Type II Secretion System Secretin PulD Localizes in Clusters in theEscherichia coliOuter Membrane

Abstract

The cellular localization of a chimera formed by fusing a monomeric red fluorescent protein to the C terminus of the Klebsiella oxytoca type II secretion system outer membrane secretin PulD (PulD-mCherry) in Escherichia coli was determined in vivo by fluorescence microscopy. Like PulD, PulD-mCherry formed sodium dodecyl sulfate-and heat-resistant multimers and was functional in pullulanase secretion. Chromosomeencoded PulD-mCherry formed fluorescent foci on the periphery of the cell in the presence of high (plasmidencoded) levels of its cognate chaperone, the pilotin PulS. Subcellular fractionation demonstrated that the chimera was located exclusively in the outer membrane under these circumstances. A similar localization pattern was observed by fluorescence microscopy of fixed cells treated with green fluorescent protein-tagged affitin, which binds with high affinity to an epitope in the N-terminal region of PulD. At lower levels of (chromosome-encoded) PulS, PulD-mCherry was less stable, was located mainly in the inner membrane, from which it could not be solubilized with urea, and did not induce the phage shock response, unlike PulD in the absence of PulS. The fluorescence pattern of PulD-mCherry under these conditions was similar to that observed when PulS levels were high. The complete absence of PulS caused the appearance of bright and almost exclusively polar fluorescent foci.

Outer Membrane Targeting, Ultrastructure, and Single Molecule Localization of the Enteropathogenic Escherichia coli Type IV Pilus Secretin BfpB

“…To test the hypothesis that BFP components localize to the cell pole, we fused either the gene for photoactivatable mCherry (PAmCherry) or mOrange to the 3= end of bfpB and examined the cellular distribution of the fusion proteins using PALM or traditional epifluorescence. These fluorescent proteins were chosen because mCherry was successfully fused to the PulD of Klebsiella oxytoca in a previous report (12) and mOrange, like mCherry, is a derivative of DsRed (71). Indeed, we found that the bfpB-mOrange fusion could complement the bfpB mutant in trans and restore autoaggregation, while the bfpB-mOrange and bfpB-PAmCherry fusions in the context of the entire BFP operon could confer the capacity to autoaggregate on the ALN92 laboratory strain of E. coli that carries a constitutively activated cell envelope sensor in the form of the cpx mutation.…”

The Essential Escherichia coli Apolipoprotein N-Acyltransferase (Lnt) Exists as an Extracytoplasmic Thioester Acyl-Enzyme Intermediate

“…After transfer onto nitrocellulose membranes, proteins were detected by incubating the membrane with antibodies against c-Myc (Sigma) followed by horseradish peroxidase conjugated secondary antibodies to rabbit immunoglobin G (Amersham). Secondary antibodies were detected by chemiluminescence (Thermo Scientific) (8). …”

Structural insights into the Type II secretion nanomachine

“…In vivo fluorescence microscopy studies indicate that secretins promote focal localization of other T2SS proteins [47,48]. Secretin monomers must pass through the peptidoglycan during transfer to the OM, in a process that may be independent of the classic OM β-barrel assembly machinery [49].…”