Etk and Etp are required for synthesis of G4C. Total polysaccharides (LPS and capsular polysaccharide) were extracted, and the capsular polysaccharide was separated from the LPS. Production of LPS O antigen was analyzed by Western blotting (A), and the amount of purified capsular polysaccharide was estimated by dot blot assay (B). In both cases, care was taken to apply amounts of material equivalent to the same amount of bacteria in all the lanes. The Western blot and the dot blots were developed with anti-O127 antiserum. The genotypes of the analyzed strains are indicated above the lanes. The etk::kan and the Δetp::Cm mutants did not produce capsules (B) but synthesized greater amounts of the LPS side chain (A). Complementation of the mutants with plasmids expressing etk or etp partially restored capsule production (B) and reduced synthesis of the LPS side chain (A). Panel C shows the resistance of the etk::kan mutant to complement-mediated killing. Early-log cultures were incubated with fresh or inactivated human serum. Wild-type EPEC cells were killed rapidly by the fresh serum (line 1) but not by the heat-inactivated serum (line 4). The EPEC etk::kan mutant (line 3) was highly resistant to fresh serum; complementation of the mutant with plasmid expressing etk (line 2) resulted in a partial increase in serum sensitivity. All experiments were carried out in triplicate, and the standard error is indicated.

Inactivation of each of the G4C operon genes results in lack of buoyancy on a Percoll step gradient. Each of the G4C operon genes was inactivated. To confirm nonpolarity, expression of Etk by the different mutants was determined using Western blot analysis with anti-Etk antibody (A). The genotype of the tested EPEC is indicated above each lane. SN40 is an EPEC strain containing a mini-Tn10kan polar insertion in ymcA. The wild-type strain is indicated as EPEC. Below the blot, the results of the buoyancy assays using the corresponding mutants (B) and the mutants complemented with plasmid expressing the corresponding gene (C) are shown. Buoyancy on Percoll is indicated as a plus sign, lack of buoyancy is indicated as a minus sign, and a slash indicates that buoyancy was not tested.

Expression of Etk and its relation to buoyant density and capsule formation. Bacterial proteins were extracted and used for immunoblotting to which an anti-Etk antibody was applied (A). Etk is expressed by wild-type EPEC (lane 1) and by the EPEC etk::kan mutant complemented with a plasmid expressing etk (lane 3), but not by the EPEC etk::kan mutant (lane 2). The bacterial buoyancy assay is shown in panel B. Bacteria were grown to an OD₆₀₀ of 0.3, and 5 ml of the cultures was underlaid with 2 ml Percoll in a transparent tube and centrifuged. Wild-type EPEC (tube 1) and an etk::kan mutant containing a plasmid expressing etk (tube 3) appear at the Percoll-medium interface (arrow), whereas the etk::kan cells (tube 2) form a pellet. Panel C shows electron microscopy images of the Etk-dependent capsule formed by EPEC O127. The etk::kan mutant appears noncapsulated, whereas the wild-type strain and the etk::kan mutant complemented with an etk-expressing plasmid form capsules (indicated by arrows).

The G4C operon. Schematic of the G4C operon and flanking genes (cspH and appA) (A). The location of the G4C operon promoter is indicated by an arrow. Mapping of the G4C promoter (B). Total RNA was extracted from exponential- and stationary-phase EPEC cultures (OD₆₀₀ of 0.3 and 1, respectively). Primer extension analysis (3 μg, total RNA) of the ymcD promoter region and the sequencing reaction were carried out using the same primer. Plasmid pAP2133 served as a template for the sequencing reaction. The transcription start site was mapped to a G residue located 166 bases upstream of the ymcD translation start site. In panel C, Western analysis with anti-Etk antibody was used to compare wild-type EPEC with strain AD2119, in which a polar cassette between ymcD and the promoter was inserted. The polar insertion abolished Etk expression, confirming that ymcD through etk are expressed by the same transcriptional unit.