Bilateral symmetry of linear streptomycete chromosomes

Here, we characterize an uncommon set of telomeres from Streptomyces rimosus ATCC 10970, the parental strain of a lineage of one of the earliest-discovered antibiotic producers. Following the closure of its genome sequence, we compared unusual telomeres from this organism with the other five classes of replicon ends found amongst streptomycetes. Closed replicons of streptomycete chromosomes were organized with respect to their phylogeny and physical orientation, which demonstrated that different telomeres were not associated with particular clades and are likely shared amongst different strains by plasmid-driven horizontal gene transfer. Furthermore, we identified a ~50 kb origin island with conserved synteny that is located at the core of all streptomycete chromosomes and forms an axis around which symmetrical chromosome inversions can take place. Despite this chromosomal bilateral symmetry, a bias in parS sites to the right of oriC is maintained across the family Streptomycetaceae and suggests that the formation of ParB/parS nucleoprotein complexes on the right replichore is a conserved feature in streptomycetes. Consequently, our studies reveal novel features of linear bacterial replicons that, through their manipulation, may lead to improvements in growth and productivity of this important industrial group of bacteria

Investigating the genome complexity of Streptomyces clavuligerus

Streptomyces clavuligerus is the producer of clavulanic acid; a β-lactamase inhibitor that isused in combination with the antibiotic amoxicillin to treat many bacterial infections. The unique genome of S. clavuligerus includes a chromosome, and four linear plasmids pSCL1, pSCL2, pSCL3 and the megaplasmid pSCL4, which is the largest linear plasmid ever identified at 1.8 Mb in size. In order to study the genomic architecture of S. clavuligerus strains, we established an optimised method of pulsed-field gel electrophoresis, which allowed visualisation of the three giant linear plasmids pSCL2, pSCL3 and pSCL4, as well as chromosomal fragments in order to establish the physical map of the S. clavuligerus genome.;Furthermore, to establish a foundation on which the genomic analyses of industrial strains of this species could be carried out, we closed the genome sequence of the S. clavuligerus type strain DSM 738, using a combination of short-read Illumina sequencing and long-read PacBio technology. Additionally, the telomeres of the five replicons were purified by a self ligation method, and identification of the sequences confirmed that the five replicons carry archetypal telomeres, with the chromosome and megaplasmid sharing identical telomeric sequences. Streptomycetes telomeres are maintained by the terminal proteins Tap and Tpg, however, while copies of the genes encoding these proteins were identified in pSCL4, pSCL3 and pSCL2, the chromosome carries none, making these the only known essential elements missing from the chromosome.;Moreover, in order to investigate the essentiality of the megaplasmid in S. clavuligerus, we cured pSCL4 by targeting parB with CRISPR/Cas9. This caused deletion of the chromosomal ends and circularisation, confirming that pSCL4 Tap-Tpg are required for maintaining the chromosome telomeres, and cannot be complemented by the proteins encoded on the other two plasmids. In addition, pSCL4-free strains were confirmed to be non-sporulating as well as presenting a phenotype of short and thin hyphae, in addition to higher branching frequency. Some mutant strains also exhibited reduced clavulanic acid production which confirmed that the loss of pSCL4 has important effects onmorphological development and secondary metabolism of S. clavuligerus. Overall, with this study we have closed the genome sequence of S. clavuligerus type strain and optimised a method for rapid screening of the S. clavuligerus mutant strains that will greatly benefit the industry for development of production strains. Additionally, we have confirmed that pSCL4 is a dispensable plasmid, but is yet necessary for maintaining optimum fitness of this bacterium.Streptomyces clavuligerus is the producer of clavulanic acid; a β-lactamase inhibitor that isused in combination with the antibiotic amoxicillin to treat many bacterial infections. The unique genome of S. clavuligerus includes a chromosome, and four linear plasmids pSCL1, pSCL2, pSCL3 and the megaplasmid pSCL4, which is the largest linear plasmid ever identified at 1.8 Mb in size. In order to study the genomic architecture of S. clavuligerus strains, we established an optimised method of pulsed-field gel electrophoresis, which allowed visualisation of the three giant linear plasmids pSCL2, pSCL3 and pSCL4, as well as chromosomal fragments in order to establish the physical map of the S. clavuligerus genome.;Furthermore, to establish a foundation on which the genomic analyses of industrial strains of this species could be carried out, we closed the genome sequence of the S. clavuligerus type strain DSM 738, using a combination of short-read Illumina sequencing and long-read PacBio technology. Additionally, the telomeres of the five replicons were purified by a self ligation method, and identification of the sequences confirmed that the five replicons carry archetypal telomeres, with the chromosome and megaplasmid sharing identical telomeric sequences. Streptomycetes telomeres are maintained by the terminal proteins Tap and Tpg, however, while copies of the genes encoding these proteins were identified in pSCL4, pSCL3 and pSCL2, the chromosome carries none, making these the only known essential elements missing from the chromosome.;Moreover, in order to investigate the essentiality of the megaplasmid in S. clavuligerus, we cured pSCL4 by targeting parB with CRISPR/Cas9. This caused deletion of the chromosomal ends and circularisation, confirming that pSCL4 Tap-Tpg are required for maintaining the chromosome telomeres, and cannot be complemented by the proteins encoded on the other two plasmids. In addition, pSCL4-free strains were confirmed to be non-sporulating as well as presenting a phenotype of short and thin hyphae, in addition to higher branching frequency. Some mutant strains also exhibited reduced clavulanic acid production which confirmed that the loss of pSCL4 has important effects onmorphological development and secondary metabolism of S. clavuligerus. Overall, with this study we have closed the genome sequence of S. clavuligerus type strain and optimised a method for rapid screening of the S. clavuligerus mutant strains that will greatly benefit the industry for development of production strains. Additionally, we have confirmed that pSCL4 is a dispensable plasmid, but is yet necessary for maintaining optimum fitness of this bacterium

