Conjugative transfer of mcr-1-bearing plasmid from Salmonella to Escherichia coli in vitro on chicken meat and in mouse gut.

Since mcr-1 was first discovered in 2015, this gene has shown excellent transmission ability and evolutionary characteristics worldwide, leading to major public health and food safety concerns. In this study, chicken meat was used as a food vehicle for the conjugation of mcr-1-bearing Salmonella at different storage temperatures (4 °C, 25 °C, and 37 °C) to simulate mcr-1 transmission during food transportation and storage and determine its efficiency and mechanism. In addition, conjugation experiments were performed in mouse gut to further confirm that mcr-1 is horizontally transferred in vivo during food consumption. 16S rDNA sequencing of mouse stool samples was performed to understand the effect of horizontal transfer of mcr-1 on mouse gut bacteria. mcr-1-bearing plasmids were characterized using pulsed-field gel electrophoresis (PFGE) and S1 nuclease-PFGE and sequenced by Illumina sequencing. Our results showed that mcr-1-bearing plasmids in donors are successfully transferred to recipients on chicken meat at not only 25 °C and 37 °C but also 4 °C with conjugation frequencies between 1.32 × 10-6 and 3.85 × 10-4 per recipient cell. In mouse gut, mcr-1 was transferred not only to the recipient bacteria introduced by intragastric administration but also to the intestinal bacteria (E. coli strain named as E6353). Horizontal transfer of mcr-1-bearing plasmid in mouse gut negatively affected the mouse intestinal microbiota. In a constant conjugative environment, plasmid replicon type is the most decisive factor affecting the conjugation frequency. The peak number of transconjugants in group D6-E. coli C600 with an IncHI2-type mcr-1-bearing plasmid (1.43 × 102 colony-forming units [CFU]/g feces) was significantly higher than that of transconjugants in group D7-E. coli C600 with an IncX4-type mcr-1-bearing plasmid (0.3 × 102 CFU/g feces). The upstream and downstream genetic environment of mcr-1 in different plasmid replicon types in Salmonella varied during conjugation in different horizontal transfer environments. An IncI2 plasmid (p25-D4R7S1_mcr-1) lost the insertion sequence ISApl1, which originally existed upstream of mcr-1, when this plasmid transferred from donor to recipient cells on chicken meat at 25 °C. An IncHI2 plasmid was more active than IncI2 and IncX4 plasmids during bacterial reproduction and evolution; an IncFIB-IncHI2 hybrid plasmid (p6176253_mcr-1) was formed in mouse gut during conjugation from pD6_tet(M) and pD6_mcr-1. mcr-1 is captured by mobile genetic elements IS26 in IncX4 plasmids and ISApl1 in IncI2, IncHI2, and IncFIB-IncHI2 hybrid plasmids and is disseminated among bacteria.

Crystal Structure Analysis of Cationic Peroxidase from Proso Millet and Identification of Its Phosphatase Active Sites.

Proso millet peroxidase (PmPOD) belongs to class III plant peroxidases, which are enzymes typically characterized by their heme coenzymes. PmPOD exhibits not only heme-dependent peroxidase activity but also heme-independent phosphatase activity. Crystal structure analysis and sequence alignment showed that PmPOD contained a phosphatase catalytic loop CXXXXXR in its ß-domain that is similar to the active site of a dual-specific phosphatase. Recombinant truncated proso millet peroxidase (tPmPOD), which contained only a conserved catalytic loop CXXXXXR of phosphatase, was found to exhibit phosphatase activity. Five tPmPOD mutants containing five different mutations in the phosphatase active sites exhibited significantly lower phosphatase activity compared to that of tPmPOD, indicating that the five amino acids play important roles in the phosphatase activity of tPmPOD. Finally, nucleophilic amino acid Cys192 formed a disulfide bond with Cys219 to protect the stability of a sulfhydryl group; thus, it may play a decisive role in the phosphatase activity of PmPOD.

Peroxidase from proso millet exhibits endonuclease-like activity.

In this study, the mechanism of DNA cleavage by cationic peroxidase from proso millet (PmPOD) was investigated. PmPOD cleaved supercoiled circular DNA into both nicked circular and linear forms via a cleavage mechanism that resembles those of native endonucleases. Inhibition and ligation studies demonstrated that reactive oxygen species and the ferriprotoporphyrin IX moiety in PmPOD are not involved in PmPOD-mediated DNA cleavage. Similar to other endonucleases, Mg ions considerably enhance the DNA cleavage activity of PmPOD. Further studies suggested that PmPOD can disrupt phosphodiester bonds in DNA and mononucleotides, indicating that it is a phosphatase. The phosphatase activity of PmPOD is higher than that of horseradish peroxidase (HRP), but the peroxidase activity of PmPOD was lower than that of HRP. PmPOD-mediated hydrolytic cleavage of DNA observed in this study is different from those reported for heme proteins. This study provides valuable insights into the distinct mechanisms underlying DNA cleavage by heme proteins.