FIGURE 4.

Electron micrographs of P. tunicata revealing presence/absence of a square grid S‐layer in WT and Δslr4 cells, respectively. (a) Verification of Δslr4 (EAR28894 gene deletion) mutant in P. tunicata PnAD3. PCR amplification of the PnAD3 genomic DNA with oligo JC470 and JC492 yielded a DNA fragment of same size (1,086 bp) as that from plasmid pJC272, but a 2814‐bp product was obtained from wild type (WT) DNA. Lane L, DNA marker; lane 1, P. tunicata PnAD3; lane 2, plasmid pJC272; lane 3, P. tunicata WT D2. (b) SDS‐PAGE analysis of whole cell extract of WT versus Δslr4 strains. (c) TEM micrographs of WT vs (d) Δslr4 cells, revealing the presence and absence of an S‐layer, respectively. A side‐view of S‐layer revealing U‐shaped subunits is indicated by an arrow in (c). Samples were derived from liquid cultures grown in marine Difco broth for 8 hr in shaking conditions. Scale bars = 100 nm