Papel del farmacéutico en el control de la Hipertensión arterial

La hipertensión arterial (HTA) es una enfermedad que afecta a una considerable parte de la población, la cual puede tener repercusiones negativas frente a diversos órganos diana, al igual que a la salud cardiovascular del paciente, ya que es un importante factor de riesgo. Al tratarse de una condición asintomática, no es infrecuente encontrar casos de hipertensión sin diagnosticar o de baja adherencia al tratamiento farmacológico. Es por ello que el farmacéutico tiene un papel crucial contribuyendo al control de esta enfermedad. El objetivo de este trabajo es analizar el papel del farmacéutico tanto en la detección de casos de hipertensión sin diagnosticar, como en el seguimiento de pacientes ya diagnosticados como hipertensos. Para ello se llevó a cabo una revisión bibliográfica de artículos y guías de actuación farmacéutica. Se concluyó que el farmacéutico, tras la realización de medidas de presión arterial, puede diagnosticar casos de HTA, así como llevar un seguimiento y control de la efectividad del tratamiento farmacológico, también puede detectar posibles problemas y resultados negativos asociados a la medicación y derivar al médico siempre que sea necesario. Además, el farmacéutico puede instaurar un tratamiento no farmacológico cuando lo considere oportuno, mediante la promoción de modificaciones del estilo de vida cotidiano y aportando educación apropiada para mejorar la salud cardiovascular del paciente.Fac. de FarmaciaTRUEunpu

Genome editing reveals that pSCL4 is required for chromosome linearity in Streptomyces clavuligerus

Streptomyces clavuligerus is an industrially important actinomycete whose genetic manipulation is limited by low transformation and conjugation efficiencies, low levels of recombination of introduced DNA, and difficulty in obtaining consistent sporulation. We describe the construction and application of versatile vectors for Cas9-mediated genome editing of this strain. To design spacer sequences with confidence, we derived a highly accurate genome assembly for an isolate of the type strain (ATCC 27064). This yielded a chromosome assembly (6.75 Mb) plus assemblies for pSCL4 (1795 kb) and pSCL2 (149 kb). The strain also carries pSCL1 (12 kb), but its small size resulted in only partial sequence coverage. The previously described pSCL3 (444 kb) is not present in this isolate. Using our Cas9 vectors, we cured pSCL4 with high efficiency by targeting the plasmid's parB gene. Five of the resulting pSCL4-cured isolates were characterized and all showed impaired sporulation. Shotgun genome sequencing of each of these derivatives revealed large deletions at the ends of the chromosomes in all of them, and for two clones sufficient sequence data was obtained to show that the chromosome had circularized. Taken together, these data indicate that pSCL4 is essential for the structural stability of the linear chromosome

ActinoBase : tools and protocols for researchers working on Streptomyces and other filamentous actinobacteria

Actinobacteria is an ancient phylum of Gram-positive bacteria with a characteristic high GC content to their DNA. The ActinoBase Wiki is focused on the filamentous actinobacteria, such as Streptomyces species, and the techniques and growth conditions used to study them. These organisms are studied because of their complex developmental life cycles and diverse specialised metabolism which produces many of the antibiotics currently used in the clinic. ActinoBase is a community effort that provides valuable and freely accessible resources, including protocols and practical information about filamentous actinobacteria. It is aimed at enabling knowledge exchange between members of the international research community working with these fascinating bacteria. Actin-oBase is an anchor platform that underpins worldwide efforts to understand the ecology, biology and metabolic potential of these organisms. There are two key differences that set ActinoBase apart from other Wiki-based platforms: [1] ActinoBase is specifically aimed at researchers working on filamentous actinobacteria and is tailored to help users overcome challenges working with these bacteria and [2] it provides a freely accessible resource with global networking opportunities for researchers with a broad range of experience in this field

ActinoBase: tools and protocols for researchers working on Streptomyces and other filamentous actinobacteria.

Actinobacteria is an ancient phylum of Gram-positive bacteria with a characteristic high GC content to their DNA. The ActinoBase Wiki is focused on the filamentous actinobacteria, such as Streptomyces species, and the techniques and growth conditions used to study them. These organisms are studied because of their complex developmental life cycles and diverse specialised metabolism which produces many of the antibiotics currently used in the clinic. ActinoBase is a community effort that provides valuable and freely accessible resources, including protocols and practical information about filamentous actinobacteria. It is aimed at enabling knowledge exchange between members of the international research community working with these fascinating bacteria. ActinoBase is an anchor platform that underpins worldwide efforts to understand the ecology, biology and metabolic potential of these organisms. There are two key differences that set ActinoBase apart from other Wiki-based platforms: [1] ActinoBase is specifically aimed at researchers working on filamentous actinobacteria and is tailored to help users overcome challenges working with these bacteria and [2] it provides a freely accessible resource with global networking opportunities for researchers with a broad range of experience in this field