Optimised Heterologous Expression and Functional Analysis of the Yersinia pestis F1-Capsular Antigen Regulator Caf1R

Abstract

The bacterial pathogen, Yersinia pestis, has caused three historic pandemics and continues to cause small outbreaks worldwide. During infection, Y. pestis assembles a capsule-like protective coat of thin fibres of Caf1 subunits. This F1 capsular antigen has attracted much attention due to its clinical value in plague diagnostics and anti-plague vaccine development. Expression of F1 is tightly regulated by a transcriptional activator, Caf1R, of the AraC/XysS family, proteins notoriously prone to aggregation. Here, we have optimised the recombinant expression of soluble Caf1R. Expression from the native and synthetic codon-optimised caf1R cloned in three different expression plasmids was examined in a library of E. coli host strains. The functionality of His-tagged Caf1R was demonstrated in vivo, but insolubility was a problem with overproduction. High levels of soluble MBP-Caf1R were produced from codon optimised caf1R. Transcriptional-lacZ reporter fusions defined the PM promoter and Caf1R binding site responsible for transcription of the cafMA1 operon. Use of the identified Caf1R binding caf DNA sequence in an electrophoretic mobility shift assay (EMSA) confirmed correct folding and functionality of the Caf1R DNA-binding domain in recombinant MBP-Caf1R. Availability of functional recombinant Caf1R will be a valuable tool to elucidate control of expression of F1 and Caf1R-regulated pathophysiology of Y. pestis.

The N-terminal helix of MarA as a key element in the mechanism of DNA binding

“…The same result was observed when using another antibody, in this case, an HRP-conjugated anti-his tag antibody, to reveal the membrane (Supplementary Figure S10). The low expression of WT MarA in shuffle T7 express cells is supported by a recent paper in which the authors observed by WB the decline of the signal corresponding to the His-tagged AraC/XylS transcriptional factor Caf1R when inducing in shuffle T7 express cells at 37°C 40 . As the fluorescence signal for WT MarA was high in the reporter system experiment (Figure 3A), we discarded the lack of overexpression of the protein and focused on its degradation.…”

MsmR1, a global transcription factor, regulates polymyxin synthesis and carbohydrate metabolism in Paenibacillus polymyxa SC2

“…MsmR1 is a typical AraC/XylS family transcriptional regulator (Egan, 2002), which can act as activators, repressors, or both, and are involved in various biological processes ranging from metabolism to stress response and virulence (Gahlot et al, 2021). In this study, a ChIP-seq experiment combined with EMSA was performed, the regulatory network of the global regulator MsmR1 was proposed (Figure 7), and the role of MsmR1 in P. polymyxa was described.…”

“…Sequence analysis revealed that MsmR1 comprises seven α-helices forming two HTH motifs at the C-terminal domain, which is the DNA binding domain, as well as a sensing or oligomerization domain of approximately 140 amino acids at the N-terminal domain. MsmR1 is a typical AraC/XylS family transcriptional regulator ( Egan, 2002 ), which can act as activators, repressors, or both, and are involved in various biological processes ranging from metabolism to stress response and virulence ( Gahlot et al, 2021 ). In this study, a ChIP-seq experiment combined with EMSA was performed, the regulatory network of the global regulator MsmR1 was proposed ( Figure 7 ), and the role of MsmR1 in P. polymyxa was described.…”

“…Previous studies focusing on the TFs of RhaR ( Kolin et al, 2008 ; Kettle, 2013 ), AraC ( Schleif, 2010 ), UreR ( Parra and Collins, 2012 ), and MelR ( Belyaeva et al, 2000 ) in Escherichia coli , MsmR ( Nakata et al, 2005 ) in Streptococcus , XylS ( Zwick et al, 2013 ) in Pseudomonas putida , and Toxt ( Lowden et al, 2010 ) in Vibrio cholerae revealed that they contain a highly conserved C-terminal DNA binding domain (DBD) and a variable ligand-binding domain (LBD) at the N-terminal ( Bustos and Schleif, 1993 ; Cortés-Avalos et al, 2021 ). The DBD contains approximately 100 amino acids, with approximately 20% sequence identity in bacteria, and seven α-helices, forming two helix-turn-helix (HTH) DNA-binding motifs ( Egan, 2002 ; Gahlot et al, 2021 ). While the DBD is in contact with RNA polymerase (RNAP) for DNA binding and transcription ( Ibarra et al, 2008 ), the LBD is involved in dimerization and effector/signal recognition ( Egan, 2002 ).…”

Unraveling the molecular determinants of the anti-phagocytic protein cloak of plague bacteria

“…The Caf1R protein regulates caf1 expression as part of a complex thermosensitive system that responds to host body temperature [ 26 , 27 ]. To ensure reproducible Caf1 levels we simplified caf1 expression in our assays by deleting caf1R , and so used only basal T7 expression from pT7-COPΔR.…”