-
Nucleic Acids Research May 2020The increasing use of CRISPR-Cas9 in medicine, agriculture, and synthetic biology has accelerated the drive to discover new CRISPR-Cas inhibitors as potential mechanisms...
The increasing use of CRISPR-Cas9 in medicine, agriculture, and synthetic biology has accelerated the drive to discover new CRISPR-Cas inhibitors as potential mechanisms of control for gene editing applications. Many anti-CRISPRs have been found that inhibit the CRISPR-Cas adaptive immune system. However, comparing all currently known anti-CRISPRs does not reveal a shared set of properties for facile bioinformatic identification of new anti-CRISPR families. Here, we describe AcRanker, a machine learning based method to aid direct identification of new potential anti-CRISPRs using only protein sequence information. Using a training set of known anti-CRISPRs, we built a model based on XGBoost ranking. We then applied AcRanker to predict candidate anti-CRISPRs from predicted prophage regions within self-targeting bacterial genomes and discovered two previously unknown anti-CRISPRs: AcrllA20 (ML1) and AcrIIA21 (ML8). We show that AcrIIA20 strongly inhibits Streptococcus iniae Cas9 (SinCas9) and weakly inhibits Streptococcus pyogenes Cas9 (SpyCas9). We also show that AcrIIA21 inhibits SpyCas9, Streptococcus aureus Cas9 (SauCas9) and SinCas9 with low potency. The addition of AcRanker to the anti-CRISPR discovery toolkit allows researchers to directly rank potential anti-CRISPR candidate genes for increased speed in testing and validation of new anti-CRISPRs. A web server implementation for AcRanker is available online at http://acranker.pythonanywhere.com/.
Topics: Bacterial Proteins; CRISPR-Associated Protein 9; Machine Learning; Prophages; Proteome; Sequence Analysis, Protein; Streptococcus
PubMed: 32286628
DOI: 10.1093/nar/gkaa219 -
Nucleic Acids Research Sep 2023Prophages control their lifestyle to either be maintained within the host genome or enter the lytic cycle. Bacillus subtilis contains the SPβ prophage whose lysogenic...
Prophages control their lifestyle to either be maintained within the host genome or enter the lytic cycle. Bacillus subtilis contains the SPβ prophage whose lysogenic state depends on the MrpR (YopR) protein, a key component of the lysis-lysogeny decision system. Using a historic B. subtilis strain harboring the heat-sensitive SPβ c2 mutant, we demonstrate that the lytic cycle of SPβ c2 can be induced by heat due to a single nucleotide exchange in the mrpR gene, rendering the encoded MrpRG136E protein temperature-sensitive. Structural characterization revealed that MrpR is a DNA-binding protein resembling the overall fold of tyrosine recombinases. MrpR has lost its recombinase function and the G136E exchange impairs its higher-order structure and DNA binding activity. Genome-wide profiling of MrpR binding revealed its association with the previously identified SPbeta repeated element (SPBRE) in the SPβ genome. MrpR functions as a master repressor of SPβ that binds to this conserved element to maintain lysogeny. The heat-inducible excision of the SPβ c2 mutant remains reliant on the serine recombinase SprA. A suppressor mutant analysis identified a previously unknown component of the lysis-lysogeny management system that is crucial for the induction of the lytic cycle of SPβ.
Topics: Bacillus Phages; Bacillus subtilis; Bacteriophages; Lysogeny; Prophages; Recombinases; Viral Proteins
PubMed: 37602373
DOI: 10.1093/nar/gkad675 -
BMC Microbiology Nov 2022Lactic acid bacteria (LAB) are used as starters in a wide variety of food fermentations. While the number of reports of phages infecting other LAB steadily increased...
BACKGROUND
Lactic acid bacteria (LAB) are used as starters in a wide variety of food fermentations. While the number of reports of phages infecting other LAB steadily increased over the years, information about phage associated with Latilactobacillus sakei, a frequently used meat starter, remains scarce.
