Modified E. coli strains enhance baculovirus production by elimination of aberrant transposition events

Baculovirus technology has been the most commonly used expression system for insect cells both due to its potential to generate a large amount of recombinant protein as well as the benefit of post-translational modifications. The most commonly used system to generate recombinant baculoviruses is the Tn7 transposition-based technology known as Bac-to-Bac. Although improvements have been made to this system to further improve quality and reproducibility of baculovirus production, recent data suggests that improved strains still have potential issues with contamination of non-recombinant baculovirus caused by improper transposition into a Tn7 site in the E. coli chromosome. Here we describe a new option for alteration of the E. coli genome to completely block the native Tn7 attachment site, leading to far fewer false positive bacmid colonies being selected and eliminating all risk of non-recombinant baculovirus production.

MDR Salmonella enterica serovar Typhimurium ST34 carrying mcr-1 isolated from cases of bloodstream and intestinal infection in children in China

Objectives Children are vulnerable to Salmonella infection due to their immature immune system. Cases of infection with mcr-1-harbouring Salmonella in child inpatients have not been reported in China before. Methods Salmonella isolates from gastroenteritis and bacteraemia were screened using primers targeting mcr-1. Complete genome sequences of mcr-1-harbouring isolates were determined using the PacBio RS II platform. The transferability of mcr-1-harbouring plasmids was verified by conjugation. Results We investigated two mcr-1-carrying polymyxin-resistant Salmonella enterica serovar Typhimurium ST34 isolates, S61394 and S44712, from bloodstream and intestinal Salmonella infection of two child inpatients, respectively. Both isolates were non-susceptible to commonly used antibiotics for children that compromised the success of clinical treatment and infection control. The mcr-1-harbouring plasmids pLS61394-MCR and pLS44712-MCR (from S61394 and S44712, respectively) were both conjugative pHNSHP45-2-like IncHI2-type epidemic plasmids carrying multiple resistance genes. Compared with pHNSHP45-2, a ∼33 kb insertion region encoding Tn7 transposition protein and heavy metal resistance proteins was identified in pLS61394-MCR, which might enhance adaptation of bacteria carrying this plasmid to various ecological niches. The phylogenetic tree of worldwide mcr-harbouring Salmonella indicated a host preference of mcr and a worldwide and cross-sectoral prevalence of the mcr-positive Salmonella ST34 clone. Conclusions To our knowledge, for the first time we report completed whole genomes of mcr-1-positive MDR Salmonella Typhimurium ST34 isolated from infected children in China, suggesting that improved surveillance is imperative for tackling the dissemination of mcr-harbouring MDR Salmonella Typhimurium ST34.

Gain-of-Function Mutations in TnsC, an ATP-Dependent Transposition Protein That Activates the Bacterial Transposon Tn7

The bacterial transposon Tn7 encodes five genes whose protein products are used in different combinations to direct transposition to different types of target sites. TnsABC+D directs transposition to a specific site in the Escherichia coli chromosome called attTn7, whereas TnsABC+E directs transposition to non-attTn7 sites. These transposition reactions can also recognize and avoid “immune” targets that already contain a copy of Tn7. TnsD and TnsE are required to activate TnsABC as well as to select a target site; no transposition occurs with wild-type TnsABC alone. Here, we describe the isolation of TnsC gain-of-function mutants that activate the TnsA+B transposase in the absence of TnsD or TnsE. Some of these TnsC mutants enable the TnsABC machinery to execute transposition without sacrificing its ability to discriminate between different types of targets. Other TnsC mutants appear to constitutively activate the TnsABC machinery so that it bypasses target signals. We also present experiments that suggest that target selection occurs early in the Tn7 transposition pathway in vivo: favorable attTn7 targets appear to promote the excision of Tn7 from the chromosome, whereas immune targets do not allow transposon excision to occur. This work supports the view that TnsC plays a central role in the evaluation and utilization of target DNAs.