Prophages integrating into prophages: A mechanism to accumulate type III secretion effector genes and duplicate Shiga toxin-encoding prophages in Escherichia coli

Bacteriophages (or phages) play major roles in the evolution of bacterial pathogens via horizontal gene transfer. Multiple phages are often integrated in a host chromosome as prophages, not only carrying various novel virulence-related genetic determinants into host bacteria but also providing various possibilities for prophage-prophage interactions in bacterial cells. In particular, Escherichia coli strains such as Shiga toxin (Stx)-producing E. coli (STEC) and enteropathogenic E. coli (EPEC) strains have acquired more than 10 prophages (up to 21 prophages), many of which encode type III secretion system (T3SS) effector gene clusters. In these strains, some prophages are present at a single locus in tandem, which is usually interpreted as the integration of phages that use the same attachment (att) sequence. Here, we present phages integrating into T3SS effector gene cluster-associated loci in prophages, which are widely distributed in STEC and EPEC. Some of the phages integrated into prophages are Stx-encoding phages (Stx phages) and have induced the duplication of Stx phages in a single cell. The identified attB sequences in prophage genomes are apparently derived from host chromosomes. In addition, two or three different attB sequences are present in some prophages, which results in the generation of prophage clusters in various complex configurations. These phages integrating into prophages represent a medically and biologically important type of inter-phage interaction that promotes the accumulation of T3SS effector genes in STEC and EPEC, the duplication of Stx phages in STEC, and the conversion of EPEC to STEC and that may be distributed in other types of E. coli strains as well as other prophage-rich bacterial species.     

GIL16, a new gram-positive tectiviral phage related to the Bacillus thuringiensis GIL01 and the Bacillus cereus pBClin15 elements

One of the most notable characteristics of Tectiviridae resides in their double-layer coats: the double-stranded DNA is located within a flexible lipoprotein vesicle covered by a rigid protein capsid. Despite their apparent rarity, tectiviruses have an extremely wide distribution compared to other phage groups. Members of this family have been found to infect gram-negative (PRD1 and relatives) as well as gram-positive (Bam35, GIL01, AP50, and phiNS11) hosts. Several reports have shown that tectiviruses infecting gram-negative bacteria are closely related, whereas no information is currently available on the genetic relationship among those infecting gram-positive bacteria. The present study reports the sequence of GIL16, a new isolate originating from Bacillus thuringiensis, and a genetic comparison of this isolate with the tectiviral bacteriophages Bam35 and GIL01, which originated from B. thuringiensis serovars Alesti and Israelensis, respectively. In contrast to PRD1 and its relatives, these are temperate bacteriophages existing as autonomous linear prophages within the host cell. Mutations in a particular motif in both the GIL01 and GIL16 phages are also shown to correlate with a switch to the lytic cycle. Interestingly, both bacterial viruses displayed narrow, yet slightly different, host spectrums. We also explore the hypothesis that pBClin15, a linear plasmid hosted by the Bacillus cereus reference strain ATCC 14579, is also a prophage. Sequencing of its inverted repeats at both extremities and a comparison with GIL01 and GIL16 emphasize its relationship to the Tectiviridae.     

The dilemma of phage taxonomy illustrated by comparative genomics of Sfi21-like Siphoviridae in lactic acid bacteria

The complete genome sequences of two dairy phages, Streptococcus thermophilus phage 7201 and Lactobacillus casei phage A2, are reported. Comparative genomics reveals that both phages are members of the recently proposed Sfi21-like genus of Siphoviridae, a widely distributed phage type in low-GC-content gram-positive bacteria. Graded relatedness, the hallmark of evolving biological systems, was observed when different Sfi21-like phages were compared. Across the structural module, the graded relatedness was represented by a high level of DNA sequence similarity or protein sequence similarity, or a shared gene map in the absence of sequence relatedness. This varying range of relatedness was found within Sfi21-like phages from a single species as demonstrated by the different prophages harbored by Lactococcus lactis strain IL1403. A systematic dot plot analysis with 11 complete L. lactis phage genome sequences revealed a clear separation of all temperate phages from two classes of virulent phages. The temperate lactococcal phages share DNA sequence homology in a patchwise fashion over the nonstructural gene cluster. With respect to structural genes, four DNA homology groups could be defined within temperate L. lactis phages. Closely related structural modules for all four DNA homology groups were detected in phages from Streptococcus or Listeria, suggesting that they represent distinct evolutionary lineages that have not uniquely evolved in L. lactis. It seems reasonable to base phage taxonomy on data from comparative genomics. However, the peculiar modular nature of phage evolution creates ambiguities in the definition of phage taxa by comparative genomics. For example, depending on the module on which the classification is based, temperate lactococcal phages can be classified as a single phage species, as four distinct phage species, or as two if not three different phage genera. We propose to base phage taxonomy on comparative genomics of a single structural gene module (head or tail genes). This partially phylogeny-based taxonomical system still mirrors some aspects of the current International Committee on Taxonomy in Virology classification system. In this system the currently sequenced lactococcal phages would be grouped into five genera: c2-, sk1, Sfi11-, r1t-, and Sfi21-like phages.     

