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RNA Biology Aug 2021The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated proteins) system represents, in prokaryotes, an adaptive and inheritable... (Review)
Review
The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated proteins) system represents, in prokaryotes, an adaptive and inheritable immune response against invading DNA. The discovery of anti-CRISPR proteins (Acrs), which are inhibitors of CRISPR-Cas, mainly encoded by phages and prophages, showed a co-evolution history between prokaryotes and phages. In the past decade, the CRISPR-Cas systems together with the corresponding Acrs have been turned into a genetic-engineering tool. Among the six types of CRISPR-Cas characterized so far, type II CRISPR-Cas system is the most popular in biotechnology. Here, we discuss about the discovery, the reported inhibitory mechanisms, and the applications in both gene editing and gene transcriptional regulation of type II Acrs. Moreover, we provide insights into future potential research and feasible applications.
Topics: Archaea; Bacteria; Bacteriophages; Biological Coevolution; Biotechnology; CRISPR-Associated Protein 9; CRISPR-Cas Systems; Clustered Regularly Interspaced Short Palindromic Repeats; Gene Editing; Humans; Prophages; RNA, Guide, CRISPR-Cas Systems; Synthetic Biology
PubMed: 32991234
DOI: 10.1080/15476286.2020.1827803 -
MBio Oct 2022The formation of membrane vesicles (MVs) by Gram-positive bacteria has gained increasing attention over the last decade. Recently, models of vesicle formation have been...
The formation of membrane vesicles (MVs) by Gram-positive bacteria has gained increasing attention over the last decade. Recently, models of vesicle formation have been proposed and involve the digestion of the cell wall by prophage-encoded or stress-induced peptidoglycan (PG) hydrolases and the inhibition of PG synthesis by β-lactam antibiotics. The impact of these mechanisms on vesicle formation is largely dependent on the strain and growth conditions. To date, no information on the production of vesicles by the lactobacilli family has been reported. Here, we aimed to characterize the MVs released by the Gram-positive bacteria BL23 and also investigated the mechanisms involved in vesicle formation. Using electron microscopy, we established that the size of the majority of BL23 vesicles ranged from 50 to 100 nm. Furthermore, we showed that the vesicles were released consistently throughout the growth of the bacteria in standard culture conditions. The protein composition of the vesicles released in the supernatant was identified and a significant number of prophage proteins was detected. Moreover, using a mutant strain harboring a defective PLE2 prophage, we were able to show that the spontaneous and mitomycin-triggered induction of the prophage PLE2 contribute to the production of MVs by BL23. Finally, we also demonstrated the influence of prophages on the membrane integrity of bacteria. Overall, our results suggest a key role of the prophage PLE2 in the production of MVs by BL23 in the absence or presence of genotoxic stress. The last few decades have demonstrated that membrane vesicles (MVs) produced by microorganisms can have a wide variety of functions. This diversity places MVs at the crossroads of major research topics in current microbiology such as antibiotic resistance, horizontal gene transfer, cell communication, biofilm development, bacteriophage resistance, and pathogenesis. In particular, vesicles produced by probiotic strains have been shown to play a significant role in their beneficial effects. Thus, the study of vesicle biogenesis is a key element for promoting and improving their release. Overall, our results suggest a key role of spontaneous and mitomycin-triggered prophage induction in MV production by the Gram-positive bacteria BL23. This phenomenon is of great interest as prophage-induced MVs could potentially influence bacterial behavior, stress resistance, and vesicle functions.
Topics: Peptidoglycan; Virus Activation; Lacticaseibacillus casei; Prophages; N-Acetylmuramoyl-L-alanine Amidase; Anti-Bacterial Agents; Mitomycins; beta-Lactams
PubMed: 36200778
DOI: 10.1128/mbio.02375-22 -
Microorganisms Jun 2022Phageomes are known to play a key role in the functioning of their associated microbial communities. The phageomes of fermented foods have not been studied thoroughly in...