RESULTS
In this study, a predictive genomic analysis of 43 Latilactobacillus sakei genomes revealed the presence of 26 intact, eleven questionable and 52 incomplete prophage sequences across all analysed genomes with a range of one to five predicted prophage sequences per strain. Screening 24 sakei strains for inducible prophages by utilising UV light or mitomycin C, we identified seven lysogenic strains showing lysis after induction during subsequent growth monitoring. Electron microscopic analysis revealed fully assembled virions in the purified lysates of four samples, thus confirming successful prophage induction. All virions featured icosahedral, isomeric heads and long, most likely non-contractile tails indicating siphoviruses. By performing phylogenetic analyses with various marker genes as well as full prophage sequences, we displayed a remarkably high diversity of prophages, that share a similar gene module organisation and six different chromosomal integration sites were identified. By sequencing viral DNA purified from lysates of Latilactobacillus sakei TMW 1.46, we demonstrate that simultaneous induction of multiple prophages is possible.
CONCLUSIONS
With this work, we not only provide data about the incidence of prophages harboured by the meat starter Latilactobacillus sakei, we also demonstrated their potential to impact growth of their host after induction, as well as forming seemingly fully assembled virions.
Topics: Prophages; Phylogeny; Genome, Bacterial; Lysogeny; Bacteriophages
PubMed: 36348293
DOI: 10.1186/s12866-022-02675-y -
Infection, Genetics and Evolution :... Sep 2023To systematically investigate the prophages carrying in Porphyromonas gingivalis (P. gingivalis) strains, analyze potential antibiotic resistance genes (ARGs) and...
To systematically investigate the prophages carrying in Porphyromonas gingivalis (P. gingivalis) strains, analyze potential antibiotic resistance genes (ARGs) and virulence genes in these prophages. We collected 90 whole genome sequences of P. gingivalis from NCBI and utilized the Prophage Hunter online software to predict prophages; Comprehensive antibiotic research database (CARD) and virulence factors database (VFDB) were adopted to analyze the ARGs and virulence factors (VFs) carried by the prophages. Sixty-nine prophages were identified among 24/90 P. gingivalis strains, including 17 active prophages (18.9%) and 52 ambiguous prophages (57.8%). The proportion of prophages carried by each P. gingivalis genome ranged from 0.5% to 6.7%. A total of 188 antibiotic resistance genes belonging to 25 phenotypes and 46 different families with six mechanisms of antibiotic resistance were identified in the 17 active prophages. Three active prophages encoded 4 virulence genes belonging to type III and type VI secretion systems. The potential hosts of these virulence genes included Escherichia coli, Shigella sonnei, Salmonella typhi, and Klebsiella pneumoniae. In conclusion, 26.7% P. gingivalis strains carry prophages, while the proportion of prophage genes in the P. gingivalis genome is relatively low. In addition, approximately 39.7% of the P. gingivalis prophage genes have ARGs identified, mainly against streptogramin, peptides, and aminoglycosides. Only a few prophages carry virulence genes. Prophages may play an important role in the acquisition, dissemination of antibiotic resistance genes, and pathogenicity evolution in P. gingivalis.
Topics: Prophages; Genome, Bacterial; Porphyromonas gingivalis; Virulence Factors; Virulence; Escherichia coli; Anti-Bacterial Agents
PubMed: 37572952
DOI: 10.1016/j.meegid.2023.105489 -
Viruses Sep 2022Bacterial infections of livestock threaten the sustainability of agriculture and public health through production losses and contamination of food products. While... (Review)
Review
Bacterial infections of livestock threaten the sustainability of agriculture and public health through production losses and contamination of food products. While prophylactic and therapeutic application of antibiotics has been successful in managing such infections, the evolution and spread of antibiotic-resistant strains along the food chain and in the environment necessitates the development of alternative or adjunct preventive and/or therapeutic strategies. Additionally, the growing consumer preference for "greener" antibiotic-free food products has reinforced the need for novel and safer approaches to controlling bacterial infections. The use of bacteriophages (phages), which can target and kill bacteria, are increasingly considered as a suitable measure to reduce bacterial infections and contamination in the food industry. This review primarily elaborates on the recent veterinary applications of phages and discusses their merits and limitations. Furthermore, using as a model, we describe the prevalence of prophages and the anti-viral defence arsenal in the genome of the pathogen as a means to define the genetic building blocks that are available for the (synthetic) development of phage-based treatments. The data and approach described herein may provide a framework for the development of therapeutics against an array of bacterial pathogens.
Topics: Anti-Bacterial Agents; Bacteria; Bacterial Infections; Bacteriophages; Farms; Humans; Prophages; Streptococcus suis
PubMed: 36146802
DOI: 10.3390/v14091996 -
Applied and Environmental Microbiology Sep 2019The fast-growing marine bacterium represents an emerging strain for molecular biology and biotechnology. Genome sequencing and quantitative PCR analysis revealed that...
