Prophage-like elements present in Mycobacterium genomes

Background

Prophages, integral components of many bacterial genomes, play significant roles in cognate host bacteria, such as virulence, toxin biosynthesis and secretion, fitness cost, genomic variations, and evolution. Many prophages and prophage-like elements present in sequenced bacterial genomes, such as Bifidobacteria, Lactococcus and Streptococcus, have been described. However, information for the prophage of Mycobacterium remains poorly defined.

Results

In this study, based on the search of the complete genome database from GenBank, the Whole Genome Shotgun (WGS) databases, and some published literatures, thirty-three prophages were described in detail. Eleven of them were full-length prophages, and others were prophage-like elements. Eleven prophages were firstly revealed. They were phiMAV_1, phiMAV_2, phiMmcs_1, phiMmcs_2, phiMkms_1, phiMkms_2, phiBN42_1, phiBN44_1, phiMCAN_1, phiMycsm_1, and phiW7S_1. Their genomes and gene contents were firstly analyzed. Furthermore, comparative genomics analyses among mycobacterioprophages showed that full-length prophage phi172_2 belonged to mycobacteriophage Cluster A and the phiMmcs_1, phiMkms_1, phiBN44_1, and phiMCAN_1 shared high homology and could be classified into one group.

Conclusions

To our knowledge, this is the first systematic characterization of mycobacterioprophages, their genomic organization and phylogeny. This information will afford more understanding of the biology of Mycobacterium.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-243) contains supplementary material, which is available to authorized users.     

Prophage genomics.

The majority of the bacterial genome sequences deposited in the National Center for Biotechnology Information database contain prophage sequences. Analysis of the prophages suggested that after being integrated into bacterial genomes, they undergo a complex decay process consisting of inactivating point mutations, genome rearrangements, modular exchanges, invasion by further mobile DNA elements, and massive DNA deletion. We review the technical difficulties in defining such altered prophage sequences in bacterial genomes and discuss theoretical frameworks for the phage-bacterium interaction at the genomic level. The published genome sequences from three groups of eubacteria (low- and high-G+C gram-positive bacteria and gamma-proteobacteria) were screened for prophage sequences. The prophages from Streptococcus pyogenes served as test case for theoretical predictions of the role of prophages in the evolution of pathogenic bacteria. The genomes from further human, animal, and plant pathogens, as well as commensal and free-living bacteria, were included in the analysis to see whether the same principles of prophage genomics apply for bacteria living in different ecological niches and coming from distinct phylogenetical affinities. The effect of selection pressure on the host bacterium is apparently an important force shaping the prophage genomes in low-G+C gram-positive bacteria and gamma-proteobacteria.     

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.     

Identification of prophages in bacterial genomes by dinucleotide relative abundance difference

Background

Prophages are integrated viral forms in bacterial genomes that have been found to contribute to interstrain genetic variability. Many virulence-associated genes are reported to be prophage encoded. Present computational methods to detect prophages are either by identifying possible essential proteins such as integrases or by an extension of this technique, which involves identifying a region containing proteins similar to those occurring in prophages. These methods suffer due to the problem of low sequence similarity at the protein level, which suggests that a nucleotide based approach could be useful.

Methodology

Earlier dinucleotide relative abundance (DRA) have been used to identify regions, which deviate from the neighborhood areas, in genomes. We have used the difference in the dinucleotide relative abundance (DRAD) between the bacterial and prophage DNA to aid location of DNA stretches that could be of prophage origin in bacterial genomes. Prophage sequences which deviate from bacterial regions in their dinucleotide frequencies are detected by scanning bacterial genome sequences. The method was validated using a subset of genomes with prophage data from literature reports. A web interface for prophage scan based on this method is available at http://bicmku.in:8082/prophagedb/dra.html. Two hundred bacterial genomes which do not have annotated prophages have been scanned for prophage regions using this method.

Conclusions

The relative dinucleotide distribution difference helps detect prophage regions in genome sequences. The usefulness of this method is seen in the identification of 461 highly probable loci pertaining to prophages which have not been annotated so earlier. This work emphasizes the need to extend the efforts to detect and annotate prophage elements in genome sequences.     

