e, Escherichia coli) (Blattner et al, 1997; Dippel & Boos, 2005

e., Escherichia coli) (Blattner et al., 1997; Dippel & Boos, 2005). Based on the sequence

annotation, the genetic information encoded on pPag3 corresponds with the previously described phenotypic characteristics of nonpigmented variants check details of P. agglomerans (i.e., thiamine deficiency, lack of maltose utilization, no pigmentation) (Chatterjee & Gibbins, 1971; Gantotti & Beer, 1982; Lindh et al., 1991), indicating that the plasmids in both species likely have similar features. In addition, when multiplying the size of pPag3 (530 kb) with the average molecular weight of a base pair (660 Da), the molecular weight of pPag3 obtained is in agreement with the observed 350 MDa plasmid reported from P. agglomerans (ex E. herbicola) Eh112Y (Gantotti & Beer, 1982). A nonpigmented variant of P. vagans C9-1, designated C9-1W, was obtained (Fig. 1). The identity of C9-1W as a derivative rather than as a contaminant was confirmed with 100%gyrB sequence identity compared with C9-1. The distinctive white colony color is attributable to loss

of the carotenoid biosynthetic gene cluster located on pPag3. Genotyping with multiple primer pairs targeting parts of the three plasmids in P. vagans C9-1 NVP-AUY922 ic50 confirmed the absence of pPag3 and the presence of pPag1 and pPag2. The plasmid sequence data identified a thiamine biosynthetic cluster (thiOSGF). Whether these are required for thiamine biosynthesis was unclear because several other genes (thiBCDEIJKLMPQ) known from pathways in prokaryotes (Begley et al., 1999; Settembre et al., 2003) are found scattered on the P. vagans C9-1 chromosome (Smits et al., 2009). When tested

on glucose-amended minimal media, C9-1W only grew with a thiamine supplement. This confirms that plasmid-borne Ixazomib solubility dmso thiOSGF are essential for thiamine autotrophy in P. vagans C9-1, and may explain thiamine auxotrophy reported for the nonpigmented variant, plasmid-cured P. agglomerans (Chatterjee & Gibbins, 1971; Gantotti & Beer, 1982). Substitution of glucose with maltose in the minimal medium resulted in no growth regardless of the presence/absence of thiamine. This further demonstrates that maltose utilization (Dippel & Boos, 2005) is conferred by genes located on pPag3 (Pvag_pPag30206–Pvag_pPag30215). When P. vagans C9-1W was grown with sucrose or sorbitol as the sole carbon sources, thiamine was again found to be the critical parameter for growth. This confirms the thiamine auxotrophy of P. vagans C9-1W, and also the retention of pPag1 (containing sucrose metabolic genes) and pPag2 (containing sorbitol metabolic genes) in the variant. Plasmid pPag3 also contains two genes with high sequence identity to a β-lactamase bla and its cognate regulator ampR (Pvag_pPag30395–Pvag_pPag30396). Ampicillin resistance has been reported to occur commonly in P. agglomerans clinical isolates (Cruz et al., 2007). Unlike the wild-type strain P.

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