SegH and Hef: two novel homing endonucleases whose genes replace the mobC and mobE genes in several T4-related phages

T4 contains two groups of genes with similarity to homing endonucleases, the seg-genes (similarity to endonucleases encoded by group I introns) containing GIY-YIG motifs and the mob-genes (similarity to mobile endonucleases) containing H-N-H motifs. The four seg-genes characterized to date encode homing endonucleases with cleavage sites close to their respective gene loci while none of the mob-genes have been shown to cleave DNA. Of 18 phages screened, only T4 was found to have mobC while mobE genes were found in five additional phages. Interestingly, three phages encoded a seg-like gene (hereby called segH) with a GIY-YIG motif in place of mobC. An additional phage has an unrelated gene called hef (homing endonuclease-like function) in place of the mobE gene. The gene products of both novel genes displayed homing endonuclease activity with cleavage site specificity close to their respective genes. In contrast to intron encoded homing endonucleases, both SegH and Hef can cleave their own DNA as well as DNA from phages without the genes. Both segH and mobE (and most likely hef) can home between phages in mixed infections. We discuss why it might be a selective advantage for phage freestanding homing endonucleases to cleave both HEG-containing and HEG-less genomes

Экономика образования: нравственный аспект

В связи с обострением социально-экономических противоречий современного общества особую актуальность приобретают вопросы изучения нравственного аспекта экономической деятельности. В статье рассматриваются предпосылки изучения нравственных основ экономики, изучается мнение ученых-экономистов о влиянии нравственности на экономику. А также делается вывод о непосредственном участии сферы высшего образования в формировании личности, разделяющей традиционные нравственные ценности общества

Direct identification of antibiotic resistance genes on single plasmid molecules using CRISPR/Cas9 in combination with optical DNA mapping.

Bacterial plasmids are extensively involved in the rapid global spread of antibiotic resistance. We here present an assay, based on optical DNA mapping of single plasmids in nanofluidic channels, which provides detailed information about the plasmids present in a bacterial isolate. In a single experiment, we obtain the number of different plasmids in the sample, the size of each plasmid, an optical barcode that can be used to identify and trace the plasmid of interest and information about which plasmid that carries a specific resistance gene. Gene identification is done using CRISPR/Cas9 loaded with a guide-RNA (gRNA) complementary to the gene of interest that linearizes the circular plasmids at a specific location that is identified using the optical DNA maps. We demonstrate the principle on clinically relevant extended spectrum beta-lactamase (ESBL) producing isolates. We discuss how the gRNA sequence can be varied to obtain the desired information. The gRNA can either be very specific to identify a homogeneous group of genes or general to detect several groups of genes at the same time. Finally, we demonstrate an example where we use a combination of two gRNA sequences to identify carbapenemase-encoding genes in two previously not characterized clinical bacterial samples

Strain-level bacterial typing directly from patient samples using optical DNA mapping

For bacterial infections, it is important to rapidly and accurately identify and characterize the type of bacteria involved so that optimal antibiotic treatment can be given quickly to the patient. However, current diagnostic methods are sometimes slow and cannot be used for mixtures of bacteria. We have, therefore, developed a method to identify bacteria directly from patient samples. The method was tested on two common species of disease-causing bacteria - Escherichia coli and Klebsiella pneumoniae - and it could correctly identify the bacterial strain or subtype in both urine samples and mixtures. Hence, the method has the potential to provide fast diagnostic information for choosing the most suited antibiotic, thereby reducing the risk of death and suffering. Nyblom, Johnning et al. develop an optical DNA mapping approach for bacterial strain typing of patient samples. They demonstrate rapid identification of clinically relevant E. coli and K. pneumoniae strains, without the need for cultivation. BackgroundIdentification of pathogens is crucial to efficiently treat and prevent bacterial infections. However, existing diagnostic techniques are slow or have a too low resolution for well-informed clinical decisions.MethodsIn this study, we have developed an optical DNA mapping-based method for strain-level bacterial typing and simultaneous plasmid characterisation. For the typing, different taxonomical resolutions were examined and cultivated pure Escherichia coli and Klebsiella pneumoniae samples were used for parameter optimization. Finally, the method was applied to mixed bacterial samples and uncultured urine samples from patients with urinary tract infections.ResultsWe demonstrate that optical DNA mapping of single DNA molecules can identify Escherichia coli and Klebsiella pneumoniae at the strain level directly from patient samples. At a taxonomic resolution corresponding to E. coli sequence type 131 and K. pneumoniae clonal complex 258 forming distinct groups, the average true positive prediction rates are 94% and 89%, respectively. The single-molecule aspect of the method enables us to identify multiple E. coli strains in polymicrobial samples. Furthermore, by targeting plasmid-borne antibiotic resistance genes with Cas9 restriction, we simultaneously identify the strain or subtype and characterize the corresponding plasmids.ConclusionThe optical DNA mapping method is accurate and directly applicable to polymicrobial and clinical samples without cultivation. Hence, it has the potential to rapidly provide comprehensive diagnostics information, thereby optimizing early antibiotic treatment and opening up for future precision medicine management