Bacteria, phages and septicemia

The use of phages is an attractive option to battle antibiotic resistant bacteria in certain bacterial infections, but the role of phage ecology in bacterial infections is obscure. Here we surveyed the phage ecology in septicemia, the most severe type of bacterial infection. We observed that the majority of the bacterial isolates from septicemia patients spontaneously secreted phages active against other isolates of the same bacterial strain, but not to the strain causing the disease. Such phages were also detected in the initial blood cultures, indicating that phages are circulating in the blood at the onset of sepsis. The fact that most of the septicemic bacterial isolates carry functional prophages suggests an active role of phages in bacterial infections. Apparently, prophages present in sepsis-causing bacterial clones play a role in clonal selection during bacterial invasion.     

Isolation of Phages for Phage Therapy: A Comparison of Spot Tests and Efficiency of Plating Analyses for Determination of Host Range and Efficacy

Phage therapy, treating bacterial infections with bacteriophages, could be a future alternative to antibiotic treatment of bacterial infections. There are, however, several problems to be solved, mainly associated to the biology of phages, the interaction between phages and their bacterial hosts, but also to the vast variation of pathogenic bacteria which implies that large numbers of different phages are going to be needed. All of these phages must under present regulation of medical products undergo extensive clinical testing before they can be applied. It will consequently be of great economic importance that effective and versatile phages are selected and collected into phage libraries, i.e., the selection must be carried out in a way that it results in highly virulent phages with broad host ranges. We have isolated phages using the Escherichia coli reference (ECOR) collection and compared two methods, spot testing and efficiency of plating (EOP), which are frequently used to identify phages suitable for phage therapy. The analyses of the differences between the two methods show that spot tests often overestimate both the overall virulence and the host range and that the results are not correlated to the results of EOP assays. The conclusion is that single dilution spot tests cannot be used for identification and selection of phages to a phage library and should be replaced by EOP assays. The difference between the two methods can be caused by many factors. We have analysed if the differences and lack of correlation could be caused by lysis from without, bacteriocins in the phage lysate, or by the presence of prophages harbouring genes coding for phage resistance systems in the genomes of the bacteria in the ECOR collection.     

Sequencing of CJIE1 prophages from Campylobacter jejuni isolates reveals the presence of inserted and (or) deleted genes

Bacteriophages capable of integrating into host bacterial genomes as prophages affect the biology and virulence of their bacterial hosts. Previously, partial sequencing of 12 prophages similar to CJIE1 from Campylobacter jejuni RM1221 did not show the presence of inserted nonphage genes. Therefore, four of these prophages were sequenced completely, and indels were found in at least two different regions of the prophage genome. Putative proteins from one indel appeared to be members of two new families of proteins, with proteins within each family related to each other by a common domain. Further heterogeneity was found adjacent to the CJE0270 homolog, creating difficulty locating the end of the prophage on this side and in determining the composition of the core prophage. These prophages appear to comprise a family that has heterogeneity in gene content resulting from insertion or deletion of additional genes at three locations in their genomes. In addition, members of the CJIE1 phage family may differ somewhat in their biology from phage Mu. Further investigations of these Campylobacter prophages can be expected to provide interesting insights into the biology of the phages themselves and into the role of these phages in the biology of their hosts.     