Widespread distribution of prophages signaling the potential for adaptability and pathogenicity evolution of Ralstonia solanacearum species complex

Integrated bacteriophages (prophages) can impact host cells, affecting their lifestyle, genomic diversity, and fitness. However, many basic aspects of how these organisms affect the host cell remain poorly understood. Ralstonia solanacearum is a gram-negative plant pathogenic bacterium that encompasses a great diversity of ecotypes regarded as a species complex (R. solanacearum Species Complex - RSSC). RSSC genomes have a mosaic structure containing numerous elements, signaling the potential for its evolution through horizontal gene transfer. Here, we analyzed 120 Ralstonia spp. genomes from the public database to identify prophage sequences. In total, 379 prophage-like elements were found in the chromosome and megaplasmid of Ralstonia spp. These elements encode genes related to host fitness, virulence factors, antibiotic resistance, and niche adaptation, which might contribute to RSSC adaptability. Prophage-like elements are widespread into the complex in different species and geographic origins, suggesting that the RSSC phages are ancestrally acquired. Complete prophages belonging to the families Inoviridae, Myoviridae, and Siphoviridae were found, being the members of Inoviridae the most abundant. Analysis of CRISPR-Cas spacer sequences demonstrated the presence of prophages sequences that indicate successive infection events during bacterial evolution. Besides complete prophages, we also demonstrated 14 novel putative prophages integrated into Ralstonia spp. genomes. Altogether, our results provide insights into the diversity of prophages in RSSC genomes and suggest that these elements may deeply affect the virulence and host adaptation and shaping the genomes among the strains of this important pathogen.     

Distribution and function of prophage phiRv1 and phiRv2 among Mycobacterium tuberculosis complex

Mycobacterium tuberculosis complex (MTBC) is notorious for causing diseases, such as tuberculosis. Tuberculosis caused by M. tuberculosis remains a global public health concern. Two prophages, phiRv1 and phiRv2, can be found among most MTBC genomes. However, no precise functions have been assigned for the two prophages. In this paper, to find out the function of these two prophages, the distribution and function of phiRv1 and phiRv2 in MTBC genomes were analyzed from multiple omics data. We found that complex insertion, deletion, and reorganization appeared on the locus of two prophages in MTBC genomes; some genes of the two prophages can be translated and are functional from proteomic data; the expression of other prophage genes, such as Rv1577c, Rv2650c, Rv2652c, Rv2659c, and Rv2658c, can vary with environmental stresses and might enhance the fitness of MTBC. These data will facilitate our in-depth understanding of their function.     

Induction of Multiple Prophages from a Marine Bacterium: a Genomic Approach

Approximately 70% of sequenced bacterial genomes contain prophage-like structures, yet little effort has been made to use this information to determine the functions of these elements. The recent genomic sequencing of the marine bacterium Silicibacter sp. strain TM1040 revealed five prophage-like elements in its genome. The genomes of these prophages (named prophages 1 to 5) are approximately 74, 30, 39, 36, and 15 kb long, respectively. To understand the function of these prophages, cultures of TM1040 were treated with mitomycin C to induce the production of viral particles. A significant increase in viral counts and a decrease in bacterial counts when treated with mitomycin C suggested that prophages were induced from TM1040. Transmission electron microscopy revealed one dominant type of siphovirus, while pulsed-field gel electrophoresis demonstrated two major DNA bands, equivalent to 35 and 75 kb, in the lysate. PCR amplification with primer sets specific to each prophage detected the presence of prophages 1, 3, and 4 in the viral lysate, suggesting that these prophages are inducible, but not necessarily to the same level, while prophages 2 and 5 are likely defective or non-mitomycin C-inducible phages. The combination of traditional phage assays and modern microbial genomics provides a quick and efficient way to investigate the functions and inducibility of prophages, particularly for a host harboring multiple prophages with similar sizes and morphological features.     

