Analysis of Factors That Affect FlgM-Dependent Type III Secretion for Protein Purification with Salmonella enterica Serovar Typhimurium

Abstract

Conditions known to affect flagellar gene expression, FlgM stability, and flagellar T3S were tested either alone or in combination to determine their effects on levels of secreted FlgM. These conditions included mutations that affect activity of the flagellar FlhD 4 C 2 master regulatory protein complex or the FlgM T3S chaperone 28 , the removal of Salmonella pathogenicity island 1 (Spi1), the removal of flagellar late secretion substrates that could compete with FlgM for secretion, and changes in the ionic strength of the growth medium. Conditions that enhanced FlgM secretion were combined in order to maximize levels of secreted FlgM. An optimized FlgM secretion strain was used to secrete and isolate otherwise difficult-to-produce proteins and peptides fused to the C terminus of FlgM. These include cysteine-rich, hydrophobic peptides (conotoxins ␦-SVIE and MrVIA), nodulespecific, cysteine-rich antimicrobial peptides (NCR), and a malaria surface antigen domain of apical membrane antigen AMA-1.

Extracellular matrix-associated proteins form an integral and dynamic system during Pseudomonas aeruginosa biofilm development

“…Key protein structural components of the biofilm matrix were identified as CdrA and CupB5, which showed the highest abundance in phase I; this result was consistent with their roles in promoting biofilm formation and auto-aggregation (Borlee et al, 2010 ; Giraud et al, 2010 ). Moreover, the FlgM protein, which is secreted into the matrix in response to flagellar hook-basal body secretion (Guo et al, 2014 ), decreased by four-fold during biofilm development, which was consistent with the notion that motility is inhibited by biofilm development. The top 20 highly abundant proteins are listed in Supplementary Table 5 , and most of them were secreted proteins such as lipoproteins and outer membrane proteins such as OmpA.…”

Under Elevated c-di-GMP in Escherichia coli, YcgR Alters Flagellar Motor Bias and Speed Sequentially, with Additional Negative Control of the Flagellar Regulon via the Adaptor Protein RssB

“…Additionally, FlgM acts as a negative regulator of flagellin synthesis by directly binding 28 , serving as a crucial checkpoint in the staggered assembly of the entire flagellin complex (11). Acting as an anti-sigma factor, FlgM is also degraded by the ClpXP complex (66), adding regulatory complexity as these components are removed or accumulate within the system.…”

The Bacterial Flagellar Type III Export Gate Complex Is a Dual Fuel Engine That Can Use Both H+ and Na+ for Flagellar Protein Export

“…On testing flagellar promoter activities, however, the flagellar gene expression levels were slightly higher in the absence of NaCl than in its presence ( S6 Fig ). It has been shown that increased ionic strength facilitates the export of a flagellum-specific anti-sigma factor, FlgM, by wild-type cells, enhancing motility in soft agar [ 20 ]. Because neither Li + , K + nor Mg 2+ affected flagellar protein export by the Δ fliH-fliI flhB(P28T) bypass mutant ( Fig 1A , middle panel, lanes 8–10), we suggest that Na + is specific for this positive impact on flagellar protein export by the bypass mutant.…”