The fast-growing marine bacterium represents an emerging strain for molecular biology and biotechnology. Genome sequencing and quantitative PCR analysis revealed that the first chromosome of ATCC 14048 contains two prophage regions (VNP1 and VNP2) that are both inducible by the DNA-damaging agent mitomycin C and exhibit spontaneous activation under standard cultivation conditions. Their activation was also confirmed by live cell imaging of an mCherry fusion to the major capsid proteins of VNP1 and VNP2. Transmission electron microscopy visualized the release of phage particles belonging to the family into the culture supernatant. Freeing from its proviral load, followed by phenotypic characterization, revealed an improved robustness of the prophage-free variant toward DNA-damaging conditions, reduced cell lysis under hypo-osmotic conditions, and an increased pyruvate production compared to wild-type levels. Remarkably, the prophage-free strain outcompeted the wild type in a competitive growth experiment, emphasizing that this strain is a promising platform for future metabolic engineering approaches. The fast-growing marine bacterium represents an emerging model host for molecular biology and biotechnology, featuring a reported doubling time of less than 10 minutes. In many bacterial species, viral DNA (prophage elements) may constitute a considerable fraction of the whole genome and may have detrimental effects on the growth and fitness of industrial strains. Genome analysis revealed the presence of two prophage regions in the genome that were shown to undergo spontaneous induction under standard cultivation conditions. In this study, we generated a prophage-free variant of Remarkably, the prophage-free strain exhibited a higher tolerance toward DNA damage and hypo-osmotic stress. Moreover, it was shown to outcompete the wild-type strain in a competitive growth experiment. In conclusion, our study presents the prophage-free variant of as a promising platform strain for future biotechnological applications.
Topics: DNA Damage; Osmotic Pressure; Prophages; Vibrio
PubMed: 31253674
DOI: 10.1128/AEM.00853-19 -
Cell Host & Microbe Nov 2021Bacteria have evolved many immune systems to combat their viral parasites (i.e., phages). In this issue of Cell Host & Microbe, Owen et al. discover a mechanism of...
Bacteria have evolved many immune systems to combat their viral parasites (i.e., phages). In this issue of Cell Host & Microbe, Owen et al. discover a mechanism of anti-phage immunity that is mediated by a phage-encoded protein, and thus provide an example of how inter-phage conflict can promote survival of the bacterial population.
Topics: Bacteria; Bacteriophages; Prophages
PubMed: 34762825
DOI: 10.1016/j.chom.2021.10.004 -
Microbial Genomics Nov 2021The human zoonotic pathogen O157:H7 is defined by its extensive prophage repertoire including those that encode Shiga toxin, the factor responsible for inducing...
The human zoonotic pathogen O157:H7 is defined by its extensive prophage repertoire including those that encode Shiga toxin, the factor responsible for inducing life-threatening pathology in humans. As well as introducing genes that can contribute to the virulence of a strain, prophage can enable the generation of large-chromosomal rearrangements (LCRs) by homologous recombination. This work examines the types and frequencies of LCRs across the major lineages of the O157:H7 serotype. We demonstrate that LCRs are a major source of genomic variation across all lineages of O157:H7 and by using both optical mapping and Oxford Nanopore long-read sequencing prove that LCRs are generated in laboratory cultures started from a single colony and that these variants can be recovered from colonized cattle. LCRs are biased towards the terminus region of the genome and are bounded by specific prophages that share large regions of sequence homology associated with the recombinational activity. RNA transcriptional profiling and phenotyping of specific structural variants indicated that important virulence phenotypes such as Shiga-toxin production, type-3 secretion and motility can be affected by LCRs. In summary, O157:H7 has acquired multiple prophage regions over time that act to continually produce structural variants of the genome. These findings raise important questions about the significance of this prophage-mediated genome contingency to enhance adaptability between environments.
Topics: Animals; Cattle; Escherichia coli O157; Genomic Structural Variation; Prophages; Shiga Toxin; Shiga Toxin 2
PubMed: 34751643
DOI: 10.1099/mgen.0.000682 -
BMC Genomics Oct 2022Ralstonia solanacearum species complex (RSSC) strains are destructive plant pathogenic bacteria and the causative agents of bacterial wilt disease, infecting over 200...