The terminally redundant, nonpermuted genome of Listeria bacteriophage A511: a model for the SPO1-like myoviruses of gram-positive bacteria

Only little information on a particular class of myoviruses, the SPO1-like bacteriophages infecting low-G+C-content, gram-positive host bacteria (Firmicutes), is available. We present the genome analysis and molecular characterization of the large, virulent, broad-host-range Listeria phage A511. A511 contains a unit (informational) genome of 134,494 bp, encompassing 190 putative open reading frames (ORFs) and 16 tRNA genes, organized in a modular fashion common among the Caudovirales. Electron microscopy, enzymatic fragmentation analyses, and sequencing revealed that the A511 DNA molecule contains linear terminal repeats of a total of 3,125 bp, encompassing nine small putative ORFs. This particular genome structure explains why A511 is unable to perform general transduction. A511 features significant sequence homologies to Listeria phage P100 and other morphologically related phages infecting Firmicutes such as Staphylococcus phage K and Lactobacillus phage LP65. Equivalent but more-extensive terminal repeats also exist in phages P100 (approximately 6 kb) and K (approximately 20 kb). High-resolution electron microscopy revealed, for the first time, the presence of long tail fibers organized in a sixfold symmetry in these viruses. Mass spectrometry-based peptide fingerprinting permitted assignment of individual proteins to A511 structural components. On the basis of the data available for A511 and relatives, we propose that SPO1-like myoviruses are characterized by (i) their infection of gram-positive, low-G+C-content bacteria; (ii) a wide host range within the host bacterial genus and a strictly virulent lifestyle; (iii) similar morphology, sequence relatedness, and collinearity of the phage genome organization; and (iv) large double-stranded DNA genomes featuring nonpermuted terminal repeats of various sizes.     

Characterization of the ELPhiS prophage from Salmonella enterica serovar Enteritidis strain LK5

Phages are a primary driving force behind the evolution of bacterial pathogens by transferring a variety of virulence genes into their hosts. Similar to other bacterial genomes, the Salmonella enterica serovar Enteritidis LK5 genome contains several regions that are homologous to phages. Although genomic analysis demonstrated the presence of prophages, it was unable to confirm which phage elements within the genome were viable. Genetic markers were used to tag one of the prophages in the genome to allow monitoring of phage induction. Commonly used laboratory strains of Salmonella were resistant to phage infection, and therefore a rapid screen was developed to identify susceptible hosts. This approach showed that a genetically tagged prophage, ELPhiS (Enteritidis lysogenic phage S), was capable of infecting Salmonella serovars that are diverse in host range and virulence and has the potential to laterally transfer genes between these serovars via lysogenic conversion. The rapid screen approach is adaptable to any system with a large collection of isolates and may be used to test the viability of prophages found by sequencing the genomes of various bacterial pathogens.     

Marine phage genomics

Marine phages are the most abundant biological entities in the oceans. They play important roles in carbon cycling through marine food webs, gene transfer by transduction and conversion of hosts by lysogeny. The handful of marine phage genomes that have been sequenced to date, along with prophages in marine bacterial genomes, and partial sequencing of uncultivated phages are yielding glimpses of the tremendous diversity and physiological potential of the marine phage community. Common gene modules in diverse phages are providing the information necessary to make evolutionary comparisons. Finally, deciphering phage genomes is providing clues about the adaptive response of phages and their hosts to environmental cues.     

New method for evaluation of genotoxicity, based on the use of real-time PCR and lysogenic gram-positive and gram-negative bacteria

A method for the detection of the SOS response as measured by the liberation of resident prophages from the genomes of their hosts is described. It is based on the use of two converging oligonucleotides that flank the attP attachment site of the phage as primers for real-time PCR. Amplification was observed only after the phage DNA became excised. The system responds to both chemicals and physical conditions. Quantitative data on the concentration and/or potency of the genotoxic condition were obtained. Results can be achieved within 1 day and are less susceptible to possible toxic effects than phage generation or other methods that require DNA synthesis. The use of both gram-positive and gram-negative bacteria widens the range of compounds that can be tested because it eliminates impermeability problems derived from the particular composition of each cell wall type.     

