Membrane Localization of Motility, Signaling, and Polyketide Synthetase Proteins in Myxococcus xanthus

Abstract

Myxococcus xanthus cells coordinate cellular motility, biofilm formation, and development through the use of cell signaling pathways. In an effort to understand the mechanisms underlying these processes, the inner membrane (IM) and outer membrane (OM) of strain DK1622 were fractionated to examine protein localization. Membranes were enriched from spheroplasts of vegetative cells and then separated into three peaks on a three-step sucrose gradient. The high-density fraction corresponded to the putative IM, the medium-density fraction corresponded to a putative hybrid membrane (HM), and the low-density fraction corresponded to the putative OM. Each fraction was subjected to further separation on discontinuous sucrose gradients, which resulted in discrete protein peaks for each major fraction. The purity and origin of each peak were assessed by using succinate dehydrogenase (SDH) activity as the IM marker and reactivities to lipopolysaccharide core and O-antigen monoclonal antibodies as the OM markers. As previously reported, the OM markers localized to the low-density membrane fractions, while SDH localized to high-density fractions. Immunoblotting was used to localize important motility and signaling proteins within the protein peaks. CsgA, the C-signalproducing protein, and FibA, a fibril-associated protease, were localized in the IM (density, 1.17 to 1.24 g cm ؊3 ). Tgl and Cgl lipoproteins were localized in the OM, which contained areas of high buoyant density (1.21 to 1.24 g cm ؊3 ) and low buoyant density (1.169 to 1.171 g cm ؊3 ). FrzCD, a methyl-accepting chemotaxis protein, was predominantly located in the IM, although smaller amounts were found in the OM. The HM peaks showed twofold enrichment for the type IV pilin protein PilA, suggesting that this fraction contained cell poles. Two-dimensional polyacrylamide gel electrophoresis revealed the presence of proteins that were unique to the IM and OM. Characterization of proteins in an unusually low-density membrane peak (1.072 to 1.094 g cm ؊3 ) showed the presence of Ta-1 polyketide synthetase, which synthesizes the antibiotic myxovirescin A.

A singular enzymatic megacomplex from Bacillus subtilis

“…One possibility is that a specific portion of every assembly line is associated with a membrane subdomain, either directly or by interaction with a membrane protein. This kind of membrane association has been observed in proteins from other assembly line enzymes (24,25). Another possibility is that the individual assembly line complexes associate with each other to form a megacomplex.…”

Combinatorial Regulation of thedevOperon by MrpC2 and FruA during Myxococcus xanthus Development

“…It involves CsgA (21), a 25-kDa protein (p25) that appears to associate with the inner membrane during cell growth (22) but with the outer membrane during development (23). Considerable evidence supports a model in which p25 is cleaved to a 17-kDa form (p17) that appears to be the C signal (19,(23)(24)(25).…”

Synthesis and Assembly of a Novel Glycan Layer in Myxococcus xanthus Spores

“…We were unable to adapt other previously employed methods for separating M. xanthus OM and CM membranes, such as outer membrane isolation (37) or sucrose density gradient fraction (38), likely because the envelope characteristics of sporulating cells differs significantly from that of the vegetative cells for which they were developed. Therefore, we set out to examine the localization patterns of the Nfs proteins produced heterologously in E. coli with the well defined and rigorous sucrose density gradient fractionation protocol (24,39).…”