A Structural Perspective of Reps from CRESS-DNA Viruses and Their Bacterial Plasmid Homologues

Rolling circle replication (RCR) is ubiquitously used by cellular and viral systems for genome and plasmid replication. While the molecular mechanism of RCR has been described, the structural mechanism is desperately lacking. Circular-rep encoded single stranded DNA (CRESS-DNA) viruses employ a viral encoded replicase (Rep) to initiate RCR. The recently identified prokaryotic homologues of Reps may also be responsible for initiating RCR. Reps are composed of an endonuclease, oligomerization, and ATPase domain. Recent structural studies have provided structures for all these domains such that an overall mechanism of RCR initiation can begin to be synthesized. However, structures of Rep in complex with its various DNA substrates and/or ligands are lacking. Here we provide a 3D bioinformatic review of the current structural information available for Reps. We combine an excess of 1590 sequences with experimental and predicted structural data from 22 CRESS-DNA groups to identify similarities and differences between Reps that lead to potentially important functional sites. Experimental studies of these sites may shed light on how Reps execute their functions. Furthermore, we identify Rep-substrate or Rep-ligand structures that are urgently needed to better understand the structural mechanism of RCR.

Two-Dimensional Gel Electrophoresis to Study the Activity of Type IIA Topoisomerases on Plasmid Replication Intermediates

DNA topoisomerases are the enzymes that regulate DNA topology in all living cells. Since the discovery and purification of ω (omega), when the first were topoisomerase identified, the function of many topoisomerases has been examined. However, their ability to relax supercoiling and unlink the pre-catenanes of partially replicated molecules has received little attention. Here, we used two-dimensional agarose gel electrophoresis to test the function of three type II DNA topoisomerases in vitro: the prokaryotic DNA gyrase, topoisomerase IV and the human topoisomerase 2α. We examined the proficiency of these topoisomerases on a partially replicated bacterial plasmid: pBR-TerE@AatII, with an unidirectional replicating fork, stalled when approximately half of the plasmid had been replicated in vivo. DNA was isolated from two strains of Escherichia coli: DH5αF’ and parE10. These experiments allowed us to assess, for the first time, the efficiency of the topoisomerases examined to resolve supercoiling and pre-catenanes in partially replicated molecules and fully replicated catenanes formed in vivo. The results obtained revealed the preferential functions and also some redundancy in the abilities of these DNA topoisomerases in vitro.

Staphylococcal phages and pathogenicity islands drive plasmid evolution

Conjugation has classically been considered the main mechanism driving plasmid transfer in nature. Yet bacteria frequently carry so-called non-transmissible plasmids, raising questions about how these plasmids spread. Interestingly, the size of many mobilisable and non-transmissible plasmids coincides with the average size of phages (~40 kb) or that of a family of pathogenicity islands, the phage-inducible chromosomal islands (PICIs, ~11 kb). Here, we show that phages and PICIs from Staphylococcus aureus can mediate intra- and inter-species plasmid transfer via generalised transduction, potentially contributing to non-transmissible plasmid spread in nature. Further, staphylococcal PICIs enhance plasmid packaging efficiency, and phages and PICIs exert selective pressures on plasmids via the physical capacity of their capsids, explaining the bimodal size distribution observed for non-conjugative plasmids. Our results highlight that transducing agents (phages, PICIs) have important roles in bacterial plasmid evolution and, potentially, in antimicrobial resistance transmission.

PlasForest: a homology-based random forest classifier for plasmid detection in genomic datasets

Background Plasmids are mobile genetic elements that often carry accessory genes, and are vectors for horizontal transfer between bacterial genomes. Plasmid detection in large genomic datasets is crucial to analyze their spread and quantify their role in bacteria adaptation and particularly in antibiotic resistance propagation. Bioinformatics methods have been developed to detect plasmids. However, they suffer from low sensitivity (i.e., most plasmids remain undetected) or low precision (i.e., these methods identify chromosomes as plasmids), and are overall not adapted to identify plasmids in whole genomes that are not fully assembled (contigs and scaffolds). Results We developed PlasForest, a homology-based random forest classifier identifying bacterial plasmid sequences in partially assembled genomes. Without knowing the taxonomical origin of the samples, PlasForest identifies contigs as plasmids or chromosomes with a F1 score of 0.950. Notably, it can detect 77.4% of plasmid contigs below 1 kb with 2.8% of false positives and 99.9% of plasmid contigs over 50 kb with 2.2% of false positives. Conclusions PlasForest outperforms other currently available tools on genomic datasets by being both sensitive and precise. The performance of PlasForest on metagenomic assemblies are currently well below those of other k-mer-based methods, and we discuss how homology-based approaches could improve plasmid detection in such datasets.