BACKGROUND
Ralstonia solanacearum species complex (RSSC) strains are destructive plant pathogenic bacteria and the causative agents of bacterial wilt disease, infecting over 200 plant species worldwide. In addition to chromosomal genes, their virulence is mediated by mobile genetic elements including integrated DNA of bacteriophages, i.e., prophages, which may carry fitness-associated auxiliary genes or modulate host gene expression. Although experimental studies have characterised several prophages that shape RSSC virulence, the global diversity, distribution, and wider functional gene content of RSSC prophages are unknown. In this study, prophages were identified in a diverse collection of 192 RSSC draft genome assemblies originating from six continents.
RESULTS
Prophages were identified bioinformatically and their diversity investigated using genetic distance measures, gene content, GC, and total length. Prophage distributions were characterised using metadata on RSSC strain geographic origin and lineage classification (phylotypes), and their functional gene content was assessed by identifying putative prophage-encoded auxiliary genes. In total, 313 intact prophages were identified, forming ten genetically distinct clusters. These included six prophage clusters with similarity to the Inoviridae, Myoviridae, and Siphoviridae phage families, and four uncharacterised clusters, possibly representing novel, previously undescribed phages. The prophages had broad geographical distributions, being present across multiple continents. However, they were generally host phylogenetic lineage-specific, and overall, prophage diversity was proportional to the genetic diversity of their hosts. The prophages contained many auxiliary genes involved in metabolism and virulence of both phage and bacteria.
CONCLUSIONS
Our results show that while RSSC prophages are highly diverse globally, they make lineage-specific contributions to the RSSC accessory genome, which could have resulted from shared coevolutionary history.
Topics: Bacteriophages; Humans; Phylogeny; Prophages; Ralstonia solanacearum; Virulence
PubMed: 36199029
DOI: 10.1186/s12864-022-08909-7 -
Journal of Bacteriology Apr 2018Bacterial viruses (bacteriophages) play a significant role in microbial community dynamics. Within the human gastrointestinal tract, for instance, associations among...
Bacterial viruses (bacteriophages) play a significant role in microbial community dynamics. Within the human gastrointestinal tract, for instance, associations among bacteriophages (phages), microbiota stability, and human health have been discovered. In contrast to the gastrointestinal tract, the phages associated with the urinary microbiota are largely unknown. Preliminary metagenomic surveys of the urinary virome indicate a rich diversity of novel lytic phage sequences at an abundance far outnumbering that of eukaryotic viruses. These surveys, however, exclude the lysogenic phages residing within the bacteria of the bladder. To characterize this phage population, we examined 181 genomes representative of the phylogenetic diversity of bacterial species within the female urinary microbiota and found 457 phage sequences, 226 of which were predicted with high confidence. Phages were prevalent within the bladder bacteria: 86% of the genomes examined contained at least one phage sequence. Most of these phages are novel, exhibiting no discernible sequence homology to sequences in public data repositories. The presence of phages with substantial sequence similarity within the microbiota of different women supports the existence of a core community of phages within the bladder. Furthermore, the observed variation between the phage populations of women with and without overactive bladder symptoms suggests that phages may contribute to urinary health. To complement our bioinformatic analyses, viable phages were cultivated from the bacterial isolates for characterization; a novel coliphage was isolated, which is obligately lytic in the laboratory strain C. Sequencing of bacterial genomes facilitates a comprehensive cataloguing of the urinary virome and reveals phage-host interactions. Bacteriophages are abundant within the human body. However, while some niches have been well surveyed, the phage population within the urinary microbiome is largely unknown. Our study is the first survey of the lysogenic phage population within the urinary microbiota. Most notably, the abundance of prophage exceeds that of the bacteria. Furthermore, many of the prophage sequences identified exhibited no recognizable sequence homology to sequences in data repositories. This suggests a rich diversity of uncharacterized phage species present in the bladder. Additionally, we observed a variation in the abundances of phages between bacteria isolated from asymptomatic "healthy" individuals and those with urinary symptoms, thus suggesting that, like phages within the gut, phages within the bladder may contribute to urinary health.
Topics: Bacteria; Bacteriophages; Coliphages; Computational Biology; Female; Genome, Bacterial; High-Throughput Nucleotide Sequencing; Humans; Microbiota; Phylogeny; Pregnancy; Prophages; Sequence Analysis, DNA; Urinary Bladder; Urinary Bladder, Overactive; Urinary Tract
PubMed: 29378882
DOI: 10.1128/JB.00738-17