Widespread distribution of a group I intron and its three deletion derivatives in the lysin gene of Streptococcus thermophilus bacteriophages

Of 62 Streptococcus thermophilus bacteriophages isolated from various ecological settings, half contain a lysin gene interrupted by a group IA2 intron. Phage mRNA splicing was demonstrated. Five phages possess a variant form of the intron resulting from three distinct deletion events located in the intron-harbored open reading frame (orf 253). The predicted orf 253 gene sequence showed a significantly lower GC content than the surrounding intron and lysin gene sequences, and the predicted protein shared a motif with endonucleases found in phages from both gram-positive and gram-negative bacteria. A comparison of the phage lysin genes revealed a clear division between intron-containing and intron-free alleles, leading to the establishment of a 14-bp consensus sequence associated with intron possession. The conserved intron was not found elsewhere in the phage or S. thermophilus bacterial genomes. Folding of the intron RNA revealed secondary structure elements shared with other phage introns: first, a 38-bp insertion between regions P3 and P4 that can be folded into two stem-loop structures (shared with introns from Bacillus phage SPO1 and relatives); second, a conserved P7.2 region (shared with all phage introns); third, the location of the stop codon from orf 253 in the P8 stem (shared with coliphage T4 and Bacillus phage SPO1 introns); fourth, orf 253, which has sequence similarity with the H-N-H motif of putative endonuclease genes found in introns from Lactococcus, Lactobacillus, and Bacillus phages.     

New Method for Evaluation of Genotoxicity, Based on the Use of Real-Time PCR and Lysogenic Gram-Positive and Gram-Negative Bacteria

A method for the detection of the SOS response as measured by the liberation of resident prophages from the genomes of their hosts is described. It is based on the use of two converging oligonucleotides that flank the attP attachment site of the phage as primers for real-time PCR. Amplification was observed only after the phage DNA became excised. The system responds to both chemicals and physical conditions. Quantitative data on the concentration and/or potency of the genotoxic condition were obtained. Results can be achieved within 1 day and are less susceptible to possible toxic effects than phage generation or other methods that require DNA synthesis. The use of both gram-positive and gram-negative bacteria widens the range of compounds that can be tested because it eliminates impermeability problems derived from the particular composition of each cell wall type.     

前噬菌体研究进展

噬菌体是地球上最多的生物实体,一直被认为是细菌的天敌。然而随着基因组学和分子生物学等技术的快速发展,人们发现噬菌体与宿主之间存在微妙而复杂的关系。前噬菌体是指溶原性细菌内存在的整套噬菌体DNA基因组,广泛分布在细菌基因组中,对调节细菌宿主生理具有重要作用,如参与调节宿主的毒力、影响生物膜形成、赋予宿主免疫力等。有趣的是,前噬菌体可以通过"监听"细菌的群体感应来调节自身的溶原–裂解状态。近年来,一些细菌中由前噬菌体编码的抗CRISPR蛋白的发现引起了人们对前噬菌体研究的关注。因此,对前噬菌体的研究可以为改造宿主和前噬菌体提供基础理论参考。本文对前噬菌体的预测、分布、分类及功能进行了综述,以期为进一步研究噬菌体与宿主间的关系提供基础。     

Prophage Tracer: precisely tracing prophages in prokaryotic genomes using overlapping split-read alignment

The life cycle of temperate phages includes a lysogenic cycle stage when the phage integrates into the host genome and becomes a prophage. However, the identification of prophages that are highly divergent from known phages remains challenging. In this study, by taking advantage of the lysis-lysogeny switch of temperate phages, we designed Prophage Tracer, a tool for recognizing active prophages in prokaryotic genomes using short-read sequencing data, independent of phage gene similarity searching. Prophage Tracer uses the criterion of overlapping split-read alignment to recognize discriminative reads that contain bacterial (attB) and phage (attP) att sites representing prophage excision signals. Performance testing showed that Prophage Tracer could predict known prophages with precise boundaries, as well as novel prophages. Two novel prophages, dsDNA and ssDNA, encoding highly divergent major capsid proteins, were identified in coral-associated bacteria. Prophage Tracer is a reliable data mining tool for the identification of novel temperate phages and mobile genetic elements. The code for the Prophage Tracer is publicly available at https://github.com/WangLab-SCSIO/Prophage_Tracer.     

Phylogenetic and complementation analysis of a single-stranded DNA binding protein family from lactococcal phages indicates a non-bacterial origin

Background

The single-stranded-nucleic acid binding (SSB) protein superfamily includes proteins encoded by different organisms from Bacteria and their phages to Eukaryotes. SSB proteins share common structural characteristics and have been suggested to descend from an ancestor polypeptide. However, as other proteins involved in DNA replication, bacterial SSB proteins are clearly different from those found in Archaea and Eukaryotes. It was proposed that the corresponding genes in the phage genomes were transferred from the bacterial hosts. Recently new SSB proteins encoded by the virulent lactococcal bacteriophages (Orf14_bIL67-like proteins) have been identified and characterized structurally and biochemically.