Phageomes are known to play a key role in the functioning of their associated microbial communities. The phageomes of fermented foods have not been studied thoroughly in fermented foods yet, and even less in fermented beverages. Two approaches were employed to investigate the presence of phages in cider, a fermented beverage made from apple, during a fermentation process of two cider tanks, one from an industrial producer and one from a hand-crafted producer. The phageome (free lytic phages) was explored in cider samples with several methodological developments for total phage DNA extraction, along with single phage isolation. Concentration methods, such as tangential flow filtration, flocculation and classical phage concentration methods, were employed and tested to extract free phage particles from cider. This part of the work revealed a very low occurrence of free lytic phage particles in cider. In parallel, a prophage investigation during the fermentation process was also performed using a metagenomic approach on the total bacterial genomic DNA. Prophages in bacterial metagenomes in the two cider tanks seemed also to occur in low abundance, as a total of 1174 putative prophages were identified in the two tanks overtime, and only two complete prophages were revealed. Prophage occurrence was greater at the industrial producer than at the hand-crafted producer, and different dynamics of prophage trends were also observed during fermentation. This is the first report dealing with the investigation of the phageome and of prophages throughout a fermentation process of a fermented beverage.
PubMed: 35744720
DOI: 10.3390/microorganisms10061203 -
International Journal of Molecular... May 2023is a common human commensal and opportunistic pathogen. In recent years, the clinical isolation and resistance rates of have shown a yearly increase, leading to a...
is a common human commensal and opportunistic pathogen. In recent years, the clinical isolation and resistance rates of have shown a yearly increase, leading to a special interest in mobile genetic elements. Prophages are a representative class of mobile genetic elements that can carry host-friendly genes, transfer horizontally between strains, and coevolve with the host's genome. In this study, we identified 15,946 prophages from the genomes of 1437 fully assembled deposited in the NCBI database, with 9755 prophages on chromosomes and 6191 prophages on plasmids. We found prophages to be notably diverse and widely disseminated in the genomes. The prophages encoded multiple putative virulence factors and antibiotic resistance genes. The comparison of strain types with prophage types suggests that the two may be related. The differences in GC content between the same type of prophages and the genomic region in which they were located indicates the alien properties of the prophages. The overall distribution of GC content suggests that prophages integrated on chromosomes and plasmids may have different evolutionary characteristics. These results suggest a high prevalence of prophages in the genome and highlight the effect of prophages on strain characterization.
Topics: Humans; Prophages; Klebsiella pneumoniae; Plasmids; Genomics; Virulence Factors; Anti-Bacterial Agents; Genome, Bacterial
PubMed: 37298067
DOI: 10.3390/ijms24119116 -
Nature Communications Oct 2023Bacteriophages (phages) are the most abundant biological entities on Earth, exerting a significant influence on the dissemination of bacterial virulence, pathogenicity,...
Bacteriophages (phages) are the most abundant biological entities on Earth, exerting a significant influence on the dissemination of bacterial virulence, pathogenicity, and antimicrobial resistance. Temperate phages integrate into the bacterial chromosome in a dormant state through intricate regulatory mechanisms. These mechanisms repress lytic genes while facilitating the expression of integrase and the CI master repressor. Upon bacterial SOS response activation, the CI repressor undergoes auto-cleavage, producing two fragments with the N-terminal domain (NTD) retaining significant DNA-binding ability. The process of relieving CI NTD repression, essential for prophage induction, remains unknown. Here we show a specific interaction between the ClpX protease and CI NTD repressor fragment of phages Ф11 and 80α in Staphylococcus aureus. This interaction is necessary and sufficient for prophage activation after SOS-mediated CI auto-cleavage, defining the final stage in the prophage induction cascade. Our findings unveil unexpected roles of bacterial protease ClpX in phage biology.
