Glycosyltransferases generally display low primary sequence similarities among each other despite high structural homology (Breton et al., 2006; Lairson et al., 2008). Amino acid sequences of glycosyltransferases Saracatinib manufacturer involved in lipopolysaccharide biosynthesis were retrieved from GenBank and compared with that of Ssg. Interestingly, Ssg of KL28 is related to WbpL (10.4% identity and 17.7% similarity) and WapR (10.4% identity and 15.1% similarity). WbpL and WapR have been described as bifunctional glycosyltransferase and rhamnosyltransferase, respectively, and play pivotal roles in P. aeruginosa PAO1 lipopolysaccharide biosynthesis (Rocchetta et al., 1998; Poon et al., 2008). An

ssg-in-frame-deletion mutant of KL28 was generated and used for phenotypic

characterization. Similar to the transposon mutant C23, the colonies of KL28Δssg(pBBR1MCS-5) exhibited a smooth, shiny surface (Fig. 2a2) as compared with the characteristic wrinkled surface of wild-type KL28. In addition, when compared with KL28, which is known for its unique, highly branched SAS, the mutant strain showed a defect in SAS development. Although wild-type and mutant strains initiated the formation of glittering domes over the same incubation period (Fig. 2b2, arrows), the mutant strain failed to form highly branched tips (Fig. 2b1 and b3, arrows), which are reservoirs of metabolically inactive ultramicrocells (Lee & Veeranagouda, CP-690550 in vitro 2009). Complementing the mutant by adding ssg in trans restored the colony morphology and SAS development as seen in the wild-type strain (Fig. 2a3 and b3). Further, we examined cell-surface-related properties such as motility on soft agar and biofilm

formation. When the level of surface BCKDHA spreading was examined on 0.3% agar, KL28(pBBR1MCS-5) exhibited a characteristic surface spreading (29.6±1.8 mm) with a wrinkled colony growth pattern. Interestingly, KL28Δssg(pBBR1MCS-5) formed a smooth colony pattern with slightly reduced surface spreading (20.8±1.9 mm). On the other hand, surface spreading on 0.8% agar by the mutant was significantly affected when compared with the wild-type strain KL28; the average colony diameters on 0.8% LB agar by the wild type with empty vector [KL28(pBBR1MCS-5)], the mutant [KL28Δssg(pBBR1MCS-5)] and the complemented strain [KL28Δssg(pSsg)] were 14±0.7, 6±0.4, and 15±2 mm, respectively (Fig. 2c1–c3). In addition, the mutant strain also failed to form characteristic wrinkling; rather it exhibited a smooth, shiny colony appearance. When strain KL28 was inoculated into LB liquid medium contained in a Petri plate, the culture formed unique circular pellicles at the air/medium interface (Fig. 2d1). These structures were robust and exhibited characteristic boundaries. The structures became fully grown to 0.46±0.04 mm in diameter within 48 h.