Methodology/Principal Findings

This study focused on the determination of phylogenetic relationships between Orf14_bIL67-like proteins and other SSBs. We have performed a large scale phylogenetic analysis and pairwise sequence comparisons of SSB proteins from different phyla. The results show that, in remarkable contrast to other phage SSBs, the Orf14_bIL67–like proteins form a distinct, self-contained and well supported phylogenetic group connected to the archaeal SSBs. Functional studies demonstrated that, despite the structural and amino acid sequence differences from bacterial SSBs, Orf14_bIL67 protein complements the conditional lethal ssb-1 mutation of Escherichia coli.

Conclusions/Significance

Here we identified for the first time a group of phages encoded SSBs which are clearly distinct from their bacterial counterparts. All methods supported the recognition of these phage proteins as a new family within the SSB superfamily. Our findings suggest that unlike other phages, the virulent lactococcal phages carry ssb genes that were not acquired from their hosts, but transferred from an archaeal genome. This represents a unique example of a horizontal gene transfer between Archaea and bacterial phages.     

一株枯草芽孢杆菌原噬菌体Bsu-yong1的基因组分析

【目的】枯草芽孢杆菌（Bacillus subtilis）是在自然界中广泛存在的革兰氏阳性菌，其抗逆性极强，能抑制大多数有害菌的繁殖，是常用的产酶菌，用其生产的蛋白酶、淀粉酶占全球工业酶产量的50%。原噬菌体（prophage）整合在宿主基因组中，可为宿主提供基因和生物学功能，非常具有研究价值。以往，有关B. subtilis原噬菌体的报道主要集中于缺陷型原噬菌体（defective prophage），本研究对一株非缺陷型活性原噬菌体（active prophage）的基因组进行解析，以扩充对非缺陷型原噬菌体的认知。【方法】使用丝裂霉素C从枯草芽孢杆菌中诱导一株噬菌体，命名为Bacillus phage Bsu-yong1（简称Bsu-yong1）。对Bsu-yong1进行负染、透射电镜（transmission electron microscopy，TEM）观察，用Illumina MiSeq测定其基因组序列、综合运用生物信息学工具对其进行基因功能注释和系统进化分析。【结果】Bsu-yong1与PBSX类缺陷型原噬菌体在形态上相似，但Bsu-yong1具有完整的噬菌体基因组，这与缺陷型原噬菌体不同，后者在包装过程中不能正确包裹自身的基因组，而是随机包裹一段宿主染色体。Bsu-yong1基因组全长为43 590 bp，G+C含量为41%，含有62个开放阅读框（open reading frame，ORF），呈模块化分布。Bsu-yong1拥有基因编码T7SS效应器LXG多态性毒素（T7SS effector LXG polymorphic toxin）、ImmA/IrrE蛋白和SMI1/KNR4蛋白。前二者为细菌毒素（toxin），后者为抗毒素（antitoxin），toxin-antitoxin是细菌免疫系统重要成员，参与菌间竞争与环境适应。此前，尚未有编码LXG polymorphic toxin的基因在噬菌体中被发现和报道。在基于全基因组比对构建的蛋白谱进化树（proteomic tree）中，Bsu-yong1与噬菌体sv105、rho14、vB_BteM-A9Y聚集形成一个独立的进化支（clade），基因组比对显示它们基因组的复制与调控模块具有高度保守性，它们共享29个核心基因（core gene），均具有PBSX样形态特征。Bsu-yong1与其他噬菌体的进化距离较远。将Bsu-yong1与所有噬菌体进行比对，得到的成对序列比较（pairwise sequence comparison，PASC）最大值为46.72%，小于属边界值（70%）。【结论】vB_Bsu-yong1在有尾纲中代表一个新的未知的属；建议构建一个新的科（family），该科由Bsu-yong1与噬菌体sv105、rho14、vB_BteM-A9Y组成。vB_Bsu-yong携带免疫相关基因，它可能有利于宿主在菌间竞争中获胜和适应环境。本研究丰富了噬菌体基因数据库，拓展了对芽孢杆菌活性原噬菌体的认知。