Comparative Genomics of Prophages Sato and Sole Expands the Genetic Diversity Found in the Genus Betatectivirus

Tectiviruses infecting the Bacillus cereus group represent part of the bacterial “plasmid repertoire” as they behave as linear plasmids during their lysogenic cycle. Several novel tectiviruses have been recently found infecting diverse strains belonging the B. cereus lineage. Here, we report and analyze the complete genome sequences of phages Sato and Sole. The linear dsDNA genome of Sato spans 14,852 bp with 32 coding DNA sequences (CDSs), whereas the one of Sole has 14,444 bp comprising 30 CDSs. Both phage genomes contain inverted terminal repeats and no tRNAs. Genomic comparisons and phylogenetic analyses placed these two phages within the genus Betatectivirus in the family Tectiviridae. Additional comparative genomic analyses indicated that the “gene regulation-genome replication” module of phages Sato and Sole is more diverse than previously observed among other fully sequenced betatectiviruses, displaying very low sequence similarities and containing some ORFans. Interestingly, the ssDNA binding protein encoded in this genomic module in phages Sato and Sole has very little amino acid similarity with those of reference betatectiviruses. Phylogenetic analyses showed that both Sato and Sole represent novel tectivirus species, thus we propose to include them as two novel species in the genus Betatectivirus.

PlasForest: a homology-based random forest classifier for plasmid detection in genomic datasets

Plasmids are mobile genetic elements that often carry accessory genes, and are vectors for horizontal transfer between bacterial genomes. The detection of plasmids in large sets of genomes is crucial to analyze their spread and quantify their role in bacteria adaptation and particularly in antibiotic resistance genes propagation. Several bioinformatics methods have been developed to detect plasmids. However, they suffer from low sensitivity (i.e., most plasmids remain undetected) or low precision (i.e., these methods identify chromosomes as plasmids), and are overall not adapted to identify plasmids in whole genomes that are not fully assembled (contigs and scaffolds). Here, we present PlasForest, a homology-based random forest classifier identifying bacterial plasmid sequences in unassembled genomes. This tool is based on the determination of homologies against a database of plasmid sequences, which allow a random forest classifier to discriminate plasmid contigs. Without knowing the taxonomical origin of the samples, PlasForest identifies contigs as plasmids or chromosomes with an accuracy of 98%. Notably, it can detect 96% of plasmid contigs over 50kb with 3.3% of false positives. PlasForest outperforms other currently available tools on test datasets by being both sensitive and precise. We implemented this tool in a user-friendly pipeline that can identify plasmids in large datasets in a reasonable amount of time.

DETEKSI Liberibacter spp. PADA JERUK SIAM BERGEJALA KLOROSIS DISERTAI BERCAK HITAM DENGAN Polymerase Chain Reaction

Liberibacter bacteria are one of the pathogenic bacteria in citrus plants. Siam Citrus Plant in Setapok Village, Singkawang City, suspected to be infected with Liberibacter spp. bacteria by showing patterns of symptoms of chlorosis accompanied by patches. This study aims to detect Liberibacter spp. on Siam Citrus leaves with symptomatic chlorosis with patches. Leaf sampling uses the roaming method in one of the citrus plantations in Setapok Village, Singkawang City. Detection of Liberibacter spp. done by PCR (Polymerase Chain Reaction) technique. Detection of bacterial DNA through several stages namely, isolation of bacterial plasmid DNA, 16S rDNA gene amplification with a thermocycler machine, and visualization using a set of electrophoresis devices. Electrophoresis results show the presence of DNA band lines, with a size of around 1100 bp for OI1 and OI2c primers, and DNA bands of 703 bp for A2 and J5 specifics. The results showed that there were Liberibacter spp. in the symptomatic chlorosis of Siam Citrus leaves accompanied by patches.

Online searching platform for the antibiotic resistome in bacterial tree of life and global habitats

ABSTRACT Metagenomic analysis reveals that antibiotic-resistance genes (ARGs) are widely distributed in both human-associated and non-human-associated habitats. However, it is difficult to equally compare ARGs between samples without a standard method. Here, we constructed a comprehensive profile of the distribution of potential ARGs in bacterial tree of life and global habitats by investigating ARGs in 55 000 bacterial genomes, 16 000 bacterial plasmid sequences, 3000 bacterial integron sequences and 850 metagenomes using a standard pipeline. We found that >80% of all known ARGs are not carried by any plasmid or integron sequences. Among potential mobile ARGs, tetracycline and beta-lactam resistance genes (such as tetA, tetM and class A beta-lactamase gene) distribute in multiple pathogens across bacterial phyla, indicating their clinical relevance and importance. We showed that class 1 integrases (intI1) display a poor linear relationship with total ARGs in both non-human-associated and human-associated environments. Furthermore, both total ARGs and intI1 genes show little correlation with the degree of anthropogenicity. These observations highlight the need to differentiate ARGs of high clinical relevance. This profile is published on an online platform (ARGs-OSP, http://args-osp.herokuapp.com/) as a valuable resource for the most challenging topics in this field, i.e. the risk, evolution and emergence of ARGs.