酱香型白酒发酵中地衣芽孢杆菌前噬菌体的鉴别

【背景】噬菌体是微生物群落的重要组成部分,但传统白酒发酵中噬菌体的分类和存在尚不清楚。【目的】通过检测公共数据库和酱香型白酒发酵中地衣芽孢杆菌(Bacillus licheniformis)基因组中的前噬菌体整合区域,探究传统酱香型白酒发酵中关键功能菌株的前噬菌体分类和侵染情况。【方法】使用未培养(细菌全基因组分析)和可培养(菌株筛选和特异性PCR反应)技术对不同环境来源和来自酱香型白酒发酵的地衣芽孢杆菌前噬菌体的分类和存在进行解析。【结果】细菌全基因组分析显示,30株来自不同环境的地衣芽孢杆菌基因组中共注释到165个前噬菌体,其中63.6%(105/165)为完整前噬菌体序列。97.1%感染地衣芽孢杆菌的噬菌体属于长尾噬菌体科(Siphoviridae),2.9%属于肌尾噬菌体科(Myoviridae),53.0%完整前噬菌体的基因功能未知。在来自酱香型白酒发酵的B. licheniformis MT-B06中检测到7个前噬菌体整合序列,其中57.1%(4/7)为完整前噬菌体序列,来自酱香型白酒发酵的地衣芽孢杆菌存在多种不同前噬菌体的共感染。来自酱香型白酒发酵的地衣芽孢杆菌前噬菌体存在来自细菌基因组上相邻CotD孢子外壳蛋白(CotD family spore coat protein)基因的水平基因转移。在26株来自酱香型白酒发酵的地衣芽孢杆菌中,69.2%(18/26)存在噬菌体编码主要衣壳蛋白的基因,100.0%(26/26)存在噬菌体编码CotD孢子外壳蛋白的基因。【结论】来自不同环境的地衣芽孢杆菌和酱香型白酒发酵的地衣芽孢杆菌中存在高水平的前噬菌体整合,来自酱香型白酒发酵的地衣芽孢杆菌前噬菌体中广泛存在来源于宿主的CotD孢子外壳蛋白基因的水平基因转移。本研究为首次对传统发酵白酒中噬菌体的分类和存在进行探究,有助于对发酵微生物群落中噬菌体-细菌相互作用加深理解。     

Comparative genomic analysis of pyrene-degrading <Emphasis Type="Italic">Mycobacterium</Emphasis> species: Genomic islands and ring-hydroxylating dioxygenases involved in pyrene degradation

The genome sequences of two pyrene-degrading bacterial strains of Mycobacterium spp. PYR10 and PYR15, isolated from the estuarine wetland of the Han river, South Korea, were determined using the PacBio RS II sequencing platform. The complete genome of strain PYR15 was 6,037,017 bp in length with a GC content of 66.5%, and contained 5,933 protein-coding genes. The genome of strain PYR10 was 5,999,427 bp in length with a GC content of 67.7%, and contained 5,767 protein-coding genes. Based on the average nucleotide identity values, these strains were designated as M. gilvum PYR10 and M. pallens PYR15. A genomic comparison of these pyrene-degrading Mycobacterium strains with pyrene-non-degrading strains revealed that the genomes of pyrene-degrading strains possessed similar repertoires of ringhydroxylating dioxygenases (RHDs), including the pyrenehydroxylating dioxygenases encoded by nidA and nidA3, which could be readily distinguished from those of pyrenenon-degraders. Furthermore, genomic islands, containing catabolic gene clusters, were shared only among the pyrenedegrading Mycobacterium strains and these gene clusters contained RHD genes, including nidAB and nidA3B3. Our genome data should facilitate further studies on the evolution of the polycyclic aromatic hydrocarbon-degradation pathways in the genus Mycobacterium.     

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.     

Spectrum of heart diseases in a new cardiac service in Nigeria: An echocardiographic study of 1441 subjects in Abeokuta

Background

The recent determination of the complete nucleotide sequence of several Mycobacterium tuberculosis (MTB) genomes allows the use of comparative genomics as a tool for dissecting the nature and consequence of genetic variability within this species. The multiple alignment of the genomes of clinical strains (CDC1551, F11, Haarlem and C), along with the genomes of laboratory strains (H37Rv and H37Ra), provides new insights on the mechanisms of adaptation of this bacterium to the human host.

