correspondence analysis of amino acid usage across the four newly sequenced Shigella bacteriophage genomes revealed perfect overlap in amino acid usage across all four bacteriophages (Figure 2), implying that their gene pools have substantially comparable signatures. This observation suggests that these phages may have common evolutionary origins or be subject to similar selective forces, resulting in convergent amino acid usage patterns. The lack of significant variation in amino acid usage among bacteriophages may possibly reflect functional constraints imposed by the need to properly infect and reproduce within Shigella hosts. When we broadened our study to look at the amino acid usage of one of the bacteriophages and its host, Shigella flexneri, we discovered a significant overlap between the two genomes (Figure 3). Specifically, 10% of the Shigella genes overlap with 17.5% of the bacteriophage genes, where mostly metabolic or structural genes are located. More than 80% of preferred amino acids are similar between the phage and its host supporting the hypothesis that phages and their bacterial hosts are co-evolving [50, 51]. This significant match in amino acid preferences may indicate that the phages are designed to interact well with host cellular machinery, implying a level of metabolic integration between the phage and host. Such integration may be required for successful phage multiplication and assembly within the host cell. These findings are consistent with the rising knowledge that bacteriophages are active players in bacterial evolution, potentially influencing host metabolism and gene transfer [52]. A notable finding is the anti-CRISPR proteins in each of the four phages that potentially play critical roles in the phage's evolutionary strategies and impact on the host. Rz-like spanin, belongs to the lysis functional category, indicates its significance in the phage's capacity to rupture host cell membranes. The SbcC-like subunit of a palindrome-specific endonuclease is involved in DNA, RNA and nucleotide metabolism, implying a more general regulatory role during the infection cycle. Both of these proteins are anti-CRISPR factors and could assist the phage bypassing the host's CRISPR-Cas defensive systems, increasing the phage's survival and multiplication inside the host. It shows that phages may evolve to circumvent bacterial immunity, influencing the dynamics of bacteria-virus interactions and perhaps facilitating horizontal gene transfer (HGT) between phages and their bacterial hosts [17]. We also found an antimicrobial resistance (AMR) gene in each of the four phages called dihydrofolate reductase (DHFR). This enzyme is highly similar to known AMR proteins including dfrA9, dfrA10 and dfrA26. The high sequence identity suggests that this protein may be involved in both the phage's fundamental metabolic processes and the host bacteria's AMR profile. Given DHFR's significance in folate metabolism and involvement in antimicrobial resistance mechanisms such as trimethoprim [53], the identification of this protein in Shigella phages raises crucial questions concerning its possible impact on the AMR evolution. It highlights the complex interplay between phages and their bacterial hosts, where phages may not only mediate the transfer of antimicrobial resistance genes but also act as potential vectors for the spread of resistance traits. Phylogenetic investigations show that these four phages share deep evolutionary ties, grouping them with other Shigella and Escherichia phages (Figure 4). It emphasizes their shared evolutionary origins and ecological niches, as well as the genetic traits shaped by common selective pressures. Core genome investigations emphasize the importance of critical proteins for genome replication, structural assembly and host interaction. These findings indicate that these phages developed from a common ancestor and adapted to specific hosts and environmental circumstances. The effect of HGT in altering phage genomes is most clear in these four phages, which has acquired auxiliary metabolic and structural genes that improve its ability to infect and reproduce within Shigella hosts. The findings of this work have important significance for both basic bacteriophage biology and applied phage therapy. Understanding the co-evolutionary dynamics of these phages and their hosts may potentially help to optimize their usage in combating antibiotic resistance. Future study should delve deeper into the functional roles of the identified proteins, investigate the ecological consequences of phage-host interactions and improve the usage of phages in combating the growing worldwide challenge of antibiotic resistance.
Conclusions:
The amazing genetic conservation of critical structural and functional proteins, emphasizing the evolutionary mechanisms that maintain efficient host recognition, infection and reproduction is shown. Furthermore, the finding of anti-CRISPR proteins in phages provides an intriguing peek into the phage's capacity to elude bacterial immune systems for increasing its survival and proliferation within Shigella. Thus, this study provides a solid platform for harnessing the potential of phages in combating bacterial infections while addressing the complexities of microbial evolution and resistance.
Acknowledgments
This research is funded by Indian Council of Medical Research through grant number 2021-10515.
No conflict of interest exists.
Edited by P Kangueane
Citation: Kayet et al. Bioinformation 20(12):2050-2061(2024)
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