Topics: Virus Activation; Staphylococcus aureus; Peptide Hydrolases; Bacteriophages; Prophages
PubMed: 37852980
DOI: 10.1038/s41467-023-42413-0 -
Frontiers in Genetics 2022As an intracellular form of a bacteriophage in the bacterial host genome, a prophage usually integrates into bacterial DNA with high specificity and contributes to...
As an intracellular form of a bacteriophage in the bacterial host genome, a prophage usually integrates into bacterial DNA with high specificity and contributes to horizontal gene transfer (HGT). With the exponentially increasing number of microbial sequences uncovered in genomic or metagenomics studies, there is a massive demand for a tool that is capable of fast and accurate identification of prophages. Here, we introduce DBSCAN-SWA, a command line software tool developed to predict prophage regions in bacterial genomes. DBSCAN-SWA runs faster than any previous tools. Importantly, it has great detection power based on analysis using 184 manually curated prophages, with a recall of 85% compared with Phage_Finder (63%), VirSorter (74%), and PHASTER (82%) for (Multi-) FASTA sequences. Moreover, DBSCAN-SWA outperforms the existing standalone prophage prediction tools for high-throughput sequencing data based on the analysis of 19,989 contigs of 400 bacterial genomes collected from Human Microbiome Project (HMP) project. DBSCAN-SWA also provides user-friendly result visualizations including a circular prophage viewer and interactive DataTables. DBSCAN-SWA is implemented in Python3 and is available under an open source GPLv2 license from https://github.com/HIT-ImmunologyLab/DBSCAN-SWA/.
PubMed: 35518360
DOI: 10.3389/fgene.2022.885048 -
Frontiers in Microbiology 2022belonging to the enterohemorrhagic (EHEC), Shiga toxin-producing (STEC) and atypical enteropathogenic (aEPEC) pathotypes are significant foodborne zoonotic pathogens...
belonging to the enterohemorrhagic (EHEC), Shiga toxin-producing (STEC) and atypical enteropathogenic (aEPEC) pathotypes are significant foodborne zoonotic pathogens posing serious health risks, with healthy cattle as their main reservoir. A representative sampling of Hungarian cattle farms during 2017-2018 yielded a prevalence of 6.5 and 5.8% for STEC and aEPEC out of 309 samples. The draft genomes of twelve STEC (of them 9 EHEC) and four aEPEC of bovine origin were determined. For comparative purposes, we also included 3 EHEC and 2 aEPEC strains of human origin, as well four commensal isolates and one extraintestinal pathogenic (ExPEC) obtained from animals in a final set of 26 strains for a WGS-based analysis. Apart from key virulence genes, these isolates harbored several additional virulence genes with arrays characteristic for the site of isolation. The most frequent insertion site of Shiga toxin encoding prophages was for the Stx1 prophage and and for Stx2. For O157:H7 strains, the locus of enterocyte effacement pathogenicity island was present at the site, with integration at for other serotypes, and in the case of O26:H11 strains. Several LEE-negative STEC and aEPEC as well as commensal isolates carried additional prophages, with an average of ten prophage regions per isolate. Comparative phylogenomic analysis showed no clear separation between bovine and human lineages among the isolates characterized in the current study. Similarities in virulence gene arrays and close phylogenetic relations of bovine and human isolates underline the zoonotic potential of bovine aEPEC and STEC and emphasize the need for frequent monitoring of these pathogens in livestock.
PubMed: 35865933
DOI: 10.3389/fmicb.2022.896296 -
MSphere Aug 2021Prophages are often involved in host survival strategies and contribute toward increasing the genetic diversity of the host genome. Prophages also drive horizontal...