Findings

The genetic variation found in six M. tuberculosis strains does not involve significant genomic rearrangements. Most of the variation results from deletion and transposition events preferentially associated with insertion sequences and genes of the PE/PPE family but not with genes implicated in virulence. Using a Perl-based software islandsanalyser, which creates a representation of the genetic variation in the genome, we identified differences in the patterns of distribution and frequency of the polymorphisms across the genome. The identification of genes displaying strain-specific polymorphisms and the extrapolation of the number of strain-specific polymorphisms to an unlimited number of genomes indicates that the different strains contain a limited number of unique polymorphisms.

Conclusion

The comparison of multiple genomes demonstrates that the M. tuberculosis genome is currently undergoing an active process of gene decay, analogous to the adaptation process of obligate bacterial symbionts. This observation opens new perspectives into the evolution and the understanding of the pathogenesis of this bacterium.     

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.     

Comparative analysis of bacterial genomes: identification of divergent regions in mycobacterial strains using an anchor-based approach

Comparative genomic approaches are useful in identifying molecular differences between organisms. Currently available methods fail to identify small changes in genomes, such as expansion of short repetitive motifs and to analyse divergent sequences. In this report, we describe an anchor-based whole genome comparison (ABWGC) method. ABWGC is based on random sampling of anchor sequences from one genome, followed by analysis of sampled and homologous regions from the target genome. The method was applied to compare two strains of Mycobacterium tuberculosis CDC1551 and H37Rv. ABWGC was able to identify a total of 104 indels including 20 expansion of short repetitive sequences and five recombination events. It included 18 new unidentified genomic differences. ABWGC also identified 188 SNPs including eight new ones. The method was also used to compare M. tuberculosis H37Rv and M. avium genomes. ABWGC was able to correctly pick 1002 additional indels (size>100nt) between the two organisms in contrast to MUMmer, a popular tool for comparative genomics. ABWGC was able to identify correctly repeat expansion and indels in a set of simulated sequences. The study also revealed important role of small repeat expansion in the evolution of M. tuberculosis strains.     

Carriage of λ Latent Virus Is Costly for Its Bacterial Host due to Frequent Reactivation in Monoxenic Mouse Intestine

Temperate phages, the bacterial viruses able to enter in a dormant prophage state in bacterial genomes, are present in the majority of bacterial strains for which the genome sequence is available. Although these prophages are generally considered to increase their hosts’ fitness by bringing beneficial genes, studies demonstrating such effects in ecologically relevant environments are relatively limited to few bacterial species. Here, we investigated the impact of prophage carriage in the gastrointestinal tract of monoxenic mice. Combined with mathematical modelling, these experimental results provided a quantitative estimation of key parameters governing phage-bacteria interactions within this model ecosystem. We used wild-type and mutant strains of the best known host/phage pair, Escherichia coli and phage λ. Unexpectedly, λ prophage caused a significant fitness cost for its carrier, due to an induction rate 50-fold higher than in vitro, with 1 to 2% of the prophage being induced. However, when prophage carriers were in competition with isogenic phage susceptible bacteria, the prophage indirectly benefited its carrier by killing competitors: infection of susceptible bacteria led to phage lytic development in about 80% of cases. The remaining infected bacteria were lysogenized, resulting overall in the rapid lysogenization of the susceptible lineage. Moreover, our setup enabled to demonstrate that rare events of phage gene capture by homologous recombination occurred in the intestine of monoxenic mice. To our knowledge, this study constitutes the first quantitative characterization of temperate phage-bacteria interactions in a simplified gut environment. The high prophage induction rate detected reveals DNA damage-mediated SOS response in monoxenic mouse intestine. We propose that the mammalian gut, the most densely populated bacterial ecosystem on earth, might foster bacterial evolution through high temperate phage activity.     