Selection of Resistant Bacteria at Very Low Antibiotic Concentrations

The widespread use of antibiotics is selecting for a variety of resistance mechanisms that seriously challenge our ability to treat bacterial infections. Resistant bacteria can be selected at the high concentrations of antibiotics used therapeutically, but what role the much lower antibiotic concentrations present in many environments plays in selection remains largely unclear. Here we show using highly sensitive competition experiments that selection of resistant bacteria occurs at extremely low antibiotic concentrations. Thus, for three clinically important antibiotics, drug concentrations up to several hundred-fold below the minimal inhibitory concentration of susceptible bacteria could enrich for resistant bacteria, even when present at a very low initial fraction. We also show that de novo mutants can be selected at sub-MIC concentrations of antibiotics, and we provide a mathematical model predicting how rapidly such mutants would take over in a susceptible population. These results add another dimension to the evolution of resistance and suggest that the low antibiotic concentrations found in many natural environments are important for enrichment and maintenance of resistance in bacterial populations

Facilitated sequence assembly using densely labeled optical DNA barcodes:A combinatorial auction approach

<div><p>The output from whole genome sequencing is a set of contigs, i.e. short non-overlapping DNA sequences (sizes 1-100 kilobasepairs). Piecing the contigs together is an especially difficult task for previously unsequenced DNA, and may not be feasible due to factors such as the lack of sufficient coverage or larger repetitive regions which generate gaps in the final sequence. Here we propose a new method for scaffolding such contigs. The proposed method uses densely labeled optical DNA barcodes from competitive binding experiments as scaffolds. On these scaffolds we position theoretical barcodes which are calculated from the contig sequences. This allows us to construct longer DNA sequences from the contig sequences. This proof-of-principle study extends previous studies which use sparsely labeled DNA barcodes for scaffolding purposes. Our method applies a probabilistic approach that allows us to discard “foreign” contigs from mixed samples with contigs from different types of DNA. We satisfy the contig non-overlap constraint by formulating the contig placement challenge as a combinatorial auction problem. Our exact algorithm for solving this problem reduces computational costs compared to previous methods in the combinatorial auction field. We demonstrate the usefulness of the proposed scaffolding method both for synthetic contigs and for contigs obtained using Illumina sequencing for a mixed sample with plasmid and chromosomal DNA.</p></div

Group I Introns and Homing Endonucleases in T-even-like Bacteriophages

Homing endonucleases are rare-cutting enzymes that cleave DNA at a site near their own location, preferentially in alleles lacking the homing endonuclease gene (HEG). By cleaving HEG-less alleles the homing endonuclease can mediate the transfer of its own gene to the cleaved site via a process called homing, involving double strand break repair. Via homing, HEGs are efficiently transferred into new genomes when horizontal exchange of DNA occurs between organisms. Group I introns are intervening sequences that can catalyse their own excision from the unprocessed transcript without the need of any proteins. They are widespread, occurring both in eukaryotes and prokaryotes and in their viruses. Many group I introns encode a HEG within them that confers mobility also to the intron and mediates the combined transfer of the intron/HEG to intronless alleles via homing. Bacteriophage T4 contains three such group I introns and at least 12 freestanding HEGs in its genome. The majority of phages besides T4 do not contain any introns, and freestanding HEGs are also scarcely represented among other phages. In the first paper we looked into why group I introns are so rare in phages related to T4 in spite of the fact that they can spread between phages via homing. We have identified the first phage besides T4 that contains all three T-even introns and also shown that homing of at least one of the introns has occurred recently between some of the phages in Nature. We also show that intron homing can be highly efficient between related phages if two phages infect the same bacterium but that there also exists counteracting mechanisms that can restrict the spread of introns between phages. In the second paper we have looked at how the presence of introns can affect gene expression in the phage. We find that the efficiency of splicing can be affected by variation of translation of the upstream exon for all three introns in T4. Furthermore, we find that splicing is also compromised upon infection of stationary-phase bacteria. This is the first time that the efficiency of self-splicing of group I introns has been coupled to environmental conditions and the potential effect of this on phage viability is discussed. In the third paper we have characterised two novel freestanding homing endonucleases that in some T-even-like phages replace two of the putative HEGs in T4. We also present a new theory on why it is a selective advantage for freestanding, phage homing endonucleases to cleave both HEG-containing and HEG-less genomes

The Role of Antibiotic Resistance Genes in the Fitness Cost of Multiresistance Plasmids

