ATCC 17978 has served as such a model system for research on pathogenic spp. of these antibiotics were partially relieved by exogenous THF addition, indicating that Smx and Tmp turn off Csu assembly by inducing folate stress. We propose that, for play particularly important roles in establishing infection (2,C4). A CU pathway for pilus biogenesis is present in most medically relevant species (5). SU9516 The genes for this CU pathway are encoded in a single six-gene operon, termed (6). In the presence of the CsuC chaperone, CsuA/B polymerizes to form the major pilus subunit (7). Additional experimental and bioinformatic analyses have provided evidence that CsuD functions as the usher and CsuE as a tip adhesin, while CsuA and CsuB may play roles as minor pilin subunits (5, 6, 8). Despite its being conserved in many sequenced strains, the role of Csu has been characterized only for ATCC 19606, Rabbit polyclonal to AEBP2 an isolate used in many laboratories (5, 6). Genetic studies in this strain have shown that mutation of or completely abolishes expression of Csu pili and prevents biofilm formation (5). Additionally, the two-component regulatory system BfmRS is involved in Csu expression, as a mutant did not express CsuA/B and was unable to form biofilms (6). Interestingly, previous genome sequence analyses suggested that other strains, including the commonly used ATCC 17978 (17978) SU9516 strain, encode a nonfunctional Csu operon due to inactivating single-nucleotide polymorphisms (9, 10). However, those observations have not been phenotypically confirmed. We recently resequenced ATCC 17978 (GenBank accession no. “type”:”entrez-nucleotide”,”attrs”:”text”:”CP012004″,”term_id”:”884898580″CP012004), which led to the identification of a previously unreported plasmid, pAB3. We found that, when cells were cultured under standard laboratory conditions, pAB3 was readily lost by a subset of 17978 cells, leading to activation of the type VI secretion system (T6SS) (11), and we suggested that this may be a differentiation process SU9516 that yields SU9516 two distinct bacterial types. Originally, these two cell types were termed Ab17978T6+ (containing no pAB3 plasmid [pAB3?]) and Ab17978T6? (containing the pAB3 plasmid [pAB3+]), in SU9516 reference to their T6SS-related phenotypes. Here, we update our nomenclature and refer to these cells as 17978pAB3? and 17978pAB3+, respectively, to indicate cells that either do not or do harbor the plasmid. Importantly, the chromosomal sequences of 17978pAB3? and 17978pAB3+ are identical. In addition to playing a regulatory role for T6SS, we sought to determine whether pAB3 regulated additional chromosomal genes, such as Csu. Here, we present data showing that Csu pili are expressed and are essential for biofilm formation in ATCC 17978. We found that subinhibitory concentrations of the antifolate antibiotics sulfamethoxazole (Smx) and trimethoprim (Tmp), which are used to maintain the pAB3 plasmid, are able to repress CsuA/B expression. This result indicates that pilus expression is tightly linked to folate metabolism. RESULTS ATCC 17978 produces Csu pili, which are required for biofilm formation. To investigate the effects of carrying the pAB3 plasmid on gene expression, we carried out transcriptome sequencing (RNA-seq) experiments to compare the transcriptomes of 17978pAB3? and 17978pAB3+. This analysis indicated that T6SS was regulated by the plasmid (12) but, unexpectedly, we found that the Csu pili were also induced 5-fold in 17978pAB3? cells. Previous comparative genomic analyses using the original 17978 sequence (GenBank accession no. “type”:”entrez-nucleotide”,”attrs”:”text”:”CP000521″,”term_id”:”126385999″CP000521) reported that the gene contained a single-base-pair insertion, resulting in a truncated coding sequence, which led to the hypothesis that 17978 cells were unable to produce functional Csu pili (9, 10). However, inspection of the recently updated genome sequence (GenBank accession no. “type”:”entrez-nucleotide”,”attrs”:”text”:”CP012004″,”term_id”:”884898580″CP012004) revealed that the entire gene is actually intact and.