Predominance of Single Prophage Carrying a CRISPR/cas System in “Candidatus Liberibacter asiaticus” Strains in Southern China

“Candidatus Liberibacter asiaticus” (CLas) is an uncultureable α-proteobacterium associated with citrus Huanglongbing (HLB, yellow shoot disease), a highly destructive disease affecting citrus production worldwide. HLB was observed in Guangdong Province of China over a hundred years ago and remains endemic there. Little is known about CLas biology due to its uncultureable nature. This study began with the genome sequence analysis of CLas Strain A4 from Guangdong in the prophage region. Within the two currently known prophage types, Type 1 (SC1-like) and Type 2 (SC2-like), A4 genome contained only a Type 2 prophage, CGdP2, namely. An analysis on CLas strains collected in Guangdong showed that Type 2 prophage dominated the bacterial population (82.6%, 71/86). An extended survey covering five provinces in southern China also revealed the predominance of single prophage (Type 1 or Type 2) in the CLas population (90.4%, 169/187). CLas strains with two and no prophage types accounted for 7.2% and 2.8%, respectively. In silico analyses on CGdP2 identified a CRISPR (clustered regularly interspaced short palindromic repeats)/cas (CRISPR-associated protein genes) system, consisting of four 22 bp repeats, three 23 bp spacers and 9 predicted cas. Similar CRISPR/cas systems were detected in all 10 published CLas prophages as well as 13 CLas field strains in southern China. Both Type 1 and Type 2 prophages shared almost identical sequences in spacer 1 and 3 but not spacer 2. Considering that the function of a CRISPR/cas system was to destroy invading DNA, it was hypothesized that a pre-established CLas prophage could use its CRISPR/cas system guided by spacer 1 and/or 3 to defeat the invasion of the other phage/prophage. This hypothesis explained the predominance of single prophage type in the CLas population in southern China. This is the first report of CRISPR/cas system in the “Ca. Liberibacter” genera.     

Prophages in Salmonella enterica: a driving force in reshaping the genome and physiology of their bacterial host?

Thanks to the exponentially increasing number of publicly available bacterial genome sequences, one can now estimate the important contribution of integrated viral sequences to the diversity of bacterial genomes. Indeed, temperate bacteriophages are able to stably integrate the genome of their host through site‐specific recombination and transmit vertically to the host siblings. Lysogenic conversion has been long acknowledged to provide additional functions to the host, and particularly to bacterial pathogen genomes where prophages contribute important virulence factors. This review aims particularly at highlighting the current knowledge and questions about lysogeny in Salmonella genomes where functional prophages are abundant, and where genetic interactions between host and prophages are of particular importance for human health considerations.     

High resolution assembly and characterization of genomes of Canadian isolates of Salmonella Enteritidis

Background

There is a need to characterize genomes of the foodborne pathogen, Salmonella enterica serovar Enteritidis (SE) and identify genetic information that could be ultimately deployed for differentiating strains of the organism, a need that is yet to be addressed mainly because of the high degree of clonality of the organism. In an effort to achieve the first characterization of the genomes of SE of Canadian origin, we carried out massively parallel sequencing of the nucleotide sequence of 11 SE isolates obtained from poultry production environments (n = 9), a clam and a chicken, assembled finished genomes and investigated diversity of the SE genome.

Results

The median genome size was 4,678,683 bp. A total of 4,833 chromosomal genes defined the pan genome of our field SE isolates consisting of 4,600 genes present in all the genomes, i.e., core genome, and 233 genes absent in at least one genome (accessory genome). Genome diversity was demonstrable by the presence of 1,360 loci showing single nucleotide polymorphism (SNP) in the core genome which was used to portray the genetic distances by means of a phylogenetic tree for the SE isolates. The accessory genome consisted mostly of previously identified SE prophage sequences as well as two, apparently full- sized, novel prophages namely a 28 kb sequence provisionally designated as SE-OLF-10058 (3) prophage and a 43 kb sequence provisionally designated as SE-OLF-10012 prophage.

Conclusions

The number of SNPs identified in the relatively large core genome of SE is a reflection of substantial diversity that could be exploited for strain differentiation as shown by the development of an informative phylogenetic tree. Prophage sequences can also be exploited for SE strain differentiation and lineage tracking. This work has laid the ground work for further studies to develop a readily adoptable laboratory test for the subtyping of SE.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-713) contains supplementary material, which is available to authorized users.