By providing the bacterial cell with protection against several antibiotics at once, multiresistance plasmids have an evolutionary advantage in situations where antibiotic treatments are common, such as in hospital environments. However, resistance plasmids can also impose fitness costs on the bacterium in the absence of antibiotics, something that may limit their evolutionary success. The underlying mechanisms and the possible contribution of resistance genes to such costs are still largely not understood. Here, we have specifically investigated the contribution of plasmid-borne resistance genes to the reduced fitness of the bacterial cell. The pUUH239.2 plasmid carries 13 genes linked to antibiotic resistance and reduces bacterial fitness by 2.9% per generation. This cost is fully ameliorated by the removal of the resistance cassette. While most of the plasmid-borne resistance genes individually were cost-free, even when overexpressed, two specific gene clusters were responsible for the entire cost of the plasmid: the extended-spectrum-beta-lactamase gene bla(CTX-M-15 )and the tetracycline resistance determinants tetAR. The bla(CTX-M-15 ) cost was linked to the signal peptide that exports the beta-lactamase into the periplasm, and replacement with an alternative signal peptide abolished the cost. Both the tetracycline pump TetA and its repressor TetR conferred a cost on the host cell, and the reciprocal expression of these genes is likely fine-tuned to balance the respective costs. These findings highlight that the cost of clinical multiresistance plasmids can be largely due to particular resistance genes and their interaction with other cellular systems, while other resistance genes and the plasmid backbone can be cost-free. IMPORTANCE Multiresistance plasmids are one of the main drivers of antibiotic resistance development and spread. Their evolutionary success through the accumulation and mobilization of resistance genes is central to resistance evolution. In this study, we find that the cost of the introduction of a multiresistance plasmid was completely attributable to resistance genes, while the rest of the plasmid backbone is cost-free. The majority of resistance genes on the plasmid had no appreciable cost to the host cell even when overexpressed, indicating that plasmid-borne resistance can be cost-free. In contrast, the widespread genes bla(CTX-M-15 ) and tetAR were found to confer the whole cost of the plasmid by affecting specific cellular functions. These findings highlight how the evolution of resistance on plasmids is dependent on the amelioration of associated fitness costs and point at a conundrum regarding the high cost of some of the most widespread beta-lactamase genes

Self-Splicing of the Bacteriophage T4 Group I Introns Requires Efficient Translation of the Pre-mRNA In Vivo and Correlates with the Growth State of the Infected Bacterium

Bacteriophage T4 contains three self-splicing group I introns in genes in de novo deoxyribonucleotide biosynthesis (in td, coding for thymidylate synthase and in nrdB and nrdD, coding for ribonucleotide reductase). Their presence in these genes has fueled speculations that the introns are retained within the phage genome due to a possible regulatory role in the control of de novo deoxyribonucleotide synthesis. To study whether sequences in the upstream exon interfere with proper intron folding and splicing, we inhibited translation in T4-infected bacteria as well as in bacteria containing recombinant plasmids carrying the nrdB intron. Splicing was strongly reduced for all three T4 introns after the addition of chloramphenicol during phage infection, suggesting that the need for translating ribosomes is a general trait for unperturbed splicing. The splicing of the cloned nrdB intron was markedly reduced in the presence of chloramphenicol or when translation was hindered by stop codons inserted in the upstream exon. Several exon regions capable of forming putative interactions with nrdB intron sequences were identified, and the removal or mutation of these exon regions restored splicing efficiency in the absence of translation. Interestingly, splicing of the cloned nrdB intron was also reduced as cells entered stationary phase and splicing of all three introns was reduced upon the T4 infection of stationary-phase bacteria. Our results imply that conditions likely to be frequently encountered by natural phage populations may limit the self-splicing efficiency of group I introns. This is the first time that environmental effects on bacterial growth have been linked to the regulation of splicing of phage introns

A bla(OXA-181)-harbouring multi-resistant ST147 Klebsiella pneumoniae isolate from Pakistan that represent an intermediate stage towards pan-drug resistance

Carbapenem resistant Klebsiella pneumoniae (CR-KP) infections are an ever-increasing global issue, especially in the Indian subcontinent. Here we report genetic insight into a bla(OXA-181) harbouring Klebsiella pneumoniae, belonging to the pandemic lineage ST147, that represents an intermediate stage towards pan-drug resistance. The CR-KP isolate DA48896 was isolated from a patient from Pakistan and was susceptible only to tigecycline and colistin. It harboured bla(OXA-181) and was assigned to sequence type ST147. Analysis from whole genome sequencing revealed a very high sequence similarity to the previously sequenced pan-resistant K. pneumoniae isolate MS6671 from the United Arab Emirates. The two isolates are very closely related with only 46 chromosomal nucleotide differences, 14 indels and differences in plasmid content. Both carry a substantial number of plasmidborne and chromosomally encoded resistance determinants. Interestingly, the two differences in susceptibility between the isolates could be attributed to DA48896 lacking an insertion of bla(OXA-181) into the mgrB gene that results in colistin resistance in MS6671 and SNPs affecting AcrAB efflux pump expression likely to result in tigecycline resistance. These differences between the otherwise very similar isolates indicate that strong selection has occurred for resistance towards these last-resort drugs and illustrates the trajectory of resistance evolution of OXA-181-producing versions of the ST147 international risk clone