Engineering large functional plasmids for biosafety

Cangelosi, Chris; Shank, Caroline; Santiago, Clayton; Wilson, James W.

doi:10.1016/j.plasmid.2013.09.002

Cited by 1 publication

(5 citation statements)

References 36 publications

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“…All experiments were performed in accordance with established biosafety guidelines and have been approved by the investigators' institutional biosafety committee. Strains were grown in Lennox broth (LB), LB A c c e p t e d M a n u s c r i p t 5 0.3M NaCl, LB pH5.5, MgM10, and MgM8 media as previously described (Lennox 1955;Wilson and Nickerson 2006a;Wilson et al 2007;Cangelosi et al 2013). Deletion of invJ, prgI, and hilA from R995 + SPI-1 and deletion of ssrAB from R995 + SPI-2 were performed using standard recombineering techniques as described previously (Datsenko and Wanner 2000).…”

Section: Bacterial Strains Plasmids and Mediamentioning

confidence: 99%

“…Analysis of SPI-1 and SPI-2 protein expression via Western blot for SipC, SseB, p25, p15, HA, and Flag was performed as described previously (Wilson and Nickerson 2006a;Wilson et al 2007;Jennings et al 2012;Cangelosi et al 2013). Preparation of secreted SPI-1 and SPI-2 proteins from bacterial cultures was performed as described previously (Wilson and Nickerson 2006a;Wilson et al 2007;Jennings et al 2012;Cangelosi et al 2013).…”

Section: Protein Techniques and Western Blot Analysismentioning

confidence: 99%

“…Preparation of secreted SPI-1 and SPI-2 proteins from bacterial cultures was performed as described previously (Wilson and Nickerson 2006a;Wilson et al 2007;Jennings et al 2012;Cangelosi et al 2013). Briefly, bacterial cultures grown for 4 hours (late exponential phase) were centrifuged and supernatants were saved for the analysis of secreted proteins.…”

Section: Protein Techniques and Western Blot Analysismentioning

confidence: 99%

“…The R995 + SPI-1 construct has been demonstrated to mediate translocation of substrate proteins from S. Typhimurium bacteria to the inside eukaryotic host cells which is an important function of the SPI-1 T3SS (Wilson and Nickerson 2006a;Jennings et al 2012;Cangelosi et al 2013). …”

Section: R995 + Spi-1 Is Defective For Translocation Activity and Formentioning

confidence: 99%

“…To study this phenomenon, previous analysis of SPI-2 gene expression has established the use of different minimal media termed MgM8 (low magnesium, pH=5.5) and MgM10 (high magnesium, pH=7.5) (Beuzon et al 1999;Deiwick et al 1999). In addition, though it is established that SPI-2 expression can be achieved in LB media, the use of LB media with low pH (pH=5.5) can induce greater SPI-2 expression and activity (Hansen-Wester et al 2004;Cangelosi et al 2013). Interestingly, cloned SPI-2 is expressed in S.…”

Section: The Role Of Key Transcriptional Regulators In Cloned Spi-1 Amentioning

confidence: 99%

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Transfer of the cloned Salmonella SPI-1 type III secretion system and characterization of its expression mechanisms in Gram negative bacteria in comparison with cloned SPI-2

Cangelosi

Hannagan

Santiago

et al. 2015

Microbiological Research

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Cloned type III secretion systems have much potential to be used for bacterial engineering purposes involving protein secretion and substrate translocation directly into eukaryotic cells. We have previously cloned the SPI-1 and SPI-2 type III systems from the Salmonella enterica serovar Typhimurium genome using plasmid R995 which can conveniently capture large genomic segments for transfer between bacterial strains. However, though expressed and functional in Salmonella strains, cloned SPI-1 was previously observed to have a serious expression defect in other Gram negative bacteria including Escherichia coli. Here we show that cloned SPI-1 expression and secretion can be detected in the secretion preps from E. coli and Citrobacter indicating the first observation of non-Salmonella SPI-1 expression. We describe a compatible plasmid system to introduce engineered SPI-1 substrates into cloned SPI-1 strains. However, a SPI-1 translocation defect is still observed in E. coli, and we show that this is likely due to a defect in SipB expression/secretion in this species. In addition, we also examined the requirement for the hilA and ssrAB regulators in the expression of cloned SPI-1 and SPI-2, respectively. We found a strict requirement for hilA for full cloned SPI-1 expression and secretion. However, though we found that ssrAB is required for full cloned SPI-2 expression in a range of media across different bacteria, it is not required for cloned SPI-2 expression in MgM8 inducing media in S. Typhimurium. This suggests that under SPI-2 inducing conditions in S. Typhimurium, other factors can substitute for loss of ssrAB in cloned SPI-2 expression. The results provide key foundational information for the future use of these cloned systems in bacteria.

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