Investigating streptomyces clavuligerus linear replicons for improved clavulanic acid production

Increasing antimicrobial resistance against ß-lactam antibiotics through bacterially produced ß-lactamases has prompted research into effective enzyme inhibitors, such as clavulanic acid. Streptomyces clavuligerus is the primary producer of clavulanic acid, which is used in various drugs such as Augmentin®. Streptomyces clavuligerus contains a dynamic genome and is composed of four giant linear plasmids (GLPs), pSCL1, pSCL2, pSCL3 and pSCL4 and its chromosome. Various genes essential for the maintenance of linear replicons, such as tap and tpg which encode telomeric terminal proteins (TPs), are found on three out of four GLPs (pSCL2, pSCL3, pSCL4). Previous work demonstrated a circularised chromosome and loss of plasmid after Cas9 mediated cleavage of the largest GLP, pSCL4, potentially due to the absence of tap-tpg. To determine the role of tap-tpg in chromosomal and plasmid linearity, we optimised and tested their inactivation using CRISPR-dCas9 multiplexing, targeting tap-tpg₄ on pSCL4, tap tpg₃ on pSCL3 and tap-tpg₂ on pSCL2. We used Illumina short-read sequencing to genotypically analyse our strains, which highlighted that the knockdown of multiple taptpg genes resulted in mutant strains with various combinations of lost plasmids and terminal ends. Noticeably, only the mutants with silenced tap-tpg4 demonstrated the loss of multiple plasmids, and pSCL2 loss was suggested to be a direct consequence of the CRISPR-dCas9 system, as all mutants and vector controls lost the plasmid. Additionally, we demonstrated chromosome circularisation and the loss of the 13-nucleotide binding site of Tap for our tap-tpg₄, tap-tpg₃ and tap-tpg₂ silenced mutant EM10, suggesting that overall tap-tpg expression levels in S. clavuligerus affect end-patching. The mutant strains were phenotypically characterised and mutants which had lost pSCL3, pSCL2 and/or pSCL1 had a significantly higher specific growth rate, therefore we confirmed that these replicons were essential for strain fitness. We also confirmed that S. clavuligerus does not require pSCL3, pSCL2 and pSCL1 for clavulanic acid production, as strains that had lost more replicons did not produce more clavulanic acid. Therefore, biotechnologically, the loss of replicons is of importance in terms of strain fitness rather than clavulanic acid production in S. clavuligerus. Moreover, to study the process of endpatching and determine the binding activity of TPs, overexpression of Tap4, Tap3 and Tap2 proteins was optimised and Tap4 was shown to potentially bind to the chromosomal ssDNA telomeres. With this study we have elucidated the importance of tap-tpg in the plasmid maintenance of S. clavuligerus and confirmed the essentiality of replicons, highlighting that whilst most replicons were dispensable, they are necessary for maintaining strain fitness and optimum growth.Increasing antimicrobial resistance against ß-lactam antibiotics through bacterially produced ß-lactamases has prompted research into effective enzyme inhibitors, such as clavulanic acid. Streptomyces clavuligerus is the primary producer of clavulanic acid, which is used in various drugs such as Augmentin®. Streptomyces clavuligerus contains a dynamic genome and is composed of four giant linear plasmids (GLPs), pSCL1, pSCL2, pSCL3 and pSCL4 and its chromosome. Various genes essential for the maintenance of linear replicons, such as tap and tpg which encode telomeric terminal proteins (TPs), are found on three out of four GLPs (pSCL2, pSCL3, pSCL4). Previous work demonstrated a circularised chromosome and loss of plasmid after Cas9 mediated cleavage of the largest GLP, pSCL4, potentially due to the absence of tap-tpg. To determine the role of tap-tpg in chromosomal and plasmid linearity, we optimised and tested their inactivation using CRISPR-dCas9 multiplexing, targeting tap-tpg₄ on pSCL4, tap tpg₃ on pSCL3 and tap-tpg₂ on pSCL2. We used Illumina short-read sequencing to genotypically analyse our strains, which highlighted that the knockdown of multiple taptpg genes resulted in mutant strains with various combinations of lost plasmids and terminal ends. Noticeably, only the mutants with silenced tap-tpg4 demonstrated the loss of multiple plasmids, and pSCL2 loss was suggested to be a direct consequence of the CRISPR-dCas9 system, as all mutants and vector controls lost the plasmid. Additionally, we demonstrated chromosome circularisation and the loss of the 13-nucleotide binding site of Tap for our tap-tpg₄, tap-tpg₃ and tap-tpg₂ silenced mutant EM10, suggesting that overall tap-tpg expression levels in S. clavuligerus affect end-patching. The mutant strains were phenotypically characterised and mutants which had lost pSCL3, pSCL2 and/or pSCL1 had a significantly higher specific growth rate, therefore we confirmed that these replicons were essential for strain fitness. We also confirmed that S. clavuligerus does not require pSCL3, pSCL2 and pSCL1 for clavulanic acid production, as strains that had lost more replicons did not produce more clavulanic acid. Therefore, biotechnologically, the loss of replicons is of importance in terms of strain fitness rather than clavulanic acid production in S. clavuligerus. Moreover, to study the process of endpatching and determine the binding activity of TPs, overexpression of Tap4, Tap3 and Tap2 proteins was optimised and Tap4 was shown to potentially bind to the chromosomal ssDNA telomeres. With this study we have elucidated the importance of tap-tpg in the plasmid maintenance of S. clavuligerus and confirmed the essentiality of replicons, highlighting that whilst most replicons were dispensable, they are necessary for maintaining strain fitness and optimum growth

In-house Optimization Radiolabeling of Recombinant scFv with 99mTc-Tricarbonyl and Stability Studies: Radiolabeling scFv with technetium tricarbonyl

His-tagged scFv fragments of monoclonal antibodies have better pharmacokinetic properties than whole antibodies. Radiolabeled scFvs are considered for targeted imaging and treatment. Technetium tricarbonyl provides radiolabeling of scFvs without losing its biological activity in a fast and easy procedure. Technetium tricabonyl was prepared as follows: A freshly eluted solution of Na99mTcO4 was added to a mixture containing sodium carbonate, sodium potassium tartarate, boranocarbonate, sodium borohydride. The mixture was heated for 30 min at 100°C. Radiochemical purity was determined using radio thin lyer chromatography. Then, technetium tricarbonyl was added to a solution of scFv in PBS buffer and incubated for 2 h at 50°C, purified by PD-10 column and radiochemical purity was determined. Results showed that radiochemical purity of technetium tricarbony was over 98%. The best conditions for radiolabeling of scFv was: scFv concentration >2 mg/mL, PBS buffer, 2 h incubation at 50°C, pH 8-9, and high activity concentration of tricarbonyl. The best radiochemical purity of scFv was 70% before purificarion. Radiolabeled scFv was stable in PBS for 24 h incubation and there was no release of technetium in competition with histidine. In this study, we optimized radiolabeling of a scFv with technetium tricarbonyl using house made boranocarbonates. The results are promising and will be used for future studies. HIGHLIGHTS Radiolabeling of scFv was done directly by 99mTc-tricarbonyl. 99mTc-tricarbonyl was prepared in house from boranocarbonate. 99mTc-Radiolabeled scFv can be used for radioimmunoscintigraphy

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