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-signal-producing 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.

FIG. 1.

Scheme for separating membranes of M. xanthus DK1622 vegetative cells. Total membranes were separated with a three-step sucrose gradient by centrifugation for 3 h at 100,000 × g (A). Each membrane enrichment was concentrated (B) and separated with separate discontinuous sucrose gradients (C). The peaks were designated peaks I through VII.

FIG. 2.

Protein contents of the heavy membrane (A), medium-density membrane (B), and light membrane peaks (C) from discontinuous sucrose gradients as a function of absorbance at 280 nm (A₂₈₀) (squares) and buoyant density (diamonds).

FIG. 3.

Immunoblots of isolated membrane fractions containing LPS lipid A (A), LPS O-antigen (B), Tgl (a lipoprotein required for assembly of TFP) (C), CglB (a lipoprotein required for adventurous gliding) (D), PilA (the structural pilin protein for TFP) (E), FibA (an extracellular matrix-associated zinc metalloprotease) (F), CsgA (a protein required for C-signaling) (G), and FrzCD (a methylated chemotaxis protein) (H). Total membrane (lane TM), IM peak I (lane I), HM peak II (lane II), HM peak IV (lane IV), OM peak V (lane V), and OM peak VI (lane VI) fractions were separated by SDS-PAGE for Western blotting. The numbers on the left indicate molecular masses of protein standards (in kilodaltons).

FIG. 4.

Two-dimensional SDS-polyacrylamide gel separated in the first dimension by pH 3.5 to 10 and in the second dimension on 10% polyacrylamide. (A) IM peak I; (B) HM peak IV; (C) OM peak V; (D) OM peak VI. Molecular masses (MW) (in kilodaltons) are indicated on the left. A series of internal, carbamylated carbonic anhydrase markers ranging from pH 5 to 7.3 are underlined on each gel. The rectangles indicate acidic proteins common to IM peak I and HM peak IV which did not resolve well. The stars in panel A indicate protein spots common to IM peak I and HM peak VI. The numbers in panels C and D indicate clusters of proteins that are differentially expressed in the two OM peaks.

FIG. 5.

Fraction VII contains a PKS. (A) Polyacrylamide (7.5%) gel of fraction VII stained with Coomassie blue. The stars indicate protein bands analyzed by MALDI-TOF MS. (B) Predicted organization of the 7,831-amino-acid Ta-1 PKS. The conserved domains are as follows: C domain, A domain, ACP, KS/AT, and KR. The domains are organized in modules, which are basic units responsible for incorporation of extender units. The scale bar indicates the amino acid numbers in the primary structure of Ta-1.