PMC

SEM of EAEC 042 and 042orf3-4 biofilms. Strains 042 (A and B) and 042orf3-4 (C and D) were visualized by SEM; samples were visualized at a magnification of ×4,000 (A and C), ×20,000 (B), or ×30,000 (D).

AggR-regulated genes located in pAA2. (A) Transcriptional levels of plasmid-borne AggR-regulated genes were quantitated by qRT-PCR in 042 (black bars), 042aggR (open bars), and 042aggR(pBAD30aggR) (gray bars). (B) Graphic representation of the genes in the pAA2 plasmid.

AggR-regulated genes located in the chromosome. (A) Transcriptional levels of AggR-regulated genes were determined by qRT-PCR in 042 (black bars), 042aggR (open bars), and 042aggR(pBAD30aggR) (gray bars). (B) Graphic representation of the chromosomal genes regulated by AggR.

Microarray analysis of the AggR regulon. RNA samples of 042 (black bars) and 042ΔaggR (open bars) were hybridized in duplicate to an EAEC oligonucleotide microarray (NimbleGen, Inc.). (A) aggR transcription was evaluated in DMEM supplemented with glucose, bicarbonate, maltose, or bile salts. (B) Transcript levels for aap and aggR were determined by RT-PCR in 042, 042aggR, 042aggR(pBAD30), and 042aggR(pBADaggR), grown as described in the text.

ORF3 and ORF4 mutants are more susceptible to defensin and polymyxin B treatment. Strains were treated with polymyxin B (panel A), defensin-1 (panel B, open bars), or PBS (panel B, black bars) for 1 h. Cultures were serially diluted in PBS, and viable counts were determined by plating. Percent survival of the original inoculum was recorded after 24 h. The difference between groups was significant (P < 0.001; Kruskal-Wallis).

Homologs for ORF3 and ORF4. (A) ECO42_pAA003 (GenBank accession number gi|284924584) and homologs in Archaea (Halobacterium salinarum R1, GenBank accession number gi|169235957; Haladaptatus paucihalophilus DX253, GenBank accession number gi|322369459) and Cyanobacteria (Synechocystis sp. PCC 6803, GenBank accession number gi|16329282) were aligned. (B) ECO42_pAA004 (GenBank accession number gi|284924585) showed homology to isopentenyl-diphosphate delta isomerase (IDI) of Salmonella enterica serovar Typhi strain CT18 (GenBank accession number gi|16761820), Citrobacter rodentium ICC168 (GenBank accession number gi|283788453), E. coli HS (GenBank accession number gi|157162349), and 042 (GenBank accession number gi|284922837).

Mutations in ORF4 result in altered surface properties. Transmission electron microscopy of negatively stained samples was performed for 042 (A to C), 042orf4 (D to F), and 042orf4(pBADorf4) (G to I). The samples were stained with 2% uranyl acetate and examined on a Jeol JEM1230 transmission electron microscope (80 kV) at the Advanced Microscopy Laboratory at the University of Virginia (AML-UVA). Samples were visualized at a magnification of ×5,000 (A, D, and G), ×10,000 (B, E, and H), or ×25,000 (C, F, and I).