Prophages are often involved in host survival strategies and contribute toward increasing the genetic diversity of the host genome. Prophages also drive horizontal propagation of various genes as vehicles. However, there are few retrospective studies contributing to the propagation of antimicrobial resistance (AMR) and virulence factor (VF) genes by prophage. We extracted the complete genome sequences of seven pathogens, including ESKAPE bacteria and Escherichia coli from a public database, and examined the distribution of both the AMR and VF genes in prophage-like regions. We found that the ratios of AMR and VF genes greatly varied among the seven species. More than 70% of Enterobacter cloacae strains had VF genes, but only 1.2% of Klebsiella pneumoniae strains had VF genes from prophages. AMR and VF genes are unlikely to exist together in the same prophage region except in and , and the distribution patterns of prophage types containing AMR genes are distinct from those of VF gene-carrying prophage types. AMR genes in the prophage were located near transposase and/or integrase. The prophage containing class 1 integrase possessed a significantly greater number of AMR genes than did prophages with no class 1 integrase. The results of this study present a comprehensive picture of AMR and VF genes present within, or close to, prophage-like elements and different prophage patterns between AMR- or VF-encoding prophage-like elements. Although we believe phages play an important role in horizontal gene transfer in exchanging genetic material, we do not know the distribution of the antimicrobial resistance (AMR) and/or virulence factor (VF) genes in prophages. We collected different prophage elements from the complete genome sequences of seven species-Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter cloacae, and Escherichia coli-and characterized the distribution of antimicrobial resistance and virulence genes located in the prophage region. While virulence genes in prophage were species specific, antimicrobial resistance genes in prophages were highly conserved in various species. An integron structure was detected within specific prophage regions such as P1-like prophage element. Maximum of 10 antimicrobial resistance genes were found in a single prophage region, suggesting that prophages act as a reservoir for antimicrobial resistance genes. The results of this study show the different characteristic structures between AMR- or VF-encoding prophages.
Topics: Anti-Bacterial Agents; Bacteria; Cross Infection; Drug Resistance, Bacterial; Enterobacter cloacae; Escherichia coli; Genome, Bacterial; Humans; Klebsiella pneumoniae; Prophages; Virulence; Virulence Factors
PubMed: 34232073
DOI: 10.1128/mSphere.00452-21 -
Nucleic Acids Research Dec 2021The 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...
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.
Topics: Animals; Anthozoa; Bacteria; Genome, Archaeal; Genome, Bacterial; Interspersed Repetitive Sequences; Prophages; Sequence Alignment; Sequence Analysis, DNA; Software
PubMed: 34551431
DOI: 10.1093/nar/gkab824 -
BMC Research Notes Oct 2019Staphylococcus aureus is considered an important pathogen with a variety of virulence factors in communities and hospitals all around the world. Prophage typing is a...
OBJECTIVE
Staphylococcus aureus is considered an important pathogen with a variety of virulence factors in communities and hospitals all around the world. Prophage typing is a practical technique for categorizing this bacterium. In this study, we focused on the detection of prophage patterns in methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) strains based on their virulence factors, antimicrobial resistance patterns, and molecular typing by rep-PCR.
RESULTS
Out of 126 S. aureus isolates, 45 (35.7%) were identified as MRSA. In total, 17 different prophage types were detected and 112 strains out of 126 strains contained at least one prophage. There was a statistically significant relationship between hld, hlg, eta and SGA, SGA, and SGFb, respectively. The results of the rep-PCR analysis revealed 14 different patterns among the MRSA and MSSA isolates. In conclusion, the presence of different prophage-encoded virulence factors and antibiotic-resistant genes among MRSA strains enables them to produce a broad range of diseases. Thus, diverse MRSA strains which have these prophages can be considered as a potential threat to the patient's health in either the hospital or the community.
Topics: Anti-Bacterial Agents; Community-Acquired Infections; Drug Resistance, Bacterial; Gene Expression; Genes, Bacterial; Humans; Iran; Methicillin-Resistant Staphylococcus aureus; Microbial Sensitivity Tests; Polymerase Chain Reaction; Prophages; Staphylococcal Infections; Staphylococcus aureus; Virulence Factors
PubMed: 31639052
DOI: 10.1186/s13104-019-4711-4